JEM Neuroscience Special Issue

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SELECTED ARTICLES NOVEMBER 2014 www.jem.org
NEUROSCIENCE
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Welcome
N e u ro s c i e n c e
The Journal of Experimental Medicine now prints topic-specific mini collections to showcase a handful
of our recent publications. In this installment, we highlight papers focusing on the mechanisms and
models of neurological disease.
Myelin destruction in multiple sclerosis (MS) is mediated by inflammatory macrophages,
but the origin of these cells has been unclear. Our collection begins with an Insight from Michael
Heneka discussing findings from Yamasaki et al. who use a mouse model of MS to distinguish
tissue-resident microglial cells from infiltrating monocytes. Using double chemokine reporter mice,
the authors find that monocyte-derived macrophages initiate myelin destruction, mainly in the
nodes of Ranvier, while microglia-derived macrophages are involved with clearing debris. In a
different type of central nervous system (CNS) injury, ODonovan et al. demonstrate that activation
of intraneuronal B-RAF kinase is sufficient to drive axon regeneration after nerve crush injury.
An Insight from Valeria Cavalli and David Holtzman discusses how reactivation of the B-RAF
pathway, which appears to be quiescent in central axons, can be utilized for regrowth.Together, these studies offer new strategies
to treat inflammatory pathologies and promote repair.
Acute cerebral ischemia reperfusion injury is mediated in part by T cells. Clarkson et al. show that brain-infiltrating
CD4+ T cells sustain neuroinflammation after stroke in mice by producing interleukin (IL)-21 and increasing neuronal death.
Treatment with an IL-21 decoy receptor or genetic lack of IL-21 protected animals from brain injury following stroke, offering
a potential target for immunotherapy. Additionally, analysis of postmortem human brain tissue confirmed that IL-21 localizes to
CD4+ T cells surrounding acute stroke lesions.
Loss of cells in the retina is one of the earliest signs of frontotemporal dementia (FTD), occurring even before behavioral
changes appear. Ward et al. show that mislocalization of CNS protein TDP-43 in eye neurons is associated with retinal thinning
and occurs before the neurologic symptoms of FTD develop. Misplaced TDP-43 appears to be due to low expression of the
TDP-43 regulating protein Ran, as boosting Ran expression corrects TDP-43 localization and increases neuron survival in the
eye. Understanding the mechanisms underlying FTD could lead to novel therapeutic targets.
Cerebral vascular abnormalities in Alzheimers disease (AD) have been shown to correlate with the degree of cognitive
impairment. Strickland and colleagues describe a small molecule, RU-505, that inhibits the interaction between amyloid-b (Ab)
and the blood clotting protein fibrinogen, reducing vascular pathologies and ameliorating cognitive impairment in mouse models
of AD. Thus targeting neurovascular pathology may offer new a therapeutic strategy for treating AD.
In AD, various biochemical functions of brain cells can also go awry, leading to progressive neuronal damage and eventual
memory loss. Impaired autophagy causes the accumulation of toxic protein plaques characteristic of the disease. Bae and colleagues
find elevated levels of acid sphingomyelinase (ASM), which breaks down cell membrane lipids in the myelin sheath that coat nerve
endings. Reducing levels of ASM in mice with AD-like disease restored autophagy, lessened brain pathology, and improved learning
and memory in the mice.
Together these studies provide new insights into the biology and mechanisms of neurologic diseases and offer insight into
therapeutics. We hope you enjoy this complimentary copy of our Neuroscience collection. We invite you to explore additional
collections at www.jem.org and to follow JEM on Facebook, Google+, and Twitter.
Selected Articles November 2014
Macrophages derived from infiltrating monocytes mediate autoimmune myelin destruction

Michael T. Heneka
Dierential roles of microglia and monocytes in the inflamed central nervous system
Ryo Yamasaki, Haiyan Lu, Oleg Butovsky, Nobuhiko Ohno, Anna M. Rietsch, Ron Cialic, Pauline M. Wu,
Camille E. Doykan, Jessica Lin, Anne C. Cotleur, Grahame Kidd, Musab M. Zorlu, Nathan Sun, Weiwei Hu,
LiPing Liu, Jar-Chi Lee, Sarah E. Taylor, Lindsey Uehlein, Debra Dixon, Jinyu Gu, Crina M. Floruta, Min Zhu,
Israel F. Charo, Howard L. Weiner, and Richard M. Ransohoff
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B-RAF unlocks axon regeneration

Valeria Cavalli and David M. Holtzman
B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured
mature CNS
Kevin J. ODonovan, Kaijie Ma, Hengchang Guo, Chen Wang, Fang Sun, Seung Baek Han, Hyukmin Kim,
Jamie K. Wong, Jean Charron, Hongyan Zou, Young-Jin Son, Zhigang He, and Jian Zhong
T cellderived interleukin (IL)-21 promotes brain injury following stroke in mice
Benjamin D.S. Clarkson, Changying Ling, Yejie Shi, Melissa G. Harris, Aditya Rayasam, Dandan Sun,
M. Shahriar Salamat, Vijay Kuchroo, John D. Lambris, Matyas Sandor, and Zsuzsanna Fabry
Early retinal neurodegeneration and impaired Ran-mediated nuclear import of TDP-43 in
progranulin-deficient FTLD
Michael E. Ward, Alice Taubes, Robert Chen, Bruce L. Miller, Chantelle F. Sephton, Jeffrey M. Gelfand,
Sakura Minami, John Boscardin, Lauren Herl Martens, William W. Seeley, Gang Yu, Joachim Herz,
Anthony J. Filiano, Andrew E. Arrant, Erik D. Roberson, Timothy W. Kraft, Robert V. Farese, Jr.,
Ari Green, and Li Gan
A novel Ab-fibrinogen interaction inhibitor rescues altered thrombosis and cognitive decline in
Alzheimers disease mice
Hyung Jin Ahn, J. Fraser Glickman, Ka Lai Poon, Daria Zamolodchikov, Odella C. Jno-Charles, Erin H. Norris,
and Sidney Strickland
Acid sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in
Alzheimers disease
Jong Kil Lee, Hee Kyung Jin, Min Hee Park, Bo-ra Kim, Phil Hyu Lee, Hiromitsu Nakauchi, Janet E. Carter,
Xingxuan He, Edward H. Schuchman, and Jae-sung Bae
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INSIGHTS
M acrophages der ived from infiltrating monocytes mediate
autoimmune myelin destruction
<doi>10.84/jem2nsight1<do>ajem.218insght</ad>uMicelT.Hnka</>AF1UiverstyofBn</AF1>crmihael.nk@ub-ode</cr>haInsigt</doce> piNws</doct>
Macrophages mediate myelin destruction in multiple sclerosis (MS), but the origin of these cells (whether derived from tissue-resident microglial cells or infiltrating monocytes) has been widely debated. Now, Yamasaki
and colleagues distinguish these cells in a mouse model of MS and show that monocyte-derived macrophages
(MDMs) mediate myelin destruction, whereas microglia-derived macrophages (MiDMs) clear up the debris.
Previous attempts to decipher the nature and role of cells involved in autoimmune demyelination have
proven challenging. Although ontogenetically distinct, it has not been possible to distinguish macrophages
derived from tissue-resident or -infiltrating cells based on morphological features (by light microscopy) or
surface phenotype. Previous attempts to address this problem include parabiosis and bone marrow transInsight from
plantation after irradiation, both strategies with substantial technical problems and limitations.
Michael Heneka
Yamasaki et al. studied double chemokine receptor (CCR2-RFP+;
CX3CR1-GFP+) mice in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Inflammatory lesions were filled with both MDMs and
MiDMs. Confocal immunohistochemistry, serial block-face scanning electron microscopy
(SBF-SEM), and subsequent 3D reconstruction revealed that myelin destruction was initiated
by MDMs, often at the nodes of Ranvier, whereas MiDMs were not detected at this site.
Disruption of MDM infiltration by CCR2 deficiency completely abolished the presence of
macrophages at the nodes of Ranvier. Gene expression profiling of both cell types at disease
onset revealed substantial differences, which correlated well with the observations obtained
by SBF-SEM. MDMs expressed genes attributable to effector functions, including those
involved in phagocytosis and cell clearance. In contrast, MiMD gene expression patterns at
disease onset were characteristic of a repressed metabolic state.
This paper sets a new standard for further studies in the field. For the first time,
MDMs and MiDMs have been clearly differentiated and their morphological relationship to axoglial structures has been analyzed. The finding that MDMs rather than
MiDMs initiate myelin destruction at disease onset should enable this cell population to
be targeted more effectively in future. The next stage is to verify these findings in human
tissue. Future research should also assess further time points over the entire disease
Nodes of Ranvier represent a prime
course, in particular to exclude that MiDMs do not join MDMs at the node of Ranvier
site of attack for MDMs at the onset
at later stages of disease. A precise distinction between local and infiltrating cell populaof EAE. This 3D reconstruction of SBFtions may also contribute to a better understanding of pathogenesis in other CNS disorSEM images shows a monocyte-derived
ders such as stroke and brain trauma and will hopefully lead to the development of new
macrophage encircling the node of
therapeutic strategies.
Ranvier, as shown by the two primary
< ID >j e m .2 1 8i n s gh t f j p e < / ID>
< I D > j em . 2 1 8i ns g h t f p < / I D >
processes (white and black arrows).
Yamasaki, R., et al. 2014. J. Exp. Med. http://dx.doi.org/10.1084/jem.20132477.
Michael T. Heneka, University of Bonn: michael.heneka@ukb.uni-bonn.de
Article
Differential roles of microglia and monocytes

in the inflamed central nervous system
Ryo Yamasaki,1 Haiyan Lu,1 Oleg Butovsky,5 Nobuhiko Ohno,2
Anna M. Rietsch,1 Ron Cialic,5 Pauline M. Wu,2 Camille E. Doykan,2
Jessica Lin,1,6 Anne C. Cotleur,1 Grahame Kidd,2 Musab M. Zorlu,1,7
Nathan Sun,8 Weiwei Hu,2,9 LiPing Liu,1 Jar-Chi Lee,3 Sarah E. Taylor,10
Lindsey Uehlein,1,6 Debra Dixon,1,11 Jinyu Gu,1 Crina M. Floruta,1,12 Min Zhu,1
Israel F. Charo,13 Howard L. Weiner,5 and Richard M. Ransohoff 1,4,11
1Neuroinflammation
Research Center and 2Department of Neurosciences, Lerner Research Institute; 3Department

of Quantitative Health Sciences; and 4Mellen Center for Multiple Sclerosis Treatment and Research, Neurological Institute,
Cleveland Clinic, Cleveland, OH 44106
5Center for Neurological Diseases, Brigham and Womens Hospital, Harvard Institutes of Medicine, Boston, MA 02115
6Ohio State University College of Medicine, Columbus, OH 43210
7Hacettepe University Faculty of Medicine, 06100 Ankara, Turkey
8Vanderbilt University, Nashville, TN 37235
9Department of Pharmacology, School of Basic Medical Sciences, Zhejiang University, Hangzhou, 310058 Zhejiang, China
10Case Western Reserve University, School of Medicine, Cleveland, OH 44106
11Cleveland Clinic Lerner College of Medicine, Cleveland, OH 44106
12Baylor University, Waco, TX 77030
13Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, San Francisco, CA 94158
In the human disorder multiple sclerosis (MS) and in the model experimental autoimmune
encephalomyelitis (EAE), macrophages predominate in demyelinated areas and their numbers correlate to tissue damage. Macrophages may be derived from infiltrating monocytes
or resident microglia, yet are indistinguishable by light microscopy and surface phenotype.
It is axiomatic that T cellmediated macrophage activation is critical for inflammatory
demyelination in EAE, yet the precise details by which tissue injury takes place remain
poorly understood. In the present study, we addressed the cellular basis of autoimmune
demyelination by discriminating microglial versus monocyte origins of effector macrophages. Using serial block-face scanning electron microscopy (SBF-SEM), we show that
monocyte-derived macrophages associate with nodes of Ranvier and initiate demyelination,
whereas microglia appear to clear debris. Gene expression profiles confirm that monocytederived macrophages are highly phagocytic and inflammatory, whereas those arising from
microglia demonstrate an unexpected signature of globally suppressed cellular metabolism
at disease onset. Distinguishing tissue-resident macrophages from infiltrating monocytes
will point toward new strategies to treat disease and promote repair in diverse inflammatory pathologies in varied organs.
CORRESPONDENCE
Richard M. Ransohoff:
ransohr@ccf.org
Abbreviations used: CNS,
central nervous system; EAE,
experimental autoimmune
encephalomyelitis; MDM,
monocyte-derived macrophage;
MiDM, microglia-derived macrophage; MS, multiple sclerosis;
SBF-SEM, serial block-face
scanning electron microscopy.
Blood-derived monocytes and resident microglia

can both give rise to macrophages in the central nervous system (CNS). In tissue sections,
macrophages derived from these two distinct
precursors are indistinguishable at the light
microscopic level both morphologically and by
surface markers. Using flow cytometry, microgliaand monocyte-derived macrophages can be
isolated separately from CNS tissue lysates and
expression profiling suggests distinct functional
R. Yamasaki, H. Lu, and O. Butovsky contributed equally to
this paper.
The Rockefeller University Press $30.00

J. Exp. Med. 2014 Vol. 211 No. 8 1533-1549
www.jem.org/cgi/doi/10.1084/jem.20132477
capacities (Gautier et al., 2012; Chiu et al., 2013;

Butovsky et al., 2014).
Microglia and monocytes are ontogenetically distinct: microglia derive from yolk-sac progenitors during embryogenesis (Ginhoux et al.,
2010; Schulz et al., 2012), whereas monocytes
continuously differentiate throughout postnatal
life from bone marrow hematopoietic stem cells
2014 Yamasaki et al. This article is distributed under the terms of an Attribution
NoncommercialShare AlikeNo Mirror Sites license for the first six months
after the publication date (see http://www.rupress.org/terms). After six months
it is available under a Creative Commons License (AttributionNoncommercial
Share Alike 3.0 Unported license, as described at http://creativecommons.org/
licenses/by-nc-sa/3.0/).
1533
(HSCs), which require the transcription factor Myb. Microglial precursors are Myb independent, and microglia self-renew
independently of bone marrow HSCs (Gomez Perdiguero
et al., 2013). Distinct developmental origin and renewal mechanisms imply that monocyte-derived macrophages (MDMs)
and microglia-derived macrophages (MiDMs) might exert different functions in pathological processes. Microglia represent
one instance of tissue-resident macrophages, which reside in all
organs. Studying the CNS as compared with other organs
may carry advantages for distinguishing tissue-resident myeloid cells from infiltrating monocytes during disease, as there
is virtually no background trafficking of monocytes in the
CNS parenchyma of healthy animals.
In EAE, which models inflammatory aspects of MS
(Williams et al., 1994; Ransohoff, 2012), macrophages dominate
the inflammatory infiltrates and their numbers correlate to
EAE severity (Huitinga et al., 1990, 1993; Ajami et al., 2011).
However the cellular mechanisms by which macrophages
promote disease progression are uncertain. Whether MiDMs
or MDMs are functionally distinct and whether the two cell
types differentially initiate demyelination or promote repair
(Steinman et al., 2002) also remains elusive (Bauer et al., 1995).
In MS autopsy tissues, prominent macrophage accumulation
correlates with active demyelination (Ferguson et al., 1997;
Trapp et al., 1998). Based on kinetics of cell accumulation and
differential marker expression, its estimated that 3050% of
activated macrophages in active MS lesions derive from microglia (Brck et al., 1995; Trebst et al., 2001). Therefore, differential functions of MDMs and MiDMs are relevant for
human demyelinating disease.
To date, no research techniques have permitted distinction
between monocytes and microglia in CNS tissue without irradiation chimerism or parabiosis, techniques that confound
interpretation or impose practical limitations (Ajami et al.,
2007, 2011; Ransohoff, 2007). When F4/80+ macrophages
were isolated from CNS and analyzed by flow cytometry

using cells from double-heterozygous Ccr2rfp::Cx3cr1gfp mice
with EAE, GFP was expressed by CD45dim/Ly6C microglia,
whereas RFP was restricted to CD45high/Ly6C+ monocytes
(Saederup et al., 2010; Mizutani et al., 2012). These findings
suggested an approach to clarifying distinct roles of MDMs
and MiDMs in EAE based on differential expression of GFP
and RFP reporters. Here, we use that strategy to extend previous findings and address the hypothesis that MDMs and
MiDMs exert different functions in neuroinflammation. We
detected detailed ultrastructural characterization of MDMs
and MiDMs at EAE onset.
Unexpectedly, this approach provided insight into the cellular basis for autoimmune demyelination, which has remained
obscure despite >80 yr of study in the EAE model. Here we
provide evidence that MDMs initiate demyelination, often at
nodes of Ranvier. In contrast, phagocytic microglia appear relatively inert at disease onset. Results from expression profiling
provided insight into mechanisms and signaling pathways underlying the disparate effector properties of MDMs and MiDMs
in EAE.The distinct functions of tissue-resident myeloid cells
as compared with infiltrating macrophages broadly underlie
disease pathogenesis in manifold circumstances and also hold
promise for innovative treatment strategies.
RESULTS
In the CNS of mice of EAE, MDMs and MiDMs
exhibit different accumulation kinetics
The histological strategy in this study is shown in Table 1. At
onset of EAE, two pools of CD11b+ mononuclear phagocytic
cells (putative red MDMs and green MiDMs) predominated
in spinal cord (Fig. 1 A), indicating that fluorochrome markers could be distinguished at this time point. Using cells isolated from Ccr2rfp/+::Cx3cr1gfp/+ spinal cords at disease onset,
flow cytometry demonstrated distinct expression of RFP and
Table 1. Histology analysis strategy

Method
Purpose
Finding
(RFP+)
from
MDMs and MiDMs can be distinguished by cell volume and primary
Confocal analysis of 0.2 mm optical
To distinguish MDMs
MiDMs (GFP+).
processes.
sections (n = 104 cells).
SBF-SEM inspection in 0.2 mm sections To detect MDMs and MiDMs in SBF- Using criteria detected in the previous step, it is possible to distinguish
MDMs and MiDMs in SBF-SEM images.
from 14 lesions, 7 mice at EAE onset. SEM using cell volume and process
criteria.
SBF-SEM inspection of ultrastructure To detect ultrastructural characteristics MDMs and MiDMs show characteristic ultrastructural differences
of MDMs and MiDMs.
of MDMs and MiDMs.
regarding their mitochondria, nuclei, osmiophilic granules and
microvilli.
To determine relationship of MDMs Most (55/75; 73%) axoglial units are contacted in limited fashion by
Quantification of relation of MDMs
MDMs and MiDMs. If one cell type is present (20/75 cells), its nearly
and MiDMs to axoglial units and
(n = 169) and MiDMs (n = 86) to
always (18/20 segments) MDMs.
axoglial units (n = 29 intact axons, characterize presence of myelin debris.
46 demyelinated axons).
To detect relationship of MDMs with In all, 49 MDMs interacting with axoglial units in absence of nearby
Reconstruction of 3D shape of four
axoglial units at EAE onset.
MiDMs, 2-3 MDMs were attached to each (n = 18) axoglial unit.
representative MDMs at axoglial
MDMs have close relationship with nodes of Ranvier (7 MDMs/75
units.
axoglial units). 3D reconstructions showed four representative MDMs
at axoglial units: show one carrying out active demyelination, three at
nodes of Ranvier.
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Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.
Ar ticle
GFP by F4/80+/CD45high MDMs and F4/80+/CD45dim

MiDMs, respectively (Fig. 1 B). Enumeration of cells recovered
from cell sorting using F4/80+RFP+ as MDMs gate and F4/
80+GFP+ as MiDMs gate indicated that MDMs and MiDMs
showed equal numbers at disease onset when explosive MDM
accumulation occurred. MiDM expansion began at peak
(Fig. 1 B). At recovery, MiDMs were found near preonset
numbers as MDM frequency continued to decline, which is
compatible with previous studies (Ajami et al., 2011). Therefore, there were unequal numbers of MiDMs/MDMs before
and after disease onset (Fig. 1 B). Morphological analyses and
definitions of relations between myeloid cells and axoglial
units were conducted at disease onset so that equal numbers
of MDMs and MiDMs could be assayed and early events in

the demyelinating disorder could be explored.
Morphological features distinguish
MDMs and MiDMs at EAE onset
Immunofluorescence staining for RFP and GFP in spinal cord
at EAE onset showed that red MDMs exhibited elongated or
spindle shape, whereas green MiDMs showed a process-bearing
morphology (Fig. 1 C). Quantification in 3D reconstructions
from 0.2-m confocal z-stack images showed that MiDMs
exhibited much larger size than MDMs along with multiple
primary processes, which were sparse in MDMs (Fig. 1 D).
Several 3D shape parameters also discriminated between MDMs
Figure 1. MDMs and MiDMs exhibit different time courses of accumulation in the CNS of mice with EAE and morphological characteristics
can distinguish them. (A) Immunohistochemistry shows expression of CD11b for red RFP+ MDMs and green GFP+ MiDMs in the spinal cords of
Cx3cr1gfp/+::Ccr2rfp/+ mice at EAE onset. Bars: 25 m. We studied 6 mice at EAE onset from 3 EAE inductions. In each EAE induction, 810 mice were used and
2 mice were selected from each induction. (B) Flow cytometric analysis of CCR2-RFP+ and CX3CR1-GFP+ populations in cells gated for F4/80 expression
(top); CD45 expression of F4/80+RFP+ MDMs and F4/80+GFP+ MiDMs populations (middle); and MDMs and MiDMs numbers at EAE onset, peak, and recovery
(bottom). We studied 3 mice for naive groups; 12 for onset; 15 for peak; 13 for recovery from 5 EAE inductions. For each induction, 810 mice were used
and 23 mice were selected at each time point (onset, peak, and recovery) for analysis. (C) Confocal microscopy assessment of myeloid cell morphology in
lumbar spinal cord from mice at EAE onset. We studied 54 MDMs and 51 MiDMs of 5 EAE onset mice from 3 EAE inductions for (CE); 2 sections/mouse;
46 cells/section; 812 cells/mouse. In each EAE induction, 810 mice were induced and 12 EAE onset mice were selected from each experiment. Bars,
25 m. (D) Cell volumes of 500 m3; surface areas of 1,000 m2; primary process numbers 3 or 5; solidity3D of 0.4; and Formfactor3D of 0.3 discriminate between MDMs and MiDMs. (E) Model plot of cell volume against primary process number to distinguish MDMs (red symbols and pink area) from
MiDMs (green symbols and green area).
JEM Vol. 211, No. 8
1535
and MiDMs (Fig. 1 D). We observed scant overlap of several

values between MDMs and MiDMs (Fig. 1 D), and entirely
nonoverlapping distributions for cell volume and primary
processes (Fig. 1 E).
MDMs and MiDMs exhibit differentiating
ultrastructural characteristics at EAE onset
We used confocal microscopy in 0.2-m optical sections to
correlate structural features of MDMs and MiDMs with RFP
or GFP fluorescence, as a bridge to characterizing cells in 0.2 m
SBF-SEM images (Table 1). Using this approach (Fig. 1 E),
MDMs and MiDMs were identified by estimating volume
and counting primary processes. Volume estimations came
from multiplying the midcell area by the number of sections in
which the cell was identified. In electromagnetic (EM) images,
quantitative analysis also demonstrated differentiating ultrastructural characteristics for mitochondria, nuclei, cytoplasmic osmiophilic granules and microvilli (unpublished data). MDMs
had shorter, thicker mitochondria than MiDMs (unpublished
data). Total mitochondrial numbers and volumes were equal
in MDMs and MiDMs (unpublished data). MDMs had bilobulated or irregular nuclei, whereas MiDMs had round nuclei (unpublished data). MDMs, but not MiDMs, frequently
contained osmiophilic granules and microvilli (unpublished
data). Collectively, these ultrastructural features provided confirmatory ultrastructural characteristics to distinguish MDMs
from MiDMs.
MDMs initiated demyelination at EAE onset
Results from confocal and EM analysis yielded a secure basis
for examining the relationships of MDMs and MiDMs to axoglial units at EAE onset (n = 7 mice; 14 lesions) using serial
block-face scanning electron microscopy (SBF-SEM), as presented diagrammatically (Table 1). We quantified contacts
made by MDMs (n = 169) and MiDMs (n = 86) with axoglial
units (n = 75; Fig. 2), and observed that most (55/75; 73%) of
all segments (both intact and demyelinated) contacted both
MDMs and MiDMs (Fig. 2). Where only one myeloid cell
type was present (20/75; 27%), nearly all axoglial units made
contacts to MDMs (Fig. 2). In particular, 8/29 intact and 10/46
Figure 2. SBF-SEM shows MDMs initiating demyelination at EAE onset. Quantitation of MiDMs and MDMs interacting with axoglial units in SBFSEM images of CNS at EAE onset. Intact (69%) and demyelinated (76%) segments interacted with MDMs and MiDMs. Red and pink, MDMs; green and
light green, MiDMs; yellow, both MDMs and MiDMs. We studied 29 intact axon segments, 46 demyelinated axon segments, 86 MiDMs, and 169 MDMs in
14 lesions of 7 EAE onset mice from 3 EAE inductions as follows: 810 mice were immunized at each experiment and 23 onset mice were selected from
each induction.
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Ar ticle
demyelinated axoglial units were contacted solely by MDMs.

We found 23 MDMs attached to each of the 18/20 (90%)
axoglial units where only MDMs were present (Fig. 2). More
than half of all analyzed MDM and MiDM cells (n = 255
total) contained myelin debris, regardless of whether axon
segments were intact or demyelinated (Fig. 2). Of the MDMs
found in sole contact with axoglial units, virtually all (>90%)
MDMs contained myelin when found in sole contact with a
demyelinated axon (Fig. 2). These findings motivated evaluation of relationships of MDMs to axoglial units by 3D reconstruction of SBF-SEM image stacks.
MDMs frequently exhibited morphological characteristics
suggesting an involvement in active demyelination. Reconstruction of one representative image stack shows MDMs with
large intracellular myelin inclusions tightly encircling a partially
demyelinated axon (Fig. 3 A). The myelin peeled away from
the axon remained in continuity with a large myelin inclusion
inside the MDMs (Fig. 3 A). Remaining myelin was undergoing

vesicular breakdown (Fig. 3 A). In contrast, a nearby MiDM
encompassed a large fragment of myelin debris (Fig. 3 B) and
contacted the nearby MDMs (Fig. 3 B), but made minimal
connection to the axoglial unit (Fig. 3 B). In our SBF-SEM
data, only MDMs seemed to be implicated in active damage
to myelin. These observations suggested that MDMs initiated
demyelination at the onset of EAE.
MDMs surrounded apposed and invaded
nodes of Ranvier at EAE onset
We analyzed axoglial units to examine the nature of contacts
with myeloid cells. Unexpectedly, 7/75 (9%) of axoglial units
demonstrated MDMs attached to nodes of Ranvier. In each
case, the contact between MDMs and node appeared to be
pathogenic. One representative monocyte surrounded a node
of Ranvier with two microvilli interposed between myelin
Figure 3. SBF-SEM shows example of

MDM-initiating demyelination at EAE
onset. (A) Representative MDMs encircles the
axoglial unit. A myelin ovoid within an intracellular phagolysosome shows physical continuity with myelin remaining attached to an
axoglial unit which is undergoing active demyelination. In serial images, disrupted myelin
shows continuity from outside to inside the
MDM. (B) Rotated view from B demonstrating
MDM-extensive attachment to axoglial unit
and MiDM nearby with limited attachment to
axon. A, axon; m, myelin; c, cytosol; n, nucleus.
Red, MDM cytosol; green, MiDM cytosol;
yellow: nuclei; blue, myelin and myelin debris;
gray, axoplasm; red line, MDM plasma membrane. We studied 14 lesions from 7 EAE onset mice from 3 EAE inductions as follows:
810 mice were immunized at each experiment and 23 EAE onset mice were selected
from each induction. Bar, 2 m.
JEM Vol. 211, No. 8
1537
and axolemma near the paranode complex (Fig. 4 A). The axoglial unit appeared otherwise healthy and no myelin debris
was found in the MDM cytosol. This observation suggested
that initial MDMaxoglial contacts might occur at nodes of
Ranvier. Further, we detected an intratubal (Stoll et al., 1989)
MDMs with myelin debris interposed between compact myelin and axolemma near a node of Ranvier (Fig. 4 B). Additionally we identified an MDM apposed to a node of Ranvier
and actively phagocytizing myelin (Fig. 4 C). At this node,
paranode loops were disrupted and surrounded by MDM cytosol (Fig. 4 C), indicating likely involvement in damaging myelin near the node. No MiDMs contacted nodes of Ranvier.
Nodal pathology without demyelination
at EAE onset in Ccr2rfp/rfp::Cx3cr1gfp/+ mice
We interpreted our ultrastructural findings to indicate that
MDMs recognized altered nodal structure and initiated demyelination at EAE onset. CCR2 is essential for monocyte recruitment to CNS tissues during immune-mediated inflammation
(Fife et al., 2000; Izikson et al., 2000; Savarin et al., 2010). To
address the role of MDMs in demyelination at EAE onset, we

investigated clinical characteristics in relation to node pathology
and demyelination in Ccr2rfp/rfp::Cx3cr1gfp/+ mice in which
MDMs were virtually absent from inflamed EAE tissues and
replaced in large part by neutrophils (Saederup et al., 2010).We
observed equivalent magnitude of weight loss in Ccr2rfp/+::
Cx3cr1gfp/+ and Ccr2rfp/rfp::Cx3cr1gfp/+ mice at preonset and
onset stages of EAE, showing that CCR2 deficiency did not
affect systemic inflammation in this model (Fig. 5 A). There was
a moderate delay in disease onset (Fig. 5 B) and slight reduction
in EAE onset severity (Fig. 5 A) in Ccr2rfp/rfp::Cx3cr1gfp/+ mice.
SBF-SEM was used to evaluate nodal pathology, myeloid
cell relations to axoglial units and demyelination at and before
EAE onset. In three distinct tissues from individual Ccr2rfp/+
::Cx3cr1gfp/+ mice with EAE preonset, we found five MDMs
attached to disrupted nodes of Ranvier. In an equivalent sample of EAE tissues from three Ccr2rfp/rfp::Cx3cr1gfp/+ mice, only
one MDM was found in contact with a node of Ranvier, despite
the presence of disrupted nodes in proximity to neutrophils.
One representative MDM from Ccr2rfp/+::Cx3cr1gfp/+ tissue
Figure 4. MDMs surrounded, apposed,

and invaded nodes of Ranvier at EAE
onset. (A) SBF-SEM images and 3D reconstruction of SBF-SEM images of MDMs with a node
of Ranvier. White and black arrow: microvillus. (B) SBF-SEM images and 3D construction
of intratubal MDMs with demyelinated axon
and node of Ranvier. (C) SBF-SEM images and
3D reconstruction of an MDM with intracellular myelin debris apposed to a node of Ranvier. Red, MDM cytosol; yellow, nucleus; blue,
myelin; gray, axoplasm. M, myelin; c, cytosol.
red line, MDM plasma membrane. We studied
14 lesions from 7 EAE onset mice collected as
follows: 810 mice were immunized at each
induction and 23 EAE onset mice were
selected from each immunization. Bar, 2 m.
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Figure 5. Nodal pathology without demyelination at EAE onset in Ccr2rfp/rfp::Cx3cr1gfp/+ mice. (A) Magnitude of weight loss in Ccr2rfp/+::Cx3cr1gfp/+ and
Ccr2rfp/rfp::Cx3cr1gfp/+ mice at preonset and onset stages of EAE. Clinical score in Ccr2rfp/+::Cx3cr1gfp/+ and Ccr2rfp/rfp::Cx3cr1gfp/+ mice at EAE onset stage. (B) Days
at disease preonset and onset stages of EAE. We studied 28 Ccr2rfp/+::Cx3cr1gfp/+ mice and 26 Ccr2rfp/rfp::Cx3cr1gfp/+ mice for A and B. Data were collected from
12 EAE inductions in Ccr2rfp/+::Cx3cr1gfp/+ mice and 19 EAE inductions in Ccr2rfp/rfp::Cx3cr1gfp/+ mice as follows: 810 mice were immunized at each induction and
13 EAE recovery mice were selected from each immunization. **, P < 0.01; ***, P < 0.001. (C) SBF-SEM imaging of MDMs with nodes of Ranvier phagocytosis in
Ccr2rfp/+::Cx3cr1gfp/+ mice at EAE preonset. Pink, MDM cytosol; red arrow, myelin inclusion of MDM connecting to the node of Ranvier. We studied 3 tissues from 3
Ccr2rfp/+::Cx3cr1gfp/+ EAE mice at preonset stage from 3 EAE inductions: 810 mice were immunized at each experiment and one EAE preonset mouse was selected
from each induction. Bar, 2 m. (D) SBF-SEM of disrupted nodes (black arrows) in preonset spinal cord tissues of Ccr2rfp/rfp::Cx3cr1gfp/+ mice. Bar, 2 m. (E) SBFSEM of neutrophil is with myelin phagocytosis from internode at preonset stage of Ccr2rfp/rfp::Cx3cr1gfp/+ mouse. Blue, neutrophil cytosol. For DE, we studied
three tissues from three Ccr2rfp/rfp::Cx3cr1gfp/+ EAE mice at preonset stage from 3 EAE inductions: 810 mice were immunized at each experiment and one EAE
preonset mouse was selected from each induction. Bar: 2 m. (F) Histochemical staining and with aurohalophosphate complexes (black gold staining) and quantification of demyelinated area of Ccr2rfp/+::Cx3cr1gfp/+ mice and Ccr2rfp/+::Cx3cr1gfp/+ mice. We studied 5 naive Ccr2rfp/+::Cx3cr1gfp/+ mice, 5 naive Ccr2rfp/rfp
::Cx3cr1gfp/+ mice, 5 onset Ccr2rfp/+::Cx3cr1gfp/+ mice, and 5 onset Ccr2rfp/rfp::Cx3cr1gfp/+ mice from 3 EAE inductions as follows: 810 mice were immunized at each
experiment and 12 onset mice were selected from each induction. **, P < 0.01. Bar: 250 m.
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Figure 6. Inflammatory signature in MiDMs versus MDMs in the CNS of Cx3cr1gfp/+::Ccr2rfp/+ mice with EAE. (A) Quantitative nCounter expression profiling of 179 inflammation related genes was performed in CNS-derived GFP+ microglia and RFP+ recruited monocytes from naive and EAE mice
at onset, peak and recovery stages. Each row of the heat map represents an individual gene and each column an individual group from pool of 5 mice at
each time point. The relative abundance of transcripts is indicated by a color (red, higher; green, median; blue, low). For AH, we studied five mice in each
time point (onset, peak, recovery) from 3 EAE inductions; 810 mice were immunized in each induction. (B) Heat map of differentially expressed microglia
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Ar ticle
having concave nucleus (Fig. 5 C, left) had multiple intracellular myelin inclusions, one of which (Fig. 5 C, left middle) was
physically connected to a myelin sheath (Fig. 5 C, right middle) at a paranode (Fig. 5 C, right), indicating active ongoing
demyelination at a node of Ranvier. By distinct contrast, EAE
onset tissues of Ccr2rfp/rfp::Cx3cr1gfp/+ mice were characterized
by nodal pathology often without cellular infiltrates (Fig. 5 D).
In one instance, we detected a neutrophil abstracting myelin
from the myelin internode (Fig. 5, left and right) despite a
nearby disrupted node (Fig. 5, left) in tissues from a Ccr2rfp/rfp::
Cx3cr1gfp/+ mouse. Importantly, there was no evidence for
neutrophil recognition of disrupted nodes of Ranvier. We interpreted these observations to suggest that MDMs specifically
recognized nodal components to initiate demyelination, and that
absence of MDMs at disrupted nodes of Ccr2rfp/rfp::Cx3cr1gfp/+
mice with EAE was caused by the virtual absence of infiltrating monocytes (Saederup et al., 2010).
To quantify the outcome of these ultrastructural differences,
we monitored demyelination using histochemical staining with
aurohalophosphate complexes at disease onset in Ccr2rfp/rfp::
Cx3cr1gfp/+ and Ccr2rfp/+::Cx3cr1gfp/+ mice. Demyelination was
significantly reduced at EAE onset in CCR2-deficient mice
(Fig. 5 F), indicating the importance of MDM recognition of
disrupted nodes for efficient inflammatory demyelination. Furthermore, as nodal pathology was equivalent in Ccr2rfp/rfp::
Cx3cr1gfp/+ (Fig. 5 D) and Ccr2rfp/+::Cx3cr1gfp/+ mice at the
preonset stage of EAE, the results suggested that inflammatory
nodal disruption could be reversible if MDMs were prevented
from initiating demyelination at those sites.
Expression profiling demonstrates differential
MiDMs and MDMs gene expression
across the time course of an EAE attack
We reasoned that different phenotypes (Fig. 1) and effector
properties (Figs. 24) of MDMs and MiDMs should be reflected in distinct gene expression profiles in the dynamic CNS
microenvironment during EAE. To address this hypothesis,
nCounter digital multiplexed gene expression analysis (Kulkarni,
2011) was performed using directly ex vivo naive microglia
and splenic F4/80+ macrophages (here termed monocytes and
considered similar to microglia by expression profiling; Gautier
et al., 2012), as well as flow-sorted MiDMs or MDMs across the
time course of an EAE attack. Microglia and MiDMs clustered together during unsupervised hierarchical clustering, as
did monocytes and MDMs (Fig. 6, A and B). In both MiDMs
and MDMs, naive and recovery-stage expression profiles were
more alike than were onset and peak-stage profiles (Fig. 6,
A and B) suggesting a return to homeostasis at EAE recovery.
We noted a subset of genes that were expressed in microglia

and highly regulated in MiDMs during EAE, but not expressed at all in monocytes or MDMs (Fig. 6, A and B). Conversely, a subset of MDM-enriched genes were dynamically
regulated in monocytes and MDMs but not in microglia
(Fig. 6, A and B). MDM-enriched genes were sharply upregulated from naive monocytes to onset and peak-stage
MDMs (Fig. 6 B), descending toward naive levels during
recovery (Fig. 6 B). In contrast, MiDM-enriched genes
were strongly expressed in naive cells, almost uniformly silenced at onset, and began a return toward naive levels at peak
and recovery (Fig. 6 B). Comparing MDM-enriched genes
with MiDM-enriched genes showed that MDMs were
more likely to express effector functions, including secreted
factors and surface molecules (18/28; 64.3% of MDMenriched genes encoded effector functions; Fig. 6 C: and
Table S1, purple genes). In contrast, only 18/48 (37.5%) of
MiDM-enriched genes encoded effector functions (Fig. 6 D,
Table S1, purple genes). These observations indicated that
MiDMs and MDMs exhibited markedly distinct expression
profiles during EAE.
Differential expression of macrophage
effector functions by MiDMs and MDMs
Our ultrastructural analysis of myeloid cells in EAE focused
on myeloid cell relationships to tissue elements. Expression
profiling also addressed the cytokine and growth factor output
of MiDMs and MDMs, potentially providing insight into disease
pathogenesis.We used k-means clustering to discriminate five
distinct patterns of MiDM gene expression during the course
of EAE (Fig. 6, E and F). The red, blue and green groups increased in MiDMs at onset, peak, and recovery, respectively.
Red group genes involved several surface molecules. Green
group genes, up-regulated at onset and transiently further
up-regulated at peak, were comprised mainly of complementsystem elements (C3aR1; C4a, C1qa, C1qa, C3, and Cfb);
mononuclear cellspecific chemokines (CCl2, 3, 4, 5, 7, and
CXCL9); proliferation related genes ( fos, jun, myc, and CSF1);
and acute inflammationrelated genes (IL1a, IL1b, TNF,
CEBP, STAT1). Cell growthrelated genes expressed at this
time point correlated to reported patterns of microglial proliferation during EAE (Ajami et al., 2011). Blue group genes
up-regulated at recovery included heterogeneous cytokines
(IFN-, IFN-, TGFB3, IL2, IL3, IL4, IL12, IL12,
PDGFA, CSF2, and CXCL2). Both yellow group and golden
group genes were strongly expressed in naive microglia, reduced drastically at onset, and either returned to preEAE levels during recovery (yellow) or failed to do so (golden).These
and monocyte genes. (C) Enriched monocyte genes as compared with resident microglia. Bars represent fold changes of gene expression across naive and
all disease stages versus resident microglia. (D) Enriched microglia genes as compared with recruited monocytes. Bars represent fold changes of gene
expression across naive and all disease stages versus recruited monocytes. (EH) K-means clustering of inflammation genes in resident microglia and
recruited monocytes. K-means clustering was used to generate 5 disease stagerelated clusters in MiDMs. Heat map (E) and expression profile (F) of inflammation genes in MiDMs are shown by generated clusters. MDM expression matrix overlaid on microglial based clusters shows (G) heat map and
(H) expression profile.
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genes included a large spectrum of intracellular signaling components from the MAP-kinase pathways, as well as TGF and
receptor, both of which are implicated in the nave microglial
phenotype (Butovsky et al., 2014).
These five gene groups were also analyzed for MDM expression patterns during EAE (Fig. 6, G and H). None of the
gene groups showed coordinate regulation patterns in MDMs,
as were observed in MiDMs (Fig. 6 H). This observation underscored disparate responses of MiDMs and MDMs to the
inflammatory CNS microenvironment of EAE, despite their
being present in close proximity (Fig. 1 A).
Expression patterns at EAE onset
in relation to MiDM and MDM function
To determine whether gene expression patterns could be informative for understanding the relationships of cells to axoglial elements in tissues at EAE onset, we interrogated naive
versus onset MDM and MiDM gene expression related to
cellular functions (Fig. 7). MiDMs showed highly significant
up-regulation of functions associated with cell movement, chemoattraction, and migration (Fig. 7 B). In the Ingenuity IPA
database, the terms cell movement, chemoattraction, and migration indicated production of chemokines such as CCL2,
CCL3, CCL4, CCL5, and CCL7, which are up-regulated at
onset and further increased at peak (Fig. 6, E and F, green
group and genes). In other respects, MiDMs exhibited a repressed metabolic and activation phenotype by comparison
to naive microglia (Fig. 7 B) including proliferation, RNA
metabolism, cytoskeletal organization, microtubule dynamics,
extension of processes, phagocytosis and generation of reactive oxygen species.
MDMs showed up-regulation of functions associated to
macrophages, including phagocytosis, calcium signaling, production of prostanoids, adhesion, autophagy, and cell clearance
(Fig. 7 B).This pathway analysis corresponded well to effector
properties displayed by MDMs in our SBF-SEM analysis
(Figs. 24). No functions were reported to be down-regulated
in MDMs at EAE onset as compared with naive monocytes.
A comprehensive listing (Table S1) of all genes regulated
by at least twofold in MiDMs or MDMs as compared with
expression levels in naive mice affirmed and extended these
interpretations. At EAE onset when SBF-SEM analyses were
performed, MiDMs predominantly suppressed the distinctive
gene expression pattern which correlates to their unique phenotype (Chiu et al., 2013), reflected by the observation that
MiDMs down-regulated far more genes than were up-regulated
(Table S1). In contrast, MDMs up-regulated far more genes
than did MiDMs and up-regulated more transcripts than were
down-regulated. Additionally, the extent of gene up-regulation
in MDMs exceeded that seen in MiDMs.
MiDM and MDM gene expression kinetics reflected
return toward homeostasis in recovery stage
These expression profiles showed consonant changes for the
vast majority of genes analyzed: if a gene was up-regulated at
any time point, then its expression level showed an increase at
1542
other time points as well. However, a substantial minority of

genes both for MDMs and MiDMs showed some dissonant
time points, at which a previously down-regulated gene might
show up-regulation (unpublished data). We show this subset of
recovered genes in Fig. 8. In virtually every case (Fig. 8, AD),
these dissonant compensatory changes took place during recovery and almost always showed an increase in a gene that had
been down-regulated during onset and peak. Both MiDMs
(Fig. 8 B) and MDMs (Fig. 8 D) demonstrated this pattern of
gene-expression kinetics.
Convergent and divergent responses to upstream
regulatory signaling by MiDMs and MDMs
Translation of observations made using expression profiles can
be enabled through identification of upstream regulators. We
used Ingenuity IPA software to identify putative upstream regulators of the gene expression alterations demonstrated by
MiDMs and MDMs at disease onset. Putative regulatory elements were then grouped in signaling modules and subjected
to pathway analysis. Cell motility pathways were clearly different in MiDMs and MDMs (unpublished data). Core elements
such as RhoA (Xu et al., 2009) were regulated divergently and
associated signaling components were predicted to be enhanced
in MDMs but depressed in MiDMs, consistent with our phenotypic characterization using SBF-SEM. Both HIF-1 (Fig. 9 A)
and TNF pathways (not depicted) were also differentially regulated in MiDMs and MDMs. By contrast, type I IFN pathway
(Fig. 9 B) was regulated virtually identically in MiDMs and
MDMs. Collectively, these data suggest that HIF-1 and TNF
signaling may partly drive pathogenic properties of MDMs.
Additionally, these data indicated that the separate ontogeny
of microglia and monocytes will lead, probably by epigenetic
influences, to divergent responses to some but not all environmental stimuli, with phenotypic consequences according to
the CNS microenvironment.
DISCUSSION
In this study, we developed a novel strategy to discriminate
MDMs from MiDMs. We used SBF-SEM to address the detailed relationships of MiDM and MDM to axoglial units in
the spinal cords of mice at EAE onset and expression profiling
to examine potential mechanisms. Selection of the EAE disease
model ensured that both recruited monocytes and resident microglia were exposed to the same intensely inflammatory environment to increase the likelihood that ambient conditions
could activate these two myeloid cell types toward a convergent
inflammatory phenotype. Instead, we found strikingly divergent relationships of MDMs and MiDMs to axoglial units, by
quantitative and qualitative ultrastructural analysis. Results
from expression profiling supported this interpretation by showing that MiDM metabolism was severely down-regulated,
whereas expression profiles of MDMs reflected the activated
phagocytic phenotype observed through SBF-SEM.
Several salient new observations emerged from these experiments. First, we showed that MDMs initiate demyelination
at EAE onset, as MDMs were the overwhelmingly dominant
Ar ticle
cells found in isolation attached to axoglial units and demonstrated destructive interactions with myelinated axons in 3D
reconstructions. Second, MDMs were unexpectedly observed
at nodes of Ranvier in 9% of axoglial units and showed remarkably invasive behavior, including extension of microvilli (Fig. 4 A)
or localization of cell soma (Fig. 4 B) between axolemma and
myelin sheath. Our observed frequency of MDMnodal interaction represents a minimum estimate as MDMs found at heminodes adjacent to a demyelinated segment (Fig. 3 B) were not
scored. Comparison of Ccr2rfp/rfp::Cx3cr1gfp/+ and Ccr2rfp/+::
Cx3cr1gfp/+ mice at and before EAE onset emphasized the
importance of MDMs for this mechanism of demyelination. In

particular, neutrophils in inflamed CNS of Ccr2rfp/rfp::Cx3cr1gfp/+
mice did not recognize disrupted nodes. These observations
are clinically pertinent: our detection of MDMs at nodes of
Ranvier is consistent with recent reports of nodal pathology
in clinical demyelinated tissues (Fu et al., 2011; Desmazires
et al., 2012).The present observations extend this concept and
provide a cellular basis for nodal pathology at the earliest stages
of demyelination. Given the presence of potential phagocytic
signals at nodes (antibodies to paranodal proteins such as contactin and neurofascins; Meinl et al., 2011); complement-derived
Figure 7. Affected functions in MiDMs and MDMs at EAE onset. nCounter inflammatory gene expression data were uploaded to IPA. Genes with
fold change (EAE onset vs. Naive) 1.5 or 1.5 were included in downstream effects analysis. (A) MDMs up-regulated functions, sorted by activation
z-score. (B) MiDMs up-regulated (left) and down-regulated (right) functions, sorted by activation z score. The bias term indicates imbalanced numbers of
up- and down-regulated genes associated with a distal function requiring significance at the P < 0.01 level. We studied pooled samples from 5 mice in
each time point (onset, peak, recovery) from 3 EAE inductions; 810 mice were immunized in each induction.
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Figure 8. Restoration of affected inflammatory genes in resident microglia and recruited monocytes at recovery stage. For each gene, fold
change of all different disease stages versus naive state were calculated. Genes that contained at least one fold change >2 or < 0.5 and average fold
change for peak and onset >2 or <0.5 were presented. (A) Microglial up-regulated; (B) Microglial down-regulated; (C) monocyte up-regulated; (D) monocyte
down-regulated. We studied pooled samples from five mice in each time point (onset, peak, recovery) from three EAE inductions; 810 mice were immunized in each induction. FC, fold change.
1544
Ar ticle
Figure 9. Function networks in MiDMs and MDMs. nCounter inflammatory gene expression data were uploaded to IPA. Genes with fold change
(EAE onset vs. Naive) 1.5 or 1.5 were included in upstream regulators analysis. Predicted upstream regulators were manually curated to form functional clusters. Clusters were uploaded to IPA using Z scores as reference value for each gene. Networks were generated for each cluster consisting of
uploaded genes and additional predicted molecules. (A) Typical example of functions with dissimilar activation pattern in MiDMs and MDMs: HIF1A.
(B) Function with similar activation pattern in MiDMs and MDMs: Type I IFN. Red object denotes positive (>2) z score and green object denotes negative
JEM Vol. 211, No. 8
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opsonins (Nauta et al., 2004); and stress-induced eat-me signals (Hochreiter-Hufford and Ravichandran, 2013), it may be
feasible to identify a direct molecular pathway for initiating demyelination in this model. Third, we characterized a molecular signature for resident microglia at EAE onset. Grouping of
regulated genes into functional categories demonstrated a
remarkable down-regulation of microglial metabolism at the
nuclear, cytoplasmic and cytoskeletal levels.
In our initial experiments we found that the presence of
myelin debris at the peak of EAE did not discriminate MDMs
from MiDMs. We considered that SBF-SEM would exhibit
advantages for spatial resolution (Denk and Horstmann, 2004)
required for characterizing relationships of myeloid cells to
axoglial units during the inflammatory demyelinating process
at EAE onset.To take advantage of this technique we developed
methods based on cell volume and process number (Fig. 1 E),
to distinguish MDMs from MiDMs in 0.2-m confocal optical
sections, and translated this approach directly to SBF-SEM
image sets at 0.2-m intervals.We also noted differential nuclear
morphology, mitochondrial shape, and osmiophilic granule
content between MDMs and MiDMs. These characteristics of
MDMs and MiDMs may not be universally present in other
pathological circumstances but demonstrate an approach
to ultrastructural distinction of myeloid cell populations in
tissue sections.
Gene expression profiling across the time course of EAE
yielded intriguing kinetics as analyzed by k-means clustering.
Five patterns were observed. Red group genes (increased at
onset) comprised the smallest number and involved several surface molecules: CCR1, CCR7, CXCR2, and CD40. Of these,
CCR7 and CD40 have been reported on activated microglia,
including those observed in MS tissue sections (Kiviskk et al.,
2004; Serafini et al., 2006). GAPDH was up-regulated in
MiDMs at onset. Although often regarded as a housekeeping
gene, GAPDH is found in complexes that limit the translation
of inflammatory gene transcripts in activated mouse macrophages (Mukhopadhyay et al., 2009; Arif et al., 2012). As previously reported (Chiu et al., 2013), MiDM gene expression
during the course of EAE did not correspond to the M1/M2
pattern of peripheral macrophage responses to infection or tissue injury. Microglial morphological transformation can be
relatively uniform regardless of the inflammatory process that
provokes it. Despite this apparent uniformity, gene expression
by morphologically identical microglia can differ drastically
contingent on context (Perry et al., 2007).
Unsupervised hierarchical clustering provided insight into
gene expression patterns of MDMs and MiDMs. Naive and
recovery patterns were similar for both cell types. At disease
onset, microglia showed drastic down-regulation of the expression profile observed in cells from healthy brain. Brisk microglial proliferation (Ajami et al., 2011) may have accelerated
a gradual return toward a homeostatic expression profile, as

suggested by MiDM up-regulation of fos, jun, myc, and CSF1
(Wei et al., 2010) at disease onset. By striking contrast, MDMs
up-regulated a large suite of inflammation-associated genes at
EAE onset, with subsequent regression to the expression phenotype of circulating monocytes.
Blood monocytes and resident microglia were exposed to
the same inflammatory environment. However, their preEAE
states were extremely distinct, with monocytes being generated from a bone marrow progenitor within weeks of entry
into CNS, whereas microglia originated during early embryogenesis and had inhabited a serum-free unique environment
from midgestation. In a recent study, we characterized resident
microglia by profiling mRNA, miRNA, and protein in comparison with infiltrated brain macrophages, nonmicroglial resident brain cells, and peripheral macrophages (Butovsky et al.,
2014). The detailed profiling after separating cells via CD45dim
status showed distinct mRNA, miRNA, and protein expression by microglia as compared with infiltrating monocytes or
neuroepithelial brain cells (Butovsky et al., 2014). The study
described transcription factors and miRNAs characteristic of
microglia in healthy brain but not in peripheral monocytes.
These findings partially explain a divergent response of these
two cell types to the same stimuli (Butovsky et al., 2014).
The strength of the study is that the dual reporter system
is sufficient to accurately distinguish monocyte versus microglial cells and thus to address the general concept that
monocytes and microglia can exert differential functions in
a CNS disease process. At the onset of EAE, the time point
at which our imaging studies were focused, we are able to
make an unequivocal distinction between resident microglia
(CX3CR1gfp) and infiltrating monocytes (CCR2rfp). Two empirical observations underline this discrimination: microglia
are uniformly CX3CR1+ from early embryonic time points
through adulthood (Cardona et al., 2006; Ginhoux et al., 2010;
Schulz et al., 2012), and CCR2+Ly6C+ cells constitute the vast
majority of infiltrating monocytes at EAE onset (Saederup
et al., 2010; Mizutani et al., 2012).
There were unavoidable limitations of our research; specifically, to address how monocytes and microglia respond to
a shared microenvironment, we focused on a single, pathogenically relevant time point: onset of EAE. For this reason, it
was beyond the scope of our study to decipher the phenotypic
fate of infiltrated monocytes. In peripheral models of inflammation, Ly6Chi/CCR2rfp monocytes down-regulate the reporter over time and show phenotypic evolution. Furthermore,
our conclusions should not be generalized beyond the present
disease paradigm: in other models, such as spinal cord contusion,
the inflammatory infiltrate includes Ly6Clow/CX3CR1gfp
monocytes, which are highly pathogenic (Donnelly et al.,
2011). Our findings carry biological and medical significance
(<2) z-score. Orange object denotes predicted activation of the network object. Blue object denotes predicted inhibition of the network object. Predicted
relationships (connecting lines): orange, leads to activation; blue, leads to inhibition; yellow, finding inconsistent with state of downstream molecule;
gray, effect not predicted.
1546
Ar ticle
by demonstrating and characterizing differential responses of

infiltrating monocytes and resident microglia in a relevant disease model at a prespecified time point, at which point pathogenic events are taking place.Therefore, we focused our analysis
on the day of EAE onset rather than subsequent events to
challenge our overall hypothesis that infiltrating monocytes
versus resident microglia respond very differently to acute inflammatory stimuli.
Activated myeloid cells are the proximate effectors of a
bewildering array of acute and chronic disorders (Wynn et al.,
2013). The technical and conceptual approach taken in this
study may be applicable to other tissues and disease processes.
In many pathological conditions, tissues harbor a mixed population of activated resident and recruited monocytes. The
therapeutic strategy will differ conclusively based on the specific effector properties of each cell type and the stage of disease. In particular, if monocytes are pathogenic, then their
trafficking should be blocked using a peripherally active agent.
The optimal application of agents that regulate leukocyte migration and intracellular signaling will be promoted by detailed examination of each individual myeloid population.
MATERIALS AND METHODS
Mice. C57BL/6 mice were obtained from the National Cancer Institute.
Ccr2rfp/+::Cx3cr1gfp/+ mice were generated by crossbreeding Ccr2rfp/rfp::C57BL/6
mice (Saederup et al., 2010) with Cx3cr1gfp/gfp::C57BL/6 mice ( Jung et al.,
2000). Ccr2rfp/rfp::Cx3cr1gfp/gfp mice were generated by breeding Ccr2rfp/+
::Cx3cr1gfp/+ mice. Ccr2rfp/rfp::Cx3cr1gfp/+ mice were generated by crossbreeding Ccr2rfp/rfp::C57BL/6 mice with Ccr2rfp/rfp::Cx3cr1gfp/gfp mice. Animal experiments were performed according to the protocols approved by the
Institutional Animal Care and Use Committee at the Cleveland Clinic following the National Institutes of Health guidelines for animal care.
EAE induction and clinical evaluation. EAE was induced in Ccr2rfp/+
::Cx3cr1gfp/+ mice and Ccr2rfp/rfp::Cx3cr1gfp/+ mice of 2428 wk of age using
myelin-oligodendrocyte-glycoprotein peptide 3555 (MOG) as previously
described (Huang et al., 2006). All mice were weighed and graded daily for
clinical stages as previously reported (Saederup et al., 2010).We defined clinical
stage of EAE as follows: pre-onset was the day sudden weight loss for 810%
occurred; onset was the day EAE signs appeared; peak was the second day score
didnt increase after sustained daily worsening; and recovery was the second
day score didnt decrease after a period of sustained daily improvement.
To address our research questions, we integrated flow cytometry, immuno
histochemistry with quantitative morphometry, cell sorting for expression
profiling, and serial block-face scanning electronic microscopy. In all, we performed 12 EAE immunizations in Ccr2rfp/+::Cx3cr1gfp/+ mice and 19 immunizations in Ccr2rfp/rfp::Cx3cr1gfp/+ mice for this project, with 810 mice in
each immunization. We selected EAE mice at onset, peak or recovery depending on the specific studies underway at that time, with the majority of
mice coming from the onset stage of EAE. Each experiment incorporated
samples from at least three separate immunizations. Details of mouse numbers
and how they were selected for each experiment were included in the figure
legends as requested.
Cell isolation and flow cytometry. Brains and spinal cords were removed
and homogenized. Mononuclear cells were separated with a 30%/70% Percoll (GE Healthcare) gradient as previously reported (Pino and Cardona,
2011). Single-cell suspensions from CNS were stained with antiF4/80-APC
(BM8; eBioscience) and antiCD45-PerCP (30-F11; BioLegend). Cells were
either analyzed on a LSR-II (BD) or sorted on a FACSAria II (BD) running
Diva6. Data were analyzed with FlowJo software (Tree Star).
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Histological and immunohistochemical analysis. Spinal columns were

removed after mice were perfused with 4% paraformaldehyde (PFA). For immunofluorescence assay, free floating sections of the lumbar spinal cord were
prepared as previously described (Huang et al., 2006). For immunofluorescence assay, sections were blocked with 10% normal serum for 2 h and stained
with primary antibodies at 4C for 2448 h. After washing with PBS-T (PBS
with 0.1% Triton X-100; Sigma-Aldrich) three times, the sections were incubated with secondary antibodies at room temperature for 2 h and mounted
in ProLong Gold antifade reagent (Invitrogen). Antibodies used include rat
anti-CD11b (BD), mouse anti-GFP (Abcam), rabbit anti-RFP (Abcam), Alexa
Fluor 488 goat antimouse IgG (Invitrogen), Alexa Fluor 594 goat antirabbit
IgG (Invitrogen), and Alexa Fluor 647 goat antirat IgG (Invitrogen). Nuclei
were labeled by DAPI. Images were collected by confocal laser-scanning microscope (SP5; Leica).
Quantitative 3D morphology. Quantitative 3D morphology of MDMs
and MiDMs was analyzed in confocal images from spinal cord of mice at
EAE onset. Free floating sections of the lumbar spinal cord were stained with
RFP for MDMs, GFP for MiDMs, and DAPI for nuclei. Stack images were
taken at 0.2-m step size along the z-direction with a 63 objective (numerical aperture [NA] = 1.4) and zoom factor 2. A square (1,024 1,024 pixels)
corresponding to 123 123 m2 was used for the analysis. Cells were 3D reconstructed by ImageJ software and all analyses were performed using ImageJ
with 3D Convex Hull plugin. The parameters analyzed include voxel (volumetric pixel), convex voxel, volume, convex volume, surface, and convex surface area. Other calculated parameters were: Solidity3D = volume/convex
volume; Convexity3D = convex surface area/surface area; Formfactor3D =
3 36 volume 2 surface area 3 . The number of primary processes was estimated visually. We included 5 mice, 54 MDMs; 51 MiDMs in this assay with
2 sections/mouse, 46 cells/section and 812 cells/mouse. Those mice came
from three EAE inductions.
SBF-SEM. Spinal cords were removed after mice were perfusion-fixed
using 4% PFA with 1% glutaraldehyde. Lumbar spinal cord sections were
made on a vibratome (Leica). Sections were stained with 0.4% OsO4, uranyl
acetate and lead aspartate, then embedded in epon resin (Electronic Microscopy Sciences). SBF-SEM images were acquired using a Sigma VP SEM
(Carl Zeiss) with 3View (Gatan). Serial image stacks of images at 100-nm
steps were obtained by sectioning 48 48 20 m3 tissue blocks (length
width depth) at a resolution of 8192 8192 pixels. Image stacks were processed for 3D reconstruction by TrakEM2 in FIJI software (National Institutes
of Health). Alternating sections from the same stacked images were chosen to
make stacks for 3D reconstructions which matched the 0.2-m step size used
for acquiring confocal stacked images. In SBF-SEM images, we discriminated
MDMs and MiDMs using the volume/primary processes model (Fig. 1 E)
generated from analyzing confocal images. Quantifications of myeloid-cell
spatial relationships to axoglial units, including myelin incorporation, were
done in SBF-SEM images.
Quantification of nuclei and mitochondria. Characterizations of nuclear shapes were conducted in SBF-SEM images. Nuclei were categorized
as follows: round, round shape and smooth surface with ratio of length/
width 1.5; elongated, elongated or oval shape with length/width >1.5, and
may have small indentations; Bilobulated: two connected lobes with single
intervening large indentation; Irregular: complicated shape with corrugated
surface, and may have multiple and variable sizable indentations. Blinded observers (n = 3) scoring the nuclear morphology from SBF-SEM images included a research student, a research fellow and a neuroscientist. Observers
were trained on the same nuclear examples in each category and practiced
using 20 nuclei comprising all shapes before scoring the nuclei. Kappa test
showed good pairwise agreement rates among observers (>0.8) and the data
from the neuroscientist are used. Quantifications were done in 3 individual
mice from 3 EAE inductions including 2835 cells from two separate lesions
from each mouse in the assay.
1547
Mitochondria of MDMs and MiDMs at EAE onset were reconstructed

from SBF-SEM images to 3D images using TrakEM2. 5 MDM and 5 MiDM
cells from 3 separate mice at EAE onset (total 10 cells) were included in the
assay.Those mice came from 3 EAE inductions. Mitochondria were quantified
for length, cross-sectional area, volume and ratio of length/cross-sectional
area using Fiji software.
Quantification of demyelination. Black-gold staining was performed according to a protocol described previously (Liu et al., 2010). In brief, 5 free
floating lumbar spinal cord sections were stained in 0.2% black-gold solution
at 65C water bath for 10 min. After staining with black-gold, sections were
pictured by 3-CCD video camera interfaced with an Image-Pro Plus Analysis System (Version 4.1.0.0; MediaCybernetics) and analyzed with ImageJ
software. Demyelinated areas are those void of black-gold staining. Mean
percentage of demyelinated areas in white matter were calculated. We included 5 mice from 3 EAE inductions in this assay.
Statistical analysis of cellular elements. Statistical analyses were performed using SAS (SAS Institute Inc.), PRISM (GraphPad Software) and
SPSS 17.5 (SPSS Inc.). Flow cytometry data were analyzed by two-way
ANOVA test and Wilcoxon matched-pairs signed rank test. Nuclear shape
quantifications were compared by paired Students t tests and logistic regression. Mitochondrial quantifications were compared by Mann-Whitney U
test and linear mixed model. Quantitative relationships of myeloid cells to
axoglial units were compared using logistic regression with generalized estimating equations (GEE). Clinical characteristics of EAE mice were analyzed
using two-way ANOVA test with Bonferroni post test. Percentage of demyelination was compared by Students t test. Data were shown as mean SEM
or median (the first quartilethe third quartile) and P < 0.05 was considered
statistically significant.
Gene expression. Mononuclear cells were prepared from brains and spinal
cords as described previously. Cells were sorted on a BD FACSAria II by gating on F4/80+GFP+ for MiDMs and F4/80+RFP+ for MDMs. RNA was
isolated from FACS-sorted cells mixed from three mice from six EAE inductions per data point in TRIzol Reagent (Ambion) according to manufacturers protocol. RNA samples were analyzed by nCounter gene expression
analysis and quantified with the nCounter Digital Analyzer (NanoString
Technologies). Expressions of 179 genes were analyzed using nCounter GX
Mouse Inflammation kit.
Nanostring data normalization. Normalization was conducted with
nSolver Analysis Software1.1. Data were normalized using positive and negative controls and housekeeping genes probes. Background level was calculated for each sample as mean of negative control probes + (x2 SD). Calculated
background was subtracted from each gene expression value. In cases where
the calculated value was <1, values were set to 1.
Hierarchical and k-means clustering analysis. Hierarchical cluster analy
sis was performed using Pearson correlation for distance measure algorithm
to identify samples with similar patterns of gene expression. MiDM samples
expression data were used in k-means clustering using Pearson correlation
for distance measures (Multi Experiment Viewer v. 4.8).
IPA (Ingenuity) analysis. Data were analyzed using IPA (Ingenuity Systems). Differentially expressed genes (EAE onset MiDMs versus naive microglia and EAE onset MDMs versus naive splenic monocytes) were used in
downstream effects and upstream regulators analyses. Uploaded dataset for
analysis were filtered using cutoff definition of 1.5-fold change. Level of confidence for analysis was set to high-predicted and experimentally observed.
Terms used in IPA analyses. The p-value is a measure of the likelihood
that the association between a set of genes in the uploaded dataset and a related function or upstream regulator is due to random association.The smaller
the p-value, the less likely it is that the association is random and the more
significant the association. In general, P < 0.05 indicate a statistically significant,
1548
nonrandom association. The p value of overlap is calculated by the Fishers

Exact Test.
The activation z-score is a value calculated by the IPA z-score algorithm.
The z-score predicts the direction of change for a function or the activation
state of the upstream regulator using the uploaded gene expression pattern
(upstream to the function and downstream to an upstream regulator). An absolute z-score of 2 is considered significant. A function is increased/upstream
regulator is activated if the z-score is 2. A function is decreased/upstream
regulator is inhibited if the z-score -2.
The bias term is the product of the dataset bias and the bias of target
molecules involved in a particular function annotation or upstream regulator
activity. A biased dataset is one where there is more up- than down-regulated
genes or vice versa. The dataset bias is constant for any given analysis and the
function/upstream regulator bias is unique for each upstream regulator/function.
When the absolute value of this term is 0.25 or higher, then that function/upstream regulators prediction is considered to be biased and the Fishers exact
p-value must be 0.01 or lower for the analysis to be considered significant.
We thank Dr. Bruce D. Trapp for invaluable suggestions. We thank Flow core
in Cleveland Clinic Foundation for the flow cytometry experiments. We thank
Aishwarya Yenepalli for help with quantification.
This research was supported by grants from the US National Institutes of
Health, the Charles A. Dana Foundation, the National Multiple Sclerosis Society, and
the Williams Family Fund for MS Research, as well as a Postdoctoral Fellowship
from National Multiple Sclerosis Society (to N. Ohno).
The authors have no competing financial interests.
Submitted: 28 November 2013
Accepted: 9 June 2014
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1549
INSIGHTS
B-RAF unlocks axon regeneration
The mechanisms that drive axon regeneration after central nervous system (CNS) injury or
disease are proposed to recapitulate, at least in part, the developmental axon growth pathways.
This hypothesis is bolstered by a new study by ODonovan et al. showing that activation of
a B-RAF kinase signaling pathway is sufficient to promote robust axon growth not only during
development but also after injury.
B-RAF was previously shown to be essential for developmental axon growth but it was
not known if additional signaling pathways are required. In this study, the authors demonstrate that activation of B-RAF alone is sufficient to promote sensory axon growth during
Insight from Valeria Cavalli (left)
development. Using a conditional B-RAF
and David Holtzman
gain-of-function mouse model, the authors
elegantly prove that B-RAF has a cell-autonomous role in the developmental
axon growth program. Notably, activated B-RAF promoted overgrowth of
embryonic sensory axons projecting centrally in the spinal cord, suggesting
that this pathway may normally be quiescent in central axons.
Could activated B-RAF also enhance axon regeneration in the adult
central nervous system? The authors found that activated B-RAF not only
enabled sensory axon growth into the spinal cord after spinal injury, but also
promoted regrowth of axons projecting in the optic nerve. Regeneration in
the injured CNS is prevented by both the poor intrinsic regrowth capacity
of axons and by inhibitory factors in the tissue environment. Importantly,
the B-RAFactivated signaling growth program was insensitive to this
repulsive environment.
Interestingly, the authors find that B-RAF synergizes with the PI3Activation of B-RAF signaling enables crushed
kinasemTOR pathway, which also functions downstream of growth factors.
sensory axons (green) to grow into the adult
This opens the possibility that combinatorial approaches that integrate these
spinal cord in both white (white arrows) and gray
(pink arrows) matter.
two pathways may heighten regenerative capacity.
This in vivo study significantly advances the understanding of the role
of MAP kinases in axon growth and suggests that reactivation of the B-RAF pathway may be exploited to promote axon
regeneration in the injured central nervous system. An exciting future avenue will be to determine the downstream mechanisms
controlled by B-RAF.
ODonovan, K.J., et al. 2014. J. Exp. Med. http://dx.doi.org/10.1084/jem.20131780.
Valeria Cavalli and David M. Holtzman, Washington University School of Medicine in Saint Louis: cavalli@pcg.wustl.edu and holtzman@neuro.wustl.edu
Article
B-RAF kinase drives developmental axon

growth and promotes axon regeneration
in the injured mature CNS
Kevin J. ODonovan,1,2 Kaijie Ma,1,2 Hengchang Guo,1 Chen Wang,3,4
Fang Sun,3,4 Seung Baek Han,5,6 Hyukmin Kim,5,6 Jamie K. Wong,7
Jean Charron,9 Hongyan Zou,7,8 Young-Jin Son,5,6 Zhigang He,3,4
and Jian Zhong1,2
1Burke
Medical Research Institute, Weill Cornell Medical College of Cornell University, White Plains, NY 10605
and Mind Research Institute, Weill Cornell Medical College of Cornell University, New York, NY 10065
3F.M. Kirby Neurobiology Center, Boston Childrens Hospital; and 4Department of Neurology; Harvard Medical School, Boston, MA 02115
5Shriners Hospitals Pediatric Research Center and 6Department of Anatomy and Cell Biology, Temple University
School of Medicine, Philadelphia, PA 19140
7Fishberg Department of Neuroscience and 8Department of Neurosurgery, Friedman Brain Institute, Icahn School of Medicine
at Mount Sinai, New York, NY 10029
9Centre de Recherche en Cancrologie de lUniversit Laval, Centre Hospitalier Universitaire de Qubec, Qubec,
Qubec G1R 2J6, Canada
2Brain
Activation of intrinsic growth programs that promote developmental axon growth may also
facilitate axon regeneration in injured adult neurons. Here, we demonstrate that conditional activation of B-RAF kinase alone in mouse embryonic neurons is sufficient to drive
the growth of long-range peripheral sensory axon projections in vivo in the absence of
upstream neurotrophin signaling. We further show that activated B-RAF signaling enables
robust regenerative growth of sensory axons into the spinal cord after a dorsal root crush
as well as substantial axon regrowth in the crush-lesioned optic nerve. Finally, the combination of B-RAF gain-of-function and PTEN loss-of-function promotes optic nerve axon
extension beyond what would be predicted for a simple additive effect. We conclude that
cell-intrinsic RAF signaling is a crucial pathway promoting developmental and regenerative
axon growth in the peripheral and central nervous systems.
CORRESPONDENCE
Jian Zhong:
jiz2010@med.cornell.edu
Abbreviations used: CGRP,
calcitonin gene-related peptide;
CNS, central nervous system;
DREZ, dorsal root entry zone;
DRG, dorsal root ganglion;
kaB-RAF, kinase-activated
B-RAF; NGF, nerve growth
factor; RGC, retinal ganglion
cell; SCI, spinal cord injury.
Axon growth is essential for the establishment

of a functional nervous system as well as for the
restoration of neuronal connectivity after injury
or disease. It has long been hypothesized that reactivation of developmental growth mechanisms
might help to achieve axon regeneration in the
injured adult nervous system (Filbin, 2006).The
role of MAP kinases in axon growth signaling
has been much studied and discussed (Markus
et al., 2002; Hanz and Fainzilber, 2006; Agthong
et al., 2009; Hollis et al., 2009). However, depending on the model systems used, the outcomes
have been controversial or even contradictory
(Pernet et al., 2005; Sapieha et al., 2006; Hollis
et al., 2009). We have shown that RAFMEK
signaling robustly promotes axon growth in primary sensory neurons in vitro (Markus et al.,
2002). In vivo, conditional gene targeting studies
K.J. ODonovan and K. Ma contributed equally to this paper.

J. Exp. Med. 2014 Vol. 211 No. 5 801-814
have shown that RAF signaling is necessary for

developing sensory neurons to arborize in their
target fields in the skin (Zhong et al., 2007).
However, it remains unknown whether RAF signaling is sufficient to enable axon growth in vivo
or whether concomitant activation of other
signaling pathways is necessary to drive longrange axon projections. Furthermore, it is unclear whether this pathway can promote axon
growth in neuronal populations beyond the sensory neurons and the extent to which it can be
harnessed to promote regeneration in the injured central nervous system (CNS). To address
these questions, we have used conditional B-RAF
gain-of-function mouse models to show that
2014 ODonovan et al. This article is distributed under the terms of an Attribution
NoncommercialShare AlikeNo Mirror Sites license for the first six months after
the publication date (see http://www.rupress.org/terms). After six months it is
available under a Creative Commons License (AttributionNoncommercialShare
Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/
by-nc-sa/3.0/).
801
Figure 1. Conditional expression of kaBRAF specifically activates MAP kinase

signaling in the nervous system. (A) E18.5
mouse embryos expressing kaB-RAF develop
grossly normally, except for substantial hydrocephalus. (B) DRG size is unaffected by
kaB-RAF expression. (C) Representative Western blots show increased levels of pMEK1/2
and pERK1/2 in the E12.5 neocortex and spinal cord. (D) Quantitation of Western blots as
in C. Amounts are normalized to corresponding III-tubulin levels. n = 4 repeats with
three animals per group for each bar. Students t test: *, P < 0.05; **, P < 0.01. (EG)
Analysis of the interaction of kaB-RAF with
other signaling pathways in the E12.5 nervous
system. Representative Western blots are
from three independent experiments, each
with two or three E12.5 embryos for each
genotype. (G) B-RAF protein levels are not
increased in E12.5 DRG and spinal cord of
LSL-kaBraf:nesCre embryos and control
littermates. Molecular mass is indicated in
kilodaltons. (H) Representative images of
nerve endings in trunk skin of E13.5 embryos.
Bars: (B) 200 m; (H) 40 m. (I) The number of
branches per nerve trunk was quantitated,
blinded to the genotypes, for a defined skin
section by counting the total number of
branch points observed along each major
nerve trunk entering the skin (Zhong et al.,
2007). n = 3 for each group. (D and I) Error
bars indicate SEM.
activation of intraneuronal RAFMEK signaling is sufficient to

promote robust axon growth in developing and regenerating
neurons in the peripheral nervous system and CNS.
RESULTS
Activation of B-RAF signaling alone is sufficient to promote
sensory axon extension during early development
In vivo, the neurotrophin nerve growth factor (NGF) signals
through its receptor kinase TrkA to promote developmental
extension of dorsal root ganglion (DRG) nociceptive TrkA+
802
peripheral axons into the epidermis. To examine whether

RAF signaling alone is sufficient to promote long-range axon
extension of TrkA-positive neurons, we set out to selectively
activate RAF kinase signaling in these neurons in a TrkAnull background.
To this end, we first conditionally activated RAF signaling
in a WT background using a genetically modified loxP-STOPloxP-BrafV600E (LSL-kaBraf) knock-in mouse line (Mercer et al.,
2005), in which a kinase-activated B-RAF (kaB-RAF) mutant
is expressed from the endogenous B-RAF locus upon Cre
recombination.We next bred LSL-kaBraf mice with a neuronal
B-RAF drives axon growth and regeneration | ODonovan et al.
Ar ticle
Figure 2. Expression of kaB-RAF substantially rescues sensory afferent growth in the absence of TrkA/NGF signaling. (A, left) Normal sensory
cutaneous innervation at E16.5. (middle) Sensory cutaneous innervation is lost in embryos lacking the NGF receptor TrkA. (right) Expression of kaB-RAF
restores cutaneous innervation. Arrowheads label the blue -galpositive (presumptive TrkA+) sensory trajectories. (B) Visualization of axon growth patterns after tissue clearing. The thoracic somatosensory innervation driven by kaB-RAF in a TrkA/ embryo (bottom; compare with middle for TrkA/
alone) is similar to that seen in a control TrkAWT/ littermate (top). White arrowheads indicate the normal pathways of peripheral axons extending from
thoracic DRGs. Red arrowheads indicate sensory projections rescued by kaB-RAF in the TrkA/ background. (C) Expression of kaB-RAF substantially rescues trigeminal TrkA+ afferent growth in the absence of TrkA/NGF signaling. Presumptive TrkA+ trigeminal axon projections (top) are lost in TrkA-deficient
mice (middle) and are rescued by kaB-RAF (bottom). Ga, great auricular nerve; Go, greater occipital nerve; Mn, mandibular branch; Mx, maxillary branch;
Op, ophthalmical branch. Images show littermates and are representative of three embryos per genotype. Bars: (A) 2 mm; (B and C) 1 mm.
nestin promoter-driven Cre deleter (nes-Cre; Tronche et al.,

1999). In DRG neurons, nes-Cremediated recombination occurs as early as embryonic day (E) 11.5 (Galabova-Kovacs et al.,
2008). Embryos heterozygously expressing kaB-RAF progressively developed macrocephaly from E13.5 onwards (Fig. 1 A)
but appeared otherwise normal, including normally sized
DRGs (Fig. 1 B). The known RAF effectors were activated
in neuronal tissues expressing Cre recombinase, as indicated by
elevated phospho-MEK1/2 (pMEK1/2) and pERK1/2 in the
neocortex and spinal cord of E12.5 LSL-kaBraf:nes-Cre mice
JEM Vol. 211, No. 5
(Fig. 1, C and D). Note that compared with MEK1/2, ERK1/2

activation appears minor in the kaB-RAFexpressing DRGs;
this is because of relatively high levels of pERK1/2 in the DRG
at baseline. B-RAF activation did not affect mTOR phosphorylation (Fig. 1 E). Levels of pAKT, pS6K, and pGSK3 were
not changed significantly in the DRG of LSL-kaBraf:nes-Cre
mice (Fig. 1 F), indicating minimal cross talk between the MAP
kinase and PI3-kinaseAKT pathways. Because the expression
of kaB-RAF is under the control of endogenous Braf promoter, the expression level of B-RAF protein is not changed in
803
Figure 3. Axon terminal innervation of

E18.5 footpad. (A, top) Normal innervation.
(middle) In the absence of TrkA, innervation is
diminished overall, and the CGRP-positive
nociceptor endings are completely absent.
Red arrowheads indicate the CGRP-positive
axon terminals in the epidermis. (bottom)
kaB-RAF expression partially rescues nociceptive innervation in the TrkA/ background.
The dashed lines indicate the dermalepidermal
border. Bar, 100 m. (B) Quantification of
axon innervation in footpad (Luo et al., 2007;
Hancock et al., 2011). Data are from three
fetuses per genotype. Error bars indicate SEM.
One-way ANOVA with post-hoc Tukeys HSD
test: **, P < 0.01.
the DRG and spinal cord at E12.5 (Fig. 1 G). At E13.5, the
branching pattern of sensory nerves in the skin was not changed
by kaB-RAF expression (Fig. 1, H and I).
B-RAF activation rescues nociceptor axon
extension in embryos lacking TrkA
To test whether kaB-RAF is sufficient to drive nociceptor
axon growth in the absence of TrkA signaling, we next mated
the LSL-kaBraf:nes-Cre line with available TrkAtaulacZ and
Bax/ lines to generate LSL-kaBraf:TrkAtaulacZ/taulacZ:Bax/:
nes-Cre mice. In TrkAtaulacZ mice, the WT TrkA gene is replaced by a taulacZ expression cassette, such that the axonal
morphology of putative TrkA+ neurons can be visualized by
-gal staining (Moqrich et al., 2004). Because TrkA expression is absent in homozygous TrkAtaulacZ/taulacZ mice, we refer
to the TrkAtaulacZ/taulacZ as TrkA/ in the text below. Removal of the Bax gene blocks apoptosis in embryonic DRG
neurons, rescuing them from cell death that is otherwise observed in the absence of TrkA signaling. The Bax/ background thus allows for the molecular dissection of signaling
pathways that specifically affect axon growth (Knudson et al.,
1995; Lentz et al., 1999; Patel et al., 2000; Markus et al., 2002;
Kuruvilla et al., 2004; Moqrich et al., 2004). In TrkA/:
Bax/ mice, DRG neurons survive, but sensory afferent innervation in the skin is completely abolished (Fig. 2, A and B,
middle; Patel et al., 2000). Compared with control littermates
804
(which include LSL-kaBraf:TrkA/:Bax/, nes-Cre:TrkA/:

Bax/, and TrkA/:Bax/ genotypes), in which we detected
no LacZ-positive fibers in the skin at E16.5, expression of kaBRAF in TrkA/:Bax/ DRG and trigeminal neurons substantially restored cutaneous sensory axon projections (Fig. 2,
AC). The morphologies of the radial thoracic trajectories derived from spinal DRGs as well as those growing from trigeminal ganglia induced by kaB-RAF in the TrkA/:Bax/
background were grossly similar to those seen in control mice.
Epidermal innervation of the footpad was partially rescued by
kaB-RAF (Fig. 3, A and B). Thus, sustained B-RAF kinase
activity can, to a large extent, substitute for TrkA-mediated
axon growth signaling in presumptive TrkA+ sensory neurons.
B-RAFmediated axon growth indirectly rescues
calcitonin gene-related peptide (CGRP) expression
in TrkA/:Bax/ neurons
CGRP expression in the peptidergic subset of nociceptive
DRG neurons is induced by skin-derived factors and therefore indicates that sensory axon peripheral innervation into
the skin is complete (Hall et al., 1997, 2001; Patel et al., 2000;
Xu and Hall, 2007). In TrkA/:Bax/ mice, DRG neurons and their centrally projecting axons are devoid of CGRP
because of the lack of cutaneous innervation as previously described (Patel et al., 2000), whereas CGRP expression in the
spinal motor neurons remains unaffected (Fig. 4 A, middle).
Ar ticle
Figure 4. Activation of B-RAF indirectly

rescues CGRP expression in TrkA/ nociceptive neurons. (A, left) Normal CGRP staining in the DRG and superficial dorsal horn.
Arrowhead indicates CGRP-expressing spinal
motoneurons. (middle) CGRP expression is
completely abolished in the DRG and its
projections in TrkA/Bax double-null mice.
CGRP staining in spinal motoneurons is not
affected by loss of TrkA signaling (arrowhead).
(right) CGRP expression in DRG is rescued by
expression of kaB-RAF, in the absence of TrkA
signaling (LSL-kaBraf:nes-Cre:TrkA/:Bax/).
Arrowhead indicates the CGRP+ motor neurons. Dashed white lines outline the spinal cord
and DRG. (B) The nociceptive projection into
the dorsal horn (left) does not depend on TrkA
(middle). Expression of kaB-RAF causes overgrowth and ectopic targeting of these fibers
(right). (A and B) Images are representative of
three embryos each. (C) Activation of B-RAF
does not directly induce CGRP expression in
cultured DRG neurons. (top) No CGRP is expressed in 7-d in vitro cultures of dissociated
E12.5 LSL-kaBraf:Bax/:nes-Cre DRG neu
rons. (bottom) NGF and conditioned medium
from skin cultures are necessary to induce
CGRP expression in E12.5 LSL-kaBraf:Bax/:
nes-Cre DRG neurons. Images are representative of three independent experiments. This
experiment has been repeated three times.
Each experiment used two embryos per genotype. Bars: (A and B) 100 m; (C) 20 m.
B-RAF gain-of-function restored CGRP expression in the

absence of TrkA (Fig. 4 A, right). Note also that in contrast
to the projections in the periphery, the growth of presumptive
TrkA-positive afferent projections in the dorsal horn is independent of TrkA signaling (Fig. 4 B, middle; Patel et al., 2000;
Harrison et al., 2004) and that kaB-RAF expression caused
overgrowth of these afferents (Fig. 4 B, right) but not of peripheral projections (Fig. 1). In vitro, kaB-RAF alone did not
induce CGRP expression in DRG neurons (Fig. 4 C, top);
this required the addition of NGF and skin-conditioned medium as previously reported (Fig. 4 C, bottom; Hall et al., 1997;
Patel et al., 2000; Xu and Hall, 2007). In addition, we have
shown previously that loss of both B- and C-RAF in DRGs
does not abrogate the CGRP expression (Zhong et al., 2007).
These data together indicate that the restoration of CGRP
expression in LSL-kaBraf:nes-Cre:TrkA/:Bax/ DRG neurons is not directly caused by the elevation of neuron-intrinsic
B-RAF activity, but indirectly through the restoration of cutaneous innervation and subsequent retrograde signaling from
skin-derived factors.
kaB-RAF causes overgrowth of nociceptive
and proprioceptive afferent fibers in the spinal cord
In WT mice, different subpopulations of sensory neurons pro
ject from the DRG to highly specific targets in the spinal cord.
JEM Vol. 211, No. 5
Specifically, nociceptive TrkA+ fibers terminate in the superficial laminae I and II of the dorsal horn, and proprioceptive parvalbumin-positive afferents project to intermediate laminae or to
the ventral spinal cord.
In B-RAF gain-of-function mice, we observed excessive
growth of both nociceptive and proprioceptive afferents (Fig. 5).
Nociceptive axons normally restricted to superficial dorsal
horn extended ectopically into deeper layers of dorsal spinal
cord, and many axons aberrantly crossed the midline (Fig. 5 A).
This kaB-RAFdriven overgrowth was substantially rescued
by concomitant elimination of MEK1/2, the canonical downstream kinases of RAF (Fig. 5 C), suggesting that the effect of
kaB-RAF expressed from the endogenous Braf locus depends
strictly on canonical signaling.
In WT mice, the central proprioceptive afferents enter the
cord medially at tightly circumscribed dorsal root entry zones
(DREZs; Fig. 5 B, left). kaB-RAF expression caused the proprioceptive sensory axons to enter the spinal cord all across
its surface and to aberrantly terminate some branches in the
superficial dorsal laminae (Fig. 5 B, right). Proprioceptive
axons in the DREZs normally are subject to repulsive guidance from Semaphorin 6C/D (Sema6) expressed in the spinal cord, acting on PlexinA1 on the sensory axons (Yoshida
et al., 2006). kaB-RAF expression did not detectably alter the
protein (Fig. 5 D) or transcript levels (RNAseq; not depicted)
805
Figure 5. Activation of B-RAF drives overgrowth of centrally projecting nociceptive and proprioceptive DRG axons in the E18.5 spinal
cord. (A) Nociceptive projections stained for TrkA. Yellow arrowheads indicate the different patterns of axon projections of WT and kaB-RAFexpressing, nociceptive neurons. (B) Proprioceptive projections stained for parvalbumin. White arrowheads indicate the different patterns of axon projections
of WT and kaB-RAFexpressing, proprioceptive neurons. Asterisks label the presumptive DREZs. (C) kaB-RAFdriven overgrowth of central nociceptive
projections (left) is abolished in the absence of the downstream effectors MEK1/MEK2 (right). (AC) n = 3 per genotype. (D) kaB-RAF does not affect
the expression of known guidance cues PlexinA1 and Sema6D in the E12.5 DRG and spinal cord. Western blot is representative of three independent experiments, each with two embryos per genotype. Molecular mass is indicated in kilodaltons. (EJ) Cross sections of P0 spinal cord at cervical
(E and H), thoracic (F and I), and lumbar (G and J) levels were stained for CGRP, parvalbumin (Parv), and Draq5 (blue). Bars: (A and B) 200 m;
(C) 100 m; (EJ) 50 m.
of these factors in E12.5 DRG and spinal cord. The overgrowth

phenotype for both nociceptive and proprioceptive afferents
was observed at all levels of the spinal cord (Fig. 5, EJ). This
806
phenotype suggested that reactivation of the B-RAF pathway in injured adult neurons might be exploited to promote regeneration.
Ar ticle
Figure 6. Activation of B-RAF signaling in mature DRG neurons elevates their growth competency. (A, top) Schematic of the brn3a-CreERT2
construct used to generate the brn3a-CreERT2 deleter mouse line. (bottom) A cross section of the spinal cord of a 10-wk-old Rosa26-lacZ:brn3aCreERT2 mouse treated with tamoxifen. Blue LacZ staining indicates CreERT2-medicated recombination in the DRG neurons. (B) Representative DRGs
from adult LSL-kaBraf:TdTom:brn3a-CreERT2 mice without (top left) and with (bottom left) tamoxifen treatment. TdTom expression indicates recom
bination in DRG neurons. Cells were counterstained with Draq5 (Dq5) to label nuclei. (C) ATF3 is induced by preconditioning lesion. Blue shows nuclear stain Draq5. (D) Representative images of adult DRG neurons derived from intact brn3a-CreERT2:TdTom (left), LSL-kaBraf:brn3aCreERT2:TdTom
JEM Vol. 211, No. 5
807
Figure 7. Activation of B-RAF signaling enables crushed sensory

axons to regenerate into adult spinal cord. (AD) Confocal views of
regenerating dorsal root axons in whole mounts (A and B) or transverse
sections (C and D) 2 wk after root crush. Axons were labeled by AAV-GFP
injected into C6 and C7 DRGs at the time of the crush injury. (A and C) Control mice (brn3a-CreERT2). (B and D) Mice expressing activated B-RAF in
DRGs (LSL-kaBraf:brn3a-CreERT2). Dashed yellow lines indicate the DREZ,
dashed gray lines indicate the border between gray and white matter, and
arrowheads indicate the extent of axon growth across the DREZ (B) and
into gray matter (D). DH, dorsal horn; DR, dorsal root; PNS, peripheral nervous system; SC, spinal cord. n = 2 DRGs from each of three animals per
genotype. Bars, 200 m.
kaB-RAF enables regeneration of injured adult

DRG central axons across the DREZ
To test whether activation of B-RAF signaling can drive mature sensory axon regeneration, we generated LSL-kaBraf:
TdTomato (TdTom):brn3a-CreERT2 mice to inducibly express
kaB-RAF in adult DRG neurons. The brn3a-CreERT2 deleter
mouse line was generated using a brn3a promoter (Eng et al.,
2001), which mediates expression selectively in sensory neurons (Fig. 6 A). We first assessed B-RAF gain-of-function in
cultured adult neurons. 12-wk-old mice were treated with
tamoxifen for a consecutive 5 d to induce kaB-RAF expression, as indicated by TdTom expression (Fig. 6 B). ATF3, a
marker whose expression is triggered by conditioning lesion
(Smith and Skene, 1997; Seijffers et al., 2007) was not induced
(Fig. 6 C). DRG neurons were cultured for up to 24 h. kaBRAF expression correlated with both greatly increased numbers of axon-bearing neurons and increased total axon length
with more branching compared with that of WT neurons

(Fig. 6, DG). Furthermore, the axonal morphology of kaBRAFexpressing neurons differs from WT neurons subject to
a preconditioning lesion, which exhibited single long axons
(Fig. 6, D and H).
Having thus ascertained the functionality of the LSLkaBraf:TdTom:brn3a-CreERT2 mouse line, we next tested
whether kaB-RAF can enable axon regeneration after dorsal
root crush injury in vivo. 12-wk-old mice were again treated
with tamoxifen for a consecutive 5 d. After 2-d rest, C58
cervical roots were crushed and AAV2-GFP was injected to
C6 and C7 DRGs to label regenerating sensory axons. After
2 wk, regeneration in the C6 and C7 roots and spinal cord
was examined in whole-mount preparations (Fig. 7, A and B)
or in transverse sections (Fig. 7, C and D). As expected, in
control brn3a-CreERT2 mice (Fig. 7, A and C), axons regenerated along the roots but stopped at the DREZ. In contrast, in
mice with kaB-RAF expression in DRG neurons, numerous
axons penetrated the DREZ and grew deeply into the spinal
cord, exhibiting dense collateral branches in the dorsal column (Fig. 7 B) and reaching superficial laminae of the dorsal
horn (Fig. 7 D). Thus, elevation of intrinsic B-RAF signaling
is sufficient, both in vitro and in vivo, to induce robust axon
regrowth of adult DRG neurons and, importantly, renders the
axons capable of overcoming growth-inhibitory signals that
are abundant at the DREZ and within the spinal cord.
kaB-RAF enables regenerative axon growth in the injured
optic nerve through an MEK-dependent pathway
To test whether activation of B-RAF kinase signaling can promote axon regeneration of injured mature CNS neurons, we
used an optic nerve regeneration model (Fig. 8, A and B; Park
et al., 2008; Benowitz and Yin, 2010). 812-wk-old LSL-kaBraf:
Bax/ mice and Bax/ controls were injected intravitreally
with AAV2-Cre to induce kaB-RAF expression in retinal ganglion cells (RGCs) and then subjected to optic nerve crush.The
Bax/ background was used to minimize apoptotic death of
retinal ganglion neurons triggered by optic nerve injury, which
may amount to 80% at 2 wk after optic nerve crush (Li et al.,
2000). 2 wk after the injury, we observed robust regenerative
axon growth up to 3 mm past the lesion site in the kaB-RAF
expressing optic nerve (Fig. 8, D and G), with very limited
growth in the control Bax/ littermates (Fig. 8 C), consistent
with previous observations that survival alone is not sufficient to
promote growth of adult RGC axons (Goldberg et al., 2002).
Combined deletion of the canonical RAF effector kinases
MEK1 and MEK2 substantially suppressed the regenerative
axon growth caused by kaB-RAF (Fig. 8 E), indicating that
kaB-RAF drives axon growth through the canonical MEK
effectors. Whereas the length of axon extension induced by
(middle), and WT preconditioning lesioned mice (right) after 24 h in vitro. TdTom is shown in green to improve contrast. Bars: (AC) 100 m; (D) 20 m.
(EH) Quantitation of axon extension in adult DRG cultures at 24 h in vitro. Data were collected from three independent experiments from three animals per genotype or condition and analyzed as described previously (Parikh et al., 2011); >100 cells were counted per group. Error bars indicate SEM.
One-way ANOVA with post-hoc Tukeys HSD test: *, P < 0.01; **, P < 0.005.
808
Ar ticle
Figure 8. Activation of B-RAF enables regenerative

axon growth in the crush-lesioned optic nerve via the
canonical effectors MEK1/2. (A) Schematic of experimental time course. (B) Intravitreal injection of AAV2-Cre induces
expression of TdTom in retinal ganglion neurons, labeling the
entire optic nerve (red). (C, top) Whole-mount image of a
crushed Bax/ optic nerve. Crush site is indicated by a red
asterisk here and in all following panels. (bottom) Confocal
fluorescence image of the same nerve. Green shows axons
anterogradely labeled with CTBAlexa Fluor 488. (D) Wholemount confocal imaging shows strong regenerative growth
in the lesioned kaB-RAFexpressing optic nerve. (inset) Axons
at 3.5 mm from the crush site (magnified from the boxed
area). Arrowheads indicate outgrowing axons. (E) Loss of
MEK1 and MEK2 abolishes the regeneration driven by kaB-RAF.
(F) Optic nerve regeneration in the absence of PTEN. Bar,
0.5 mm. (CF) Images are representative of three optic
nerves per genotype. (G) Quantitation of axon regenerative
growth in the optic nerve 2 wk after nerve crush; genotypes
as shown in BE. At 1.6 mm from the crush site, the density of regenerating axons is more than threefold greater in
the LSL-kaBraf:Bax/ genotype than in the Ptenf/f:Bax/
genotype. Data are from three nerves per genotype. Optic
densities were acquired from the whole-mount optic nerves
using an LSM710NLO two-photon confocal microscope with
the ZEN2009 software. Data were normalized by setting the
baseline OD, as measured 0.2 cm proximal to the crush site
in all nerves, to the same (arbitrary) level.
B-RAF gain-of-function is comparable with the maximal

axon growth reported in PTEN deletion mice (Fig. 8 F; Park
et al., 2008), in a direct comparison, we found up to a 3.9-fold
JEM Vol. 211, No. 5
higher density of regenerating axons in the LSL-kaBraf:Bax/

mice 1.5 mm distal to the crush site than is seen in the crushed
Pten/:Bax/ optic nerve (Fig. 8, D, F, and G).
809
Figure 9. Combined B-RAF activation and PTEN deletion enables

long-range axon regeneration. (A) Representative longitudinal sections of regenerating optic nerve 2 wk after crush injury. Genotypes are
as indicated. Crush site is indicated by a red asterisk. Bar, 200 m. (B) Quantitation of data as shown in A. Axons were counted as described in Park
et al. (2008). n = 6 nerves per genotype. Error bars indicate SEM. Twoway ANOVA tests comparing LSL-kaBraf with Ptenf/f, LSL-kaBraf and
Ptenf/f with WT, or LSL-kaBraf and Ptenf/f with LSL-kaBraf:Ptenf/f all resulted in p-values < 0.001.
kaB-RAF expression and PTEN deletion synergize

to increase axon regenerative growth
To test whether combined activation of the B-RAF and PI3kinasemTOR pathways can further boost axon growth capacity in injured adult retinal ganglion neurons, we performed
the optic nerve regeneration experiments using double LSLkaBraf:Ptenf/f mice. Both the numbers and length of regenerating axons were increased in the optic nerve of the LSL-kaBraf:
Ptenf/f mice compared with those in single LSL-kaBraf or in
Ptenf/f mice (Fig. 9 A). The synergistic effect of activating both
B-RAF and PI3-kinase signaling is most apparent at the long
est lengths of axon regeneration (Fig. 9 B). Note that the mice
in these experiments were from a Bax WT background to enable direct comparison with the original PTEN deletion data
(Park et al., 2008); however, this means that possible survivalpromoting effects of the mutant alleles cannot be excluded.
810
DISCUSSION
An understanding of the mechanisms that drive axon growth
is important, both to decipher how connectivity develops in
the nervous system and to develop therapeutic strategies for
nervous system repair after injury or disease.We show that the
RAFMEK axis plays a key role in axon growth signaling.
Activation of B-RAF in neurons is sufficient to drive sensory
axon growth in the embryo, to enable adult sensory axons to
regenerate across the DREZ and further into the spinal cord,
and to induce robust regeneration of adult retinal axons in
the injured optic nerve. Both developmental DRG axon
overgrowth in the spinal cord and mature RGC axon regeneration in the optic nerve were abrogated by concomitant
ablation of MEK1 and MEK2. We thus establish classical cellautonomous RAFMEK signaling as a fundamental driver of
axon growth. We should note that this pathway seems to be
selective to axon growth signaling because we have never observed that B-RAF activation supports neuronal survival (unpublished data).
In vitro work has long suggested a potential role for RAF
MEK signaling in axon growth. Previous in vivo data, however,
have been scarce and controversial. In the retina, for example,
pharmaceutical inhibition of MEKERK signaling abrogated
optic nerve regeneration supported by FGF2 (Sapieha et al.,
2006). Two putative intracellular activators of RAF signaling,
BAG1 and Mst3b, have been shown to promote regenerative
axon growth in the optic nerve (Planchamp et al., 2008; Lorber
et al., 2009), but the expression of a constitutively active MEK1
did not drive any regeneration in the optic nerve (Pernet et al.,
2005). Others have concluded that ERK activity promotes
RGC axon regeneration via an indirect mechanism dependent
on glial cells (Mller et al., 2009). Although it is likely that mul
tiple mechanisms, direct as well as indirect, will contribute to
axon regeneration in the inhibitory environment of the CNS,
the current cacophonic state of the field is likely caused by the
mainly indirect approaches of incomplete penetrance that have
been taken by various laboratories.When using small molecule
inhibitors or transient viral overexpression of interfering or activating constructs, it is difficult to accurately titrate the dose for
the entire duration of an experiment. We believe that we have
applied a stringent approach toward activation of RAF signaling in RGCs, and our data argue strongly for a direct positive
effect of RAFMEK signaling on axon growth and regeneration of RGCs, as well as in DRG neurons. Possible downstream
mechanisms beyond the MEK kinases remain speculative at
this point. Stabilization of microtubules improves axon regeneration in a spinal cord injury (SCI) model through both neuron-intrinsic and -extrinsic mechanisms (Hellal et al., 2011),
and it is likely that activation of RAFMEK signaling will directly affect microtubule stability in injured axons via its effects
on microtubule-regulating enzymes such as HDAC6 (Williams
et al., 2013). Furthermore, B-RAF has been shown to directly
interact with tubulin (Bonfiglio et al., 2011). Activation of
B-RAF signaling is also likely to trigger the expression of axon
growthenhancing gene sets in injured neurons. The elucidation of exact mechanisms awaits further study.
Ar ticle
The developmental phenotypes we observed in the

B-RAF gain-of-function embryos were generally complementary to those previously observed in B-RAF/C-RAF
loss-of-function mice (Zhong et al., 2007). In contrast to
nociceptors peripheral projections, the development of their
central projections does not depend on NGF/TrkA signaling
(Patel et al., 2000; Harrison et al., 2004; Zhong et al., 2007).
Notably, we found that activation of B-RAF resulted in overgrowth of both proprioceptive and nociceptive axons in the
spinal cord, whereas the expression of two known repulsive
signaling molecules in the dorsal cord and DRG, Sema6 and
PlexinA1, remained unaltered. Thus, kaB-RAF appears to activate a normally quiescent axon growth signaling pathway in the
central sensory axons that seems to be unaffected by repulsive
guidance cues.
The importance of this effect, the lack of response to repulsive or inhibitory cues, becomes clear in the context of regeneration of central sensory branches after dorsal root crush
injury. Sensory axons expressing kaB-RAF robustly regenerated into the DREZ and spinal cord.
The regeneration failure of DRG axons after dorsal root
avulsion injuries has been variously attributed to the lack of
intrinsic growth capacity, to extrinsic growth barriers such as
glia-associated growth inhibitors at the DREZ, and to premature synaptic differentiation (Han et al., 2012; Smith et al., 2012).
Application of neurotrophic factors acting via tyrosine kinase
receptors has shown substantially enhanced regeneration (Ramer
et al., 2000;Wang et al., 2008; Harvey et al., 2010), even functional recovery with the systemic administration of artemin
(Wang et al., 2008), although these results await independent
replication. Future studies will test whether a combination of
RAF activation with trophic growth factors can further enhance axon regeneration and reinnervation of presumptive targets in the spinal cord.
Compared with spinal cord lesions, the optic nerves simple structure allows for clear evaluation of both lesion and regeneration. In recent years, the optic nerve model has revealed
several intracellular signaling pathways that can drive CNS
axon regeneration, most prominently the PI3-kinasemTOR
and the JAKSTAT pathways, engaged by growth factor tyrosine kinase receptors and cytokines (Park et al., 2008; Smith
et al., 2009; Buchser et al., 2012; Leibinger et al., 2013; Pernet
et al., 2013). Combined deletion of endogenous inhibitors of
these two pathways enhanced regeneration above the level
reported for deletion of either gene alone (Sun et al., 2011).
Activation of PI3-kinasemTOR via PTEN deletion also enhanced regenerative growth in an SCI model (Liu et al., 2010),
suggesting that results obtained in the optic nerve crush model
may generally translate to SCI models. Here, we show that
the classic growth factor signaling module RAFMEK enables
axon regeneration in the optic nerve at least as powerfully as
any previously reported single molecule manipulation and that
the combination of kaB-RAF with activation of PI3-kinase
mTOR via PTEN deletion enhances optic nerve axon regeneration even more strongly than would be expected for a simple
additive effect.
JEM Vol. 211, No. 5
The extent of regeneration achieved by direct genetic activation of specific intracellular signaling pathways, including
B-RAF, DLK, PI3-kinasemTOR, KLF, and JAKSTAT pathways (Smith et al., 2009; Yan et al., 2009; Park et al., 2010;
Blackmore et al., 2012; Shin et al., 2012; Lang et al., 2013), compares favorably with what has been reported for application of
growth factors such as NGF, BDNF, GDNF, and CNTF (Lykissas
et al., 2007; Zhang et al., 2009; Allen et al., 2013; Leibinger
et al., 2013). Growth factors as promoters of regeneration are
hobbled by two major issues. First, the signaling machinery that
enables growth factors to drive axon growth in the developing
nervous system is not expressed at sufficient levels in the adult
nervous system (Hollis et al., 2009). Indeed, growth inhibitory
signaling molecules such as the SOCS family or phosphatases
are up-regulated upon maturation (Lu et al., 2002; Smith et al.,
2009; Park et al., 2010; Gatto et al., 2013). Therefore, the most
promising studies using growth factors have combined them
with genetic intervention to up-regulate growth factor receptors
or down-regulate their intrinsic inhibitors (Hollis et al., 2009;
Sun et al., 2011). The second issue is that of undesirable side effects, especially that of neuropathic pain caused by neurotrophin
administration (Obata et al., 2006; Jankowski and Koerber, 2009).
Development of painless neurotrophins (Capsoni et al., 2011)
may improve the usefulness of this family of growth factors in the
context of regeneration. Future combined activation of several
growth signaling pathways with blockade of growth inhibitory
pathways may lead to realistic treatment options for patients with
loss of vision, sensation, or locomotion.
Mouse models. Mouse breeding and genotyping were performed as described previously (Mercer et al., 2005; Zhong et al., 2007). The animal protocol was approved by the Institutional Animal Care and Use Committee at Weill
Cornell Medical College, and experiments were conducted in accordance with
the National Institutes of Health Guide for the Use and Care of Laboratory
Animals.The LSL-kaBraf mouse line was provided by C.A. Pritchard (University of Leicester, Leicester, England, UK; Mercer et al., 2005). The TrkAtaulacZ
mouse was provided by L. Reichardt (University of California, San Francisco,
San Francisco, CA; Moqrich et al., 2004). Nestin-Cre deleter and Bax-null
mice were generated in R. Kleins laboratory (Max Planck Institute of Neurobiology, Martinsried, Germany;Tronche et al., 1999) and S.J. Korsmeyers laboratory (Dana-Farber Cancer Institute, Boston, MA; Knudson et al., 1995),
respectively. The brn3a-CreERT2 deleter mouse line was generated by J. Zhong
in W.D. Sniders laboratory (University of North Carolina at Chapel Hill,
Chapel Hill, NC). All mice were on a mixed 129Sv and C57BL/6 background.
We used littermates as controls throughout.
Generation of the brn3a-CreERT2 deleter mouse line. Brn3a is a POU
domain transcription factor that is selectively expressed in DRG neurons.
Using a brn3a promoter construct (Eng et al., 2001), we generated a brn3a-CreERT2 deleter mouse line using the pronuclear injection technique (Fig. 6 A).
Western blotting and immunohistochemical staining. Western blotting and immunohistochemical staining were performed as described previously (Zhong et al., 2007). An equal amount of total protein was loaded in
each lane. The antibodies used were as follows: TrkA, Brn3a, and PlexinA1
antibodies were provided by L. Reichardt, E. Turner (University of California, San Diego, La Jolla, CA), and T. Jessell (Columbia University, New York,
NY), respectively. Antibodies against MEK1/2 (9122), pMEK1/2 (9121),
ERK1/2 (9102), pERK1/2 (9101), pAKT (9271 and 9275), phospho-p70S6K
811
(9206), phospho-mTOR Ab (2971), and pGSK3 (9336) were obtained

from Cell Signaling Technology. III-Tubulin (AA10) was purchased from
Invitrogen; Sema6D (S-16) was purchased from Santa Cruz Biotechnology, Inc.; C-RAF antibody (610151) was purchased from BD. Parvalbumin
antibody (PV28) was obtained from Swant. CGRP antibodies (AB5920 and
AB5705) were obtained from EMD Millipore. All Western blot and immunohistochemical experiments were repeated with tissue from at least three
embryos for each genotype, and these embryos were obtained from a different litter for each experiment. Littermate controls were used throughout.
LacZ staining. E16.5 embryos were fixed in 4% paraformaldehyde and
stained with X-gal using EMD Millipores Tissue Base staining solution according to the manufacturers protocol. After imaging of axon skin innervation,
embryos were dehydrated using a methanol in PBS dilution series (2550%,
7595%, and 100%), followed by incubation in 50% methanol: 50% benzoyl
alcohol/benzoyl benzoate (BABB), and subsequently cleared in 100% BABB
(Sigma-Aldrich). Specimens were imaged with a M205A stereomicroscope
equipped with a DFC310FX color digital camera system (Leica).
DRG culture. For CGRP expression assay, E12.5 DRGs derived from
LSL-kaBraf:Bax/:nes-Cre embryos were cultured with 100 ng/ml NGF
(Alomone Labs) and skin-conditioned medium for 8 d. DRG neurons
cultured in N2-supplied MEM (without NGF) were used as control. Both
cultures where treated with FdU (5-fluoro-2-deoxyuridine; Sigma-Aldrich).
Culture media were changed every 12 h. The cells were fixed 8 d thereafter,
and the cells were stained for CGRP and TrkA. For axon growth and neuron
survival assays, DRGs were dissected from E12.5 embryos with the desired
genotypes as described previously (Markus et al., 2002; Zhong et al., 2007).
The cells were dissociated and plated on laminin-coated coverslips. Cells were
then cultured in serum-free media supplemented with 1 N2 (Invitrogen)
and 1% BSA. FdU, NGF, or AAV2-Cre (Vector Laboratories) were added as
described previously (Markus et al., 2002; Zhong et al., 2007).
Dorsal root crush. Surgeons and all other personnel performing experiments and analyses were blinded as to genotypes. 1014-wk-old mice were
injected s.c. with tamoxifen (5 g/10 g body weight) for a consecutive 5 d.
After another 2 d, i.e., 1 wk from the first tamoxifen injection, crush injury
of the C5, C6, C7, and C8 dorsal roots was performed using a fine forceps
(Dumont #5) as described previously (Di Maio et al., 2011). AAV2-GFP
(1010 GC, 7004;Vector Laboratories) was then injected to C6 and C7 DRGs.
2 wk after the crush, mice were perfused, and axon regeneration through the
C6 and C7 dorsal roots was analyzed in a thin dorsal slice preparation of whole
spinal cord or in cryostat sections.
Sciatic nerve crush. Mice were injected with tamoxifen (2 g/10 g body
weight) for a consecutive 5 d, followed by 5 d of rest. Unilateral sciatic nerve
crush was then performed as described previously (Zhong et al., 1999). Adult
DRGs were collected and cultured as described previously (Zou et al., 2009).
Images were taken using a Carl Zeiss LSM710NLO confocal microscope. Axon
length was quantified as described previously (Zou et al., 2009).
Optic nerve crush. Surgeons and all other personnel performing experiments and analyses were blinded as to genotypes.The crush-regeneration and
axon counting protocol is adapted from Park et al. (2008). Whole-mount
optic nerves were treated with FocusClear (CelExplorer Labs) and scanned
using an LSM710NLO multiphoton confocal microscope. OD was determined with the Carl Zeiss ZEN2009 software.
We would like to thank Louis Reichardt for the TrkAtaulacZ mice and TrkA antibody,
Catrin A. Pritchard for the LSL-kaBraf mice, Eric Turner for the Brn3a promoter, and
Thomas Jessell for the Plexin1A antibody. Larry Benowitz provided experimental
advice on the optic nerve injury model. Rajiv Ratan, David Ginty, William D. Snider,
and Annette Markus are acknowledged for insightful discussion and suggestions.
This work was supported by startup funds from the Burke Foundation as well
as Whitehall Foundation research grant 2010-08-61, grants 1R01EY022409 and
812
3R01EY022409-01S1 from the National Eye Institute (NEI), and grant ZB1-1102-1
from the Christopher & Dana Reeve Foundation to J. Zhong. Z. He is supported by
grants 5R01EY21526 and EY021342 from NEI. Y.-J. Son is supported by grant
1R01NS079631 from the National Institute of Neurological Disorders and Stroke
and grants from Shriners Hospitals for Children and the Muscular Dystrophy
Association. H. Zou is supported by grants from the National Institutes of Health
(1R01NS073596) and the Irma T. Hirschl/Monique Weill-Caulier Foundation.
K.J. ODonovan is a Goldsmith fellow.
The authors declare no competing financial interests.
Submitted: 24 August 2013
Accepted: 18 March 2014
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Brief Definitive Report
T cellderived interleukin (IL)-21 promotes

brain injury following stroke in mice
Benjamin D.S. Clarkson,1,3 Changying Ling,1 Yejie Shi,2 Melissa G. Harris,1,4
Aditya Rayasam,4 Dandan Sun,2,5 M. Shahriar Salamat,1 Vijay Kuchroo,6
John D. Lambris,7 Matyas Sandor,1 and Zsuzsanna Fabry1
1Department
of Pathology and Laboratory Medicine, 2Department of Neurological Surgery, 3Department of Cellular

and Molecular Pathology, 4Neuroscience Training Program, School of Medicine and Public Health, University
of Wisconsin-Madison, Madison, WI 53792
5Veterans Affairs Pittsburgh Health Care System, Geriatric Research, Educational and Clinical Center, Pittsburgh, PA 15213
6Center for Neurological Diseases, Brigham and Womens Hospital Harvard Medical School, Boston, MA 02115
7Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104
T lymphocytes are key contributors to the acute phase of cerebral ischemia reperfusion
injury, but the relevant T cellderived mediators of tissue injury remain unknown. Using a
mouse model of transient focal brain ischemia, we report that IL-21 is highly up-regulated
in the injured mouse brain after cerebral ischemia. IL-21deficient mice have smaller
infarcts, improved neurological function, and reduced lymphocyte accumulation in the
brain within 24 h of reperfusion. Intracellular cytokine staining and adoptive transfer
experiments revealed that brain-infiltrating CD4+ T cells are the predominant IL-21 source.
Mice treated with decoy IL-21 receptor Fc fusion protein are protected from reperfusion
injury. In postmortem human brain tissue, IL-21 localized to perivascular CD4+ T cells in the
area surrounding acute stroke lesions, suggesting that IL-21mediated brain injury may be
relevant to human stroke.
CORRESPONDENCE
Zsuzsanna Fabry:
zfabry@facstaff.wisc.edu
Abbreviations used: BA, basilar
artery; ECA, external carotid
artery; ICA, internal carotid
artery; I/R, ischemia/reperfusion; MCA, middle cerebral
artery; PCA, posterior cerebral
artery; PComA, posterior communicating artery; RAG,
recombination activating
gene; rCBF, regional cerebral
blood flow; tMCAO, transient
MCA occlusion; TTC, 2,3,5triphenyltetrazolium chloride;
VA, vertebral artery.
Stroke is one of the leading causes of death and

disability worldwide. Clinical and preclinical experimental studies highlight the importance of
inflammation in both acute and delayed neuronal tissue damage after ischemic stroke; however,
the mechanisms and cells involved in this neuroinflammation are not fully understood. There is
currently no available treatment targeting the
acute immune response that develops in the
brain after transient focal ischemia. Therefore,
we sought to identify novel T cellderived cytokines that contribute to acute cerebral reperfusion using the mouse model of transient middle
cerebral artery occlusion (tMCAO).
During the reperfusion of infarcted brain tissue, leukocytes accumulate in the injured brain
where, in addition to clearing cell debris, they
promote secondary tissue injury (Yilmaz and
Granger, 2010). Within the acute phase of ischemic reperfusion (I/R) injury there are multiple
waves of cell infiltration of macrophages, neutrophils, and lymphocytes (Gelderblom et al., 2009).
Brain-infiltrating T cells have also been widely
reported in stroke and animal models of stroke
and are thought to have acute detrimental and
delayed protective effects (Magnus et al., 2012).

J. Exp. Med. 2014 Vol. 211 No. 4 595-604
Conventionally, the protective role of T cells has

been attributed to the accumulation of regulatory T cells within the CNS in later stages of reperfusion injury. These T cells produce a variety
of cytokines including TGF and IL-10, which
are both antiinflammatory and neuroprotective.
(Liesz et al., 2009; Stubbe et al., 2013). In addition to having an established role in delayed
neuroprotection, Kleinschnitz et al. (2013) have
recently shown that CD4+ CD25+ regulatory
T cells also promote acute ischemic injury through
interaction with the cerebral vasculature. The
acute detrimental effects can be further divided
into early (24 h) and late (72 h) phases, with
IL-17 production by nonconventional T cells
(less common T cell subset associated with mucosal tissues) possibly accounting for the latter by
promoting neutrophil accumulation (Gelderblom
et al., 2012).
The mechanisms of the early detrimental effects of T cells after cerebral ischemia are least
2014 Clarkson et al. This article is distributed under the terms of an Attribution
595
understood. Several laboratories have reported reduced neurological deficit and infarct volumes at 2448 h reperfusion in
T celldeficient mice after tMCAO (Yilmaz and Granger, 2010).
After tMCAO, recombination activating gene 1deficient
(RAG1 KO) mice, which lack T and B lymphocytes, have significantly smaller brain injury compared with controls; whereas,
adoptive transfer of WT CD4+ helper T cells or CD8+ cyto
toxic T cells increases stroke infarct volumes within 24 h after
ischemia in these mice (Kleinschnitz et al., 2010). Additionally,
TCR-transgenic mice and mice lacking co-stimulatory TCR
signaling molecules were fully susceptible to acute I/R injury,
indicating that T cell involvement at early time points is antigen-independent (Kleinschnitz et al., 2010). These data demonstrate that conventional CD4+ or CD8+ T cells exacerbate
acute injury after cerebral ischemia independently of TCR ligation, and this effect seems to be concomitant with an early
increase in T cell infiltration into the postischemic brain, which
many have reported to be between 3 and 48 h (Yilmaz et al.,
2006; Gelderblom et al., 2009).
Recent findings suggest that, in the postischemic brain,
within hours of reperfusion T cells accumulate in postcapillary
segments of periinfarct inflamed cerebral microvasculature
characterized by high endothelial expression of chemokines
and adhesion molecules. These postcapillary venules have been
postulated to allow accumulating immune cells to activate each
other and promote platelet adhesion in a process termed
thrombo-inflammation (Nieswandt et al., 2011). Much research
has been devoted to identifying T cell factors that promote
thrombo-inflammation (Barone et al., 1997; Hedtjrn et al.,
2002; Yilmaz et al., 2006; Shichita et al., 2009; Gelderblom et al.,
2012); however, to our knowledge no study has yet identified
the T cellderived factors responsible for the early increase in
infarct volumes at 24 h reperfusion. Here, we present data that
identify IL-21 as a key CD4+ T cellderived inflammatory factor that contributes to increased early ischemic tissue injury.
RESULTS AND DISCUSSION
Robust up-regulation of IL-21 during cerebral I/R injury
IL-21 is closely related to IL-2 and IL-15 and signals through
the IL-21 receptor, which is comprised of an IL-21specific
subunit and a common subunit shared with IL-2, IL-7, IL-9,
and IL-15. IL-21 is known to regulate immune responses by
promoting antibody production, T cellmediated immunity,
and NK cell and CD8+ T cell cytotoxicity. Recently, others
have shown that stress signals from necrotic tissue can induce
rapid IL-21 production from naive T cells (Holm et al., 2009),
and co-stimulation with TLR3 ligands during polyclonal T cell
activation significantly increases IL-21 secretion that contributes
to small intestine localized pediatric celiac disease (van Leeuwen
et al., 2013).To test whether IL-21 is up-regulated in brain after
ischemic necrosis induced by MCAO and to better understand
the cytokines involved in T cellmediated cerebral I/R injury,
we measured changes in inflammatory gene expression in the
brain within 24 h after tMCAO in mice using PCR-based
gene array analysis. In addition to verifying the up-regulation
596
of several previously reported inflammatory genes, we found

that IL-21 was one of the most highly expressed inflammatory
genes among those measured (Fig. 1 a). Gene expression levels
from arrays were normalized to interquartile spot intensity.
Arrays did not differ systemically in gene expression levels before or after normalization (not depicted).This increase in IL-21
gene expression was confirmed by real-time (RT) PCR analysis, which detected a >24-fold relative increase in IL-21 gene
expression in the ipsilateral ischemic brain tissues compared
with the contralateral hemisphere at 24 h reperfusion (Fig. 1 b).
IL-21 was not detectable by this method in healthy brain tissue (Fig. 1 b).
IL-21deficient mice are protected from acute
neuronal injury after cerebral I/R injury
Whether IL-21 contributes to ischemic tissue injury had not
been directly studied. However, in the last few years it has become evident that IL-21 expression is associated with acute
rejection in mice after kidney, heart, or liver allograft (Baan et al.,
2007; Hecker et al., 2009; Xie et al., 2010). Because these
models also involve reperfusion of ischemic tissues, these findings support the potential role of IL-21 in I/R injury.
We evaluated the levels of cerebral I/R injury in IL-21
deficient (IL-21 KO) mice. Infarct volumes in IL-21 KO mice
were reduced to 35% of the infarct volumes observed in congenic C57BL6/J WT mice as early as 24 h after tMCAO, as
measured by triphenyltetrazolium chloride (TTC; Fig. 1 c).
Similar effects were also seen at 4 d (not depicted) and 7 d after
tMCAO (Fig. 1 d), indicating that IL-21 contributes to both
immediate and delayed brain injury and suggesting that in the
absence of IL-21 tissue repair can occur. Analysis of intracranial vascular anatomy revealed no differences between WT and
IL-21 KO mice in the patency of the posterior communicating
artery that would account for the observed differences in tissue
injury (Fig. 1 g). Nor did we observe differences between WT
and IL-21 KO mice in heart rate, or blood pressure before or
after tMCAO (Fig. 1 h). WT and IL-21 KO CD4+ T cells,
CD8+ T cells, and CD11b+ myeloid cells showed no difference
in IL-2, IL-17A, IFN-, or TNF production when stimulated
in vitro (Fig. 2, gl). The reduction in infarct volumes corresponded with less weight loss (unpublished data), less spleen
atrophy (Fig. 2 c), and improved neurological functioning in
IL-21 KO mice compared with WT mice as assessed by both
grip strength (Fig. 1 e) and Bederson (Fig. 1 f) scoring 1, 4, and
7 d after tMCAO.
We measured accumulation of monocytes and lymphocytes in the brain after tMCAO in IL-21 KO and WT animals
(gating strategy in Fig. 2 a).We did not observe significant differences in the rate of accumulation of monocytes (CD45high
CD11b+Ly6chigh), microglia (CD45intCD11b+), B cells (B220+),
T cells (TCR+), NK, or NKT cells (NK1.1+) in the
ischemic brain of WT and IL-21 KO mice after 1, 4, and 7 d
of reperfusion (unpublished data). In contrast, as early as 1 d
after tMCAO, IL-21 KO mice showed significantly diminished cerebral accumulation of CD4+ T cells and CD8+ T cells
IL-21 promotes brain injury after stroke | Clarkson et al.
Br ief Definitive Repor t
Figure 1. IL-21 is up-regulated early in

mouse brain, and IL-21deficient mice are
protected after tMCAO. GeArray S Series
Mouse Autoimmune and Inflammatory Response gene array of transcripts expressed in
pooled brain tissues 24 h after tMCAO or
sham procedure (n = 36 mice per group).
(a) Bar graphs show PCR array spot intensity
of genes with a greater than sixfold difference
in gene expression in tMCAO compared with
sham, normalized to the interquartile mean
spot intensity. (b) IL-21 mRNA expression
level in ipsilateral hemisphere relative to contralateral hemisphere 24 h after tMCAO (n = 3
per group). Mann-Whitney rank sum test
*, P < 0.05. Plots show median, lower, and upper
quartile (box) and range (error bars). Infarct
volumes of WT and IL-21 KO mice 24 h after
tMCAO (c) and 7 d after tMCAO (d). Representative images of TTC-stained 2-mm brain
slices shown below (n = 57 mice per group).
WT and IL-21 KO mice grip strength (e) and
Bederson score (f) at 1, 4, and 7 d after 1 h
tMCAO (n = 78 for each group). (g) Brain
vasculature of C57BL/6 and IL-21KO mice
perfused transcardially with carbon lampblack
(C198-500; Thermo Fisher Scientific) in 20%
gelatin ddH2O. Arrows indicate anterior cerebral (ACA), MCA, posterior cerebral (PCA),
basilar (BA), and vertebral arteries (VA), showing point of occlusion (white circle). High
magnification images demonstrate no difference in patency of the posterior communicating artery (PComA, arrows). (h) Heart rate and
blood pressure of WT and IL-21 KO mice before and after tMCAO (n = 34 mice per
group). Data are representative of 23 independent experiments. **, P < 0.01; ***, P <
0.001; ****, P < 0.0001 by Students t test
(single comparison) or one-way ANOVA (multiple comparisons). Error bars indicate SD.
after tMCAO compared with WT mice (Fig. 2 e) and these

differences persisted at day 7 (Fig. 2 f ). These differences were
not reflected in the spleen before or after tMCAO (Fig. 2 b).
IL-21 has also been shown to be produced by and modulate
the function of regulatory T cells (Peluso et al., 2007; Battaglia
et al., 2013), which begin to accumulate in the brain after
tMCAO. Thus, we compared the frequency of regulatory
CD4 T cells expressing the marker Foxp3 in the brain and
spleen 24 h after tMCAO in WT and IL-21 KO mice. IL-21
KO mice exhibited no difference in regulatory T cell abundance
JEM Vol. 211, No. 4
in either tissue compared with WT experimental animals. Nor

did we observe a difference between WT and IL-21 KO mice
in the frequency of lymphocytes producing the antiinflammatory cytokine IL-10 among B cells (B220+), CD8+ T cells,
or CD4+ T cells (unpublished data). These data demonstrate
that IL-21 deficiency is protective at acute time points after
tMCAO and IL-21 levels in the CNS correlate with early infiltration of T cells without affecting regulatory T or B cell
accumulation or IL-10 cytokine production during the acute
period (day 14).
597
Figure 2. Lymphocyte recruitment to brain is diminished in IL-21 deficient mice. (a) Gating strategy for leukocytes isolated from brain after
MCAO. (b) WT and IL-21 KO spleen cells 24 h after tMCAO or sham procedure (n = 3 mice per group). (c) Relative change in spleen weight of WT and IL-21
KO mice after tMCAO (n = 37 mice per group). (d) Percentage of blood and spleen CD4+ T cells expressing IL-21 after 5-h ex vivo stimulation with PMA
(10 ng/ml) and Ionomycin (1 g/ml) 4 d after MCAO or control treatment. (e and f) Leukocyte accumulation in the brain of WT mice compared with IL-21
KO mice 1, 4, and 7 d after tMCAO (n = 36 mice per group). (gk) In vitro cytokine expression by WT and IL-21 KO CD4+ and CD8+ T cells after 5-h
stimulation under indicated conditions with or without recombinant mouse IL-21 (100 ng/ml). (l) TNF production by CD11b+ myeloid cells stimulated with
LPS (500 ng/ml) for 5 h with or without recombinant mouse IL-21 (100 ng/ml). Cells isolated from n = 3 mice per group. Data are representative of two to
four independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by Students t test (single comparison) or one-way ANOVA (multiple
comparisons). Error bars indicate SD (bd and gl) and SEM (e, f).
598
IL-21 is primarily produced

by brain-infiltrating CD4+ T cells
We measured intracellular IL-21 production by various cell
populations. IL-21producing cells were not detected by flow
cytometry among cells isolated from healthy brain, but could
be detected among mononuclear cells isolated from ischemic brain 24 h after tMCAO. Gating on IL-21+ cells revealed
that the majority of these cells were CD4+ T cells (Fig. 3 a).
IL-21producing CD4+ T cells were also detected at low levels among cells isolated from blood, but not spleen, of healthy
WT mice, and these levels were unaffected after transient cerebral ischemia (Fig. 2 d), indicating that the increase in IL-21
production was limited to CD4+ T cells recovered from the
postischemic brain. Next, we adoptively transferred WT or
IL-21 KO CD4+ T cells into lymphocyte-deficient RAG2
KO mice. Purity of transferred CD4+ T cells was confirmed
by flow cytometry to be 95% (Fig. 3 b). As shown previously (Kleinschnitz et al., 2010), we observed markedly reduced infarcts in RAG KO mice compared with WT mice,
and infarct volumes could be restored to WT levels in RAG2
KO by adoptively transferring WT CD4+ T cells. Most importantly, RAG2 KO mice that received WT CD4+ T cells
had significantly larger infarcts than those receiving IL-21
KO CD4+ T cells (Fig. 3 c).
IL-21 blockade is protective in tMCAO

We treated WT mice with IL-21 receptor Fc protein (IL-21R.
Fc) using a previously described protocol (Jang et al., 2009;
McGuire et al., 2011; Spolski et al., 2012). We administered
500 g of IL-21R.Fc i.p. 1 h before tMCAO. As measured by
TTC staining, treated mice showed significantly reduced infarct volumes compared with control-treated mice 24 h after
tMCAO (Fig. 4 a). We found a similarly protective effect in
mice treated with IL-21R.Fc protein (500 g i.p.) 2 h after initiation of reperfusion (Fig. 4 a). These differences were associated with decreased locomotor function (decreased resistance
to lateral push and increased circling behavior) in controltreated mice compared with those treated with IL-21R.Fc
(Fig. 4 b and Video 1). Although we cannot exclude the possibility that IL-21R.Fc exhibits its blocking effect in peripheral immune compartments, using ELISA for human IgG4 we
were able to observe thatupon i.p. injectionsoluble IL-21R.
Fc accumulates in the CNS of mice after tMCAO (Fig. 4 c).
IL-21 presence localizes with CD4 staining
in human stroke tissue
IL-21+ cells were recently detected in human brain tissue
during different neuroinflammatory conditions (Tzartos et al.,
2011).Thus, we stained groups of postmortem brain tissue from
Figure 3. IL-21 is primarily produced by brain-infiltrating CD4+ T cells. (a) Intracellular cytokine staining of lymphocytes isolated from n = 5
pooled healthy WT, ischemic IL-21/, or ischemic WT mouse brains 24 h after tMCAO or sham procedure showing IL-21 versus CD8 expression. Histograms show CD4, NK1.1, and TCR expression on IL-21+ cells from ischemic WT brain. (b) CD45, CD4, and LFA-1 expression by negative fractions purified
from WT and IL-21/ lymph node cells by CD4+ negative selection using magnetic cell separation before transfer into RAG2/ recipients. (c) Infarct
volume in WT mice (n = 4), RAG2/ mice (n = 4), RAG2/ mice + WT CD4 T cells (n = 10), and RAG2/ mice + IL-21/ CD4 T cells (n = 10) 24 h
after tMCAO. Representative TTC-stained 2-mm mouse brain slices shown on top. Data are representative of two independent experiments. **, P < 0.01;
***, P < 0.001; ****, P < 0.0001 one-way ANOVA. Error bars indicate SD.
JEM Vol. 211, No. 4
599
Figure 4. Blockade of IL-21 signaling before or after tMCAO reduces infarct size in WT mice. (a) Infarct volumes 24 h after tMCAO in WT mice
treated with 500 g recombinant mIL-21R.Fc or PBS 1 h before (pretreatment) or 2 h after (posttreatment) surgery. Representative TTC-stained brain
slices shown on left (n = 34 mice per group). (b) Still image from Video 1 depicting behavioral differences between WT mice posttreated with IL-21R.Fc
or PBS. (c) IL-21R.Fc protein levels in the indicated organs 2024 h after tMCAO in WT mice injected with 500 g IL-21R.Fc 2 h after start of reperfusion
(n = 24 mice per group). N.D., not detected. Data are representative of two independent experiments. **, P < 0.01; ***, P < 0.001, by Students t test. Error
bars indicate SEM. Representative images of postmortem paraffin-embedded human acute stroke lesions stained with control sera (d), or primary antibodies against CD4 (e and g [ii-iii]), IL-21 (f and g [iii]), or eosin (g [i]) visualized with Fast Red (d, e, and g) and/or DAB (d, f, and g [iii]) and counterstained
with hematoxylin. High magnification images are shown on right. Arrows indicate positive staining. Bars, 50 m.
600
patients with acute and chronic stroke lesions. In acute infarcts,

rare CD4+ T cells were found in the necrotic brain parenchyma
(Fig. 4 g, ii, arrows), which was predominantly infiltrated by
foamy macrophages (Fig. 4 g, i). In contrast, CD4+ T cells were
consistently found within the Virchow Robins space of vessels
bordering acute infarcts (not depicted) and in the subarachnoid
space adjacent to meningeal vessels (arrows, Fig. 4 e, i). IL-21
staining was limited almost exclusively to these perivascular
spaces. Compared with control stained tissue (Fig. 4 d, i), anti
IL-21 staining labeled cells extensively in the subarachnoid
space of deep sulci penetrating the infarcted tissueshowing
a similar distribution to CD4+ cells in serial sections (arrows,
Fig. 4 f ). Additionally, double staining with antibodies for
CD4 and IL-21 revealed the presence of CD4+ IL-21+ cells
in this perivascular niche (arrow, Fig. 4 g, iii). In summary, CD4+
T cells that can secrete IL-21 were detected within the

CSF-filled subarachnoid and perivascular spaces during cerebral infarction in humans.
Neuronal cells express IL-21 receptor and up-regulate
autophagy genes in response to IL-21
RT-PCR analysis of primary mouse neurons and murine neuronal cell lines (Neuro2A) indicated that IL-21R expression
was higher on neuronal cells than on other brain cells, including astrocytes and endothelial cells (Fig. 5, a and c). This is consistent with another report where in situ hybridization detected
neuron-restricted IL-21R expression in inflamed human brain
tissue (Tzartos et al., 2011). Moreover, treating Neuro2a cells
with IL-21 after in vitro oxygen glucose deprivation (OGD)
significantly increased cell death as measured by XTT cell
Figure 5. IL-21 promotes autophagy expression in neuronal cells after hypoxia/ischemia.

(a) Il21r mRNA expression relative to GAPDH
expression levels is shown in normoxic and hypoxic primary mouse neurons after OGD or control treatment. (b) Viability of Neuro2A cells
treated with the indicated doses of IL-21 after
OGD. (c) Il21r mRNA expression relative to
GAPDH in neuronal (Neura2A), astrocytic, and
endothelial cell lines (MB114) expressed relative
to BMDC expression. (d) ATG6 expression in primary neurons treated with PBS, etoposide, or
32256 ng/ml rIL-21 for 4 h after 12 h oxygen
glucose deprivation as measured by RT-PCR. Cells
treated in triplicate. (e) Number of ATG6+ cells per
field in the same regions of WT and IL-21 KO
mouse brains after tMCAO as assessed by immune staining (n = 3 mice per group). Arrows
indicate ATG-6+ cells in periinfarcted brain tissue
of WT and IL-21 KO mice. Bars, 100 m. Data
are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001;
****, P < 0.0001 by Students t test (single comparison) or one-way ANOVA (multiple comparisons). Error bars indicate SEM.
JEM Vol. 211, No. 4
601
viability assay (Fig. 5 b). In subsequent studies we found that

treatment of primary neurons with IL-21 up-regulated mRNA
levels of the autophagy associated gene ATG6 (Fig. 5 d). These
data suggest that IL-21 could directly affect neuronal autophagy during ischemic injury, which has been implicated in neuronal death in infarcted and periinfarcted brain tissue.Thus, we
stained WT and IL-21 KO postischemic brain tissues for ATG6.
We observed significantly fewer ATG6+ cells in infarcted brain
tissue of IL-21 KO mice compared with WT, suggesting that
IL-21 may contribute to increased cerebral autophagy after
stroke (Fig. 5 e).
In conclusion, we implicate IL-21 as a lymphocyte-derived
factor with a pronounced effect on brain injury after focal ischemia in mice. We also present data demonstrating that IL-21
producing CD4+ T cells are present in the brain of patients
with acute stroke.These data warrant investigation of the therapeutic potential of IL-21modifying treatments in isolation
and combination with current anti-thrombotic treatments for
ischemic stroke.
Ethics statement. C57BL/6 WT mice were obtained from The Jackson
Laboratory. IL-21deficient mice (IL-21tm1Lex) were purchased from the Mutant Mouse Regional Resources Center. All mice underwent 1 h tMCAO
and 24 h reperfusion. All animal procedures used in this study were conducted in strict compliance with the National Institutes of Health Guide for
the Care and Use of Laboratory Animals and approved by the University of
Wisconsin Center for Health Sciences Research Animal Care Committee.
All mice (25 g) were anesthetized with 5% halothane for induction and
1.0% halothane for maintenance vaporized in N2O and O2 (3:2), and all efforts were made to minimize suffering.
Regional cerebral blood flow (rCBF) measurement. Changes in rCBF
at the surface of the left cortex were recorded using a blood perfusion monitor
(Laserflo BPM2;Vasamedics) with a fiber optic probe (0.7 mm diam). The tip
of the probe was fixed with glue on the skull over the core area supplied by the
MCA (2 mm posterior and 6 mm lateral from bregma). Changes in rCBF after
MCAO were recorded as a percentage of the baseline value. Mice included in
these investigations had >80% relative decrease in rCBF during MCAO.
Investigation of intracranial vasculature. WT and IL-21 KO mice were
anesthetized with ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.).
After thoracotomy was performed, a cannula was introduced into the ascending aorta through the left ventricle. Transcardial perfusion fixation was performed with 2 ml saline and 2 ml of 3.7% formaldehyde. Carbon lampblack
(C198-500; Thermo Fisher Scientific) in an equal volume of 20% gelatin in
ddH2O (1 ml) was injected through the cannula. The brains were removed
and fixed in 4% PFA overnight at 4C. Posterior communicating arteries
(PComA) connect vertebrobasilar arterial system to the Circle of Willis and
internal carotid arteries, and its development affects brain sensitivity to ischemia among different mouse strains (Barone et al., 1993). Development of
PComA in both hemispheres was examined and graded on a scale of 03, as
reported previously (Majid et al., 2000). 0, no connection between anterior
and posterior circulation; 1, anastomosis in capillary phase (present but poorly
developed); 2, small truncal PComA; 3, truncal PComA.
Focal ischemia model. Focal cerebral ischemia in mice was induced by occlusion of the left MCA, as described previously (Longa et al., 1989). Operators
performing surgeries were masked to experimental groups. In brief, the left
common carotid artery was exposed, and the occipital artery branches of the
external carotid artery (ECA) were isolated and coagulated. After coagulation
602
of the superior thyroid artery, the ECA was dissected distally and coagulated
along with the terminal lingual and maxillary artery branches.The internal carotid artery (ICA) was isolated, and the extracranial branch of the ICA was then
dissected and ligated. A standardized polyamide resin glue-coated 6.0 nylon
monofilament (3021910; Doccol Corp) was introduced into the ECA lumen,
and then advanced 99.5 mm in the ICA lumen to block MCA blood flow.
During the entire procedure, mouse body temperature was kept between 37
and 38C with a heating pad. The suture was withdrawn 60 min after occlusion. The incision was closed, and the mice underwent recovery.
Infarction size measurement. After 24 h reperfusion, mice were sacrificed
and brains were removed and frozen at 80C for 5 min. 2-mm coronal
slices were made with a rodent brain matrix (Ted Pella, Inc.). The sections
were stained for 20 min at 37C with 2% TTC (Sigma-Aldrich). Infarction
volume was calculated with the method reported by Swanson et al. (1990) to
compensate for brain swelling in the ischemic hemisphere. In brief, the sections were scanned, and the infarction area in each section was calculated by
subtracting the noninfarct area of the ipsilateral side from the area of the
contralateral side with National Institutes of Health image analysis software,
ImageJ. Infarction areas on each section were summed and multiplied by section thickness to give the total infarction volume.
Gene array and RT-PCR. Ipsilateral brain hemispheres were dissected and
stored in RNAlater (QIAGEN) at 4C until further use. Total RNA was extracted and purified with RNeasy Protect Mini kit (QIAGEN) according to
the manufacturers instructions. Purified RNA samples were analyzed by
GeArray S Serious Mouse Autoimmune and Inflammatory Gene array
(SuperArray; Bioscience Corporation). Results from GeArray were filtered
for genes with spot intensities higher than the mean local background of the
bottom 75% of nonbleeding spots. For RT-PCR, 1 g total RNA from each
sample was reverse transcribed using SuperScript II first strand cDNA synthesis kit (Invitrogen). RT-PCR was performed on a Smart Cycler (Model SC
1001; Cepheid) using IL-21 TaqMan gene expression assay (Mm00517640_m1;
Applied Biosystems), RT2 qPCR Primer assay for mouse Becn1
(PPM32434A; SABiosciences), or RT2 qPCR Primer Assay for Mouse IL21r
(PPM03762A; SABiosciences). The data were normalized to an internal reference gene, GAPDH.
Mononuclear cell isolation and flow cytometry. Brains were removed
from perfused animals, weighed, minced, transferred to Medicon inserts, and
ground in a MediMachine (BD) for 2030 s.The cell suspension was washed
with HBSS, and cells were resuspended in 70% Percoll (Pharmacia) and overlaid with 30% Percoll. The gradient was centrifuged at 2,250 g for 30 min at
4C without brake. The interface was removed and washed once for further
analysis. CD11b-positive and -negative fractions were isolated using Imag
anti-CD11b magnetic particles (BD), following the manufacturers protocol.
A total of 106 cells were incubated for 30 min on ice with saturating concentrations of labeled antibodies with 40 g/ml unlabeled 2.4G2 mAb to block
binding to Fc receptors, and then washed 3 times with 1% BSA in PBS.
Single-cell suspensions from various tissues were cultured at 37C in
10% FBS in RPMI 1640 media supplemented with GolgiStop (BD) in the presence of either phorbol myristate acetate (50 ng/ml) and ionomycin (1 g/ml)
for 5 h. After surface staining with antibodies against CD4, NK1.1, and
TCR, cell suspensions were fixed and permeabilized by Cytofix/Cytoperm solution (BD), followed by staining with antiIL-21 antibodies.
Fluorochrome-labeled antibodies against CD45, CD11b, Ly6c, B220, CD4,
CD8a, NK1.1, IFN-, and appropriate isotype controls were purchased from
BD. Fluorochrome-labeled antibody against IL-21 and TCR was purchased from eBioscience. Cell staining was acquired on a FACSCalibur or
LSRII (BD) and analyzed with FlowJo (Tree Star) software version 5.4.5.
Neurofunctional assessment. Neuromuscular coordination was assessed by
grip strength test, as previously described (Kleinschnitz et al., 2010). For this
test, mice were placed on a horizontal string midway between two supports.
Mice were scored from 0 to 5 as follows: 0, falls off within 2 s; 1, hangs on with
forepaw(s); 2, hangs on with forepaws and moves laterally on string; 3, hangs

onto string with forepaws and hindpaw(s); 4, hangs onto string with forepaws,
hindpaw(s) and tail; 5, escape to supports. Mice were allowed to rest between
trials. Scores for each mouse were determined by averaging 510 trials (each
lasting 15 s). Global neurological deficit was determined by a modified Bederson scoring system: 0, no deficit; 1, forelimb flexion; 2, unidirectional circling
after being lifted by tail; 3, spontaneous unidirectional circling; 4, longitudinal
rolling upon being lifted by tail; 5, spontaneous longitudinal rolling.
Generation of IL-21 receptor Fc fusion protein. Chinese hamster ovary
cell line (Korn et al., 2007) expressing the extracellular domain (aa 20236)
of mouse IL-21R fused to the fragment crystallizable (Fc) portion of human
IgG4 (IL-21R.Fc) were maintained in UltraCHO (BioWhittaker). IL-21R.
Ig was purified from the culture supernatant by passage through a protein
GSepharose column and concentrated by ultrafiltration. Concentration was
determined spectrophotometrically. Purity and molecular weight were confirmed by sodium dodecyl-sulfate PAGE and human-IgG4 ELISA (eBioscience) following the manufacturers instructions. The IL-21R.Fc reagent was
tested in vitro for its ability to suppress IL-21induced T cell proliferation.
Immunohistochemistry. Paraffin-embedded postmortem brain tissue sections from individuals with acute and chronic stroke lesions were obtained
from the Neuropathology Laboratory of the University of Wisconsin Department of Pathology. After rehydration and deparaffinization, sections underwent heat-induced antigen retrieval in 10 mM sodium citrate, pH 6.0, for
surface antigens or Tris-EDTA (10 mM/1 mM) with 0.05% Tween-20 for
intracellular antigens. Sections were blocked for 30 min with secondary
serum (10% in Tris-buffered saline) and then stained with primary antibodies,
0.5% chicken antiIL-21 (Lifespan Biosciences) or prediluted mouse antiCD4 ([1F6]; ab17131; Abcam) for 12 h at 37C or overnight at 4C. Normal primary sera (510%) were used for negative control. After several washes,
secondary antibodies (biotin-labeled goat antichicken or biotin-labeled
goat antimouse;Vector Laboratories) were applied to sections and incubated
for 2 h at room temperature. Staining was developed using the VECTASTAIN ABC-HRP kit (Vector Laboratories) with diaminobenzidine substrate (BD) or streptavidin-alkaline phosphatase with Fast Red substrate
(Laboratory Vision), following the manufacturers instructions. Slides were
lightly counterstained with hematoxylin, rinsed with running tap water, and
mounted. For frozen mouse sections WT and IL-21 KO mice underwent 1-h
tMCAO and 17-d reperfusion. Brains were perfused with PBS and 3% formalin, embedded in OCT, and cut into 8-m frozen sections for immuno
histochemistry. Frozen sections were thawed for 10 min at room temperature
and blocked with 5% goat serum solution in PBS for 15 min. Sections were
stained with rabbit polyclonal antibodies for ATG6 for 1 h at room temperature, followed by phycoerythrin-labeled goat antirabbit IgG (Santa Cruz
Biotechnology, Inc.). Images were acquired on a BX40 microscope equipped
with a Q-Color 3 camera using Q-Capture software (Olympus). Digital
images were processed and analyzed using Photoshop CS4 software (Adobe).
Color balance, brightness, and contrast settings were manipulated to generate
final images. All changes were applied equally to entire image.
Neuronal cell oxygen glucose deprivation. Primary neuronal cultures
derived from embryonic day 1418 mouse cortices were grown to 80% confluency in neural basal media supplemented with B27 (2%) and penicillin/
streptomycin (1%), as previously described (Kintner et al., 2010). Astrocytic
and microglial contamination was excluded based on the absence of GFAP+
and CD11b+ cells when stained by immunocytochemistry. For OGD, media
was replaced with neural basal media with or without glucose and placed in
a hypoxic chamber or under normoxic conditions for 2 h at 37C. Afterward,
cells were lysed and mRNA isolated using RNeasy mini kit (QIAGEN). For
XTT viability assay, Neuro2A underwent OGD and were treated with dose
curve of IL-21 immediately after return to normoxic media and incubated
for 4 h at 37C. XTT labeling mixture (50 l per well; Roche) was added according to manufacturers instructions, and at18 h fluorescence was read on
a GENious Microplate reader (Tecan). Cell number was calculated using a
standard curve of known untreated cells kept under normoxic conditions.
JEM Vol. 211, No. 4
Statistical analyses and quality standards. All surgeries were performed

in a blinded manner by a third party and measurements masked where possible. Infarct volume measurements from TTC stained sections were averaged
from two to three independent blinded observers. Based on power calculations, n = 310 sex- and age-matched mice were used for each experiment
and group assignment was randomized. Among animals receiving MCAO
procedure, 86.5% of WT mice, 93.5% of IL-21KO mice, and 100% of
RAG2KO mice were included in analysis. Mice were excluded due to premature death (13.5% of WT mice, 3.2% of IL-21 KO mice) or vessel variation
(3.2% of IL-21KO mice). Results are given as means 1 SD. Multiple comparisons were made using one-way ANOVA. Where appropriate, two-tailed
Students t test analysis was used for comparing measures made between two
groups. For comparison of RT-PCR data, nonparametric Mann-Whitney
rank sum analysis was used. P-values <0.05 were considered significant.
Online supplemental material. Video 1 shows groups of WT C57BL6
mice treated with 500 g IL-21R.Fc or PBS control via i.p. injection. Online supplemental material is available at http://www.jem.org/cgi/content/
full/jem.20131377/DC1.
We thank Satoshi Kinoshita for expert histopathology services, Guoqing Song for
assisting in the surgical procedures, Dr. Wenda Gao for providing reagents and
protocols for the purification of IL-21R.Fc protein, and members of our laboratory
for helpful discussions and constructive criticisms of this work. We also thank Khen
Macvilay and Sinarack Macvilay for their expertise provided for cytofluorimetry and
immunohistochemistry studies and Samuel (Joe) Ollar for assisting in the OGD procedure.
This work was supported by awards from the American Heart Association (predoctoral fellowship #12PRE12060020 to B.D.S. Clarkson) and the National Institutes
of Health (NS037570 and NS076946 to ZF, AI048087 to M.S. Salamat, and
AI068730 to J.D. Lambris).
The authors have no competing financial interests.
Submitted: 1 July 2013
Accepted: 24 February 2014
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Brief Definitive Report
Early retinal neurodegeneration and impaired

Ran-mediated nuclear import of TDP-43
in progranulin-deficient FTLD
Michael E. Ward,1,2 Alice Taubes,1 Robert Chen,2 Bruce L. Miller,2
Chantelle F. Sephton,5 Jeffrey M. Gelfand,2 Sakura Minami,1
John Boscardin,3 Lauren Herl Martens,4 William W. Seeley,2 Gang Yu,5
Joachim Herz,5 Anthony J. Filiano,6 Andrew E. Arrant,6 Erik D. Roberson,6
Timothy W. Kraft,7 Robert V. Farese, Jr.,4 Ari Green,2 and Li Gan1,2
1Gladstone
Institute of Neurological Diseases, 2Department of Neurology, 3Department of Medicine, 4Gladstone Institute

of Cardiovascular Disease, University of California, San Franciso, San Francisco, CA 94158
5Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75390
6Departments of Neurology and Neurobiology and 7Department of Vision Sciences, University of Alabama at Birmingham,
Birmingham, AL 35294
Frontotemporal dementia (FTD) is the most common cause of dementia in people under
60 yr of age and is pathologically associated with mislocalization of TAR DNA/RNA
binding protein 43 (TDP-43) in approximately half of cases (FLTD-TDP). Mutations in the
gene encoding progranulin (GRN), which lead to reduced progranulin levels, are a significant cause of familial FTLD-TDP. Grn-KO mice were developed as an FTLD model, but lack
cortical TDP-43 mislocalization and neurodegeneration. Here, we report retinal thinning as
an early disease phenotype in humans with GRN mutations that precedes dementia onset
and an age-dependent retinal neurodegenerative phenotype in Grn-KO mice. Retinal
neuron loss in Grn-KO mice is preceded by nuclear depletion of TDP-43 and accompanied
by reduced expression of the small GTPase Ran, which is a master regulator of nuclear
import required for nuclear localization of TDP-43. In addition, TDP-43 regulates Ran
expression, likely via binding to its 3-UTR. Augmented expression of Ran in progranulindeficient neurons restores nuclear TDP-43 levels and improves their survival. Our findings
establish retinal neurodegeneration as a new phenotype in progranulin-deficient FTLD, and
suggest a pathological loop involving reciprocal loss of Ran and nuclear TDP-43 as an
underlying mechanism.
CORRESPONDENCE
Ari Gree:
agreen@ucsf.edu
OR
Li Gan:
lgan@gladstone.ucsf.edu
Abbreviations used: AD,
Alzheimers disease; CDR,
clinical disease rating; FTD,
frontotemporal dementia;
FTLD, frontotemporal lobar
degeneration; GCC, ganglion
cell complex; GRN, progranulin;
INL, inner nuclear layer; OCT,
optical coherence tomography;
ONL, outer nuclear layer; PD,
Parkinsons disease; PSP,
progressive supranuclear palsy;
RGC, retinal ganglion cell;
RNFL, retinal nerve fiber layer;
PhNR, photopic negative
response; TDP-43, TAR DNA/
RNA binding protein 43.
Approximately 50% of (frontotemporal dementia) FTLD cases are characterized by cellular

aggregation and mislocalization of TDP-43 (i.e.,
FTLD-TDP). TDP-43 normally localizes to
the nucleus and regulates transcriptional control, splicing, and RNA processing (Sephton et al.,
2011; Polymenidou et al., 2011). In FTLDTDP, nuclear depletion of TDP-43 occurs, often
in neurons containing cytoplasmic TDP-43 aggregates (Neumann et al., 2006). The mechanisms underlying TDP-43 mislocalization in
FTLD have not been characterized, and whether
TDP-43 mislocalization plays a causal role in
neurodegeneration remains controversial (Lee
et al., 2012). FTLD has sporadic and familial
forms. Mutations in the GRN gene that cause
progranulin haploinsufficiency are a common

J. Exp. Med. 2014 Vol. 211 No. 10 1937-1945
cause of familial FTLD-TDP (Baker et al., 2006).

Aged Grn-KO mice exhibit FTD-like behavioral abnormalities but lack TDP-43 mislocalization or neurodegeneration in cortical regions
(Ahmed et al., 2010; Yin et al., 2010; Martens
et al., 2012).
Retinal abnormalities are documented in
Alzheimers disease (AD), progressive supranuclear palsy (PSP), Parkinsons disease, and multiple systems atrophy (Hinton et al., 1986; Bayer
et al., 2002; Tamura et al., 2006; Paquet et al.,
2007; Albrecht et al., 2012; Helmer et al., 2013).
2014 Ward et al. This article is distributed under the terms of an Attribution
Share Alike 3.0 Unported license, as described at http://creativecommons
.org/licenses/by-nc-sa/3.0/).
1937
Due to the clinical accessibility of the retina, new retinal

imaging techniques under development hold promise as
potential diagnostic and prognostic modalities for neurodegenerative diseases (Koronyo-Hamaoui et al., 2011). However,
whether retinal abnormalities are an early or late disease phenomenon has not been established. Here, we identify retinal
neurodegeneration as a novel disease-related phenotype in
human subjects with GRN mutations before clinical symptoms of dementia. In Grn-KO mouse, retinal neuronal loss
is preceded by depletion of nuclear TDP-43. We further
explore the role of Ran, a central regulator of nuclear trafficking, in TDP-43 nuclear depletion and degeneration in
Grn-KO neurons. Our findings suggest a novel relationship
between TDP-43 and Ran-mediated nuclear trafficking in
FTLD pathogenesis.
RESULTS AND DISCUSSION

Early retinal abnormalities in humans
with GRN mutations and Grn-KO mice
Because retinal neuron loss occurs in other neurodegenerative
diseases, we suspected that retinal neurons could be a vulnerable
neuronal population in humans with progranulin haploinsuf
ficiency secondary to GRN mutations. Using optical coherence tomography (OCT), we measured retinal nerve fiber layer
(RNFL) thickness and macular volume in living human control
subjects and subjects with GRN mutations. The RNFL constitutes the axonal compartment of retinal ganglion cells (RGCs)
and, as such, is a surrogate measurement of RGC number. Macular volume is a combined measurement of all of the layers of the
retina within the macula. We observed significant reductions in
RNFL thickness and macular volume in subjects with GRN
Figure 1. Progranulin deficiency causes retinal neuron loss in humans with GRN mutations and retinal neuron loss/dysfunction in a progranulindeficient mouse model of familial FTLD. (A and B) RNFL thinning (A) and macular volume loss (B) occur in humans with progranulin haploinsufficiency
caused by GRN mutations and precede dementia onset. Each dot represents value of an individual eye, and bars represent median values. Asymptomatic
GRN-mutation carriers (CDR = 0) are shown in blue and symptomatic GRN mutation carriers (CDR score 0.5) are shown in red. Control age- and sexmatched subjects are represented by gray dots. (CF) Macular ganglion cell loss occurs in GRN mutation carriers and precedes dementia onset. An automated
segmentation algorithm (C) was used to determine the volumes of GCC (D), INL (E), and ONL (F) in the maculae of control and GRN mutation carriers (DF).
Results represent a single cohort of n = 24 control subjects and 12 GRN mutation carrier subjects (7 asymptomatic, 5 symptomatic), and p-values were generated via mixed-effects linear regression analyses. (G) Progranulin expression occurs in the GCL and photoreceptor inner and outer segments (IS, OS) of
mouse retinas. Immunostaining of progranulin and DAPI staining of nuclei are shown in a representative retinal cross section. (H) RNFL thinning and loss of
inner retinal neurons shown in H&E-stained retinal cross sections from 18-mo-old Grn KO mice. Representative sections equidistant to the optic nerve are
shown. (I) Quantification of RNFL layer thickness. n = 57 mice/age/genotype; *, P < 0.05, one-way ANOVA with Tukeys post-hoc analysis, 2 independent
experiments. (J) Loss of Neu-Npositive neurons in the GCL of 18-mo Grn KO retinas. Neu-Npositive cells in the GCL were quantified in sections equidistant
to the optic nerve head. n = 57 mice/age/genotype; **, P < 0.01, one-way ANOVA with Tukeys post-hoc analysis, 2 independent experiments. (K) Impaired
light-evoked RGC electrophysiological responses in aged Grn KO mice. Electroretinograms (ERGs) were performed on 12- and 18-mo-old mice, and the amplitude of the photopic-negative response (PhNR, a RGC-specific waveform) was quantified. n = 6 mice/age/genotype; ***, P < 0.001 at 18 mo of age via repeated measures two-way ANOVA with Bonferronis multiple comparison test; 2 independent experiments. Bars: (C, G, and H) 50 m.
1938
Retinal thinning and TDP-43 mislocalization in FTLD | Ward et al.
mutations compared with controls, indicating that retinal neuron loss occurred in these subjects (Fig. 1, A and B; and Fig. S1).
A substantial number of the GRN mutation carriers enrolled
in our trial (7/12) were cognitively asymptomatic. Intriguingly, we found that this subgroup of asymptomatic GRN
mutation carriers also had significant reductions in RNFL
thickness and macular volume (Fig. 1, A and B). We then analyzed individual neuronal layers of the macula via an automated segmentation algorithm, to determine the volumes of
the ganglion cell complex (GCC), inner nuclear layer (INL),
and outer nuclear layer (ONL) in the maculae of control and
GRN-mutation carriers (Fig. 1 C). Significant thinning of the
GCC and INL, but not ONL, was observed in GRN mutation carriers (Fig. 1, DF). The volume of GCC was significantly reduced even in asymptomatic carriers, further implicating
RGC loss as an early neurodegenerative phenotype in subjects with progranulin deficiency (Fig. 1 D).
We then determined if a similar phenotype occurred in
Grn-KO mice.Total retinal progranulin expression levels were
similar to those in the brain, based on ELISA (unpublished
data), with prominent expression in RGCs and photoreceptors (Fig. 1 G). Despite a lack of significant neurodegeneration
in the brain (Ahmed et al., 2010; Yin et al., 2010), we observed progressive, substantial thinning of the RNFL and a
loss of ganglion cell layer (GCL) cells in 18-mo-old Grn-KO
mice (Fig. 1, HJ). In agreement with the pathological alterations, electrophysiological abnormalities in Grn-KO mouse
retinas paralleled GCL neuron loss. At 18 mo, but not 12 mo,
of age, Grn-KO mice had substantially reduced amplitudes
of photopic negative responses (PhNRs), a measure of RGC
function (Fig. 1 K). Amplitudes of a- and b-wave responses
were also significantly reduced in an age-dependent manner
in Grn-KO mice, indicating additional dysfunction of inner
nuclear layer neurons and photoreceptors (unpublished data).
Nuclear depletion of TDP-43 in Grn-KO
retinal neurons precedes neurodegeneration
Loss of nuclear TDP-43 is commonly observed in postmortem brain tissue from patients with FTLD-TDP (Neumann
et al., 2006; Davidson et al., 2007), including FTLD associated
with GRN mutations (Fig. 2 A). However, the mechanism of
nuclear depletion of TDP-43 in FTLD is unknown, and it
remains unclear if loss of nuclear TDP-43 plays a causal role in
FTLD pathogenesis. At 18 mo of age, levels of nuclear TDP-43
Figure 2. Nuclear depletion of TDP-43 occurs in

retinal neurons before neurodegeneration. (A) Postmortem brain tissue from a human GRN mutation carrier was co-stained with the nuclear marker DAPI (blue)
and an antiTDP-43 antibody (green). Normal TDP-43
distribution is still observed in some neurons (arrowhead), but other neurons exhibit nuclear depletion of
TDP43 (arrow) with or without apparent cytoplasmic
inclusions. Bar, 10 m. (B) Immunofluorescence confocal microscopy of TDP-43 in 18-mo-old retinal GCL
neurons. Brightness of the TDP-43 channel was increased in insets to highlight loss of nuclear TDP-43 in
Grn KO neurons. Bar, 1 m. (C) Scatter plot of nuclear
and cytoplasmic intensities of TDP-43 in GCL neurons
of 18-mo-old Grn KO mice. n = 121174 cells from
6 mice/genotype; ***, P < 0.001, mixed-effects multivariate linear regression mode; 2 independent experiments.
Bars represent median values. (D) Significant reduction
in the nuclear/cytoplasmic TDP-43 ratio was observed
in 12-mo-old Grn-KO GCL neurons (**, P < 0.01) and
18-mo-old Grn-KO GCL neurons (**, P < 0.01). n = 217357
cells from 57 mice/age/genotype, mixed-effects
multivariable linear regression model; 2 independent
experiments. Mean SEM is shown.
JEM Vol. 211, No. 10
1939
were strikingly reduced in Grn-KO retinal GCL neurons,

whereas levels of cytoplasmic TDP-43 were unchanged (Fig. 2,
B and C). Depletion of nuclear TDP-43 also occurred in
12-mo-old Grn-KO mice, before significant GCL neuron
loss (Fig. 2 D). Interestingly, neither nuclear nor cytoplasmic
TDP-43 inclusions were found in the >100 Grn-KO GCL
neurons we examined (Fig. 2 B). Thus, in progranulindeficient FTLD-TDP, nuclear depletion of TDP-43 and neu
rodegeneration can occur independent of cytoplasmic TDP-43
accumulation/aggregation. These results are consistent with
observations that TDP-43, especially nuclear TDP-43, is required for neuron survival (Wegorzewska and Baloh, 2011;
Igaz et al., 2011; Arnold et al., 2013).
TDP-43 regulates Ran mRNA levels
and requires Ran for nuclear localization
We then explored how nuclear clearing of TDP-43 occurs in
FTLD.The small GTPase Ran is a master regulator of nuclear
transport (Melchior et al., 1995), and Ran accessory proteins
are necessary for nuclear TDP-43 localization (Nishimura
et al., 2010). We hypothesized that Ran expression might be
altered in our retinal FTLD model and contribute to nuclear
TDP-43 depletion. Indeed, nuclear Ran was significantly depleted in Grn-KO GCL neurons (Fig. 3, A and B), and nuclear
TDP-43 levels correlated with those of nuclear Ran (Fig. 3 C).

Moreover, in the inferior frontal gyrus of three patients with
FTLD-TDP due to GRN mutations, we found a significant
correlation between nuclear depletion of TDP-43 and Ran
(Fig. 3, D and E).
To understand the mechanisms underlying the intimate
correlation between nuclear TDP-43 and Ran, we next assessed whether Ran mRNA is altered in the brains of FTLDTDP-43 patients, in which TDP-43 is mislocalized. By
mining an existing mRNA-expression database comparing
healthy control versus GRN-mutation-carrying FTD subjects (Chen-Plotkin et al., 2008), we found that cortical Ran
expression was reduced by 60% in human subjects carrying a
GRN mutation (P = 0.04). As TDP-43 regulates the expression
of thousands of genes, in many cases by binding directly to
mRNAs and altering their stability (Polymenidou et al., 2011),
we explored the possibility that Ran mRNA is a substrate of
TDP-43. Indeed, analyses of a published unbiased screen of the
TDP-43RNA interactions (Sephton et al., 2011) revealed
that TDP-43 binds to the 3 UTR of Ran mRNA (Fig. 4 A).
Moreover, inhibiting TDP-43 expression by shRNA-mediated
knockdown significantly reduced levels of Ran mRNA
(Fig. 4 B) and protein (Fig. 4, CD) in N2A cells. Ran mRNA
levels were also reduced in retinas of aged Grn KO mouse
(Fig. 4 E), consistent with our observations of nuclear depletion
Figure 3. Nuclear clearing of TDP-43

and Ran are pathologically associated in
FTLD-TDP. (A) 18-mo-old GCL neurons from
WT and Grn-KO retinas were co-stained for
TDP-43 and Ran. Nuclei were labeled with
DAPI. (B) Nuclear Ran levels in 18-mo-old GCL
neurons. n = 165278 cells from 6 mice/genotype; *, P = 0.019, linear regression model;
2 independent experiments. Scatter plot of
individual cell intensities with medians shown.
(C) Nuclear Ran and TDP-43 intensities are
correlated in Grn-KO GCL neurons. Each dot
represents a single cell. n = 165 cells from 6
Grn-KO mice; r = 0.8963; P < 0.001, Spearmans rho; 2 independent experiments.
(D) Immunofluorescence co-staining of GRN
mutant human cortex shows depletion of Ran
and TDP-43 in the same neuron (noted with
an arrow; compare to neurons with high levels of TDP-43 and Ran [arrowhead]). (E) TDP-43
and Ran levels correlate in cortical neurons
from human GRN-mutation carriers. Shown
are the correlation analyses of nuclear Ran
and TDP-43 intensities of individual neurons
from post-mortem brain. n = 111141 cells
from each of 3 subjects;, r = 0.56; P < 0.001.
The serum progranulin levels were 19.321.2
ng/ml for R493X carrier (control patients: 41.3
15.5 ng/ml). Spearmans rho. Bars: 2 m (A),
10 m (D).
1940
Figure 4. Maintenance of functional TDP-43 and Ran by an interdependent feedback loop and improved survival of Grn-KO neurons by exogenous
Ran expression. (A) Snapshot of unique reads from the TDP-43 RIP library mapped to the Ran gene shown. Reads mapped to the 3-UTR of Ran indicate TDP-43
binding. No reads mapped to the Ran gene from the Ctrl RIP library. (B) TDP-43 knockdown in N2A cells results in a reduction in steady-state Ran mRNA levels, as
measured by Q-PCR. n = 12 wells/group; ***, P < 0.001, Students t test; 3 independent experiments. (C) Representative Western blot showing reduced Ran protein
levels after TDP-43 knockdown. (D) Quantification of (C). n = 9 wells/group, ***, P < 0.001, Students t test; 2 independent experiments). (E) Ran mRNA expression
is reduced in aged Grn KO retinas. Q-PCR results from homogenized whole retinas from 1012-mo-old mice shown. n = 1516 mice/genotype; *, P = 0.014; Students t test; 2 independent experiments. Bars, 10 m. (FG) Ran is necessary for nuclear localization of TDP-43. (F) Representative images showing subcellular
localization of TDP-43 in cortical neurons cotransfected with TDP-43-GFP and either empty vector, mCherry-Ran, mCherry-RanQ69L, or mCherry-RanT24N. TDP43-GFP is present in the nuclei of neurons transfected with mCherry and mCherry-Ran, but is significantly reduced in nuclei of neurons transfected with either of
the Ran mutants (arrowheads, dashed lines). (G) Quantification of the ratios of nuclear/cytoplasmic TDP-43-GFP. n = 21 cells/transfection; ***, P < 0.001, one-way
ANOVA with Tukeys post-hoc analysis; 2 independent experiments. (HI) N2A cells were transfected with siRNA against Grn. Levels of Ran (H) and TDP-43 (I) were
quantified via Western blot 7 d after transfection. n = 6 wells/group; **, P = 0.002 (TDP-43); **, P = 0.006 (Ran); 2 independent experiments. (J) Living wild-type or
Grn KO primary neurons transfected with GFP + empty vector (control) or GFP + Ran were imaged longitudinally by automated microscopy at 2448-h intervals for
79 d. Kaplan-Meir survival analysis was used to create cumulative risk of death functions for each population of transfected neurons. ***, P < 0.001 (log-rank
test); n = 423 neurons (WT Ctrl), 518 neurons (KO Ctrl), 427 neurons (WT Ran), and 463 neurons (KO Ran); 3 independent experiments pooled. (K) Primary cortical
neurons from wild-type or Grn KO mice were transduced with AAV-GFP (control) or AAV-GFP-P2A-Ran. 1 wk later, neurons were fixed and processed for TDP-43
immunostaining. Nuclear TDP-43 levels were quantified via Volocity. n = 101478 cells imaged from 612 wells of a 96-well dish; *, P < 0.05 (mixed-effects
multivariate linear regression model); 3 independent experiments. Means SEM shown (B, D, E, GI, and K).
of TDP-43 in the retinas of Grn KO mice (Fig. 3 A).These findings suggest that nuclear depletion of TDP-43 in progranulindeficient neurons could down-regulate Ran.
Ran is required for nuclear transport of the majority of proteins that shuttle between the nucleus and cytoplasm (Stewart,
2007).To determine if inactivation of Ran is sufficient to cause
1941
nuclear depletion of TDP-43, we expressed dominantnegative

RanQ69L (which cannot hydrolyze GTP) or RanT24N (which
is nucleotide-free or GDP-bound) in cortical neurons. Both of
these Ran mutants caused TDP-43 mislocalization (Fig. 4,
F and G), indicating that TDP-43 requires functional Ran for
import into the nucleus.
Enhancing Ran expression improves the survival
of progranulin-deficient neurons
The results of the prior experiments suggested a model of a
reciprocal depletion of nuclear TDP-43 and Ran: loss of nuclear TDP-43 down-regulates Ran mRNA, and Ran dysfunction depletes nuclear TDP-43. Indeed, acute knockdown
of progranulin levels in N2A cells via siRNA reduced both
TDP-43 and Ran protein expression (Fig. 4, H and I). To
directly test this model, we hypothesized that augmenting
Ran expression would increase nuclear TDP-43 and improve
the survival of Grn-KO neurons. An automated microscopy
approach was used to quantify the effect of Ran expression
on neuron survival (Arrasate and Finkbeiner, 2005). In this
assay, individual GFP-transfected neurons are repeatedly imaged over multiple days, thus generating longitudinal survival
curves for cohorts of neurons from different genetic backgrounds and/or those expressing different plasmids. Previous
studies have established that progranulin-deficient cortical
neurons exhibit enhanced vulnerability in culture (Guo et al.,
2010; De Muynck et al., 2013). Indeed, primary Grn-KO
cortical neurons had shorter lives than wild-type neurons
(Fig. 4 J). Expression of exogenous Ran enhanced the survival of Grn-KO neurons, but not wild-type neurons (Fig. 4 J).
Consistent with our findings in the retina of Grn-KO mice,
primary Grn-KO cortical neurons had decreased nuclear
TDP-43 expression, which was increased by expression of
exogenous Ran (Fig. 4 K).
In conclusion, our findings provide strong evidence of
early retinal abnormalities in GRN mutation carriers. Retinal thinning occurred in cognitively asymptomatic GRN
mutation carriers (who are at high risk for future dementia),
indicating that retinal neuron loss is an early phenomenon
that can precede clinical symptoms in FTD. Because small
changes in retinal thickness can be reproducibly measured
longitudinally via OCT, retinal imaging could be a useful
modality for assessing response to therapeutics in early disease stages of progranulin-deficient FTD. Further study will
be needed to establish the rate of retinal degeneration in
GRN mutation carriers. The reasons why RGCs are particularly susceptible to death in GRN mutation carriers are unclear. RGCs have long axons and due to their central role in
vision they have a high metabolic demand; both of these characteristics may contribute selective vulnerability in the setting of progranulin deficiency.
We also observed a retinal neurodegenerative phenotype
in Grn-KO mice that parallels our observations in humans,
establishing the retina as a new model system in which to
study mechanisms of neurodegeneration in FTLD-TDP.
Our findings support a pathogenic loop involving reciprocal
1942
depletion of nuclear TDP-43 and Ran as a potential mechanism of neurodegeneration in FTLD-TDP. In this model,
loss of function of TDP-43 via nuclear depletion contributes
to neurodegeneration and can occur without cytoplasmic
TDP-43 aggregation. Loss of Ran expression, potentially in
combination with other associated nuclear transport factors,
impairs transport of TDP-43 to the nucleus (Nishimura et al.,
2010). In turn, loss of nuclear TDP-43 lowers Ran levels,
which could further deplete nuclear TDP-43. These data
may point toward novel therapeutic strategies aimed at restoring nucleocytoplasmic transport as a means to improve
neuronal survival in neurodegenerative diseases.
Human subjects. Subjects enrolled through the UCSF Memory and Aging
Center in whom GRN mutations were identified and age- and sex-matched
control subjects without a history of neurological disease were invited to participate in our study. A standardized clinical evaluation was performed on all
GRN mutation carriers at the UCSF Memory and Aging Center by boardcertified neurologists who had additional training in behavioral neurology. For
GRN mutation carriers, based on the results of this clinical evaluation, subjects
were then subgrouped into asymptomatic GRN mutation carriers (CDR = 0,
n = 7) and symptomatic GRN mutation carriers (CDR 0.5, n = 5). One
GRN mutation carrier had a prior diagnosis of age-related macular degeneration. This subject was included in the analysis (exclusion of this subject from
analysis did not meaningfully affect statistical significance of RNFL thinning or
macular volume loss). No other control subjects or GRN mutation carriers had
a history of ophthalmological disease or ocular surgery.
Written informed consent was obtained from all participants with capacity. Written informed consent was obtained from a designated surrogate
decision maker in subjects deemed unable to provide informed consent due
to diminished capacity, but we only enrolled subjects who were able to assent. The UCSF Committee on Human Research (CHR) approved this protocol, and the study was performed in accordance with the Declaration
of Helsinki.
Retinal imaging. We performed spectral domain optical coherence tomography (OCT) at the UCSF Neurodiagnostics Center using a Heidelberg
Spectralis instrument (Heidelberg Engineering, Heidelberg, Germany).
A trained technician blinded to patient diagnosis and to genotype (when relevant) performed all scans and repeated each measurement at least three times.
Mean RNFL thickness was determined using a peripapillary B-scan 3.4-mm
from the center of the papilla. Images were evaluated by a blinded technician
to meet prespecified image quality criteria, including signal intensity and
beam uniformity. For this analysis, we analyzed and averaged the RNFL
thickness and macular volume of all interpretable scans. RNFL thickness and
macular volume was measured using automated software provided by Heidelberg. Segmentation analysis of macular scans was then performed to determine the volume of individual neuronal layers via a proprietary, validated
computerized algorithm (Heidelburg Engineering, Heidelburg, Germany).
Layers analyzed included the ganglion cell complex (GCC; comprising
ganglion cell neuronal cell bodies, their dendrites, and axons projecting from
underlying inner nuclear layer neurons), the inner nuclear layer (INL), and
the outer nuclear layer (ONL).
Statistical analysis for human subjects. RNFL thickness, macular volumes, and segmented macular volume were analyzed in human subjects. We
used multiple linear regression analysis to compare differences between
GRN mutation carriers and unaffected controls. Adjustment for age and sex
did not meaningfully change the results, so we elected to report unadjusted
values. To account for inter-eye correlations, when two eyes from the same
individual were analyzed, the standard error was adjusted using the clustered
sandwich estimator. P < 0.05 was considered significant. Analyses were performed using STATA 12.0.
Mice. Wild-type and Grn-KO mice were obtained from R.V. Fareses laboratory (University of California, San Francisco, CA; Martens et al., 2012).
Mice used in experiments shown in Figs. 13 were of a mixed background
consisting of 62.5% C57BL/6J, 12.5% 129Sv/Jae, and 25% FVB. Mice used
in experiments shown in Fig. 4 were fully backcrossed into C57BL/6J. Ageand sex-matched mice from the same genetic background were used as controls for Grn KO mice.
RNFL and GCL neuron quantification. 16-m transverse sections of
WT and Grn-KO mouse eyes were made and stained with H&E/Neu-N,
and the center sections (as determined by the center of the optic nerve head)
were imaged via light/fluorescent microscopy, respectively. The area of the
RNFL was measured in ImageJ and divided by the length of the RNFL
across the field of view to determine thickness (equidistant from the optic
nerve head across mice). For GCL neuron quantification, sections were
stained with anti-NeuN antibody and subsequently imaged via fluorescence
microscopy. The number of Neu-N positive cells in the GCL in individual
fields of view were counted and divided by the length of the GCL using sections equidistant from the optic nerve head across mice. Statistical analysis
was conducted with a one-way ANOVA followed by Tukey multiple comparison test.
Electroretinography. After overnight dark adaptation, mice were anesthetized under dim red illumination with 0.1 mg/kg ketamine and 10 mg/kg
xylazine. Under anesthesia, both eyes were treated with 0.5% proparacaine
followed by a mixture of 2.5% phenylephrine and 1% tropicamide for pupil
dilation. The mice were kept warm using a 37C heating pad (Deltaphase
Isothermal Pads; Braintree Scientific). A gold reference electrode was electrically connected to the cornea of one eye and a platinum wire, mounted on
a fiber-optic cable, was connected to the cornea of the other eye. Electrical
continuity was made using hydroxypropyl methylcellulose (Goniosol). Light
stimuli were delivered directly into the eye through the tip of the fiber optic.
Stimulus intensity was controlled by calibrated neutral density filters, and
stimulus wavelength was 500 nm (5 nm; narrow band filter) or 505 nm
(17 nm; broad band filter). Responses were recorded from threshold up to
light 1,000,000 fold brighter in darkness, and the photopic responses were
recorded in the presence of rod-saturating background lights. Electrical responses were amplified (Astro-med CP122W; DC-300Hz) and digitized at
2 KHz (Real-Time PXI Computer; National Instruments).
Antibodies used. Rabbit anti-TDP-43 (Protein Tech), 1:1,000; rabbit
C-terminal antiTDP-43 (produced by G. Yu; UT Southwestern, Dallas,
TX), 1:1,000 each; goat anti-Ran (Santa Cruz Biotechnology, Inc.), 1:200;
and mouse anti-Ran (BD), 1:2501:1,000.
Immunostaining. 16-m transverse sections of WT and Grn-KO mouse
eyes were made from paraffin embedded tissue and mounted on silanized
slides. After de-waxing and rehydration, sections were blocked for 1 h at
room temperature in PBS/0.5% Triton/10% donkey serum. Primary antibodies were incubated with sections overnight at 4C, and then slides were
washed and stained with secondary Alexa Fluorconjugated antibodies
(1:300; Invitrogen) for 2 h at room temperature. After washing, samples were
mounted with #1.5 coverslips using Prolong-Gold antifade reagent with
DAPI (Invitrogen). All fluorescent imaging was performed on an inverted
confocal Ti microscope (Eclipse; Nikon) with a Nipkow spinning disk attachment and EM camera (Hamamatsu).
Quantification of TDP-43 and Ran. Sections from equal retinal eccentricity were imaged with a spinning disk confocal microscope, and fields of
view of a similar distance from the center of the retinal sections were imaged.
Acquisition settings were identical between samples, and all samples used for
quantification were stained on the same day. Regions of interest were drawn
around the DAPI-stained nuclei at the z-position center of the nuclei, and
used to quantify mean intranuclear TDP-43 intensity. Cytoplasmic intensity
of TDP-43 was determined by drawing a perinuclear region of interest in
the cytoplasm. This mode of cytoplasmic intensity quantification was found
to be more accurate than tracing around the entire cell soma, given the relatively
high density of ganglion cells. Nuclear/cytoplasmic intensity ratios represent
the mean nuclear intensity/mean cytoplasmic intensity per cell. For Ran quantification, intranuclear intensity was determined by drawing a ROI slightly
inside of the nuclear envelope. Data were transformed into log10 intensity,
and a mixed-model regression of the intensity variable versus genotype
and/or age that controlled for clustering by mouse was applied to assess statistical significance in STATA.
Transfection of cortical neurons with Ran mutants. Postnatal day 0 rat
cortical neurons were isolated and cultured in Neurobasal-A with B27. 35 d
after isolation, they were transfected with human TDP-43-GFP + mCherry,
mCherry-huRan, mCherry-huRanT24N, or mCherry-huRanQ69L. 1 d
after transfection, neurons were fixed and imaged on a spinning disk confocal
microscope. Images of mCherry-positive neurons were taken with the same
acquisition settings across transfection groups, and the TDP-43-GFP signal in
the nucleus and cytoplasm was quantified. Differences in nuclear/cytoplasmic ratio across groups was assessed with one-way ANOVA with Tukeys
post-hoc analysis.
Immunostaining of postmortem brains from human subjects with
GRN mutations. De-identified post-mortem brain tissue (left inferior
frontal gyrus) from FTLD subjects with previously documented GRN mutations, deemed nonhuman subject material as per UCSF CHR guidelines,
was obtained from the UCSF Neurodegenerative Disease Brain Bank. Tissue was embedded in paraffin and 10-m sections were made. Antigen retrieval was performed using IHC Worlds antigen retrieval solution as per
manufacturers guidelines, followed by Sudan Black treatment and primary
antibody incubation overnight at 4C (antiTDP-43, 1:3,000; Protein Tech;
anti-Ran, 1:1,000; BD), followed by secondary antibody and DAPI-staining.
For quantification of staining, confocal images of DAPI-stained nuclei, TDP43, and Ran were taken at equal intensities across multiple fields of view in
the cortex. Nuclear TDP-43 and Ran levels were quantified and analyzed as
was done in mouse RGCs.
TDP-43 RIP. The TDP-43-RNA immunoprecipitation dataset was generated from pull-down experiments conducted as part of a previous study
using the methods described therein (Sephton et al., 2011).
TDP-43 knockdown, progranulin knockdown, Q-RT-PCR, and
Western blot analysis. N2A cells were grown in DMEM (low glucose) + 10%
FCS and transfected with Mission TDP-43 shRNA construct #752 or control shRNA construct (Sigma-Aldrich) via Lipofectamine 2000. 68 d after
transfection, N2A cells were harvested for RNA or protein analysis. RNA
was prepared via RNeasy columns (QIAGEN), transcribed into cDNA, and
Ran or cyclophilin (control) RNA levels were analyzed via Q-RTPCR.
Equal amounts of total protein from knockdown and control cells were immunoblotted against tubulin (loading control), TDP-43, or Ran, and then
quantified using a Licor imaging system, with relative levels of Ran/tubulin
quantified for each sample. For progranulin knockdown experiments, N2A
cells were transfected with control siRNA (Thermo Fisher Scientific) or
Grn#1 siRNA. Greater than 95% knockdown was observed by Western blot
and progranulin ELISA by 5 d after transfection. Samples were processed for
Ran and TDP-43 quantification, as above. For Ran mRNA analysis of mouse
retinas, whole retinas were isolated from freshly perfused mice and nonneuroretinal tissue was dissected away. RNA preparation and Q-RTPCR was
performed as above.
Longitudinal neuronal survival analysis. Longitudinal survival analysis
of individual GFP-transfected neurons was essentially performed as described
previously (Barmada et al., 2010), with the following modifications: cortical
1943
neurons from postnatal day 0 wild-type or Grn KO mice were dissociated

and plated on PDL-coated 96 well-dishes (microclear bottom dishes;
Greiner) at a density of 90,000 cells/well in Neurobasal-A + B27 supplement. 35 d after plating, neurons were transfected via Lipofectamine 2000
with GFP + empty vector or GFP + mouse Ran cDNA at a ratio of 1:3 to
ensure that all fluorescent cells co-expressed the second plasmid. The day
after transfection, wells containing GFP-expressing neurons were imaged
at 5 magnification with an automated microscope (Array Scanner XTI;
Thermo Fisher Scientific). The same fields of view were reimaged every
2448 h for a total of 79 d after transfection. Adjacent fields of view from
individual wells were stitched together into montages via ImageJ, and a
time series across days for each well was then generated using a custommade macro in ImageJ. The survival of individual GFP-transfected neurons over time was then assessed by loss of GFP fluorescence. Kaplan-Meier
and cumulative risk of death curves were made with R software, and statistical significance of differences in survival between cohorts of neurons
was determined with the log-rank test. Tukey multiple comparison test
was used for comparisons involving more than two groups.
Rescue of neuronal TDP-43 expression. Primary cortical neurons from
postnatal day 0 wild-type or Grn KO pups were isolated and plated as described above. Neurons were transduced with AAV2-GFP (Virovek) or
AAV2-GFP-P2A-human Ran (in which bicistronic GFP and untagged Ran
co-expression is driven by a single chicken--actin-CMV promoter, with a
P2A sequence separating GFP and Ran sequences). 7 d after transduction,
neurons were fixed and immunostained with antiTDP-43 antibody. Individual wells were imaged, and regions of interest of nuclei from GFP-positive
cells were generated via Volocity from background-subtracted images. Using
these regions of interest, integrated density measurements of total TDP-43
levels in each nuclei were then calculated. A mixed-model regression of the
intensity variable versus genotype and AAV-vector that controlled for clustering by well was applied to assess statistical significance in STATA.
Statistics. Design and execution of statistical tests were done in collaboration with a professional biostatistician (J. Boscardin, University of California,
San Francisco, CA). For human retinal imaging data and for nuclear and cytoplasmic TDP-43 and Ran immunostaining in retinal neurons and in cultured cortical neurons, significance was determined via mixed-effects linear
regression analyses, accounting for interindividual, within-eye correlations
in the human studies and intramouse clustering in rodent models. In experiments involving two comparison groups, unpaired two-tailed Students
t tests were used to assess for differences. In experiments involving more than
two comparison groups, ANOVA test with post-hoc tests (Tukey or Bonferroni, as specified in the text) were used. For longitudinal neuronal survival
analysis, cumulative risk of death curves were generated with R software,
and statistical significance was determined with the log-rank test. P < 0.05
was considered significant. Unless otherwise noted, statistical testing was
performed using Prism and Stata 12.0 software.
Study approval. For retinal imaging, written informed consent was obtained from all participants with capacity; in subjects deemed unable to provide informed consent due to diminished capacity, written consent was
obtained from a designated surrogate decision-maker and subjects provided
assent. The study protocol was approved by the UCSF Committee on Human
Research (IRB # 1105333). For studies involving mice, all procedures
were approved by the Institutional Animal Care and Use Committee at
UCSF (#AN087501-02A) and UAB (#101109282, #130309617).
Online supplemental material. Fig. S1 shows clinical characteristics of
human subjects who underwent retinal imaging. Online supplemental material
is available at http://www.jem.org/cgi/content/full/jem.20140214/DC1.
We would like to thank Dr. Sami Barmada for his thoughtful input on the project
and assistance with analysis of neuronal survival experiments, Yungui Zhou and
Marcel Alavi for technical assistance, Dr. Anna Karydas for her assistance with
genetic analysis of human subjects, and Dr. Laura Mitic for her feedback.
1944
This project was funded by the Consortium for Frontotemporal Dementia

Research (L.G., G.Y., E.D.R. J.H., B.V.F), The Bluefield Project to Cure FTD (M.W.,
fellowship), R01 AG036884 (L.G), the UCSF Resource Allocation Program (A.G.), the
UCSF Alzheimers Disease Research Center (M.W.), #P50 AG023501 (B.F),
R01NS079796 (G.Y.), R01NS075487 (E.D.R), T32HD071866 (A.E.A), K08EY023610,
(M.W) the Chartrand Foundation and Clinical & Science Translational Institute
(M.W., A.G.), the Howard Hughes Medical Institute (A.G.), the Alzheimers Association
(G.Y.), the Welch Foundation (G.Y.), R37HL63762 (J.H), the Brightfocus Foundation
(J.H), the Alzheimers Drug Discovery Foundation (B.M).
The authors report no competing financial interests.
Submitted: 31 January 2014
Accepted: 5 August 2014
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1945
Article
A novel A-fibrinogen interaction inhibitor

rescues altered thrombosis and cognitive
decline in Alzheimers disease mice
Hyung Jin Ahn,1 J. Fraser Glickman,2 Ka Lai Poon,1
Daria Zamolodchikov,1 Odella C. Jno-Charles,1 Erin H. Norris,1
and Sidney Strickland1
1Laboratory
of Neurobiology and Genetics and 2High Throughput Screening Resource Center, The Rockefeller University,
New York, NY 10065
Many Alzheimers disease (AD) patients suffer from cerebrovascular abnormalities such as
altered cerebral blood flow and cerebral microinfarcts. Recently, fibrinogen has been identified as a strong cerebrovascular risk factor in AD, as it specifically binds to -amyloid
(A), thereby altering fibrin clot structure and delaying clot degradation. To determine if
the Afibrinogen interaction could be targeted as a potential new treatment for AD, we
designed a high-throughput screen and identified RU-505 as an effective inhibitor of the
Afibrinogen interaction. RU-505 restored A-induced altered fibrin clot formation and
degradation in vitro and inhibited vessel occlusion in AD transgenic mice. Furthermore,
long-term treatment of RU-505 significantly reduced vascular amyloid deposition and
microgliosis in the cortex and improved cognitive impairment in mouse models of AD. Our
studies suggest that inhibitors targeting the Afibrinogen interaction show promise as
therapy for treating AD.
CORRESPONDENCE
Sidney Strickland:
strickland@rockefeller.edu
Abbreviations used: AD, Alz
heimers disease; BBB, blood
brain barrier; CAA, cerebral
amyloid angiopathy; FP, fluor
escence polarization; HTS,
high-throughput screen; SPR,
surface plasmon resonance;
TAMRA, 5-carboxy-tetramethylrhodamine; tPA, tissue plasminogen activator.
Accumulating evidence suggests that cerebrovascular risk factors play an important role in
Alzheimers disease (AD) pathophysiology. Many
AD patients suffer from altered cerebral blood
flow, damaged cerebral vasculature, and increased
cerebral microinfarcts (de la Torre, 2004; Brundel
et al., 2012), and a majority of patients with dementia present with both AD and vascular pathologies (MRC CFAS, 2001;Viswanathan et al.,
2009). Furthermore, cerebral amyloid angiopathy
(CAA), which is the deposition of the -amyloid
(A) peptide within cerebral blood vessels, results in degenerative vascular changes (Thal et al.,
2008; Smith and Greenberg, 2009). Patients
with both CAA and neurological pathology
including neurofibrillary tangles and neuritic
plaques have more severe cognitive impairment
than patients with only AD pathology or CAA
alone (Pfeifer et al., 2002), and reduction of CAA
levels in AD transgenic mice leads to memory
improvement (Park et al., 2013). Interestingly,
the Nun Study showed that one-third of the
participants who had neurological AD pathology were actually not demented at the time of
death, but when AD pathology was concomitant
with brain infarcts, there was a high prevalence

J. Exp. Med. 2014 Vol. 211 No. 6 1049-1062
of dementia found in participants (Snowdon

et al., 1997; Mortimer, 2012). Thus, the identification of a molecular association between these
vascular and neurological pathologies could aid
in more efficient diagnoses and effective treatments for AD.
Recent studies have suggested that fibrinogen, a primary protein component of blood
clots, serves as a molecular link between the
vascular and neurological abnormalities observed in AD patients. Normally, fibrinogen is
found in the blood and is excluded from the
brain via the bloodbrain barrier (BBB). However, it has been shown that: 1) fibrinogen is
often localized to CAA in the brains blood vessels and brain parenchyma in AD patients and
in mouse models of AD (Paul et al., 2007; Ryu
and McLarnon, 2009; Cortes-Canteli et al., 2010;
Klohs et al., 2012); 2) fibrin deposition in the
vasculature increases BBB dysfunction and neurovascular damage in AD mice (Paul et al., 2007;
2014 Ahn et al. This article is distributed under the terms of an Attribution
1049
Cortes-Canteli et al., 2010); 3) A binds specifically to fibrinogen; and 4) fibrin clots formed in the presence of A have an
abnormal structure, making them resistant to degradation by
fibrinolytic enzymes (Ahn et al., 2010; Cortes-Canteli et al.,
2010). Overall, these results indicate that in the presence of A,
any fibrin clots formed might be more persistent and may exacerbate neurovascular damage and cognitive impairment.
Therefore, molecules that block this interaction without affecting clotting in general could restore altered thrombosis and fibrinolysis and protect against vascular damage in AD patients,
and could be used as therapeutic agents.
RESULTS
Hit identification and optimization
using high-throughput screening
To investigate this idea, we designed a high-throughput screen
(HTS) to identify small molecules that inhibit the interaction
between A and fibrinogen. Low molecular weight compounds
were screened using fluorescence polarization (FP) and AlphaLISA assays in a complementary fashion to cross check the
activity of the hit compounds and to ensure the removal of
false-positive artifacts. Primarily, 93,000 compounds were
screened using FP, which measured the changes in the anisotrophy induced by binding of a 5-carboxy-tetramethylrhodamine
(TAMRA)labeled A peptide to fibrinogen (Fig. 1 A). Then,
hits from FP were screened using AlphaLISA to independently
confirm the activity of the inhibitors identified in the FP assay
(Fig. 1 B). After both steps, we selected only drug-like compounds using Lipinskis Rule of Five, which allowed us to determine which chemical compounds have pharmacological
properties that would make them likely orally active drugs in
humans (Lipinski et al., 2001). We also filtered out artifactual
compounds using a quenching assay, which identifies insoluble
compounds, singlet oxygen quenchers, and biotin mimetics interfering with the AlphaLISA signal.We identified several candidate compounds with half-maximal inhibitions (IC50) between
10 and 50 M from the dose-response assays using both FP and
AlphaLISA assays (Table 1).
To expand and improve our candidate compounds, we
purchased a focused analogues compound library, based on
combinatorial variations of scaffolds from the primary hit
compounds. These analogues were screened at three different
concentrations (5, 10, and 20 M) using AlphaLISA. Next, we
selected only drug-like compounds using Lipinskis Rule of
Five and also included the quenching assay. If inhibition by
quenching was >30%, the compounds were removed from further analyses because these compounds were more likely to be
false positives. Finally, we screened the active nonquenching
compounds in concentration-response experiments with freshly
dissolved powders using both FP and AlphaLISA assays. We
identified five drug-like compounds with IC50 < 3 M by FP
and IC50 < 10 M by AlphaLISA (Fig. 1 C and Table 2).
In some cases, the maximum inhibition of several compounds in AlphaLISA was lower than that of FP. There are
several possible reasons for these differences. First, some hit
compounds showed negative quenching values, which increases
1050
the background AlphaLISA signal. Therefore, the actual inhibitory efficacy of these compounds could be higher than
the results from doseresponse experiments of AlphaLISA.
In addition, avidity effects may cause higher IC50 values in
AlphaLISA than FP. There could be multiple Afibrinogen
interactions between acceptor bead and donor bead in AlphaLISA (Fig. 1 B), and therefore blocking one interaction may
not reduce the signal.
Because both AlphaLISA and the FP assay are based on
optical measurements, colored compounds could significantly
modify the measurement through inner filter effects.Thus, we
confirmed the potency of our candidates using a pull-down
assay. All five compounds showed inhibitory effects, whereas
RU-505 had significant inhibitory efficacy (Fig. 2 A). These
combined experiments show that the compounds identified
are inhibitors of the Afibrinogen interaction.
Soluble oligomeric A has been hypothesized to be the
primary toxic species in AD (Cleary et al., 2005).Therefore, we
tested which form of A, monomer or oligomer (prepared as
in Stine et al. [2011]), interacts with fibrinogen and whether
RU-505 can selectively inhibit the interaction of one or the
other. Using the AlphaLISA assay, we found that both A42
monomer and oligomer interact with fibrinogen, but the affinity of oligomer for fibrinogen binding is 4 times higher than
that of the monomer (Fig. 2 B). RU-505 inhibits the inter
action of both monomer and oligomer with fibrinogen, but
has higher inhibitory efficacy against the monomerfibrinogen
interaction than the oligomer (Fig. 2 C).
Validation of hit compounds using in vitro clotting assay
Because the interaction between A42 and fibrinogen induces a structurally abnormal fibrin clot and delays fibrin clot
degradation during fibrinolysis (Ahn et al., 2010; CortesCanteli et al., 2010; Zamolodchikov and Strickland, 2012),
one of the main objectives of our study was to identify compounds that restore A-induced delayed fibrinolysis. When
fibrinogen associates into a fibrin meshwork after cleavage by
thrombin, the fine structure of this fibrin clot scatters light
and the solution increases in turbidity. Thus, the kinetics of
turbidity can be used as a read-out to analyze fibrin network
formation and degradation. We tested whether our hits restored A-induced altered thrombosis and fibrinolysis in vitro.
Each hit compound (20 M) or vehicle (0.4% DMSO) was
incubated for 10 min with purified human fibrinogen and
plasminogen in the presence or absence of A42. Fibrin clot
formation and degradation were analyzed by measuring turbidity immediately after adding thrombin and tissue plasminogen activator (tPA) to the mixture. In the presence of A42,
the maximum turbidity of the fibrin clot was decreased because A altered fibrin clot structure and the dissolution of
the fibrin clot was delayed (Fig. 2 D; red). RU-505 restored
the A-induced decrease in turbidity during fibrin clot formation (Fig. 2 D; green) and significantly reduced the delay in
fibrin degradation in the presence of A (Fig. 2 E). We also
tested other hit compounds, including RU-965, using the
turbidity assay, but none had significant effects (Fig. 2 F and
A-fibrin interaction inhibitor as AD treatment | Ahn et al.
Ar ticle
Figure 1. The chemical structure and

doseresponse curve of Afibrinogen
interaction inhibitors. (A) TAMRAlabeled
A peptide was bound to fibrinogen and the
test compound, and the anisotropy of
TAMRAAfibrinogen binding was determined by FP. (B) Biotin-labeled A42, which
binds a streptavidin donor, was incubated
with fibrinogen, which binds a protein A acceptor bead coated with antifibrinogen antibody. A42 and fibrinogen interactions bring
the beads in close proximity, resulting in the
excitation of the donor beads and release of
singlet oxygen molecules that triggers light
emission in acceptor beads (AlphaLISA [AL]).
(C) The half-maximal inhibitory concentration
(IC50) values of the indicated compounds were
determined by doseresponse FP and AL experiments and are indicated inside the panel
(red, FP; blue, AL). A quenching test was also
performed to calculate how much each hit
compound interfered with the AL signal at
10 M concentration. Quenching values are
indicated below the doseresponse curve.
n = 34 repeats per assay and all error bars
indicate SEM. Data are representative of at
least three independent experiments.
not depicted). Moreover, RU-505 did not have any effect

on fibrin clot formation and degradation in the absence of
A (Fig. 2 D, purple). This result suggests that RU-505 could
effectively restore A-induced altered fibrin clot structure
and delayed degradation without affecting normal clot formation and fibrinolysis.
The interaction between A and RU-505
To elucidate how RU-505 inhibits the Afibrinogen interaction, surface plasmon resonance (SPR) was used to analyze
the binding characteristics of RU-505 (Fig. 2 G). Hexafluoroisopropanol-treated monomerized A42 was immobilized
JEM Vol. 211, No. 6
to the sensor chip surface, and RU-505 was injected for 2 min
at 30 l/min. Sulindac sulfide was used as positive control, and
sulindac was used as negative control (Richter et al., 2010).To
analyze the correlation between HTS and SPR, we used an
analogue of RU-505, RU-4180 (Fig. 2 H), which did not inhibit the Afibrinogen interaction in AlphaLISA assay. Although RU-4180 weakly binds to A42 (green; Fig. 2 G),
RU-505 showed strong binding to A42 (blue; Fig. 2 G). Furthermore, because it is known that sulindac sulfide binds A,
we tested whether it could inhibit the Afibrinogen interaction by AlphaLISA and found that it had no effect.These results
suggest that RU-505 inhibits the Afibrinogen interaction
1051
Table 1. Workflow of high-throughput primary screening

Step
Test compound
concentration
Total library compound

Primary assay hits
using FP
Secondary assay hits using AlphaLISA (AL)
Filtering using quenching (AL)
Lipinskis rule
Validation/doseresponse Hits (FP and AL)
Doseresponse using fresh compound (FP
and AL)
Number of compounds
% of picked
compounds
Inhibition cut-off (%)
20 M
93,716
3,010
3.21
>75
12.5 M
12.5 M
167
97
87
26
10
0.18
0.1
0.09
0.028
0.011
>50
<30
1 violation
IC50 <50 M
IC50 <50 M
0.3140 M
0.0740 M
through A binding, but RU-4180 does not inhibit the interaction because its affinity for A is too weak. Moreover, from
the case of sulindac sulfide, A binding itself is not enough to
inhibit the interaction between A and fibrinogen. To inhibit
the Afibrinogen interaction, a compound requires at least
two features: 1) an A-specific binding moiety and 2) a moiety responsible for inhibiting fibrinogens binding to A.
RU-505 restored altered thrombosis in AD mice
To assess whether our lead compound could restore Ainduced altered thrombosis and fibrinolysis in vivo, we examined cerebral blood flow and thrombosis in a transgenic
mouse model of AD, Tg6799 mice (Oakley et al., 2006), with
or without long-term treatment of RU-505. Blood flow and
thrombosis were analyzed by a FeCl3-induced thrombosis
model combined with intravital microscopy (Cortes-Canteli
et al., 2010). We administered RU-505 or vehicle (35 mg/kg
dose, every other day) to 4-mo-old Tg6799 and WT littermates for 4 mo (analyzed at 8 mo of age). Brains of 8-mo-old
Tg6799 or WT mice were exposed by craniotomy, and blood
flow was observed using injected fluorescence-conjugated dextran (Fig. 3 A). Three concentrations of FeCl3 (5, 10, and 15%)
were incrementally administered to the brain surface to induce thrombosis. Clot formation was revealed by the appearance of an enlarging shadow superimposed on normal blood
flow (Fig. 3 A and Videos 14). The length of all visible vessels
with >20 m diam was measured before FeCl3 treatment,
and the length of occluded vessels was measured 5 min after

the addition of each concentration of FeCl3 for both Tg6799
and WT. There was no significant difference in the percentage
of occluded vessels before FeCl3 treatment or after 5 and 10%
FeCl3 treatment among groups (Fig. 3 B). However, there was
a significant difference between the percentage of occluded
vessels after 15% FeCl3 treatment in vehicle-treated WT and
Tg6799 mice (Fig. 3, B and C). Approximately half (52.7
12.1%) of the vessels were occluded in vehicle-treated WT
mice, but 95.6 3.5% of vessels were occluded in vehicletreated Tg6799 mice. RU-505 treatment significantly lowered
the vessel occlusion in Tg6799 mice to 60.7 8.7%, but did
not change vessel occlusion in WT mice (54.2 11.8%).These
results suggest that our lead compound significantly restored
altered thrombosis and fibrinolysis in AD mice without affecting normal thrombosis and fibrinolysis in WT littermates.
Treatment with RU-505 reduced vascular A deposition
CAA has been implicated in vascular degeneration of AD
(Chen et al., 2006; Okamoto et al., 2010). Our previous studies showed that the Afibrinogen interaction increases A
fibrillization (Ahn et al., 2010).Thus, we investigated whether
treatment of Tg6799 mice with RU-505 for 4 mo could decrease A deposition in blood vessels.A deposits were stained
using Congo red, and blood vessels were labeled using laminin (green; Fig. 4 A). We quantified CAA area in the cortex
Table 2. Workflow of high-throughput screening using a focused library

Step
Test compound
concentration
Number
of compounds
% of picked
compounds
Inhibition cut-off
Library compound
AlphaLISA (AL) assay hits
5, 10, and 20 M
2,092
327
15.6
Inhibition >35% at 5 M
and >50% at 10 M
Quenching <27% at 10 M
and inhibition >55% at 10 M
1 violation
IC50 < 3 M (FP) and
<10 M (AL)
Filtering using quenching (AL)

Lipinskis rule
Validation/dose response hits (FP
and AL)
10 M
58
2.77
0.0120 M
50
5
2.39
0.24
Selection criteria and number of compounds selected during each step of screening.
1052
Ar ticle
Figure 2. RU-505 inhibited the Afibrinogen interaction and restored A-induced altered fibrin clot formation and degradation. (A) Candidate compounds (10 M) were incubated with biotinylated A42 and fibrinogen, and pull-down assays were performed using streptavidinSepharose.
All samples were analyzed by Western blot. Dot blots were performed to control for amounts of A pulled down. Control (Ctrl) lane contains only A and
fibrinogen without any compound (one-way ANOVA and Bonferroni post-hoc test; *, P < 0.05; n = 34 independent experiments). (B) The binding affinity
between fibrinogen and monomeric or oligomeric biotinylated A42 was measured using the AL assay. (n = 34 experiments, data are representative of
three independent experiments). (C) The inhibitory efficacy of RU-505 on the interaction between fibrinogen and monomeric or oligomeric biotin-LCA42 was accessed in doseresponse experiments using the AL assay. (n = 34 experiments, data are representative of three independent experiments).
(D) RU-505 or DMSO was incubated with fibrinogen in the presence or absence of A42, followed by plasminogen, thrombin, tPA, and CaCl2. Fibrin clot
formation was assessed by measuring turbidity (n = 3 experiments, data are representative of three independent experiments). (E and F) The time to fibrin
clot degradation was analyzed by measuring time to half lysis. Control clot half lysis time was set to 100% for each experiment and all other values were
calculated relative to controls. (***, P < 0.001; n = 3 experiments, data are representative of three independent experiments). (G and H) A42 was immobilized on the SPR sensor chip surface, and the interaction of the indicated compounds with A42 was analyzed using Biacore 3000. Sulindac sulfide
(known to bind A42) was a positive control, and sulindac was negative control. (H) Chemical structure of RU-4180. Data are representative of three to
four independent experiments. All values are means and SEM.
by measuring Congo red deposits inside blood vessels, and

A plaque deposition was quantified by measuring Congo
red outside blood vessels.The CAA area of RU-505treated
Tg6799 mice (0.025 0.006%, cortex) was significantly decreased from that of vehicle-treated Tg6799 mice (0.046
0.004%, cortex; Fig. 4 C). However, there was no significant
difference in A plaque area in the cortex between RU-505
and vehicle-treated Tg6799 mice (Fig. 4, B and D). WT mice
did not exhibit any CAA-specific pattern of Congo red staining.
JEM Vol. 211, No. 6
This result indicates that inhibition of the Afibrinogen interaction by RU-505 reduced A deposits in blood vessels of
AD mice.
Treatment with RU-505 improved cognitive
impairment of AD mice
Because RU-505 restored A-induced altered thrombosis
and impaired fibrinolysis in vitro and in vivo, we explored
whether long-term RU-505 treatment could have behavioral
1053
Figure 3. RU-505 prevented altered thrombosis and fibrinolysis in AD transgenic mice. (A) After craniotomy, three concentrations of FeCl3
(5, 10, and 15%) were incrementally administered to the surface of the brains of vehicle- or RU-505treated WT and Tg6799 mice (Videos 14), and clotting
of cerebral blood vessels (>20 M) was imaged (bars, 200 m). Representative intravital images shows the surface of the brains of vehicle- or RU-505
treated WT and Tg6799 mice before FeCl3 treatments or 5 min after 15% FeCl3 treatments. (B and C) Frequency of clotted vessels was calculated at increasing concentrations of FeCl3 (B) and was plotted for 15% FeCl3 treatment (C; ***, P < 0.001; n = 5 mice per group). All values are means and SEM.
Results are from two independent experiments.
effects on AD mice. 7-mo-old Tg6799 mice treated for 3 mo

with RU-505 were tested using contextual fear conditioning
to assess possible cognitive changes. RU-505 treatment had
no effect on baseline freezing behavior in WT and Tg6799
mice (Fig. 5 A). When we evaluated contextual memory of
Tg6799 and WT mice 24 h after training, vehicle-treated
Tg6799 mice showed a severe memory deficit compared with
vehicle-treated WT mice (Fig. 5 B). RU-505treated Tg6799
mice exhibited significantly improved memory compared
with their vehicle-treated AD counterparts, whereas longterm treatment of RU-505 in WT mice did not impact basal
freezing behavior or contextual memory.
We also explored cognitive performance of RU-505
treated Tg6799 mice with the Barnes maze, a behavioral test
that assesses spatial learning and memory in rodents (Walker
et al., 2011). Vehicle-treated Tg6799 mice took a significantly longer amount of time to find the target hole compared with vehicle-treated WT and RU-505treated Tg6799
mice (Fig. 5 C). During the memory retention test in the
probe trials, vehicle-treated Tg6799 mice also had significantly
1054
longer latency to reach the closed target hole (Fig. 5 D) and

significantly fewer visits to the target hole compared with vehicle-treated WT and RU-505treated Tg6799 mice (Fig. 5 E).
These results suggested that vehicle-treated Tg6799 mice
have impaired spatial learning and memory, and RU-505 treatment restored the cognitive impairment of Tg6799 mice.
When we measured total distance traveled during probe trials,
Tg6799 mice moved significantly less than WT mice. However, there was no significant difference in distance traveled
between RU-505treated and untreated Tg6799 mice (Fig. 5 F).
This result suggests that the better performance of RU-505
treated Tg6799 mice compared with untreated Tg6799 mice
is likely caused by memory improvement and not effects
on locomotion.
To further address any possible issue of hypoactivity in the
Tg6799 mice and to test whether RU-505 treatment had a
similar effect on a different strain of AD transgenic mice, we
administered RU-505 to 4-mo-old TgCRND8 mice (Chishti
et al., 2001) for 3 mo (analyzed at 7 mo-of-age) as a pilot experiment. During training, RU-505 treatment did not lead to
Ar ticle
Figure 4. CAA pathology in AD transgenic mice was reduced after long-term

treatment with RU-505. (A) A deposits
within cortical blood vessels of vehicle- or
RU-505treated Tg6799 mice were visualized
using Congo red and laminin (green) labeling
(bars, 100 m). (B) Representative pictures
showing parenchymal A deposition in untreated and treated mice (bars, 100 m).
(C and D) CAA and A plaques in (A) and (B)
were quantified from 710 sections per
mouse (n = 5 mice per group; *, P < 0.05). All
values are means and SEM. Results are from
two independent experiments.
improvement in spatial learning in TgCRND8 mice (Fig. 6 A);

however, this treatment significantly reduced the latency to reach
the target hole during the probe trial compared with vehicletreated TgCRND8 mice (Fig. 6 B). Furthermore, the number of
visits to the target hole during the probe trial was significantly
higher in RU-505treated TgCRND8 mice compared with
vehicle-treated TgCRND8 mice (Fig. 6 C). In addition, vehicletreated TgCRND8 mice showed similar locomotor activity
during probe trials (Fig. 6 D), indicating that the impaired performance of vehicle-treated TgCRND8 mice in Barnes maze
test is more likely caused by deficits in spatial memory.These results suggest that treatment of RU-505 substantially improved
the deficits in spatial memory of TgCRND8 mice.
JEM Vol. 211, No. 6
Long-term treatment with RU-505 decreased the level

of infiltrated fibrinogen and microgliosis in AD mice
To understand the mechanisms underlying improvement
in cognitive function of RU-505treated AD mice, we ana
lyzed cortical fibrinogen infiltration and microgliosis in
Tg6799 mice after four months of RU-505 treatment.
BBB permeability is increased in mouse models of AD
(Paul et al., 2007) and infiltrated fibrinogen might bind to
A and become resistant to degradation in the parenchyma. If RU-505 can inhibit the interaction between infiltrated fibrinogen and A in the parenchyma, the level of
infiltrated fibrinogen could be decreased in the brain of AD
mice. In addition, activation of microglia is highly increased
1055
Figure 5. RU-505 restored cognitive function in Tg6799 mice. (A) Freezing behavior was measured before electric foot shock during the training
day to assess the basal freezing tendency of each group of mice. (n = 810 mice per group). (B) Contextual memory was assessed by measuring freezing
behavior upon reexposure to the training chamber 24 h after fear conditioning training. (*, P < 0.05; **, P < 0.01; n = 810 mice per group). Results are
from two independent experiments. (CE) Spatial learning and memory retention of WT and Tg6799 mice was assessed using the Barnes maze after 3 mo
of treatment with RU-505 or vehicle. One target hole was connected to a hidden escape chamber. (C) During training trials, latency to poke the target
hole was measured. Significance was assessed using two-way ANOVA analysis with repeated measure (WT/vehicle vs. Tg6799/vehicle: F[1,120] = 40.47;
P < 0.001; Tg6799/vehicle vs. Tg6799/RU-505: F[1,108] = 11.97; P < 0.01; n = 1014 mice per group). Differences in latency were assessed by Bonferroni post hoc
analysis. (DF) During the Barnes maze probe trial, latency to reach the closed target hole (D), number of visits to the target hole (E), and total traveled
distance (F) were measured ([E] *, P < 0.05; **, P < 0.01; ***, P < 0.001; n = 1014 mice per group; [F] ***, P < 0.001; n = 1014 mice per group). All results
of the Barnes maze are from three independent experiments.
in AD patients and mouse models of AD, and an increase of

inflammation in the brain is correlated with memory impairment (Bayer et al., 1999; Dhawan and Combs, 2012;
Vom Berg et al., 2012).
Therefore, we measured the level of infiltrated fibrinogen
and microgliosis in the cortex of Tg6799 or WT littermate
mice after RU-505 treatment. We quantified fibrinogen
deposition (green; Fig. 7 A) outside the endothelial cells of

blood vessels that were labeled using CD31 (red; Fig. 7 A), and
the area of activated microglia that were labeled using CD11b
(red; Fig. 7 B). The levels of infiltrated fibrinogen and microgliosis were highly increased in the cortex of Tg6799 compared with WT mice (Fig. 7, C and D), and these increases
were significantly decreased by RU-505 (Fig. 7, C and D).
Figure 6. RU-505 restored spatial retention memory

in TgCRND8 mice without affecting motor behavior.
(A) The spatial memory of vehicle- or RU-505treated WT
and TgCRND8 mice was assessed using Barnes maze.
(B and C) Spatial memory of RU-505treated WT and
TgCRND8 mice was tested using the Barnes maze probe
trials. Time to reach the target hole (B), the number of visits
to the closed target hole (C), and total distance traveled (D)
were assessed (n = 711 mice per group). The results corroborate those in Fig. 5 and are from one experiment. All
values are means and SEM.
1056
Ar ticle
DISCUSSION
The present study shows that the novel compound, RU-505,
restored A-induced altered thrombosis and delayed fibri
nolysis in vitro and in vivo by inhibiting the Afibrinogen
interaction. We also demonstrate that long-term RU-505
treatment can reduce vascular amyloid deposits, infiltrated
fibrinogen, and microgliosis in the cortex of a transgenic
mouse model of AD. Finally, this novel Afibrinogen inter
action inhibitor improved the cognitive decline of two different
strains of AD transgenic mice.
Using pharmacokinetics, we found that RU-505 is highly
permeable to the BBB because RU-505 levels in the brain
were equal to or greater to that in the blood over a 24-h
period after single subcutaneous injection. The half-life of
RU-505 was 3.7 h in the blood and 12.4 h in the brain.
Therefore, the intravascular and the tunica media of arterioles
are the most likely regions for RU-505 action. Several studies
have shown that the amount of soluble A significantly increases in the vicinity of amyloid deposits in blood vessels
(Shinkai et al., 1995; Suzuki et al., 1994), and the intravascular area near CAA might be a major target of inhibition by
RU-505. In addition, the increased infiltrated fibrinogen in

AD mice may interact with A in the tunica media of arterioles, which could be another target region for inhibition of
A-induced exacerbated thrombosis, as well as for prevention
of CAA formation.
One question that arises from our results is why RU-505
treatment reduced vascular amyloid deposits, but not parenchymal plaque. Amyloid accumulates in the tunica media of
arterioles in CAA, and the tunica media is much closer to
the intravascular region than the parenchyma. Therefore, the
fibrinogen levels in the tunica media should be much higher
than in the parenchyma. For this reason, the Afibrinogen
interaction could be a major factor of A fibrillization in CAA,
but have only minor effects on A fibrillization on parenchymal plaques.
We primarily investigated the interaction between A42
and fibrinogen in this study, but the ratio of A40 to A42
is higher in CAA. Another question is how RU-505, an
inhibitor of the A42fibrinogen interaction, could reduce
CAA, which is primarily composed of A40. One possibility
is that A40 also interacts with fibrinogen even though its
Figure 7. Long-term treatment with RU-505 reduced the level of infiltrated fibrinogen and microgliosis in the cortex of Tg6799 mice.
(A) Fibrinogen localized outside of endothelial cells of blood vessels was labeled with FITC-conjugated antifibrinogen antibody (green), and endothelial
cells were labeled using anti-CD31 antibody (red; bars, 50 m). (B) Activated microglia were visualized by staining for CD11b (red). DAPI staining (blue)
was used to show integrity of tissue (bars, 100 m). (C and D) Total fibrinogen area (C) and microgliosis (D) were quantified from 3 sections per mouse
(n = 34 mice per group; *, P < 0.05; ***, P < 0.001). All values are means and SEM. Results are from two independent experiments.
JEM Vol. 211, No. 6
1057
affinity is 10 times less than A42, and RU-505 has a strong

inhibitory efficacy on A40fibrinogen interaction (unpublished data). Therefore, RU-505 could reduce CAA through
inhibition of both A42- and A40fibrinogen interaction.
Second, despite the higher levels of A40 in vascular amyloid, A42 is also essential for vascular amyloid deposition
in transgenic mice overexpressing human APP (Van Dorpe
et al., 2000; McGowan et al., 2005) and AD human patients
(Roher et al., 1993; Shinkai et al., 1995). A42 could act as
a nucleation seed of amyloid deposit in the vessel walls and
accelerate deposition of A40 (Van Dorpe et al., 2000;Yoshiike
et al., 2003; McGowan et al., 2005). Therefore, even though
the ratio of A40 to A42 is higher in vascular amyloid, the
A42fibrinogen interaction could be critically involved in
CAA formation.
Fibrinogen is a proinflammatory mediator in several diseases and induces the activation of microglia in the nervous
system (Adams et al., 2007; Davalos and Akassoglou, 2012).
Our study showed that RU-505 treatment reduced the level
of infiltrated fibrinogen and activated microglia in the brain
of AD transgenic mice. One possible mechanism for this reduction is that RU-505 binds A, inhibits the Afibrinogen
interaction, and facilitates fibrinogen degradation.The decreased
level of infiltrated fibrinogen could result in the decrease of
microgliosis. The other possible mechanism is that long-term
RU-505 treatment reduced vascular amyloid deposits and
prevented BBB leakage.This recovery of a healthy BBB could
reduce fibrinogen infiltration and inflammation in the parenchyma of Tg6799 mice.
Increased levels of plasma fibrinogen are associated with
cognitive deficits (Xu et al., 2008), AD risk (van Oijen et al.,
2005), and brain atrophy (Thambisetty et al., 2011), and increased levels of fibrinogen have also been found in the CSF
of AD patients (Craig-Schapiro et al., 2011; Vafadar-Isfahani
et al., 2012). Moreover, several studies have shown that anticoagulant treatment improves cognition in mouse models of
AD and dementia patients (Ratner et al., 1972; Walsh et al.,
1978; Cortes-Canteli et al., 2010;Timmer et al., 2010). However, anticoagulant therapy can cause severe problems in
elderly patients who have a more fragile vasculature because
it may increase the incidence of major systemic bleeding.Therefore, drugs should specifically block the Afibrinogen interaction so that A-induced altered blood clot formation and
degradation can be restored without affecting general hemostasis. RU-505 successfully targeted only A-induced altered
blood clot formation and did not affect general clot formation and degradation (Fig. 2 D and Fig. 3).
The maximum tolerated dose of RU-505 after single intravenous dose in mice was between 100 and 200 mg/kg.
When we treated Tg6799 mice and WT littermates with two
doses (100 and 50 mg/kg) of RU-505 every other day for 3 mo,
we found that 100 mg/kg for long-term treatment was toxic
to the AD mice, but 50 mg/kg showed no clinical signs of
toxicity except local chronic inflammation at the injection
site.To address the issue of local inflammation, we lowered the
dose to 35 mg/kg for Tg6799 or 25 mg/kg for TgCRND8
1058
over 3 mo, which minimized this issue. Our future direction

would modify RU-505, and find less toxic analogues with similar or better efficacy.
For more than a decade, A has been the major target for
developing AD therapies. Most of these efforts focused on using
antibodies to lower A levels, preventing A aggregation, or reducing A production. However, none of these methods were
successful as the treatments did not show clinical efficacy or
caused serious adverse side effects such as aseptic meningoencephalitis (Gilman et al., 2005; Mangialasche et al., 2010). However, numerous studies still support the hypothesis that A plays
an important role in the pathogenesis of AD (Tanzi and Bertram,
2005; Jonsson et al., 2012).Therefore, new strategies for anti-A
therapy are necessary for developing novel treatments for AD. Inhibiting the interaction between A and its binding proteins
could be an alternative therapeutic approach, and our study
shows that a small molecule, bioavailable inhibitor of the A
fibrinogen interaction, RU-505, significantly restored altered
thrombosis and improved cognitive deficits observed in AD
transgenic mouse models. Therefore, treatment of the neurovascular pathology observed in AD using an inhibitor of the
Afibrinogen interaction may be a valuable strategy for developing novel AD therapeutics.

Animals
Tg6799 mice (The Jackson Laboratory) are double transgenic mice for APP/
Presenilin 1 that coexpress five early onset familial AD mutations on a mixed
background C57BL/6 x SJL (Oakley et al., 2006). TgCRND8 mice (provided by A. Chishti and D. Westaway, University of Toronto, Canada) have
three APP mutations (K670N, M671L, and V717F) driven by the human
prion protein promoter on a mixed background C57 x C3H/C57 (Chishti
et al., 2001). RU-505 was prepared in 2.5% EtOH, 4.5% Cremophor RH40
(Sigma-Aldrich), and 14% D5W (5% dextrose in water) in saline. We administered 35 mg/kg dose of RU-505 or vehicle to Tg6799 mice and 25 mg/kg
dose or vehicle to TgCRND8 mice subcutaneously every other day. Nontransgenic (WT) littermates were used in all experiments.The assigned genotype of all the mice used in the experiments throughout the paper was
double-checked by taking tail tissue the day of sacrifice. Only male mice
were used in experiments, and all animals were maintained in The Rockefeller University Comparative Biosciences Center and treated in accordance
with protocols approved by The Rockefeller University Institutional Animal
Care and Use Committee.
Primary compound screening

Approximately 93,000 compounds were screened using HTS. Compound
screening libraries that include known off-patent drugs, natural products, and
combinatorially elaborated active pharmacophores were purchased from
several vendors listed in Table 3. The primary assay used FP to measure the
changes in the anisotropy induced by binding of TAMRA-labeled A42
(Anaspec) to fibrinogen. TAMRA-A42 (2 nM) was mixed with 300 nM
fibrinogen (EMD Millipore) and 20 M compounds (dissolved in 1% DMSO
[final]) in 50 mM PBS, pH 7.4, 0.001% Tween 20, and 0.001% BSA as 50 l
final volume in black 384-well plates (Greiner) at RT. After binding reached
equilibrium, polarization measurements were recorded with a Perkin-Elmer
EnVision plate reader with excitation at 490 nm and emission at 535 nm.The
FP response was monitored and plotted as milli-Polarization (mP) units.
Compounds that showed >75% inhibition of the Afibrinogen interaction in the FP assay were selected for screening by AlphaLISA as a secondary assay. Compounds (12.5 M) were plated in white 384-well plates
Ar ticle
(Greiner) and were incubated with 10 nM biotinylated A42 (Anaspec) and

1 nM fibrinogen for 30 min at RT in final volume of 10 l assay buffer
(25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20, and 0.1%
BSA). The mixture was incubated with antifibrinogen antibody (Dako),
20 g/ml streptavidin-conjugated donor, and protein Aconjugated acceptor beads (PerkinElmer) for 90 min at RT. Samples were read by a Perkin
Elmer EnVision plate reader.
Hit compounds from the secondary assay were evaluated using Lipinskis
Rule of Five to determine whether each chemical compound has properties
that make it a potential usable drug. If compounds violated more than one of
Lipinskis Rule of Five, those compounds were removed from our list. The
AlphaScreen TruHits kit (PerkinElmer) was used to detect those compounds
that react with singlet oxygen and thus unspecifically quench the assay. The
AlphaScreen TruHits kit also allows for the identification of color quenchers,
light scatterers (insoluble compounds), and biotin mimetics interfering with
the AlphaLISA signal. If inhibition by quenching was more than 30% at 10 M
compound, those compounds were removed from our list. After completing
the quenching test, we screened hit compounds in a doseresponse experiment
with various compound concentrations (0.0120 M) using FP and AlphaLISA.The data were fitted to sigmoidal doseresponse equation (Y = Bottom +
(Top Bottom)/1 + 10(logIC50 X) Hill coefficient)) using GraphPad Prism 4 to
calculate half-maximal inhibition (IC50) of each compound. Compounds with
IC50 < 50 M in both FP and AlphaLISA were purchased as powder and were
retested in doseresponse experiments using both assays.
Analogue compound screening

To improve our candidate compounds, we had access to the ChemNavigator
database, which has 50 million commercially available compounds and software
for Tanimoto-based similarity searching. We purchased 2,000 analogue compounds through ChemNavigator or directly from Albany Molecular Research
Inc. These analogues were tested using AlphaLISA at 5, 10, and 20 M. We selected compounds which have >50% inhibition at 10 M and a proportional inhibitory effect at 5 or 20 M. Drug-like compounds were evaluated using
Lipinskis Rule of Five, and false-positive compounds were filtered out using the
AlphaScreen TruHits kit (PerkinElmer) as described above. Compounds with
IC50 < 10 M in both FP and AlphaLISA were selected using doseresponse experiments. Selected compounds were purchased as powder and were retested in
doseresponse experiments using both assays. Finally, we identified hit compounds of with IC50 < 3 M in FP and IC50 < 10 M AlphaLISA assay.
Pull-down assay
Hit compounds were tested using a pull-down assay as described previously
(Ahn et al., 2010). In brief, compounds at 10 M were incubated with 100 nM
Table 3. Vendor list for primary screening library

Provider
ChemDiv
Prestwick
Cerep
ChemBridge
Microsource
AMRI
Biofocus
GreenPharma
Sigma LOPAC
Prof. Derek Tan (Memorial SloanKettering Cancer Center, New
York, NY)
Total
JEM Vol. 211, No. 6
No. of compound
from each provider
21,986
1,110
4,000
5,000
2,000
50,000
7,750
240
1,280
350
93,716
biotinylated A42 (Anaspec) and 5 nM fibrinogen (EMD Millipore) for 1 h

at room temperature in 500 l of binding buffer (50 mM Tris-HCl, pH 7.4,
150 mM NaCl, 0.1% Nonidet P-40, 0.1% BSA, and protease inhibitor mixture). The samples were gently rotated for 1 h at room temperature with
30 l streptavidinSepharose high performance beads (GE Healthcare). After
incubation, the beads were washed five times with binding buffer, and nonreducing sample buffer was added to the beads for elution. Western blots
were performed using antifibrinogen antibody (Dako). Dot blots were performed using anti-A antibody 4G8 (Covance) to show comparable amounts
of A were also being pulled down.
The binding assay between fibrinogen

and monomeric or oligomeric A42
Biotin-A42 monomers and oligomers were prepared as in (Stine et al.,
2011). In brief, biotin-LC-A42 (Anaspec) was monomerized by treatment
with hexafluoroisopropanol, dissolved to 5 mM with dimethyl sulfoxide,
then diluted to 100 M with cold PBS, and sonicated. Monomeric biotinLC-A42 was incubated at 4C for 24 h for oligomeric preparation. 1 nM
fibrinogen was mixed with increasing concentrations of monomeric or
oligomeric biotin-LC-A42 (0.520 nM) for 30 min at room temperature
and the binding affinity was measured using AlphaLISA assay. The inhibitory
efficacy of RU-505 on the interaction between fibrinogen and monomeric
or oligomeric biotin-LC-A42 was accessed in doseresponse experiments
using AlphaLISA assay.
In vitro thrombosis and fibrinolysis assay

To test whether hit compound have an effect on fibrin clot formation and
lysis, 20 M of each compound (dissolved in 0.4% DMSO [final]) or DMSO
control was incubated with fibrinogen (1.5 M) in the presence or absence
of A42 (3 M) for 10 min and then mixed with plasminogen (0.25 M) in
20 mM Hepes buffer (pH 7.4) with 137 mM NaCl. Fibrin clot formation
and degradation was analyzed measuring turbidity right after adding thrombin (0.5 U/ml), tPA (0.15 nM), and CaCl2 (5 mM) in a final volume of
150 l. Assays were performed at RT in High Binding 96-well plates
(Thermo Fisher Scientific) in triplicate and were measured at 450 nm using
a Spectramax Plus384 reader (Molecular Devices).
SPR
SPR experiments were performed to test whether our lead compounds bind
to A42 as described previously (Richter et al., 2010). Biacore 3000 instrument and CM5 sensor chips (GE Healthcare) were used for this assay. Hexafluoroisopropanol-treated monomerized A42 was immobilized to the
sensor chip surface by amine coupling. Compounds were diluted to 40 M
from DMSO stock solutions in PBS as running buffer (final 2% DMSO) and
injected for 2 min at a flow rate of 30 l/min using the KINJECT command.
After the dissociation phase the chip was rinsed with 20 mM HCl. Corresponding DMSO dilutions were used as a buffer blank, and a solvent correction assay was performed to correct the difference of DMSO response
between empty reference surface and protein-immobilized surface. Sulindac
sulfide and sulindac were used as positive control and negative controls, respectively (Richter et al., 2010).
In vivo toxicity study

Maximum tolerated dose studies were performed to determine the toxicity
of RU-505 (AMRI) and to identify the optimal dose for in vivo assays. Single injection toxicity was performed at Absorption Systems LP (Exton, PA),
and four different doses (200, 100, 50, and 20 mg/kg mouse) of RU-505,
along with saline and vehicle, were injected into male and female CD-1
mice intravenously. Mortality and overt clinical signs of toxicity were monitored for 2 d. All animals dosed with 200 mg/kg were found dead after single
intravenous injection, and no clinical signs of toxicity were observed after
single dose of 20, 50, or 100 mg/kg for 2 d after injection. Therefore, the
maximum tolerated dose of RU-505 after single intravenous dose in mice
was established as 100 mg/kg.
1059
Because AD treatment would be long-term and toxicity of long-term

treatment can be different from toxicity after a single injection, we treated
Tg6799 mice and WT littermates with two doses (100 and 50 mg/kg) of
RU-505 every other day for 3 mo, and overt clinical signs of toxicity were
monitored. After 3 mo, mice were sent to the Laboratory of Comparative
Pathology at Memorial Sloan-Kettering Cancer Center for complete necropsy and hematology reports to determine the effects of our lead compound
after long-term treatment.
Pharmacokinetics
The pharmacokinetics of and BBB permeability to RU-505 were determined by assessing the drugs decay in blood plasma and brain homogenates
over a 24-h period after subcutaneous injection (35 mg/kg) into WT mice of
the same genetic background as Tg6799. Blood was collected in heparinized
tubes after cardiac puncture 0.5, 1, 2, 4, 6, and 24 h after RU-505 administration. After perfusion, brains were collected and homogenized with PBS.
Plasma and brain homogenates were sent to Apredica and were analyzed by
LC/MS/MS using an Agilent 6410 mass spectrometer coupled with an Agilent 1200 HPLC. This analysis revealed that RU-505 penetrates the blood
brain barrier, and RU-505 levels in the brain were equal to or greater than in
the blood (3 M).
In vivo thrombosis assay

To observe blood circulation and to induce thrombosis, a cranial window was
prepared as described previously (Cortes-Canteli et al., 2010). In brief, a cranial window was prepared over the parietal cortex of 7-mo-old Tg6799 mice
and WT littermates which were treated with RU-505 (35 mg/kg) or vehicle
for three months (n = 5 per group). Mice were anesthetized by i.p. injection
of 500 mg/kg tribromethanol and 0.04 mg/kg atropine and placed in a custom built restraint system. A 2.5-mm circular craniotomy was prepared using
510 circular brush strokes with a fine dental drill bit, and a 4-mm plastic
ring surrounding the window was attached with dental acrylic and cyano
acrylate adhesive. Sterile saline was applied periodically to protect the brain
surface and prevent drying. For imaging of blood flow, 100 l of 5 mg/ml
2 MDa FITC-conjugated dextran (Sigma-Aldrich) dissolved in PBS was administered by retroorbital injection. During the entire imaging session, the
body temperature of mice was kept at 37.5C using a TC-1000 Mouse complete temperature control system (CWE Inc.).
Increasing concentrations of FeCl3 (5, 10, and 15%) were added directly
to the brain surface with an interval of 5 min, and thrombosis was recorded
using Olympus IX71 microscope equipped with Hamamatsu Orca ER B/W
digital camera. MetaMorph acquisition software and 5 objective lens (NA =
0.25) were used for image collection.The total length of vessels with a >20 m
diam before FeCl3 treatment and the total length of occluded vessels at
5 min after each FeCl3 treatment (5, 10, and 15%) were measured. All analysis
was performed using National Institutes of Health ImageJ software with the
analyzer blinded to genotype and treatment of mice.
Immunohistochemistry for infiltrated

fibrinogen and microgliosis
Mice were saline/heparin-perfused, and 20 m coronal brain cryostat sections
were fixed with 50% methanol and 50% acetone. For fibrinogen and endothelial cell staining, brain sections were incubated with FITC-conjugated antifibrinogen antibody (Dako) and anti-CD31 antibody (BD) overnight. For
activated microglia staining, brain sections were incubated with anti-CD11b
antibody (DSHB) overnight. After immunohistochemistry, brain sections
were analyzed with a confocal microscope (Inverted DMI 6000; Leica)
equipped with HyD detectors and HCX PL APO CS (10 NA 0.4 and 20
NA 0.7) objective lenses at room temperature. The imaging medium was air
for both the objective lenses used and Leica Application Suite Advanced Fluor
escence was used for image collection as software. Each set of stained sections
was processed under identical gain and laser power setting and under identical
brightness and contrast settings. Images of brain section were acquired and
thresholded using ImageJ. The total area of infiltrated fibrinogen or activated
microglia was analyzed as percentage of total cortex area with the analyzer
blinded to treatment of mice. The average of 3 different sections from each
mouse was determined (n = 34 mice per group).
Behavioral analysis
All behavioral experiments were performed and analyzed with a researcher
blinded to genotype and treatment. We administered 35 mg/kg of RU-505
or vehicle to 4-mo-old Tg6799 mice and WT littermates and 25 mg/kg or
vehicle to 4-mo-old TgCRND8 mice and WT littermates subcutaneously
every other day for three months (analyzed at 7 mo of age). Mice were handled and allowed to acclimate to the testing room for 10 min per day for at
least 5 d.
Contextual fear conditioning

During training, Tg6799 mice and WT littermates (n = 810 per group)
were allowed to explore the training chamber (Med Associates, Inc.) for the
first 2 min, and then received three mild footshocks (2 s, 0.7 mA) spaced
1 min apart. Mice were removed from the training chamber 30 s after the last
foot shock. Contextual learning was assessed 24 h after training by reexposing mice to the same training chamber for 3 min. Mouse behavior during
training and testing was recorded, and freezing behavior was measured by
observing mice every 5 s.
Barnes maze
Immunohistochemistry for CAA and A plaques
The Barnes maze apparatus (TAP Plastics) consisted of a white circular platform (92 cm diam) with 20 equally spaced holes (5 cm in diameter; 7.5 cm
between holes). Among these holes, one hole (target hole) was connected to
a hidden black escape chamber. Bright lights (600 lx) were used to motivate
the mice to find the target hole and enter into the escape chamber. Visual
clues surrounded the maze. To remove any lingering scent on the maze from
the previous animal, the platform and escape box were cleaned using 50%
ethanol between mice. The entire experiment was recorded and analyzed
using the Ethovision video tracking system (Noldus).
Mice were saline/heparin-perfused, and 20-m coronal brain cryostat sections were fixed with 4% paraformaldehyde. Brain sections were incubated
with rabbit antilaminin antibody (Sigma-Aldrich) overnight and stained for
30 min with 0.2% Congo Red (Sigma) in 70% isopropanol. After immunohistochemistry, brain sections were analyzed with a microscope (Axiovert
200; Carl Zeiss) equipped with Plan-Neofluar (10 NA 0.3, and 20 NA
0.5) objective lenses at room temperature. The imaging medium was air for
both the objective lenses used. The AxioCam color camera (Carl Zeiss) and
AxioVision software (Carl Zeiss) were used for image collection. Each set
of stained sections was processed under identical gain and laser power setting and under identical brightness and contrast settings. Images of all the
areas with CAA and A plaques were acquired and thresholded using
Image J. The total area of CAA and A plaques was analyzed as percentage
of total cortex area with the analyzer blinded to treatment of mice. The average of 710 different sections from each mouse was determined (n = 5
mice per group).
Tg6799 mice. Training consisted of two training trials per day over a period of 7 d (n = 1014 per group). During each trial, mice were placed in the
center of the maze in a black starting box for 30 s. After 30 s, the box was removed, and mice were allowed to freely explore and find the target hole
within 2 min. Latency to poke the target hole was recorded. If mice did not
enter into the escape chamber within 2 min, they were gently guided into
the escape chamber and placed in the chamber for 30 s. To assess memory
retention, a probe trial was conducted 24 h and 3 d after the last training.
The target hole was closed like the other 19 holes, and the escape chamber
was removed. Holes were kept in the same position as during the training.
Mice were placed in the center of the maze in a black starting box for 30 s.
After 30 s, the box was removed, and mice were allowed to freely explore
for 90 s. The number of visits into each hole and the latency to reach the
target hole were recorded. For analysis, scores of each mouse from both
probe trials were combined and averaged.
1060
Ar ticle
TgCRND8 mice. Training consisted of a trial per day over a period of

12 d (n = 711 per group). During training, mice were placed in the center
of the maze in a black starting box for 30 s. After 30 s, the box was removed,
and mice were allowed to freely explore and find the target hole for 5 min.
To assess memory retention, probe trials were conducted 24 h and 6 d after
the last training. Mice were allowed to freely explore for 2 min during probe
trials. The number of visits into each hole and the latency to reach the target
hole were recorded. For analysis, scores of each mouse from both probe trials
were combined and averaged.
Statistical analysis
All numerical values presented in graphs are mean SEM. Statistical significance of most experiments was determined using two-tailed t test analysis
comparing control and experimental groups. The pull-down assay (Fig. 2 A)
was analyzed using one-way ANOVA and Bonferroni post hoc test. Comparison of training curves from the Barnes maze (Fig. 5 C and Fig. 6 A) was
analyzed using two-way ANOVA with repeated measure and Bonferroni
post hoc test.
Online supplemental material

Video S1S4 show intravital visualization of blood flow and blood vessel
occlusion in Tg6799 mice or WT littermates that were treated with RU-505
or vehicle as increasing concentrations of FeCl3 (5%, 10%, and 15%) were
added directly to the brain surface. Online supplemental material are available at http://www.jem.org/cgi/content/full/jem.20131751/DC1.
The authors thank The Rockefeller University Bio-Imaging Resource Center for
technical assistance, as well as Dr. Marta Cortes-Canteli, Dr. Zu-Lin Chen, and
members of the Strickland Laboratory for discussion.
This work was supported by the Thome Memorial Medical Foundation,
Alzheimers Drug Discovery Foundation, National Institutes of Health (NS50537),
Woodbourne Foundation, Mellam Family Foundation, May and Samuel Rudin Family
Foundation, and the Blanchette Hooker Rockefeller Fund.
The authors have no conflicting financial interests.
Submitted: 20 August 2013
Accepted: 27 March 2014
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Article
Acid sphingomyelinase modulates

the autophagic process by controlling
lysosomal biogenesis in Alzheimers disease
Jong Kil Lee,1,2,3 Hee Kyung Jin,1,4 Min Hee Park,1,2,3 Bo-ra Kim,1,2,3
Phil Hyu Lee,5 Hiromitsu Nakauchi,6 Janet E. Carter,7 Xingxuan He,8
Edward H. Schuchman,8 and Jae-sung Bae1,2,3
1Stem
Cell Neuroplasticity Research Group, 2Department of Physiology, Cell and Matrix Research Institute, School of Medicine,
of Biomedical Science, BK21 Plus KNU Biomedical Convergence Program, 4Department of Laboratory Animal
Medicine, College of Veterinary Medicine, Kyungpook National University, Daegu 702-701, Korea
5Department of Neurology and Brain Research Institute, Yonsei University College of Medicine, Seoul 120-752, Korea
6Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science,
University of Tokyo, Tokyo 108-8639, Japan
7Mental Health Sciences Unit, Faculty of Brain Sciences, University College London, London WC1E 6DE, England, UK
8Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029
3Department
CORRESPONDENCE
Jae-sung Bae:
jsbae@knu.ac.kr
Abbreviations used: A, amyloid-;
AC, acid ceramidase; AD,
Alzheimers disease; ALP,
autophagylysosome pathway;
AMI, amitriptyline-hydrochloride;
AP, alkaline phosphatase;
ApoE4, apolipoprotein E4;
APP, amyloid precursor protein;
ASM, acid sphingomyelinase;
AV, autophagic vacuole; CM,
conditioned medium; EM,
electron microscope; FAD,
familial AD; i.c., intracerebral;
iPSC, induced pluripotent stem
cell; Lamp1, lysosomal-associated
membrane protein 1; LBPA,
lysobisphosphatidic acid; LC3,
microtubule-associated protein 1
light chain 3; M6P, mannose-6phosphate; NPD, NiemannPick disease; PD, Parkinsons
disease; PS1, presenilin 1;
SA--gal, senescence-associated-galactosidase; TFEB, transcription factor EB.
In Alzheimers disease (AD), abnormal sphingolipid metabolism has been reported, although
the pathogenic consequences of these changes have not been fully characterized. We show
that acid sphingomyelinase (ASM) is increased in fibroblasts, brain, and/or plasma from
patients with AD and in AD mice, leading to defective autophagic degradation due to
lysosomal depletion. Partial genetic inhibition of ASM (ASM+/) in a mouse model of familial AD (FAD; amyloid precursor protein [APP]/presenilin 1 [PS1]) ameliorated the autophagocytic defect by restoring lysosomal biogenesis, resulting in improved AD clinical and
pathological findings, including reduction of amyloid- (A) deposition and improvement
of memory impairment. Similar effects were noted after pharmacologic restoration of ASM
to the normal range in APP/PS1 mice. Autophagic dysfunction in neurons derived from FAD
patient induced pluripotent stem cells (iPSCs) was restored by partial ASM inhibition.
Overall, these results reveal a novel mechanism of ASM pathogenesis in AD that leads to
defective autophagy due to impaired lysosomal biogenesis and suggests that partial ASM
inhibition is a potential new therapeutic intervention for the disease.
Alzheimers disease (AD) is the most common

form of dementia. It is characterized clinically
by progressive loss of memory, and pathologically by the presence of neuritic plaques and
neurofibrillary tangles (Selkoe, 2001). There are
profound biochemical alterations in multiple
pathways in the AD brain, including changes in
amyloid- (A) metabolism, tau phosphorylation, and lipid regulation, although to date the
underlying mechanisms leading to these complex
abnormalities, as well as the downstream consequences, remain largely unknown (Yankner et al.,
2008; He et al., 2010; Mielke et al., 2012).
Sphingolipid metabolism is an important process for tissue homeostasis that regulates the
formation of several bioactive lipids and second
messengers that are critical in cellular signaling
J.K. Lee and H.K. Jin contributed equally to this paper.

J. Exp. Med. 2014 Vol. 211 No. 8 1551-1570
(Lahiri and Futerman, 2007; Wymann and

Schneiter, 2008). In the brain, the proper balance of sphingolipid metabolites is essential for
normal neuronal function, and subtle changes in
sphingolipid homeostasis may be intimately involved in neurodegenerative diseases including
AD (Cutler et al., 2004; Grimm et al., 2005;
Hartmann et al., 2007; Grsgen et al., 2010;
Haughey et al., 2010; Mielke and Lyketsos, 2010;
Di Paolo and Kim, 2011; Tamboli et al., 2011).
Recently, our studies and those of others
(Katsel et al., 2007; He et al., 2010) have shown
that the activity of several sphingolipid metaboliz
ing enzymes, including acid sphingomyelinase
2014 Lee et al. This article is distributed under the terms of an Attribution
1551
Figure 1. ASM is increased in AD and complete ASM gene deficiency exacerbates pathology of APP/PS1 mice. (A and B) ASM was estimated in
the blood plasma (A; control, n = 30; AD, n = 40; and PD, n = 20) and fibroblast (B; control, n = 24; PS1-FAD, n = 24; ApoE4, n = 24; and PD, n = 12) with
AD, PD, or normal controls. (C) ASM activity did not show passage differences between AD and normal fibroblasts (n = 8 per passage group). (D) Detection
of sphingomyelin, ceramide, and AC in plasma (control, n = 2022; and AD, n = 3335) and fibroblast (control, n = 12; PS1-FAD, n = 18; and ApoE4, n = 18).
(E) Crossing scheme to generate WT, APP/PS1, ASM/, and APP/PS1/ASM/ mice. PCR-based genotyping to detect WT, APP/PS1, ASM/, and APP/PS1/
ASM/ mice. (F) Survival curves of WT (n = 26), APP/PS1 (n = 30), ASM/ (n = 30), and APP/PS1/ASM/ (n = 25) mice. (G) Body weights of WT,
1552
Role of ASM in the pathogenesis of AD | Lee et al.
Ar ticle
(ASM), are abnormal in the brains of AD patients. ASM is expressed by almost all cell types and has an important housekeeping role in sphingolipid metabolism and membrane
turnover. It is mainly located within the endosomal/lysosomal
compartment but is associated with the cellular stress response
and may become preferentially transported to the outer leaflet of the cell membrane under conditions of cell stress ( Jenkins
et al., 2009). Mutations in the ASM gene (SMPD1) lead to
the type A and B forms of the lysosomal storage disorder
Niemann-Pick disease (NPD). In addition to its role in NPD,
the importance of ASM in numerous signaling processes,
including cell death, inflammation, and autophagy, has been
extensively documented in several pathological conditions
(Santana et al., 1996; Grska et al., 2003; Petrache et al., 2005;
Lang et al., 2007; Smith and Schuchman, 2008; Teichgrber
et al., 2008; Sentelle et al., 2012). However, the role of ASM
in AD and the cellular mechanisms that link ASM and AD
have not been fully characterized. This lack of understanding
between the correlation of altered ASM levels and AD pathophysiology led us to explore the mechanisms underlying
ASMs role in AD pathogenesis. Here, we show for the first
time that increased ASM activity in AD causes a defect of autophagic degradation due to disruption of lysosomal biogenesis and integrity, and that partial inhibition of ASM activity
leads to restoration of autophagy and improvement of pathological and clinical findings in AD mice.
RESULTS
ASM activity is increased in AD patients
We first sought to confirm whether sphingolipid metabolism
is altered in AD patient samples. We examined ASM and acid
ceramidase (AC) activities, and the levels of several sphingolipids, including sphingomyelin and ceramide, in samples from
normal individuals and AD patients. Consistent with previous
results (He et al., 2010), ASM was significantly increased in
plasma and fibroblasts from individuals with AD compared
with normal aged individuals (Fig. 1, A and B). To assess
whether increased ASM activity was an AD-specific signature,
we analyzed ASM activity in samples from individuals with
Parkinsons disease (PD). The activity of ASM was not elevated in PD-derived samples compared with normal (Fig. 1,
A and B). ASM activity also did not show passage differences
between AD and normal fibroblasts (Fig. 1 C). Sphingomyelin
levels were decreased in the AD plasma compared with normal (Fig. 1 D). No significant differences in the ceramide and
AC levels were found between the two groups (Fig. 1 D).
These results confirmed that elevation of ASM, an important
sphingolipid-modulating factor, is AD specific and may influence disease progression and/or pathogenesis.
Partial ASM inhibition in AD mice reduces pathology

To investigate the influence of ASM on AD pathology, we
first generated amyloid precursor protein (APP)/presenilin 1
(PS1) double mutant and APP/PS1/ASM/ triple mutant
mice (Fig. 1 E). AD-related pathologies in APP/PS1 mice
normally begin at 67 mo of age; however, our APP/PS1/
ASM/ mice died young (Fig. 1 F). We presume that the
early death of the APP/PS1/ASM/ animals was due to
their ASM/ phenotype because these animals (originally
developed as a model of the neurodegenerative type A NPD)
usually die by 68 mo of age.The APP/PS1/ASM/ mice
showed significantly decreased body weight compared with
APP/PS1 mice (Fig. 1 G), and indicators of brain injury, such
as cell death and inflammation, were significantly increased
(Fig. 1, HJ). These data demonstrated that complete deletion
of ASM in APP/PS1 mice exacerbated brain pathology, and
that APP/PS1/ASM/ mice were not suitable to examine
the correlation of ASM and AD pathology.
To overcome these obstacles, we generated APP/PS1/
ASM+/ triple mutant mice (with partial genetic deletion of
the ASM gene; Fig. 2, A and B). Similar to AD patients, ASM
activity was elevated in plasma, brain, and fibroblasts of 9-mo-old
APP/PS1 mice (Fig. 2 C), likely due to the stress response related to the progression of AD-like disease in these animals.
Next, to further investigate the cell contribution of increased
ASM activity in AD mouse brain, we isolated neurons and
microglia from the brain. Although ASM activity was slightly
increased in APP/PS1 microglia compared with WT microglia, the degree of ASM increase was greater in neurons than
microglia (Fig. 2 D), indicating that neurons were the main
contributor of elevated ASM activity in AD mouse brain.
Importantly, ASM activity in age-matched APP/PS1/
ASM+/ mice was significantly decreased compared with the
APP/PS1 mice to levels within the normal range or lower
(Fig. 2 C). Other sphingolipid factors were unaltered in the
APP/PS1/ASM+/ mice except sphingomyelin, which was
modestly reduced in APP/PS1 mice and elevated in the triple
mutant animals (Fig. 2 E).
To determine whether the reduced ASM activity in the
APP/PS1/ASM+/ mice affected AD pathology, we first
determined the A profile. Thioflavin S staining, immuno
fluorescence, and ELISA results of A40 and A42 showed
significantly lower A levels in the 9-mo-old APP/PS1/
ASM+/ mice compared with age-matched APP/PS1 mice
(Fig. 2, FI). In APP/PS1/ASM+/ mice, cerebral amyloid angiopathy and C-terminal fragment of APP were also reduced
(Fig. 2 J; and see Fig. 4, D and F). There were no significant
differences of tau hyperphosphorylation between the two
groups (Fig. 2 K).
APP/PS1, ASM/, and APP/PS1/ASM/ mice were determined at the indicated ages (n = 67 per group). (HJ) Brain sections from 7-mo-old mice were
immunostained with antiactive caspase3 (H; n = 4 per group; bars, 50 m), anti-GFAP (I; n = 4 per group; bars, 100 m), and antiIba-1 (J; n = 4 per
group; bars, 100 m). Data are representative of three independent experiments. AD and G, Students t test. HJ, one-way ANOVA, Tukeys post hoc test.
*, P < 0.05; **, P < 0.01. All error bars indicate SEM.
JEM Vol. 211, No. 8
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Figure 2. Partial genetic inhibition of ASM leads to decreased AD pathology in the APP/PS1 mice. (A) Generation of the APP/PS1/ASM+/ mice. (B) Body
weights of WT, APP/PS1, ASM+/, and APP/PS1/ASM+/ mice were determined at 9 mo of age (n = 14 per group). (C) ASM activity in blood plasma (n = 1415 per
group), brain (n = 1314 per group), and fibroblast (n = 8 per group) derived from WT, APP/PS1, ASM+/, and APP/PS1/ASM+/ mice. (D) ASM activity was assessed
in neuron and microglia isolated from mouse brain (WT, n = 8; APP/PS1, n = 6; and APP/PS1/ASM+/, n = 6). (E) Detection of sphingomyelin, ceramide, and AC in
1554
Ar ticle
Figure 3. Partial genetic inhibition of ASM prevents memory impairments in APP/PS1 mice. (A) Learning and memory was assessed by Morris
water maze test in the WT (n = 13), APP/PS1 (n = 12), ASM+/ (n = 12), and APP/PS1/ASM+/ (n = 12) mice (BF) Probe trial day 11. (B) Time spent in
target platform and other quadrants was measured. (C and D) Path length (C) and swim speed (D) were analyzed. (E) The number of times each animal
entered the small target zone during the 60-s probe trial. (F) Representative swimming paths at day 10 of training. (G) The freezing response during the
training session. Bars show exposure to the tone and arrows the application of the footshock. (H) The results of contextual and tone tasks (WT, n = 14;
APP/PS1, n = 14; ASM+/, n = 13; and APP/PS1/ASM+/, n = 13). Data are representative of three independent experiments. A, C, D, E, and H, one-way
ANOVA, Tukeys post hoc test. B, Students t test. *, P < 0.05; **, P < 0.01. All error bars indicate SEM.
Next, to assess the potential effect of partial genetic ASM

inhibition on learning and memory in APP/PS1 mice, we performed the Morris water maze task and fear conditioning. Aged
APP/PS1 mice showed severe deficits in memory formation
and APP/PS1/ASM+/ mice were largely protected from this
defect (Fig. 3). Collectively, these results suggested that restored ASM activities to the normal range in APP/PS1 mice
decreased A load and improved learning and memory.
Partial ASM inhibition reverses

defective autophagy in AD mice
A reduction in APP/PS1/ASM+/ mouse cerebral amyloidosis could be due to a decreased inflammatory response, attenuated APP expression, or activation of proteases involved in
A degradation. We first assessed the apoptotic and inflammatory responses in brain samples derived from APP/PS1 and
APP/PS1/ASM+/ mice but did not detect differences between
plasma (n = 810 per group), brain (n = 79 per group), and tail (n = 56 per group) fibroblast. (F) Mice brain sections were stained with thioflavin S in APP/PS1 and
APP/PS1/ASM+/ mice. The relative area occupied by A plaques were determined (n = 67 per group; bars, 100 m). (GI) Analysis of A40 and A42 depositions
from the mice brain samples using immunofluorescence staining (G and H; n = 67 per group; bars, 200 m) and ELISA kits (I; n = 8 per group). (J and K) Confocal
laser microscope images and quantification of cerebral amyloid angiopathy (J; n = 6 per group; bars, 50 m) and tau hyperphosphorylation (K; n = 6 per group;
bars, 20 m) in APP/PS1 and APP/PS1/ASM+/ mice. Data are representative of two (D and K), three (B, C, and E), or four (FJ) independent experiments. BE, oneway ANOVA, Tukeys post hoc test. FK, Students t test. *, P < 0.05; **, P < 0.01; ***, P < 0.005. All error bars indicate SEM.
JEM Vol. 211, No. 8
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Figure 4. Genetic inhibition of ASM does not affect inflammatory pathway and processing of APP. Brain sections of APP/PS1 and APP/PS1/ASM+/ mice
were stained with active caspase3 (A; n = 5 per group; bars, 50 m; arrows indicate active caspase3-positive cells) and GFAP antibody (B; n = 6 per group; bars,
100 m). (C) mRNA levels of proinflammatory cytokines or antiinflammatory cytokines (n = 45 per group). (D) Mouse brain lysates were tested for APP and -CTF
levels using Western blot analysis. (E and F) Quantification of APP (E) and -CTF (F) levels (n = 6 per group). (G) Western blot analysis for Bace-1 levels (n = 6 per
1556
Ar ticle
the two strains (Fig. 4, AC). To determine whether reduction of ASM activity affected APP expression, we compared
the levels of APP in the two strains. We found that partial genetic inhibition of ASM did not influence the overall expression levels of APP (Fig. 4, D and E).We also examined the A
generating enzyme Bace-1 using brain homogenates. Bace-1
was slightly decreased in APP/PS1/ASM+/ mice compared
with APP/PS1 mice, but the reduction did not reach statistical significance (Fig. 4 G). To address A clearance by microglia, we analyzed microglia activation and A degrading
enzyme release by microglia but again did not detect differences (Fig. 4, H and I). Similarly, these changes did not show
any differences between APP/PS1 and APP/PS1/ASM+/
mice in 5-mo-old young mice (Fig. 4, JM). There were also
no significant differences of brain pathology, such as apoptosis, inflammation, and A deposition, between the WT and
ASM+/ mice (Fig. 4, AI). Overall, these data suggested that
the partial inhibition of ASM in APP/PS1/ASM+/ did not
alter major inflammatory pathways or expression of APP.
Dysfunction of the normal proteolytic degradation system
also could affect AD pathogenesis and lead to enhanced A
deposition (Lee et al., 2010b). Autophagy, a major degradative
pathway of the lysosomal system, is known to be markedly
impaired in AD (Boland et al., 2008). We found that the
microtubule-associated protein 1 light chain 3 (LC3)II levels
were significantly increased in human AD-derived fibroblasts
compared with control fibroblasts (Fig. 5, AC). Increased
LC3-II levels could stem from overinduction of autophagy or
may be a product of reduced autophagic turnover and defects
in the latter stages of autophagic degradation. We therefore
measured the level of beclin-1 expression in the AD cells,
which is part of a kinase complex responsible for autophagy
induction (Zeng et al., 2006), and found that it did not vary
between the groups (Fig. 5, A and B).
To examine autophagic turnover of protein, we then analyzed proteolysis of long-lived proteins (Lee et al., 2010b) in
control and AD fibroblasts.When autophagic/lysosomal degradation was induced through serum withdrawal, proteolysis was
increased in control fibroblasts but not significantly changed in
PS1 and apolipoprotein E4 (ApoE4)derived AD patient fibroblasts (Fig. 5 D). Lysosome stability relating to autophagy also
could be affected by lysobisphosphatidic acid (LBPA) binding
with ASM (Kirkegaard et al., 2010). We therefore measured
LBPA immunofluorescence intensity in control and AD fibro
blasts, and found that it did not vary between the groups (Fig. 5 E).
Collectively, these data indicated that the autophagosome accumulation in AD is due to dysregulation of autophagic protein degradation, similar to previous results (Lee et al., 2010b).
To examine how genetic inhibition of ASM affected the

autophagic pathway in AD, we also analyzed fibroblasts and
brain samples derived from 9-mo-old WT, APP/PS1, APP/
PS1/ASM+/, and ASM+/ mice. Compared with WT, APP/
PS1 mice showed increased LC3-II, similar to human AD
fibroblasts. This enhanced LC3-II level was reduced in APP/
PS1/ASM+/ mice. Beclin-1 expression did not vary between
the groups (Fig. 5, F, G, K, and L). Metabolic analysis of protein turnover (Cuervo et al., 2004) was assessed using fibroblasts from WT, APP/PS1, and APP/PS1/ASM+/ mice.
Under culture conditions that induced autophagy (absence of
serum), degradation of long-lived proteins was significantly
lower in cells from APP/PS1 mice compared with WT mice
but was increased in cells derived from APP/PS1/ASM+/
mice (Fig. 5 I). The differences of cell senescence levels in
cultured mice fibroblasts were not found between the groups,
indicating that these changes were not related to the cell senescence (Fig. 5 J). The levels of cathepsin D, a lysosomal hydrolase, were elevated in APP/PS1 mice compared with WT.
Enhanced cathepsin D level was ameliorated in APP/PS1/
ASM+/ mice (Fig. 5, F, G, K and L). However, the activity of
cathepsin D was not changed between the groups (Fig. 5,
H and M). This result indicates that the elevated levels of cathepsin D in APP/PS1 mice did not ultimately translate into
a significant increase of enzyme activity. We also analyzed the
expression of p62, indicator of autophagic turnover. Increased
p62 levels in APP/PS1 mice were reduced in APP/PS1/
ASM+/ mice (Fig. 5, F, G, K and L).
To corroborate the immunoblotting results, we performed
electron microscope (EM) analysis using mouse brain samples.
As previously reported (Yu et al., 2005), APP/PS1 mouse brain
regions showed an increased number of autophagic vacuoles
(AVs), whereas brains of APP/PS1/ASM+/ mice showed a
reduced number of these vesicles, albeit still higher than WT
mice (Fig. 5 N).
The endocytic pathway is also considered a major contributor to A deposition in AD (Ginsberg et al., 2010; Li
et al., 2012).To determine whether the endocytic pathway was
affected by partial ASM inhibition, we examined Rab5 and
Rab7 expression in our animals.The expression pattern of these
proteins showed no difference between the groups (Fig. 5 O).
Although additional studies of endocytic pathway are required
to identify the exact mechanism, our results showed that endocytic pathway was not a main mechanism by ASM inhibition. Collectively, these results revealed dysfunctional changes
in the turnover of AVs in the APP/PS1 mice, and that partial
genetic ASM inhibition could reverse this abnormality and
improve autophagic degradation of proteins.
group). (H) Immunofluorescence images of Iba-1 in the APP/PS1 and APP/PS1/ASM+/ mouse brain (bars, 100 m). The relative area occupied by Iba-1positive cells
was quantified (n = 6 per group). (I) The expression of NEP, IDE, and MMP9 was measured in the brain with quantitative real-time RT-PCR (n = 45 per group).
(J) Immunofluorescence images of GFAP-positive cells in the 5-mo-old WT, APP/PS1, and APP/PS1/ASM+/ mouse brain (bars, 100 m). The relative area occupied by
GFAP-positive cells was quantified (n = 67 per group). (K) Western blot analysis and quantification for APP and -CTF levels in the 5-mo-old mice (n = 6 per
group). (L) Western blot analysis for Bace-1 levels in the 5-mo-old mice (n = 6 per group). (M) Immunofluorescence images of Iba-1 in the in the 5-mo-old mouse
brain (bars, 100 m). The relative area occupied by Iba-1positive cells was quantified (n = 6 per group). Data are representative of three (AH) or two (JM) independent experiments. AC, GJ, L, and M, one-way ANOVA, Tukeys post hoc test. E, F, and K, Students t test. *, P < 0.05. All error bars indicate SEM.
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Figure 5. Partial genetic inhibition of

ASM reverses defective autophagy in
APP/PS1 mice. (A) Western blot analysis of
LC-3 and beclin-1 levels in controls, PS1-FAD,
and ApoE4 fibroblasts. (B) LC3-II and beclin-1
levels were quantified (n = 4 per group).
(C) Immunocytochemistry for LC3 in controls,
PS1-FAD, and ApoE4 fibroblast (n = 45 per
group; bars, 20 m). (D) Degradation of longlived proteins was measured in controls, PS1FAD, and ApoE4 fibroblasts (n = 6 per group).
(E) Representative images and quantification
of LBPA in control, PS1-FAD, and ApoE4
fibroblast (n = 4 per group; bars, 50 m).
(F) Western blot analyses for LC3, beclin-1,
p62, and cathepsin D in tail fibroblast derived
from WT, APP/PS1, ASM+/, and APP/PS1/
ASM+/ mice. (G) Densitometric analysis of
LC-3-II, beclin-1, p62, and cathepsin D (n =
78 per group). (H) Cathepsin D activity in
mice tail fibroblast (n = 4 per group). (I) Rates
of proteolysis of long-lived proteins in fibroblasts (n = 6 per group). (J) Representative
images and quantification data of SA--gal
staining in the mice tail fibroblasts (n = 5 per
group; bars, 50 m). (K) Western blot analyses for LC3, beclin-1, p62, and cathepsin D in
the brains of 9-mo-old WT, APP/PS1, ASM+/,
and APP/PS1/ASM+/ mice. (L) Densitometric
quantification of LC-3-II, beclin-1, p62, and
cathepsin D (n = 68 per group). (M) Cathepsin D activity in brain extracts of WT, APP/
PS1, ASM+/, and APP/PS1/ASM+/ mice (n = 4
per group). (N) EM images and quantification data of cortical region. Higher magnification of boxed area shows detail of AVs
(arrow; n = 5 per group; bars: [low magnification] 2 m, [high magnification] 1 m).
(O) Western blot analysis of Rab5 and Rab7
levels in the brain lysates (n = 5 per group).
Data are representative of two (AE and N) or
three (FM and O) independent experiments.
BO, one-way ANOVA, Tukeys post hoc test.
*, P < 0.05. All error bars indicate SEM.
ASM elevation causes defective autophagic

degradation by lysosomal depletion
To gain more direct insights into the relationship of ASM and
autophagic dysfunction, we treated human fibroblasts and neurons with recombinant 110 M ASM and determined the
LC3-II and p62 levels. ASM strongly accelerated LC3-II and
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p62 levels in human fibroblasts and neurons in a concentrationdependent manner (Fig. 6, AC). The level of beclin-1 expression was not affected by ASM (Fig. 6, A and C), indicating
that the accumulation of autophagosomes was not due to the
biogenesis pathway. ASM is found in a secretory and a lysosomal
form ( Jenkins et al., 2009), and the mannose-6-phosphate (M6P)
Ar ticle
Figure 6. Autophagic processes are affected by lysosomal ASM. (A and C) Western blot analysis of LC3, Beclin-1, and p62 in human fibroblast
(A; n = 7 per group) and human neuron (C; n = 6 per group). (B) Immunocytochemistry and quantification for LC3 after ASM treatment (n = 78 per
group; bars, 20 m). (D) ASM activity was assessed in the ASM-treated fibroblast with or without M6P (n = 6 per group). (E) Confocal microscopic analysis
of Lamp1- and ASM-positive vesicles (bars, 20 m). (F and G) The effect of lysosomal ASM on LC3-II expression. (F) 10 M ASM was added to fibroblast
for 24 h with or without 10 mM M6P. LC3-II expression was determined by Western blot analysis (n = 56 per group). (G) LC3-II levels were examined in
10 M ASM-treated fibroblast with or without M6P receptor suppression using siRNA (n = 56 per group). (H) The effect of 10 M ASM on cell viability
was estimated by MTT assay (n = 56 per group). (I and J) Representative images and quantification data of LC3 (I; bars, 20 m) and SA--gal staining
(J; bars, 100 m) in P5, P10, and P20 human fibroblasts. NH4Cl and H2O2 were used for positive control (n = 5 per group). Data are representative of three
(B, E, I, and J) or four (A, C, D, and FH) independent experiments. A, C, F, G, I, and J, one-way ANOVA, Tukeys post hoc test. B, D, and H, Students t test.
*, P < 0.05; **, P < 0.01; ***, P < 0.005. All error bars indicate SEM.
receptor system is involved in trafficking of ASM to the lysosome (Dhami and Schuchman, 2004).To elucidate which ASM
form affected lysosomal/autophagic dysfunction, cells were
incubated with ASM alone, or in the presence of M6P. As expected, the activity of ASM was significantly increased in
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ASM-treated cells compared with nontreated cells. This enhanced ASM activity was reduced in the presence of M6P
(Fig. 6 D).To confirm whether the ASM treatment reached the
lysosomes, we also examined the colocalization of ASM and lysosomes using immunocytochemistry. Double immunostaining
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Figure 7. ASM causes abnormal autophagic protein degradation by altering ALP. (A) Autophagic flux assay. Human fibroblasts were cultured in:
(1) complete medium with or without 10 M ASM in the presence or absence of NH4Cl (left), (2) complete medium or starvation condition in the presence
or absence of NH4Cl (middle), or (3) complete medium or starvation condition with or without 10 M ASM (right). The LC3-II levels were examined by Western blotting (n = 67 per group). (B) The accumulation of p62 was assessed in the human fibroblast cultured with 10 M ASM, 20 mM NH4Cl, or starvation
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of ASM and lysosomal-associated membrane protein 1 (Lamp1)

showed that most ASM-positive vesicles were colocalized
with Lamp1-positive vesicles, indicating that treated ASM was
located in lysosome (Fig. 6 E).The ASM-induced autophagosome accumulation was significantly decreased by inhibition
of lysosomal ASM uptake using M6P or M6P receptor siRNA
(Fig. 6, F and G). These results suggested that elevation of lysosomal ASM may lead to autophagic dysfunction in AD. ASM
treatment did not affect the cell survival (Fig. 6 H). The levels
of LC3 and cell senescence in the fibroblast also did not show
the differences with passage number, indicating that ASM
caused the abnormal autophagy (Fig. 6, I and J).
As discussed above, the accumulation of AVs in cells can
result from either autophagy induction or the blockade of autophagic degradation.To further distinguish between these possibilities, we performed an autophagic flux assay (Rubinsztein
et al., 2009) in the presence or absence of NH4Cl that blocks
autophagic degradation but does not affect autophagosome
formation. It was hypothesized that if ASM treatment enhanced autophagy induction, in the presence of NH4Cl
(which inhibits degradation) a considerable increase in LC3II would be expected due to the combined effects of blocking
degradation and enhancing induction. However, compared
with NH4Cl treatment alone, dual treatment of fibroblasts
with ASM and NH4Cl did not show any significant changes
of LC3-II (Fig. 7 A, left). In contrast, the addition of NH4Cl
(Fig. 7 A, middle) or ASM (Fig. 7 A, right) in serum starvation
culture resulted in a significant but similar increase of LC3-II
levels. Furthermore, we measured the autophagic flux by detecting the abundance of p62. The levels of p62 were markedly increased in the cells treated with ASM or NH4Cl
(Fig. 7 B). We also performed LC3 flux assay in human AD
fibroblasts and 9-mo-old WT, APP/PS1, and APP/PS1/
ASM+/ mice fibroblasts. Autophagic flux was measured by
assessing the changes of LC3-II in the presence and absence
of NH4Clmediated lysosomal inhibition. Under basal condition, human AD and APP/PS1 fibroblasts showed significantly increased LC3-II levels compared with normal cells.
NH4Clinduced lysosome inhibition led to marked increase
of LC3-II levels in the normal fibroblasts, but this increase
was significantly less in the AD cells (Fig. 7, C and D). APP/
PS1/ASM+/ fibroblast showed similar pattern in LC3-II
increase compared with normal cell (Fig. 7 D). Collectively,
these results indicated that enhanced lysosomal ASM in AD
caused a defect of autophagic degradation but not induction.
To further investigate the relationship of elevated ASM

and defective lysosomal/autophagic degradation, we evaluated alteration in lysosomal pH using the acidotropic dye
LysoTracker red. H2O2- and NH4Cl-treated cells were used
as positive and negative controls, respectively. Flow cytometry
and fluorescent microscopic analysis did not show any differences of lysosomal pH between ASM- and vehicle-treated
fibroblast (Fig. 7 E). Recently, the transcription factor EB
(TFEB) was identified as a master regulator of the autophagy
lysosome pathway (ALP) and lysosome biogenesis (Settembre
et al., 2011). Enhancement of TFEB function is able to stimulate ALP function and promote protein clearance. To examine whether ASM could affect the ALP and lysosome
biogenesis, we tested endogenous levels of TFEB. ASMtreated fibroblasts and neurons showed significantly decreased
TFEB levels (Fig. 7, F and G). Also, the levels of the lysosomal
structural protein Lamp1 were decreased in ASM-treated cells
(Fig. 7, FH). To further validate our observation, we investigated TFEB subcellular localization after ASM treatment.
Interestingly, ASM-treated cells showed a reduced TFEB
expression in the nuclear compartment (Fig. 7 I). Similarly,
the expression levels of TFEB target genes related to lysosome were significantly decreased in ASM-treated fibroblasts
(Fig. 7 J). Conversely, to determine whether autophagic degradation affected ASM, we evaluated ASM activity in fibroblasts after NH4Cl treatment. Blocking of autophagic degradation
via NH4Cl did not show any significant changes of ASM activity (Fig. 7 K). These results further suggested that lysosomal ASM acts not as an inducer but rather as an inhibitor
of autophagic protein degradation by reducing ALP function
and lysosome biogenesis.
To examine the in vivo effect of ASM activation on autophagic dysfunction, we introduced conditioned medium
(CM) from cultured ASM-overexpressing cells into C57BL/6
mice via intracerebral (i.c.) and intravenous (i.v.) injections.
ASM-CMtreated mice showed elevated ASM activity in the
brain and plasma (Fig. 8, A and B), as well as increased LC3-II
without changes of beclin-1 expression in the brains (Fig. 8,
C and D). Cathepsin D level was increased in the ASM-CM
(i.c.)treated mice compared with control mice, but actual
activity was not changed (Fig. 8, C and E). p62 also was increased in the ASM-CM (i.c.)treated mice compared with
control mice (Fig. 8 C). ASM-CM (i.c.)treated mice further
exhibited abnormal ALP function, indicative of decreased
TFEB and Lamp1 levels (Fig. 8 F). Although ASM-CM (i.v.)
treated mice showed slightly increased cathepsin D and p62
condition (n = 4 per group). (C) Western blot analysis of LC3-II levels in controls, PS1-FAD, and ApoE4 fibroblasts in the presence or absence of NH4Cl (n = 6
per group). (D) Western blot analysis for LC3-II levels in fibroblasts derived from WT, APP/PS1, and APP/PS1/ASM+/ mice in the presence or absence of
NH4Cl (n = 6 per group). (E) Effect of ASM on lysosomal pH. FACS and histological analysis of fibroblasts stained with LysoTracker red (n = 5 per group; bars,
20 m). H2O2- and NH4Cl-treated cells were used as positive and negative controls, respectively. (F and G) Western blot analyses for TFEB and Lamp1 in
human fibroblasts (F; n = 6 per group) and neurons (G; n = 6 per group) after treatment with ASM. (H) Immunocytochemistry of Lamp1 in control and
ASM-treated fibroblast (n = 5 per group; bars, 20 m). (I) Western blot analysis for nuclear localization of TFEB in ASM-treated cells (n = 5 per group).
(J) Quantitative real-time PCR analysis of TFEB-target gene expression in normal (n = 6) and ASM-treated (n = 10) fibroblasts. (K) ASM activity was
estimated in the fibroblast with or without NH4Cl (n = 5 per group). Data are representative of two (E, H, and I) or three (AD, F, G, J, and K) independent
experiments. A, B, and EG, one-way ANOVA, Tukeys post hoc test. C, D, and HK, Students t test. *, P < 0.05; **, P < 0.01. All error bars indicate SEM.
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Figure 8. ASM causes autophagic dysfunction in vivo by sequestrating ALP function. (A and B) ASM was estimated in the brain and blood plasma
of C57BL/6 mice after ASM-CM treatment into the hippocampus (A; i.c., n = 6 per group) or tail vein (B; i.v., n = 6 per group). (C and D) Western blot analy
ses for LC3, beclin-1, p62, and cathepsin D in the brains of C57BL/6 mice after ASM-CM treatment into the hippocampus (C; n = 56 per group) or tail
vein (D; n = 45 per group). (E) Cathepsin D activity in the brain extracts of C57BL/6 mice after ASM-CM treatment (n = 4 per group). (F and G) Protein
expression of TFEB and Lamp1 in the brains after ASM-CM treatment into the hippocampus (F; n = 56 per group) or tail vein (G; n = 5 per group).
(H) Protein expression of TFEB and Lamp1 in the brains of 9-mo-old WT, APP/PS1, ASM+/, and APP/PS1/ASM+/ mice (n = 67 per group). Data are representative of three independent experiments. AG, Students t test. H, one-way ANOVA, Tukeys post hoc test. *, P < 0.05. All error bars indicate SEM.
levels and decreased ALP function proteins, this did not reach
statistical significance (Fig. 8, D and G).The activity of cathepsin D was also not changed (Fig. 8 E). These relatively modest
effects of ASM-CM (i.v.) treatment on autophagy dysfunction
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might be due to presence of the bloodbrain barrier because

only a slight increase of ASM activity in the brain was achieved
by these treatments, and the activity of ASM did not reach
those of APP/PS1 mice.
Ar ticle
Figure 9. Pharmacological restoration of ASM to the normal range improves pathology in AD mice. (A) Protocol of AMI treatment in APP/PS1
mice. (B) ASM was estimated in the blood plasma (n = 1214 per group) and brain (n = 910 per group) of APP/PS1 mice after AMI treatment. (C) Sphingomyelin, ceramide, and AC were determined using UPLC based methods in the plasma (n = 9 per group) and brain (n = 8 per group). (D) Mice brain sections were stained with thioflavin S to detect A (bars, 200 m). The relative area occupied by A plaques were determined (n = 6 per group). (EG) A40
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To examine whether partial genetic inhibition of ASM

affected the ALP in APP/PS1 mice, we also analyzed TFEB
and Lamp1 levels in the brain samples derived from WT,
APP/PS1, ASM+/, and APP/PS1/ASM+/ mice. Compared
with WT, APP/PS1 mice showed significantly decreased TFEB
and Lamp1 expression, which were increased in APP/PS1/
ASM+/ mice (Fig. 8 H). Together, these findings show for
the first time a direct correlation of lysosomal ASM and the
function of the ALP, and suggest that abnormal autophagic
degradation in AD may be due to the effects of elevated ASM
expression on this pathway.
Pharmacological restoration of ASM to the
normal range improves pathology in AD mice
The ASM-mediated lysosomal/autophagic dysfunction in AD
prompted us to examine possible therapeutic implications of
this pathway.To decrease ASM in APP/PS1 mice, we undertook
pharmacological inhibition using amitriptyline-hydrochloride
(AMI) for 4 mo (Fig. 9 A). AMI is a known inhibitor of ASM
that can cross the bloodbrain barrier. At 9 mo of age, AMItreated APP/PS1 mice exhibited decreased ASM activity
compared with vehicle-treated mice (Fig. 9 B). Other sphingolipid metabolites were not changed (Fig. 9 C). A levels were
decreased in the AMI-treated APP/PS1 mice compared with
the nontreated littermates (Fig. 9, DG).The levels of LC3-II,
p62, and cathepsin D were decreased in the AMI-treated APP/
PS1 mice (Fig. 9, H and I). Actual activity of cathepsin D was
not changed by AMI treatment (Fig. 9 J). AMI treatment significantly increased TFEB and Lamp1 protein levels in APP/
PS1 mice (Fig. 9, H and I). Similarly, APP/PS1 mice treated
with AMI showed recovery of memory function (Fig. 9, KP).
Overall, these positive but relatively moderate results (e.g., A
levels) in AMI-treated APP/PS1 mice might be due to under
dosing of the animals.We speculate that this may be improved
in the future by adjusting the dose or using modified, more
potent drugs of a similar class.
Restoration of ASM ameliorates autophagic
dysfunction in the AD patientspecific cells
To further validate our observation made by partial ASM inhibition in AD mice, we studied possible changes in autophagy
dysfunction in human AD fibroblast after ASM inhibition.
Elevated ASM levels in human AD fibroblasts (PS1familial
AD [FAD] and ApoE4) were restored to normal range by ASM
siRNA treatment (Fig. 10 A). ASM siRNA-treated human
AD fibroblasts (PS1-FAD and ApoE4) showed decreased
LC3-II and p62 accumulation compared with control siRNAtreated cells (Fig. 10 B). Also, ASM siRNA was able to increase
lysosome levels (as judged by Lamp1 expression) by activating
TFEB in the human AD fibroblasts (Fig. 10 C).
Many insights into the pathogenesis in neurodegenerative
disease have come from investigating postmortem brain tissues due to the difficulty of invasive access to living human
CNS. The recent developments in induced pluripotent stem
cells (iPSCs) and induced neurons have allowed investigation
of pathogenesis of neurological diseases in vitro (Kondo et al.,
2013). To explore whether the observed effects of ASM in
previous results are paralleled by similar alterations in AD human
neurons, we first established iPSCs with PS1 mutation (PS1
iPSC-2, -4, and -21) by transduction of human fibroblast with
retroviruses encoding OCT4, SOX2, KLF4, and c-Myc. The
PS1-iPSC cell line was shown to be fully reprogrammed
to pluripotency, as judged by colony morphology, alkaline
phosphatase (AP) staining, expression of pluripotency-associated
transcription factors and surface markers, karyotype stability,
and generation of teratomas (Fig. 10, DG). To establish
whether the PS1 mutation may affect neuronal differentiation, PS1 iPSC and control iPSC lines were induced to differentiate into neurons for 10 d. Consistent with previous
results (Kondo et al., 2013), no obvious differences in the ability to generate neurons were observed between control and
PS1-iPSCs (Fig. 10 H). A42 secretion level was increased in
PS1 iPSC-derived neurons compared with control iPSCderived neuron (Fig. 10 I).
Next, we investigated whether elevated ASM in fibroblasts
was also evident increased in PS1 iPSC and iPSC-derived
neurons. The ASM activity in PS1 iPSC was not changed except for PS1-4 iPSC in comparison to those in control iPSC,
but the activity of ASM was significantly higher in PS1 iPSCderived neurons compared with control iPSC-derived neuron (Fig. 10 J). Elevated ASM levels in PS1 iPSC-derived
neurons were restored to normal range by ASM siRNA treatment (Fig. 10 J). Neurons from PS1-4 iPSCs also had significantly higher abnormal autophagic markers than neurons
from control iPSC (Fig. 10 K). ASM siRNA treatment signifi
cantly decreased the protein level of abnormal autophagic
markers in PS1 iPSC-derived neurons (Fig. 10 K).To corroborate the immunoblotting results, we performed EM analysis
using control and PS1 iPSC-derived neurons. As expected,
PS1 iPSC-derived neurons exhibited increased AV accumulation, whereas ASM siRNA-treated PS1 iPSC-derived neurons showed a reduced number of these vesicles (Fig. 10 L).
and A42 in the brains of AMI treated or nontreated APP/PS1 mice were assessed using immunofluorescence staining (E and F; n = 8 per group; bars,
200 m) and ELISA kits (G; n = 6 per group). (H and I) Western blot analyses and quantification for LC3, Beclin-1, p62, cathepsin D, TFEB, and Lamp1 in
the brains of APP/PS1 mice treated with AMI or control (n = 68 per group). (J) Cathepsin D activity in the brain extracts of AMI-treated or nontreated
APP/PS1 mice (n = 4 per group). (K) Escape latencies of APP/PS1 mice treated with AMI or control over 10 d (WT, n = 14; nontreated APP/PS1, n = 10; and
AMI-treated APP/PS1, n = 12). (LO) Probe trial day 11. (L and M) Path length (L) and swim speed (M) were recorded and analyzed. (N) Time spent in target
platform and other quadrants was measured. (O) The number of times each animal entered the small target zone during the 60-s probe trial. (P) Representative swimming paths at day 10 of training. Data are representative three independent experiments. BJ and N, Students t test; KM and O, one-way
ANOVA, Tukeys post hoc test. *, P < 0.05; **, P < 0.01. All error bars indicate SEM.
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Figure 10. Restoration of ASM to the normal level reverses impaired autophagy in the AD patient-specific cells. (A) SMPD1 gene suppression
by ASM-siRNA in human fibroblasts. ASM activity was assessed after ASM siRNA treatment in the control and AD fibroblast (n = 6 per group). (B) LC3-II
and p62 levels were examined in human AD fibroblast with or without ASM inhibition. siRNA-mediated suppression of ASM reduced LC3-II and p62 levels
in PS1-FAD (left; n = 7 per group) and ApoE4 fibroblast (right; n = 6 per group). (C) Protein expression of TFEB and Lamp1 in the PS1-FAD and ApoE4
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Decreased TFEB target genes in PS1 iPSC-derived neurons

were also significantly increased by ASM siRNA treatment
(Fig. 10 M). These results confirm that abnormal autophagy
observed in AD mice and human fibroblasts by ASM also
occur in AD patient neurons, and restoration of ASM back to
normal levels is able to ameliorate autophagic dysfunction by
restoring lysosomal biogenesis in AD patient cells.
DISCUSSION
Although the exact causes of AD are unknown, the complex
interactions of genetic and environmental factors are likely play
important roles in the pathogenesis. ASM activity is known to
be increased by environmental stress and in various diseases,
and is elevated in AD patients (He and Schuchman, 2012).
One downstream consequence of increased ASM is elevated
ceramide, contributing to cell death, inflammation, and other
common disease findings. Although elevated ASM is known
to occur in AD, the cellular mechanisms that link ASM and
AD have not been fully characterized. The data presented
here suggest a previously unknown role of ASM in the downregulation of lysosomal biogenesis and inhibition of lysosomedependent autophagic proteolysis. The findings also establish
proof of concept for ASM inhibitor therapy in AD.
Our previous study showed that sphingolipid metabolism
was severely impaired in the human AD brain, and that ASM
activity was positively correlated with the A levels (He et al.,
2010). Consistent with our previous study, we found that ASM
was significantly increased in fibroblasts, brain, and/or plasma
from patients with AD and in AD mice, although other sphingolipid factors were unaltered.There are some differences between the previous and this study. For example, the previous
results showed increased ceramide level in AD, but we could
not found significant changes of sphingolipid factors including ceramide in AD compared with normal samples. These
differences might be related to the fact that once formed, ceramide can rapidly enter several metabolic pathways. It may
be used for either the biosynthesis of complex lipids or broken down into sphingosine, which itself is rapidly converted
to sphingosine 1 phosphate.
Accumulation of abnormal AVs has been observed in AD
(Lee et al., 2010b; Nixon and Yang, 2011), and the autophagy
pathway is increasingly regarded as an important contributor
to A-mediated pathogenesis in AD (Yu et al., 2005; Nixon,
2007). Moreover, it has been shown that the abnormal autophagic flux in AD may be due to dysfunction at the late autophagy stage associated with the lysosome (Lai and McLaurin,
2012; Zhou et al., 2012). Similar to previous results (Lee et al.,
2010b; Lai and McLaurin, 2012), we found that the impaired
autophagic flux in AD was associated with reduced autophagic degradation due to decreased ALP function. In addition,
we show for the first time that this is directly linked to elevated ASM activity. Suppression of ASM expression or inhibition of its uptake and delivery to lysosomes using M6P reversed
abnormal autophagic degradation. These findings indicate
that increased lysosomal ASM plays a negative role in AD by
causing autophagic dysfunction, suggesting that therapeutic
strategies to restoring ASM activity to the normal range may
be beneficial for AD pathology.
This was further studied in the AD mice by finding that
partial genetic or systemic inhibition of ASM activities in these
animals largely reversed autophagic pathology by restoring
ALP function, as well as reducing the accumulation of incompletely digested substrates within the autophagic-lysosomal
compartments (e.g., LC3-II and p62). A accumulation also
was reduced in response to ASM inhibition, as was cathepsin D
expression. There are several challenges associated with interpretation of cathepsin D levels in AD. Although some reports
have shown that cathepsin D activities were decreased in AD
(Lee et al., 2010b), many studies indicate that cathepsin D is
elevated in AD and contributes to the pathogenesis, such as A
formation (Cataldo et al., 2004; Lai and McLaurin, 2012; Zhou
et al., 2012). A recent paper suggested that COP9 signalosome
deficiency increased cathepsin D levels but reduced the autophagic degradation. They suggested that these results were
associated with a failure of lysosomal assembly of cathepsin D
because only a lysosomal cathepsin D could affect autophagic
degradation (Su et al., 2011). In this study, we have found that
maturation of cathepsin D was increased in AD mice, but the
actual enzyme activity was not changed between the groups.
This result indicated that the elevated levels of cathepsin D did
not ultimately translate into a significant increase of enzyme
activity. Based on these papers and our data, a plausible interpretation of increased cathepsin D in our AD mice is that AD
microenvironment attempts to increase cathepsin D synthesis,
but this does not have a direct impact on lysosomal function
because the activity of the enzyme is unchanged. Therefore,
fibroblast after ASM inhibition (n = 56 per group). (DG) Generation of PS1 iPSC lines from patient fibroblast. (D) Established iPSCs showed embryonic
stem celllike morphology (Phase; bar, 1 mm), AP activity (bar, 200 m), and expressed pluripotent stem cell markers SSEA4 (bar 100 m), TRA1-60 (bar
100 m), and TRA1-81 (bar 100 m). (E) Normal karyotype of PS1 iPSC. (F) Quantitative real-time PCR analysis of hESC marker gene of PS1 iPSC (n = 3 per
group). (G) Gross morphology and hematoxylin-eosin staining of representative teratomas generated from PS1-4 iPSCs (bars, 50 m). (H) Estimation of
neural differentiation from control and PS1-4 iPSCs. Representative images of immunocytochemical staining the -III tubulin after neural differentiation
(bars, 50 m). (I) The amount of A42 secreted from control iPSC-derived neuron and PS1 iPSC-derived neuron (n = 5 per group). (J) Characterization of
ASM activity in the control and PS1 iPSC and iPSC-derived neurons (n = 6 per group). (K) Western blot analyses for LC3, beclin-1, p62, TFEB, and Lamp1 in
the control and PS1-4 iPSCderived neuron after ASM siRNA treatment (n = 56 per group). (L) EM images and quantification data of control and PS1
iPSC-derived neurons. Higher magnification of boxed area shows detail of AVs (arrow; n = 4 per group; bars: [low magnification] 1 m, [high magnification] 500 nm). (M) Quantitative real-time PCR analysis of TFEB-target gene expression in iPSC-derived neurons after ASM siRNA treatment (n = 56 per
group). Data are representative of two (A, DG, I, and L), or three (B, C, H, J, K, and M) independent experiments. A, C, F, I, J, and M, Students t test. B, K,
and L, one-way ANOVA, Tukeys post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. AK, error bars indicate SEM. L and M, Error bars indicate SD.
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enhanced cathepsin D level in our AD mice induced by increased ASM is more likely a compensatory response to an
impaired lysosome system. The present study also provides the
first evidence of increased ASM activity and autophagic dysfunction in living human (iPSC-derived) neurons derived
from AD patients and that restoring normal levels of ASM in
AD neurons effectively blocks abnormal autophagy.
Overall, the data presented here show that increased ASM
activity in AD contributes to the abnormal lysosomal/autophagic process by leading to dysfunction of ALP.This results
in an inability to break down appropriate substrates during
the autophagy process. Restoration of ASM effectively blocks
AD progression by increasing autophagic degradation. Although the involvement of other ASM-related mechanisms in
AD remains to be explored, the data in this study demonstrate
that inhibition of ASM improves A clearance and rescues
impaired memory in a validated mouse model of AD, suggesting this as a potential therapy for AD patients in the future.
Mice. Transgenic mouse lines overexpressing the hAPP695swe (APP) and
presenilin-1M146V (PS1) mutations, respectively, were generated at Glaxo
SmithKline by standard techniques as previously described (Howlett et al.,
2004). In brief, a Thy-1APP transgene was generated by inserting the 695 aa
isoform of human cDNA (APP695) harboring the Swedish double familial
mutation (K670N; M671L) into a vector containing the murine Thy-1 gene.
The Thy-1PS-1 transgene was generated by inserting the coding sequence
of human PS-1 cDNA harboring the M146V familial mutation into a vector
containing the murine Thy-1 gene. Transgenic lines were generated by pronuclear microinjection into fertilized oocytes from either C57BL/6xC3H
mice in the case of Thy-1-APP transgene, or into fertilized oocytes from pure
C57BL/6 mice in the case of Thy-1PS-1 transgene.Thy-1 APPswe mice were
generated and backcrossed onto a pure C57BL/6 background before crossing
with TPM (PS-1 M146V) mice to produce heterozygote double mutant mice.
ASM+/ mice (C57BL/6 background; Horinouchi et al., 1995) were bred
with APP/PS1 mice to generate APP/PS1/ASM+/ mice. Because APP/PS1
mice show sex difference in disease progression, we used only male mice. Data
analysis of APP/PS1 mice was done at 5 or 9 mo old. Block randomization
method was used to allocate the animals to experimental groups. To eliminate
the bias, we were blinded in experimental progress such as data collection and
data analysis. SCID Beige mice (Charles River) were used for teratoma formation assay. Mice were housed at a 12 h day/12 h night cycle with free access to
tap water and food pellets. Mouse studies were approved by the Kyungpook
National University Institutional Animal Care and Use Committee (IACUC).
Plasma collection. Human plasma samples were obtained from individuals
with AD, PD, and age-matched, non-AD controls from Yonsei University
Severance Hospital (Table S1). Informed consent was obtained from all subjects according to the ethics committee guidelines at the Yonsei University
Severance Hospital.
Cell culture. Human fibroblast lines (normal, PS1, ApoE4, and PD) acquired from the Coriell Institute were maintained in DMEM with 15% FBS.
The human cortical neuronal cell line HCN-2 was acquired from ATCC.
Cells with a passage number 1015 were used in this study. To obtain CM
containing ASM, 5 105 Chinese hamster ovary cells overexpressing human
ASM (He et al., 1999) were cultured in DMEM for 2 d. Cells were washed
with PBS and changed with new media. 24 h later, the CM was collected,
centrifuged, and filtered using a 0.22 m filter. We isolated WT, APP/PS1,
APP/PS1/ASM+/, and ASM+/ mouse tail fibroblasts as previously described (Takahashi et al., 2007a) from 9-mo-old mice. For some experiments,
JEM Vol. 211, No. 8
cells were treated with purified, recombinant ASM or ASM siRNA to measure autophagy regulation. NH4Cl was used to inhibit the autophagic flux.
For the inhibition of lysosomal ASM uptake, 10 mM M6P or M6P receptor
siRNA were added to the fibroblast culture media at the same time as ASM.
Drug or CM treatments. 4-mo-old APP/PS1 mice received 100 g/g body
weight AMI (Sigma-Aldrich) per os in their drinking water for 4 mo, and a
control group received water without drug. 3-mo-old C57BL/6 mice were treated
with ASM-CM via i.v. (100 l) or i.c. (3 l) injections on 10 consecutive days.
Immunofluorescence. Thioflavin S staining was done according to previously described procedures (Lee et al., 2012). We used anti-20G10 (mouse,
1:1,000, provided by D.R. Howlett, GlaxoSmithKline, Harlow, Essex, UK)
for A 42, anti-G30 (rabbit, 1:1,000, provided by D.R. Howlett) for A40,
rabbit antiIba-1 (1:500; Wako), rabbit anti-GFAP (1:500, Dako), mouse
anti-SMA (1:400; Sigma-Aldrich), rabbit anti-AT8 (1:500; Thermo Fisher
Scientific), and rabbit antiactive caspase3 (1:50; EMD Millipore). The sections were analyzed with a laser-scanning confocal microscope (FV1000;
Olympus) or with a BX51 microscope (Olympus). MetaMorph software
(Molecular Devices) was used to quantification.
Ab ELISA. For measurement of A40 and A42, we used commercially
available ELISA kits (BioSource). Hemispheres of mice were homogenized
in buffer containing 0.02M guanidine. ELISA was then performed for A40
and A42 according to the manufacturers instructions.
Behavioral studies. We performed behavioral studies to assess spatial learning and memory in the Morris water maze as previously described (Lee et al.,
2012). Animals were given four trials per day for 10 d to learn the task. At 11 d,
animals were given a probe trial in which the platform was removed. Fear
conditioning was conducted as previously described techniques (Kojima et al.,
2005). On the conditioning day, mice were individually placed into the conditioning chamber. After a 60-s exploratory period, a tone (10 kHz, 70 dB)
was delivered for 10 s; this served as the conditioned stimulus (CS).The CS coterminated with the unconditioned stimulus (US), a scrambled electrical footshock (0.3 mA, 1 s).The CS-US pairing was delivered twice at a 20-s intertrial
interval. On day 2, each mouse was placed in the fear-conditioning chamber
containing the same exact context, but with no administration of a CS or foot
shock. Freezing was analyzed for 5 min. On day 3, a mouse was placed in a test
chamber that was different from the conditioning chamber. After a 60-s exploratory period, the tone was presented for 60 s without the footshock. The
rate of freezing response of mice was used to measure the fear memory.
Quantitative real-time PCR. RNA was extracted from the brain homogenates and cell lysates using the RNeasy Lipid Tissue Mini kit and RNeasy
Plus Mini kit (QIAGEN) according to the manufacturers instructions.
cDNA was synthesized from 5 g of total RNA using a commercially available kit (Takara Bio Inc.). Quantitative real-time PCR was performed using
a Corbett research RG-6000 real-time PCR instrument. Used primers are
described in Table S2.
EM. Brain tissues and cells were fixed in 3% glutaraldehyde/0.1 M phosphate
buffer, pH 7.4, and postfixed in 1% osmium tetroxide in Sorensens phosphate
buffer. After dehydration in ethyl alcohol, the tissue and cells were embedded
in epon (Electron Microscopy Sciences). Samples were cut serially and placed
on copper grids and analyzed using a transmission EM (Tecnai). Images were
captured on a digital camera and Xplore3D tomography software.
Intracellular protein degradation measurement. Total protein degradation in cultured cells was measured by pulse-chase experiments with 48 h
pulse with 2 Ci/ml [3H]-leucine for 48 h to preferentially label long-lived
proteins (Lee et al., 2010b).
Western blotting. Samples were immunoblotted as previously described
(Settembre et al., 2011; Lee et al., 2012). Primary antibodies to the following
1567
proteins were used: BACE-1 (mouse, 1:1,000; Millipore), LC3 (rabbit,

1:1,000; Cell Signaling Technology), Beclin-1 (rabbit, 1:1,000; Cell Signaling
Technology), p62 (rabbit, 1:1,000; Cell Signaling Technology), rab5 (rabbit,
1:1,000; Cell Signaling Technology), rab7 (rabbit, 1:1,000; Cell Signaling
Technology),TFEB (rabbit, 1:1,000; Cell Signaling Technology), Lamp1 (rabbit, 1:1,000; Abcam), TFEB (rabbit, 1:500; Novus Biologicals), cathepsin
D (goat, 1:500; R&D Systems), 6E10 (mouse, 1:500; Signet), Histon H3 (rabbit, 1:1,000; Cell Signaling Technology), and -actin (1:1,000; Santa Cruz
Biotechnology, Inc.). We performed densitometric quantification using the
ImageJ software (National Institutes of Health).
ASM and AC activity assays. We performed the measurements as previously described methods using a UPLC system (Waters; He et al., 2010).
Lipid extraction and ceramide/sphingomyelin quantification. We
prepared samples for lipid extraction as previously described (Lee et al.,
2010a). To quantify the sphingomyelin and ceramide levels, the dried lipid
extract was resuspended in 0.2% Igepal CA-630 (Sigma-Aldrich) and the
levels of each lipid were determined using the UPLC system.
Isolation of neuron and microglia. We isolated neuron and microglia
from the mouse brain as previously described (Brewer and Torricelli, 2007).
In brief, 9-mo-old mice cerebrum were minced in HibernateA (Brainbits
LLC)/B27 (Invitrogen) medium and dissociated using papain solution. After
tissue trituration, cells were separated by density gradient centrifugation.
Fractionated cells were used for ASM activity assay.
LysoTracker labeling and quantification. LysoTracker red (Invitrogen) was
used at a final concentration of 75 nM.The cells were trypsinized, resuspended
in PBS, and analyzed on a FACSCalibur using FACSDiva software (BD).
Cell viability assays. Cell viability was quantified by using MTT (3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; Sigma-Aldrich) reagent. In brief, human fibroblast was seeded onto 96-well plates at a density of
1.5 103 cells per well. 10 M ASM was added to the culture media, and the
cells were incubated for 24 h. MTT stock solution (5 mg/ml) was prepared in
PBS (Gibco) and added to the culture media at a final concentration of 1 mg/ml.
After 90 min incubation, the media was removed, and the chromogen in the
cells was dissolved in DMSO containing 0.01 N NaOH. The absorbance was
measured at 570 nm using a 96-well microplate spectrophotometer.
Measurement of activity of cathepsin D. Enzyme activity of cathepsin
D was determined with cathepsin D activity fluorometric assay kit according
to the manufacturers protocol (Abcam).
Generation of iPSCs. PS1-iPSCs were established from the PS1 patients
skin fibroblasts (Coriell Institute) as previously described (Okita et al., 2007),
with slight modifications. In brief, PS1 fibroblasts were seeded at 3 105 cells
in 60 mm2 dishes coated with gelatin. On day 1, the VSV-G pseudotyped retroviral vector system carrying OCT4, SOX2, KLF4, and c-Myc was added to
fibroblast cultures. On day 2, cells were subjected to the same transduction
procedures and harvested 24 h later.Transduced cells were replated on mouse
embryonic fibroblast (MEF) layers in 100 mm2 dishes containing the fibroblast medium. On the next day, the medium was changed to complete ES
medium with 0.5 mM valproic acid (Sigma-Aldrich), and thereafter changed
every other day. After 20 d, ES-like colonies appeared and were picked up to
be reseeded on new MEF feeder cells. Cloned ES-like colonies were subjected to further analysis. Normal iPSC line (HPS0063) was obtained from
the RIKEN Bioresource Center (Takahashi et al., 2007b).
In vitro differentiation of human iPSCs. Neural differentiation of iPSCs
was performed as described previously (Okada et al., 2008). iPSC colonies
were detached from feeder layers and cultured in suspension as embryonic
body for 30 d in bacteriological dishes. Embryonic bodies were then enzymatically dissociated into single cells and the dissociated cells cultured in suspension
1568
in serum-free media hormone mix media (Okada et al., 2008) for 1014 d to
allow the formation of neurospheres. Neurospheres were passaged repeatedly
by dissociation into single cells followed by culture in the same manner.Typically, neurospheres between passages 3 and 8 were used for analysis. For
terminal differentiation, dissociated neurospheres were allowed to adhere to
poly-l-ornithine and laminin-coated coverslips and cultured for 10 d.
AP, senescence-associated--galactosidase (SA--gal), and immunocytochemical staining. AP staining was performed using an ES-AP detection kit (EMD Millipore) according to manufacturers recommendations.
SA--gal activity was detected using SA--gal staining kit (Cell Signaling
Technology) according to manufacturers protocol. For immunocytochemical
analysis, we used anti-SSEA4, TRA-1-60, TRA-1-81 (mouse, 1:100; EMD
Millipore), anti-III-tubulin (mouse, 1:400; EMD Millipore), rabbit antiLC3B (1:200; Cell Signaling Technology), rabbit anti-ASM (1:1,000, Abcam),
mouse anti-LAMP1 (1:100; Abcam), and mouse anti-LBPA (1:500; Echelon).
Teratoma formation and histological analysis. Established iPSCs were
prepared at 107 cells/ml in PBS. Suspended cells (13 106) were injected
into testes of anesthetized male SCID Beige mice. 8 wk after transplantation,
mice were sacrificed and tumors were dissected.Tumor samples were fixed in
10% formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin.
Statistical analysis. Comparisons between two groups were performed
with Students t test. In cases where more than two groups were compared
with each other, a one-way analysis of variance (ANOVA) was used, followed
by Tukeys HSD test. All statistical analysis was performed using SPSS statistical software. P < 0.05 was considered to be significant.
Online supplemental material. Table S1 shows subjects characteristics.
Table S2 shows sequences of primer pairs. Online supplemental material is
available at http://www.jem.org/cgi/content/full/jem.20132451/DC1.
This work was supported by the Bio & Medical Technology Development Program
(2010-0020234, 2011-0019356, 2012M3A9C6049913, and 2012M3A9C6050107) of
the National Research Foundation (NRF) of Korea funded by the Ministry of Science,
ICT & Future Planning, Republic of Korea.
The authors declare no competing financial interests.
Author contributions: J.K. Lee, H.K. Jin, M.H. Park, B.R. Kim, P.H. Lee, H. Nakauchi,
J.E. Carter, and X. He performed experiments and analyzed data, J.K. Lee, H.K. Jin,
and J.S. Bae designed the study and wrote the paper. E.H. Schuchman and J.S. Bae
interpreted the data and reviewed the paper. All authors discussed results and
commented on the manuscript.
Submitted: 26 November 2013
Accepted: 20 June 2014
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MICROMANIPULATION
The most powerful aspect of the Multi-Link Position Control
software is the ability to link and coordinate movement
of multiple devices that can be connected to our MPC-200
controller. You can combine manipulators, translators,
microscopes (MOM or SOM) and stages to give you complete
control over your complex experiments. Bring all devices to
memorized positions or bring pipettes to a working location in
seconds. Available as a free download on our web site.
MICROPIPETTE FABRICATION
Sutter Instrument, the recognized leader in micropipette
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the P-1000 micropipette puller with an intuitive, full-featured
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OPTICAL PRODUCTS
Our latest products include the Lambda HPX, a new
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moving microscope optimized to allow in vivo and in vitro
experimentation in one set-up. As with our two-photon
MOM microscope, the SOM can robotically position the
objective over the sample and focus, opening up experimental
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MICROINJECTION
The XenoWorks microinjection system has been designed
to meet the needs of a wide variety of applications that require
the manipulation of cells and embryonic tissues including ICSI,
ES Cell Microinjection, and Adherent Cell Microinjection.
Highly-responsive movement and excellent ergonomics
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yield saving time and resources.
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