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Something wicked this way comes: huntingtin

Albert R La Spada

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2014 Nature America, Inc. All rights reserved.

Does cell-to-cell spreading of misfolded proteins occur in all neurodegenerative disorders? A study in this issue of
Nature Neuroscience now demonstrates propagation of mutant huntingtin in brain slice cultures and in vivo, thereby
extending the process of cell-to-cell propagation of misfolded proteins to Huntingtons disease.
One goal of a criminal justice system is to
imprison malevolent offenders so that they
can no longer gain access to law-abiding citizens. An important question in the neurodegenerative disease field is whether a misfolded
protein produced in one neuron can leave
that cell to enter an unaffected neuron, with
recent work suggesting that bad actor misfolded proteins are not confined to the cells
in which they originate, but can instead move
to other cells, like unrestrained criminal
agents of chaos and destruction. A study by
Pecho-Vrieseling et al. in this months issue
of Nature Neuroscience provides convincing
in vitro data, as well as the first definitive in vivo
evidence, for spreading of misfolded huntingtin
protein between neurons1, and thus supports
an emerging model of cell-to-cell propagation
as an essential element of disease progression
in all neurodegenerative disorders.
Production of misfolded proteins is a defining feature of all neurodegenerative disorders.
This paradigm-shifting realization about
20 years ago marked a turning point in our
understanding of neurodegeneration, and
the last two decades of research have further
reinforced the view that all neurodegenerative
disorders result from the accumulation of misfolded proteins that form aggregates, detectable as inclusion bodies at the light microscope
level. This realization linked prion diseases,
whose agents are proteins that confer their own
misfolding on normal conformers through
a process of templated change2, to the entire
spectrum of neurodegenerative disorders,
Albert R. La Spada is in the Departments of
Cellular & Molecular Medicine, Neurosciences and
Pediatrics, Division of Biological Sciences, Institute
for Genomic Medicine, and Sanford Consortium
for Regenerative Medicine, University of California,
San Diego, La Jolla, California, USA, and at Rady
Childrens Hospital, San Diego, California, USA.
e-mail: alaspada@ucsd.edu

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including Alzheimers disease, Parkinsons disease, Huntingtons disease, amyotrophic lateral

sclerosis and tauopathies. However, in the last
decade, compelling research has also suggested
that, besides accumulation of misfolded proteins, another common feature of neurodegeneration is the propensity of misfolded proteins
to spread from one cell to another, and that
this propagation is a key feature of disease
pathogenesis (reviewed in ref. 3). Although
mounting in vivo evidence for prion-like
propagation has emerged for Alzheimers and
Parkinsons diseases4,5, the issue of whether
this propagation phenomenon might apply to
Huntingtons disease and other polyglutamine
repeat diseases had remained unresolved.
One compelling aspect of the new study from
Pecho-Vrieseling et al.1 is that they took three
different, progressively more sophisticated
approaches to examine cell-to-cell spreading of mutant huntingtin (mHtt) protein. The
initial system was based on the introduction
of human embryonic stem cells (hESCs) into
organotypic brain slice cultures obtained from
either wild-type mice or Huntingtons disease
model R6/2 mice. In the slice culture milieu,
hESCs readily differentiate into neurons and
form functional connections with existing
neurons, as the authors nicely demonstrate
through combined optogenetic and pharmacological experimentation. Having established
this powerful system, they assessed hESCderived neurons located in both the striatal and
cortical regions of slice cultures and found that
they developed huntingtin aggregate pathology, initially in the cytosol and subsequently
in the nucleus, but only when placed in the
R6/2 context. Elaboration of aggregate pathology in hESC-derived neurons was not innocuous: such neurons exhibited signs of toxicity,
including reductions in neurite number and
neurite length. Notably, neurite degeneration
phenotypes were accentuated when hESCderived neurons were situated in R6/2 slices

containing huntingtin aggregates in comparison with R6/2 slices lacking aggregates.

After completing careful studies with
the hESC neuronslice culture system,
Pecho-Vrieseling et al.1 pursued a more representative brain slice system to test the hypo
thesis of huntingtin propagation by dissecting
out cortex or striatum from wild-type mice or
R6/2 mice and then placing the cortical region
of one mouse adjacent to the striatal region
of a different mouse to create mixed cortico
striatal brain cultures1. Of the various possible
permutations, the authors chose to carefully
study R6/2 cortex and wild-type striatum
versus wild-type cortex and wild-type striatum, as they could only document functional
corticostriatal circuit connections when mixed
corticostriatal brain slices employed wild-type
striatum. Here again, mHtt aggregates materialized in wild-type striatal neurons only when
placed adjacent to R6/2 cortex. Consequently,
these two different brain slice culture systems
revealed the capacity for mHtt to spread from
one neuron to another, naive neuron, and corroborated earlier in vitro studies in which mHtt
placed in the culture medium or expressed in
one cell in culture was found to gain access to
normal cells not expressing mHtt6,7.
However, demonstrating the capacity of
mHtt to move from a diseased cell to a normal cell, even when employing sophisticated
organotypic slice culture approaches, may
not faithfully represent what is happening in corticostriatal circuits in vivo. Thus,
Pecho-Vrieseling et al.1 ultimately performed the key experiment necessary for
testing mHtt propagation by co-injecting,
into the cortex of wild-type mice, a lenti
virus with an expression construct carrying
polyglutamine-expanded (Q72) huntingtin
exon 1 and an adeno-associated virus containing a synaptophysin-GFP fusion vector
(to mark transduced neurons). They then
documented the presence of huntingtin

volume 17 | number 8 | august 2014 nature neuroscience

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a

Blood

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2014 Nature America, Inc. All rights reserved.

vessels

Gut

Muscle

Figure 1 Potential cellular pathways for misfolded protein propagation. (a) Adjacent spread. Neurons in
functional contact with one another, as shown here, or in direct physical contact via intercellular bridges
(for example, tunneling nanotubes) may be subject to cell-to-cell transmission of misfolded proteins.
(b) Paracrine propagation. Neurons (or other cell types) may expel misfolded proteins in vesicles from the
autophagy or endosome pathways, as shown here, or may release misfolded proteins extracellularly by
exocytosis. Once proteins are released in this way, neurons in the vicinity may take up the misfolded proteins
by fusion with membrane-bound structures or simply by endocytosis of diffusible extracellular protein.
(c) Distant dissemination (prion-like). Certain misfolded proteins and peptides (for example, prions,
amyloid-42, -synuclein) may travel great distances, perhaps originating in non-neural peripheral tissues such
as the gut, to enter the CNS by crossing the blood-brain barrier and then moving into neurons and other cells.

aggregates in the majority of striatal medium

spiny neurons located in regions innervated
by virally transduced cortical projection neurons that synapse with the striatal medium
spiny neurons, thereby providing persuasive
evidence that misfolded huntingtin conformers can propagate in the mammalian
brain in a transneuronal fashion1, which is
consistent with recent work demonstrating
the interaction of the cortex and striatum in
Huntingtons disease pathogenesis8.
Although this latest advance further underscores the existence of common pathogenic
processes at work in neurodegeneration, it is
important to not overemphasize the commonality of the propagation phenomenon, as the
underlying mechanisms driving cell-to-cell
spreading still defy explanation and may vary
substantially in different neurodegenerative
diseases. Indeed, another interesting aspect of
the work by Pecho-Vrieseling et al.1 was their
attempt to define the basis for mHtt propagation in their hESC neuronR6/2 slice culture
system by treating their cultures with two
different botulinum toxin strains that cleave
distinct components of the synaptic vesicle
docking fusion complex to prevent the release
of synaptic vesicles. Either strain of botulinum

toxin markedly blunted the accumulation of

huntingtin aggregates in hESC-derived neurons cultured in R6/2 slices, but each treatment was effective only if applied before the
first wave of aggregation, which produced
cytosolic aggregates. Botulinum toxin treatment after detection of cytosolic huntingtin
aggregates had no appreciable effect on the
extent of huntingtin aggregate accumulation
in cocultured hESC neurons.
Although the necessity of synaptic vesicle
release for huntingtin protein propagation
requires validation with genetic knockdown
approaches, given the many possible off-target
effects of botulinum toxin, and also needs to
be extended to the in vivo setting, the implication of synaptic vesicle delivery as the basis for
mHtt propagation raises important questions,
as one can envisage at least three distinct pathways by which misfolded proteins may transit
from one cell to another: adjacent spread, paracrine propagation and distant dissemination.
Adjacent spread, as is reported here for huntingtin protein, is a process involving cells that
are directly connected to one another physically
or as part of a functional neural circuit (Fig.1a).
Thus, the transmission of misfolded protein
occurs by virtue of the cells physical and/or

nature neuroscience volume 17 | number 8 | august 2014

functional connection (for example, tunneling

nanotubes9 or synapses), as hypothesized for
the Huntingtons disease corticostriatal synaptic
junction in the present work. However, in a process of paracrine propagation, cell-to-cell propagation may also occur between neurons and
other cells that are not functionally or physically
connected, but rather simply nearby10, when a
misfolded protein is released from a diseased
cell (Fig. 1b). Finally, for the prion diseases,
and likely also in Alzheimers and Parkinsons
diseases, it appears that misfolded proteins can
traverse great distances, sometimes even from
the gut or elsewhere in the periphery3,4,10,11, to
finally end up at their ultimate neuronal destination in the CNS (Fig. 1c). This distant dissemination of misfolded proteins is a defining
feature of prion disease; hence, the term prionlike should be reserved for neurodegenerative
diseases that exhibit this capacity for distant dissemination. Indeed, if cell-to-cell propagation
of mHtt protein in Huntingtons disease occurs
only at synaptic junctions (adjacent spread),
and not via other pathways, then this feature
of Huntingtons disease pathogenesis and progression would have strong implications for
the molecular basis of the Huntingtons disease transmission process, which may involve
extracellular release and uptake, or delivery
in membrane-bound structures3. As it turns
out, however, analysis of fetal neural allografts
in post-mortem Huntingtons disease patient
brain has uncovered the presence of huntingtin aggregates in the allograft tissue, albeit only
in extracellular matrix12, which suggests an
extracellular release process consistent with
paracrine propagation. Clearly, future work
should focus on defining the underlying cellular pathways by which cell-to-cell spreading of
misfolded proteins occurs in different neuro
degenerative diseases. As multiple pathways for
cell-to-cell spreading may be imagined, defining how misfolded protein propagation occurs
in different disorders will have crucial implications for therapy development.
COMPETING FINANCIAL INTERESTS
The author declares no competing financial interests.
1. Pecho-Vrieseling et al. Nat. Neurosci. 17, 10641072
(2014).
2. Pan, K.M. et al. Proc. Natl. Acad. Sci. USA 90,
1096210966 (1993).
3. Garden, G.A. & La Spada, A.R. Neuron 73, 886901
(2012).
4. Eisele, Y.S. et al. Science 330, 980982 (2010).
5. Luk, K.C. et al. Science 338, 949953 (2012).
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doi:10.1002/ana.24174 (6 May 2014).

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