Reversing Mitochondrial Dysregulation in a Murine Model of HIV associated
Neurocognitive Disorder using a TrKB Agonist
Sanketh Andhavarapu, Udit Gupta, Naomi Baig
University of Maryland School of Medicine, Department of Neurology
Introduction
The introduction of combined antiretroviral therapy (cART) significantly
increased the lifespan of HIV patients, turning a fatal disease to a
chronic one. However, as a lower but persistent level of HIV infection
remains, the susceptibility of HIV-associated neurocognitive disorder
(HAND) is increased. HIV-associated neurocognitive disorder (HAND)
collectively describes the cognitive impairment that results from
neurologic involvement of HIV (3,4). While the mechanisms through
which HIV damages the CNS remain obfuscated, postmortem brains of
HIV-positive individuals with cognitive deficits revealed dysregulated
mitochondrial morphology and impaired mitochondrial metabolism in
comparison to HIV-positive patients with no cognitive deficits (9,10).
Mitochondrial dysfunction results in reduced energy production,
impairing neuronal function and thus playing a role in
neurodegenerative diseases (13). Accordingly, research in HAND has
increased focus on mitochondrial dynamics in relation to neuronal
degeneration. The search for new set of drugs that reduce neuronal
injury requires a better understanding of the mechanisms and the
plethora of steps involved in the pathogenic cascade. Therefore, novel
therapies should be developed that can be used adjunctively with cART
to prevent deterioration or restore normal cognitive function. To
develop innovative treatments, animal models are used for preclinical
testing.
Brain derived neurotrophic factor (BDNF) is a growth factor in the
family of neurotrophins that is essential in maintaining neuronal
modulation of dendritic branching and spines in the cortex and
long-term potentiation in the hippocampus (14,15). BDNF also plays an
important role in learning, memory and the preservation of cortical
circuits. Treatment with BDNF can potentially reduce or abolish
neuronal death in several brain areas of various neurological diseases;
moreover, BDNF prevents degeneration of motor descending
pathways (18,19) and prevent synaptic simplifications of neurons
exposed to the gp120 protein (20). In the immune system, BDNF also
decreases the apoptosis of T cells, which are depleted in AIDS (21,22).
Thus, there exists great potential in a therapy that combines BDNF and
antiretroviral therapy (cART), as such treatment can delay AIDS by
maintaining appropriate numbers of immune cells. Interestingly, this
also can have broader implications in CNS function. The neuronal and
astrocytic presence of chemokine receptors CXCR4 and CCR5 plays a
key role in the neuropathogenesis HAND (23). One of the key
neuroprotective mechanisms of BDNF is its ability to downregulate
CXCR4, as demonstrated by vitro and in vivo data (26,27). In HIV, the
role of CCR5 in neurologic outcomes is more difficult to assign since
CCR5 is a major cofactor for the infection process. The loss of CCR5
reduces long-term inflammatory injury, potentially through increases
in BDNF levels (32,33). HIV also reduces proBDNF processing through
gp120 by affecting furin levels and therefore causing an altered
balance between anti-apoptotic and pro-apoptotic neurotrophins (27).
Thus, from a pharmacological point of view, BDNF is an important
neuroprotective compound to potentially treat HIV or gp120
neurotoxicity caused by the CXCR4 receptors. Therefore, BDNF could
be a potential therapeutic agent for the treatment of diseases
characterized by loss of synaptic plasticity such as HAND. However, the
use of BDNF therapy is limited due to poor ability to penetrate the
blood-brain-barrier and its short half-life (34). Therefore, an alternative
molecule which mimics BDNF function and is also easily administered
is optimal.
7, 8 -dihydroxyflavone (DHF) is a small molecule that has recently been
identified as a high affinity tropomyosin receptor kinase B (TrkB)
agonist with neuroprotective abilities (35,36). Treatment with DHF
improves neurological impairments in rodent models of stress,
depression, aging, and Alzheimer disease (37-40). Therefore, it is
hypothesized that neuronal dysregulation in HAND could be improved
by modulating not only the CXCR4 and CCR5 chemokine receptors but
also mitochondrial metabolism through activation of TrkB by DHF. This
study examines the role of DHF in HAND using a TG26 mice model.
Molecular Structure of
7,8 Dihydroxyflavone
Methodology
Animals
HIV-1 transgenic mice (Tg26) expressing high levels of HIV protein such
as Tat, rev, nef, vif, vpr, and vpu, were established using the 7.4 kb
transgene construct lacking the 3 kb sequence overlapping the gag/pol
region of provirus pNL4-3 (Mouse Genome Informatics identification
number 3771187) as described previously (41). Tg26 mice in the FVB/N
background were backcrossed eight generations to a C57BL/6
background by Dr. Roy L. Sutliff (Veterans Affairs Medical Center,
Atlanta, GA). Wild-type (WT) mice generated from the same litter of Tg26
mice were used as controls for these studies. Female Transgenic (Tg26)
mice were housed under pathogen-free conditions at the animal facility
of the Institute of Human Virology, University of Maryland School of
Medicine, Baltimore. All experimental procedures were conducted
following NIH guidelines under an Institutional Animal Care and Use
Committee-approved protocol from the University Of Maryland School
Of Medicine, Baltimore.
Drug
We administered DHF (Tokyo Chemical Industry) intraperitoneally at a
dose of 5 mg/kg in 200 μl vehicle of 0.2% dimethyl sulfoxide (DMSO) in
phosphate-buffered saline (PBS). Mice were divided into 3 groups
untreated-Tg26 mice and DHF-treated Tg26 mice (Tg + DHF) and Wild
type mice. Tg26 + DHF mice received a single dose once daily from day
90 and continued for one month. Vehicle (200 μl of 0.2% DMSO in PBS)
was administered to Tg26 mice.
Methodology (Cont.)
Tissue pathology
Mice were euthanized after one-month treatment. Brains were removed
for analysis. Paraffin sections of brain were prepared. 7 μm thick
sections were stained. Slides were examined using standard bright-field
microscopy.
Immunohistochemistry
Immunohistochemistry of was performed as previously described
(Makar et al., 2012) using VECTASTAIN ABC kits (Vector Laboratories,
Burlingame, CA, USA). 7 μm thick sections were used. Nuclei were
counterstained with hematoxylin. Slides were examined using standard
bright field microscopy.
Analysis of histological images using ImageJ
Hippocampal and cortex regions of brain sections were selected from all
mice for pathology and immunohistochemistry. Histological
quantification was performed by using Image J. All cell labeling
experiments (GFAP, CXCR4, CCR5, P-AKT, P-TRKB, P-STAT3, SIRT-3, SUR-1,
TRPM-4, Citrate Synthase, PGC1α, MFN2, FIS-1) were quantified based on
the number of positive cells/field. (Each field = 40 × magnification
picture). All fields covering the hippocampus and cortex were analyzed
from each brain. Antibodies are listed below.
Statistical analysis
Statistical analyses were done using Prism software (GraphPad, San
Diego, CA). Values are expressed as means ± SEM. Statistical analysis
was performed with one-way ANOVA. Newman-Keuls Multiple
Comparison Post Test was used for one-way ANOVA. Statistical
significance was accepted at the 95% confidence level (p < 0.05).
Table 1. List of Primary Antibodies
Antibody Target Vendor Concentration
Glial Fibrillary Acidic Protein EMD Millipore, Billerica, MA, USA 1:100 (IHC)
Anti GFAP
Anti P-TRKB Phospho-Tropomyosin Receptor Abcam, Cambridge, MA, USA 1:500 (IHC)
Kinase B
Anti P-AKT Phospho-Protein Kinase B Cell Signaling Technology, Boston, MA, USA 1:50 (IHC)
Anti SUR-1 Sulfonylurea Receptor 1 Santa Cruz Biotechnology, Dallas, TX, USA 1:250 (IHC)
Anti CXCR4 C-X-C Chemokine Receptor Abcam, Cambridge, MA, USA 1:20 (IHC)
Type 4
C-C Chemokine Receptor Type 5 Abcam, Cambridge, MA, USA 1:20 (IHC)
Anti CCR5
Anti TRPM4 Transient Receptor Potential
Cation Channel Subfamily M
Sigma Aldrich, St. Louis, MO, USA 1:200 (IHC)
Member 4
Anti Signal Transducer & Activator of Cell Signaling Technology, Boston, MA, USA 1:50 (IHC)
Transcription 3
P-STAT3
Anti PGC1α Peroxisome proliferator-activated Abcam, Cambridge, MA, USA 1:100 (IHC)
receptor gamma coactivator 1 α
Anti MFN2 Mitofusin 2 Genetex, Irvine, CA, USA 1:500 (IHC)
Anti SIRT3 Mitochondrial protein Cell Signaling Technology, Boston, MA, USA 1:100 (IHC)
Anti FIS1 Mitochondrial fission 1 protein Genetex, Irvine, CA, USA 1:200 (IHC)
Anti-Citrate Enzyme marker for intact Genetex, Irvine, CA, USA 1.500 (IHC)
mitochondria
Synthase
Results
DHF treatment downregulates CXCR4 and CCR5:
1 treated Tg26 mice in comparison to DHF
Infection with HIV-1 within the brain
has long been known to be
associated with neurodegeneration
and neurocognitive disorder. HIV-1
genes interact with host CD4 and
chemokine co-receptors CXCR4 and
CCR5 to initiate infection in
macrophages and lymphocytes.
Therefore, HIV co-receptors play a
direct and indirect part in the
development of HAND. On this note,
2 Tg26 mice expressing HIV genes in
the brain share several pathological
hallmarks with HAND. We found that
in the cortex and hippocampus of
Tg26 mice, CXCR4 (Fig. 1) and CCR5
(Fig. 2) expression was significantly
upregulated in comparison to
normal mice, and DHF treatment
significantly downregulated these
co-receptors.
DHF treatment activates TrkB signaling
3
To determine whether DHF activated
TrkB and downstream signaling
pathways in Tg26 mice, the levels of
phosphorylated TrkB (P-TrkB), AKT
(P-AKT) and STAT3 (P-STAT3) were
examined in the mouse brains by
immunohistochemical labeling. As
shown in Fig. 3, DHF treatment
significantly (P < 0.001) increased the
expression of P-TrkB in the
hippocampal and cortical regions in
comparison to those of the Tg26
4 Psychiatr Res 41:387–394
mice without DHF treatment. The
Akt, or PI3K-Akt, pathway is a signal
transduction pathway that promotes
survival and growth in response to
extracellular signals. The expression
of phospho-AKT (P-AKT) was also
increased significantly (P < 0.001) in
the DHF group compared to the Tg26
mice without DHF treatment (Fig. 4).
Signal transduction activator
transcription factor 3 (STAT-3) is
involved in different signal
5 transduction processes. The
activated form P-STAT-3 was
significantly (P < 0.001) increased in
the brain of Tg26 + DHF mice when
compared to normal and Tg26 mice
(Fig. 5). These results show that the
activation of the hippocampal
TrkB-Akt-STAT-3 signaling is involved
in the neuroprotective-like effects of
DHF.
Results (Cont.)
DHF treatment balance calcium homeostasis by regulating
Sur1-Trpm4:
6 Trpm4 contributes to the formation of
cytotoxic edema, and it functions as an
end-executioner in accidental necrotic
death. On the other hand, the sulfonylurea
receptor 1 (Sur1)-regulated NC (Ca-ATP)
channel is a nonselective cation channel
that is regulated by intracellular calcium
and adenosine triphosphate. SUR1-TRPM4
channels, which conduct monovalent but
not divalent cations, are opened by
intracellular Ca2+ via calmodulin and a
calmodulin binding site at the C terminus
of TRPM4, resulting in Na+ influx. Cell
7 in transmitting Th1 and/or Th17 cytokine-mediated signals, so it
depolarization due to Na+ influx reduces
the inward electrochemical driving force
for Ca2+, thus acting as a negative regulator
to reduce Ca2+ influx by channels such as
TRPC3 (44). This raised the possibility that
SUR1-TRPM4 could be involved in the brain
of Tg26 mice linked to Ca2+ influx. There
was a significant increase of Sur-1 (Fig. 6)
and Trpm-4 (Fig. 7) upregulation in the
Tg26 mice, and DHF treatment
downregulated these channels, indicating
that DHF treatment restores calcium
homeostasis.
DHF Treatment protects mitochondrial toxicity:
8 channels in the hippocampal and cortical regions of the Tg26 mice.
Mitochondria are vital to the survival of
neuronal cells, as neurons have a high
energy demand. Citrate synthase is an
enzyme-marker for intact mitochondria.
Interestingly, we found a significant
decrease in the expression of citrate
synthase in the Tg26 brain in comparison
to the brains of the normal mice, and
DHF treatment significantly increased its
9 expression, indicative of an improvement
in mitochondrial function (Fig. 9). Sirtuin
3 (SIRT3) is a mitochondrial deacetylase
that is enriched in metabolically active
brain. SIRT3 regulates many aspects of
mitochondrial biology including ATP
generation, mitochondrial dynamics, and
the mitochondrial-unfolded protein
response (UPR) (45,46). Peroxisome
proliferator-activated receptor gamma
coactivator 1-alpha (PGC-1α) is a major
regulator of mitochondrial function,
10 oxygen consumption and mitochondrial
biogenesis (47). SIRT3 has recently been
discovered to be the target of PGC-1α
and is important in mitochondrial
processes, such as mitochondrial
biogenesis, and energy metabolism (48).
Interestingly, we found Sirt3 (Fig. 10) and
PGC-1α (Fig. 11) are increased in DHF
untreated Tg26 mice, indicating that DHF
improved mitochondrial function by
increasing mitochondrial biogenesis.
Mitochondrial biogenesis is crucial for the
11 regeneration of CNS tissue and is tightly
controlled by the PGC-1α signaling
network. Mitochondrial dynamics are
regulated by a complex system of
proteins representing the mitochondrial
quality control (MQC). MQC balances the
antagonistic forces of fusion and fission,
which is crucial in determining
mitochondrial cell fates. We found that
DHF treatment enhances mitochondrial
fusion-2 (MFN-2) (Fig. 12) and
downregulates mitochondrial fission
12
(FIS-1) (Fig. 13) in the brains of Tg26 mice,
which indicates improved mitochondrial
quality. Overall, it can be concluded that
DHF treatment downregulates
mitochondrial toxicity by improving
mitochondrial function, biogenesis and
dynamics.
Discussion
HIV patients suffer from HAND and HAD, which is characterized
pathologically by neuronal loss and dysfunction (1). It has long been
known that the hippocampus is critical for the encoding and retrieval
of long-term declarative memories, more recently, the region has
been shown to play a central role in other cognitive processes such
as working memory. Hippocampal neurons express high levels of HIV
chemokine co-receptors, activation of which causes injury or death in
vitro. To determine if there in vivo expression correlates with injury,
we evaluated neuronal CXCR4 and CCR5 immunoreactivity and
reactive gliosis in the hippocampus of Tg26 mice before and after
DHF treatment. BDNF exhibits neuroprotection in vitro against two
strains of gp120 that binds to CXCR4 and CCR5 (49). Moreover, BDNF
+/− mice are more sensitive to gp120 toxicity (49). In these Tg26 mice,
we found an increase of CXCR4 and CCR5 in hippocampus and cortex
brain areas and DHF treatment downregulates the accumulation of
CXCR4 and CCR5 immunoreactivity in the brains of Tg26 mice
perhaps by stimulating the internalization of the receptors.
Previously it has been shown that BDNF down-regulates CXCR4 and
CCR5 levels in vitro and in vivo (49). Based on these findings, we
propose that down-regulation of those co-receptor is a mechanism
that may account for the neuroprotective property of BDNF/TrkB
against HIV. Pharmacological data has shown that TrkB is the main
BDNF receptor that modulates the neuroprotective property of BDNF
by decreasing CXCR4 levels. And our data supports that BDNF/TrkB
signaling has a crucial role in hippocampal neurogenesis (49).
Discussion (Cont.)
Overall, the data suggests that CXCR4 and CCR5 levels may serve as a
proxy for ongoing risk of HIV-associated brain injury and
neurocognitive disorder, but further longitudinal studies are needed.
Therefore, it may be worth speculating that a balance between
BDNF/TrkB and chemokine receptors may contribute to modulate
adult neurogenesis.
Previous studies have shown that DHF treatment was
neuroprotective in traumatic brain injury activation of the
TrkB-mediated PI3K/Akt signal pathway. Our study demonstrates that
DHF activates neuroprotection in an animal model of HAND by
triggering TrkB/PI3K/Akt signal pathway and activating downstream
transcription factor STAT3. STAT family proteins have essential roles
would be interesting for future study to see the effect of DHF on
inflammatory mediators as well. To determine if these
neuroprotective effects might be due to the stabilization of
intracellular calcium we measured calcium changes in response to
treatment of DHF. Both SUR1 and TRPM4 were upregulated in the
brain cortex and hippocampus of Tg26 mice and down regulated by
DHF treatment. Previous studies have shown that SUR1 and TRPM4
often co-assemble to form an ion channel that plays a significant role
in central nervous system (CNS) injury and perturbation of calcium
homeostasis. In fact, SUR1-TRPM4 channel opening results in Na +
influx; cell depolarization due to Na+ influx reduces the inward
electrochemical driving force for Ca2+, acting as a negative regulator
for Ca2+ influx. DHF treatment upregulated the SUR1 and TRPM4
Therefore, DHF plays a significant role in restoring perturbed calcium
homeostasis.
Ca2+ uptake by mitochondria plays an important role for the
organelle and for the whole cell. In this context, we found calcium
homeostasis changes as well as mitochondrial dysfunction associated
with inhibition of mitochondrial biogenesis, imbalance of
mitochondrial dynamics in the brain of TG26 mice. On the other
hand, DHF treatment improved mitochondrial stabilization and
function. A potential action of DHF on cell energy homeostasis was
corroborated by the normalization in levels of PGC-1α, TFAM, COII,
AMPK and SIRT1 in animals subjected to TBI (52). Our findings
suggest a potential mechanism by which DHF counteracts HAND
pathology via activation of the TrkB receptor and engaging the
interplay between cell energy management and gliosis. Since
metabolic dysfunction is an important risk factor for the development
of HAND, these results set a precedent for the therapeutic use of DHF
in a larger context.
Conclusion
As antiretroviral therapy leads to persistent chronic infection of HIV, a
mounting number of various commodities have become a focus in
the research world, one of these being HAND. While BDNF exemplies
impressive ameliorative and neuroprotective effects as a possible
means of treatment, its inability to penetrate the blood-brain-barrier
hinders its potential. In this study, we show that the small molecule
7,8 dihydroxyflavone, a TrkB agonist (like BDNF) shows great promise
as a future treatment for HAND. In this study, DHF was shown to
activate TrkB and its downstream signalling pathways as well. One
characteristic pathological mechanism during the pathophysiology of
HAND is mitochondrial dysregulation. Interestingly, DHF not only
effectively downregulates HIV chemokine coreceptor CXCR4 and
CCR5, but also improves the expression of downregulated/
upregulated mitochondrial proteins in conjunction with the
SUR1-TRPM4 calcium channel. Currently, we are continuing this
research with a focus on inflammatory mediators, and the data in
progress in this focus have also shown great promise. Overall, DHF
presents itself as a potential efficacious treatment for HAND.
Acknowledgements
We would like to express gratitude towards Dr. Tapas Makar for
providing us guidance throughout the entirety of the project. Also, a
special thanks to the Institute of Human Virology for maintaining the
mice and providing us with the fixed tissue blocks of the mice brains.
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