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Mitochondrial pyruvate carrier regulates autophagy, inflammation, and

neurodegeneration in experimental models of Parkinsons disease

Article  in  Science translational medicine · December 2016

DOI: 10.1126/scitranslmed.aag2210

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PARKINSON’S DISEASE 2016 © The Authors,

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Mitochondrial pyruvate carrier regulates autophagy, exclusive licensee

American Association
inflammation, and neurodegeneration in experimental for the Advancement
of Science.
models of Parkinson’s disease
Anamitra Ghosh,1 Trevor Tyson,1 Sonia George,1 Erin N. Hildebrandt,1 Jennifer A. Steiner,1
Zachary Madaj,2 Emily Schulz,1 Emily Machiela,1 William G. McDonald,3 Martha L. Escobar Galvis,1
Jeffrey H. Kordower,1,4 Jeremy M. Van Raamsdonk,1 Jerry R. Colca,3 Patrik Brundin1*

Mitochondrial and autophagic dysfunction as well as neuroinflammation are involved in the pathophysiology of
Parkinson’s disease (PD). We hypothesized that targeting the mitochondrial pyruvate carrier (MPC), a key controller
of cellular metabolism that influences mTOR (mammalian target of rapamycin) activation, might attenuate neuro-
degeneration of nigral dopaminergic neurons in animal models of PD. To test this, we used MSDC-0160, a compound
that specifically targets MPC, to reduce its activity. MSDC-0160 protected against 1-methyl-4-phenylpyridinium (MPP+)
insult in murine and cultured human midbrain dopamine neurons and in an a-synuclein–based Caenorhabditis elegans

Downloaded from http://stm.sciencemag.org/ on December 7, 2016

model. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)–treated mice, MSDC-0160 improved locomotor behav-
ior, increased survival of nigral dopaminergic neurons, boosted striatal dopamine levels, and reduced neuroinflamma-
tion. Long-term targeting of MPC preserved motor function, rescued the nigrostriatal pathway, and reduced
neuroinflammation in the slowly progressive Engrailed1 (En1+/−) genetic mouse model of PD. Targeting MPC in
multiple models resulted in modulation of mitochondrial function and mTOR signaling, with normalization of autoph-
agy and a reduction in glial cell activation. Our work demonstrates that changes in metabolic signaling resulting from
targeting MPC were neuroprotective and anti-inflammatory in several PD models, suggesting that MPC may be a
useful therapeutic target in PD.

INTRODUCTION of TZDs or genetically—results in a compensatory increase in utilization

People with Parkinson’s disease (PD) exhibit a range of nonmotor and
motor symptoms (1, 2), with the latter being strongly linked to the
degeneration of nigral dopamine neurons. Whereas the disease mecha-
nisms are incompletely understood, evidence suggests that mitochon-
drial deficits, failure of autophagy, and neuroinflammation each plays
a role (3–5). Disease-modifying therapies are not available for PD,
and several trials targeting individual pathways implicated in PD patho-
genesis have failed (6–14). Therefore, a potentially more powerful
strategy is the targeting of molecules that are upstream in the signaling
cascades that modulate altered cellular functions and that affect both
neurons and glia in the brain.
The metabolism of all nutrients flows through various molecular
pathways, but ultimately, all are linked to the metabolism of pyruvate
(15). Recently, a protein complex for transporting pyruvate into the mito-
chondria, mitochondrial pyruvate carrier (MPC), was discovered in
the internal mitochondrial membrane (16–18). MPC contains the proteins
MPC-1 and MPC-2, is conserved in yeast, flies, worms, and mammals
(16–18), and is essential for cellular function (17–21).
The MPC complex is the target of a group of compounds known
as the thiazolidinedione (TZD) insulin sensitizers (22–25). TZDs slow
the entry of pyruvate across the mitochondrial membrane (24), and
the effects of TZDs on gluconeogenesis require the MPC complex
(21). Knockout of the MPC complex in Drosophila eliminated the pos-
itive effects of TZD treatment in flies on a high-sucrose diet (22). Slow-
ing the entry of pyruvate at the MPC—for example, by administration
1
Center for Neurodegenerative Science, Van Andel Research Institute, Grand Rapids,
MI 49503, USA. 2Bioinformatics and Biostatistics Core, Van Andel Research Institute,
Grand Rapids, MI 49503, USA. 3Metabolic Solutions Development Company, Kalama-
zoo, MI 49007, USA. 4Center for Brain Repair, Department of Pathology, Rush Medical
College, Chicago, IL 60612, USA.
*Corresponding author. Email: patrik.brundin@vai.org
of other substrates, that is, amino acids and fatty acids, resulting in
alterations in cellular metabolism (20, 21, 26, 27).
For the past two decades, the general consensus has been that TZDs
activate the transcription factor peroxisome proliferator–activated
receptor-g (PPARg) and have multiple effects on mitochondrial func-
tion, including partial inhibition of complex I [for review, see (28)].
The identification of MPC, however, suggests that some of the anti-
diabetic effects and other actions of TZDs might be mediated through
this protein complex (29). The use of TZDs in diabetes has declined
over recent years because of several side effects driven by the activa-
tion of nuclear receptor PPARg, including fluid retention, weight gain,
and concern over a potential increased risk for urinary bladder cancer
(30) and other cancers (31). Notwithstanding the risk for dose-limiting
and sometimes serious side effects, TZDs have recently garnered great
attention in PD. A large retrospective study demonstrated that diabetic
subjects prescribed with TZDs have a 29% reduced risk of developing
PD within 14 years (32), suggesting that these compounds might affect
molecular mechanisms involved in PD. Furthermore, TZDs are pro-
tective in PD animal models (33, 34). Recent research has shown that
the disease processes in diabetes and PD share several features, includ-
ing metabolic perturbations and inflammation (35–38).
MSDC-0160 is a member of a new class of compounds that mod-
ulate MPC and act as insulin sensitizers without activating PPARg
(22–24, 39). Therefore, MSDC-0160 lacks the negative side effects of
the first-generation insulin sensitizers (40). In a phase 2b trial in diabe-
tes, MSDC-0160 effectively lowered glucose and, importantly, caused
minimal fluid retention and weight gain (41). Furthermore, MSDC-
0160 preserved cerebral 2-deoxyglucose uptake after 3 months of use
in Alzheimer’s disease patients, suggesting that it engaged the target
MPC in the brain after oral administration (42). Given this background,
we hypothesized that modulating MPC function could normalize

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SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE
molecular pathways that are perturbed in PD and reduce neurodegen-
eration in cellular and animal models of PD.
RESULTS
Protective role of MPC modulation in cellular and
nematode models
Previous studies have shown that micromolar concentrations of
MSDC-0160 acutely modulate the metabolism of pyruvate in multiple
cells types, including neurons, through a direct interaction with MPC
(24). We hypothesized that this modulation of MPC function by
MSDC-0160 would protect compromised dopaminergic neurons in
models of PD both in vitro and in vivo. We used several model
systems to evaluate the potential pathways involved.
We found that modulation of MPC shielded cultured human,
murine, and invertebrate dopaminergic neurons from 1-methyl-4-
phenylpyridinium (MPP+) toxicity (Fig. 1, B, G, and K, and fig. S1, A
and B). Specifically, MSDC-0160 (10 mM) pretreatment (1 hour) pre-
vented the MPP+ (10 mM)–induced loss of both tyrosine hydroxylase
(TH)–immunoreactive differentiated Lund human mesencephalic
(LUHMES) cells (Fig. 1, A and B, and fig. S1A) (43) and TH-immuno-
reactive mouse primary mesencephalic neurons in culture (Fig. 1, F and
G) (44). We determined that about 11% of the neurons in differentiated
LUHMES cultures and 6% of the neurons in the primary murine mes-
encephalic culture expressed TH. Further, we found that MSDC-0160
protected only TH-immunoreactive neurons (no change in the number
of TH-negative cells was observed), which is consistent with the selected
concentration of MPP+ primarily being toxic to dopamine neurons. In
addition, MSDC-0160 counteracted both MPP+-induced shortening of
neurite length and reduced branching in both LUHMES cells (Fig. 1, C
to E) and primary dopaminergic cultures of mouse midbrain tissue (Fig.
1, H to J). To observe the effects of MPC modulation in a whole orga-
nism, we exclusively treated Caenorhabditis elegans nematodes
expressing green fluorescent protein (GFP) in dopaminergic neurons
with 0.75 mM MPP+ and quantified the resulting neuron loss according
to established protocols (45, 46). Treatment with MSDC-0160 (10 or
100 mM) prevented the loss of GFP-fluorescent dopaminergic neurons
induced by MPP+ (0.75 mM) in nematodes (Fig. 1K and fig. S1B) (P =
0.0001), whereas 1 mM MSDC-0160 did not. These results indicate that
dopaminergic neurons were protected by MSDC-0160 treatment.
Protective role of MPC modulation in mouse models of PD
To determine whether MPC targeting was neuroprotective in mammals,
we examined the effects of MSDC-0160 on motor behavior and neuro-
degeneration in mouse models of PD. To confirm that MSDC-0160
effectively enters the brain, we pretreated C57BL/6J mice with MSDC-
0160 (30 mg/kg per day via oral gavage) and euthanized the mice 2 or
4 hours later. Our measurements revealed MSDC-0160 and its hydro-
xymetabolite MSDC-0037 both in plasma and in brain tissue (fig. S2,
A and B), confirming that orally administered MSDC-0160 was able
to gain access to the brain. When expressed as nanogram per gram in
the brain and nanogram per milliliter in the plasma, at 2 hours after
an oral dose, there was parent drug (278 ng/g) and hydroxymetabolite
(21,100 ng/ml) in the brain, and at the same time point, the plasma
concentrations were 164 ng/ml for the parent drug and 51,600 ng/ml
for the metabolite. This suggested that oral administration was suffi-
cient to provide direct targeting of MPC in brain cells.
We treated a separate cohort of mice with the neurotoxin 1-methyl-
4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a model of PD (47).
Ghosh et al., Sci. Transl. Med. 8, 368ra174 (2016) 7 December 2016
Some mice were pretreated with MSDC-0160, whereas controls received
vehicle. Twenty-four hours later, the mice were subjected to a subacute
MPTP regimen, that is, five daily injections of MPTP (25 mg/kg per day
intraperitoneally) (48, 49). During this time, mice received daily oral
gavage of MSDC-0160 or vehicle for a total of 11 days (fig. S4A). Four
days after the last MPTP injection, we tested the mice for spontaneous
locomotion in an open-field arena and for motor coordination on the
rotarod test. Consistent with previous reports (48, 50, 51), MPTP injec-
tions caused significant impairment on both tests, with decreases in dis-
tance traveled, speed, and mobility time in the open field, as well as time
spent on the rotating rod. However, MSDC-0160 pretreatment attenu-
ated or prevented these deficits (Fig. 2, A and B, and fig. S4B). The
brains from treated mice were subjected to immunohistochemistry, ste-
reological counting of neurons, and high-performance liquid chroma-
tography (HPLC) and Western blot analyses. As expected (47, 48),
MPTP treatment induced a loss of dopaminergic neurons and terminals
in the substantia nigra and striatum, respectively (Fig. 2, C to E), a re-
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duction of cresyl violet–positive nigral neurons (fig. S4C), depletion of
striatal dopamine and its metabolite 3,4-dihydroxyphenylacetic acid
(DOPAC) (Fig. 2, F and G), and a loss of TH expression in the nigro-
striatal pathway (Fig. 2, H to J). These toxic effects of MPTP were atte-
nuated or blocked by modulation of MPC through pretreatment with
MSDC-0160 (Fig. 2, C to J).
To address the possibility that the neuroprotection induced by
MSDC-0160 was due to inhibition of the conversion of MPTP to
the neurotoxic metabolite MPP+ by glia, we measured MPP+ concen-
trations in striatum 3 hours after the fourth MPTP injection, with or
without MSDC-0160 treatment. We found that MSDC-0160 did not
affect striatal MPP+ concentrations (fig. S4D).
Next, we tested whether modulation of MPC could halt neurode-
generation in the MPTP mouse model after the insult has already been
triggered. We found that starting MSDC-0160 administration 2 days
after the initial dose of MPTP (in the subacute MPTP regimen) still
mitigated motor behavioral deficits (fig. S4G), rescued the loss of do-
paminergic neurons and terminals (Fig. 2, K and L), increased the
numbers of Nissl-stained neurons (fig. S4F), and restored neuro-
transmitter concentrations (Fig. 2, M and N). Collectively, these results
demonstrate that modulation of MPC both protected against and
could rescue MPTP-induced neurodegeneration in the subacute MPTP
mouse model of PD.
To validate the effects of modulating MPC in a progressive PD
model, we turned to the Engrailed1 heterozygous (En1+/−) mouse.
En1+/− mice represent a chronic degeneration genetic model with a
PD-like pattern of dopaminergic neurodegeneration that exhibits de-
creased autophagy and increased neuroinflammation. The En1+/−
mice have a normal complement of dopaminergic neurons at birth,
but at 8 weeks after birth, the mice exhibit the first signs of a progres-
sive loss of nigral dopaminergic neurons, reaching a plateau at 24 weeks
(52). Furthermore, En1+/− mice display motor impairment at 24 to
27 weeks of age in the open-field and rotarod tests. Here, we chose to
initiate the modulation of MPC function at two different ages to de-
termine whether the drug would be effective when pathology is mild
or modest. Treatment starting at 3 weeks after birth served as a “mild
pathology stage,” at which there is axonal pathology but no cell death
(Fig. 3A) (53). Treatment starting at 8 weeks after birth served as a
“modest pathology stage,” at which 20% of TH-immunoreactive neu-
rons have been lost in En1+/− mice (Fig. 3B).
At the mild pathology stage, En1+/− mice were fed a diet formu-
lated to deliver MSDC-0160 (30 mg/kg) starting at week 3 after birth
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Fig. 1. MSDC-0160 protects dopaminergic neurons against MPP+-induced toxicity in cell cultures and C. elegans. LUHMES cells were pretreated with 10 mM MSDC-
0160 (0160) for 1 hour followed by 10 mM MPP+ treatment for 24 hours. (A) Double-label immunocytochemistry for TH and Tuj1 in LUHMES cells. (B) Number of TH-positive
dopaminergic neurons, (C) mean neurite length (mm), (D) neurite branching points in each neuron, and (E) longest neurite length (mm). Primary mesencephalic mouse neurons
were pretreated with 10 mM MSDC-0160 for 1 hour followed by 10 mM MPP+ treatment for 24 hours. (F) Double-label immunocytochemistry for TH and Tuj1 in primary
mesencephalic culture. (G) Number of TH-positive dopaminergic neurons, (H) mean neurite length (mm), (I) neurite branching points in each neuron, and (J) longest neurite
length (mm). C. elegans were synchronized, and L1 larvae were placed in 96-well plates containing OP50 bacteria in liquid culture (6 mg/ml) and MPP+ (0.75 mM) with MSDC-
0160 (1, 10, and 100 mM) for 48 hours. (K) Quantification of percent dopaminergic neuron loss in C. elegans. Scale bars, 50 mm. Data are means ± SEM of three independent
experiments. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, analyzed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test.

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Fig. 2. MSDC-0160 improves motor behavior, protects nigrostriatal neurons, and suppresses disease progression in the MPTP mouse model of PD. (A) Open-field
parameters: distance traveled (cm), mean speed (cm/s), and time spent mobile (s). (B) Time spent on rotarod (s). (C) Diaminobenzidine immunohistochemistry for TH in
substantia nigra (SN; upper panel) and striatum (ST; lower panel) of MPTP mice. (D) Stereological counting of TH-positive neurons from the SN. (E) Relative density of TH-positive
neuronal fibers in the ST. Quantification of (F) dopamine (ng/mg) and (G) the dopamine metabolite DOPAC (ng/mg) by HPLC in the ST. (H) Representative Western blots
illustrating the expression of TH in SN and ST. Bar graph showing mean Western blot TH/b-actin in SN (I) and in ST (J). For the disease progression studies, mice were administered
MSDC-0160 (30 mg/kg per day) starting 3 days after MPTP treatment, with continuing treatment for another 7 days. (K) Stereological counting of TH-positive neurons from SN. (L)
Relative density of TH-positive neuronal fibers in ST. Quantification of striatal (M) dopamine and (N) DOPAC by HPLC. Scale bars, 50 mm (SN) and 200 mm (ST). Data are means ±
SEM of 7 to 10 mice per group. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test.

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Fig. 3. MSDC-0160 improves motor behavior in the open-field and rotarod tests in the En1+/− genetic mouse model of PD. (A) Schedule for MSDC-0160 treatment of
En1+/− mice in the mild pathology stage. (B) Schedule for MSDC-0160 treatment of En1+/− mice in the modest pathology stage. (C) Distance traveled (cm), (D) mean speed (cm/s),
(E) maximum speed (cm/s), and (F) time on rotarod (s) in the mild pathology stage. WT, wild type. (G) Distance traveled (cm), (H) mean speed (cm/s), (I) maximum speed (cm/s),
and (J) time on rotarod (s) in the modest pathology stage. Data are means ± SEM of 8 to 12 mice per group. ***P < 0.001, **P < 0.01, *P < 0.05, oP < 0.05 to 0.1. Data were analyzed
using linear mixed-effects regression analysis with false discovery rate (FDR)–corrected P values.

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until euthanasia at week 28 or week 48. At the modest pathology stage,
En1+/− mice were fed the same diet starting at week 8 until euthanasia
at week 16 or week 28. At each time point, we assayed motor behavior,
and after euthanasia, we performed immunohistochemistry, Western
blotting, and HPLC analysis to examine dopaminergic markers and neu-
rotransmitter concentrations. For motor behaviors, including sponta-
neous activity in the open-field and coordination testing on the rotarod,
the MSDC-0160–treated group exhibited improvements on all mea-
sures (Fig. 3, C to G, fig. S5, and table S1).
For all outcomes, the En1+/− non–MSDC-0160–treated control
group of mice performed significantly worse than did the wild-type
control mice (see the En1 genotype variable in table S1 for adjusted
and unadjusted P values). Treatment with MSDC-0160 did not signif-
icantly affect the behavior of wild-type mice. However, the En1+/−
group treated with MSDC-0160 showed evidence of improvement (af-
ter false discovery multiple testing adjustments) in mean maze speed
for the groups that started on MSDC-0160 at 3 and 8 weeks of age
(3 weeks, P = 0.032; 8 weeks, P = 0.027), mean distance traveled (group
that started on treatment at 8 weeks of age; P = 0.021), and time spent
mobile in the maze (group that started on treatment at 8 weeks of age;
P = 0.003). There was little evidence that treatment of En1+/− mice
with MSDC-0160 reduced the amount of time spent frozen in either
of the groups that started MSDC-0160 treatment at 3 or 8 weeks of age.
All other behavioral measures for the En1+/− groups treated with MSDC-
0160 exhibited significant improvement (P < 0.05) before the multiple
testing adjustments, but these apparent improvements did not remain
significant after adjustment (see the En1 genotype * MSDC treatment
variable in table S1 for adjusted and unadjusted P values).
Consistent with the results of the behavioral testing, stereological
analyses of immunohistochemically stained sections of the brains of
the En1+/− mice revealed that treatment with this MPC modulator pro-
tected the TH-immunoreactive (Fig. 4, A, B, and G to I) and Nissl-
stained (fig. S6, A to C) dopaminergic neurons of the substantia nigra.
In addition, MPC modulation with MSDC-0160 increased the expres-
sion of TH and restored striatal dopamine and DOPAC content in all
groups at each studied time point in the En1+/− mice [Fig. 4, C and D
(P = 0.0018), E (P = 0.0018), F (P = 0.0168), J to L (P = 0.0001), M (P =
0.0001), N (P = 0.0014), O (P = 0.0001), and P (P = 0.0009), and fig. S6,
E (P = 0.0002) and F (P = 0.0001)]. Collectively, these data demonstrate
that modulation of MPC was neuroprotective in the slowly progressing
En1+/− mouse model of PD.
The effects of MPC modulation on degeneration of
dopaminergic neurons
MPC, the target of MSDC-0160, is present in nigral dopaminergic
neurons (fig. S7, A and B) (17). Thus, we set out to dissect the molecular
mechanisms underpinning the neuroprotective effect of MPC modula-
tion using C. elegans and cultured cells, as well as the MPTP and En1+/−
mouse models of PD. C. elegans expresses brain protein 44–like
(BRP44L), which is an ortholog of MPC-1. In the C. elegans model,
we used worms expressing both GFP and A53T mutant a-synuclein
in dopaminergic neurons (a point mutation that causes autosomal dom-
inant PD in humans). Aggregated a-synuclein is a major component of
Lewy bodies in PD (54), and a-synuclein is genetically linked to both
inherited and sporadic forms of PD (55). We visualized dopaminergic
neurons using GFP in worms expressing A53T a-synuclein. These worms
showed marked degeneration of dopaminergic neurons after 8 days (P =
0.05), which was rescued when BRP44L function was modulated by treat-
ment with 100 mM MSDC-0160 (P = 0.0001) (Fig. 5, A and B).
Ghosh et al., Sci. Transl. Med. 8, 368ra174 (2016) 7 December 2016
On the basis of the literature, we hypothesized that the mammalian
target of rapamycin (mTOR) pathway is involved downstream of
MPC (29, 56–59). In nematodes, we knocked down genes of interest
related to MPC and to the mTOR pathway by RNA interference and
evaluated whether the knockdown of these genes affected the protec-
tion of A53T a-synuclein–induced dopaminergic neurons from degen-
eration by MSDC-0160. MSDC-0160 treatment prevented neuron loss
due to the A53T mutation in a-synuclein in control worms, which were
fed bacteria expressing the empty vector L4440 (Fig. 5C). Knockdown
of BRP44L (the ortholog of MPC-1) prevented neuroprotection by
MSDC-0160 (Fig. 5D). Furthermore, knockdown of AKT-1 (a serine/
threonine kinase that functions upstream of RHEB-1; Fig. 5E), RHEB-
1 (a guanosine triphosphatase that is a stimulator of the mTOR
pathway; Fig. 5F), or LET-363 (the C. elegans ortholog of the human
mTOR protein; Fig. 5G) also prevented the neuroprotection exerted by
MSDC-0160 in this dopaminergic neuronal loss assay. By contrast,
knockdown of AAK-1, which is a homolog of adenosine monopho-
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sphate (AMP)–activated protein kinase, mimicked the control condition
(Fig. 5H), suggesting that BRP44L does not control this pathway. RNA
interference knockdown of the genes tested above was confirmed by
quantitative polymerase chain reaction (fig. S1C). The conclusion of
these experiments is that engaging MPC and the mTOR pathway is ne-
cessary for the neuroprotective effect of MSDC-0160 in C. elegans.
In a second set of experiments, we used the mitochondria isolated
from rat brain and intact cultured LUHMES cells to define the time
course of the effects of MSDC-0160 on the different components of
the mTOR pathway. Initially, we demonstrated that MSDC-0160 de-
creased pyruvate oxidation in isolated brain mitochondria in a direct
and immediate fashion (fig. S2, C and D). We then measured the ef-
fect of MSDC-0160 on cellular respiration in LUHMES neuronal cells
treated with the mitochondrial toxin MPP+ and found that when
MSDC-0160 was added 1 hour before or after MPP+ addition,
MSDC-0160 normalized oxygen consumption (fig. S2E). These data
show that modulation of MPC has immediate effects on mitochondri-
al function. On the other hand, when LUHMES cells were treated for
2, 6, or 24 hours with MSDC-0160, no changes in mTOR activity
[monitored as the ratio of phosphorylated mTOR (p-mTOR)/mTOR]
could be detected (fig. S3, A and B). These data indicated that the
changes in nutrient-sensing pathways were downstream of the meta-
bolic action of MPC and upstream of the demonstrated neuroprotec-
tive effect (fig. S2C). Together, the data obtained in nematodes and
cultured neurons showed that when modulating MPC, functional
changes in the mTOR pathway (described in detail below) appear with
a delay after the immediate effects seen on mitochondrial function.
Next, we queried the role of the mTOR pathway on the neuropro-
tection observed in vivo after MPC modulation in the MPTP and En1+/−
mouse models of PD. In the MPTP model, using Western blotting of
substantia nigra samples, we measured mTOR, p-mTOR (Ser2448), S6,
and phosphorylated S6 (pS6) (Ser235/Ser236), normalized to b-actin
(Fig. 5I). As expected, MPTP injections increased p-mTOR measured
as the p-mTOR/mTOR ratio relative to control conditions (P = 0.04)
(Fig. 5J). MPTP treatment also increased the ratio of pS6/S6 (P = 0.03)
(Fig. 5K). Although there was little to no effect under control con-
ditions, MPC modulation using MSDC-0160 pretreatment reduced
the activation of both mTOR and phosphorylation of its downstream
substrate, indicating that this regulation was a component of neuro-
protection (P = 0.032) (Fig. 5, I to K).
We also returned to the En1+/− mouse model to validate further
the involvement of the mTOR pathway downstream of MPC. Using
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Fig. 4. MSDC-0160 prevents dopaminergic neurodegeneration in the En1+/− genetic mouse model of PD. (A) TH immunostaining in the SN. (B) Stereological counting
of TH-immunoreactive (TH+) neurons in the SN. (C) Representative Western blot illustrating the expression of TH in the SN. (D) Bar graph showing mean Western blot TH/
b-actin ratios in SN, quantification of striatal (E) dopamine (ng/mg), and (F) DOPAC (ng/mg) by HPLC in the mild pathology stage. (G) TH immunostaining in SN (upper panel
pictures are from 16 weeks of age, and lower panel pictures are from 28 weeks of age). Scale bars, 50 mm. (H) Stereological counting of TH-positive neurons in SN from 16 weeks of
age. (I) Stereological counting of TH-positive neurons in SN from 28 weeks of age. (J) Representative Western blot illustrating the expression of TH in SN from 16 weeks of age (top)
and 28 weeks of age (bottom), and bar graphs showing mean Western blot TH/b-actin ratios in SN at 16 weeks (K) and at 28 weeks of age. (L) Quantification of striatal (M) dopamine
and (N) DOPAC by HPLC at 16 weeks of age. Quantification of striatal (O) dopamine and (P) DOPAC by HPLC at 28 weeks of age in the modest pathology stage. Data are means ± SEM
of six to eight mice per group. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Data were analyzed by two-way ANOVA with Bonferroni’s multiple comparison test.

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Fig. 5. MSDC-0160 modulates mTOR signaling in C. elegans and the MPTP mouse model of PD. (A) Images show dopaminergic neuron loss in worm strains BY250 and
JVR203 at 12 days of age. Scale bar, 10 mm. (B) Quantification of dopaminergic neuron loss (percentage of total number of dopamine neurons). (C) Quantification of dopa-
minergic neuron loss when C. elegans were fed bacteria expressing the empty vector L4440 as control. Quantification of dopaminergic neuron loss in C. elegans after knocking
down BRP44L/MPC-1 (D), AKT-1 (E), RHEB-1 (F), LET-363/mTOR (G) and AAK-1/AMPK (H). Mice were coadministered MPTP and MSDC-0160. After 1 day of pretreatment with
MSDC-0160 (30 mg/kg, by oral gavage) and 5 days of cotreatment with MSDC-0160 (30 mg/kg, by oral gavage) and MPTP (25 mg/kg per day intraperitoneally), mice were
euthanized, and SN tissue was dissected and analyzed by Western blot. (I) Representative Western blot illustrating the expression of p-mTOR (Ser2448), total mTOR, pS6 (Ser235/
Ser236), and total S6 in the SN. Bar graphs showing mean Western blot p-mTOR/mTOR ratios relative to b-actin (J) and mean Western blot p-S6/S6 ratios relative to b-actin
(K). (A to H) Data are means ± SEM (n = 3 experiments) and were analyzed by unpaired Student’s t test except (B), which was analyzed by two-way ANOVA with Bonferroni’s
multiple comparison test. (I and J) Data are means ± SEM of six to eight mice per group and were analyzed by two-way ANOVA with Bonferroni’s multiple comparison test.
***P < 0.001, **P < 0.01, *P < 0.05.

Western blotting of substantia nigra samples, we quantitated mTOR,
p-mTOR (Ser2448 and Ser2481), p70S6kinase, phosphorylated p70S6kinase
(Thr389), pS6 (Ser235/Ser236), S6, AKT, phosphorylated AKT (Thr308),
regulated in development and DNA damage responses 1 (REDD1),
LC3b, and p62 (Fig. 6, A to E, and fig. S7). These data show that all
of these pathways were perturbed in the En1+/− mice versus wild-type
animals (P value between 0.01 and 0.0001). Treatment with MSDC-
0160 moved all of these changes toward the values observed in wild-
type mice. The changes included those in mTOR signaling—increases
in p-mTOR Ser2448/mTOR/b-actin, phosphorylated p70S6kinase/
p70S6kinase/b-actin, pS6/S6/b-actin (Fig. 6, B to D, F, G, I, and J)—
and also REDD1/b-actin ratios (fig. S7, C and F). As in the MPTP
experiments described above, MSDC-0160 treatment of the wild-
type mice did not affect mTOR signaling proteins (fig. S7). Notably,
En1+/− mice also had reduced LC3b/b-actin (Fig. 6, E, H, and K) and
p62/b-actin ratios (fig. S7G) relative to the wild-type mice, suggest-
ing that the genetic model has a disturbance in autophagy. Treat-
ment of the En1+/− mice with MSDC-0160 returned the amounts
of LC3b and p62 proteins to those seen in wild-type mice. Again,
there was no significant effect of MSDC-0160 treatment on the expres-
sion of these autophagy markers in wild-type mice (Fig. 6, E and K,
and fig. S7, C to G).
Together, the results indicate that modulation of MPC leads to an
immediate effect on mitochondrial metabolism associated with a later

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Fig. 6. MSDC-0160 down-regulates mTOR signaling and restores autophagy in the En1+/− genetic mouse model of PD. (A) Representative Western blot illustrating the
expression of p-mTOR (Ser2448), mTOR, phosphorylated p70S6kinase (p-p70S6K) (Thr389), p70S6kinase, pS6 (Ser235/Ser236), S6, and LC3b in the SN. Bar graphs showing mean
Western blot p-mTOR/mTOR ratios relative to b-actin (B), mean Western blot p-p70S6K/p70S6K ratios relative to b-actin (C), mean Western blot pS6/S6 ratios relative to b-actin
(D), and mean Western blot LC3b/b-actin ratio (E). (F) Immunostaining for TH and p-mTOR (Ser2448) in SN; insets demonstrate overlap of TH and p-mTOR. (G) Immunostaining
for TH and pS6 (Ser235/Ser236) in the SN. (H) Immunostaining for TH and LC3b. DAPI, 4′,6-diamidino-2-phenylindole. Intensity of p-mTOR (Ser2448) (I), pS6 (Ser235/Ser236)
(J), and LC3b (K) in the TH-positive neurons [a.u. (arbitrary units)]. Scale bars, 10 mm. Data are means ± SEM of six to eight mice per group. ****P < 0.0001, ***P < 0.001,
**P < 0.01, *P < 0.05. Data were analyzed by two-way ANOVA with Bonferroni’s multiple comparison test.

Ghosh et al., Sci. Transl. Med. 8, 368ra174 (2016) 7 December 2016 9 of 17

SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE
reduction in the overactivation of the mTOR pathway. The data sug-
gest that MPC modulation induces autophagy as part of the response
that prevents neurodegeneration.
Role of inflammation in the effects of MPC modulation
Given that neuroinflammation is also considered a contributory factor
to PD pathogenesis and occurs in several PD animal models (60), we
were curious whether modulation of MPC with MSDC-0160 treatment
would directly reduce inflammation. We found that MSDC-0160 re-
duced glial fibrillary acidic protein (GFAP; an astrocyte marker), io-
nized calcium-binding adapter molecule 1 (Iba-1; a microglial
marker), and inducible nitric oxide synthase (iNOS) expression in
the substantia nigra of MPTP-treated mice (Fig. 7, A to D, and fig.
S8D). Similarly, in En1+/− mice, treatment with MSDC-0160 at the
modest pathology stage attenuated expression of Iba-1, GFAP, and
iNOS in the ventral midbrain, as assayed by immunoblot (Fig. 7, E
to G, and fig. S8F). In addition, immunohistochemical analysis for
Iba-1 and GFAP in sections through the substantia nigra of En1+/−
mice showed that MSDC-0160 reduced elevated astrogliosis and mi-
crogliosis (Fig. 7H and fig. S8G).
To determine in a less complex model whether modulation of
MPC directly affected inflammatory cells, we moved to a microglia
cell line. As expected (see above), we observed expression of MPC-
1 and MPC-2 in mouse BV2 microglia cells (fig. S8, A and B) (61).
To induce inflammation in these cells, we used pretreatment (1 hour)
with lipopolysaccharide (LPS; 1 mg/ml), which is an endotoxin that in-
duces inflammation in vitro and in vivo. We found that in BV2 cell ly-
sates, 10 mM MSDC-0160 blocked LPS-induced increases in iNOS
expression (fig. S8C). We also confirmed our findings in mouse primary
microglia cells. After harvesting primary microglia (62), we used immu-
nofluorescence to determine that MSDC-0160 pretreatment (1 hour)
blocked LPS-induced iNOS expression (Fig. 7I). Next, we performed a
functional Griess assay on supernatant from the treated BV2 cells to de-
tect the concentration of nitrite and found that MSDC-0160 blocked the
LPS-induced increase in nitrite (Fig. 7J). Promoter regions of proinflam-
matory molecules contain the DNA binding site for nuclear factor kB
(NF-kB), a transcription factor involved in inflammatory responses. The
inhibition of NF-kB activation reduces the induction of proinflamma-
tory molecules in PD animal models (63). Therefore, we monitored
whether MPC modulation blocked the entry of p65 into the nucleus.
Immunofluorescence of BV2 cells stained with antibodies against Iba-
1 and phosphorylated (Ser276) p65 showed that pretreatment with
MSDC-0160 prevented nuclear accumulation of phosphorylated p65
(Fig. 7K). Consistent with the immunofluorescence data, we found
increased expression of phosphorylated (Ser276) p65 in the nuclear frac-
tion of BV2 microglial cells (Fig. 7L). Next, we analyzed supernatants
from BV2 cells on a Meso Scale platform to detect multiple cytokines.
We determined that MSDC-0160 pretreatment lessened the LPS-related
induction of expression of IL-1b (Fig. 7M), TNF-a (Fig. 7N), and IL-6
(Fig. 7O). Together, our results from BV2 cell cultures indicated that
modulating MPC by MSDC-0160 directly in microglia prevented in-
flammation induced by LPS.
Consistent with the direct effect of MSDC-0160 on mitochondrial
function in LUHMES cells subjected to MPP+ (fig. S2), MSDC-0160
normalized oxygen consumption in LPS-activated BV2 cells when
added 1 hour before or after LPS addition, demonstrating its immediate
action on mitochondrial function (fig. S9A). Also consistent with the
effects in the LUHMES neuronal cultures (fig. S3, A and B), no early
changes (2 and 6 hours) in the p-mTOR/mTOR ratio could be detected
Ghosh et al., Sci. Transl. Med. 8, 368ra174 (2016) 7 December 2016
in LPS-activated BV2 cells treated with MSDC-0160. A reduction in
mTOR activation caused by LPS was only observed after 24 hours of
incubation with MSDC-0160 (Fig. 7P and fig. S9, B and C). Subse-
quently, LPS-induced activation of S6 was also attenuated by
MSDC-0160 in BV2 cells (Fig. 7Q). However, no changes in the
AKT pathway (pAKT/AKT ratio) were detected at any time point
(fig. S9, B and C). This supports the notion that the anti-inflammatory
effects of modulating MPC are downstream of the immediate effects
on metabolism. Modulation of MPC had anti-inflammatory
consequences in cellular and mouse models of inflammation and
PD. The earliest measured effects involved direct modulation of pyr-
uvate metabolism and protection of oxidative metabolism followed by
changes in the mTOR/AKT pathways (increases in mTOR phospho-
rylation while AKT activation was reduced).
DISCUSSION
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We show that modulating the MPC consistently normalized metabo-
lism and downstream molecular pathways that are dysregulated in a
range of cell, nematode, and mouse models of PD (both toxin-induced
and genetic). As a consequence, autophagy and neuroinflammatory
changes were normalized, and survival of substantia nigra dopamin-
ergic neurons was promoted.
Modulation of MPC function has been tested as a therapeutic stra-
tegy in diabetes. The disease processes in diabetes and PD share sev-
eral features, with metabolic alterations and inflammation being
common to both diseases (35–38, 64) and specific metabolic genes be-
ing linked to PD (64). Attempts to modify the course of PD with an
antidiabetic agent of the TZD class (pioglitazone) have yielded
conflicting results, possibly because of inadequate engagement of the
target in the brain (8, 32). A safety and efficacy trial of the antidiabetic
agent, exendin-4, a glucagon-like peptide-1 agonist, suggested a reduc-
tion in motor and cognitive decline in PD patients, which was main-
tained even 12 months after cessation of treatment (65, 66). These
findings tentatively support the idea that disease mechanisms in diabe-
tes and PD have common features, warranting further exploration of
molecular therapeutic targets that could be common to the two diseases.
Here, modulation of MPC by MSDC-0160 normalized mTOR ac-
tivity in response to multiple insults (Figs. 5 and 6). mTOR signaling is
known to be activated in clinical samples from PD patients (67, 68).
Aberrant mTOR signaling is also found in several models of PD, in-
cluding the MPTP toxin rodent model (69–71) and recently in the
En1+/− genetic mouse model (53). The mTOR pathway interacts with
a stress response protein, RTP801/REDD1, and has been suggested to
control neuronal death in PD (72). Rapamycin, which is an inhibitor
of mTOR signaling, promotes longevity, protects dopaminergic and
other neurons, and stimulates autophagy in the MPTP mouse model
of PD (72). In the mTOR signaling complex 1 (mTORC1), mTOR is
predominantly phosphorylated on Ser2448, whereas in the mTORC2, it
is predominantly autophosphorylated on Ser2481. In our PD models,
we observed increased phosphorylation of mTOR at Ser2448 and
Ser2481. We found that MSDC-0160 reduced phosphorylation at
Ser2448, but not at Ser2481, mimicking the effects of rapamycin treatment,
which inhibits signaling by mTORC1 but not mTORC2. The mTORC1
complex also phosphorylates the ribosomal protein p70S6kinase, an
AGC kinase family member, on its hydrophobic motif site, Thr389 (73).
Our studies showed that the En1+/− mouse exhibits greater phos-
phorylation of p70S6kinase and its downstream target molecule ribo-
somal S6 and that modulation of MPC reduced the phosphorylation
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Fig. 7. MSDC-0160 attenuates inflammation in animal models and in mouse microglial cells. (A) Representative Western blot of Iba-1 and iNOS in the SN. Bar graphs
show mean Iba-1/b-actin (B) and mean iNOS/b-actin in SN (C). (D) Iba-1 immunostaining in SN. Bar graph shows number of Iba-1–positive cells in SN. (E) Representative blot of
Iba-1 and iNOS in SN. Bar graph shows mean Iba-1/b-actin (F) and mean iNOS/b-actin in SN (G). (H) Iba-1 immunostaining in SN. Bar graph shows number of Iba-1–positive
cells in SN. (I) Immunocytochemistry of Iba-1 and iNOS in primary mouse microglial cells. (J) Nitrite measurement and (K) immunocytochemistry of Iba-1 and NF-kB–p65 in BV2
mouse microglial cells. (L) Representative Western blot of NF-kB–p65 from cytosol and nucleus in BV2 cells. Concentration of interleukin-1b (IL-1b) (pg/ml) (M), tumor necrosis
factor–a (TNF-a) (pg/ml) (N), and IL-6 (pg/ml) (O) in BV2 cells. Immunocytochemistry of Iba-1 and p-mTOR (P) and Iba-1 and pS6 (Q) in BV2 cells. Scale bars, 50 mm (D, H, and I),
5 mm (K, P, and Q). (A to H) Data are means ± SEM of six to eight mice per group. (I to Q) Data are means ± SEM (n = 3 experiments). ****P < 0.0001, ***P < 0.001, **P < 0.01, *P <
0.05. Data were analyzed by two-way ANOVA with Bonferroni’s multiple comparison test.

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SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE
to wild-type levels. We also observed increased expression of REDD1
protein and inhibition of AKT phosphorylation at Thr308 in the En1
+/−
mice. These changes are all relevant to the pathophysiology of PD
because dopaminergic neurons in PD exhibit down-regulation of the
Ser473- and Thr308-phosphorylated forms of AKT (67). REDD1, which
functions upstream of AKT, is induced during the neurodegenerative
process (72). Modulation of MPC by MSDC-0160 reduced REDD1
(fig. S7, C and F). Although this could be due to attenuation of
p70S6kinase activation, REDD1 was increased in response to multiple
stresses, and it is likely that the upstream modulation of stress path-
ways is a component of the metabolic modulation.
Part of the metabolic modulation by MSDC-0160 in all of the
models we examined included increased phosphorylation of AKT at
Thr308. Although the precise mechanism underlying this change is not
clear, reduction in endoplasmic reticulum stress might play a role (74).
Some of the effects we observed after modulation of MPC function
were consistent with previous work using mTOR inhibitors (72). No-
tably, whereas those inhibitors directly inhibit mTOR, the action of
MSDC-0160 is on upstream metabolic changes deriving from the
change in the mitochondrial handling of pyruvate (20, 21, 29). Thus,
the effects of MSDC-0160 are mainly to prevent the activation of
mTOR produced by the metabolic changes rather than to directly in-
hibit mTOR kinase activity. This is consistent with our observations of
an immediate effect of MPC modulation on pyruvate utilization and ox-
ygen consumption, followed by a delayed inhibition of the mTOR
pathway.
It is well established that mTOR signaling is intricately tied to au-
tophagy, which is a conserved homeostatic process by which un-
wanted cellular components are degraded by the lysosome (75).
Several studies have implicated changes in the lysosome autophagy
pathway in both idiopathic and certain rare genetic forms of PD
(76, 77). By boosting lysosomal biogenesis and inducing autophagy,
rapamycin restores normal lysosomal activity and mitigates dopamin-
ergic neurodegeneration in the MPTP mouse model of PD (78). We
show here that MSDC-0160 attenuated the activation of mTOR and
its downstream signaling pathway in the subacute MPTP mouse
model (Fig. 5, I to K). Similarly, we also found down-regulation of
mTOR signaling along with reduced LC3b and p62 expression in
En1+/− mice (Fig. 6 and fig. S6) and that modulation of MPC in this
slowly progressing mouse model of PD reversed these changes. Together,
we found that modulation of MPC normalized autophagy in multiple
models of PD, which was likely to be key for preserving the nigral
dopaminergic neurons.
Autophagic signals modulate inflammatory pathways (79–81), and
neuroinflammation is an integral part of the pathogenesis of PD (82–84).
Neuroinflammation was not only evident after LPS or MPTP treat-
ment but was also found in the substantia nigra of En1+/− mice.
We hypothesized that modulation of MPC influences inflammatory
pathways. We found that modulation of MPC mitigated inflammation
in all of the models we examined. In both MPTP-treated mice and
En1-deficient mice, modulation of MPC attenuated the activation of
GFAP-positive astrocytes and Iba-1–positive microglia. The reduced
inflammatory response was likely not just the result of reduced neu-
rodegeneration but also the result of direct effects of MSDC-0160 on
the glial cells. We deduced this from our observations in cultured
mouse BV2 cells, in which we found that modulation of MPC by
MSDC-0160 blocked nuclear transport of p65 and reduced cytokine
release in response to LPS administration. Given that mTOR also
plays a crucial role in the regulation of the innate immune system
Ghosh et al., Sci. Transl. Med. 8, 368ra174 (2016) 7 December 2016
(85, 86), and because mTOR inhibitors can inhibit release of inflam-
matory cytokines from activated macrophages (85, 86), we speculated
that MPC modulation would also mitigate mTOR activation in stimu-
lated microglia. In LPS-stimulated BV2 cells, exposure to MSDC-0160
caused a down-regulation of p-mTOR (Ser2448) and its target mole-
cule, pS6 (Fig. 7). As was the case for neurons, these effects were tem-
porally downstream from the direct effects of mitochondrial
metabolism in the microglia. These results are key because they
showed that MPC was engaged in both neurons and glial cells and
that down-regulating the mTOR signaling pathway was a consequence
in both cell types. On the basis of our observations, we propose that
strategies that modulate MPC can have dual beneficial effects in PD,
by both improving autophagy in neurons and reducing microglia
activation (fig. S10). We suggest that modulation of MPC by MSDC-
0160 reduced the effects of PD-causing insults, whether a toxin, a mis-
folded protein (a-synuclein), or an En1+/− mutation. The direct interaction
of MSDC-0160 with the MPC complex has recently been modeled,
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showing a potentially unique pattern of interaction for both compo-
nents of the complex (87). Downstream events may involve specific
substrates, posttranslational modifications, and redox signals.
There are limitations to our study. It is not yet clear exactly how
modulation of MPC results in the downstream changes that we have
documented. In the rodent PD models, we cannot determine whether
the neuroprotection was primarily the consequence of modulation of
MPC in the neurons or the microglia, or both, because our cell culture
studies clearly demonstrated that the MPC modulator MSDC-0160
could positively affect either cell population when they are grown in
isolation. Similar to what has been published in flies (22), we found
that knockdown of MPC-1 in the worm model blocked the response
to MSDC-0160, although the knockdown itself did not protect against
neurodegeneration. This illustrates a difference between modulating
MPC by reversible inhibition and completely diminishing its activity
by genetic manipulation. Notably, although the protective effects
produced by MSDC-0160 treatment were consistent in all of the
PD models we tested, some dopaminergic neurons still died in the
different PD models, suggesting that it may not be possible to fully
prevent neurodegeneration by MPC modulation alone.
Antidiabetic agents have recently been proposed as possible disease-
modifying therapies for PD (36, 88). Diabetes, insulin resistance, inflam-
mation, and PD are all linked (89), and PD patients exhibit an increased
prevalence of diabetes (90). A recent phase 2, multicenter study using
pioglitazone, a member of the first generation of TZDs, failed to show a
disease-modifying effect in PD (8). A retrospective epidemiological
study, however, determined that persons with diabetes with a prescrip-
tion for a TZD insulin sensitizer drug were less likely to develop PD
(32), suggesting that intervention with TZD insulin sensitizers early
in the PD disease process, or its prodromal phase, would be more ef-
fective. First-generation TZDs, such as pioglitazone, not only modu-
late the MPC complex. In contrast to MSDC-0160, they are also direct
activators of the nuclear transcription factor PPARg, which drives
dose-limiting side effects (29, 91). Consequently, the safety profile of
MSDC-0160 is more favorable, which allows dosing to higher expo-
sures than achieved with pioglitazone and thus makes it more likely to
significantly engage the target MPC in the brain. The safety of MSDC-
0160 has been demonstrated in people with diabetes and Alzheimer’s dis-
ease during 3 months of exposure (41, 42), with tentative evidence of
target engagement in the brain from [18F]deoxyglucose imaging scans.
In these trials, exposure to circulating MSDC-0160 and its metabolite
was 50% higher than could be achieved with the highest approved dose
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of pioglitazone (45 mg, once daily). In response to MSDC-0160, hemo-
globin A1c and fasting blood glucose in diabetic patients fell to levels
similar to those of people taking pioglitazone, but fluid retention and
other side effects were less prominent with MSDC-0160 treatment (41).
Given the effects of MPC modulation by MSDC-0160 in several
different PD models in this study and its favorable safety profile in
humans, we believe that targeting MPC in PD is warranted. MPC as
a target is attractive because it affects multiple processes that are im-
plicated in PD pathogenesis, including autophagy and neuroinflam-
mation, through actions on both neurons and glia. These findings
should stimulate the development of other MPC modulators as
potential disease-modifying therapeutic agents in PD and related
neurodegenerative disorders.
MATERIALS AND METHODS
Study design
The goal of this study was to establish evidence, using multiple PD
models, that modulation of the mitochondrial pyruvate carrier
complex via MSDC-0160 is a viable approach to modify disease pro-
gression in PD. All experiments were blinded. Typically, one individ-
ual would randomize the animals, plates, and slides, and another
would analyze them. The minimum sample size for all experiments
was held at six mice per group based on the design of previous studies
(51, 92). To improve our power, and thus our ability to statistically
detect smaller effects, many of our analyses included more rodents
per group and/or repeated measures. Further experimental details
and protocols of each model, including animal care/handling and
the number of biological/technical replicates, are in this section, in
the Supplementary Materials, and in the figure legends.
C. elegans strains and protocols
C. elegans strains were cultured and maintained on nematode growth
medium containing a lawn of OP50 bacteria at 20° or 16°C using
established techniques (93). The transgenic strain BY250 (vtIs7
[pDAT::GFP(pRB490)]) was provided by the laboratory of R. Blakely
(Florida Atlantic University, Jupiter, FL). The strain TU3401 (sid-1(pk3321)
V; uIs69[pCFJ90(myo-2p::mCherry) + unc-119p::sid-1] V) was obtained
from the Caenorhabditis Genetics Center, which is funded by the Na-
tional Institutes of Health Office of Research Infrastructure Programs
(P40 OD010440). The a-synuclein–expressing strain JVR107 was
produced by transforming a Pdat-1::a-syn (A53T) construct into wild-
type C. elegans (N2 Bristol strain), generated by T. Iwatsubo (University
of Tokyo, Japan). Additional strains were generated by crossing these
strains to generate JVR203 (Pdat-1::a-syn(A53T), Pges-1::RFP; vtIs7
[Pdat-1::GFP(pRB490)]), JVR325 (Pdat-1::a-syn(A53T), Pges-1::RFP;
vtIs7[Pdat-1::GFP(pRB490)]; sid-1(pk3321) V; uIs69[pCFJ90(myo-2p::
mCherry) + unc-119p::sid-1] V) and JVR326 (vtIs7[Pdat-1::GFP
(pRB490)]; sid-1(pk3321) V; uIs69[pCFJ90(myo-2p::mCherry) + unc-
119p::sid-1] V).
C. elegans MPP+ neurodegeneration assay
Worms were synchronized, and L1 larvae (BY250 strain, GFP expres-
sion in dopaminergic neurons) were placed in 96-well plates
containing OP50 bacteria in liquid culture (6 mg/ml) and MPP+
(0.75 mM) with MSDC-0160 (1, 10, or 100 mM). Wells containing
no MPP+ [substituted with water or vehicle (methylcellulose)] and
with MSDC-0160 alone were also included. Because in these
experiments worms were soaked in solution containing MSDC-0160
Ghosh et al., Sci. Transl. Med. 8, 368ra174 (2016) 7 December 2016
of varying concentrations, the exact dose ingested by the worm cannot
be accurately calculated. Therefore, the maximum concentration that
had a neuroprotective effect was used in subsequent experiments to
ensure efficacy. About 50 worms were added per well, and the plate
was incubated at 20°C for 48 hours. After 48 hours, worms were moved
and allowed to recover on unseeded nematode growth medium plates
for several minutes before being mounted on an agarose pad with 3 mM
levamisole and analyzed microscopically. Each worm was scored for
the presence of GFP-fluorescent dopaminergic neurons in the anterior
of the worm (CEP and ADE neurons). A minimum of 25 worms were
analyzed for each condition in three independent trials.
Measurement of brain and plasma drug concentrations
C57BL/6J mice were orally dosed with MSDC-0160 (30 mg/kg)
suspended in 1% low-viscosity methylcellulose with 0.01% Tween
80 in distilled water (10 ml/kg) and then euthanized 2 and 4 hours
later. Plasma and whole-brain homogenates were extracted and
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subjected to LC/mass spectrometry measurement of both active
drug and active alcohol metabolite (MSDC-0037), as previously de-
scribed (41).
MPTP mouse model
Ten- to 12-week-old male C57BL/6J mice weighing 24 to 28 g were
housed under standard conditions: constant temperature (22°C), hu-
midity (relative, 30%), and a 12-hour light/dark cycle, with free access
to food and water. Procedures were performed during daylight hours
and were approved and supervised by the Institutional Animal Care
and Use Committee at the Van Andel Research Institute. Mice were
administered MSDC-0160 (30 mg/kg) by oral gavage beginning 24 hours
before MPTP (Sigma-Aldrich) treatment. Next, mice received five
consecutive doses of MPTP via intraperitoneal injection at 25 mg/kg
per day (subacute regimen) along with coadministration of MSDC-
0160, followed by 6 days of MSDC-0160 treatment. MSDC-0160
was dissolved in 1% methylcellulose with 0.01% Tween 80. Control
mice received vehicle treatment (1% methylcellulose with 0.01%
Tween 80). In the modest pathology stage, mice were administered
MSDC-0160 (30 mg/kg per day) by oral gavage for 7 days starting
3 days after MPTP (25 mg/kg per day) treatment. Seven days after
MPTP treatment, mice were euthanized, and tissues were pro-
cessed for further evaluation. Mice were randomized to the exper-
imental groups.
En1+/− mouse model
The En1+/− heterozygous mice were maintained on an OF1 genetic
background (52) and under the same conditions as the C57BL/6J
mice. In the mild pathology stage, En1+/− mice were fed a diet of chow
formulated to deliver MSDC-0160 (30 mg/kg) starting at 3 weeks of
age (at this point, no nigral cell death is detectable) and were eutha-
nized at two different time points: 28 and 48 weeks. In the modest
pathology stage, En1+/− mice were fed a diet of chow formulated to
deliver MSDC-0160 (30 mg/kg) starting at 8 weeks of age (at this
point, 15 to 20% nigral cell death is detectable) and were euthanized
at either 16 or 28 weeks. Mice were randomized to the groups, and
control mice were fed regular chow.
Behavioral experiments
Spontaneous activity data were collected and analyzed by an ANY-maze
analyzer (Stoelting). The dimensions of the activity chamber (San Diego
Instruments) were 109 cm by 109 cm by 38 cm. Before any treatment,
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mice were placed daily inside the chamber for 10 min for two consec-
utive days. Open-field activities were recorded for 10-min test sessions.
We evaluated a total of seven parameters: total distance traveled, mean
speed, maximum speed, time frozen, time mobile, time immobile, and
head distance traveled. For the rotarod experiment, a speed of 18 rpm
for C57BL/6J mice and 10 rpm for En1+/− mice was used. Before testing,
each mouse was trained on the rotarod for 5 min on three consecutive
days. Mice were given a 7- to 10-min rest between rotarod recording
sessions. All behavior experiments were conducted in a blinded fashion.
Measurement of fluorescence intensity and densitometry
A total of three to four sections per brain containing the substantia
nigra and three mice per group were stained with antibodies directed
against TH, p-mTOR, pS6, and LC3b. After immunofluorescence
staining, we took five 40× images from each section, blind-coded
them, and used the NIS-Elements AR 4.00.08 software (Nikon) to
quantify mean intensity of fluorescence of p-mTOR, pS6, LC3b, and
TH in TH-immunoreactive neurons, as well as the number of LC3b-
immunoreactive punctae in TH-positive nigral neurons (53).
HPLC analysis for striatal monoamine detection
Striatal levels of dopamine and DOPAC were quantified as described
previously (48). Briefly, striata tissues were collected, immediately fro-
zen on dry ice, and stored at −80°C until analysis. On the day of anal-
ysis, tissues were sonicated in 0.2 M perchloric acid containing
isoproterenol (internal standard), and the homogenates were centri-
fuged at 20,000g for 15 min at 4°C. Dopamine and DOPAC were
separated isocratically in a C18 reversed-phase column using an HPLC
system with an automatic sampler equipped with a refrigerated tem-
perature control and electrochemical detector (Thermo Fisher Scientif-
ic). Data acquisition and analysis were performed using the Chromeleon
HPLC Software.
Pyruvate oxidation assay
The effect of MSDC-0160 on oxygen consumption rates in brain
mitochondria was analyzed using MitoXpress Xtra (Luxcel). This flu-
orescence reagent, which is quenched by oxygen, allows for the direct,
real-time analysis of oxygen consumption. Mitochondria were
prepared by differential centrifugation of freshly prepared Sprague-
Dawley rat brain homogenates and resuspended in respiration buffer
containing 250 mM sucrose, 15 mM KCl, 1 mM EGTA, 5 mM MgCl2,
and 30 mM K2HPO4 (pH 7.4). MSDC-0160 concentration curves (0,
0.1, 0.3, 1.0, 3.0, and 10 mM) were prepared in dimethyl sulfoxide
(0.2% final) and assayed in duplicate with 50 mg of mitochondrial pro-
tein per well in a 96-well format (Corning). Malate (5 mM)/pyruvate
(5 mM)/adenosine 5′-diphosphate (1 mM), determined empirically
to be optimal substrate concentrations, were included in the reac-
tion. Time-resolved fluorescence (excitation/emission, 380/645; time-
resolved 30-ms delay; 100-ms read) was measured over a 2-hour time
course on a BioTek Synergy 2 plate reader. Respiration curves and
kinetic analysis were conducted using R version 3.2.2 and GraphPad
Prism software, respectively.
Seahorse assay for cellular oxygen consumption
Basal cellular oxygen consumption was measured using the Seahorse
XFe96 Analyzer. Two days before experiments, BV2 cells were plated
at 1.5 × 105 cells/ml (seeding density) in Seahorse 96-well utility plates
and supplemented with RMPI 1640 medium. LUHMES cells were
plated at 5 × 105 cells/ml (seeding density) in Seahorse 96-well utility
Ghosh et al., Sci. Transl. Med. 8, 368ra174 (2016) 7 December 2016
plates and supplemented with Advanced Dulbecco’s modified Eagle’s
medium/F12 medium. At 24 hours before the assay, 10 mM MPP+ or
10 mM LPS was added to appropriate wells, and Seahorse probes were
hydrated with Seahorse calibrant solution (pH 7.4). The day of the
assay, medium was removed and replaced with Seahorse media (pH
7.4). One hour before the assay, cells were treated with 10 mM MSDC-
0160 (posttreatment conditions). Cells were then washed twice in Sea-
horse medium and calibrated in a 37°C non-CO2 incubator for 1 hour.
Oxygen consumption was measured twice basally. Injections of vehi-
cle, 10 mM MSDC, or 10 mM MPP+/LPS (pretreatment conditions)
were then added, and six oxygen consumption rate measurements
of 3 min each were taken in a blinded manner. Measurements were
normalized to 50,000 cells per well. A total of 15 wells per condition
were measured, and three independent experiments for each cell line
were performed.
Statistical analysis
Downloaded from http://stm.sciencemag.org/ on December 7, 2016
Except for the behavioral data from En1+/− mice, data were analyzed
with Prism 3.0 software (GraphPad Software). Raw data were first
analyzed using either one-way or two-way ANOVA, and then either
Tukey’s or Bonferroni’s multiple comparison test was performed to
compare all treatment groups. Differences with P < 0.05 were
considered significant. Linear mixed-effects models (LMMs) (94) were
used to determine whether the interaction between the En1+/− geno-
type and the MSDC drug treatment was significant.
There are two major advantages to using LMMs: first, they appro-
priately adjust for repeated measures but allow for the inclusion of
mice without repeated measures, unlike repeated-measures ANOVA;
second, LMMs adjust for a portion of the within-subject variation via
random effects. Both of these features should result in superior model
performance and accuracy (95). Each LMM fit included independent
intercepts to adjust for some of the within-subject variation observed
between mice at the initial time points. Random slopes were also
considered, but there were not enough data to include this effect.
Analyses for each outcome were stratified by the week in which
chow treatment was initiated (that is, 3 or 8 weeks). For each outcome,
the LMMs were first fit with a three-way interaction between genotype
(wild-type/En1+/−), time point (28 and 44 weeks or 16 and 28 weeks
for 3- and 8-week treatment start times, respectively), and treatment
(control/MSDC). If this interaction was not found to be significant,
then it was removed from the model, and the model was then refit,
including all two-way interactions (that is, genotype × treatment,
genotype × time, and treatment × time). If the genotype × time inter-
action was not significant, then it was removed; likewise, for the treat-
ment × time interaction. The genotype × treatment interaction was
retained regardless of significance because this interaction pertained
to one of the primary questions of interest, namely, does MSDC-
0160 improve the En1+/− measures. Thus, the minimal final model
included at least the following variables: genotype, treatment, time,
and the genotype × treatment interaction.
An FDR correction was used to adjust all of the P values calculated
by the 18 LMMs. FDR corrections are less reliant upon the need for all
tests to be independent (96), which is particularly important because
many of our outcome measurements are highly correlated and, thus,
not independent.
Observations were determined to be outliers if they were more
than 3 SDs away from the mean of the residuals. All observations that
met this criterion were investigated to ensure that the data were recorded
accurately (for example, not miskeyed) and that the measurements were
14 of 17
SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE
within the realm of plausibility. Measurements found to have record-
ing errors were corrected. No observations were removed. All LMM
analyses were performed using R version 3.2.0 (97).
SUPPLEMENTARY MATERIALS
www.sciencetranslationalmedicine.org/cgi/content/full/8/368/368ra174/DC1
Materials and Methods
Fig. S1. MSDC-0160 protects dopaminergic neurons against MPP+-induced toxicity in LUHMES
cells and C. elegans.
Fig. S2. Modulation of MPC improves mitochondrial function.
Fig. S3. Time-course treatment of MSDC-0160 in LUHMES cells.
Fig. S4. MSDC-0160 improves behavioral parameters in the MPTP mouse model.
Fig. S5. MSDC-0160 improves behavioral activities in the En1+/− mice.
Fig. S6. MSDC-0160 prevents dopaminergic neurodegeneration in the En1+/− mice.
Fig. S7. MSDC-0160 down-regulates mTOR signaling pathway and restores autophagy in the
En1+/− mice.
Fig. S8. MSDC-0160 attenuates inflammation in BV2 cells and in vivo animal models.
Fig. S9. MSDC-0160 improves mitochondrial function and blocks mTOR activation in BV2 cells.
Fig. S10. Schematic diagram of proposed mechanism of action of MSDC-0160 in glial cells and
dopaminergic neurons.
Table S1. LMM results.
Reference (98)
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Acknowledgments: We thank R. Wyse, Cure Parkinson’s Trust, for helpful discussions. We
acknowledge the technical expertise of C. Cole, D. Dues, L. Kefene, and D. Marckini. We also
acknowledge excellent service from the Vivarium Core and the Biostatistics and Bioinformatics
Core of the Van Andel Research Institute. We thank D. Nadziejka for manuscript editing
assistance. We also thank J. Das and J. L. Leasure from the University of Houston for assistance
with the StereoInvestigator software and Western blot settings in their laboratories. Funding:
The work presented here was supported by Cure Parkinson’s Trust (to P.B.), Campbell
Foundation (to P.B.), Spica Foundation (to P.B.), and Van Andel Research Institute (to P.B. and
J.M.V.R.). Author contributions: A.G., J.R.C., J.M.V.R., and P.B. designed the study. A.G., T.T., S.G.,
E.N.H., E.M., E.S., and W.G.M. carried out the experiments: A.G. did all of the experiments except
the following: C. elegans studies (T.T.), microglia cultures (E.N.H.), Seahorse assay (E.M.),
drug pharmacodynamics (W.G.M.), and stereological cell counts in some experiments (E.S.).
A.G., T.T., S.G., E.N.H., Z.M., W.G.M., M.L.E.G., J.A.S., J.R.C., and J.H.K. performed analyses and
Downloaded from http://stm.sciencemag.org/ on December 7, 2016
figure preparation. A.G., J.A.S., T.T., Z.M., M.L.E.G., J.H.K., J.R.C., and P.B. wrote the manuscript.
Competing interests: JRC is the cofounder and significant owner of Metabolic Solutions
Development Company (MSDC), which is currently developing MSDC-0160 as a potential
treatment for Alzheimer’s disease. MSDC-0160 is protected by several patents, including US
8389556 B2 and EP 2001468 B1, which are assigned to MSDC. P.B. has received paid support as
a consultant from Renovo Neural Inc., Roche, Teva Pharmaceutical Industries, Lundbeck A/S,
AbbVie Inc., IOS Press Partners, and Versant Ventures. In addition, P.B. has received support for
his research from Renovo, Teva, and Lundbeck. P.B. has ownership interests in Acousort AB
and ParkCell AB. J.H.K. has received paid commercial support as a consultant from Cellular
Dynamics International Inc., Michael J. Fox Foundation, nLife Therapeutics S.L., NsGene,
Clintrex, NeuroDerm, and BrainEver. Data and materials availability: All enquiries regarding
MSDC-0160 should be directed to J.R.C.; MSDC-0160 will be made available through a material
transfer agreement.
Submitted 22 July 2016
Accepted 17 November 2016
Published 7 December 2016
10.1126/scitranslmed.aag2210
Citation: A. Ghosh, T. Tyson, S. George, E. N. Hildebrandt, J. A. Steiner, Z. Madaj, E. Schulz,
E. Machiela, W. G. McDonald, M. L. Escobar Galvis, J. H. Kordower, J. M. Van Raamsdonk,
J. R. Colca, P. Brundin, Mitochondrial pyruvate carrier regulates autophagy, inflammation,
and neurodegeneration in experimental models of Parkinson’s disease. Sci. Transl. Med. 8,
368ra174 (2016).
17 of 17
 Mitochondrial pyruvate carrier regulates autophagy,
inflammation, and neurodegeneration in experimental models of
Parkinson's disease
Anamitra Ghosh, Trevor Tyson, Sonia George, Erin N. Hildebrandt,
Jennifer A. Steiner, Zachary Madaj, Emily Schulz, Emily Machiela,
William G. McDonald, Martha L. Escobar Galvis, Jeffrey H.
Kordower, Jeremy M. Van Raamsdonk, Jerry R. Colca and Patrik
Brundin (December 7, 2016)
Science Translational Medicine 8 (368), 368ra174. [doi:
10.1126/scitranslmed.aag2210]
Editor's Summary

A mitochondrial target for slowing Parkinson's disease

Currently, there are no disease-modifying treatments to stall progression of Parkinson's disease
(PD). A drug in development to treat diabetes might provide a new way to slow the progression of PD
according to new work by Ghosh and colleagues. The drug, MSDC-0160, targets a recently identified

Downloaded from http://stm.sciencemag.org/ on December 7, 2016

carrier of pyruvate (a major substrate for energy production) into the mitochondria. Ghosh et al. now
show that this drug, which attenuates the mitochondrial pyruvate carrier, blocks neurodegeneration in
several different cellular and animal models of PD. Furthermore, cellular autophagy was restored, and
neuroinflammation was reduced in two mouse models of PD. These results support continued
investigations into whether the mitochondrial pyruvate carrier will be a useful therapeutic target in PD.

The following resources related to this article are available online at http://stm.sciencemag.org.
This information is current as of December 7, 2016.

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Supplemental "Supplementary Materials"

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 www.sciencetranslationalmedicine.org/cgi/content/full/8/368/368ra174/DC1

Supplementary Materials for

Mitochondrial pyruvate carrier regulates autophagy, inflammation, and
neurodegeneration in experimental models of Parkinson’s disease
Anamitra Ghosh, Trevor Tyson, Sonia George, Erin N. Hildebrandt, Jennifer A. Steiner,
Zachary Madaj, Emily Schulz, Emily Machiela, William G. McDonald,
Martha L. Escobar Galvis, Jeffrey H. Kordower, Jeremy M. Van Raamsdonk,
Jerry R. Colca, Patrik Brundin*

*Corresponding author. Email: patrik.brundin@vai.org

Published 7 December 2016, Sci. Transl. Med. 8, 368ra174 (2016)

DOI: 10.1126/scitranslmed.aag2210

This PDF file includes:

Materials and Methods

Fig. S1. MSDC-0160 protects dopaminergic neurons against MPP+-induced
toxicity in LUHMES cells and C. elegans.
Fig. S2. Modulation of MPC improves mitochondrial function.
Fig. S3. Time-course treatment of MSDC-0160 in LUHMES cells.
Fig. S4. MSDC-0160 improves behavioral parameters in the MPTP mouse model.
Fig. S5. MSDC-0160 improves behavioral activities in the En1+/− mice.
Fig. S6. MSDC-0160 prevents dopaminergic neurodegeneration in the En1+/−
mice.
Fig. S7. MSDC-0160 down-regulates mTOR signaling pathway and restores
autophagy in the En1+/− mice.
Fig. S8. MSDC-0160 attenuates inflammation in BV2 cells and in vivo animal
models.
Fig. S9. MSDC-0160 improves mitochondrial function and blocks mTOR
activation in BV2 cells.
Fig. S10. Schematic diagram of proposed mechanism of action of MSDC-0160 in
glial cells and dopaminergic neurons.
Table S1. LMM results.
Reference (98)
Supplementary Materials

Materials and Methods

Cell culture

Lund human mesencephalic (LUHMES) cells are a subclone of the tetracycline-

controlled, v-myc-over-expressing human mesencephalic-derived cell line MESC2.10

(ATCC, (43)). The LUHMES cells were plated and grown as described by (98 ). Briefly,

plastic cell culture flasks and multi-well plates were pre-coated with 50 μg/mL poly-L-

ornithine and 1 μg/mL fibronectin (Sigma-Aldrich, St. Louis, MO, USA). Cells were

grown at 37°C in a humidified 95% air, 5% CO2 atmosphere using proliferation medium

consisting of Advanced Dulbecco’s modified Eagle’s medium/F12, 1x N-2 supplement

(Invitrogen, Carlsbad, CA), 2 mM L-glutamine (Gibco, Rockville, MD, USA) and 40

ng/mL recombinant basic fibroblast growth factor (R&D Systems, Minneapolis, MN,

USA). Proliferating cells were enzymatically dissociated with trypsin and differentiated

using medium consisting of Advanced Dulbecco’s Modified Eagle’s Medium

(DMEM)/F12, N-2 supplement, 2 mM L-glutamine (all reagents were purchased from

Invitrogen, USA), 1 mM dibutyryl cAMP (Sigma-Aldrich), 1 μg/mL tetracycline (Sigma-

Aldrich) and 2 ng/mL recombinant human GDNF (R&D Systems). We used LUHMES

cells in all experiments at 5-6 d post-differentiation. First, LUHMES cells were pretreated

with 10 μM MSDC-0160 for 1 h followed by 10 μM 1-methyl-4-phenyl-

tetrahydropteridine (MPP+, Sigma-Aldrich) treatment for 24 h. Cells were collected and

processed for immunocytochemistry. For the time course experiments, LUHMES cells
were treated with 10 μM MSDC-0160 for 2, 6, or 24 h. Cells were collected at these time

points and processed for western blotting.

Primary mesencephalic neuronal cultures were prepared from the ventral mesencephalon

of gestational 12- to 13-day-old mouse embryos as described previously (44). Briefly,

mesencephalic tissues were dissected and maintained in ice-cold calcium-free Hanks

balanced salt solution (HBSS) and then dissociated in HBSS containing 0.25% trypsin-

EDTA for 20 min at 37°C. Cultures were maintained in Neurobasal medium with 50x B-

27 supplement, 500 mM L-glutamine, 100 IU/ml penicillin, and 100 μg/mL streptomycin

(all reagents were purchased from Invitrogen). The cells were maintained in a humidified

CO2 incubator (5% CO2 and 37°C) for 24 h. Five days post-culture, primary

mesencephalic neurons were pre-treated with 10 μM MSDC-0160 for 1 h followed by 10

μM MPP+ treatment for 24 h. Cells were collected and processed for

immunocytochemistry.

BV2 cells were purchased from ICLC (Catalog number ATL03001) (61). The media in

which BV2 cells proliferate contained RPMI 1640 supplemented with 10% heat-

inactivated FBS, 2 mM L-glutamine, 50 IU/mL penicillin, and 50 μg/ml streptomycin.

BV2 cells were plated at a density of 200 cells per mm2. After one day, FBS was

removed from the media and the cells were pretreated with 10 μM MSDC-0160. One

hour later, they were treated with 1 μg/mL lipopolysaccharide (LPS) (Escherichia coli

0111:B4, endotoxin content 6.6000000 EU/mg, Sigma-Aldrich). After 24 h of LPS

treatment, supernatant was collected for cytokine analysis or Griess assay and cells were

processed for immunocytochemistry. For the timecourse experiments, BV2 cells were
treated simultaneously with 10 μM MSDC-0160 and 1 μg/mL LPS for 2, 6, or 24 h. Cells

were collected at these time points and processed for western blotting.

For primary microglia isolation, we followed the procedure as described by (62). Briefly,

cortical tissues were dissected out from postnatal-day-3- to day-5-old mouse pups. Tissue

was dissociated using Miltenyi Biotec Neural Tissue Dissociation Kit (P, Miltenyi

Biotec, Bergisch-Gladbach, Germany). The single-cell suspension was incubated in

CD11b microbeads and separated using the magnetic column OctoMACS

Separator. Cells were then plated in 24-well dishes for treatments. Media for primary

microglia contains DMEM/F12 supplemented with 10% heat-inactivated FBS, 50 IU/ml

penicillin and 50 μg/ml streptomycin. Primary microglial cells were plated at a density of

200 cells per mm2. After one day, FBS was removed from the media and the cells were

pretreated with 10 μM MSDC-0160. One hour later, they were treated with 1 μg/mL

LPS. After 24 h of LPS treatment, supernatant was collected for cytokine analysis or

Griess assay and cells were fixed for immunocytochemistry.

C. elegans α-syn-induced neurodegeneration assay and RNAi functional analysis

Worm strains BY250 (GFP expression in dopaminergic neurons) and JVR203 (GFP + α-

syn (A53T) expression in dopaminergic neurons) were synchronized to L4 stage and

grown in liquid culture (6 mg/mL OP50, 120 μM FUDR) treated or untreated with 100

μM MSDC-0160 for 12 d. After this time, worms were scored for neurodegeneration as

described above. RNAi experiments were carried following an identical paradigm using

strains JVR325 and JVR326, in which RNAi is only effective in neuronal cells. Bacterial
RNAi clones were obtained from the Ahringer RNAi library (Source Bioscience). NGM

plates were supplemented with 100 µg/mL carbenicillin and 1 mM IPTG. Bacterial

clones were grown overnight in LB with 100 µg/mL carbenicillin. The following day,

IPTG was added to a final concentration of 1 mM and the bacteria were allowed to grow

(shaking at 37 °C) for another hour. 50 µL of culture was seeded onto each plate. Worms

were synchronized and 100 L1s were added to each NGM RNAi plate and incubated at

20 °C in the dark for 48 h. L4s/early adults were washed from the plates and 50 worms

per well were incubated at 20 °C in M9 with RNAi bacteria (6 mg/mL), FUDR (120 uM),

IPTG (1 mM) and either MSDC-0160 or vehicle (methoxy cellulose). Freshly grown

RNAi bacteria in LB (with 0.1 mM IPTG and 100 μg/mL carbenicillin) was added to the

wells every 3 d. Worms were removed from liquid culture and scored for

neurodegeneration as above after 10 d.

Knockdown of mRNA levels by RNAi was determined using single-worm RT-PCR.

cDNA was made from individual worms using the CellsDirectTM One-Step qRT-PCR

Kit (Invitrogen). qPCR was performed with Power SYBR green from Applied

Biosystems and cycled on an Applied Biosystems Step One Plus system. All reactions

were repeated multiple times: three technical replicates for each of two biological

replicates were performed and all data were normalized to act-1.

Immunocytochemistry

The LUHMES cells, primary mesencephalic neurons, BV2 cells, or primary microglia

cells were fixed with 4% paraformaldehyde in 1X PBS for 20 min and processed for

immunocytochemistry. First, nonspecific sites were blocked with 0.2% bovine serum
albumin, 0.5% Triton X-100, and 0.05% Tween 20 in PBS for 1 h at room temperature.

Cells were then incubated with different primary antibodies: TH (1:1600, EMD

Millipore, Billerica, MA, USA); Tuj-1 (1:1000, Abcam, Cambridge, MA, USA); iNOS

(1:300, Santacruz, Dallas, TX, USA); Iba-1 (1:800, WAKO, Richmond, VA, USA); p-

mTOR (1:300, Cell Signaling, Danvers, MA, USA); p-S6 (1:1000, Cell Signaling,

Danvers, MA, USA) at 4°C overnight. Appropriate secondary antibodies (Alexa Fluor

488 or 594, Invitrogen, Carlsbad, CA, USA) were used followed by incubation with

DAPI to stain the nucleus. The coverslip-containing stained cells were washed twice with

PBS and mounted on slides. Cells were viewed under a NIKON Eclipse Ni-U

fluorescence microscope (Nikon, Melville, NY, USA); images were captured with a

Retiga Exi digital camera using NIS Elements AR 4.00.08 software (Nikon).

Cytokine analysis

Supernatant from treated BV2 cells was collected and stored at -80°C until

analysis. Analysis was performed using the V-PLEX Proinflammatory Panel 1 (mouse)

kit from Meso Scale Discovery (Rockville, MA, USA). Calibration solutions and buffers

were prepared as described by the kit. Supernatants were thawed and 25 μL of sample

was diluted 1:1 with 25 μL Diluent 41 from the kit. Sample was loaded onto the reading

plate and sealed before incubation at RT for 2 h with shaking. After 3 washes with 150

μL wash buffer, 25 μL of detection antibody solution was added to each well. Again, the

plate was sealed and incubated at RT for 2 h with shaking. An additional 3 washes were

performed followed by the addition of 150 μL 2X Read Buffer T. The plate was then read

on the MesoScale QuickPlex SQ120.

Griess assay

Standards in triplicate were used for each plate. Standard mix was made up of 1 mL

media and 1 μL sodium nitrite and added to wells in volumes increasing by 5 μL from 0-

35 μL. Media was added to each well to bring volume up to a total of 100 μL. Sample

supernatant was added in triplicate to remaining wells in 100 μL amounts. Then, 100 μL

of Griess reagent (Sigma, USA) was added to each well and incubated at RT for 10

minutes on a rotator. Absorbance at 540 nm was detected in a Synergy NEO plate reader

(BioTek, Winooski, VT, USA).

Immunohistochemistry

Mice were anesthetized with sodium pentobarbital (130 mg/kg; Sigma) and perfused

transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were

removed, postfixed, and cryoprotected. Immunohistochemistry was performed on free-

floating cryomicrotome-cut sections (40 µm in thickness) encompassing substantia nigra

as described previously (51, 53). For tyrosine hydroxylase (TH)-immunohistochemistry

(IHC), free-floating sections were washed with 0.1 M PBS (pH 7.4), incubated with 3%

H2O2 for 10 min to quench endogenous peroxidase activity, and were blocked for 1 h

with 10% normal goat serum and 0.25% Triton X-100. Next, the sections were incubated

overnight at 4 °C with different antibodies: TH (1:1600, EMD Millipore); Iba-1 (1:800,

WAKO); GFAP (1:1000, EMD Millipore). Then, the sections were incubated with

appropriate biotinylated secondary antibodies (goat anti-rabbit, goat anti-mouse, 1:500,

Vector Laboratories, Burlingame, CA, USA) followed by incubation with Avidin/Biotin

ABC reagent (Vector Laboratories). Immunolabeling was revealed using

diaminobenzidine (DAB) which yielded a brown-colored stain visible in bright-field light

microscopy. For double-labeling immunofluorescence, the sections were blocked with

0.2% bovine serum albumin, 0.5% Triton X-100, and 0.05% Tween 20 in PBS for 1 h at

room temperature. Next, sections were incubated with different antibodies: TH (1:1500,

EMD Millipore); p-mTOR (1:200, Cell Signaling); p-S6 (1:800, Cell Signaling); LC3B

(1:500, Cell Signaling), phosphorylated alpha-synuclein serine 129 (1:10000, Abcam).

Appropriate secondary antibodies (Alexa Fluor 488 or 594, Invitrogen) were used

followed by incubation with DAPI to stain the nucleus. The sections were viewed under a

Nikon Eclipse Ni-U fluorescence microscope (Nikon); images were captured with a

Retiga Exi digital camera using NIS Elements AR 4.00.08 software (Nikon).

Stereology and striatal optical density

For the purpose of cell counting, we stained sections with 0.1% cresyl violet. Stained

cells were counted using unbiased sampling and blinded stereology. Following TH-DAB

immunostaining, the TH-ir neurons were counted using unbiased sampling and blinded

stereology as mentioned previously (51, 53). Briefly, the substantia nigra region was

outlined based on the Allen mouse brain atlas (Allen Institute, Seattle, WA, USA) using a

2x objective. Quantitative estimation of TH-ir neurons was performed in every 6th

section of the substantia nigra using the optical fractionator probe in Stereo Investigator

software (MBF Bioscience, Williston, VT, USA). The parameters used include a 60× oil

objective, a counting frame size of 100 × 100, a sampling site of 140 × 140, a dissector

height of 18 μm, 2 μm guard zones, and the Schaffer second estimated co-efficient error

was less than 0.1. In addition, the Gunderson estimated coefficient error was less than
0.1. A total of 6 to 9 animals per group were used and 5 to 7 sections per animal were

counted. Quantification of striatal TH fibers was performed using NIS Elements AR

4.00.08 software (Nikon) in a blinded manner. Briefly, striatal pictures were first taken at

2x magnification, then thresholded. After that, the optical density of fibers was counted

using the NIS Elements AR 4.00.08 software (Nikon).

Western blotting

Cells or brain tissues were collected and resuspended in modified

radioimmunoprecipitation (RIPA) assay buffer containing protease and phosphatase

inhibitor mixture. Cell suspensions were sonicated after resuspension, whereas mouse

brain tissues were homogenized, sonicated, and then centrifuged at 14,000 x g for 45 min

at 4°C. Protein concentrations were estimated using a BCA kit (Thermo Scientific,

Waltham, MA USA). Lysates were separated on 7.5%–15% SDS-polyacrylamide

electrophoresis gels (Bio-Rad). After the separation, proteins were transferred to a

nitrocellulose membrane, and nonspecific binding sites were blocked by treating with

either Odyssey blocking buffer (LI-COR, Lincoln, NE, USA) or TBS with 5% bovine

serum albumin (BSA) followed by antibody incubation: TH (1:1500, EMD Millipore);

phospho (Ser2448)-mTOR (1:1000, Cell Signaling); phospho (Ser2481)-mTOR (1:1000,

Cell Signaling); mTOR (1:1000, Cell Signaling); phospho (Thr389)-p70S6 kinase

(1:1000, Cell Signaling); p70S6 kinase (1:1000, Cell Signaling); phospho (Thr308) AKT

(1:1000, Cell Signaling); AKT (1:1000, Cell Signaling); phospho (Ser235/236)-S6

(1:1500, Cell signaling); S6 (1:1500, Cell signaling); LC3B (1:500, Cell signaling);

REDD1 (1:2000, Bethyl Laboratories, Montgomery, TX, USA); p62 (1:500, Abcam);
iNOS (1:300, Santa Cruz); GFAP (1:1000, EMD Millipore); Iba-1 (1:500, WAKO);

NFκB p65 (1:300, Santa Cruz); and β-actin (1:10,000, Sigma). Secondary IR-680-

conjugated anti-mouse (1:10,000, donkey anti-mouse; Molecular Probes, USA), IR-680-

conjugated anti-goat (1:10,000, donkey anti-goat, Molecular Probes, USA), IRDye 800

donkey anti-rabbit (1:10,000, Rockland, Pottstown, PA, USA) were used. New blot

stripping buffer (LI-COR, USA) was used to reprobe the membrane. Western blot images

were captured with a LI-COR Odyssey machine (LI-COR, USA). The western blot bands

were quantified using ImageJ software (National Institutes of Health, USA). An antibody

that detects β-actin was used to visualize loading controls.

Supplementary Fig. 1

Supplementary Fig. 1. MSDC-0160 protects dopaminergic neurons against MPP+-

induced toxicity in LUHMES cells and C. elegans. Six days post-differentiation,

LUHMES cells were pre-treated with 10 μM MSDC-0160 for 1 h followed by 10 μM

MPP+ treatment for 24 h. (A) TH-diaminobenzidine immunocytochemistry in LUHMES

cells. Blue arrows show degenerating TH-ir dopaminergic neurons in the MPP+ treatment

group. C. elegans were synchronized and L1 larvae were placed in 96-well plates

containing OP50 bacteria in liquid culture (6 mg/mL) and MPP+ (0.75 mM) with MSDC-

0160 (1 μM, 10 μM and 100 μM) for 48 h. (B) GFP expressing dopaminergic neurons in

C. elegans. Numbering on the images (1-6) denotes individual dopaminergic neurons.

White asterisk denotes degenerating dopaminergic neurons. (C) Relative mRNA

expression of different genes in C. elegans. Data are means ± SEM (n=3 experiments).

Scale bars, 25 μm.

Supplementary Fig.
F 2

Supp
plementary Fig. 2. Mod
dulation of MPC
M improoves mitochoondrial funcction. Mice

were orally dosed

d with 30 mg
g/kg MSDC--0160 and thhen euthanizzed at 2 or 4 hours after

dosin
ng. The plasm
ma (black do
otted lines; ng/mL)
n and bbrain homoggenates (blacck continuouus

lines;; ng/g) were extracted an

nd analyzed by LC/MS ffor the conceentrations off both parentt

drug (A) and actiive alcohol metabolite

m (B
B). Data forr are means ± SEM (n=3
experiments). Effect of increasing concentrations of MSDC-0160 (one experiment run in

duplicate) on rat brain mitochondria incubated with MitoXpress Xtra reagent (described

in the Materials and Methods section). The concentrations of MSDC-0160 shown were

added at the start of the reaction which contained 5 mM pyruvate. (C) The loss of oxygen

from the medium was measured over time as the increase in relative fluorescence (RFU).

Mean RFU is plotted at each time point. LOESS curves with 95% confidence ribbons are

plotted over top to show smoothed behavior over time. (D) Calculated changes in the

initial rate of oxygen consumption and SEM at the given concentration of MSDC-0160.

Data for are means ± SEM (n=3 experiments). (E) Measurement of oxygen consumption

in differentiated LUHMES cells. Cells were either pre-treated with 10 μM MPP+ or

MSDC-0160 and oxygen consumption was measured basally and for 30 minutes after

injection of vehicle, MSDC-0160, or MPP+. Mean oxygen consumption rate is plotted

for each experimental group at each time point (n=3 experiments). LOESS curves with

95% confidence ribbons are plotted over top to show smoothed behavior over time.
Supplementary Fig. 3

Supplementary Fig. 3. Time-course treatment of MSDC-0160 in LUHMES cells. Six

days post-differentiation, LUHMES cells were treated with 10 μM MSDC-0160 for 2, 6

or 24 h. (A) Representative western blot of phospho-mTOR (Ser 2448), mTOR and β-

actin in LUHMES cells. (B) Mean western blot phospho-mTOR/mTOR ratios relative to

β-actin. Data are means ± SEM (n=3 experiments). Data were analyzed by one-way

ANOVA with Tukey’s multiple comparison test.

Supplementary Fig. 4

Supplementary Fig. 4. MSDC-0160 improves behavioral parameters in the MPTP

mouse model. (A) Treatment schedule of MPTP-injected mice with MSDC-0160 (Pre-

treatment of MSDC-0160). (B) Traced movement of mice using ANY-maze software.

(C) Stereological counting of cresyl violet (CV)-stained neurons in SN in the MSDC-

0160 pre-treatment schedule (D) Measurement of striatal MPP+ (ppb). (E) Treatment

schedule of MPTP-injected mice in which mice were administered MSDC-0160 (30

mg/kg/day) by oral gavage two days post MPTP treatment (25 mg/kg/day) and continued

the treatment for another 7 days (Post-treatment of MSDC-0160). Four days after the last

MPTP injection, mice were tested for spontaneous open field behavioral activities. (F)

Stereological counting of CV-stained neurons in SN in the MSDC-0160 post-treatment

schedule (G) Distance traveled (cm), time mobile (sec), time immobile (sec) and time

freeze (sec). Data are means ± SEM of seven to ten mice per group. ****p < 0.0001,

***p < 0.001, **p < 0.01, *p < 0.05. Data were analyzed by one-way ANOVA with

Tukey’s multiple comparison test except (D) which was analyzed by unpaired student’s t-

test.
Supplementary Figure
F 5

Supp
plementary Fig. 5. MS
SDC-0160 improves
i b
behavioral activities in
n the En1++/−

mice. At the milld pathology 1+/- mice weere fed a dieet of chow fformulated tto
y stage, En1

deliver MSDC-0160 (30 mg//kg) starting at week 3 an

and were testted for motoor functions aat
28 weeks and 44 weeks. (A) Time freeze (sec), (B) Time mobile (sec), (C) Time

immobile (sec), (D) Head distance traveled (cm). At the modest pathology stage, En1+/-

mice were fed a diet mixed with MSDC-0160 (30 mg/kg) starting at week 8 weeks and

were tested for motor functions at 16 weeks and 28 weeks. (E) Time freeze (sec), (F)

Time mobile (sec), (G) Time immobile (sec), (H) Head distance traveled (cm). Data are

means ± SEM of eight to twelve mice per group. ***p < 0.001, **p < 0.01, *p < 0.05,
o
p < 0.05-0.1. Data were analyzed using linear mixed-effects regression analysis false

discovery rate corrected p-value.

Supplementary Fig. 6

Supplementary Fig. 6. MSDC-0160 prevents dopaminergic neurodegeneration in the

En1+/− mice. (A) Stereological counting of cresyl violet (CV)-stained neurons in SN from
3W-28W group. (B) Stereological counting of CV-stained neurons in SN from 8W-16W

group. (C) Stereological counting of CV-stained neurons in SN from 8W-28W group. (D)

Representative western blot illustrating the expression of TH in striatum from 16W (top)

and 28W (bottom). (E) Bar graph showing mean western blot TH/β-actin ratios in ST at

16W. (F) Bar graph showing mean western blot TH/β-actin ratios in ST at 28W. Data are

means ± SEM of six to eight mice per group. ****p < 0.0001, ***p < 0.001, **p < 0.01,

*p < 0.05. Data were analyzed by two-way ANOVA with Bonferroni’s multiple

comparison test.
Supplementary Fig. 7

Supplementary Fig. 7. MSDC-0160 down-regulates mTOR signaling pathway and

restores autophagy in the En1+/− mice. En1+/- mice were fed a diet formulated to

deliver MSDC-0160 (30 mg/kg) starting at week 8 (8W) and were euthanized at week 16

(16W). Substantia nigra (SN) tissues were processed for western blotting. (A)

Representative western blot for MPC-1 and MPC-2 in SN. (B) Immunohistochemistry of

TH and MPC-1 in SN after treatment with MSDC-0160 or control in WT and En1+/-

mice. (C) Representative western blot illustrating the expression of phospho-mTOR (Ser

2481), mTOR, phospho-AKT (Thr 308), AKT, REDD1 and p62 in SN after treatment

with MSDC-0160 or control in WT and En1+/- mice. (D) Bar graph showing mean

western blot phospho-mTOR (Ser 2481)/mTOR ratios in SN relative to β-actin. (E) Bar

graph showing mean western blot phospho-AKT/AKT ratios in SN relative to β-actin. (F)
Bar graph showing mean western blot REDD1/β-actin ratios in SN at 16W. (G) Bar

graph showing mean western blot p62/β-actin ratios in SN at 16W. Scale bars, 5 μm. Data

are means ± SEM of six mice per group. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p

< 0.05. Data were analyzed by two-way ANOVA with Bonferroni’s multiple comparison

test.
Supplementary Fig. 8
Supplementary Fig. 8. MSDC-0160 attenuates inflammation in BV2 cells and in vivo

animal models. (A) Representative western blot illustrating the expression of MPC-1 and

MPC-2 in BV2 cells. (B) Immunocytochemistry of Iba-1 and MPC-1/MPC-2 in BV2

cells. (C) Western blotting of iNOS in BV2 cells. In the MPTP model, MPTP and

MSDC-0160 administration was accomplished as described in Materials and Methods.

(D) Representative western blot illustrating the expression of GFAP in SN. Bar graph

showing mean western blot GFAP/β-actin ratios in SN. (E) DAB immunostaining of

GFAP in SN. In the En1+/- model, MSDC-0160 was administered as described in

Materials and Methods. (F) Representative western blot illustrating the expression of

GFAP in SN. Bar graph showing mean western blot GFAP/β-actin ratios in SN. (G)

DAB immunostaining of GFAP in SN. Scale bars, 5 μm. (A-C) Data are means ± SEM

(n=3 experiments). (D-G) Data are means ± SEM of six to eight mice per group. ****p <

0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. Data were analyzed by two-way ANOVA

with Bonferroni’s multiple comparison test.

Supplementary Fig.
F 9

Supp
plementary Fig. 9. MSD
DC-0160 improves mitoochondrial ffunction and blocks

mTO
OR activatio
on in BV2 ceells. (A) Measurement oof oxygen consumption
n in BV2

cells.. Cells were either pre-trreated with 10

1 μM LPS oor MSDC-01160 and oxyggen

consu
umption wass measured basally
b and for
f 30 minut es after injecction of vehiicle, MSDC--

0160, or LPS. Mean

M oxygen consumptio
on rate is plootted for eachh experimenntal group at

each time point (n

n=3/injected
d group). LO
OESS curvess with 95% cconfidence rribbons are

plotteed over top to

t show smo
oothed behav me.  (B) Timee course treeatment of
vior over tim

MSD
DC-0160 in BV2
B cells. BV2 M LPS and 10 μM
B cells weere co-treateed with 10 μM
MSDC-0160 for 2, 6 or 24 h. Representative western blot of phospho-mTOR (Ser 2448),

mTOR and β-actin in BV2 cells. (C) Mean western blot phospho-mTOR/mTOR ratios

relative to β-actin. Data are means ± SEM (n=3 experiments). *p < 0.05. Data were

analyzed by one-way ANOVA with Tukey’s multiple comparison test.

Supplementary Fig. 10
Supplementary Fig. 10. Schematic diagram of proposed mechanism of action of

MSDC-0160 in glial cells and dopaminergic neurons. MSDC-0160 has a dual effect on

MPC in microglia and dopaminergic neurons resulting in pro-survival signaling.

Tables:

Table 1. LMM results.

Outcome FDR
Estimated Unadjusted 95% CI 95% CI
Measure: Variable Adjusted
Effect P-Values Lower Upper
Trial Start P-Values
Open field
Distance
Wildtype 459.237 < 0.001 < 0.001 381.290 537.184
Traveled: 3
Weeks
EN1 Genotype -199.067 < 0.001 < 0.001 -288.721 -109.413
MSDC Treatment -48.301 0.334 0.431 -145.066 48.464
Weeks -2.942 0.022 0.044 -5.323 -0.561
EN1 Genotype *
145.926 0.033 0.061 16.401 275.451
MSDC Treatment
Open field
Distance
Wildtype 378.459 < 0.001 < 0.001 338.598 418.320
Traveled: 8
Weeks
EN1 Genotype -167.07 < 0.001 < 0.001 -220.541 -113.599
MSDC Treatment -3.501 0.907 0.937 -61.827 54.825
Weeks -0.927 0.488 0.585 -3.518 1.664
EN1 Genotype *
109.261 0.009 0.021 29.822 188.700
MSDC Treatment
Open field
Mean Speed: Wildtype 0.775 < 0.001 < 0.001 0.652 0.898
3 Weeks
EN1 Genotype -0.359 < 0.001 < 0.001 -0.496 -0.222
MSDC Treatment -0.07 0.362 0.461 -0.219 0.079
Weeks -0.006 0.006 0.015 -0.010 -0.002
EN1 Genotype *
0.257 0.015 0.032 0.059 0.455
MSDC Treatment
Open field
Mean Speed: Wildtype 0.593 < 0.001 < 0.001 0.526 0.660
8 Weeks
EN1 Genotype -0.283 < 0.001 < 0.001 -0.373 -0.193
MSDC Treatment -0.002 0.973 0.983 -0.100 0.096
Weeks -0.001 0.512 0.595 -0.005 0.003
EN1 Genotype *
0.174 0.012 0.027 0.041 0.307
MSDC Treatment
Open field
Time
Wildtype 554.888 < 0.001 < 0.001 519.488 590.288
Mobile: 3
Weeks
EN1 Genotype -69.87 < 0.001 < 0.001 -103.813 -35.927
 MSDC Treatment 12.922 0.49 0.585 -23.434 49.278
Weeks -1.954 0.011 0.025 -3.381 -0.527
EN1 Genotype *
43.958 0.086 0.129 -5.036 92.952
MSDC Treatment
Open field
Time
Wildtype 528.576 < 0.001 < 0.001 502.298 554.854
Mobile: 8
Weeks
EN1 Genotype -102.43 < 0.001 < 0.001 -136.961 -67.899
MSDC Treatment -1.137 0.953 0.974 -38.798 36.524
Weeks -1.234 0.19 0.256 -3.059 0.591
EN1 Genotype *
87.686 0.001 0.003 36.457 138.915
MSDC Treatment
Open field
Max Speed: Wildtype 5.35 < 0.001 < 0.001 4.397 6.303
3 Weeks
EN1 Genotype -1.743 0.005 0.013 -2.907 -0.579
MSDC Treatment -0.63 0.332 0.431 -1.888 0.628
Weeks -0.043 0.002 0.005 -0.067 -0.019
EN1 Genotype *
1.92 0.031 0.059 0.238 3.602
MSDC Treatment
Open field
Max Speed: Wildtype 3.82 < 0.001 < 0.001 3.355 4.285
8 Weeks
EN1 Genotype -0.973 0.002 0.005 -1.559 -0.387
MSDC Treatment 0.075 0.819 0.886 -0.562 0.712
Weeks -0.027 0.131 0.19 -0.062 0.008
EN1 Genotype *
0.933 0.037 0.067 0.071 1.795
MSDC Treatment
Open field
Time Frozen: Wildtype 466.815 < 0.001 < 0.001 387.288 546.342
3 Weeks
EN1 Genotype 36.526 0.506 0.595 -70.341 143.393
MSDC Treatment -121.337 0.043 0.075 -235.809 -6.865
Weeks -2.781 0.158 0.219 -6.525 0.963
EN1 Genotype *
86.573 0.277 0.368 -67.779 240.925
MSDC Treatment
EN1 Genotype *
3.92 0.144 0.206 -1.170 9.010
Weeks
MSDC Treatment
5.199 0.067 0.104 -0.111 10.509
* Weeks
EN1 Genotype *
MSDC -8.047 0.041 0.073 -15.383 -0.711
Treatment:Weeks
Open field
Wildtype 401.237 < 0.001 < 0.001 369.667 432.807
Time Frozen:
 8 Weeks
EN1 Genotype 90.281 < 0.001 < 0.001 49.770 130.792
MSDC Treatment 4.857 0.83 0.887 -39.294 49.008
Weeks -0.37 0.755 0.836 -2.691 1.951
EN1 Genotype *
-58.434 0.06 0.096 -118.334 1.466
MSDC Treatment
Rotarod
Latency: 3 Wildtype 151.333 < 0.001 < 0.001 112.976 189.690
Weeks
EN1 Genotype -107.618 < 0.001 < 0.001 -144.092 -71.144
MSDC Treatment -4.371 0.817 0.886 -41.148 32.406
Weeks -1.791 0.049 0.083 -3.524 -0.058
EN1 Genotype *
57.653 0.031 0.059 7.034 108.272
MSDC Treatment
Rotarod
Latency: 8 Wildtype 175.049 < 0.001 < 0.001 139.067 211.031
Weeks
EN1 Genotype -139.038 < 0.001 < 0.001 -185.367 -92.709
MSDC Treatment 5.161 0.839 0.887 -44.523 54.845
Weeks -2.127 0.146 0.206 -4.971 0.717
EN1 Genotype *
66.548 0.056 0.091 -0.702 133.798
MSDC Treatment

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