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Multiple system atrophy (MSA) is a rare and extremely debilitating progressive neurodegenerative disease characterized by variable combinations of parkinsonism, cerebellar ataxia, dysautonomia, and pyramidal dysfunction. MSA is a unique synucleinopathy, in which alpha synuclein-rich aggregates are present in the cytoplasm of oligodendroglia. The precise origin of the alpha synuclein (aSyn) found in the glial cytoplasmic inclusions (GCIs) as well the mechanisms of neurodegeneration in MSA remain unclear. Despite this fact, cell and animal models of MSA rely on oligodendroglial overexpression of aSyn. In the present study, we utilized a novel oligotrophic AAV, Olig001, to overexpress aSyn specifically in striatal oligodendrocytes of rats and nonhuman primates in an effort to further characterize our novel viral vector-mediated MSA animal models. Using two cohorts of animals with 10-fold differences in Olig001 vector titers, we show a dose-dependent formation of MSA-like pathology in rats. High titer of Olig001-aSyn in these animals were required to produce the formation of pS129+ and proteinase K resistant aSyn-rich GCIs, demyelination, and neurodegeneration. Using this knowledge, we injected high titer Olig001 in the putamen of cynomolgus macaques. After six months, histo-logical analysis showed that oligodendroglial overexpression of aSyn resulted in the formation of hallmark GCIs throughout the putamen, demyelination, a 44% reduction of striatal neurons and a 12% loss of nigral neurons. Furthermore, a robust inflammatory response similar to MSA was produced in Olig001-aSyn NHPs, including microglial activation, astrogliosis, and a robust infiltration of T cells into the CNS. Taken together, oligodendroglial-specific viral vector-mediated overexpression of aSyn in rats and nonhuman primates faithfully reproduces many of the pathological disease hallmarks found in MSA. Future studies utilizing these large animal models of MSA would prove extremely valuable as a pre-clinical platform to test novel therapeutics that are so desperately needed for MSA.
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Neurobiology of Disease 148 (2021) 105184
Available online 19 November 2020
0969-9961/© 2020 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Viral-based rodent and nonhuman primate models of multiple system
atrophy: Fidelity to the human disease
David J. Marmion
a
,
b
, Angela A. Rutkowski
a
, Diptaman Chatterjee
a
, Benjamin M. Hiller
a
,
Milton H. Werner
c
, Erwan Bezard
d
,
e
, Deniz Kirik
f
, Thomas McCown
g
,
h
, Steven J. Gray
i
, Jeffrey
H. Kordower
a
,
*
a
Department of Neurological Sciences, Rush University Medical Center, Chicago, IL 60612, USA
b
Parkinsons Disease Research Unit, Department of Neurobiology, Barrow Neurological Institute, Phoenix, AZ, United States
c
Inhibikase Therapeutics, Inc., Atlanta, GA, USA
d
University of Bordeaux, Neurodegenerative Diseases Institute, UMR 5293, F-33000 Bordeaux, France
e
CNRS, Neurodegenerative Diseases Institute, UMR 5293, F-33000 Bordeaux, France
f
Brain Repair and Imaging in Neural Systems (B.R.A.I.N.S) Unit, Department of Experimental Medical Science, Lund University, Lund 221 00, Sweden
g
Gene Therapy Center, University of North Carolina, Chapel Hill, NC, USA
h
Department of Psychiatry, University of North Carolina, Chapel Hill, NC, USA
i
Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, United States
ARTICLE INFO
Keywords:
Multiple system atrophy
Alpha synuclein
Nonhuman primate
Models
Oligodendroglia
ABSTRACT
Multiple system atrophy (MSA) is a rare and extremely debilitating progressive neurodegenerative disease
characterized by variable combinations of parkinsonism, cerebellar ataxia, dysautonomia, and pyramidal
dysfunction. MSA is a unique synucleinopathy, in which alpha synuclein-rich aggregates are present in the
cytoplasm of oligodendroglia. The precise origin of the alpha synuclein (aSyn) found in the glial cytoplasmic
inclusions (GCIs) as well the mechanisms of neurodegeneration in MSA remain unclear. Despite this fact, cell and
animal models of MSA rely on oligodendroglial overexpression of aSyn. In the present study, we utilized a novel
oligotrophic AAV, Olig001, to overexpress aSyn specically in striatal oligodendrocytes of rats and nonhuman
primates in an effort to further characterize our novel viral vector-mediated MSA animal models. Using two
cohorts of animals with 10-fold differences in Olig001 vector titers, we show a dose-dependent formation of
MSA-like pathology in rats. High titer of Olig001-aSyn in these animals were required to produce the formation
of pS129+and proteinase K resistant aSyn-rich GCIs, demyelination, and neurodegeneration. Using this
knowledge, we injected high titer Olig001 in the putamen of cynomolgus macaques. After six months, histo-
logical analysis showed that oligodendroglial overexpression of aSyn resulted in the formation of hallmark GCIs
throughout the putamen, demyelination, a 44% reduction of striatal neurons and a 12% loss of nigral neurons.
Furthermore, a robust inammatory response similar to MSA was produced in Olig001-aSyn NHPs, including
microglial activation, astrogliosis, and a robust inltration of T cells into the CNS. Taken together,
oligodendroglial-specic viral vector-mediated overexpression of aSyn in rats and nonhuman primates faithfully
reproduces many of the pathological disease hallmarks found in MSA. Future studies utilizing these large animal
models of MSA would prove extremely valuable as a pre-clinical platform to test novel therapeutics that are so
desperately needed for MSA.
1. Introduction
Multiple system atrophy (MSA) is a rapidly progressing neurode-
generative disorder that is extremely debilitating and fatal. MSA is
devastating, but rare, with an estimated mean incidence of 0.60.7 cases
per 100,000 person-years and increasing to 3 per 100,000 per year in the
age-group above 50 years, thus classifying it as a rare disease (Bower
et al., 1997). MSA affects both sexes equally, symptoms tend to emerge
in the fth to sixth decade of life (Wenning et al., 2008), and MSA
progresses extremely rapidly in most cases, causing patients to be
* Corresponding author at: Department of Neurological Sciences, Rush University Medical Center, 1735 West Harrison Street, Chicago, IL 60612, USA
E-mail address: Jeffrey_kordower@rush.edu (J.H. Kordower).
Contents lists available at ScienceDirect
Neurobiology of Disease
journal homepage: www.elsevier.com/locate/ynbdi
https://doi.org/10.1016/j.nbd.2020.105184
Received 28 July 2020; Received in revised form 30 October 2020; Accepted 11 November 2020
Neurobiology of Disease 148 (2021) 105184
2
wheelchair bound and succumb to the disease after an average of 69
years following symptomatic onset (Low et al., 2015; Papapetropoulos
et al., 2007; Schrag et al., 2008; Wenning et al., 2008). MSA presents
clinically with varying combinations of extrapyramidal dysfunction,
dysautonomia, parkinsonism, and cerebellar ataxia (Gilman et al., 2008;
Low et al., 2015; Wenning et al., 2008). Based on clinical symptom-
atology and the distribution of pathology, MSA can be subdivided into a
parkinsonian variant, MSA-P, with predominant nigrostriatal degener-
ation and Parkinsonian motor features, and a cerebellar variant, MSA-C,
characterized by olivopontocerebellar atrophy with ataxia (Wenning
et al., 2008). There are no currently available potent therapies either
disease modifying or symptomatic, for MSA (Krismer and Wenning,
2017; Lopez-Cuina et al., 2018).
MSA belongs to a larger family of neurodegenerative diseases termed
synucleinopathies which include Parkinsons disease (PD) and dementia
with Lewy bodies (DLB), in which the protein alpha-synuclein (aSyn)
forms brillary aggregates throughout selectively vulnerable brain re-
gions. Synucleinopathies can be very difcult to distinguish from each
other clinically, especially early in the disease process, as patients can
present with overlapping symptomatology depending on the distribu-
tion of lesions or other underlying pathologies. Unlike the typical aSyn-
rich neuronal aggregates found in PD and DLB, MSA is a unique synu-
cleinopathy in which aSyn inclusions are predominantly formed in the
cytoplasm of oligodendroglia (Martí et al., 2003; Papp et al., 1989).
Therefore, aSyn+glial cytoplasmic inclusions (GCIs) are the patholog-
ical hallmark of MSA and post-mortem identication of GCIs is required
for a denitediagnosis of MSA (Gilman et al., 2008). aSyn inclusions
isolated from the brains of individuals with PD, DLB, and MSA have been
shown to contain structurally different aSyn laments, and aSyn from
GCIs have been reported to be three orders of magnitude more potent in
seeding aggregation of aSyn than aSyn from Lewy bodies (Peng et al.,
2018; Schweighauser et al., 2020). Apart from the formation of GCIs,
evidence from post-mortem and experimental studies have shown that
the neuropathology observed in MSA includes myelin degeneration,
axonal damage, loss of neurotrophic support, neuronal degeneration,
astrogliosis, microglial activation, and, recently, regional inltration of
T cells (Jellinger, 2014; Rydbirk et al., 2017; Stefanova et al., 2007; Ubhi
et al., 2010; Williams et al., 2020). Subsequent studies have shown that
the accumulation of GCIs seem to drive the pathology of MSA, as the
degree of GCI burden strongly correlates with the level of neuronal loss
and demyelination, and that the formation of GCIs occurs prior to the
degeneration of axons or neurons (Ozawa et al., 2004; Papp and Lantos,
1994).
Based on the evidence linking oligodendroglial aSyn with disease
pathogenesis, animal models of MSA have been developed by primarily
overexpressing aSyn in oligodendrocytes via different mechanisms,
variably recapitulating aspects of the disease with inherent weaknesses
in each model. Transgenic (tg) mouse models have been established by
genetically overexpressing human aSyn using different oligodendrocyte-
specic promoters proteolipid protein (PLP) (Kahle et al., 2002),
myelin basic protein (MBP) (Shults et al., 2005), and 2,3-cyclic
nucleotide 3-phospho-diesterase (CNP) (Yazawa et al., 2005), or using a
Cre-loxP system to express inducible oligodendroglial aSyn (Tanji et al.,
2019). We and others have used viral vector-mediated overexpression of
aSyn which recently has been characterized in rodents and nonhuman
primates (NHPs) to produce GCIs in MSA-specic brain regions in adult
animals and induce a higher level of neuropathology than tg mouse
models (Bassil et al., 2017; Mandel et al., 2017; Williams et al., 2020).
In the present study, we further develop and characterize our pre-
viously established viral vector-mediated overexpression model of MSA,
in which we showed 95% specicity of the novel oligotropic AAV,
Olig001, to form insoluble, aSyn-rich GCIs specically in oligoden-
droglia of rats and NHPs resulting in demyelination and microglial
activation (Mandel et al., 2017). We now show dose-dependent myelin
loss and neurodegeneration in the striatum of rats after injection of
different viral titers Olig001-aSyn. Then, using the high titer Olig001-
aSyn injected in the putamen of cynomolgus macaques, we demon-
strate long-term robust formation GCIs and demyelination throughout
its rostro-caudal putamenal extent with neuronal loss observed in both
the putamen as well as the substantia nigra. Moreover, microglial acti-
vation, astrogliosis, and inltration of T cells coincide specically with
regions of pathologic oligodendroglial-aSyn expression as we now
demonstrate in human MSA samples. Our data highlights the ability of
viral vector-mediated overexpression of aSyn in rats and NHPs to
faithfully recapitulate many neuropathological hallmarks of MSA-P and
describe a valuable large animal model to be utilized for the develop-
ment of disease modifying therapies for MSA.
2. Materials and methods
2.1. Olig001 AAV vector
Detailed information of the development, construction, purication,
and quality control of the Olig001 AAVs are described elsewhere
(Mandel et al., 2017; Powell et al., 2016). Briey, AAV-Olig001 vectors
with a high tropism for oligodendrocytes were packaged with a self-
complementary genome with transgene expression mediated by the
CBh promoter and bovine growth hormone polyA (Gray et al., 2011).
The AAV vectors were produced by the University of North Carolina
Vector Core facility by triple-transfecting production plasmids into
HEK293 cells. AAV vectors were then puried from the cells by iodix-
anol gradient centrifugation, followed by ion-exchange
chromatography.
2.2. Rodents
Eight-week-old female Sprague-Dawley rats (Harlan, Indianapolis,
IN) were used for the rodent Olig001 experiments. Upon arrival, animals
were quarantined for one week prior to any testing and randomized into
groups. Animals were cared for in accordance with the principles of the
Guide to the Care and Use of Experimental Animals. Rats were housed
two per cage with a 12:12 h light:dark cycle (07:0019:00 h). Food and
water were available ad libitum throughout the study.
2.3. Rodent stereotaxic surgery
All rats received a single 2
μ
l injection of Olig001 in each hemisphere
at a rate of 0.5
μ
l/min. Rats received Olig001 in three different viral
titers, 2.4 ×10
11
vg/ml (n =8/group), 2.3 ×10
12
vg/ml (n =8/group),
and 3.72 ×10
12
vg/mL (n =4). The coordinates for injection were AP +
0.7 mm and ML +/2.7 mm from bregma, DV -5.0 mm from dura.
Following the completion of each injection the micropipette was
retracted 1 mm and left in place for an additional 2 min before being
slowly removed from the brain. All procedures were approved by the
Rush University Institutional Animal Care and Use Committee and
accredited by the Association for Assessment and Accreditation of Lab-
oratory Animal Care.
2.4. Primates
Eight adult male cynomolgus macaques (Macaca fascicularis) were
utilized for this study and randomized to receive either Olig001-GFP or
Olig001-aSyn. Animals were pair-housed on a 12-h light/12-h dark
cycle. All procedures were approved by the University of Illinois Chicago
Institutional Animal Care and Use Committee and the Rush University
Institutional Animal Care and Use Committee and accredited by the
Association for Assessment and Accreditation of Laboratory Animal
Care. Animal care was supervised by veterinarians skilled in the care and
maintenance of non-human primates.
D.J. Marmion et al.
Neurobiology of Disease 148 (2021) 105184
3
2.5. Primate stereotaxic surgery
Nonhuman primates underwent surgical procedures as previously
described (Mandel et al., 2017). Surgical targets were identied using
pre-operative MRI and intraoperative surgical navigation using a
Stealthstation Neuronavigation system. Olig001-GFP (3.75 ×10
12
vg/
ml) or Olig001-aSyn (3.75 ×10
12
vg/ml) was injected bilaterally into
the putamen (rostral putamen 20
μ
l, middle putamen 20
μ
l, caudal pu-
tamen 10
μ
l, 50
μ
l total per hemisphere) and infused at a rate of 1
μ
l/
min.
2.6. Nonhuman primate behavior analysis
Activity Monitoring: Animals received activity monitoring to assess
general motor function. Animals were tted with a collar containing
an Actical activity monitor (manufactured by Philips Respironics
Murrysville, PA) which senses and records any excessive acceleration.
The monitor senses acceleration that exceeds 0.05 G, recorded up to 30
times per second. The number of pulses within a pre-selected time
period are then recorded. Animals were acclimated to the collars for at
least 3 days prior to any data collection, recording took place for 7 days
per collection interval, after which time the animals will be sedated as
above and the collars were removed. Data was collected at baseline,
three months post-injection, and prior to sacrice (six months post-
injection).
Fine Motor Skills Test (Hand Reach Task): Each monkey was tested
for ne motor performance in both upper limbs using a hand reach task
(HRT) as previously described (Emborg et al., 1998). Animals were
transported to a modied testing cage from their home cage and pre-
sented with a 3 ×3 well matrix plexiglass testing board. Six pieces of
food were be placed within the wells for each trial, and time will be
recorded for how long it took the animal to retrieve them. The board is
congured in such a manner that only one limb will be tested at a time.
Monkeys underwent 10 trials per limb, with each trial being alternated
between the left and right limbs. All animals were tested every two
weeks by the same blinded investigator throughout the entirety of the
study.
2.7. Rat and primate necropsy and tissue processing
4-weeks (rat), 5-months (rat), or 6-months (NHP) post-surgery, ani-
mals were deeply sedated and euthanized by transcardial saline perfu-
sion. The brain was removed from the calvarium and was post-xed in
4% paraformaldehyde solution for 1248 h then transferred to a sucrose
gradient. Coronal slices (40
μ
m) were sectioned on a freezing-stage
sliding knife microtome and stored in cryoprotectant solution at
20 C until processed.
2.8. Human brain samples
Post-mortem brain tissue of MSA subjects were obtained from Rush
University Medical Center- approved research tissue depository with
ethical approval granted by Rush University Medical Center IRB.
Samples (n =3) underwent a complete neuropathological evaluation
conrming the presence of GCIs and diagnosis of MSA as previously
described (Chu et al., 2006; Jellinger et al., 2005; Williams et al.,
2020).
2.9. Immunohistochemistry
Free oating sections of brain tissue were rinsed of cryoprotectant
and quenched with endogenous peroxidases using a 20-min incubation
in a 0.1 M sodium periodate solution. Non-specic background staining
was blocked for 1 h in 3% normal (goat or horse) serum and 2% bovine
serum albumin. Sections were incubated with primary antibody (mouse
anti-Living Colors JL-8 (GFP), 1:2000 dilution [632,380, Clontech];
mouse anti-tyrosine hydroxylase (TH), 1:10,000 dilution [22,941;
Immunostar]; rabbit anti-Alpha-synuclein (phospho S129, EP1536Y),
1:5001:1000 dilution [ab51253, Abcam]; mouse anti-Alpha-synuclein
(LB 509), 1:5001:1000 dilution [180,215, Thermo Fischer Scientic];
rabbit anti-Alpha-synuclein (phospho Y39), 1:100 dilution [Donated by
Dr. Milton Werner]; mouse anti-NEUN (A60), 1:500 dilution [MAB
3777, EMB Millipore]; rabbit anti-Glial brillary acidic protein (GFAP),
1:2000 dilution [Z033401-2, Dako]; rabbit anti-CD3, 1:200 dilution
[A0452, Dako];mouse anti-HLA-DR (LN3), 1:200 dilution [MA5-11966,
Thermo Fischer Scientic]), 1% bovine serum albumin, 1% serum, and
0.4% Triton-X at 4 C for 18 h. The sections were then washed, incu-
bated with appropriate secondary antibodies (biotinylated goat anti-
rabbit, 1:200 dilution [BA-1000, Vector Laboratories]; biotinylated
horse anti-mouse, 1:200 dilution [BA-2000, Vector Laboratories]) for 1
h, washed again, and incubated with avidin-biotin complex (Vector
Laboratories, PK-6100) for 2 h. Tissues were then incubated in
imidazole-acetate buffer, pH 7.3, for 30 min before they were visualized
with 33-diaminobenzidine tetrahydrochloride in 0.01% hydrogen
peroxide with 2% nickel enhancement. The sections were allowed to dry
overnight, dehydrated through increasing alcohol concentrations and
xylenes, and coverslipped with cytoseal (23,244,257; Fisher Scientic
International).
2.10. Immunouorescence double labeling
Free oating sections of brain tissue were rinsed of cryoprotectant
and non-specic background staining was blocked for 1 h in 3%
normal (goat) serum and 2% bovine serum albumin. Sections were
incubated with primary antibody (mouse anti-Living Colors JL-8
(GFP), 1:2000 dilution [632,380; Clontech]; mouse anti-tyrosine hy-
droxylase (TH), 1:10,000 dilution [22,941; Immunostar]; rabbit anti-
tyrosine hydroxylase (TH), 1:3000 dilution [AB152; EMB Millipore];
mouse anti-Alpha-synuclein (LB 509), 1:5001:1000 dilution
[180,215; Thermo Fischer Scientic]; rabbit anti-Alpha-synuclein
(phospho S129, EP1536Y), 1:5001:1000 dilution [ab51253,
Abcam]; rabbit anti-alpha synuclein (rodent specic, D37A6), 1:500
dilution [4179S; Cell Signaling]; rabbit anti-p25
α
, 1:500 dilution
[generously donated by Dr. Poul Henning Jensen]; rabbit anti-Olig2
[EPR2673], 1:200 dilution [ab109186; Abcam]), 1% bovine serum
albumin, 1% serum, and 0.4% Triton-X at 4 C for 18 h. The sections
were then washed, incubated with appropriate secondary antibodies
(Alexa Fluor® 488 AfniPure Goat Anti-Mouse IgG, 1:200 [115545-
003; Jackson Immuno Research Laboratories]; Alexa Fluor® 647
AfniPure Fab Fragment Goat Anti-Mouse IgG, 1:200 [115607-003;
Jackson Immuno Research Laboratories]; Alexa Fluor® 488 AfniPure
Goat Anti-Rabbit IgG, 1:200 dilution [111545-003; Jackson Immuno
Research Laboratories]; Alexa Fluor® 647 AfniPure Goat Anti-Rabbit
IgG, 1:200 dilution [111605-003; Jackson Immuno Research Labo-
ratories]) for 1 h, washed again, and mounted on glass slides. The
sections were allowed to dry overnight, dehydrated through increasing
alcohol concentrations and xylenes, and coverslipped with DPX
mounting medium (Sigma, 44,581).
2.11. Proteinase K digestion
Proteinase K (PK) digestion was used to determine whether the aSyn
seen in oligodendrocytes was soluble (non-aggregated) or insoluble
(aggregated), the remaining signal detecting the later. Striatal sections
containing the injection site were mounted onto gelatin-coated slides
and dried for at least 8 h at 55 C. After wetting with TBS-T (10 mM
TrisHCl, pH 7.8; 100 mM NaCl; 0.05% Tween-20), the sections were
digested with 10
μ
g/ml PK (Invitrogen) in TBS-T (10 mM TrisHCl, pH
7.8; 100 mM NaCl; 0.1% Tween-20) for 30 min at 55 C. The sections
were xed with 4% paraformaldehyde for 10 min. After several washes,
the sections were processed for anti-Alpha-synuclein (LB509) IHC as
described (Mandel et al., 2017).
D.J. Marmion et al.
Neurobiology of Disease 148 (2021) 105184
4
2.12. Luxol fast blue
For analysis of myelin integrity, level matched sections of both
Olig001-GFP and Olig001-aSyn injected rats and monkeys were moun-
ted on glass slides and allowed to dry overnight. Sections were placed in
a 1:1 alcohol/chloroform solution to defat the tissue, rinsed in 70%
alcohol, and placed in a 0.1% Luxol Fast Blue solution overnight at
56 C. Sections were destained using 0.05% lithium carbonate solution
and 70% alcohol until gray matter was clear and white matter was
clearly dened.
2.13. Stereology
Unbiased stereological counting methods (West and Gundersen,
1990) using the Stereo Investigator optical fractionator probe (Micro-
brighteld Bioscience, Version10.40) were utilized to assess the number
of GCIs, neuronal degeneration, and T cell populations in Olig001
injected rats and NHPs. Briey, the region of interest was outlined at
1.25×magnication and a systematic sampling of serial sections was
employed where cells were counted using a 60×oil-immersion objective
from a random starting point at regular predetermined intervals. Details
for specic counting parameters are found in Supplemental Table 1.
2.14. Optical density
Eight level matched serial sections of Olig001 injected monkeys
stained for HLA-DR and GFAP were used in these analyses. The putamen
was imaged using 4×objective on a Nikon eclipse Ti2 microscope using
a Nikon D5-Ri2 colour camera and NIS elements AR software version
5.10.01. Quantied images were all taken with identical acquisition
settings. The acquired image les were analyzed by a blinded rater and
quantied with mean gray value units using imageJ (NIH) software.
Briey, the region of interest was outlined and converted to gray scale.
The image was then inverted, vessels and holes in the tissue were
excluded, and the background was subtracted using rolling ball radius of
25 prior to data collection.
2.15. In situ hybridization
A combination of in situ hybridization (ISH) for Olig001 and IHC of
either Olig2 or NeuN using RNAscope® (Advanced Cell Diagnostics) to
determine the cellular specicity of the Olig001 AAV as previously
described (Grabinski et al., 2015; Wang et al., 2012). The ISH probe was
designed to recognize the non-transcribed region within the promoter
region of Olig001. The ISH signal was detected using 3, 3-dia-
minobenzidine to produce brown punctate staining. Following RNA-
scope ISH labeling, the sections were processed for either Olig2 or NeuN
IHC (as described above) using Vector slate-gray to produce blue-gray
labeling of oligodendrocytes or neurons.
2.16. Thioavin S
Thioavin S histochemistry was performed to determine whether
aSyn antibody-labeled inclusions contained amyloid brils with beta-
pleated sheet structures analogous to pathology in human synucleino-
pathies. Striatal sections of Olig001-aSyn NHPs were mounted on
gelatin-coated slides and dried at room temperature. Mounted sections
were defatted in equal parts of chloroform and 100% ethanol for 2 h and
stained with 0.1% thioavin S for 10 min in the dark (Kordower et al.,
2008).
2.17. Images
Confocal images were obtained from either an Olympus laser-
scanning microscope with Fluoview software or a Nikon Eclipse Ti2
confocal microscope using a Nikon A1RHD camera using NIS elements
AR software version 5.10.01 and stored as tiff les. Conventional light
microscopic images were acquired using an Olympus microscope (BX61)
attached to a Nikon digital camera DXM1200 or a Nikon eclipse Ti2
microscope using a Nikon D5-Ri2 colour camera and NIS elements AR
software version 5.10.01. All compared images were taken using the
same intensity and exposure time. All gures were prepared using
Photoshop 8.0 graphics software. Only minor adjustments of brightness
or contrast were made.
3. Statistical analyses
All data are presented as mean ±SEM. All statistical tests were
conducted using GraphPad Prism 8 software. The specic statistical test
and statistical details for individual experiments can be found in the
corresponding gure legends.
4. Data availability
The data that support the ndings of this study are available from the
corresponding author, upon reasonable request.
5. Results
5.1. Olig001 titer comparison, GCI formation, and neuronal loss in rats
To investigate the dose dependency of aSyn overexpression in oli-
godendrocytes, two titers of AAV Olig001 were injected in the striatum
of Sprague Dawley rats. Five months following injection, the rats were
sacriced and transduction of oligodendroglia was evaluated by histo-
logical analysis. GFP expression was observed in oligodendroglia
throughout the striatum and corpus callosum (CC) of Olig001-GFP
injected rats (Fig. 1a, a, 2.4 ×10
11
2.3 ×10
12
vg/ml), however aSyn
transgene expression greatly differed between low titer (LT) (Fig. 1b, b,
2.4 ×10
11
vg/ml) and high titer (HT) (Fig. 1c, c, 2.3 ×10
12
vg/ml)
Olig001-aSyn injected animals using human aSyn (LB509, Supplemen-
tary Fig. 1) and pathological, phosphorylated Serine-129 (pS129) aSyn
antibodies. In LT Olig001-aSyn injected rats, sparse aSyn+inclusions
were seen around the needle tract in the striatum (Fig. 1b, b), while
widespread, prominent aSyn+inclusions were seen throughout the CC
and abundant in white matter bundles throughout the striatum in HT
Olig001-aSyn injected rats (Fig. 1c, c). Human-specic aSyn immuno-
reactivity (LB509) was also observed in other structures such as the
globus pallidus, thalamus, and substantia nigra (Supplementary
Fig. 1ad). Unbiased stereological counts of pS129 aSyn+inclusions in
the striatum showed a signicant 210% increase in HT titer aSyn
injected animals compared to LT animals (Fig. 1d, HT Olig001-aSyn:
23,828 ±2455; LT Olig001-aSyn: 7662 ±2074, **P 0.01).
The extent and localization of aSyn-rich GCIs has been shown to be
highly correlative with the degree of neuronal loss and demyelination in
post-mortem human MSA cases (Ozawa et al., 2004; Papp et al., 1989).
Therefore, we sought to investigate if this aspect of the disease was
recapitulated in the novel Olig001 MSA rat model and if differences in
AAV titer inuenced the observed neuropathology. In order to investi-
gate the degree of neuronal loss, rat brain sections were stained for
neuronal marker NeuN and evaluated using unbiased stereology in the
striatum. Corresponding with aSyn pathology, a titer-dependent loss of
NeuN+striatal neurons was found in LT Olig001-aSyn (14.7%) and HT
Olig001-aSyn (23.8%) groups compared to Olig001-GFP injected rats
(Fig. 1e, LT Olig001-aSyn: 116,407 ±1945; HT Olig001-aSyn: 104,035
±5484; Olig001-GFP: 136,500 ±6247, Tukeys multiple comparison
GFP vs LT aSyn: *P 0.05; GFP vs HT aSyn: ***P 0.001; LT aSyn vs HT
aSyn: ns). As neuronal loss is also observed in the substantia nigra of
subjects with MSA, nigral sections of Olig001-injected rats were stained
with dopaminergic marker tyrosine hydroxylase (TH). Similar to striatal
neurodegeneration, unbiased stereological counting revealed a titer-
dependent loss of TH+neurons in LT Olig001-aSyn (21.7%) and HT
D.J. Marmion et al.
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D.J. Marmion et al.
Neurobiology of Disease 148 (2021) 105184
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Olig001-aSyn (35.9%) groups compared to Olig001-GFP injected rats
(Fig. 1f, LT Olig001-aSyn: 6372 ±651.6; HT Olig001-aSyn: 5222 ±
279.8; Olig001-GFP: 8148 ±456.9, Tukeys multiple comparison GFP vs
LT aSyn: *P <0.033; GFP vs HT aSyn: **P <0.002; LT aSyn vs HT aSyn:
ns).
GCIs composed of insoluble aSyn aggregates are critical MSA hall-
marks to replicate within a relevant disease model. To evaluate the
solubility of aggregates within GCIs in the Olig001-aSyn MSA rat model,
striatal sections of Olig001-aSyn injected rats were subjected to
proteinase-K (PK) digestion followed by human-specic LB509 aSyn
staining. Striatal sections without PK treatment displayed robust
LB509+aggregates throughout the CC and striatum as well as granular
LB509+staining in oligodendrocytes processes (Fig. 1g and g).
Following PK digestion, much of the LB509+staining in the processes
degraded, while substantial staining of the LB509+aggregates in the
oligodendroglia remained (Fig. 1h and h), indicating the formation
insoluble aSyn rich aggregates in the Olig001-aSyn MSA rat model,
similar to clinical MSA. The use of HT Olig001-aSyn did not result in off-
target transduction and remained oligotropic, as human-specic
LB509+aSyn aggregates (Fig. 1i, green) are shown surrounding the
nuclear signal of oligodendroglial marker Olig2 (Fig. 1j, red), conrm-
ing the cytoplasmic localization of aSyn and formation of GCI-like aSyn
rich inclusions (Fig. 1k). Double staining utilizing human-specic aSyn
antibody LB509 with pS129 aSyn antibody (Supplementary Fig. 2ac)
and human-specic aSyn antibody LB509 with rodent-specic aSyn
antibody D37A6 (Supplementary Fig. 2df) indicates that exogenously
expressed human aSyn is phosphorylated at serine 129, while endoge-
nous rodent aSyn is not a component of the GCI-like formations.
Under physiological conditions, the oligodendroglia-specic phos-
phoprotein p25
α
/tubulin polymerization promoting protein (TPPP) is
found in the nucleus and myelin sheath where it interacts with myelin
basic protein (MBP) and tubulin to facilitate myelination and the sta-
bilization of microtubules. In human MSA, it has been shown that p25
α
relocates to the cytoplasm of oligodendroglia early in the disease course
where it is involved in the recruitment of aSyn to form GCIs and is
another major component of GCIs (Mavroeidi et al., 2019; Ota et al.,
2014). Immunouorescent double labeling revealed that p25
α
(Fig. 1l,
green) co-localized with LB509+aSyn aggregates (Fig. 1m, red)
following Olig001-aSyn injection, replicating the presence of p25
α
in
GCIs of clinical MSA.
5.2. aSyn overexpression destabilizes myelin
Since the relocation of p25
α
to the cytoplasm destabilizes myelin,
Luxol Fast Blue (LFB) staining and electron microscopy (EM) were used
to evaluate the myelin integrity. Regions of pS129+aSyn staining in the
striatum of HT Olig001-aSyn rats (Fig. 2a) displayed robust demyelin-
ation of striosomes as shown by decreased LFB staining in level matched
sections (Fig. 2b, arrowheads). In contrast, Olig001-GFP injected rats
displayed oligodendroglial tropism (Fig. 2c) but no demyelination
(Fig. 2d). Furthermore, EM of striatal sections of Olig001-aSyn injected
rats showed atypical myelination (Fig. 2e, red arrows), where the myelin
sheath became unraveled when compared to tightly-wound pattern of
myelination observed around unaffected, neighboring axons (Fig. 2e,
black arrow). Taken together, these results show that a higher titer of
Olig001-aSyn elicits strong MSA-like pathology with respect to oligo-
dendroglial function.
5.3. Oligodendrocyte-to-neuron transfer of aSyn in substantia Nigra
In the HT Olig001-aSyn injected group, aSyn+aggregates were seen
in the substantia nigra (Fig. 3a, a), as well as in the globus pallidus and
thalamus (Supplementary Fig. 1). To determine if the pS129+inclusions
found in nigral neurons was a result of neuronal uptake of the Olig001
AAV or transport of aSyn, RNAscope® in situ hybridization was per-
formed using a probe specic to a non-transcribed region within the
promoter region of Olig001, followed by immunohistochemistry to
detect either neurons or oligodendroglia. Positive staining of the RNA-
scope® probe (Fig. 3b, brown staining) was seen to co-localize with
oligodendrocyte marker Olig2 (Fig. 3b, blue staining) in the substantia
nigra. Positive staining of the RNAscope® probe was not observed
within NeuN+neurons in the substantia nigra (not shown), indicating
that the neurons that contained pS129+inclusions did not uptake the
Olig001-aSyn vector, but suggests limited oligodendrocyte-to-neuron
“transferof aSyn in this model. Immunouorescent double labeling
showed that the pS129+aggregates (Fig. 3c, green) co-localized with
tyrosine hydroxylase+(TH+) neurons (Fig. 3d, e, red), indicating aSyn
uptake specically in dopamine neurons of the substantia nigra. Ste-
reological counting of TH+neurons and pS129+aggregates indicate
that 11% of TH+neurons contained pS129+inclusions. Human-specic
LB509 aSyn immunoreactivity in TH+nigral neurons (Fig. 3fh) in-
dicates exogenous accumulation of vector-derived aSyn, while the lack
of colocalization with rodent-specic aSyn and TH rules out the
recruitment of endogenous rat aSyn (Fig. 3ik).
5.4. GCI formation in Olig001 injected NHPs
Using the high-titer of Olig001 dose that yielded more pronounced
MSA-like pathology, cynomolgus macaques received three injections
bilaterally in the putamen of either Olig001-aSyn or Olig001-GFP (n =4
per group). Six months post-injection, all monkeys showed remarkable
transgene expression. Extensive LB509+aSyn immunoreactivity was
observed throughout the rostral-caudal extent of the putamen in
Olig001-aSyn NHPs and displays the classical morphology of GCIs in
clinical MSA (Fig. 4a, a). Double staining using LB509 and Olig2 dis-
played the specic cytoplasmic localization of aSyn in oligodendroglia
in Olig001-aSyn injected NHPs (Fig. 4bd). Increasing evidence has
shown elevated levels of the tyrosine kinase c-Abl in PD (Brahmachari
et al., 2016; Brahmachari et al., 2019; Imam et al., 2011; Ko et al., 2010;
Mahul-Mellier et al., 2014) and other neurodegenerative diseases
Fig. 1. Olig001 transduction of rat striatal oligodendrocytes. Representative photomicrographs of striatum of rats injected with Olig001-GFP (a and a), LT Olig001-
aSyn (b and b), and HT Olig001-aSyn (c and c) illustrating oligodendroglial transduction of transgene. Scale bars: a, b, and c =1000
μ
m; a, b, and c =50
μ
m.
Stereological analysis (d) revealed a signicant 210% increase in the density of pS129+inclusions in HT Olig001-aSyn injected animals compared to LT Olig001-
aSyn (HT Olig001-aSyn: 23,828 ±2455; LT Olig001-aSyn: 7662 ±2074, **P 0.01, Two-tailed Mann Whitney Test, n =8/group). Stereological analysis (e)
revealed a titer-dependent loss of striatal NeuN+neurons was found in LT Olig001-aSyn and HT Olig001-aSyn groups compared to Olig001-GFP injected rats (LT
Olig001-aSyn: 116,407 ±1945; HT Olig001-aSyn: 104,035 ±5484; Olig001-GFP: 136,500 ±6247, Tukeys multiple comparison GFP vs LT aSyn: *P 0.05; GFP vs
HT aSyn: ***P 0.001 LT aSyn vs HT aSyn: ns, n =8/group). Stereological analysis (f) revealed a titer-dependent loss of nigral TH+neurons was found in LT
Olig001-aSyn and HT Olig001-aSyn groups compared to Olig001-GFP injected rats (LT Olig001-aSyn: 6372 ±651.6; HT Olig001-aSyn: 5222 ±279.8; Olig001-GFP:
8148 ±456.9, Tukeys multiple comparison GFP vs LT aSyn: *P 0.033; GFP vs HT aSyn: **P 0.002 LT aSyn vs HT aSyn: ns, n =8/group). LB509 aSyn stained
Olig001-aSyn sections without (g and g) and with PK digestion (h and h) illustrating insoluble aSyn aggregates. Scale bars: g and h =100
μ
m, gand h=10
μ
m.
Confocal microscopy of HT Olig001-aSyn injected animals illustrates LB509+aSyn staining (i and k, green) surrounding the nuclear oligodendroglial Olig2+signal (j
and k, red) conrms the cytoplasmic location of the aSyn aggregates in the cytoplasm of oligodendrocytes. Scale bar: e-g =50
μ
m. Confocal microscopy of HT
Olig001-aSyn injected animals illustrates the co-localization of microtubule stabilizing protein p25
α
(l and n, green) with LB509+aSyn inclusions (m and n, red) in
the cytoplasm of oligodendroglia. Scale bar: j-l =50
μ
m. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version
of this article.)
D.J. Marmion et al.
Neurobiology of Disease 148 (2021) 105184
7
(Alvarez et al., 2008; Cancino et al., 2008; Jing et al., 2009; Schlatterer
et al., 2011; Tremblay et al., 2010). In PD, c-Abl has been found to
phosphorylate parkin, which inhibits the ubiquitin ligase activity and
neuroprotective functions of parkin, and aSyn has been identied as a
substrate for c-Abl and catalyzes the phosphorylation of aSyn at tyrosine
39 (pY39), whereby pY39 increases aSyns propensity to aggregate
(Brahmachari et al., 2016; Brahmachari et al., 2019; Hebron et al., 2013;
Mahul-Mellier et al., 2014). To investigate if this pathologic post-
translational modication of aSyn is present in MSA, post-mortem
brain sections from pathologically conrmed MSA cases were immu-
nostained using antibodies specic to pS129 and pY39 motifs on aSyn.
Widespread staining of pS129 and pY39 aSyn+GCIs was observed
throughout the putamen (Fig. 4e and h) and substantia nigra (Fig. 4f and
i) of post-mortem MSA cases. Similar staining patterns for pS129 and
pY39 were seen in the striatum of Olig001-aSyn injected NHPs (Fig. 4g
and j), indicating that c-Abl-mediated phosphorylation occurs in MSA as
well as PD. Unbiased stereological counts in the region of transduction
demonstrated that no signicant differences were observed between the
number of GFP+or aSyn+cells, indicating that the specic accumula-
tion of aSyn specically, not the number of transduced cells, is respon-
sible for any observed pathology (Fig. 4k, Olig001-GFP NHPs: 25,529 ±
3758 GFP+cells/mm
3
; Olig001-aSyn NHPs: 28,830 ±1410 LB509+
cells/mm
3
and 34,162 ±6161 pS129+cells/mm
3
). Furthermore,
striatal sections from Olig001-aSyn injected NHPs contain GCIs that
stain positive for Thioavin S (Fig. 4l), indicating the formation of beta-
pleated sheets akin to GCIs in MSA.
Fig. 2. aSyn induced myelin disorganization in rats.
Photomicrograph of pS129 aSyn immunoreactivity in
Olig001-aSyn injected rats (a) correspond to regions
of demyelination in level matched sections shown by
loss of LFB staining (b). GFP expression in Olig001-
GFP rats (c) does not induce demyelination (d) n =
8/group. Scale bars: a-d =500
μ
m. LFB =Luxol Fast
Blue. Electron microscopy image of Olig001-aSyn
injected rat striatum (e). Red arrowheads denote
unwound myelin sheath, black arrowheads denote
few remaining intact myelin sheaths. Scale bar: 2
μ
m.
N =4. (For interpretation of the references to colour
in this gure legend, the reader is referred to the web
version of this article.)
D.J. Marmion et al.
Neurobiology of Disease 148 (2021) 105184
8
5.5. aSyn overexpression causes myelin loss and neurodegeneration in
NHPs
We next examined the neuronal and axonal integrity seen in Olig-001
injected monkeys. In Olig001-aSyn NHPs, LB509+aSyn staining in the
CC (Fig. 5a) and putamen (Fig. 5c) corresponded to areas of decreased
LFB staining observed in the CC (Fig. 5b) and white matter bundles
throughout the putamen (Fig. 5d). Conversely, in regions of GFP
expression (Fig. 5e and g), no changes in myelin were observed (Fig. 5f
and h) in any NHP injected with the Olig001-GFP control vector.
As with rats, NeuN was used as a neuronal marker to evaluate the
presence of degenerating neurons in the putamen of Olig001-injected
monkeys. Stereological estimates revealed a decreased density and
staining intensity of NeuN immunoreactivity throughout the entire pu-
tamen of Olig001-aSyn monkeys (Fig. 5j), whereas Olig001-GFP NHPs
displayed a loss of NeuN+staining only within in the immediate pe-
riphery of the site of injection (Fig. 5n). Unbiased stereological cell
counts throughout the rostral-caudal extent of the putamen revealed a
43.4% loss of NeuN+putamenal neurons in Olig001-aSyn injected
monkeys when compared to Olig001-GFP controls (Fig. 5q, Olig001-
Fig. 3. Transfer of aSyn from oligodendrocytes to nigral neurons. Photomicrographs of the substantia nigra of HT Olig001-aSyn rats illustrates pS129+aSyn
immunoreactivity in neurons of the substantia nigra (a and a
). Scale bar: a =500
μ
m, a=25
μ
m. RNAscope in situ hybridization (b) of Olig001 AAV genome
(brown) shows co-localization (arrowhead) with Olig2+oligodendrocytes (blue) in the substantia nigra. Scale bar: 25
μ
m. Confocal microscopy illustrates immu-
noreactivity of pS129 (c) and TH (d) co-localization in nigral dopamine neurons (e). Scale bar: 50
μ
m. Co-localization (arrows) of TH (f) with human-specic LB509
aSyn (g) showing exogenous human origin of aSyn. Scale bar: 50
μ
m. Lack of co-localization of rodent-specic aSyn (i) with TH (j). Scale bar: 50
μ
m. (For inter-
pretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)
D.J. Marmion et al.
Neurobiology of Disease 148 (2021) 105184
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D.J. Marmion et al.
Neurobiology of Disease 148 (2021) 105184
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aSyn: 26,177 ±2050 NeuN+cells/mm
3
, Olig001-GFP: 46,267 ±411.9
NeuN+cells/mm
3
, *P 0.05). Using TH as a marker for nigral dopa-
mine neurons, a loss of TH+bers and neurons were observed in the
substantia nigra of Olig001-aSyn NHPs (Fig. 5kl) compared to Olig001-
GFP controls (Fig. 5op). Unbiased stereological cell counts throughout
the rostral-caudal extent of the substantia nigra revealed an 11% loss of
TH+neurons in Olig001-aSyn injected monkeys when compared to
Olig001-GFP controls (Fig. 5r, Olig001-aSyn: 4692 ±118.1 TH+cells/
mm
3
, Olig001-GFP: 5260 ±202.9 TH+cells/mm
3
, *P 0.05). Taken
together, these results indicate that sustained overexpression of oligo-
dendroglial aSyn in NHPs is able to elicit striatonigral degeneration that
is observed in the pathogenesis of MSA-P.
5.6. Neuroinammation associated with Oligodendroglial aSyn
overexpression
The presence of neuroinammation has been well established in
synucleinopathies and recent work continues to highlight the immune
system as a key driver of disease progression. In MSA, microgliosis and
astrogliosis are prevalent pathologies observed post-mortem in primary
sites of aSyn+GCIs (Ishizawa et al., 2004; Refolo et al., 2018; Williams
et al., 2020). HLA-DR (LN3), a MHC class II cell surface receptor, is a
commonly used marker that is upregulated on microglia during activa-
tion. Microglial activation shown by increased HLA-DR immunoreac-
tivity was observed to coincide with areas of aSyn expression throughout
the entire putamen of Olig001-aSyn NHPs (Fig. 6a, a, and c), while HLA-
DR immunoreactivity seemed to be restricted to the specic injection
site and needle tract in Olig001-GFP injected NHPs (Fig. 6b and d).
Densitometric measurements revealed a statistically signicant increase
in HLA-DR staining in Olig001-aSyn injected monkeys compared to
Olig001-GFP injected controls, indicating a greater prevalence of acti-
vated microglia due to the overexpression of aSyn in oligodendroglia
(Fig. 6i, Olig001-aSyn: 18.54 ±0.692 AFU, Olig001-GFP: 12.03 ±1.032
AFU, *P 0.05). The HLA-DR+cells in Olig001-aSyn NHPs displayed
morphological features typical of activated microglia, featuring large
cell bodies with short, thick processes (Fig. 6c). Similar to the observed
increased activation of microglia, Olig001-aSyn injected monkeys were
found to display prominent astrogliosis in the putamen where aSyn+
GCIs were formed (Fig. 6e and e), which was not observed in Olig001-
GFP NHPs (Fig. 6f and f). A signicant increase in GFAP+staining in-
tensity was observed in Olig001-aSyn injected monkeys compared to
Olig001-GFP injected controls (Fig. 6j, Olig001-aSyn: 16.5 ±2.045 AFU,
Olig001-GFP: 10.84 ±1.110 AFU, *P 0.05). Recently, our group has
shown that in addition to increased activation of microglia and astroglia,
there is a signicant inltration of T cells into the CNS of post-mortem
MSA samples in brain regions containing aSyn-rich GCIs that drives
disease progression (Williams et al., 2020). Histological analysis
conrmed a robust inltration of CD3+T cells in the putamen of
Olig001-aSyn injected NHPs (Fig. 6g and g), where minimal CD3+cells
were observed with GFP injection (Fig. 6h and h). Unbiased stereo-
logical counts of CD3+T cells indicate a 470% increase in Olig001-aSyn
NHPs compared to Olig001-GFP controls (Fig. 6k, Olig001-aSyn: 17,543
±1017 CD3+cells/mm
3
, Olig001-GFP: 3076 ±696 CD3+cells/mm
3
,
*P 0.05). These results indicate that viral vector-mediated over-
expression of aSyn in oligodendrocytes of NHPs causes reactive, neu-
roinammaory phenotypes that mimic both the innate and adaptive
immune responses observed in clinical MSA.
6. Discussion
Previous work by our group utilizing the novel oligotropic AAV
Olig001 to model MSA resulted in 95% efciency to specically trans-
duce striatal oligodendroglia in both rats and NHPs. Short term (3-
month) overexpression of aSyn in NHPs produced the formation of
insoluble, pS129+GCIs throughout the caudate and putamen, causing
demyelination and microglial activation in regions of aSyn over-
expression. Critically, no evidence of neuronal loss was observed at this
short time point (Mandel et al., 2017). The objective of the present study
was to build upon our previous work and to characterize the neuropa-
thology following long term viral vector-mediated overexpression of
aSyn in rats and NHPs. Here we show viral titer-dependent formation of
MSA-like pathology in rats indicating that a ~ 10-fold higher titer of
Olig001-aSyn is required to elicit myelin disruption and neuronal loss,
and that high titers do not lead to off-target transduction. Six months
following Olig001-aSyn injection in NHPs, hyperphosphorylated, thio-
avin S+GCIs were formed throughout the rostrocaudal extent of the
putamen, which elicited demyelination, astrogliosis, microglial activa-
tion, inltration of T cells into the CNS, and neuronal loss in both the
putamen and substantia nigra. The comprehensive array of neuropath-
ological features observed in this model following Olig001-aSyn trans-
duction faithfully recapitulate an MSA-P-like phenotype in both rats and
nonhuman primates (Table 1). Many non-motor and features of dysau-
tonomia, such as heart rate variability, impaired respiratory control,
bladder dysfunction, and REM sleep behavior disorder, have been
observed in the PLP-aSyn mouse, however these aspects of MSA were
not addressed in the present study (Boudes et al., 2013Boudes et al.,
2013; Fernagut et al., 2014; Flabeau et al., 2014; Kuzdas et al., 2013;
Stefanova et al., 2005; Stemberger et al., 2010). Additionally, the failure
to observe neuronal loss in our previous short term (3 month) studies,
and the presence of striatal and nigral loss in the present longer term (6
month) experiments, provides a temporal framework for which pro-
gressive neuronal loss between these two time points can be used for
therapeutic interventions.
The Olig001-aSyn viral vector overexpression model of MSA, like
transgenic mouse models of MSA, is based on the specic overexpression
of human aSyn in oligodendroglia (Bassil et al., 2017; Kahle et al., 2002;
Shults et al., 2005; Tanji et al., 2019; Yazawa et al., 2005). These similar
strategies originated around the 1989 discovery of GCIs in patients with
striatonigral degeneration, olivopontocerebellar atrophy, and Shy-
Drager syndrome, uniting these three syndromes under the common
disease MSA (Papp et al., 1989; Quinn, 1989). Filamentous aSyn was
later identied as a major component of GCIs (Spillantini et al., 1998)
and the notion that GCIs are central to the pathogenesis of MSA arose as
the distribution of GCIs strongly correlated with the degree neuronal
loss (Ozawa et al., 2004; Papp and Lantos, 1994). Post-mortem and
experimental studies have shown that the aSyn present in GCIs has
undergone a number of post-translational modications, such as phos-
phorylation and nitration of tyrosine, and serine phosphorylation
(Beyer, 2006; Beyer and Ariza, 2007; Duda et al., 2000a; Duda et al.,
2000b; Fujiwara et al., 2002; Giasson et al., 2000; Kahle et al., 2002). In
the present study, we have demonstrated that the aSyn present in the
GCIs of Olig001-aSyn injected rats and monkeys has undergone post-
Fig. 4. Olig001 transduction and GCI formation in post-mortem MSA and Olig001 nonhuman primate brains. Photomicrographs of LB509 aSyn immunoreactivity
throughout the rostro-caudal putamen of Olig001-aSyn injected NHPs (A). High magnication inset illustrating LB509+GCIs (A). Scale bar: A=25
μ
m. LB509+
aSyn staining (b and d, green) surrounding the nuclear oligodendroglial Olig2+signal (c and d, red) conrms the cytoplasmic location of the aSyn aggregates in NHP
oligodendrocytes. Scale bar: b-d =20
μ
m. Photomicrographs of post-translational modications of aSyn shown by pS129 and pY39 aSyn immunoreactivity in post-
mortem MSA putamen (E and H), substantia nigra (F and I), and Olig001-aSyn injected NHPs (G and J). Scale bars: E-J =25
μ
m. Stereological analysis of the area of
transduction in the putamen (K) revealed no signicant difference in inclusions formed in Olig001 injected NHPs. Thioavin S+inclusions formed in Olig001-aSyn
injected NHPs (L). Scale bar: L =10
μ
m. Acb =nucleus accumbens, ac =anterior commissure, Cd =caudate, GPe =globus pallidus externus, GPi =globus pallidus
interus, ic =internal capsule, Pu =putamen. N =4/group. (For interpretation of the references to colour in this gure legend, the reader is referred to the web
version of this article.)
D.J. Marmion et al.
Neurobiology of Disease 148 (2021) 105184
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D.J. Marmion et al.
Neurobiology of Disease 148 (2021) 105184
12
translational modications, as demonstrated by the abundant pS129 and
pY39 immunoreactivity. Additionally, resistance to proteinase K diges-
tion and thioavin S staining indicates the formation of insoluble ag-
gregates that have formed beta-pleated sheets as seen in authentic GCIs.
Abnormal accumulation of brillary
α
Syn has also been reported in
neuronal cytoplasm (NCIs) and nuclei (NNIs) as well as in neurites in
post-mortem human MSA brains and in Olig001-aSyn injected animals
(Papp and Lantos, 1994; Yoshida, 2007). NCIs are widely distributed
throughout the brain, however unlike GCIs, their frequency does not
seem to relate to the degree of neuron loss, highlighting the particular
importance of GCIs to disease progression (Ozawa et al., 2004).
Despite the disruption of myelin observed in regions of GCI forma-
tion, the presence of aSyn inclusions in oligodendroglia is believed to
disrupt the normal function of oligodendrocytes, but not oligoden-
droglial degeneration, as only minor changes to oligodendrocyte
numbers have been reported in post-mortem MSA cases (Ettle et al.,
2016; Nykjaer et al., 2017; Salvesen et al., 2015; Salvesen et al., 2017).
In line with this concept, overt loss of oligodendroglia was not seen
following aSyn overexpression using Olig001 in either rats or NHPs,
while oligodendrocyte dysfunction was evident with respect to myelin
integrity as observed by LFB staining and EM in areas of induced aSyn
expression.
Another important oligodendroglial pathology during MSA patho-
genesis is the accumulation of p25
α
/tubulin polymerization-promoting
protein (TPPP) in GCIs in oligodendrocytes from patients with MSA
(Kov´
acs et al., 2004; Orosz et al., 2004; Takahashi et al., 1991). Under
physiological conditions, p25
α
is localized in the myelin sheath and is
functionally involved in the stabilization of microtubules and myelina-
tion, while relocation of p25
α
from the myelin sheath to the cytoplasm
of oligodendroglia has been observed in MSA patients (Ovadi and Orosz,
2009; Song et al., 2007). This relocation correlates with a dramatic
decrease of total MBP and an increase of degraded MBP (Kragh et al.,
2009). Using antibodies directed towards p25
α
, the co-localization of
p25
α
with LB509+inclusions was seen in oligodendrocytes 1-month
following injections of the highest titer of Olig001-aSyn in rats, which
temporally corresponded with demyelination. Highlighting the rele-
vance of p25
α
relocation and aSyn accumulation in GCIs, it has recently
been shown that the overexpression of either p25
α
or aSyn in oligo-
dendrocytes can enhance the recruitment of endogenous aSyn to form
insoluble inclusions (Mavroeidi et al., 2019).
Despite the known link between aSyn, GCIs, and the progression of
pathogenesis in MSA, the source of aSyn and the mechanism by which it
accumulates in oligodendroglia remains heavily debated. In healthy
brains, aSyn is found in nerve terminals and is believed to play an
important role in synaptic transmission (Burre et al., 2014; Emamzadeh,
2016). Following the prion hypothesis of PD, one school of thought
suggests aggregated aSyn can act as a prion that propagates throughout
the neuraxis in MSA (Peng et al., 2018; Prusiner et al., 2015; Watts et al.,
2013; Woerman et al., 2018). Experimentally, in vitro (Reyes et al.,
2014) and in vivo (Rockenstein et al., 2012; Kaji et al., 2018) studies
have shown that aSyn is able to transfer from neurons to oligodendro-
cytes. However, none of these studies have been able to replicate the
formation of GCIs in wildtype animals (Dhillon et al., 2019). Another
belief is that MSA is a primary oligodendrogliopathy with secondary
neurodegeneration (Jellinger, 2015; Wenning et al., 2008). Conicting
studies exist showing the absence (Jin et al., 2008; Miller et al., 2005;
Ozawa et al., 2001) or presence (Asi et al., 2014) of aSyn mRNA in
oligodendroglia. Developmental studies using embryonic stem cells and
induced pluripotent stem cells have shown the presence of aSyn protein
and mRNA in oligodendrocyte precursor cells (OPCs) that diminishes
upon cellular maturation (Djelloul et al., 2020), and that sustained
expression of aSyn in OPCs hinders development of oligodendroglia and
their ability to myelinate (Ettle et al., 2014; May et al., 2014). The notion
that continuous expression of aSyn hinders oligodendrocyte develop-
ment and function may raise questions as to the mechanism of neuro-
degeneration observed in tg MSA mice and its faithfulness to model the
pathological progression of clinical MSA, as overexpression is contin-
uous since birth in these models (Kahle et al., 2002; Shults et al., 2005;
Yazawa et al., 2005). Viral vector-mediated overexpression (Bassil et al.,
2017; Mandel et al., 2017) as well as inducible aSyn expression (Tanji
et al., 2019) may get around this potential pitfall by overexpressing aSyn
in adult animals, although viral vector-mediated induction avoids po-
tential recombinant leakinessof Cre transgenic models. It will be
important to further characterize the dynamics of synuclein aggregation
within oligodendrocytes in this model, as putative synuclein strains
derived from MSA patients have been suggested to produce a more toxic
and aggregate-prone proteoforms (Stroh¨
aker et al., 2019; Yamasaki
et al., 2019).
A growing body of work has highlighted the importance of the im-
mune system playing a role in neurodegenerative diseases and synu-
cleinopathies in particular. The aSyn-containing Lewy bodies and Lewy
neurites in PD have been associated with increased HLA-DR+microglia,
and CD4+and CD8+T lymphocytes have been observed surrounding
neuromelanin-laden nigral neurons, providing evidence for a aSyn-
driven neuroinammatory response (Allen Reish and Standaert, 2015;
Brochard et al., 2009; Caggiu et al., 2019). Circulating T cells in the
blood of PD patients have recently been shown to recognize aSyn pep-
tides and produce increased release of pro-inammatory cytokines
compared to healthy controls (Sulzer et al., 2017). Studies in post-
mortem MSA tissue and animal models of MSA have provided evi-
dence of increased pro-inammatory cytokines in the CSF and brain
parenchyma, as well as microgliosis and astrogliosis in brain regions
affected by the disease (Compta et al., 2019; La Holton and Revesez,
2013; Li et al., 2018; Refolo et al., 2018; Rydbirk et al., 2017; Stefanova
et al., 2007; Stefanova et al., 2012; Yamasaki et al., 2017). The robust
activation of microglia and astrogliosis observed in the current study, as
shown by increased HLA-DR+and GFAP+staining in the putamen of
Olig001-aSyn injected monkeys, parallels the growing body of evidence
pointing towards an inammatory response in MSA driven by aSyn and
supports this model as faithfully reproducing this important feature of
the human disease. Moreover, recent work by our group and others has
shown robust inltration of T lymphocytes into the brain parenchyma of
MSA patients compared to controls (Rydbirk et al., 2017; Williams et al.,
2020), which is recapitulated in the present study by showing a signif-
icant increase in the number of CD3+T-cells in the putamen of Olig001
NHPs specic to aSyn.
It has been proposed that accumulation of aSyn in oligodendroglia
leads to disruption of normal function, such as maintenance of myelin
and neurotrophic support, resulting in secondary neuronal degenera-
tion. As previously mentioned, accumulating aSyn causes glial activa-
tion, T cell inltration, and release of pro-inammatory cytokines, all of
which contribute to neurodegeneration observed in MSA. Our previous
work using Olig001 resulted in the formation of early stage neuropa-
thology observed in MSA (Mandel et al., 2017). By sacricing at later
Fig. 5. Olig001-aSyn induced neurodegeneration in nonhuman primates. Photomicrograph of LB509 aSyn immunoreactivity in Olig001-aSyn injected NHPs (A and
C) correspond to regions of demyelination in the corpus callosum and putamen (B and D respectively, arrowheads) in level matched sections shown by loss of LFB
staining. GFP expression in Olig001-GFP NHPs (E and G) does not induce demyelination (f and h). Scale bars: A-H =2000
μ
m. Photomicrograph of LB509+aSyn
staining (I) which correspond to decreased NeuN immunoreactivity in the putamen of Olig001-aSyn injected NHPs (J), whereas GFP expression (M) does not result in
diminished NeuN immunoreactivity in Olig001-GFP NHPs (N). Scale bars: I and M =1000
μ
m; J and N =500
μ
m. Diminished TH+ber and neuronal staining in the
substantia nigra of Olig001-aSyn NHPs (K,L) compared to Olig001-GFP NHPs (O, P). Scale bar K and O =2000
μ
m, L and P =100
μ
m. Stereological analysis revealed
a signicant loss of NeuN+neurons in the putamen (Q, *P 0.05, Two-tailed Mann Whitney test) and TH+neurons in the substantia nigra (R, *P 0.05, Two-tailed
Mann Whitney test) of Olig001-aSyn NHPs compared to Olig001-GFP NHPs. N =4/group.
D.J. Marmion et al.
Neurobiology of Disease 148 (2021) 105184
13
(caption on next page)
D.J. Marmion et al.
Neurobiology of Disease 148 (2021) 105184
14
time points and increasing the volume of Olig001, we were able to
induce neuronal loss in both the putamen and substantia nigra in the
present study. Using activity monitors and the hand reach test to assess
overall behavioral changes and ne motor dysfunction in monkeys, we
did not observe any signicant motor decits in the present study
(Supplementary Fig. 3). Given that we only observed an ~12% loss of
nigral dopaminergic neurons in these monkeys, it is understandable that
a behavioral decit was not seen at this time, as the threshold for the
appearance of motor symptoms was shown to require an ~80% loss of
striatal dopamine content and ~43% nigral dopaminergic neuron
degeneration experimentally in MPTP-lesioned NHPs and in post-
mortem PD cases (Bezard et al., 2001; Kordower et al., 2013). TH cell
loss was less than wat is typically seen in MSA-P, but future studies will
utilize larger doses of Olig001 vector, which will a greater amount of
neuronal loss, thus producing an observable motor decit.
Unfortunately, there are currently no available potent symptomatic
or disease modifying therapies for MSA and symptomatic therapies have
not drastically changed over the last decade (Krismer and Wenning,
2017; Lopez-Cuina et al., 2018). Dysautonomia can be managed with
drugs marketed to treat orthostatic hypotension and urinary dysfunction
and antiparkinsonian dopaminergic medications may work on a subset
of MSA-P patients (Perez-Lloret et al., 2015). Using proper animal
models in pre-clinical testing of experimental therapeutics is a necessity
for adequate translation into clinical trials. Studying neurological dis-
orders in NHPs provides advantages over the use of rodents. Anatomi-
cally, the striatum in rodents is a single entity pierced by cortical white
matter bers, whereas in humans and NHPs, the striatal mass is sepa-
rated into the caudate nucleus and putamen by the internal capsule. It is
known that white matter tracts affect the distribution of intracerebrally
injected compounds, thus studies involving striatal injections may offer
more accurate translation into humans when modeled in NHPs (Mar-
mion and Kordower, 2018). Furthermore, the size and organization of
the NHP brain, as well as the glia-to-neuron ratio, is much more similar
to humans than that of a mouse brain, which is important when studying
glial diseases and in therapies that will attempt to clear or prevent the
formation of GCIs in a larger brain. The Olig001 NHP model of MSA has
several neuropathological features, such as myelination, neuro-
inammation, GCI formation, and neuronal counts, which can be used
as outcome measures in pre-clinical studies testing the efcacy of dis-
ease modifying therapies of MSA.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.nbd.2020.105184.
Author contributions
DJM participated in the design, rodent and nonhuman primate sur-
geries, collecting data, data analysis, and writing the manuscript. AAR,
BMH and DC participated in collecting the data. MHW, EB, and DK
participated in the design, data analysis, and writing the manuscript. TM
and SJG developed and produced the Olig001 vector. JHK participated
in the design, nonhuman primate surgery, data analysis, and writing the
manuscript.
Ethical approval and consent to participate
Ethical approval for the collection and use of post-mortem brain
tissue used in this study was obtained from the Rush University Medical
Center IRB.
Ethical approval for the use of animals and all procedures were
approved by the University of Illinois Chicago Institutional Animal Care
and Use Committee and the Rush University Institutional Animal Care
and Use Committee and accredited by the Association for Assessment
and Accreditation of Laboratory Animal Care. Animals were cared for in
accordance with the principles of the Guide to the Care and Use of
Experimental Animals. Animal care was supervised by veterinarians
skilled in the care and maintenance of non-human primates.
Consent for publication
Not applicable.
Funding
This work was supported by a grant to JHK from the MSA Coalition.
Declaration of Competing Interest
Drs. Gray and McCown are inventors on a patent for the Olig001
capsid, and have received royalty income from Asklepios Biopharma
related to this invention.
Acknowledgements
We would like to acknowledge Scott Muller and Rachel Harker for
their assistance with nonhuman primate surgery and behavior and Dr.
Poul Henning Jensen for his generous donation of p25
α
antibody.
Fig. 6. Olig001-aSyn induced neuroinammation in nonhuman primates. Photomicrographs showing transgene expression (A-B), and associated microgliosis
illustrated by HLA-DR immunoreactivity (C-D), astrogliosis shown by GFAP immunoreactivity (E-F), and T-cell response shown by CD3 immunoreactivity (G-H) in
level matched sections of Olig001-aSyn and Olig001-GFP NHPs. Arrowheads denote transduction area and region of high magnication image. Scale bars: A-H =
2000
μ
m; AH=25
μ
m. Optical density measurements of HLA-DR stained tissue (I) revealed a signicant increase of microglial activation in Olig001-aSyn NHPs
compared to Olig001-GFP NHPs *P 0.05, Two-tailed Mann Whitney test). Optical density measurements of GFAP stained tissue (J) revealed a signicant increase of
astrogliosis in Olig001-aSyn NHPs compared to Olig001-GFP NHPs *P 0.05). Stereological analysis of CD3+cells (K) revealed a signicant increase in T-cell
inltration in the putamen of Olig001-aSyn NHPs compared to Olig001-GFP NHPs *P 0.05, Two-tailed Mann Whitney test). N =4/group.
Table 1
Comparison of pathological features observed in the Olig001 NHP model of MSA
and clinical MSA in humans.
Olig001-aSyn NHP
(3 month)
Olig001-aSyn NHP (6
Month)
Human
MSA
GCIs Yes Yes Yes
PK Resistant aSyn Yes Yes Yes
Thioavin S+
Inclusions
N/A Yes Yes
pS129+aSyn Yes Yes Yes
pY39+aSyn N/A Yes Yes
p25
α
Relocation N/A Yes Yes
Demyelination Yes Yes Yes
Neuronal Loss No Yes; Striatum &
Substantia Nigra
Yes
Microglial Activation Yes Yes Yes
Astrogliosis N/A Yes Yes
T-Cells in CNS Yes Yes Yes
Oligodendroglial
Loss
No No No
Motor Symptoms No No Yes
Autonomic
Dysfunction
N/A N/A Yes
N/A: Not assessed, GCIs: Glial Cytoplasmic Inclusions, PK: proteinase K, pS129:
phosphorylated serine at residue 129, pY39: phosphorylated tyrosine at residue
39.
D.J. Marmion et al.
Neurobiology of Disease 148 (2021) 105184
15
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D.J. Marmion et al.
... Using a novel modified AAV in which human α-syn is overexpressed in oligodendroglia (Olig001-SYN) 7,8 we observed significant neuroinflammation, demyelination, and neurodegeneration. Thus, effectively modeling MSA in rodents and non-human primates 9 . Using this Olig001-SYN model of MSA, we also demonstrated significant MHCII induction on CNS resident microglia and infiltrating macrophages, along with the infiltration of CD4 and CD8 T cells, similar to that observed in post-mortem brains 6 . ...
... The Olig001 vector is a modified AAV capsid generated via directed evolution that has been characterized previously [7][8][9] . Briefly, the Olig001 capsid has a >95% tropism for oligodendrocytes and the vectors utilized contained the CBh promoter and bovine growth hormone polyA, controlling the expression of either transgene (human α-syn or GFP as control). ...
... To determine if IFNγ neutralization attenuates Olig001-SYN mediated neuroinflammation and demyelination, an IFNγ neutralizing antibody was used to globally deplete IFNγ. WT mice (8)(9)(10)(11)(12) week old) were pre-treated with either an IFNγ neutralizing antibody (XMG1.2; 200ng) or an isotype control (IgG1; 200ng) intraperitoneally (i.p.). ...
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Multiple system atrophy (MSA) is a rare and fatal synucleinopathy characterized by insoluble alpha-synuclein (α-syn) cytoplasmic inclusions located within oligodendroglia. Neuroinflammation, demyelination, and neurodegeneration are correlated with areas of GCI pathology, however it is not known what specifically drives disease pathogenesis. Recently in a mouse model of MSA, CD4+ T cells have been shown to drive neuroinflammation and demyelination, however the mechanism by which this occurs also remains unclear. In this study we use genetic and pharmacological approaches in a novel model of MSA to show that the pro-inflammatory cytokine interferon gamma (IFNγ) drives neuroinflammation and demyelination. Furthermore, using an IFNγ reporter mouse, we found that infiltrating CD4+ T cells were the primary producers of IFNγ in response to α-syn overexpression in oligodendrocytes. Results from these studies indicate that IFNγ expression in CD4 T cells drives α-syn-mediated neuroinflammation and demyelination, and strategies to target IFNγ expression may be a potential disease modifying therapeutic strategy for MSA.
... Following striatal injection of Olig001 expressing aSyn or GFP under the constitutive CBh promoter, we found > 95% oligodendroglial-specific tropism in rats and 90-94% in rhesus macaques (Mandel et al. 2017). In rats, neurodegeneration corresponded with aSyn expression levels, where greater levels of demyelination and neuronal loss in both the striatum and SN were observed when using a tenfold higher titer of Olig001 (Marmion et al. 2021). In both species, widespread aSyn-rich GCIs were present, in which aSyn was phosphorylated at serine 129 and tyrosine 39. ...
... These aggregates were resistant to proteinase K, and were Thioflavin S positive, demonstrating the formation of GCIs similar to those observed in MSA. Titer-dependent degeneration was observed with a 14.7% loss of striatal NeuN + neurons and 21.7% loss of TH + nigral neurons in low-titer Olig001-aSyn injected rats (2.4 × 10 11 vg/ml), which increased to a 23.8% and 35.9% loss of neurons in the striatum and SN of high-titer Olig001-aSyn injected rats (3.2 × 10 12 vg/ml), respectively (Marmion et al. 2021). Progressive degeneration was observed in the Olig001-aSyn NHP model of MSA, with demyelination observed but no cell loss after 3 months (Mandel et al. 2017). ...
... However, there was a ~ 44% loss of NeuN + neurons in the putamen and a 11% loss of TH + nigral neurons was reported 6 months following injection of Olig001-aSyn, recapitulating the progressive nature of MSA. Robust increases in neuroinflammatory markers, such as HLA-DR, GFAP, and CD3, were observed in aSyn expressing monkeys (Marmion et al. 2021). ...
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Multiple system atrophy (MSA) is a progressive neurodegenerative disorder characterized by striatonigral degeneration (SND), olivopontocerebellar atrophy (OPCA), and dysautonomia with cerebellar ataxia or parkinsonian motor features. Isolated autonomic dysfunction with predominant genitourinary dysfunction and orthostatic hypotension and REM sleep behavior disorder are common characteristics of a prodromal phase, which may occur years prior to motor-symptom onset. MSA is a unique synucleinopathy, in which alpha-synuclein (aSyn) accumulates and forms insoluble inclusions in the cytoplasm of oligodendrocytes, termed glial cytoplasmic inclusions (GCIs). The origin of, and precise mechanism by which aSyn accumulates in MSA are unknown, and, therefore, disease-modifying therapies to halt or slow the progression of MSA are currently unavailable. For these reasons, much focus in the field is concerned with deciphering the complex neuropathological mechanisms by which MSA begins and progresses through the course of the disease. This review focuses on the history, etiopathogenesis, neuropathology, as well as cell and animal models of MSA.
... Previous reviews have summarized the limitations of rodents and nonhuman primates in modeling PD [151] and MSA [152]. The animal models might not recapitulate the prion-like propagation of pathologic αSyn in PD patient brains. ...
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Alpha-synucleinopathies, such as Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy, are characterized by aberrant accumulation of alpha-synuclein and synaptic dysfunction leading to motor and cognitive deficits. Animal models of alpha-synucleinopathy have greatly facilitated the mechanistic understanding of the disease and the development of therapeutics. Various transgenic, alpha-synuclein fibril-injected, and toxin-injected animal models of Parkinson’s disease and multiple system atrophy that recapitulate the disease pathology have been developed and widely used. Recent advances in positron emission tomography have allowed the noninvasive visualization of molecular alterations, underpinning behavioral dysfunctions in the brains of animal models and the longitudinal monitoring of treatment effects. Imaging studies in these disease animal models have employed multi-tracer PET designs to reveal dopaminergic deficits together with other molecular alterations. This review focuses on the development of new positron emission tomography tracers and studies of alpha-synuclein, synaptic vesicle glycoprotein 2 A, neurotransmitter receptor deficits such as dopaminergic receptor, dopaminergic transporter, serotonergic receptor, vesicular monoamine transporter 2, hypometabolism, neuroinflammation, mitochondrial dysfunction and leucine rich repeat kinase 2 in animal models of Parkinson’s disease. The outstanding challenges and emerging applications are outlined, such as investigating the gut-brain-axis by using positron emission tomography in animal models, and provide a future outlook.
... The third strategy to model MSA has been the overexpression of a-syn in oligodendrocytes either in constitutive or inducible transgenic mice [75][76][77][78][79] or by AAV targeted a-syn overexpression in the substantia nigra and striatum of mice, rats, or primates [80][81][82][83]. In all overexpression models, irrespective of the mode of overexpression, a delayed progressive neurodegeneration with variable phenotype and intensity has been identified to accompany the formation of GCI-like structures in parallel to signs of neuroinflammation. ...
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Multiple system atrophy (MSA) is a rare neurodegenerative disorder with unclear etiology, currently difficult and delayed diagnosis, and rapid progression, leading to disability and lethality within 6 to 9 years after symptom onset. The neuropathology of MSA classifies the disease in the group of a-synucleinopathies together with Parkinson’s disease and other Lewy body disorders, but features specific oligodendroglial inclusions, which are pathognomonic for MSA. MSA has no efficient therapy to date. Development of experimental models is crucial to elucidate the disease mechanisms in progression and to provide a tool for preclinical screening of putative therapies for MSA. In vitro and in vivo models, based on selective neurotoxicity, a-synuclein oligodendroglial overexpression, and strain-specific propagation of a-synuclein fibrils, have been developed, reflecting various facets of MSA pathology. Over the years, the continuous exchange from bench to bedside and backward has been crucial for the advancing of MSA modelling, elucidating MSA pathogenic pathways, and understanding the existing translational gap to successful clinical trials in MSA. The review discusses specifically advantages and limitations of the PLP-a-syn mouse model of MSA, which recapitulates motor and non-motor features of the human disease with underlying striatonigral degeneration, degeneration of autonomic centers, and sensitized olivopontocerebellar system, strikingly mirroring human MSA pathology.
... 11 More recently, viralmediated overexpression of a-syn in rats and non-human primates has been proposed for modelling MSA pathology. 12,13 The injection of pathological forms of a-syn obtained from brain homogenates of a diseased animal or human patient has been used to model synucleinopathies across species. To date, the toxic effects of human-derived samples from patients with various synucleinopathies have been assessed mostly for Parkinson's disease, where Lewy bodies-enriched fractions derived from patients have been used to induce a Parkinson's disease-like pathology in both mice and non-human primates (NHPs). ...
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Synucleinopathies encompass several neurodegenerative diseases, which include Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. These diseases are characterized by the deposit of α-synuclein aggregates in intracellular inclusions in neurons and glial cells. Unlike Parkinson’s disease and dementia with Lewy bodies, where aggregates are predominantly neuronal, multiple system atrophy is associated with α-synuclein cytoplasmic inclusions in oligodendrocytes. Glial cytoplasmic inclusions are the pathological hallmark of multiple system atrophy and are associated with neuroinflammation, modest demyelination and, ultimately, neurodegeneration. To evaluate the possible pathogenic role of glial cytoplasmic inclusions, we inoculated glial cytoplasmic inclusion-containing brain fractions obtained from multiple system atrophy patients into the striatum of non-human primates. After a 2-year in vivo phase, extensive histochemical and biochemical analyses were performed on the whole brain. We found loss of both nigral dopamine neurons and striatal medium spiny neurons, as well as loss of oligodendrocytes in the same regions, which are characteristics of multiple system atrophy. Furthermore, demyelination, neuroinflammation and α-synuclein pathology were also observed. These results show that the α-synuclein species in multiple system atrophy-derived glial cytoplasmic inclusions can induce a pathological process in non-human primates, including nigrostriatal and striatofugal neurodegeneration, oligodendroglial cell loss, synucleinopathy and gliosis. The present data pave the way for using this experimental model for MSA research and therapeutic development.
... Indeed, activated c-Abl is markedly increased in models of PD and in brain samples derived from PD patients. 28,62,73 These data suggest that c-Abl activation followed by formation of pY39 may be a required step for neurodegeneration to occur, whereas phosphorylation at Ser 129 may just be a marker of misfolded α-synuclein, but not a modification critical to the neurodegenerative disease process itself. The failure of poorly brain penetrant c-Abl inhibitors like nilotinib to show a consistent neuroprotective phenotype in model studies 62 or clinical trials (see below), is likely to be a result of incomplete inhibition of c-Abl activation, not a sign that c-Abl is insignificant to the disease process. ...
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Parkinson's disease (PD) is the second most prevalent neurodegenerative disease of the central nervous system, with an estimated 5 000 000 cases worldwide. Historically characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta, PD pathology is now known to be widespread and to affect serotonin, cholinergic and norepinephrine neurons as well as nerve cells in the olfactory system, cerebral hemisphere, brain stem, spinal cord, and peripheral autonomic nervous system. PD pathology is characterized by the accumulation of misfolded α‐synuclein, which is thought to play a critical role in the etiopathogenesis of the disease. Animal models of PD suggest that activation of the Abelson tyrosine kinase (c‐Abl) plays an essential role in the initiation and progression of α‐synuclein pathology and neurodegeneration. These studies demonstrate that internalization of misfolded α‐synuclein activates c‐Abl, which phosphorylates α‐synuclein and promotes α‐synuclein pathology within the affected neurons. Additionally, c‐Abl inactivates parkin, disrupting mitochondrial quality control and biogenesis, promoting neurodegeneration. Post‐mortem studies of PD patients demonstrate increased levels of tyrosine phosphorylated α‐synuclein, consistent with the activation of c‐Abl in human disease. Although the c‐Abl inhibitor nilotinib failed to demonstrate clinical benefit in two double‐blind trials, novel c‐Abl inhibitors have been developed that accumulate in the brain and may inhibit c‐Abl at saturating levels. These novel inhibitors have demonstrated benefits in animal models of PD and have now entered clinical development. Here, we review the role of c‐Abl in the neurodegenerative disease process and consider the translational potential of c‐Abl inhibitors from model studies to disease‐modifying therapies for Parkinson's disease. © 2021 Inhibikase Therapeutics, Inc. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society. The emergence of misfolded α‐synuclein in the substantia nigra as a result of oxidative/nitrosative stress, protein mutation, impaired clearance, and/or genetic factors leads to internalization of misfolded α‐synuclein, which activates the non‐receptor Abelson tyrosine kinase (c‐Abl), a key event in the initiation and progression of neurodegeneration in Parkinson's disease. c‐Abl activation both inactivates the ubiquitin E3 ligase parkin and creates the pathological form of α‐synuclein on the inside of the affected neurons, both events following specific tyrosine phosphorylation by c‐Abl. Treatment with a c‐Abl inhibitor reverses these processes, reactivating parkin and driving clearance of misfolded and pathological α‐synuclein through lysosomal and/or proteasomal processes. In model systems, these events result in substantial functional recovery in the brain and gastrointestinal (GI) tract. Although initial clinical trials of c‐Abl inhibitors failed, novel c‐Abl inhibitors thought to accumulate in the brain at concentrations sufficient to block c‐Abl are now being tested in the clinic and offer the opportunity to evaluate the potential of c‐Abl inhibition as a disease‐modifying therapy for patients with PD.
... The study found D330 cl-caspase-9 in oligodendrocytes and in neuronal and glial cytoplasmic inclusions, supporting involvement of both caspase-9 and caspase-3 in neurodegenerative pathology. Rodent and nonhuman primate models of multiple system atrophy (Lee et al., 2019;Marmion et al., 2021) offer experimental tools to investigate cell-specific consequences of caspase-9 activation in neurons and glia and enable studies to test the therapeutic potential of caspase-9 inhibitors as a neuroprotective strategy in MSA. ...
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Multiple system atrophy is a rapidly progressive and fatal neurodegenerative disorder. While numerous preclinical studies suggested efficacy of potentially disease modifying agents, none of those were proven to be effective in large-scale clinical trials. Three major strategies are currently pursued in preclinical and clinical studies attempting to slow down disease progression. These target α-synuclein, neuroinflammation, and restoration of neurotrophic support. This review provides a comprehensive overview on ongoing preclinical and clinical developments of disease modifying therapies. Furthermore, we will focus on potential shortcomings of previous studies that can be avoided to improve data quality in future studies of this rare disease.
Thesis
Mon projet de thèse s’inscrit dans l’étude des synucléinopathies, une famille de maladies neurodégénératives. Les trois principales synucléinopathies sont la maladie de Parkinson, l’atrophie multisystématisée et la démence à corps de Lewy. Ces maladies sont caractérisées par une perte de neurones dans des régions cérébrales spécifiques et la présence d’inclusions intra-cytoplasmiques positives pour l’α-synucléine dans les neurones (Corps de Lewy) ou dans les oligodendrocytes (Inclusions gliales cytoplasmiques). Les causes d’induction de ces maladies restent encore inconnues et les traitements curatifs sont inexistants. L’objectif de mon travail de thèse visait à étudier les mécanismes neurodégénératifs et de potentielles cibles thérapeutiques dans le contexte des synucléinopathies. Je me suis tout d’abord intéressée aux mécanismes impliqués dans la transmission de l’α-synucléine issue de patients atteints de l’atrophie multisystématisée. Ce travail nous a permis de développer un potentiel nouveau modèle de l’atrophie multisystématisée chez la souris et le primate non-humain, par la transmission de l’α-synucléine dans le cerveau. Dans un deuxième temps, nous nous sommes intéressés à des cibles thérapeutiques éventuelles pour la maladie de Parkinson dans un même modèle animal de la pathologie. Nous avons pu vérifier l’efficacité et la pertinence de trois différentes stratégies ciblant plusieurs mécanismes affectés dans la maladie de Parkinson dans le but d’induire une protection des neurones dopaminergiques de la substance noire des souris. Nous avons pu démontrer une dérégulation des niveaux de zinc au cours de la pathologie qui a suscité l’intérêt de cibler son homéostasie dans le cerveau à travers une molécule chélatrice du zinc. Ensuite, la surexpression d’un facteur de transcription impliqué dans la survie des neurones dopaminergiques ainsi que dans le stress oxydatif et le protéasome a montré son intérêt comme cible thérapeutique de la maladie de Parkinson. Enfin, une molécule anti-agrégative a aussi démontré sa capacité à induire une neuroprotection. En résumé, ces travaux montrent d’abord l’importance de l’α-synucléine dans la mise en place et la progression des synucléinopathies, mais aussi la nécessité de cibler d’autres mécanismes dérégulés dans ces pathologies pour proposer des nouvelles stratégies thérapeutiques.
Chapter
Animal models are essential to study the pathophysiology of Parkinson's disease and to develop disease-modifying therapies. The identification of α-synuclein as a disease-associated gene and the discovery of this protein as one of the major constituents of Lewy bodies, have prompted the generation of animal models based on α-synuclein transgenesis or viral vector-mediated α-synuclein overexpression. Later, new insights on the “prion-like” properties of α-synuclein led to a new generation of models for Parkinson's disease. These transmission models are developed by intracerebral inoculation of α-synuclein fibrils. Technical advancements and new insights into α-synuclein pathology have led to a wide array of available rodent models that have greatly contributed to our understanding of different aspects of Parkinson's disease. Each of these models has their own characteristics with certain advantages and limitations. The best choice of the animal model will be dependent on the specific question or scientific hypothesis to be tested. In this chapter, we give a comprehensive overview of the different α-synuclein-based rodent models that have been developed.
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Multiple system atrophy (MSA) is a progressive neurodegenerative disorder characterized by abnormal accumulation of alpha-synuclein (α-syn) in oligodendrocytes accompanied by inflammation, demyelination, and subsequent synapse and neuronal loss. Little is known about the mechanisms of neurodegeneration in MSA. However, recent work has highlighted the important role of the immune system to the pathophysiology of other synuclein-related diseases such as Parkinson’s disease. In this study, we investigated postmortem brain tissue from MSA patients and control subjects for evidence of immune activation in the brain. We found a significant increase of HLA-DR+ microglia in the putamen and substantia nigra of MSA patient tissue compared to controls, as well as significant increases in CD3+, CD4+, and CD8+ T cells in these same brain regions. To model MSA in vivo, we utilized a viral vector that selectively overexpresses α-syn in oligodendrocytes (Olig001-SYN) with > 95% tropism in the dorsal striatum of mice, resulting in demyelination and neuroinflammation similar to that observed in human MSA. Oligodendrocyte transduction with this vector resulted in a robust inflammatory response, which included increased MHCII expression on central nervous system (CNS) resident microglia, and infiltration of pro-inflammatory monocytes into the CNS. We also observed robust infiltration of CD4 T cells into the CNS and antigen-experienced CD4 T cells in the draining cervical lymph nodes. Importantly, genetic deletion of TCR-β or CD4 T cells attenuated α-syn-induced inflammation and demyelination in vivo. These results suggest that T cell priming and infiltration into the CNS are key mechanisms of disease pathogenesis in MSA, and therapeutics targeting T cells may be disease modifying.
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Parkinson’s disease (PD) and Multiple System Atrophy (MSA) are clinically distinctive diseases that feature a common neuropathological hallmark of aggregated α-synuclein. Little is known about how differences in α-synuclein aggregate structure affect disease phenotype. Here, we amplified α-synuclein aggregates from PD and MSA brain extracts and analyzed the conformational properties using fluorescent probes, NMR spectroscopy and electron paramagnetic resonance. We also generated and analyzed several in vitro α-synuclein polymorphs. We found that brain-derived α-synuclein fibrils were structurally different to all of the in vitro polymorphs analyzed. Importantly, there was a greater structural heterogeneity among α-synuclein fibrils from the PD brain compared to those from the MSA brain, possibly reflecting on the greater variability of disease phenotypes evident in PD. Our findings have significant ramifications for the use of non-brain-derived α-synuclein fibrils in PD and MSA studies, and raise important questions regarding the one disease-one strain hypothesis in the study of α-synucleinopathies. Parkinson’s disease (PD) and Multiple System Atrophy (MSA) are characterized by the pathological accumulation of α-synuclein. Here the authors employ fluorescent probes, electron microscopy and NMR spectroscopy to study the properties of α-synuclein aggregates that were amplified from patient brain extracts and observe a greater structural diversity among PD patients compared to MSA patients.
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α-Synuclein misfolding and aggregation plays a major role in the pathogenesis of Parkinson's disease. Although loss of function mutations in the ubiquitin ligase, parkin, cause autosomal recessive Parkinson's disease, there is evidence that parkin is inactivated in sporadic Parkinson's disease. Whether parkin inactivation is a driver of neurodegeneration in sporadic Parkinson's disease or a mere spectator is unknown. Here we show that parkin in inactivated through c-Abelson kinase phosphorylation of parkin in three α-synuclein-induced models of neurodegeneration. This results in the accumulation of parkin interacting substrate protein (zinc finger protein 746) and aminoacyl tRNA synthetase complex interacting multifunctional protein 2 with increased parkin interacting substrate protein levels playing a critical role in α-synuclein-induced neurodegeneration, since knockout of parkin interacting substrate protein attenuates the degenerative process. Thus, accumulation of parkin interacting substrate protein links parkin inactivation and α-synuclein in a common pathogenic neurodegenerative pathway relevant to both sporadic and familial forms Parkinson's disease. Thus, suppression of parkin interacting substrate protein could be a potential therapeutic strategy to halt the progression of Parkinson's disease and related α-synucleinopathies.
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α-synuclein (αS) is the major component of several types of brain pathological inclusions that define neurodegenerative diseases termed synucleinopathies. Central nervous system (CNS) inoculation studies using either in vitro polymerized αS fibrils or in vivo derived lysates containing αS aggregates to induce the progressive spread of αS inclusion pathology in animal disease models have supported the notion that αS mediated progressive neurodegeneration can occur by a prion-like mechanism. We have previously shown that neonatal brain inoculation with preformed αS fibrils in hemizygous M20+/− transgenic mice expressing wild type human αS and to a lesser extent in non-transgenic mice can result in a concentration-dependent progressive induction of CNS αS pathology. Recent studies using brain lysates from patients with multiple system atrophy (MSA), characterized by αS inclusion pathology in oligodendrocytes, indicate that these may be uniquely potent at inducing αS pathology with prion-like strain specificity. We demonstrate here that brain lysates from MSA patients, but not control individuals, can induce αS pathology following neonatal brain inoculation in transgenic mice expressing A53T human αS (M83 line), but not in transgenic expressing wild type human αS (M20 line) or non-transgenic mice within the timeframe of the study design. Further, we show that neuroanatomical and immunohistochemical properties of the pathology induced by MSA brain lysates is very similar to what is produced by the neonatal brain injection of preformed human αS fibrils in hemizygous M83+/− transgenic mice. Collectively, these findings reinforce the idea that the intrinsic traits of the M83 mouse model dominates over any putative prion-like strain properties of MSA αS seeds that can induce pathology. Electronic supplementary material The online version of this article (10.1186/s40478-019-0733-3) contains supplementary material, which is available to authorized users.
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Multiple system atrophy (MSA) is characterized by the presence of distinctive glial cytoplasmic inclusions (GCIs) within oligodendrocytes that contain the neuronal protein alpha-synuclein (aSyn) and the oligodendroglia-specific phosphoprotein TPPP/p25α. However, the role of oligodendroglial aSyn and p25α in the formation of aSyn-rich GCIs remains unclear. To address this conundrum, we have applied human aSyn (haSyn) pre-formed fibrils (PFFs) to rat wild-type (WT)-, haSyn-, or p25α-overexpressing oligodendroglial cells and to primary differentiated oligodendrocytes derived from WT, knockout (KO)-aSyn, and PLP-haSyn-transgenic mice. HaSyn PFFs are readily taken up by oligodendroglial cells and can recruit minute amounts of endogenous aSyn into the formation of insoluble, highly aggregated, pathological assemblies. The overexpression of haSyn or p25α accelerates the recruitment of endogenous protein and the generation of such aberrant species. In haSyn PFF-treated primary oligodendrocytes, the microtubule and myelin networks are disrupted, thus recapitulating a pathological hallmark of MSA, in a manner totally dependent upon the seeding of endogenous aSyn. Furthermore, using oligodendroglial and primary cortical cultures, we demonstrated that pathology-related S129 aSyn phosphorylation depends on aSyn and p25α protein load and may involve different aSyn “strains” present in oligodendroglial and neuronal synucleinopathies. Importantly, this hypothesis was further supported by data obtained from human post-mortem brain material derived from patients with MSA and dementia with Lewy bodies. Finally, delivery of haSyn PFFs into the mouse brain led to the formation of aberrant aSyn forms, including the endogenous protein, within oligodendroglia and evoked myelin decompaction in WT mice, but not in KO-aSyn mice. This line of research highlights the role of endogenous aSyn and p25α in the formation of pathological aSyn assemblies in oligodendrocytes and provides in vivo evidence of the contribution of oligodendroglial aSyn in the establishment of aSyn pathology in MSA.
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Parkinson's disease is a neurodegenerative disorder characterized by progressive loss of dopaminergic neurons of the substantia nigra pars compacta with a reduction of dopamine concentration in the striatum. The complex interaction between genetic and environmental factors seems to play a role in determining susceptibility to PD and may explain the heterogeneity observed in clinical presentations. The exact etiology is not yet clear, but different possible causes have been identified. Inflammation has been increasingly studied as part of the pathophysiology of neurodegenerative diseases, corroborating the hypothesis that the immune system may be the nexus between environmental and genetic factors, and the abnormal immune function can lead to disease. In this review we report the different aspects of inflammation and immune system in Parkinson's disease, with particular interest in the possible role played by immune dysfunctions in PD, with focus on autoimmunity and processes involving infectious agents as a trigger and alpha-synuclein protein (α-syn).
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Parkinson's disease (PD)§ and multiple system atrophy (MSA) are distinct clinical syndromes characterized by the pathological accumulation of α-synuclein (α-syn) protein fibrils in neurons and glial cells. These disorders and other neurodegenerative diseases may progress via prion-like mechanisms. The prion model of propagation predicts the existence of “strains” that link pathological aggregate structure and neuropathology. Prion strains are aggregated conformers that stably propagate in vivo and cause disease with defined incubation times and patterns of neuropathology. Indeed, tau prions have been well defined, and research suggests that both α-syn and amyloid beta may also form strains. However, there is a lack of studies characterizing PD- vs MSA-derived α-syn strains or demonstrating stable propagation of these unique conformers between cells or animals. To fill this gap, we used an assay based on fluorescence resonance energy transfer that exploits a HEK293T “biosensor” cell line stably expressing α-syn (A53T)-CFP/YFP fusion proteins to detect α-syn seeds in brain extracts from PD and MSA patients. Both soluble and insoluble fractions of MSA extracts had robust seeding activity, while only the insoluble fractions of PD extracts displayed seeding activity. The morphology of MSA-seeded inclusions differed from PD-seeded inclusions. These differences persisted upon propagation of aggregation to second-generation biosensor cells. We conclude that PD and MSA feature α-syn conformers with very distinct biochemical properties that can be transmitted to α-syn monomers in a cell system. These findings are consistent with the idea that distinct α-syn strains underlie PD and MSA and offer possible directions for synucleinopathy diagnosis.
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Introduction: Neuroinflammation is a potential player in neurodegenerative conditions, particularly the aggressive ones, such as multiple system atrophy (MSA). Previous reports on cytokine levels in MSA using serum or cerebrospinal fluid (CSF) have been inconsistent, including small samples and a limited number of cytokines, often without comparison to Parkinson's disease (PD), a main MSA differential diagnosis. Methods: Cross-sectional study of CSF levels of 38 cytokines using a multiplex assay in 73 participants: 39 MSA patients (19 with parkinsonian type [MSAp], 20 with cerebellar type [MSAc]; 31 probable, 8 possible), 19 PD patients and 15 neurologically unimpaired controls. None of the participants was under non-steroidal anti-inflammatory drugs at the time of the lumbar puncture. Results: There were not significant differences in sex and age among participants. In global non-parametric comparisons FDR-corrected for multiple comparisons, CSF levels of 5 cytokines (FGF-2, IL-10, MCP-3, IL-12p40, MDC) differed among the three groups. In pair-wise FDR-corrected non-parametric comparisons 12 cytokines (FGF-2, eotaxin, fractalkine, IFN-α2, IL-10, MCP-3, IL-12p40, MDC, IL-17, IL-7, MIP-1β, TNF-α) were significantly higher in MSA vs. non-MSA cases (PD + controls pooled together). Of these, MCP-3 and MDC were the most significant ones, also differed in MSA vs. PD, and were significant MSA-predictors in binary logistic regression models and ROC curves adjusted for age. CSF levels of fractalkine and MIP-1α showed a strong and significant positive correlation with UMSARS-2 scores. Conclusion: Increased CSF levels of cytokines such as MCP-3, MDC, fractalkine and MIP-1α deserve consideration as potential diagnostic or severity biomarkers of MSA.
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Multiple system atrophy (MSA) is an adult-onset neurodegenerative disorder clinically characterized by autonomic failure in addition to various combinations of symptoms of parkinsonism, cerebellar ataxia, and pyramidal dysfunction. Despite extensive research, the mechanisms underlying the progression of MSA remain unknown. Animal models of human diseases that recapitulate their clinical, biochemical and pathological features are indispensable for increasing our understanding of their underlying molecular mechanisms, which allows preclinical studies to be advanced. Because the onset of MSA occurs in middle age, an animal model that first manifests abnormal protein aggregates in adulthood would be most appropriate. We therefore used the Cre-loxP system to express inducible α-synuclein (Syn), a major component of the pathological hallmark of MSA, to generate a mouse model of MSA. Beginning in adulthood, these MSA model mice express excessive levels of Syn in oligodendrocytes, resulting in abnormal Syn accumulation and modifications similar to those observed in human MSA pathology. Additionally, MSA model mice exhibit some clinical features of MSA, including decreased motor activity. These findings suggest that this new mouse model of MSA represents a useful tool for analyzing the pathophysiological alterations that underlie the progression of this disease.