Introduction
Multiple system atrophy (MSA) is a fatal neurodegenerative disorder that presents clinically with varying combinations of autonomic, parkinsonian, cerebellar, and pyramidal dysfunction [
15]. It is considered to be an orphan disease, with an estimated mean incidence of 0.6–0.7 cases per 100,000 person-years [
5]. Symptoms tend to emerge in the fifth to sixth decade of life [
63], and unlike Parkinson’s disease (PD) progresses extremely rapidly, with a mean survival of 6–9 years following symptom onset [
35,
41,
50,
62]. The distribution of pathology allows for stratification into two MSA subtypes, a cerebellar variant (MSA-C), characterized by olivopontocerebellar atrophy, and a parkinsonian variant (MSA-P), characterized by nigrostriatal and striatonigral degeneration [
39,
65]. MSA, like PD, involves a prodromal premotor phase, with symptoms including urinary incontinence or retention, sexual dysfunction, orthostatic hypotension, and rapid-eye-movement sleep behavior disorder [
27]. Following the prodromal phase, motor symptoms in MSA-P develop, including slowness of movement, rigidity, postural instability, and shuffling gait [
18,
32,
38,
60]. Lack of “pill-rolling” rest tremor, symmetrical presentation of symptoms, and limited levodopa responsiveness can often distinguish MSA-P from PD [
18,
32,
38,
60].
MSA is a synucleinopathy, however, unlike PD and dementia with Lewy bodies, in which α-syn accumulates and aggregates in neurons, MSA is characterized neuropathologically by α-syn-positive glial cytoplasmic inclusions (GCIs) in oligodendroglia [
1,
42,
54,
58,
59]. The precise pathogenesis of MSA remains largely unknown, however, evidence from human postmortem cases and experimental models suggest that, in addition to GCIs, myelin degeneration, axonal damage, loss of neurotrophic support, neuronal loss, astrogliosis and microglial activation are pathological events seen in multiple regions of the central nervous system (CNS) [
28,
56]. The primary cause of degeneration is still under debate- whether cell loss in MSA is secondary to disruptions in the oligo-myelin-axon complex [
29,
64], or if MSA is a primary neuron disease, with the secondary formation of GCIs following pathological accumulation of α-syn that is neuronal in origin [
55]. Experimentally, α-syn has been shown to transfer from neurons to oligodendrocytes in vitro [
47], and inoculation of MSA brain homogenates into transgenic mice supports the hypothesis of prion-like spread in MSA [
45,
61]. However, formation of GCI-like aggregation was not reported in these transfer experiments [
47]. Supporting a primary oligodendrogliopathy, GCIs, and not neuronal inclusions, are a pathology first observed in regions where significant neurodegeneration occurs, with GCI density correlating with the degree of neuron loss [
40,
43,
68]. Moreover, GCIs are the hallmark of MSA and are not seen as frequently in PD, even though both diseases share similar lesion patterns in many overlapping circuits [
23].
Transgenic (tg) mouse models overexpressing human α-syn under different oligodendroglia-specific promoters, such as proteolipid protein (PLP) [
31], myelin basic protein (MBP) [
51], and 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) [
67], have been developed to study MSA. The resulting tg mouse lines developed widespread GCIs, however varying degrees of demyelination, neurodegeneration, and behavioral changes have been reported [
4,
16,
31,
33,
50‐
53,
67]. To date, none of the available tg mouse lines have been able to recapitulate the specific striatonigral degeneration or olivopontocerebellar atrophy as seen in the human disorder [
3].
Adeno-associated virus (AAV) has been successfully used to overexpress α-syn in dopaminergic neurons to create useful animal models to study PD in rodents and nonhuman primates [
19,
57]. AAV is a small, encapsulated parvovirus with a simple genome encoding 2 genes for packaging and replication. In 1984, Hermonat and Muzyczka showed that the entire genome could be removed and DNA could be packaged in replication deficient AAV, allowing for the transfer genes to be ectopically expressed in transduced cells [
24]. A number of AAV capsid mutants have been created, leading to improvements in target cell specificity, efficiency of transduction, and reduced immunogenicity [
22]. In the CNS, most wild-type AAV capsid serotypes transduce neurons at a much higher rate than any other neural cell type, making it difficult to manipulate glial cells. Moreover, the AAV capsid cell tropisms obtained in rodents are not always predictive of the transduction specificity in primates. For example, in our own work, AAV1, 5 and 8 almost exclusively transduce neurons in rat striatum whereas, in the macaque putamen AAV1, 5, and 8 transduced glial cells at higher rates [
11].
In an effort to develop a viral-vector mediated model of MSA, we utilized a novel AAV capsid termed Olig001 and developed by co-authors in our group (SG and TM). The vector was engineered by capsid shuffling and directed evolution to transduce oligodendrocytes in the striatum and corpus callosum of rodents [
44]. In the present study, we showed that 4-weeks following intrastriatal injection of Olig001 expressing GFP transgene leads to overwhelming oligodendroglia-specific tropism in both rats and monkeys, with little to no expression in neurons or astrocytes. We then used this vector to overexpress human α-syn in the striatum and corpus callosum of rhesus monkeys. After 3-months, we observed widespread expression of α-syn in the white matter of the striatum, again overwhelmingly within oligodendroglia. These α-syn GCIs were phosphorylated at serine-129 (pSer-129), resistant to proteinase K (PK) digestion, resulted in demyelination in the striatum and corpus callosum and activated microgla in the substantia nigra. Together, these data indicate the development of the first pathologically-based nonhuman primate model of MSA-P.
Materials and methods
AAV vector
The AAV-Olig001 capsid was developed in the laboratories of Steven Gray and Thomas McCown at the University of North Carolina at Chapel Hill, through a directed evolution screening process as described [
20,
44]. Preliminary studies demonstrated a strong preferential tropism of this capsid for oligodendrocytes following intracranial administration in rodents (data not shown). The AAV-Olig001 vector used in these studies packaged a self-complementary (sc) genome with transgene expression mediated by the CBh promoter and bovine growth hormone polyA [
21]. AAV vectors were produced using methods developed by the University of North Carolina Vector Core facility, as described [
9]. In brief, the production plasmids (pXX6–80, pTRS-ks-CBh-EGFP, and AAV pXR-Olig001) were triple-transfected into suspension HEK293 cells. AAV vectors were purified from the cells by iodixanol gradient centrifugation, followed by ion-exchange chromatography. The purified AAV was dialyzed in PBS supplemented with 5% D-Sorbitol and an additional 212 mM NaCl (350 mM NaCl total). The titer was determined by quantitative PCR and confirmed by polyacrylamide gel electrophoresis (PAGE) and silver stain. Quality control measures were in place that the qPCR titer and PAGE/silver stain titer match within 2-fold, that no contaminating proteins are visible by PAGE, and that the viral capsid proteins migrate at the expected size with a 10:1:1 VP3:VP2:VP1 ratio.
Rodents
Eight-week-old female Sprague-Dawley rats (Harlan, Indianapolis, IN) were used for the rodent rAAV-Olig001-GFP experiment. Upon arrival, animals were quarantined for one week prior to any testing. Animals were cared for in accordance with the principles of the Guide to the Care and Use of Experimental Animals, and all procedures were approved by University of Florida at Gainesville Institutional Animal Care and Use Committee. Rats were housed two per cage with a 12:12 h light:dark cycle (07:00–19:00 h). Food and water were available ad libitum throughout the study.
Rodent stereotaxic surgery
All surgical procedures were performed as previously described [
36]. All animals received unilateral striatal injections 15 min after receiving intraperitoneal injections of mannitol (3 ml sterile 25% mannitol in 0.9% saline / 100 g body weight). Each rat received 1 injection of 2 μl of Olig001-GFP (1 × 10
13 vg/ml) at a rate of 0.5 μl/min [
6]. The coordinates were AP +/− 0.0 mm and ML +/− 2.7 mm from bregma, DV −4.0 mm from dura. One minute following the completion of each injection the micropipette was retracted 1 mm and left in place for an additional 4 min before being slowly removed from the brain.
Rodent necropsy and tissue processing
Four-weeks following the intracerebral rAAV-Olig001 injection all animals were deeply anesthetized with pentobarbital and perfused through the ascending aorta with sterile Tyrode’s solution, followed by 350 ml of ice-cold 4% paraformaldehyde in 0.01 M PBS buffer. Brains were rapidly removed and post-fixed for 12–18 h in the same paraformaldehyde solution, and then transferred to a 30% sucrose in 0.01 M PBS solution. After sinking in the sucrose solution, the brains were cut into 40 μm thick sections using a freezing stage sliding microtome and then processed for immunocytochemistry as described below.
Primates
Sixteen nonhuman primates (eight adult male and eight adult female rhesus monkeys (Macaca mulatta) served as subjects and/or were analyzed for this study. Monkeys were placed in 4 groups of three monkeys each; Group 1: Olig001-GFP sacrificed at 1 month, Group 2: Olig001-α-syn sacrificed at 3 months; Group 3: intrastriatal AAV injections of neurturin sacrificed at 3 months; Group 4: intrastriatal stem cell grafted animals sacrificed at 3 months. Group 5: untreated control.
Since the Olig001-GFP (1 month) and Olig001-α-syn (3 months) were sacrificed at different time points, and to minimize the number of monkeys required for experimentation, Groups 3 and 4 were historical controls in one of our labs (JHK) and served to control for the effects of 1) anesthesia, 2) surgery, 3) needle penetration to the striatum, and delivery of 4) AAV and 5) other bioactive bioactive materials and all were sacrificed at the same post-operative time as Olig001-α-syn treated animals.
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 nonhuman primates.
Primate stereotaxic surgery
Animals were tranqilized and then intubated on the day of surgery with ketamine (10 mg/kg, IM) and propofol (2–5 mg/kg, IV) and placed in sterotaxic frames in the surgical suite. All surgical procedures were conducted under isoflurane anesthesia (1–3% maintenance, inhalation) and sterile field conditions. Sufentanil (0.005–0.3 μg/kg/min, IV infusion) or hydromorphone (0.05–0.2 mg/kg, IV) was administered pre-operatively, as was Cefazolin (25 mg/kg, IV). Surgical targets were identified using pre-operative MRI and intraoperative surgical navigation using a Stealthstation Neuronavigation system (generously donated by Medtronics Inc.). A 50-μl Hamilton syringe fitted with a 22 G needle was loaded with either Olig001-GFP (1 × 1013 vg/ml) or Olig001-α-syn (3.75 × 1012 vg/ml). Animals received injections unilaterally into the putamen (rostral putamen 10 μl, caudal putamen 5 μl) and caudate nucleus (rostral caudate 10 μl, caudal caudate 5 μl) and infused at a rate of 1 μl/min to minimize injectate reflux, inflammation or damage to the parenchyma. After the injection, the needle was left in place for 2 min, then slowly retracted. Bupivicane (1 mg/kg, SQ) was administered to the incision site prior to closure. Animals received analgesics (Buprenex SR, 0.2 mg/kg SQ, once post surgery; Meloxicam, 0.2 mg/kg SQ, once post surgery, then 0.1 mg/kg SQ, SID for 2 days post surgery) and antibiotics (Cephazolin, 25 mg/kg IM, BID for 3 days post surgery).
Primate necropsy and tissue processing
One or three-months post-surgery, animals were deeply sedated and euthanized by transcardial saline perfusion. The brain was removed from the calvarium and was post-fixed in 4% paraformaldehyde solution for 48 h then transferred to a sucrose gradient. Coronal slices (40 μm) were sectioned on a freezing-stage sliding knife microtome, and stored in cryoprotectant solution at 20 °C until processed.
Immunohistochemistry
Free floating 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-specific 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]; rabbit anti-Alpha-synuclein (phospho S129), 1:500–1:1000 dilution [ab51253; Abcam]; mouse anti-Alpha-synuclein (LB 509), 1:500–1:1000 dilution [180,215; Thermo Fischer Scientific]; mouse anti-HLA-DR (LN3), 1:200 dilution [MA5–11966, Thermo Fischer Scientific]), 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 (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 3–3′-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 Scientific International).
Immunofluorescence double labeling
Free floating sections of brain tissue were rinsed of cryoprotectant and non-specific 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-Alpha-synuclein (LB 509), 1:500–1:1000 dilution [180,215; Thermo Fischer Scientific]; rabbit anti-Olig2 [EPR2673], 1:200 dilution [ab109186; Abcam]; rabbit anti-GFAP, 1:2000 dilution [Z033401–2; Dako]; mouse anti-NeuN (A60), 1:500 dilution [MAB 3777; EMB Millipore]), 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 AffiniPure Goat Anti-Mouse IgG, 1:200 [115–545-003; Jackson Immuno Research Laboratories]; Alexa Fluor® 647 AffiniPure Fab Fragment Goat Anti-Mouse IgG, 1:200 [115–607-003; Jackson Immuno Research Laboratories]; Alexa Fluor® 488 AffiniPure Goat Anti-Rabbit IgG, 1:200 dilution [111–545-003; Jackson Immuno Research Laboratories]; Alexa Fluor® 647 AffiniPure Goat Anti-Rabbit IgG, 1:200 dilution [111–605-003; Jackson Immuno Research Laboratories]) 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).
Proteinase K digestion
PK digestion was used to determine whether the α-syn seen in oligodendrocytes was soluble (non-aggregated) or insoluble (aggregated). 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 Tris–HCl, pH 7.8; 100 mM NaCl; 0.05% Tween-20), the sections were digested with 10 μg/ml PK (Invitrogen) in TBS-T (10 mM Tris–HCl, pH 7.8; 100 mM NaCl; 0.1% Tween-20) for 30 min at 55 °C. The sections were fixed with 4% paraformaldehyde for 10 min. After several washes, the sections were processed for α-syn immunostaining as described above.
Luxol Fast Blue
For analysis of myelin integrity, level matched sections of both Olig001-GFP and Olig001-α-syn injected monkeys were mounted 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 defined.
Stereology
In order to estimate the number cells transduced by Olig001, GFP+ or pSer-129+ cells in rats and monkeys in an unbiased fashion, stereological counting methods [
66] using the optical fractionator probe in Stereo Investigator (Microbrightfield Bioscience, Version10.40) were applied. A random and systematic sampling of sections were employed in both rats and monkeys with every 12th section being analyzed. Eight equispaced sections per rat and 20 equispaced sections per monkey were evaluated. The transduction area in the striatum was outlined using a 1.25× objective. Cells were counted from a random starting point at regular predetermined intervals (
x = 300 μm,
y = 300 μm) with a counting frame (70 μm × 70 μm) using a 60× oil immersion objective. The coefficients of error (CE) were calculated according to the procedure of Gunderson and colleagues as estimates of precision [
66]. The values of CE were 0.047 ± 0.006667 (Gunndersen m = 0) for pSer-129+ cells and 0.063 ± 0.012 (Gunndersen m = 0) for GFP+ cells in NHPs, and 0.105 ± 0.0087 GFP+ cells for rats. The volume of the striatum occupied by the transduced cells was also measured and recorded using the Stereo Investigator optical fractionator probe in the same manner by which the cells were counted. To assess the specificity of transduction and to determine any potential off-target transduction, double-label immunofluorescence was employed, as described above, using GFP combined with Olig2, NeuN, or GFAP. The percentage of double-labeling was determined by counting GFP transduced cells in the striatum of rats and nonhuman primates using the Stereo Investigator optical fractionator probe. Serial sections of the entire rostral-caudal striatum containing the transduction area were used, counting every 12th section (8 sections counted per rat, 20 sections counted per monkey).
Images
Confocal images were exported from the Olympus laser-scanning microscope with Fluoview software and stored as tiff files. Conventional light microscopic images were acquired using an Olympus microscope (BX61) attached to a Nikon digital camera DXM1200 and stored as tif files. All compared images were taken using the same intensity and exposure time. All figures were prepared using Photoshop 8.0 graphics software. Only minor adjustments of brightness were made.
Discussion
Using silver impregnation (Gallyas method), Papp and Lantos first described the accumulation of insoluble protein aggregates in oligodendrocytes, naming GCIs as the pathological hallmark of MSA [
42]. These inclusions were later described to be composed mainly of α-syn [
43]. While the precise pathological mechanism(s) remain unknown, the positive correlation between GCI density and neuronal loss highlight the importance of α-syn in the disease process [
40]. The drastic demyelination observed in MSA patients is not accompanied by a severe loss of mature oligodendrocytes [
49,
14], indicating that accumulation of α-syn leads to dysfunction, rather than overt loss of oligodendroglia. Additionally, alterations in lipid composition are restricted to affected regions in human MSA samples [
12]. Moreover, decreased neurotrophic support of oligodendroglia, as shown by reductions of glial derived neurotrophic factor expression levels in MSA tg mice and human MSA samples [
56], furthers the idea that oligodendrocyte dysfunction is an early pathological event leading to secondary neurodegeneration related to retrograde axonal disease.
The question of whether α-syn is internalized by oligodendroglia or if it is pathologically overexpressed in oligodendrolia in MSA is still heavily debated, with conflicting reports of elevated α-syn expression [
2] and others arguing an absence of elevated α-syn expression in oligodendrocytes in normal individuals [
30,
37,
39]. It has been shown that α-syn is transiently expressed in oligodendroglia precursor cells during development, but reduced in mature oligodendrocytes [
30,
37,
48]. In contrast, oligodendrocytes differentiated from induced pluripotent stem cells (iPSCs) derived from MSA patients expressed α-syn, whereas iPSCs from PD and healthy controls do not [
10] suggesting that the accumulation and aggregation of α-syn in oligodendroglia is specific to the disease process. As such, experimental modeling of MSA critically relies on the overexpression of α-syn in oligodendroglia.
Currently available animal models of MSA are limited to 3 tg mouse lines overexpressing human α-syn under proteolipid protein (PLP) promoter [
31], myelin basic protein (MBP) promoter [
51], and 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) promoter [
67]. Initial studies using PLP-driven expression reported formation of GCIs, however demyelination and neurodegeneration was lacking [
31]. Later studies using the same PLP promoter demonstrated subtle motor impairment and a 31.4% loss of nigral neurons [
16,
52]. Additional reports of degeneration in non-motor regions of MSA [
53], changes in cardiac function [
33], and bladder dysfunction [
4] have been reported. Mice utilizing CNP-driven overexpression displayed progressive motor impairments and neurodegeneration localized in the spinal cord, with no observed loss in the cerebellum [
67]. Overexpression of α-syn using the MBP promoter showed the most classical distribution of pathology, with both the basal ganglia and cerebellum displaying extensive pathology [
51]. The degree of GCI accumulation, neurodegeneration, and motor impairments varied significantly with α-syn expression levels, where high expressing lines demonstrated the most significant neuropathological and behavioral deficits [
51]. While displaying certain aspects of MSA-like pathology and providing substantial insight of potential disease mechanisms, tg mouse models of MSA harbor inherent limitations. Variability of pathology is seen across the 3 mouse lines, with none of the models being able to model the distinct SND or OPCA observed in MSA patients [
3]. Moreover, the constitutive expression of α-syn under oligodendroglia-specific promoters may also incorporate developmentally expressed α-syn in the pathology observed in these models. In support of this, overexpression of α-syn in cell culture models dramatically impaired the maturation of 2 separate oligodendrocyte precursor cell lines, shown by significant reductions of MBP during maturation [
13].
The variable pathology and potential problem of constitutively expressing α-syn in tg mouse models, along with the lack of rodent and primate models of MSA, lead us to utilized a novel oligodendrocyte-directed AAV capsid, Olig001 [
44], in order to develop a viral vector based model of MSA. Olig001 was developed using capsid shuffling and directed evolution, resulting in a chimeric capsid composed of AAV1, 2, 6, 8, and 9, which exhibits oligo-specific tropism 9 fold higher than wild-type AAVs [
44]. The high level of oligodendroglia tropism allowed transgene expression to be driven by a constitutive chicken beta-actin hybrid (CBh) promoter. Previous studies by other groups target oligodendroglia using wild-type rAAVs with expression driven by MBP promoters, however the overall transduction of oligodendrocytes was still low [
8,
34]. In the present study, intrastriatal injection of Olig001-GFP in rats successfully transduced oligodendroglia 94–97% of the time, with less than 5% off-target transduction of either neurons or astrocytes, demonstrating a 19:1specificity for oligodendrocytes.
With translational studies in mind, it is important to demonstrate consistent tropism of a particular rAAV, as examples where the tropism of a given rAAV capsid does not remain consistent in rodents and higher animals have been described [
7]. In the current study, we observed that the affinity of Olig001 to transduce oligodendroglia was conserved between species. Intrastriatal injection of Olig001-GFP in rhesus macaques demonstrates the same preference for oligodendrocytes as in rodents [F(1,5) = 3.4,
p > 0.12]. Furthermore, there was slightly less co-localization of GFP and NeuN in primates than in rodents [3.7% rodents vs. 0.7% primates (F1,5) = 31.7,
p = 0.002], indicating that Olig001 produced less off target transduction in nonhuman primates.
Given the high level of specificity of Olig001 for oligodendroglia in both rats and primates, we transduced rhesus macaques with Olig001 containing the α-syn transgene in an attempt to develop the first nonhuman primate model of MSA. Intrastriatal injection of Olig001-α-syn produced widespread expression of α-syn throughout white matter regions of the striatum. Inclusions of α-syn were found to localize inside oligodendroglia, shown by co-localization of LB509 and oligodendroglia marker Olig2. Furthermore, the inclusions were phosphorylated at Serine-129 and resistant to proteinase K digestion, recapitulating many characteristics of GCIs found in human MSA samples.
Early pathological changes seen in MSA were demonstrated in the monkeys 3-months following transduction. Most notably, marked demyelination was observed throughout striatal white matter, demonstrated by reduced Luxol Fast Blue staining in the corpous callosum and striatal white matter bundles. Importantly, the observed demyelination was due to the accumulation of α-syn in oligodendroglia, as areas of pSer-129 immunoreactivity highly correlated with regions of myelin loss. Moreover, this was not a result of the AAV, as no reductions in myelin were observed in the Olig001-GFP transduced animals. While the differing post-injection time course of the Olig001-GFP and Olig001-α-syn injected monkeys does not allow for a perfect comparison, there is an enormous amount of data from numerous NHP studies following striatal injection of different transgenes that never reported demyelination or α-syn aggregation. Luxol Fast Blue and pSer-129 staining of rhesus macaques 3-months after receiving intrastriatal injections of AAV2-neurturin [
25] or stem cells, as well as untreated animals, displayed no demyelination or aggregated α-syn which supports the specificity of effects observed with Olig001-GFP and Olig001-α-syn. In line with post-mortem human MSA cases, the demyelination observed in our model is not associated with any obvious reductions in oligodendroglia, further indicating that the loss of myelin is a result of oligodendrocyte dysfunction and not an overt loss of oligodendroglia.
Our model recapitulated other pathological events observed in MSA, such as immune and inflammatory activation. It has been reported that activated microglia are found within white matter tracts and areas of neurodegeneration in patients with MSA, and are believed to release proinflammatory cytokines and possibly play a role in neurodegeneration [
26,
53]. Widespread HLA-DR+ activated microglia are seen covering α-syn transduced areas of striatal white matter, whereas minimal activation was seen specifically at the injection site in GFP transduced monkeys. Activated microglia were also observed in the substantia nigra of α-syn transduced monkeys, whereas a ‘non-activated phenotype was observed following GFP transduction. In our previous work, we have seen that a capsid and/or transgene related inflammatory response peaks at 2-weeks post-injection and then retracts to the needle track [
46], similarly to what is seen with the Olig001-GFP injected monkeys. Importantly, this suggests that the inflammatory response seen in monkeys 3-months after injection of Olig001-α-syn is directly related to virally delivered α-syn expression, and not an ongoing inflammatory response due to the rAAV injection.
This model is presently ripe for replication and extension. Due to the exploratory nature of our initial experiment, we only examined the effects of Olig001 gene delivery over a short post-surgical time course. We are currently studying longer time courses and hypothesize that neurodegeneration would occur later than 3-months after administration of our vector. The accumulation of activated microglia in demyelinated areas of the striatum and in the substantia nigra could be the pathological event immediately preceding neuronal loss. Many groups have proposed the hypothesis that accumulation of α-syn in oligodendroglia leads to loss of myelin, and that the now unmyelinated axons are exposed and vulnerable to proinflammatory cytokines released by activated microglia seen in the vicinity of GCIs. These early pathologic events, with the addition of reduction of neurotrophic support, lead to axonal damage and subsequent cell death, implicating MSA as a retrograde axonal disorder. Our model recapitulates early pathogenesis seen in MSA, as many of the pathological features induce dysfunction of the oligodendroglia-myelin-axon-neuron complex [
28].