Human tissue processing and postmortem evaluation
Post-mortem brain tissue of MSA and control subjects were obtained from Rush University Medical Center and Banner Health. Autopsies of MSA subjects (n = 3) performed at Rush University Medical Centeras were performed as follows: the brains were removed from the calvarium and processed as described previously . Briefly, each brain was cut into 1 cm coronal slabs and then hemisected. The slabs were fixed in 4% paraformaldehyde for 5 days at 4 °C. The brain slabs from one side were used for pathological diagnosis. The brain slabs from the other side were cryoprotected in 0.1 M phosphate‐buffered saline (PBS) pH 7.4 containing 2% dimethyl sulphoxide, 10% glycerol for 48 h followed by 2% dimethyl sulphoxide and 20% glycerol in PBS for at least 2 days before sectioning. The fixed slabs containing substantia nigra and striatum were cut into 18 adjacent series of 40-µm-thick sections on a freezing sliding microtome. All sections were collected and stored in a cryoprotectant solution before processing.
A complete neuropathologic evaluation was performed  confirming the presence of GCI as well as other neuropathology. These details can be found in Supplemental Table 1. Dissection of diagnostic blocks included a hemisection of brain, including the substantia nigra, striatum, cerebellar peduncle, and cerebellum. Glial cytoplasmic inclusions were examined with hematoxylin and eosin staining and further identified with antibodies to α-syn using alkaline phosphatase as the chromogen. A “definite” diagnosis of MSA was based on the presence of glial cytoplasmic inclusions, as well as a lack of Lewy bodies and Lewy neurites, and moderate or severe nigral neuronal loss, which corresponded with clinical diagnosis. Representative images confirming MSA glial cytoplasmic inclusion staining can be found in Supplementary Fig. 1a, online resource.
MSA (n = 3) and Control tissue (n = 6) from age-matched subjects were obtained from the Arizona Study of Aging and Neurodegenerative Disorders and Brain and Body Donation Program and collected as previously described . All cases underwent a full neuropathological assessment at autopsy, evaluating relevant pathological proteins such as Tau, amyloid beta, and alpha-synuclein. “Neurological Control” is a subject without dementia or Parkinsonism during life and without a major neuropathological diagnosis. While pathology of tau or amyloid beta was present in some of the cases, it never reached a level sufficient to diagnosis any disease. Of note, our analysis (MSA cases and controls) were limited to one section per case due to the availability of tissue supplied from the brain bank, where only one level is sectioned per brain region.
A list of subject demographics is given in Supplemental Table 1.
Immunohistochemistry of human samples
Free-floating striatal and nigral sections of control (n = 5) and MSA (n = 5) brain tissue were first rinsed of cryoprotectant solution and then underwent citric acid heat mediated antigen retrieval. Nonspecific background staining was blocked by a 1-h incubation in a solution containing 2% bovine serum albumin and 3% of either goat or horse serum. Tissue sections were incubated at room temperature overnight in the following primary antibodies: rabbit anti-Human CD3 (polyclonal, Dako A0452), mouse anti-CD4 (clone RIV6, Invitrogen MA1-7631), rabbit anti-CD8 (polyclonal, Abcam ab4055), and mouse anti-HLA-DR (clone LN3, Invitrogen MA5-11966). Sections were washed of primary antibody, then incubated with appropriate secondary antibodies (biotinylated goat anti-rabbit Vector Laboratories BA-1000; biotinylated horse anti-mouse Vector Laboratories BA-2000; for 1-h, washed again, and incubated with avidin–biotin complex (Vector Laboratories PK-6100) for 75-mins. The immunohistochemical reaction was completed with 0.05% 3,3′-diaminobenzidine (DAB) with 2% nickel enhancement and 0.005% H2O2. Sections were mounted on gelatin-coated slides, dehydrated through graded alcohol, cleared in xylene, and coverslipped with Cytoseal™ (Richard-Allan Scientific™). 40 μm sections of cynomolgus macaque spleen were used as positive controls for T cell staining (Supplementary Fig. 2, online resource).
Immunofluorescence of human samples
Free-floating striatal and nigral sections of control (n = 3) and MSA (n = 3) brain tissue were first rinsed of cryoprotectant solution and then underwent citric acid heat mediated antigen retrieval. Nonspecific background staining was blocked by a 1-h incubation in a solution containing 2% bovine serum albumin and 3% of donkey serum. Tissue sections were incubated at room temperature overnight in the following primary antibodies: rabbit anti-Human CD3 (polyclonal, Dako A0452), mouse anti-HLA-DR (clone LN3, Invitrogen MA5-11966), rabbit anti-alpha-synuclein (phosphoS129) [EP1536Y] (polyclonal, abcam ab51253), mouse anti-alpha-synuclein (phosphoS129) [P-syn/81A] (monoclonal, abcam ab184674), rabbit anti-DARPP-32 [EP720Y] (polyclonal, abcam ab40801). Sections were washed of primary antibody, then incubated with appropriate Alexa-conjugated secondary antibodies (Life Technologies) at room temperature for 1 h. Sections were mounted on gelatin-coated slides, dehydrated through graded alcohol, cleared in xylene, and coverslipped with DPX mounting medium (Sigma-Aldrich). All images were obtained on a Nikon Eclipse Ti2 confocal microscope using a Nikon A1RHD camera. All figures were prepared using Photoshop 8.0 graphics software. Only minor adjustments of brightness and/or contrast were made.
To estimate the number CD3+, CD4+, and CD8+ T cells in control and MSA postmortem brain tissue in an unbiased fashion, stereological counting methods  using the optical fractionator probe in Stereo Investigator (Microbrightfield Bioscience, Version 10.40) were applied in a blinded fashion. The putamen or substantia nigra were outlined using a 1.25× objective and a random and systematic sampling of sections was employed where cells were counted from a random starting point at regular predetermined intervals (Putamen: x = 700 μm, y = 700 μm; Substantia Nigra: x = 400 μm, y = 400 μm) with a counting frame (80 μm × 80 μm) using a 60 × oil immersion objective.
HLA-DR imaging and quantification
Free-floating striatal and nigral sections of control (n = 4) and MSA (n = 4) brain tissue were processed as noted above. The entire region of the putamen and substantia nigra were outlined and imaged using 4× objective on a Nikon eclipse Ti2 microscope using a Nikon D5-Ri2 color camera and NIS elements AR software version 5.10.01. The acquired image files were blinded and quantified by the mean gray value using imageJ (NIH) software. Briefly, 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.
All images were obtained on a Nikon Eclipse Ti2 microscope using a Nikon D5-Ri2 color camera. All figures were prepared using Photoshop 8.0 graphics software. Only minor adjustments of brightness and/or contrast were made.
Male and female C57BL/6 (#000664 Jackson Laboratories), Ccr2 reporter knock in (B6.129(Cg)-Ccr2tm2.1Ifc/J, #017586 Jackson Laboratories) Tcrb−/− (B6.129P2-Tcrbtm1Mom/J, #002118 Jackson Laboratories), and Cd4−/− mice (B6.129S2-Cd4tm1Mak/J, #002663Jackson laboratories) were used for these studies and maintained on a congenic background. All researches conducted on animals were approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham.
More detailed information of the development, construction, purification, and quality control of the Olig001 AAVs are described elsewhere [37, 43]. Briefly, the AAV-Olig001 vectors which have a high tropism for oligodendrocytes are packaged with a self-complementary genome with transgene expression mediated by the CBh promoter and bovine growth hormone polyA . The AAV vectors were produced by the University of North Carolina Vector Core facility by triple-transfecting production plasmids (pXX6-80, pTRS-ks-CBh-EGFP, and AAV pXR-Olig001) into HEK293 cells. AAV vectors were then purified from the cells by iodixanol gradient centrifugation, followed by ion-exchange chromatography.
All surgical procedures on male and female C57BL/6 (WT), Ccr2 reporter knock in, Tcrb−/−, or Cd4−/− mice were performed similar to previously described work [25, 26, 64]. Briefly, mice were anesthetized with isoflurane and unilaterally (immunohistochemistry experiments) or bilaterally (flow cytometry experiments) injected with 2 μl of Olig001-GFP (1 × 1013 vg/ml) or Olig001-SYN (1 × 1013 vg/ml) into the dorsolateral striatum at a rate of 0.5 μl/min with a Hamilton syringe. The needle was left in the injection site for an additional 2 min and then slowly retracted over the course of 2 min. The stereotaxic coordinates used from bregma were AP + 0.2 mm, ML +/ − 2.0 mm, and DV − 2.7 mm from dura.
Immunohistochemistry of mouse samples
At 4 weeks post viral transduction, mice were anesthetized, euthanized, and brains were collected for processing as previously described . Briefly, animals were perfused with heparinized 0.01 M PBS, followed by 4% paraformaldehyde, drop-fixed overnight and transferred for cryoprotection to 30% sucrose in PBS pH 7.4. Brains were frozen on dry ice and coronal sections 40-μm thick were serially collected using a sliding microtome. Sections were stored in 50% glycerol in 0.01 M PBS at − 20 °C.
For fluorescent analyses, free-floating sections were washed with Tris-buffered saline (TBS) and labeled as previously published [25, 26, 64]. Briefly, sections were blocked in appropriate 5% normal serum and then labeled with anti-MHCII (clone M5/114.15.2, eBiosciences), anti-alpha-synuclein (phospho-Serine129 (pSer129), clone EP1536Y, Abcam), anti-tyrosine hydroxylase (TH, clone AB152, Sigma-Aldrich), anti-IBA1 (Wako), anti-TMEM119 (clone 28-3, Abcam), anti-CD3 (clone 17A2, eBioscience), anti-CD4 (clone RM4-5, BD Bioscience), anti-CD8 (clone 4SM15, eBioscience), or anti-MBP (clone SMI 99, BioLegend) antibodies diluted in 1% normal serum in TBS-Triton (TBST) overnight at 4 °C. Appropriate Alexa-conjugated secondary antibodies (Life Technologies) or LI-COR infrared secondary antibodies (LI-COR Biosciences) were applied at room temperature for 2.5 h. Sections were mounted onto coated glass slides, and cover slips were added using hard set mounting medium (Vector Laboratories).
For diaminobenzidine (DAB) staining, anti-MHCII (clone M5/114.15.2; eBiosciences) and anti-alpha-synuclein (pSer129, clone EP1536Y, Abcam) antibodies were diluted in 1% normal serum in TBST and incubated with sections overnight at 4 °C. Appropriate biotinylated secondary antibody (Vector Laboratories) were applied at room temperature for 2-h. Then, R.T.U. Vectastain ABC reagent (Vector Laboratories) and DAB kit (Vector Laboratories) were used according to the manufacturer's instructions to develop HRP reactions. Sections were mounted onto coated glass slides, dehydrated, and coverslipped using Permount mounting medium (Fisher).
For myelin staining, sections were stained and processed according to the Millipore Black Gold II Myelin Staining Kit per the manufacturer’s instructions.
Confocal images were acquired using a Leica TCS-SP5 laser scanning confocal microscope. Images were saved using the Leica LASAF software, exported and processed using Adobe Photoshop/Illustrator.
MHCII, myelin, TH, and pSer129 imaging and quantification (mice)
Images were acquired using a Nikon Eclipse Ti-E. For myelin, MHCII (I-A/I-E), and pSer129 staining quantification, slides were blinded, scanned, and quantified by the mean gray value method via imageJ (NIH) as previously described . Three striatal sections per animal encompassing the dorsal striatum injection site and pSer129 expression were chosen for quantification. The myelin, MHCII, or pSer129 mean gray value (average pixel value in a selected region) was calculated for both ipsilateral and contralateral contours (drawn to match pSer129 expression) of the dorsal striatum and corpus callosum (normalized to non-tissue slide background mean gray value). Myelin, MHCII, or pSer129 fold induction was determined by dividing ipsilateral mean gray values by contralateral mean gray values and an average fold induction was determined per animal, n = 3–6 animals were quantified per treatment group.
For TH densitometry, LI-COR labeled striatal sections were blinded and then scanned with the Odyssey CLx system (LI-COR Biosciences). Three striatal sections per animal encompassing the dorsal striatum injection site were selected in Image Studio (LI-COR Biosciences) and the TH signal was calculated for both ipsilateral and contralateral contours of the dorsal striatum. TH fold expression was determined by dividing ipsilateral contour signal by the contralateral signal and an average fold induction was determined per animal, n = 3 animals quantified for treatment group.
For pSer129 positive cell counting, a 35,000 area per point grid was overlaid on the three striatal sections. Five random grids were assigned per ipsilateral section and an individual blinded to the genotypes counted the number of pSer129 positive cell bodies per random grid. The average of the counts per random field of view (grid) was used for the final quantifications between genotypes.
Mononuclear cell isolation and flow cytometry
Mononuclear cells were isolated 4 weeks post-transduction from striatum or lymph nodes (LNs) of mice bilaterally transduced with Olig001-SYN or Olig001-GFP control according to published methods [26, 45, 64]. Briefly, striatums were triturated and digested with 1 mg/mL Collagenase IV (Sigma) and 20 μg/mL DNAse I (Sigma) diluted in RPMI 1640 with 10% heat inactivated fetal bovine serum, 1% l-glutamine (Sigma), and 1% Penicillin–Streptomycin (Sigma). Mononuclear cells were separated out using a 30/70% percoll gradient (GE) as previously described . For deep cervical lymph nodes, lymph nodes were isolated and subsequently disassociated through a 70 μm filter. For intracellular cytokine staining, isolated mononuclear cells were stimulated with PMA (50 ng/mL, Fisher BioReagents) and ionomycin (750 ng/mL, Millipore Sigma) in the presence of GolgiStop (1:1000, BD Biosciences) for 4 h at 37 °C/5% CO2. For all staining, isolated cells were blocked with anti-Fcy receptor (clone 2.4G2 BD Biosciences) and then surfaced stained accordingly with fluorescent-conjugated antibodies against CD45 (clone 30-F11, eBioscience), CD11b (clone M1/70, BioLegend), MHCII (clone M5/114.15.2, BioLegend), Ly6C (clone HK 1.4, BioLegend), TCR-ß (clone H57-597, BioLegend), CD4 (clone GK1.5, BioLegend), CD8a (clone 53-6.7, BioLegend), CD44 (clone IM7, BD Biosciences), or CD62L (clone MEL-14, eBioscience). A fixable viability dye was used to distinguish live cells from debris per manufacturer’s instructions (Fixable Near-IR LIVE/DEAD Stain Kit, Invitrogen). For intracellular transcription factor and cytokine staining, cells were further processed using the Foxp3/Transcription Factor Staining Kit (eBioscience) or the BD Cytofix/Cytoperm Staining Kit (BD Biosciences) respectively and then stained accordingly with fluorescent-conjugated antibodies against FOXP3 (clone FJK-16s, eBioscience), T-bet (clone 4B10, BioLegend), GATA3 (clone 16E10A23, BioLegend), RORγt (clone Q31-378, BD Biosciences), IFN-γ (clone XMG1.2, eBioscience), IL-4 (clone 11B11, BioLegend), IL-17a (clone eBio17B7, eBioscience), or IL-10 (clone JES5-16E3, BioLegend). Samples were analyzed by flow cytometry using an Attune Nxt flow cytometer (Thermo Fisher Scientific) and FlowJo software (Tree Star).
All graphs and corresponding statistical tests were generated or performed using Prism software (GraphPad).
For all analysis of human data, 4–5 subjects were analyzed per group. An unpaired, nonparametric Mann–Whitney test was used to compare groups. Graphs display the mean ± SEM. *p < 0.05, **p < 0.01.
For DAB MHCII staining quantification, four animals were analyzed per group. A one-way ANOVA with Dunnett’s test was used to compare groups. Graphs displayed the mean ± SEM. ***p < 0.001. For myelin staining quantification, 3–6 mice were analyzed per group. An unpaired t-test or a one-way ANOVA with Dunnett’s test was used to compare groups accordingly. Graphs displayed the mean ± SEM. *p < 0.05. Flow cytometry experiments utilized three to four independent samples per group, with two ventral midbrains pooled per sample. Therefore, each experiment used a total of 12–16 mice. Data was analyzed using an unpaired t-test or one-way ANOVA with Dunnett’s test was used to compare groups accordingly. Graphs displayed the mean ± SEM. *p < 0.05, ** < 0.01, ***p < 0.005, ****p < 0.001.