Biological Characterization of aSyn Aggregates

1. Neurotoxicity

a. Neuronal precursor cells derived from human induced pluripotent stem cells were generated and characterized as previously described [1]. These cells were maintained in neural precursor expansion medium (NPEM) as previously described.

b. Plate approximately 5,000 cells per well in a 96-well plate, pre-coated with Geltrex LDEV-free reduced growth factor basement membrane matrix (1:100, Invitrogen), and incubated at 37 °C for 24 h.

c. After 24 h, cells were treated for 48 h with different concentration(s) of aSyn aggregates either purified from aSynucleinopathy patient brain tissues or amplified by SAA from biological samples of aSynucleinopathy patients.

d. Cell viability was determined by the MTT assay according to the manufacturer’s protocol.

2. Seeding assay in HEK cells

a. Plate HEK293T α-syn-CFP/YFP biosensor cells [2] in 24-well plate at 100,000 cells per well and incubate overnight.

b. The next day, sonicate 5 uL of aSyn aggregates briefly and mix with 45 uL of Opti-MEM medium.

c. Mix 2 uL of Lipofectmine 2000 (Invitrogen) with 48 uL of Opti-MEM medium, then add it to the fibril-Opti-MEM mixture, and incubated at room temperature for 20 minutes.

d. Add the 100 uL of fibril-Lipofectmine 2000-Opti-MEM mixture dropwise to one well of cells.

e. Keep cells in culture for 4 more days and image cells live every day to monitor the seeding of aSyn aggregates.

3. Seeding assay in transgenic animals

3.1 Inoculum preparation

3.1.1Human brain tissues

a. Make 40% (w/v) brain homogenates in homogenization buffer (1x PBS with 1x protease inhibitor (EDTA free) and 1x phosphatase inhibitor) using the Precellys tubes with bead homogenizer.

b. Store brain homogenates at -80 °C.

c. Thaw and briefly sonicate brain homogenates right before inoculation.

3.1.2 SAA amplified aSyn fibrils

a. Centrifuge 100 uL of SAA amplified aSyn aggregates at 100,000 x g for 1 hour at 4 °C.

b. Resuspend the pellet containing aSyn aggregates in 100 uL of PBS and store at -80 °C.

c. Thaw and briefly sonicate aSyn aggregates right before inoculation.

3.2 Stereotaxic injection

a. Anesthetize 6- to 8-week-old TgM83 mice (B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J, hemizygous) with isoflurane (5% in the induction chamber and 2% for maintenance).

b, Stereotaxically inject anesthetized mouse with 10 uL of 40% brain homogenate, purified aSyn aggregates, or SAA amplified aSyn aggregates into striatum (AP: 0.2 mm, ML: ±2 mm, DV: 2.6 mm) bilaterally (5 uL per side), using a Hamilton model 80366 syringe mounted in a Stoelting™ Quintessential Stereotaxic Injector with a 2 uL/min rate.

c. After inoculation, provide analgesia by the administration of carprofen at a 5 mg/kg dose subcutaneously, once a day for three days.

d. Monitor animals 3 times a week for the following clinical signs: kyphosis, ataxia, lethargy, cachexia, and paralysis.

e. Sacrifice animals with in 1 or 2 days after observing neurological signs, due to fast progression of disease.

f. Immediately after sacrifice, transcardially perfuse animals 1x PBS containing 5 mM EDTA and collect brains.

g. Snap-freeze the right sagittal hemisphere in liquid Nitrogen and stored at -80⁰C for biochemical analyses. Keep the left hemisphere 10% formalin for immunohistochemical studies.

3.3 Immunohistochemistry (IHC)

a. Dehydrate and embed formalin fixed brain tissues in paraffin wax.

b. Rehydrate 10 mm-thick sections sequentially in Xylene (twice, 7 minutes each time), 100% EtOH (twice, 3 minutes each time), 95% EtOH, 90% EtOH, 80% EtOH, 70% EtOH (3 minutes each solution), and rinsed 3 times in PBS (5 minutes each time).

c. Perform antigen retrieval in Sodium Citrate buffer (pH 6.0) containing 0.05% Tween-20 at 80 ºC for 20 min.

d. Rinse slides with PBS 3 times (5 minutes each time), then treat slides with methanol and H2O2 (1:1) for 20 minutes with agitation to eliminate endogenous peroxidase activity.

e. Rinse slides with PBS 3 times (5 minutes each time), then block the slides with 2.5% normal goat serum for 20 minutes at room temperature.

f. Immediately afterwards, incubate slides with primary anti-phosphorylated alpha-synuclein (phosphoS129) antibody (EP1536Y, Abcam) (1:2000) in PBS containing 0.2% Triton X-100 and 3% BSA overnight at room temperature.

g. The following day, wash slides with PBS 3 times (5 minutes each time), then incubate slides with the secondary antibody (ImmPRESS® Goat Anti-Rabbit IgG Polymer kit, Vector Laboratories, Burlingame, CA, USA) for 30 minutes at room temperature.

h. Rinse slides with PBS 3 times (5 minutes each time), then develop the slides for 1 minute using a DAB peroxidase substrate kit (Vector Laboratories, Burlingame, CA, USA).

i. Rinse slides with distilled water for 5 minutes, then counterstain slides with hematoxylin for 1 min and rinse with tap water for 5 min.

j. Dehydrated the slides sequentially in 70% EtOH, 80% EtOH, 90% EtOH, 95% EtOH (3 minutes each solution), 100% EtOH (twice, 3 minutes each time), and Xylene (twice, 7 minutes each time).

k. Mount the slides with DPX (Electron Microscopy Sciences, Hatfield, PA, USA) and coverslips.

3.4 Imaging and histopathological analyses

a. For pathological aSyn burden analyses, acquire whole sagittal section images (10x objective) using Leica Stellaris 5 Microscope (Mannheim, Germany).

b. For each animal, scan 3 sections with 100 mm interval.

c. Quantify the pathological aSyn burden by expressing the pS129-positive signals as percentage of a predefined brain region using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The following brain regions were examined: cortex, hippocampus, hypothalamus, thalamus, midbrain, cerebellum, and pons-medulla.

d. Present the percentage values as means ±E.M. and compare the values using two-way ANOVA analyses followed by Sidak’s multiple comparisons test in Graph Pad Prism (Version 9.3.1, GraphPad, La Jolla, CA, USA).

e. For morphological characterization of the pS129-positive aSyn deposits, acquire midbrain images (40x objective) using Leica Stellaris 5 Microscope (Mannheim, Germany).

f. For each animal, scan 3 sections with 100 mm interval.

g. Quantify the deposits by counting numbers of the ring-like and Lewy body (LB)-like aSyn aggregation as described previously [3].

h. Express ring-like or LB-like deposits as percentage of total deposits and present the percentage values as mean ±E.M..

i. Compare the values using one-way ANOVA analyses followed by Sidak’s multiple comparisons test using Graph Pad Prism(Version 9.3.1, GraphPad, La Jolla, CA, USA).

j. For statistical analyses, significance threshold was set at P=0.05.

Reference:

  1. Armijo E, Gonzalez C, Shahnawaz M, Flores A, Davis B, Soto C. Increased susceptibility to Aβ toxicity in neuronal cultures derived from familial Alzheimer’s disease (PSEN1-A246E) induced pluripotent stem cells. Neurosci Lett. 2017 Feb 3;639:74-81. doi: 10.1016/j.neulet.2016.12.060. Epub 2016 Dec 26. PMID: 28034781.
  2. Yamasaki TR, Holmes BB, Furman JL, Dhavale DD, Su BW, Song ES, Cairns NJ, Kotzbauer PT, Diamond MI. Parkinson’s disease and multiple system atrophy have distinct α-synuclein seed characteristics. J Biol Chem. 2019 Jan 18;294(3):1045-1058. doi: 10.1074/jbc.RA118.004471. Epub 2018 Nov 26. PMID: 30478174; PMCID: PMC6341389.
  3. Lau A, So RWL, Lau HHC, Sang JC, Ruiz-Riquelme A, Fleck SC, Stuart E, Menon S, Visanji NP, Meisl G, Faidi R, Marano MM, Schmitt-Ulms C, Wang Z, Fraser PE, Tandon A, Hyman BT, Wille H, Ingelsson M, Klenerman D, Watts JC. α-Synuclein strains target distinct brain regions and cell types. Nat Neurosci. 2020 Jan;23(1):21-31. doi: 10.1038/s41593-019-0541-x. Epub 2019 Dec 2. PMID: 31792467; PMCID: PMC6930