Ab92434

Cells were fixed in 4% paraformaldehyde for 15 min, followed by three washes of DPBS, blocked and permeabilized in PBS containing 0.1–0.2% Triton X-100 and 10% horse serum. The coverslips were incubated with primary antibodies; for NPCs: rabbit anti-PAX6 (CST, mAb#60433, 1:250) and mouse anti-NESTIN (CST, mAb#33475,1:2000); for Neurons: chicken anti-MAP2 (Abcam, ab92434, 1:500), rabbit anti-TBR1 (Abcam, ab183032, 1:250), rabbit anti-VGLuT1 (Abcam, ab227805, 1:500) and Mouse anti-GABA (Abcam, ab86186, 1:400) in the blocking solution overnight at 4 °C. On the next day, they were washed in DPBS and incubated with DAPI (Abcam, ab228549, 1:1000) and corresponding secondary antibodies (Abcam, ab150084, ab150117, ab175711, 1:250) for 60 min at room temperature. Then the coverslips were washed three times, mounted on glass slides using Fluromount-G (mounting medium), and dried overnight while being protected from light. Fluorescence signals were detected using a Leica THUNDER imager and analyzed using ImageJ and MATLAB.

Coverslips containing the co-cultures were first fixed in 2% PFA and 5% sucrose (10 min), then washed in PBS, and blocked for 1 h at RT in BSA 1%. Staining with primary antibodies rabbit anti-GFAP (DakoCytomation, Glostrup, Denmark, Z0334, 1: 500) or goat anti-GFAP (Santa Cruz Biotechnology, California, United States of America, sc-6170, 1:300), anti-MAP2 antibody (Abcam, ab92434; 1:500), and anti-caspase 3 antibody, active (cleaved) form (AB3623, Sigma-Aldrich; 1:100) was performed overnight at 4°C. Fluoroshield (Sigma Aldrich, MO, United States #F6057) containing DAPI was used to mount the coverslip, and a Leica SP2 confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with an HCX PL APO 63.0X OIL (NA = 1.40) objective was used to acquire images.

Healthy, human induced pluripotent stem cell-derived neural stem cells were propagated and differentiated using Induction/DOPA Differentiation kit (XCell Science), as previously described [30 (link)]. At day 38 of differentiation, neurons were exposed to human recombinant α-syn S129A PFFs (14 μg/mL) added to the cell culture medium. After 24 hours, cells were carefully washed in PBS, and allowed to grow for an additional 6 days in fresh medium before fixation in 4% PFA at day 45. 4 hours prior to fixation, cells were treated with 1 μM BI2536 for inhibition of PLK1-3 [17 (link)] or DMSO as a vehicle control. Immunostaining was carried out as described in [23 (link)], using the following primary antibodies: chicken polyclonal MAP2 (#ab92434, Abcam, 1:2000, RRID: AB_2138147), mouse monoclonal pS129-α-syn (11A5, 1:10,000) and tyrosine hydroxylase (TH, #AB152, Merck Millipore, 1:1000, RRID: AB_390204). Appropriate secondary Alexa Flour antibodies (Life Technologies and Abcam) were diluted 1:1000 and DAPI (TH.GEYER, 5 μg/mL) was used for nuclear staining. Coverslips were mounted with DAKO fluorescent mounting medium (DAKO, #S3023) and edges sealed with nail polish.

Cytotoxicity in neuroblastoma M17 αSyn 3K-GFP model was assessed by caspase 3 and 7 activity via a commercial kit (Promega, G8090). Before lysis and caspase measurement cell counts were taken by staining with Hoechst and Opera Phenix high-content imaging. Caspase activity was normalized to cell counts. Viability in rodent cortical neurons and in NGN2 neurons was assessed by CellTiter-Glo (Promega, G7571). In addition, neuronal and astrocytic viability in rat cortical neurons was determined after fixation, permeabilization (0.1% Triton X-100 for 20 min), and staining with MAP2 (Abcam, ab92434) and GFAP (Abcam, ab7260) by neuron and astrocyte counting in the Opera Phenix high-content imaging system. MAP2 and GFAP were visualized by Alexa Fluor 647 and 488 secondary antibodies.

Coverslips with DA neurons were fixed in 4% paraformaldehyde (PFA) for 15 min at 37 °C. After washing with DPBS, the cells were blocked and permeabilized in a solution of DPBS, 0.2% Molecular Grade Triton X-100, and 10% Donor Horse Serum for one hour. Primary antibodies were added to the blocking solution at 4 °C overnight, with the following dilutions for DA neurons: TH (Abcam ab129991) (1:500) and MAP2 (Abcam ab92434) (1:500) (please note that while MAP2 is mainly expressed in neurons, there may be some low expression in glial cells). The next day, the coverslips were washed with DPBS and incubated with Alexa Fluor secondary antibodies, followed by counterstaining with DAPI staining solution (1:3000) for one hour at room temperature. The coverslips were rewashed, mounted on slides using Fluoromount-G mounting medium (0100-01, Southern Biotech), and allowed to dry overnight in the dark. The fluorescence signals were visualized using a Nikon A1-R confocal microscope, and images were processed using NIS elements 5.21 (Nikon) and microscopy image analysis software Imaris 9.8 (Oxford Instruments).

The following primary antibodies were used: Early endosome antigen 1 (EEA1) at 1:500 (#610456; BD Biosciences); amyloid precursor protein (APP) at 1:500 (#ab32136; Abcam); microtubule-associated protein 2 (MAP2) at 1:1000 (ab92434; Abcam); Ras-related protein Rab-7a (Rab7) at 1:1000 (ab50533; Abcam); Ras-related protein Rab-11 (Rab11) at 1:250 (#610656; BD Biosciences); Lysosome-associated membrane protein-1 (LAMP1) at 1:250 (#sc 2011; Santa Cruz); Tropomyosin receptor kinase B (TRKB; # ab18987; abcam) at 1:1000, GLUA1(# MAB2263; Millipore sigma) at 1:500 and VPS35 (Abcam; #ab97545) at 1:500. DAPI was used at a final concentration of 0.1 µg/ml (Alfa Aesar).

The plasmid encoding Mettl3 was generated by amplifying total mouse brain cDNA using the forward primer 5′-ATAGTCGACGATGTCGGACACGTGGAGCTCT-3′ and the reverse primer 5′- TTGCGGCCGCGTGCGTCTATAAATTCTTAGGTT-3′. The PCR products were digested with SalI and NotI (New England Biolabs) and ligated into the cut pRK5-myc vector. Plasmids encoding V5-tagged Tau (wild-type and P301L mutant) were generously provided by Prof. Jürgen Götz (Li and Götz, 2017 (link)).
Antibodies were purchased from commercial sources as follows: anti-METTL3 (ab195352, Abcam), anti-RBM15B (2249-1-AP, Proteintech), anti-METTL14 (HPA038002, Sigma), anti-Tau (MN1000, Thermo Scientific), anti-V5 (V8137, Sigma or clone7/4, Biolegend), anti-myc (clone 9E10, Bio-Rad Laboratories or clone A-14, Santa Cruz Biotechnology), anti-MAP2 (ab92434, Abcam), or anti-β-actin (sc-47 778, Santa Cruz Biotechnology).