Ab218819

Mouse neurons were cultured as described, with AraC added to feeding medium at Div 0, except as follows. At Div 4, cell media was collected, and recombinant 1 µg/ml human α-Synuclein protein aggregate (Abcam; ab218819) was added, vortexed and replaced on cells. Neurons were maintained till DiV 15 by the replacement of 50% media with Neurobasal-A + B27 every 3 days. Cells were then moved into TMo, stimulated for 8 h with 1 µM Trichostatin A, then 24 h with 250 nM CDDO-TFEA. Neurons were then either processed for western blot as described below or fixed, permeabilised and probed with fibril-conformation specific anti- α-Synuclein antibody (Abcam 1:5000, MJFR-14-6-4-2 ab209538) or anti phospho-Serine 129 α-Synuclein (Abcam, 1:500; MJF-R13(8-8), ab168381); images were analysed using ImageJ, with DAPI images used to mask nuclear stain to analyse specific neurite stain only.

Recombinant human α-syn protein aggregates were purchased from Abcam (ab218819). These aggregates are advertised as preformed fibrils (PFFs) which was confirmed using negative-stain electron microscopy. Since we wanted to introduce smaller oligomeric species of α-syn into neurons, the PFFs were first broken down into smaller aggregates before recordings were made. PFF samples were sonicated (as in Polinski et al., 2018 (link)) for 15 min (50–60 Hz) at room temperature using a Grant Ultrasonic XUBA1 bath. Recombinant human α-syn protein in monomeric form (ab218818) was used as a control and negative-stain electron microscopy was used to confirm it was not aggregated.

Differentiated SH-SY5Y cells were treated with human α-syn protein aggregates (active) (ab218819, 4 μg/ml; Abcam) and human α-syn protein monomer (active) (ab218818; Abcam). Immunostainings were performed as described previously. Differentiated SH-SY5Y cells were fixed with 4% PFA and subsequently incubated with primary antibodies against phosphorylated S129 α-syn (α-syn-P; 1:1,000; ab184674) at 4°C for 24 h. After washing 3× with PBS, the cells were incubated with Alexa Fluor 647 anti-mouse secondary antibody (1/500) for 1 h at RT, counterstained with Hoechst (blue) nuclear stain (1/4,000), and mounted on slides with antifade reagent. Images were acquired by a Nikon AIR confocal microscope using a 60× objective.

Primary astrocyte was cultured as described previously [27 (link)]. The neonatal midbrain (P0-3) was trypsinized with 0.25% trypase at 37 °C for 10 min and then centrifuged for 5 min at 1000g centrifugation. The cells were resuspended in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 medium containing 10% fetal bovine serum (FBS, GIBCO, Gaithersburg, MD, USA) and plated onto T-75 flasks at 50,000 cells/cm². After 10 d, confluent mixed glial cultures were shaken at 220 rpm for 6 h at 37 °C to remove unwanted cell types (microglia, oligodendrocytes, neurons, and fibroblasts). The purity of astrocytes was > 95% as determined with GFAP immunocytochemistry. To induce premature senescence model, astrocytes were pretreated with metformin at the indicated concentration for 1 h and then stimulated with MPP⁺ (200 μM, Sigma, St. Louis, MO, USA) for 24 h or α-synuclein aggregate (α-Syn PFF, 1 μg/mL, ab218819, abcam, USA) for 48 h. To induce naturally senescence model, astrocytes were cultured for 40 days and then treated with metformin for 10 days in vitro.

The inhibitor of lipid peroxidation Trolox-C (#238813), the iron chelator deferoxamine (D9533), the NMDA glutamate receptor blocker MK-801 (M107), the anti-inflammatory drug dexamethasone (D4902), the antimitotic cytarabine (Ara-C; C6645) and the DA uptake inhibitor GBR12909 (D052) were all purchased from Sigma Aldrich (L’Isles d’Abeau Chesnes, France). The two fluorogenic dyes used to monitor oxidative stress and mitochondrial membrane potential, dihydrorhodamine-123 (DHR-123; #D23806) and tetramethylrhodamine methyl ester (TMRM; #T668), were both obtained from ThermoFisher Scientific (Courtaboeuf, France). The TLR2 agonist PAM 3CSK3 (#tlrl-pms) was purchased from Invivogen (San Diego, CA, USA). To promote seeded αS aggregation, we used commercially available fibril seeds of recombinant human αS (ab218819; Abcam, Cambridge, UK).