Salubrinal

Alpha-Syn transfectant 3D5 was derived from a human neuroblastoma cell line (BE2-M17D) and characterized previously [13 (link),14 (link)]. The transfectant expresses wild-type human α-Syn and displays neuronal phenotypes upon TetOff induction and incubation with RA [14 (link)]. Cultures of 3D5 were maintained in DMEM/10% fetal bovine serum with 2 μg/mL Tet (referred to as non-induced or α-Syn-) or without (referred to as induced or α-Syn+) at 37°C and 5% CO₂. Those intended for biochemical analysis were seeded at 0.5 × 10⁶ cells/plate (100 × 20 mm, BD Biosciences, San Jose, CA) or 1.5 × 10⁵ cells/well in 6-well plates. For immunofluorescence or spectrophotometric assay, 3D5 cells were seeded at 2 × 10⁴ cells/well on coverslips in 24-well plates and 1 × 10⁴ in 48-well plates (Bellco Glass Inc, Vineland, NJ), respectively. Media were replaced the next day with Neurobasal medium (Invitrogen, Carlsbad, CA), 2% B-27 supplement (antioxidant free, Invitrogen), 2 mM L-glutamine (Sigma) and 10 μM RA (Sigma-Aldrich, St Louis, MO). After 10 ds of differentiation and induced α-Syn overexpession, cells were treated with HDAC inhibitors [SB (sigma), VPA (Sigma) and Agk2 (EMD Biosciences, La Jolla, CA)], a derivative of short-chain fatty acid [sodium acetate (SA)], an endoplasmic reticulum (ER) stress inducer [tunicamycin (TM, Sigma)], an inhibitor of phosphatases that act on the eukaryotic translation factor 2 subunit alpha (EIF2α) [salubrinal (Sal, Tocris Bioscience, Ellisville, Missouri)], a protein synthesis inhibitor [cycoheximide (CHX, Sigma), or a pan caspase inhibitor (CI) [Z-VAD (OMe)-FMK (EMD Biosciences)]. The cultures were treated with different drug concentrations for durations described in the Result section. Some cultures were treated with two of the aforementioned drugs or with SB and tetracycline (2 μg/ml, for blocking α-Syn induction).
Primary neuronal cultures were prepared from hippocampal and cortical brain tissues of postnatal (day 1) mice (see below) with or without transgenic expression of wild-type human α-Syn, according to a protocol reported previously [61 (link)]. They were plated at 6 ~ 7 × 10⁵ cells per well on poly-D-lysine (Sigma) coated 6-well plate and maintained for 7 ds before treatment in the absence or presence of different drugs for different durations.

Tunicamycin (TUN), N-methyl-2-pyrrolidone (NMP), Kolliphor® HS15, methylcellulose and clonidine hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO). Salubrinal was purchased from Tocris, now part of Bio-Techne Corporation (Minneapolis, MN). Sephin1 acetate was purchased from Otava chemicals (Concord, ON, Canada). GSK2606414 was synthesized by Perfemiker Canspec China (Shanghai, China). Refer to S1 Fig for the compound structures of cited compounds and to S2 Fig for confirmatory pharmacokinetic studies indicating that GSK2606414, salubrinal, and Sephin1 can penetrate into the central nervous system in measurable quantities.