Objective: To determine if enhancement of protein palmitoylation benefits alpha-synuclein (aS) dependent vesicular trafficking defects and cytotoxicity.

Background: Recent studies suggest that vesicular trafficking defects caused by aS accumulation play a key role in PD. The mechanism is unknown. Interestingly, palmitoylation, post-translational addition of the fatty acid palmitate to cysteines, is involved in trafficking by targeting several SNARE proteins to membranes where they mediate vesicle fusion. aS itself is not palmitoylated but binds to membrane vesicles via an amphipathic helix. Amphipathic helices may regulate palmitoylation, which occurs enzymatically at membranes. Here, we hypothesize that abnormal aS accumulation and membrane binding disrupt palmitoylation of SNAREs and subsequent trafficking. We asked whether enhancing palmitoylation might rescue these phenotypes. To that end, we increased cellular palmitoylation using inhibitors of acyl protein thioesterase-1 (APT1), a de-palmitoylating enzyme, followed by assessment of aS-associated cellular pathologies.

Methods: Our lab previously found that “amplification” of the E46K familial PD aS mutant with 2 additional E-to-K mutations in the adjacent KTK(E)GV repeat motifs (E35K, E61K) causes cytotoxicity and aS-rich cytoplasmic inclusions. YFP-tagged versions of these aS “3K” mutants were expressed in M17D human neuroblastoma cells and inclusions measured by automated image analysis (Incucyte). Cytotoxicity was assessed by adenylate kinase release. Phosphorylated aS was examined by Western blotting with a Ser129 phospho-specific antibody. Acyl resin-assisted capture (RAC) was used to measure palmitoylation.

Results: Inclusions in aS 3K M17D cells are comprised of clusters of aS plus many membrane vesicles indicative of disrupted trafficking. Enhancing palmitoylation with the structurally distinct APT1 inhibitors palmostatin B (PSB) and ML348 reduced inclusions. PSB also reduced cell death and levels of phosphorylated aS, a marker of cytotoxicity. In transgenic mice expressing 3K aS, which exhibit a robust parkinsonian phenotype, we found decreased palmitoylation of the vesicular proteins SNAP-25, syntaxin-1, and synaptotagmin-1.

Conclusions: Our findings support the novel hypothesis that impaired protein palmitoylation can contribute to aS-dependent vesicular trafficking defects. Enhancing palmitoylation may be cytoprotective by partially correcting these deficits.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/enhancing-protein-palmitoylation-is-protective-in-alpha-synuclein-dependent-cytotoxicity/