Palmitoylation of the Parkinson's disease-associated protein synaptotagmin-11 links its turnover to α-synuclein homeostasis.

Synaptotagmin-11 (Syt11) is a vesicle-trafficking protein that is linked genetically to Parkinson's disease (PD). Likewise, the protein α-synuclein regulates vesicle trafficking, and its abnormal aggregation in neurons is the defining cytopathology of PD. Because of their functional similarities in the same disease context, we investigated whether the two proteins were connected. We found that Syt11 was palmitoylated in mouse and human brain tissue and in cultured cortical neurons and that this modification to Syt11 disrupted α-synuclein homeostasis in neurons. Palmitoylation of two cysteines adjacent to the transmembrane domain, Cys39 and Cys40, localized Syt11 to digitonin-insoluble portions of intracellular membranes and protected it from degradation by the endolysosomal system. In neurons, palmitoylation of Syt11 increased its abundance and enhanced the binding of α-synuclein to intracellular membranes. As a result, the abundance of the physiologic tetrameric form of α-synuclein was decreased, and that of its aggregation-prone monomeric form was increased. These effects were replicated by overexpression of wild-type Syt11 but not a palmitoylation-deficient mutant. These findings suggest that palmitoylation-mediated increases in Syt11 amounts may promote pathological α-synuclein aggregation in PD.

Upregulation of Cellular Palmitoylation Mitigates α-Synuclein Accumulation and Neurotoxicity.

BACKGROUND: Synucleinopathies, including Parkinson's disease (PD), are characterized by α-synuclein (αS) cytoplasmic inclusions. αS-dependent vesicle-trafficking defects are important in PD pathogenesis, but their mechanisms are not well understood. Protein palmitoylation, post-translational addition of the fatty acid palmitate to cysteines, promotes trafficking by anchoring specific proteins to the vesicle membrane. αS itself cannot be palmitoylated as it lacks cysteines, but it binds to membranes, where palmitoylation occurs, via an amphipathic helix. We hypothesized that abnormal αS membrane-binding impairs trafficking by disrupting palmitoylation. Accordingly, we investigated the therapeutic potential of increasing cellular palmitoylation. OBJECTIVES: We asked whether upregulating palmitoylation by inhibiting the depalmitoylase acyl-protein-thioesterase-1 (APT1) ameliorates pathologic αS-mediated cellular phenotypes and sought to identify the mechanism. METHODS: Using human neuroblastoma cells, rat neurons, and iPSC-derived PD patient neurons, we examined the effects of pharmacologic and genetic downregulation of APT1 on αS-associated phenotypes. RESULTS: APT1 inhibition or knockdown decreased αS cytoplasmic inclusions, reduced αS serine-129 phosphorylation (a PD neuropathological marker), and protected against αS-dependent neurotoxicity. We identified the APT1 substrate microtubule-associated-protein-6 (MAP6), which binds to vesicles in a palmitoylation-dependent manner, as a key mediator of these effects. Mechanistically, we found that pathologic αS accelerated palmitate turnover on MAP6, suggesting that APT1 inhibition corrects a pathological αS-dependent palmitoylation deficit. We confirmed the disease relevance of this mechanism by demonstrating decreased MAP6 palmitoylation in neurons from αS gene triplication patients. CONCLUSIONS: Our findings demonstrate a novel link between the fundamental process of palmitoylation and αS pathophysiology. Upregulating palmitoylation represents an unexplored therapeutic strategy for synucleinopathies. © 2020 International Parkinson and Movement Disorder Society.