Objective: Our aim is to elucidate how selegiline drives extracellular secretion of α-synuclein (αS) and whether selegiline-induced αS secretion affects cell-to-cell transmission of αS.

Background: Extracellular αS secretion play a key role in developing cell-to-cell transmission of αS pathology in progressive neurodegeneration of α-synucleinopathies. Previous studies have demonstrated that αS is secreted under physiological conditions in neuronal cell lines and primary neurons. We have previously shown that monoamine oxidase-B inhibition facilitates extracellular αS secretion in SH-SY5Y cells (1). However, its molecular mechanisms and its effect to cell-to-cell transmission of αS remain unclear.

Method: Using mouse primary cortical neurons or SH-SY5Y cells stably expressing αS, we assessed effects of selegiline, monoamine oxidase-B inhibitor, on extracellular secretion of αS via an autophagic mechanism. To investigate the effect of selegiline-induced αS secretion on cell-to-cell transmission of αS, we treated primary neurons with the conditioned medium of PBS or selegiline-treated SH-SY5Y cells.

Results: Selegiline enhanced αS secretion in mouse primary cortical neurons. Secretion of αS induced by selegiline was suppressed by intracellular calcium chelator, BAPTA-AM. siRNA-mediated knockdown of Atg5 inhibited selegiline-induced αS and p62 secretions in SH-SY5Y cells stably expressing αS. Selegiline increased LC3-II to β-actin ratio and decreased intracellular p62 levels in primary neurons. Immunofluorescence analysis revealed that selegiline induced colocalization of αS and LC3-II in primary neurons. Blocking amphisome formation by tetanus toxin significantly reduced selegiline-induced αS and p62 secretions. Selegiline didn’t affect colocalization of αS and LAMP1 in primary neurons. While selegiline increases αS secretion in non-exosome enriched extracellular vesicle (EV) fraction, selegiline didn’t affect αS secretion in EV fraction. When primary neurons were exposed with the conditioned medium of PBS or selegiline-treated SH-SY5Y cells, selegiline didn’t affect αS uptake, solubility and viability of recipient neurons compared to PBS.

Conclusion: These findings show that selegiline drives autophagy-based secretion of αS through affecting intracellular Ca²⁺ concentration and amphisome formation. Selegiline don’t affect exosomal and lysosomal αS secretions. Additionally, selegiline-induced αS secretion don’t facilitate cell-to-cell transmission of αS.

References: 1. Nakamura Y, Arawaka S, Sato H, Sasaki A, Shigekiyo T, Takahata K, Tsunekawa H, Kato T. Monoamine oxidase-B inhibition facilitates α-synuclein secretion in vitro and delays its aggregation in rAAV-based rat models of Parkinson’s disease. Journal of Neuroscience. 2021;41:7479-91.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/selegiline-drives-autophagic-secretion-of-%ce%b1-synuclein/