Alpha-Synuclein defects autophagy by impairing SNAP29-mediated autophagosome-lysosome fusion

Qilin Tang,Thomas Koeglsperger,Matthias Höllerhage,Thomas Arzberger,Günter Höglinger,Jochen Herms,Pan Gao

doi:10.1038/s41419-021-04138-0

Abstract

Dopaminergic (DA) cell death in Parkinson’s disease (PD) is associated with the gradual appearance of neuronal protein aggregates termed Lewy bodies (LBs) that are comprised of vesicular membrane structures and dysmorphic organelles in conjunction with the protein alpha-Synuclein (α-Syn). Although the exact mechanism of neuronal aggregate formation and death remains elusive, recent research suggests α-Syn-mediated alterations in the lysosomal degradation of aggregated proteins and organelles – a process termed autophagy. Here, we used a combination of molecular biology and immunochemistry to investigate the effect of α-Syn on autophagy turnover in cultured human DA neurons and in human post-mortem brain tissue. We found α-Syn overexpression to reduce autophagy turnover by compromising the fusion of autophagosomes with lysosomes, thus leading to a decrease in the formation of autolysosomes. In accord with a compensatory increase in the plasma membrane fusion of autophagosomes, α-Syn enhanced the number of extracellular vesicles (EV) and the abundance of autophagy-associated proteins in these EVs. Mechanistically, α-Syn decreased the abundance of the v-SNARE protein SNAP29, a member of the SNARE complex mediating autophagolysosome fusion. In line, SNAP29 knockdown mimicked the effect of α-Syn on autophagy whereas SNAP29 co-expression reversed the α-Syn-induced changes on autophagy turnover and EV release and ameliorated DA neuronal cell death. In accord with our results from cultured neurons, we found a stage-dependent reduction of SNAP29 in SNc DA neurons from human post-mortem brain tissue of Lewy body pathology (LBP) cases. In summary, our results thus demonstrate a previously unknown effect of α-Syn on intracellular autophagy-associated SNARE proteins and, as a consequence, a reduced autolysosome fusion. As such, our findings will therefore support the investigation of autophagy-associated pathological changes in PD

Highlights

IntroductionMacroautophagy (here referred to as autophagy) is a genetically regulated intracellular nutrient recycling process involving lysosomal degradation of unwanted cellular proteins and defective organelles
Macroautophagy is a genetically regulated intracellular nutrient recycling process involving lysosomal degradation of unwanted cellular proteins and defective organelles
In order to achieve viral overexpression of α-Syn or GFP as a fractions, we found that bafilomycin A1 (Baf) resulted in an additional increase in control, adenoviruses serotype 5 (AV5)-α-Syn or AV5-GFP at a oligomeric α-Syn fractions at 37, 53, 72, and 85 kDa (Fig. 1j, k) multiplicity of infection (MOI) of 2.15 was added to the cell culture suggesting that these α-Syn fractions are subjected to autophagic medium 24 h after plating as described previously [33]

Summary

Introduction

Macroautophagy (here referred to as autophagy) is a genetically regulated intracellular nutrient recycling process involving lysosomal degradation of unwanted cellular proteins and defective organelles. The formation of autophagosomes and autolysosomes is orchestrated by multimeric protein complexes, including the unc-51-like kinase 1 (ULK1) complex in addition to numerous autophagy-regulated genes (ATGs). MTORC1 and AMP-activated protein kinase (AMPK), regulate autophagy initiation by phosphorylating ULK1 at different sites [3]. Autophagosomes are thought to form randomly throughout the cytoplasm, before moving to the perinuclear region on microtubule tracks, where the fusion with lysosomes takes place [4]. In cooperation with Rasrelated protein in brain (Rab) proteins (Rab7, 33b, 2), ATG8 family proteins, membrane-tethering factors (HOPS, ATG14), and regulatory molecules (RILP, TECPR1, BRUCE, PLEKHM1, Pacer), soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) eventually mediate the fusion of autophagosomes and amphisomes to lysosomes [5]

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Results

Conclusion