SIGMAR1/Sigma-1 receptor ablation impairs autophagosome clearance.

Autophagosome-lysosome fusion is a common critical step in various forms of macroautophagy/autophagy including mitophagy, the selective degradation of mitochondria. Regulations of this fusion process remain poorly defined. Here we have determined the role of SIGMAR1, a unique endoplasmic reticulum membrane protein. Knockout of Sigmar1 impaired mitochondrial clearance without altering the PINK1-PRKN/Parkin signaling, in mouse retinal explants and cultured cells treated with carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for induction of mitophagy. SIGMAR1 depletion also caused accumulation of autophagosome markers LC3-II and SQSTM1, but did not change the levels of BECN1 and ATG7, proteins associated with autophagosome biogenesis. Lysosomal pH and protease activities were not negatively affected. However, sigmar1 knockout partially compromised autophagosome-lysosome fusion in CCCP-treated NSC34 cells, as revealed by reduced GFP fluorescence quenching of GFP-RFP-LC3-II puncta and co-localization of lysosomes with mitochondria. Furthermore, SIGMAR1 co-immunoprecipitated with ATG14, STX17, and VAMP8 (but not SNAP29), proteins key to autophagosome-lysosome membrane fusion. Re-expressing SIGMAR1 in the null background rescued clearance of mitochondria and autophagosomes. In summary, we started out finding that sigmar1 knockout impaired the clearance of mitochondria and autophagosomes, and then narrowed down the SIGMAR1 modulation to the autophagosome-lysosome fusion step. This study may shed new light on understanding autophagy-associated cyto-protection and disease mechanisms. Abbreviations: APEX2, a genetically engineered peroxidase; BiFC, bimolecule fluorescence complementation; CCCP, a mitophagy inducing compound; CRISPR, clustered regularly interspaced short palindromic repeats; EM, electron microscopy; ER, endoplasmic reticulum; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; SIGMAR1, sigma non-opioid intracellular receptor 1.