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Huan Yang, Timur Mavlyutov, Lianwang Guo; An essential role of the sigma-1 receptor in mitophagy and autophagosome-lysosome fusion. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1603.
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© ARVO (1962-2015); The Authors (2016-present)
Mitophagy is a lysosome-mediated autophagic process to cleanse dysfunctional mitochondria thus critical for cell survival. The sigma-1 receptor (S1R) is a unique endoplasmic reticulum protein involved in cellar stress responses. Its regulations in mitophagy remain largely unknown. We hypothesize that S1R plays an essential role in mitophagy and lysosomal organelle fusion.
Retinal explants were isolated from wild type (WT) and S1R knockout (KO) mice. Permanent S1R KO and induced S1R knockdown (KD) were generated using the CRISPR-Cas9 technology in neuronal cell lines. Mitophagy was induced with CCCP, a mitochondrial uncoupler, and detected as decrease of mitochondrial protein markers (VDAC1 and TIM23) by Western blotting. Autophagic lysosomal organelle fusion was assessed via fluorescent microscopy of cells transfected with a pH-sensitive LC3-II-GFP-RFP construct or organelle markers. S1R protein interactions were studied with co-immunoprecipitation (co-IP) assay.
Compared to WT control, S1R KO impaired mitophagy in retinal explants and NSC34 cells (permanent S1R KO) and SH-SY5Y cells (doxycycline-induced KD), but did not affect parkin recruitment to mitochondria, an early event of mitophagy. Re-expressing S1R in the NSC34 cells with a S1R KO background rescued mitophagy. The deficiency of S1R in these tissue or cell samples also caused acumulation of LC3II and SQSTM1 (autophagosome markers), but did not alter the levels of Beclin1 and ATG7, proteins involved in autophagosome biogenesis. Lysosome pH and protease activities were not negatively affected by S1R KO. However, co-localization of the label of lysosomes with that of autophagosomes or mitochondria was disrupted, in S1R KO NSC34 cells in contrast to WT control, supporting an important role of S1R in lysosomal organelle fusion. Moreover, S1R was found to be in a complex with the proteins key to autophagosome/lysosome fusion, including ATG14, STX17, and VAMP8. S1R interactions with these proteins were confirmed by co-IP.
S1R plays an important role in CCCP-induced mitophagy. Its molecular function in this context can be narrowed down to autophagosome/lysosome fusion, likely through its interactions with the protein complex essential for this autophagic organelle fusion.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
S1R localization at the ER-lysosome contact site
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