Author:

Keywords:

Parkin, mitochondria, mitophagy

Abstract:

Mutations in the gene encoding Parkin are the most common cause of recessive Parkinson’s disease. In Parkin-transfected HeLa cells, Parkin translocates from the cytosol to depolarized mitochondria and mediates mitophagy. However, the molecular mechanisms underlying Parkin translocation and Parkin-mediated mitophagy are not clear. Using tandem affinity purification of Parkin and its binding partners from HEK293 cells and mass spectrometry, we have previously identified the autophagy-promoting protein Ambra1 (activating molecule in Beclin1-regulated autophagy) as a Parkin interactor (Van Humbeeck C, Hofkens H, Mandemakers W, Gevaert K, De Strooper B, Vandenberghe W. Parkin mediates mitophagy through interaction with Ambra1. Abstract 461.26; 2010 Meeting of the Society for Neuroscience, San Diego, CA). Moreover, we have previously found that Ambra1 is required for Parkin-mediated mitophagy in Parkin-transfected HeLa cells. Here, we investigated whether Ambra1 also binds to Parkin at the endogenous protein level and mediates mitophagy in neural cells. Endogenous Parkin and Ambra1 were co-immunoprecipitated from SH-SY5Y cells, a dopaminergic neural cell line. Interestingly, prolonged (12 h) treatment of SH-SY5Y cells with the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) potently increased the interaction of endogenous Parkin and Ambra1. Moreover, prolonged CCCP treatment of SH-SY5Y cells triggered a redistribution of endogenous Ambra1 to the mitochondria. We asked whether Ambra1 was required for the translocation of endogenous Parkin to depolarized mitochondria. Exposure to CCCP for 3 h substantially increased the percentage of SH-SY5Y cells showing clusters of endogenous Parkin immunoreactivity that colocalized with mitochondria. However, siRNA-mediated knockdown of Ambra1 did not inhibit mitochondrial translocation of endogenous Parkin. We then tested for involvement of endogenous Ambra1 in CCCP-induced mitophagy. Treatment of untransfected SH-SY5Y cells with CCCP for 24 h resulted in a significant reduction of mitochondrial mass and staining, and this effect was blocked by the lysosomal inhibitor bafilomycin. Interestingly, siRNA-mediated Ambra1 knockdown suppressed CCCP-induced mitochondrial clearance, indicating that this process was dependent on Ambra1. These data further support a key role for Ambra1 in the clearance step of Parkin-mediated mitophagy.