Molecular investigation of mitophagy in neuronal cell models

Abstract

Autophagy functions as a cellular recycling and quality control pathway and is likely responsible for maintenance of viable mitochondria by mitophagy, even though exact mechanisms remain unclear. Mitophagy is characterised by accumulation of the protein kinase PINK1 on damaged mitochondria from where it recruits the E3-ligase Parkin, leading to ubiquitination of damaged mitochondrial proteins, marking them for autophagic engulfment. PINK1 and PARK2 (Parkin) mutations are linked to early-onset Parkinson?s disease (PD), suggesting defects in mitophagy could contribute to PD pathogenesis. Considering most research regarding mitophagy and PD are carried out in non-neuronal cell models, this study sought to identify the mechanisms of mitochondrial degradation and whether this involved the autophagy pathway in cell models of neuronal origin (SH-SY5Y cells), and in mouse embryonic stem cells (mESC), and human induced pluripotent stem cells (hiPSC) that have the potential to become differentiated towards the neuronal lineage.

In SH-SY5Y cells, treatment with the mitochondrial uncoupler FCCP led to activation of autophagy and degradation of mitochondrial markers TOM20, TIM23, and VDAC1. Inhibition of ULK1 or PIK3C3 did not affect degradation of damaged mitochondria, suggesting canonical autophagy is not involved. Proteasomal degradation was important instead. FCCP did not lead to mitochondrial degradation in mESC and likely inhibited autophagy. In presumptive neuronal precursor hiPSC, FCCP induced autophagy, but not classical mitophagy. hiPSC differentiated into presumptive dopamine (DA) neurons showed FCCP-mediated loss of mitochondrial markers in control cells, which was not affected by autophagic or proteasomal inhibition. PD hiPSC had fragmented mitochondria and increased susceptibility to FCCP cytotoxicity, suggesting mitochondria could be intrinsically damaged. Combined, the findings show mitophagy and involvement of autophagy are differentially regulated in different cell types of neuronal origin, underscoring the importance of appropriate selection of cell models for PD related research. The data also supports the notion that mitophagy/autophagy are differentially regulated in presumptive DA neurons obtained from hiPSC of normal and PD patients, suggesting that hiPSC cells may provide a good model system for further investigation of mitophagy in PD.