AKT signalling selectively regulates PINK1 mitophagy in SHSY5Y cells and human iPSC-derived neurons

Marc P M Soutar,Mark R Cookson,Liam Kempthorne,Mark Carlton,Selina Wray,Hélène Plun-Favreau,Julian Downward,Shuichi Miyakawa,Jasmine Harley,Daniela Melandri,David C Hancock,Gregory A O’Sullivan,Emily Annuario

doi:10.1038/s41598-018-26949-6

Abstract

The discovery of mutations within genes associated with autosomal recessive Parkinson’s disease allowed for the identification of PINK1/Parkin regulated mitophagy as an important pathway for the removal of damaged mitochondria. While recent studies suggest that AKT-dependent signalling regulates Parkin recruitment to depolarised mitochondria, little is known as to whether this can also regulate PINK1 mitochondrial accumulation and downstream mitophagy. Here, we demonstrate that inhibition of AKT signalling decreases endogenous PINK1 accumulation in response to mitochondria depolarisation, subsequent Parkin recruitment, phosphorylation of ubiquitin, and ultimately mitophagy. Conversely, we show that upon stimulation of AKT signalling via insulin, the mitophagy pathway is increased in SHSY5Y cells. These data suggest that AKT signalling is an upstream regulator of PINK1 accumulation on damaged mitochondria. Importantly, we show that the AKT pathway also regulates endogenous PINK1-dependent mitophagy in human iPSC-derived neurons.

Highlights

Much of what we know about the mitophagy pathway is based on the investigation of genetic forms of Parkinson’s disease (PD), where mutations were found at recessive loci PARK6 (PINK1 (PTEN-induced putative kinase 1)) and PARK2 (Parkin)[8,9]
Western blotting (WB) of enriched cytoplasmic fractions demonstrated that insulin induced a robust phosphorylation of AKT at Ser[473] in SHSY5Y FLAG-Parkin cells, which was blocked by MK2206 pre-treatment (Fig. 1A,B)
We show that AKT signalling regulates endogenous PINK1 accumulation, and subsequent efficient clearance of damaged mitochondria in SH-SY5Y cells and induced pluripotent stem cell (iPSC) neurons

Summary

Introduction

Much of what we know about the mitophagy pathway is based on the investigation of genetic forms of PD, where mutations were found at recessive loci PARK6 (PINK1 (PTEN-induced putative kinase 1)) and PARK2 (Parkin)[8,9]. The mitochondrial kinase PINK1 and ubiquitin E3 ligase Parkin act in concert to regulate the mitophagy process[10,11]. PINK1 selectively accumulates on the outer membrane of damaged mitochondria, where it facilitates Parkin recruitment from the cytoplasm to the mitochondria and activates its E3 ligase activity causing ubiquitination of mitochondrial membrane proteins[12,13,14,15,16,17,18,19,20]. We show that selective AKT inhibition in SHSY5Y FLAG-Parkin over-expressing neuroblastoma cells attenuates PINK1 accumulation in response to mitochondria depolarisation. This effect correlates with reduced recruitment of FLAG-Parkin to damaged mitochondria, reduced ubiquitin phosphorylation www.nature.com/scientificreports/. Our results further strengthen the role of the PI3K/AKT pathway in modulating PINK1/Parkin-dependent mitophagy

Methods

Results

Conclusion