Inhibition of PIKfyve by YM-201636 Dysregulates Autophagy and Leads to Apoptosis-Independent Neuronal Cell Death

Sally Martin,Elizabeth J Coulson,Linda M May,Frederic A Meunier,Callista B Harper,Shona L Osborne,Stefan Strack

doi:10.1371/journal.pone.0060152

Sally Martin, Elizabeth J Coulson + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0060152

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Journal: PloS one	Publication Date: Mar 27, 2013
Citations: 81	License type: CC BY 4.0

Affiliation: Queensland Eye Institute, University of Queensland

Abstract

The lipid phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P 2), synthesised by PIKfyve, regulates a number of intracellular membrane trafficking pathways. Genetic alteration of the PIKfyve complex, leading to even a mild reduction in PtdIns(3,5)P 2, results in marked neurodegeneration via an uncharacterised mechanism. In the present study we have shown that selectively inhibiting PIKfyve activity, using YM-201636, significantly reduces the survival of primary mouse hippocampal neurons in culture. YM-201636 treatment promoted vacuolation of endolysosomal membranes followed by apoptosis-independent cell death. Many vacuoles contained intravacuolar membranes and inclusions reminiscent of autolysosomes. Accordingly, YM-201636 treatment increased the level of the autophagosomal marker protein LC3-II, an effect that was potentiated by inhibition of lysosomal proteases, suggesting that alterations in autophagy could be a contributing factor to neuronal cell death.

Highlights

Phosphoinositides are important lipid regulators of membrane trafficking and cellular signalling
To investigate the mechanism contributing to this neuronal cell death pathway, we used the specific PIKfyve inhibitor YM-201636 in isolated hippocampal neurons
Quantitative measurement of cell survival after 24 h showed a marked reduction of,50% following PIKfyve inhibition (Figure 1A,B). Together these data suggest that acute inhibition of PIKfyve closely mimics the neuronal cell death phenotype observed in Fig42/2 and Vac142/2 mice [1,12,13]

Summary

Introduction

Phosphoinositides are important lipid regulators of membrane trafficking and cellular signalling. PIKfyve is part of an active complex regulating PtdIns(3,5)P2 levels, which includes the lipid phosphatase Fig and accessory protein Vac14 [1,2]. Despite being a minor component of cellular lipids, PtdIns(3,5)P2 and/or PIKfyve has been implicated in many cellular processes, including trafficking through the endolysosomal system, exocytosis, ion channel regulation and autophagy [3,4,5,6,7,8,9,10]. Genetic ablation of PIKfyve results in pre-implantation lethality [11], while mutations or genetic ablation of Fig or Vac results in decreased levels of PtdIns(3,5)P2 and a prominent vacuolar phenotype in the central nervous system, accompanied by marked spongiform degeneration [12,13]. Targeted re-expression of Fig in neurons of Fig42/2 mice clearly demonstrates a primary role for neuronal PIKfyve activity in preventing spongiform degeneration [15]

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