Unique properties of PTEN-L contribute to neuroprotection in response to ischemic-like stress

Magdalena C E Jochner,Ulrich Dirnagl,Gisela Lättig-Tünnemann,Marieluise Kirchner,Britta J Eickholt,Alina Dagane,Christoph Harms,Junfeng An,Gunnar Dittmar

doi:10.1038/s41598-019-39438-1

Abstract

Phosphatase and tensin homolog (PTEN) signalling might influence neuronal survival after brain ischemia. However, the influence of the less studied longer variant termed PTEN-L (or PTENα) has not been studied to date. Therefore, we examined the translational variant PTEN-L in the context of neuronal survival. We identified PTEN-L by proteomics in murine neuronal cultures and brain lysates and established a novel model to analyse PTEN or PTEN-L variants independently in vitro while avoiding overexpression. We found that PTEN-L, unlike PTEN, localises predominantly in the cytosol and translocates to the nucleus 10–20 minutes after glutamate stress. Genomic ablation of PTEN and PTEN-L increased neuronal susceptibility to oxygen-glucose deprivation. This effect was rescued by expression of either PTEN-L indicating that both PTEN isoforms might contribute to a neuroprotective response. However, in direct comparison, PTEN-L replaced neurons were protected against ischemic-like stress compared to neurons expressing PTEN. Neurons expressing strictly nuclear PTEN-L NLS showed increased vulnerability, indicating that nuclear PTEN-L alone is not sufficient in protecting against stress. We identified mutually exclusive binding partners of PTEN-L or PTEN in cytosolic or nuclear fractions, which were regulated after ischemic-like stress. GRB2-associated-binding protein 2, which is known to interact with phosphoinositol-3-kinase, was enriched specifically with PTEN-L in the cytosol in proximity to the plasma membrane and their interaction was lost after glutamate exposure. The present study revealed that PTEN and PTEN-L have distinct functions in response to stress and might be involved in different mechanisms of neuroprotection.

Highlights

Phosphatase and tensin homolog -long (PTEN-L or PTENα) is a longer variant of the lipid and protein phosphatase PTEN
In order to unequivocally characterise the www.nature.com/scientificreports upper band as PTEN variant, we tested if it was sensitive to genetic knockout of PTEN in neurons derived from conditional PTEN knockout mice
In response to Cre delivery via transduction with lentiviral particles (LVPs), both the 57 kD band and bands of higher molecular weight (~70–75 kD) started to fade at day in vitro three (DIV 3) and disappeared at DIV 9 (Fig. 1b), indicating that the upper band detected by the PTEN antibody truly represents a PTEN variant

Summary

Introduction

Phosphatase and tensin homolog -long (PTEN-L or PTENα) is a longer variant of the lipid and protein phosphatase PTEN. It is thought that nuclear localisation of PTEN is a dynamic process that correlates with cell cycle progression and the cellular differentiation state, which can be triggered by cellular insults such as ischemia[3,4] To date it is unclear whether nuclear translocation of PTEN is beneficial or detrimental for cellular survival after ischemic-like stress[5,6,7]. We used oxygen-glucose deprivation and exposure to 50 μM glutamate to apply ischemic-like stress to neurons in vitro Both methods lead to neurodegeneration and cell death, which is mostly caused by intracellular calcium overload triggered by excess influx of extracellular calcium through NMDA receptor gated channels[10,11]. PTEN and PTEN-L had a distinct protein interactome in response to ischemic-like stress, which indicates that the intrinsically disordered N-terminal extension of PTEN-L might incorporate unique regulatory properties

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Results