SOD1 activity threshold and TOR signalling modulate VAP(P58S) aggregation via reactive oxygen species-induced proteasomal degradation in a Drosophila model of amyotrophic lateral sclerosis.

Kriti Chaplot,Balaji Ramalingam,Siddhesh S Kamat,Girish S Ratnaparkhi,Lokesh Pimpale,Senthilkumar Deivasigamani

doi:10.1242/dmm.033803

Abstract

ABSTRACTFamilial amyotrophic lateral sclerosis (ALS) is an incurable, late-onset motor neuron disease, linked strongly to various causative genetic loci. ALS8 codes for a missense mutation, P56S, in VAMP-associated protein B (VAPB) that causes the protein to misfold and form cellular aggregates. Uncovering genes and mechanisms that affect aggregation dynamics would greatly help increase our understanding of the disease and lead to potential therapeutics. We developed a quantitative high-throughput Drosophila S2R+ cell-based kinetic assay coupled with fluorescent microscopy to score for genes involved in the modulation of aggregates of the fly orthologue, VAP(P58S), fused with GFP. A targeted RNA interference screen against 900 genes identified 150 hits that modify aggregation, including the ALS loci Sod1 and TDP43 (also known as TBPH), as well as genes belonging to the mTOR pathway. Further, a system to measure the extent of VAP(P58S) aggregation in the Drosophila larval brain was developed in order to validate the hits from the cell-based screen. In the larval brain, we find that reduction of SOD1 levels or decreased mTOR signalling reduces aggregation, presumably by increasing the levels of cellular reactive oxygen species (ROS). The mechanism of aggregate clearance is, primarily, proteasomal degradation, which appears to be triggered by an increase in ROS. We have thus uncovered an interesting interplay between SOD1, ROS and mTOR signalling that regulates the dynamics of VAP aggregation. Mechanistic processes underlying such cellular regulatory networks will lead to better understanding of the initiation and progression of ALS.This article has an associated First Person interview with the first author of the paper.

Highlights

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease characterized by loss of motor neurons resulting in muscular atrophy, gradual paralysis and death of the patient within 2-5 years post diagnosis (Cleveland and Rothstein, 2001; Tarasiuk et al, 2012)
We found that levels of several species of oxidized phospholipids were higher with Tor knockdown with or without neuronal overexpression of VAMP-associated Protein B (VAP)(P58S) in third instar larval brains to levels similar to Superoxide dismutase 1 (SOD1) knockdown (Fig. 7A). mTOR pathway downregulation has recently been shown to activate autophagy and ubiquitin proteasomal machinery (Zhao et al, 2015) via Mpk1/ERK5 pathway in yeast and humans (Rousseau and Bertolotti, 2016)
First and foremost, we found ALS loci, SOD1 and TDP-43 as modifiers of VAP(P58S) aggregation, which we had previously identified as VAP genetic interactors (Deivasigamani et al, 2014)

Summary

Introduction

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease characterized by loss of motor neurons resulting in muscular atrophy, gradual paralysis and death of the patient within 2-5 years post diagnosis (Cleveland and Rothstein, 2001; Tarasiuk et al, 2012). In ~10% of the cases, the disease occurs due to inheritance of altered gene(s) (F-ALS). About 50 potential genetic loci (Taylor et al, 2016) have been identified in ALS through Genome-wide association (GWAS), linkage and sequencing studies. While several studies have demonstrated the wide-range of consequences of the genetic alterations on cellular function, no clear unifying mechanism has emerged that might explain the pathogenesis of the disease (Andersen and Al-Chalabi, 2011; Mulligan and Chakrabartty, 2013; Taylor et al, 2016; Turner et al, 2013; Walker and Atkin, 2011)

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