Clusterin protects neurons against intracellular proteotoxicity

Jenna M Gregory,Sandeep Satapathy,Janet R Kumita,Justin J Yerbury,Mark R Wilson,Colin Smith,Daniel R Whiten,Teresa P Barros,Karina Mcdade,Leila M Luheshi,Christopher M Dobson,Rebecca A Brown

doi:10.1186/s40478-017-0481-1

Abstract

It is now widely accepted in the field that the normally secreted chaperone clusterin is redirected to the cytosol during endoplasmic reticulum (ER) stress, although the physiological function(s) of this physical relocation remain unknown. We have examined in this study whether or not increased expression of clusterin is able to protect neuronal cells against intracellular protein aggregation and cytotoxicity, characteristics that are strongly implicated in a range of neurodegenerative diseases. We used the amyotrophic lateral sclerosis-associated protein TDP-43 as a primary model to investigate the effects of clusterin on protein aggregation and neurotoxicity in complementary in vitro, neuronal cell and Drosophila systems. We have shown that clusterin directly interacts with TDP-43 in vitro and potently inhibits its aggregation, and observed that in ER stressed neuronal cells, clusterin co-localized with TDP-43 and specifically reduced the numbers of cytoplasmic inclusions. We further showed that the expression of TDP-43 in transgenic Drosophila neurons induced ER stress and that co-expression of clusterin resulted in a dramatic clearance of mislocalized TDP-43 from motor neuron axons, partially rescued locomotor activity and significantly extended lifespan. We also showed that in Drosophila photoreceptor cells, clusterin co-expression gave ER stress-dependent protection against proteotoxicity arising from both Huntingtin-Q128 and mutant (R406W) human tau. We therefore conclude that increased expression of clusterin can provide an important defense against intracellular proteotoxicity under conditions that mimic specific features of neurodegenerative disease.

Highlights

Protein misfolding, aggregation and deposition are unifying features of a wide range of neurodegenerative diseases [1]
We studied the aggregation of the 43 kDa transactive response DNA-binding protein (TDP-43), which is implicated in the pathology of amyotrophic lateral sclerosis (ALS), where it translocates from its normal location in the nucleus and forms insoluble inclusions in the cytoplasm
When cells were 60-80% confluent they were transfected as indicated with either pCAG-EGFP/RFP (encoding the wild-type TDP-43-tdTomato fusion protein (Addgene plasmid 28,205) using X-tremeGENE HP (Roche), pRc/CMV-HT7, pEGFP-N1-TDP-CTF (encoding a ~ 20 kDa C-terminal fragment of human TDP-43 fused to enhanced green fluorescent protein (EGFP); Addgene plasmid #28197) or pCMV6-AC-(M337 V)TDP-43-tGFP (Origene; M337 V mutation introduced into the wild type human TDP-43 sequence, fused at the C-terminus to turboGFP) using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. 48 h after transfection, cells were treated for 10 h with 2.5 μM A23187, 2.75 μM Tg and/or 10 μM MG132

Summary

Introduction

Aggregation and deposition are unifying features of a wide range of neurodegenerative diseases [1]. While the mechanism by which CLU translocates from the ER lumen to the cytosol in response to stress is yet to be elucidated, it appears that the release may form a part of a larger strategy in which many different chaperones are released from the ER to the cytosol to defend the cell against intracellular stresses. This has been suggested by the demonstration that, in response to cytosolic foreign DNA or inhibition of the proteasome, BiP is released to the cytosol [18]. The previous literature points to a potential involvement of CLU, especially during ER stress, in facilitating the degradation of specific cell proteins via both of the primary cell degradative pathways, the proteasome and autophagy

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Discussion

Conclusion