ALS-Associated TDP-43 Induces Endoplasmic Reticulum Stress, Which Drives Cytoplasmic TDP-43 Accumulation and Stress Granule Formation

Adam K Walker,Malcolm K Horne,Julie D Atkin,Sonam Parakh,Kai Y Soo,Peter J Crouch,Manal A Farg,Yi Ma,Robyn H Wallace,Vinod Sundaramoorthy,Bradley J Turner

doi:10.1371/journal.pone.0081170

Adam K Walker, Malcolm K Horne + Show 9 more

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https://doi.org/10.1371/journal.pone.0081170

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In amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration, TAR DNA binding protein 43 (TDP-43) accumulates in the cytoplasm of affected neurons and glia, where it associates with stress granules (SGs) and forms large inclusions. SGs form in response to cellular stress, including endoplasmic reticulum (ER) stress, which is induced in both familial and sporadic forms of ALS. Here we demonstrate that pharmacological induction of ER stress causes TDP-43 to accumulate in the cytoplasm, where TDP-43 also associates with SGs. Furthermore, treatment with salubrinal, an inhibitor of dephosphorylation of eukaryotic initiation factor 2-α, a key modulator of ER stress, potentiates ER stress-mediated SG formation. Inclusions of C-terminal fragment TDP-43, reminiscent of disease-pathology, form in close association with ER and Golgi compartments, further indicating the involvement of ER dysfunction in TDP-43-associated disease. Consistent with this notion, over-expression of ALS-linked mutant TDP-43, and to a lesser extent wildtype TDP-43, triggers several ER stress pathways in neuroblastoma cells. Similarly, we found an interaction between the ER chaperone protein disulphide isomerase and TDP-43 in transfected cell lysates and in the spinal cords of mutant A315T TDP-43 transgenic mice. This study provides evidence for ER stress as a pathogenic pathway in TDP-43-mediated disease.

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TAR DNA-binding protein 43 (TDP-43) is a protein constituent of pathologic cytoplasmic and intranuclear inclusions in neurons and glia of patients with sporadic and familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) [1,2]
In contrast to the diffuse cytoplasmic staining of mCherry, transiently transfected TDP-43-mCherry constructs resulted in punctate fluorescence confined to, but dispersed throughout, the nucleus with exclusion from nucleoli (Fig. 1A) This distribution is similar to previous observations for endogenous TDP-43 [5,49,50] and confirms correct targeting of the fusion proteins (Fig. 1A)
We demonstrate that endoplasmic reticulum (ER) stress leads to accumulation of both wildtype and ALS-linked TDP-43 mutants in the cytoplasm and to incorporation of TDP-43 into cytoplasmic stress granules (SGs), We demonstrate that C-terminal TDP-43 inclusions, induced by proteasome inhibition, are closely associated with protein disulphide isomerise (PDI) and the ER-Golgi compartments, suggesting potential disturbances to these organelles in TDP-43-linked disease

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Introduction

TAR DNA-binding protein 43 (TDP-43) is a protein constituent of pathologic cytoplasmic and intranuclear inclusions in neurons and glia of patients with sporadic and familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) [1,2]. While predominantly a nuclear protein, a proportion of TDP-43 is cytoplasmic, even under normal conditions [3,4,5]. When nuclear localisation sequences of TDP-43 are genetically ablated, the protein accumulates in the cytoplasm and forms inclusions that are similar to those seen in disease [4,6]. It was shown that under cellular stress, TDP-43 accumulates in the cytoplasm and forms cytoplasmic stress granules (SGs) [7,8,9].

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