Chronic oxidative stress promotes GADD34-mediated phosphorylation of the TAR DNA-binding protein TDP-43, a modification linked to neurodegeneration

Abstract

Oxidative and endoplasmic reticulum (ER) stresses are hallmarks of the pathophysiology of ALS and other neurodegenerative diseases. In these stresses, different kinases phosphorylate eukaryotic initiation factor eIF2α, enabling the translation of stress response genes; among these is , the protein product of which recruits the α-isoform of protein phosphatase 1 catalytic subunit (PP1α) and eIF2α to assemble a phosphatase complex catalyzing eIF2α dephosphorylation and resumption of protein synthesis. Aberrations in this pathway underlie the aforementioned disorders. Previous observations indicating that GADD34 is induced by arsenite, a thiol-directed oxidative stressor, in the absence of eIF2α phosphorylation suggest other roles for GADD34. Here, we report that arsenite-induced oxidative stress differs from thapsigargin- or tunicamycin-induced ER stress in promoting transcription and the preferential translation of its mRNA in the absence of eIF2α phosphorylation. Arsenite also stabilized GADD34 protein, slowing its degradation. In response to oxidative stress, but not ER stress, GADD34 recruited TDP-43, and enhanced cytoplasmic distribution and cysteine modifications of TDP-43 promoted its binding to GADD34. Arsenite also recruited a TDP-43 kinase, casein kinase-1ϵ (CK1ϵ), to GADD34. Concomitant with TDP-43 aggregation and proteolysis after prolonged arsenite exposure, GADD34-bound CK1ϵ catalyzed TDP-43 phosphorylations at serines 409/410, which were diminished or absent in GADD34 cells. Our findings highlight that the phosphatase regulator, GADD34, also functions as a kinase scaffold in response to chronic oxidative stress and recruits CK1ϵ and oxidized TDP-43 to facilitate its phosphorylation, as seen in TDP-43 proteinopathies.

Induction Of Chronic Stress Reveals An Interplay Of Stress Granules And TDP-43 Pathological Aggregates In Human ALS Fibroblasts And iPSC-Neurons

“…Consistent with our findings, while TDP-43 is protected by phosphorylation when it is recruited within SG, as previously hypothesized [23,25], when the stress persists becoming chronic as in neurodegeneration, cytoplasmic fulllength TDP-43 protein may be in part released from SG and undergo phosphorylation by protein kinases, such as GADD34/CK1ε [47]. This may trigger formation of P-TDP-43 inclusions, driving a pathological aggregate transition and leading to an engulfment of the autophagy and other systems involved in protein quality control.…”

Endoplasmic Reticulum Stress Signalling Induces Casein Kinase 1-Dependent Formation of Cytosolic TDP-43 Inclusions in Motor Neuron-Like Cells

“…Here we have focussed on chemical induction of ER stress, showing that tunicamycin-induced ER stress signalling can cause profound accumulation of TDP-43 in NSC-34 cells. In both this study and the work by Leggett et al [61], tunicamycin induces the formation of a small number of large cytoplasmic inclusions, rather than the numerous puncta seen with some other stressors [23]. ER stress has been strongly implicated in the pathogenesis of MND, and our study is among the first to show that ER stress can induce cytoplasmic accumulation of endogenous TDP-43, a major pathological hallmark of MND, although this has been shown using thapsigargin, which also induces ER stress [62,63] This process is partially blocked by pharmacological inhibition of CK1, suggesting a key role for CK1.…”

“…It is possible that, in patients with sporadic disease, different cellular stresses, for example ER stress and oxidative stress, lead to TDP-43 aggregation through distinct pathways and this may account for the recent finding of different TDP-43 inclusion morphologies in MND and frontotemporal dementia, namely the rounded and circumferential morphologies [58]. Furthermore, the differences in underlying stress may account the multiplicity of signalling pathways and TDP-43 kinases implicated in neurodegeneration, ranging from CK1 to cyclin-dependent kinase and glycogen synthase kinase 3 [22,23,25,69,70].…”

“…The mechanisms underlying TDP-43 aggregation in sporadic MND are incompletely characterized, although phosphorylation of TDP-43 is likely to be important [20]. TDP-43 is a substrate for a number of protein kinasescasein kinase 1 (CK1) has been identified as a direct TDP-43 kinase, including at S409/410 [21][22][23][24][25], and its overexpression can induce TDP-43 phosphorylation [26]. Furthermore, custom synthesized CK1δ inhibitors were able to reduce TDP-43 phosphorylation and protect cultured cells against ethacrynic acid challenge, which induces neuroblastoma cell death by mediating the phosphorylation of TDP-43 [27].…”

The Pathobiology of TDP-43 C-Terminal Fragments in ALS and FTLD

“…Although numerous other studies have substantiated the effect of cellular stress on TDP-43 cleavage, they did not examine whether this occurred via caspase activation. For instance, arsenite-induced oxidative stress generated CTF-35, an effect which was dependent on increased translation of the stress response gene GADD34 (Goh et al, 2018). Inhibition of the ubiquitin-proteasome system by treatment with epoxomicin or MG132 was associated with cytoplasmic TDP-43 fragments of approximately 32–35 kDa, and relatively low levels of CTF-25, in both immortalized neuronal cell lines and primary hippocampal and cortical cultures (Ayala V. et al, 2011; van Eersel et al, 2011).…”