Fine tuning of the unfolded protein response by ISRIB improves neuronal survival in a model of amyotrophic lateral sclerosis

Ricardo Bugallo,Tomás Aragón,Roberto Ferrero,Elías Marlin,Estefanía Toledo,Ana Baltanás,Laura Larrea,Rodrigo Vinueza-Gavilanes,Montserrat Arrasate

doi:10.1038/s41419-020-2601-2

Abstract

Loss of protein folding homeostasis features many of the most prevalent neurodegenerative disorders. As coping mechanism to folding stress within the endoplasmic reticulum (ER), the unfolded protein response (UPR) comprises a set of signaling mechanisms that initiate a gene expression program to restore proteostasis, or when stress is chronic or overwhelming promote neuronal death. This fate-defining capacity of the UPR has been proposed to play a key role in amyotrophic lateral sclerosis (ALS). However, the several genetic or pharmacological attempts to explore the therapeutic potential of UPR modulation have produced conflicting observations. In order to establish the precise relationship between UPR signaling and neuronal death in ALS, we have developed a neuronal model where the toxicity of a familial ALS-causing allele (mutant G93A SOD1) and UPR activation can be longitudinally monitored in single neurons over the process of neurodegeneration by automated microscopy. Using fluorescent UPR reporters we established the temporal and causal relationship between UPR and neuronal death by Cox regression models. Pharmacological inhibition of discrete UPR processes allowed us to establish the contribution of PERK (PKR-like ER kinase) and IRE1 (inositol-requiring enzyme-1) mechanisms to neuronal fate. Importantly, inhibition of PERK signaling with its downstream inhibitor ISRIB, but not with the direct PERK kinase inhibitor GSK2606414, significantly enhanced the survival of G93A SOD1-expressing neurons. Characterization of the inhibitory properties of both drugs under ER stress revealed that in neurons (but not in glial cells) ISRIB overruled only part of the translational program imposed by PERK, relieving the general inhibition of translation, but maintaining the privileged translation of ATF4 (activating transcription factor 4) messenger RNA. Surprisingly, the fine-tuning of the PERK output in G93A SOD1-expressing neurons led to a reduction of IRE1-dependent signaling. Together, our findings identify ISRIB-mediated translational reprogramming as a new potential ALS therapy.

Highlights

Loss of protein folding homeostasis has been identified as a common pathological feature of most neurodegenerative diseases (NDs), including Parkinson’s, Alzheimer’s disease, or amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder caused by degeneration of upper and lower motor neurons
Rat primary cortical neurons were transfected with plasmids encoding wild-type (WT) (SOD1Ch) or ALS mutant SOD1 (G85RSOD1Ch, G93ASOD1Ch) alleles expressed as fusion proteins with a C-terminal monomeric Cherry fluorescent protein (Ch) tag; as a control, a plasmid encoding non-toxic protein Ch was transfected
Since the expression levels of all SOD1 variants were similar (Fig. 1c), differences in survival result from the intrinsic toxicity/conformation that family history of the disease (fALS) mutations convey to SOD1

Summary

Introduction

Loss of protein folding homeostasis has been identified as a common pathological feature of most neurodegenerative diseases (NDs), including Parkinson’s, Alzheimer’s disease, or amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder caused by degeneration of upper and lower motor neurons. Official journal of the Cell Death Differentiation Association. Bugallo et al Cell Death and Disease (2020)11:397. Three independent UPR mechanisms initiate a transcriptional response to restore proteostasis, and remodel translation to (1) reduce the load of proteins to be folded in the ER, and (2) enhance the translation of a select subset of stress-regulated mRNAs4. The translational arm of the UPR is regulated by the ER stress sensor PERK (PKR-like ER kinase) via eIF2 α-subunit (eIF2α) phosphorylation. EIF2α phosphorylation (p-eIF2α) prevents the recycling of the eIF2 complex, thereby repressing translation initiation of most cellular messenger RNAs (mRNAs). The versatile translational/transcriptional program established by PERK is initiated by three other eIF2α kinases, PKR, HRI, and GCN2, that are activated either by viral infections, and heme or amino acid deprivation, respectively. By providing a common output to distinct stresses, this pathway is known as the integrated stress response (ISR)[9,10,11]

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