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Genome-Wide CRISPR/Cas9 Screen shows that inhibiting GET4 is neuroprotective and increases mitochondrial-endoplasmic reticulum contact sites

ABSTRACT: Organelles form contacts between each other allowing for transfer of molecules and signals. Mitochondria-ER Contact sites (MERCS) are lengths of the Mitochondria and ER membrane which come together but do not fuse. They have been implicated in many diseases, including neurodegenerative, metabolic, and cardiac disease. MERCS have been highly researched, however, a lot is still left to explore. To uncover novel regulators of MERCS we conducted a genome-wide, flow cytometry-based screen using an engineered MERCs reporter cell line. We found 410 genes whose downregulation promotes MERCS and 230 genes whose downregulation decreases MERCS. From these, 29 genes were selected from each population for arrayed screening. 14/29 validated from the higher population and 7/29 from the low population. GET4 and BAG6 were highlighted as the top 2 genes that upon suppression increase MERCS from both the pooled and arrayed screen, and were subjected to further investigation. Loss of GET4 or BAG6 increased mVenus puncta, Proximity Ligation Assay (PLA) puncta and increased the percentage of ER in contact with mitochondria. In addition, GET4 and BAG6 were observed to interact with known MERCS proteins, inositol 1,4,5-trisphosphate receptors (IP3R) and Glucose-regulated protein 75 (GRP75). In addition, we found that loss of GET4 increased Calcium concentration [Ca2+] in the mitochondria and mitochondrial respiration. Finally, we show that loss of GET4 rescues motor ability, neurodegeneration and improves lifespan in a Drosophila model of Alzheimer's Disease (Aβ42Arc). Together, these results suggest that GET4 is involved in decreasing MERCS and that its loss is neuroprotective.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Robin Antrobus

LAB HEAD: Emmanouil Metzakopian

PROVIDER: PXD042710 | Pride | 2024-05-24

REPOSITORIES: Pride

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Genome-wide CRISPR/Cas9 screen shows that loss of GET4 increases mitochondria-endoplasmic reticulum contact sites and is neuroprotective.

Wilson Emma L EL Yu Yizhou Y Leal Nuno S NS Woodward James A JA Patikas Nikolaos N Morris Jordan L JL Field Sarah F SF Plumbly William W Paupe Vincent V Chowdhury Suvagata R SR Antrobus Robin R Lindop Georgina E GE Adia Yusuf M YM Loh Samantha H Y SHY Prudent Julien J Martins L Miguel LM Metzakopian Emmanouil E

Cell death & disease 20240311 3

Organelles form membrane contact sites between each other, allowing for the transfer of molecules and signals. Mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) are cellular subdomains characterized by close apposition of mitochondria and ER membranes. They have been implicated in many diseases, including neurodegenerative, metabolic, and cardiac diseases. Although MERCS have been extensively studied, much remains to be explored. To uncover novel regulators of MERCS, we conducted a g ...[more]

PMID: 38467609

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Project description:SEPN1 is a type II protein of the endoplasmic reticulum (ER) whose loss of function gives rise to a collection of debilitating autosomal recessive myopathies gathered under the umbrella term of SEPN1-related myopathy (RM). At the moment, SEPN1-RM lacks an effective pharmacological treatment; thus, the medical management of the disease is only supportive. The potentially fatal diaphragmatic weakness leading to respiratory insufficiency in patients still ambulant is the main reason for medical concerns. Thus, studies on SEPN1-RM pathogenesis are necessary to implement a targeted pharmacological therapy aimed at relieving the general muscle weakness and the more worrisome diaphragmatic dysfunction. Here, we show a physical and functional interaction between SEPN1 and the ER stress mediator ERO1 alpha (henceforth, ERO1). Both SEPN1 and ERO1 are involved in the redox regulation of proteins into the ER, although in an opposite way SEPN1 imposes a less oxidant ER poise while ERO1 a more oxidant one, conceivable with a reductase function of SEPN1 and an oxidase one of ERO1. Furthermore, both are mainly localized in a region of the ER in close contact with mitochondria termed mitochondria-associated membranes (MAMs) and their loss impacts oppositely on the short-range MAMs, thereby impinging ER-mitochondria Ca2+ dynamics, OXPHOS, and bioenergetics. We find that ERO1 depletion restores the impaired short-range MAMs due to SEPN1 loss together with mitochondrial ATP. ERO1 knockout in a mouse background of SEPN1 loss blunts ER stress and the consequent Unfolded Protein Response (UPR) while it rescues the diaphragmatic weakness by improving ER/mitochondria contacts, calcium dynamics, and OXPHOS. Importantly, treatments of SEPN1 knock out mice with the chemical chaperone tauroursodeoxycholic acid (TUDCA) mimic the results of ERO1 loss improving calcium dynamics, OXPHOS, ER/mitochondria contacts, thereby rescuing diaphragmatic weakness as well. In addition, TUDCA-treated SEPN1-RM patients-derived myoblasts show a dynamic dose-dependent increase in ATP levels under ER stress conditions suggesting improved mitochondrial function. Thus, our findings point to the ERO1 axis in the pathogenesis of SEPN1-RM thereby impinging on MAMs and mitochondria bioenergetics and recall for the efficacy of a pharmacology therapy with ad hoc ER stress/ERO1 inhibitors for SEPN1-RM.

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