In mammalian cells, mitochondrial dysfunction triggers the integrated stress response (ISR), in which eIF2α phosphorylation induces the transcription factor ATF4 1 - 3 . However, how mitochondrial stress is relayed to ATF4 is unknown. We found that HRI is the eIF2α kinase necessary and sufficient for this relay. In a genome-wide CRISPRi screen, we identified factors upstream of HRI: OMA1, a mitochondrial stress-activated protease, and DELE1, a little-characterized protein we found to be associated with the inner mitochondrial membrane. Mitochondrial stress stimulates OMA1-dependent cleavage of DELE1, leading to its accumulation in the cytosol, where it interacts with HRI and activates its eIF2α kinase activity. Additionally, DELE1 is required for ATF4 translation downstream of eIF2α phosphorylation. Blockade of the OMA1-DELE1-HRI pathway triggers an alternative response inducing specific molecular chaperones. Therefore, this pathway is a potential therapeutic target enabling fine-tuning of the ISR for beneficial outcomes in diseases involving mitochondrial dysfunction.
Microglia are emerging as key drivers of neurological diseases. However, we lack a systematic understanding of the underlying mechanisms. Here, we present a screening platform to systematically elucidate functional consequences of genetic perturbations in human induced pluripotent stem cell-derived microglia. We developed an efficient 8-day protocol for the generation of microglia-like cells based on the inducible expression of six transcription factors. We established inducible CRISPR interference and activation in this system and conducted three screens targeting the ‘druggable genome’. These screens uncovered genes controlling microglia survival, activation and phagocytosis, including neurodegeneration-associated genes. A screen with single-cell RNA sequencing as the readout revealed that these microglia adopt a spectrum of states mirroring those observed in human brains and identified regulators of these states. A disease-associated state characterized by osteopontin (SPP1) expression was selectively depleted by colony-stimulating factor-1 (CSF1R) inhibition. Thus, our platform can systematically uncover regulators of microglial states, enabling their functional characterization and therapeutic targeting.
In mammalian cells, mitochondrial dysfunction triggers the integrated stress response (ISR), in which eIF2α phosphorylation upregulates the transcription factor ATF4. However, how mitochondrial stress is relayed to the ISR is unknown. We found that HRI is the eIF2α kinase necessary and sufficient for this relay. Using an unbiased CRISPRi screen, we identified factors upstream of HRI: OMA1, a mitochondrial stress-activated protease, and DELE1, a littlecharacterized protein we found to be associated with the inner mitochondrial membrane. Mitochondrial stress stimulates the OMA1-dependent cleavage of DELE1, leading to its accumulation in the cytosol, where it interacts with HRI and activates its eIF2α kinase activity.
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