Mitochondrial Triggers of the Integrated Stress Response: Disentangling the Bioenergetic Rubik’s Cube

Abstract

Mitochondria are hubs of metabolism and signaling in eukaryotic cells whose dysfunction underlies a class of devastating genetic disorders and is also frequently associated with common conditions, such as neurodegeneration, diabetes, cancer and the ageing process. Mitochondrial dysfunction, particularly breakdown of the electron transport chain (ETC) and oxidative phosphorylation, yields a perplexingly variable spectrum of consequences at the cellular, tissue and whole-organism level. Deciphering the context-dependent pathophysiology of mitochondrial dysfunction is thus a major challenge in both basic and translational biomedical research. Studies over the past decade have revealed that a prominent molecular signature of mitochondrial dysfunction in vivo is activation of the integrated stress response (ISR), a gene expression program eukaryotic cells engage upon different types of insults. How mitochondrial dysfunction is sensed to trigger the ISR and whether the response serves a protective role or contributes to pathology remain far from understood. The work in this thesis sought to delineate functional parameters tied to the ETC, such as ATP synthesis or NADH oxidation, that can lead to ISR activation. We used chemical and genetic tools to perturb ETC functions in mouse muscle cells while specifically compensating for some of the resulting metabolic effects. We then monitored the impact of these interventions on ISR-dependent gene expression by RNA sequencing. Our results revealed that in proliferating cells, the increase in the cytosolic [NADH]/[NAD+] ratio during complex I dysfunction potently triggered the ISR, mostly by sharply depressing aspartate levels and activating the amino acid sensitive eIF2α kinase GCN2. Strikingly, this route to ISR activation became inoperative in terminally-differentiated myotubes where only ATP synthase inhibition elicited a significant response. The path to ISR activation in the latter case was dependent on residual ETC activity and could be abolished by co-inhibition of complex I, mild uncoupling or mild hypoxic preconditioning. Finally, our data suggests dysfunction of mitochondrial genome expression is not directly sensed to trigger the ISR. These results shed light on the complicated interplay between mitochondrial dysfunction and the ISR. They implicate diverse metabolic and bioenergetic routes to its activation whose relevance in vivo should be carefully evaluated in future work.