The mitochondrial stress response and its impact on prohibitin-mediated aging phenotypes

Zusammenfassung

Aging is a natural progression that affects all living organisms from birth, through maturity, to old age. It is characterized by a decline of the physiological integrity and it is accompanied by a higher incidence of age-related diseases, which represent nowadays the most common reason of death. Understanding the aging process has always been of interest. Initially it was thought to be the result of stochastic degradation but, nowadays, it is widely acknowledged that aging is controlled, at least in part, by genetic pathways and biochemical mechanisms. In this work, we focus in mitochondrial dysfunction as one of the most important processes regulating aging. Proper mitochondrial activity is preserved through regulation of mitochondrial dynamics and mitophagy and through proper maintenance of mitochondrial homeostasis. Under proteotoxic conditions the mitochondrial unfolded protein response, UPRmt, is activated to maintain mitochondrial proteostasis by inducing the expression of mitochondrial chaperones and proteases that control protein folding, assembly and degradation. The mitochondrial prohibitin, PHB, complex is a ring-like structure sitting in the inner mitochondrial membrane. It is composed of two subunits, PHB-1 and PHB-2, that are highly evolutionary conserved. PHBs have been related with many different functions: maintenance of nucleoids organization and stability, protection of newly synthesized mitochondrial proteins, assistance in folding and formation of the respiration super-complexes or acting as scaffold protein. Lack of PHB results in embryonic lethality in Caenorhabditis elegans, while homozygous PHB deletion mutants develop into sterile adults due to maternal contribution. In addition, deletion of PHB induces a very strong mitochondrial stress response. In this work, we show that the induction of UPRmt upon PHB deletion does not require the canonical components of the stress response, suggesting an alternative signalling mechanism. We present a sorting strategy capable of selecting homozygous mutants carrying the UPRmt-GFP stress reporter from GFP-balanced animals at the second larval stage. Because sorting is not completely error-free, we developed an automated high-throughput image analysis protocol that identifies and discards animals carrying the chromosome balancer. This method allows the study of balanced lethal mutations in a high-throughput manner. It can be easily adapted depending on the user’s requirements and should serve as a useful resource for the C. elegans community for probing new biological aspects of essential nematode genes as well as the generation of more comprehensive genetic networks. In a chromosome-wide RNAi screen for C. elegans genes having human orthologues, we uncovered both known and new PHB genetic interactors affecting the UPRmt and growth. Interestingly, depletion of PHB shows an opposite effect on aging: it shortens lifespan in wild-type worms while it dramatically extends the longevity of the already long-lived insulin/IGF-1 signaling (IIS) pathway mutants. Moreover, the strong mitochondrial stress response elicited upon PHB depletion is remarkably reduced in IIS mutants. We aim at identifying new pathways involved in the regulation of the PHB-mediated mitochondrial stress response, as well as mechanisms responsible for the opposite longevity outcomes of PHB depletion. Towards this aim, we carried out genome-wide RNAi screens in PHB-depleted wild type animals and PHB-depleted IIS mutants. By performing GO term enrichment analysis, we identify inhibition of protein degradation, disruption of mitochondrial integrity and impairment of ATP synthesis as processes increasing the mitochondrial stress response. Moreover, we report a boost in the mitochondrial stress response as a mechanism to cope with the inhibition of other stress responses, such as nutrient sensing or defense response. Besides, inhibition of processes that are very energy consuming, such as protein biogenesis or ATP hydrolysis, reduced the mitochondrial stress response. In particular, we describe two new regulators of the mitochondrial stress response, two transcription factors, C16A3.4 and TLF-1. Furthermore, a new role for chromatin organization emerge in the regulation of the UPRmt and lifespan in an insulin dependent manner. We identify USP-48, a deubiquitinase, as an important factor for the enhanced lifespan of daf-2 mutants. Additionally, we establish a histone H2A, HIS-65, as a modulator of the mitochondrial stress response and aging in PHB-depleted daf-2 mutants. In this work thus, we pinpoint new players involved in the regulation of the mitochondrial stress response. These results will unveil the molecular pathways regulating mitochondrial quality control mechanisms and shed some light on the processes contributing to the differential effect in aging of PHB depletion in wild type and metabolically compromised animals.