Glycogen synthase kinase-3a role in ageing and metabolism

Abstract

Prohibitins (PHBs) are a class of conserved mitochondrial proteins that profoundly influence ageing. PHB depletion shortens the lifespan of wild type animals, while it causes a dramatic extension in metabolically compromised daf-2(e1370) mutants. This opposing lifespan phenotype is attributed to alterations in mitochondrial function and metabolism, but the exact function of PHBs is yet to be deciphered. This project was developed to better understand the function of the essential mitochondrial prohibitins in the regulation of ageing. To elucidate novel signalling mechanisms mediating the metabolic adjustments that lead to opposite ageing outcomes in response to PHB depletion, we performed a kinase RNAi screen using prohibitin deletion mutants. First, we characterized prohibitin deletion mutants. As these mutants are sterile, they are maintained balanced heterozygous. We accomplished a sorting protocol for selection of homozygous PHB mutants. We used vital Nile Red (NR) staining as a read-out as PHB depletion reduces NR staining. In order to quantify the intensity of NR staining, we developed an image analysis protocol. From the screen, we identified the conserved Glycogen Synthase Kinase-3 (GSK-3), as a strong suppressor of the reduced NR staining phenotype caused by prohibitin deletion mutants. Beyond its role as a regulator of insulin-dependent glycogen synthesis, GSK-3 also controls critical cellular functions. We investigated how GSK-3 influences longevity in conditions of compromised insulin signalling and mitochondrial impairment. We demonstrate that GSK-3 depletion decreases wild type lifespan but does not affect phb-2 mutants. However, the long lived daf-2 and phb-2;daf-2 mutants show strong suppression in lifespan upon loss of GSK-3. We show that GSK-3 is ubiquitously expressed via CRISPR-Cas9 endogenous gene tagging. We examined several parameters, including alterations in energy stores - glycogen/triglycerides, mitochondrial respiration and lipid composition to deduce how metabolic alterations upon GSK-3 depletion influence lifespan and found that these varied in a genetic background specific manner. Additionally, we also prove that the activity of GSK-3 is essential in the intestine for normal ageing and especially for the long lived daf-2 mutants. Our data thus, delineates a novel role for GSK-3 in metabolism and its interplay with IIS and mitochondrial metabolism in ageing regulation.