Glycogen Synthase Kinase-3: a Role in Ageing and Metabolism

Programa de Doctorado en Biotecnología, Ingeniería y Tecnología QuímicaLínea de Investigación: Modelos Animales en Biotecnología y BiomedicinaClave Programa: DBICódigo Línea: 22Prohibitins (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.Universidad Pablo de Olavide de Sevilla. Departamento de Biología Molecular e Ingeniería BioquímicaPostprin

Loss of MTCH-1 suppresses age-related proteostasis collapse through the inhibition of programmed cell death factors

The age-related loss of protein homeostasis (proteostasis) is at the heart of numerous neurodegenerative diseases. Therefore, finding ways to preserve proteome integrity in aged cells may be a powerful way to promote long-term health. Here, we show that reducing the activity of a highly conserved mitochondrial outer membrane protein, MTCH-1/MTCH2, suppresses age-related proteostasis collapse in Caenorhabditis elegans without disrupting development, growth, or reproduction. Loss of MTCH-1 does not influence proteostasis capacity in aged tissues through previously described pathways but instead operates by reducing CED-4 levels. This results in the sequestration of HSP-90 by inactive CED-3, which in turn leads to an increase in HSF-1 activity, transcriptional remodeling of the proteostasis network, and maintenance of proteostasis capacity with age. Together, our findings reveal a role for programmed cell death factors in determining proteome health and suggest that inhibiting MTCH-1 activity in adulthood may safeguard the aging proteome and suppress age-related diseases

Combined flow cytometry and high-throughput image analysis for the study of essential genes in Caenorhabditis elegans

Background: Advances in automated image-based microscopy platforms coupled with high-throughput liquid workflows have facilitated the design of large-scale screens utilising multicellular model organisms such as Caenorhabditis elegans to identify genetic interactions, therapeutic drugs or disease modifiers. However, the analysis of essential genes has lagged behind because lethal or sterile mutations pose a bottleneck for high-throughput approaches, and a systematic way to analyse genetic interactions of essential genes in multicellular organisms has been lacking. Results: In C. elegans, non-conditional lethal mutations can be maintained in heterozygosity using chromosome balancers, commonly expressing green fluorescent protein (GFP) in the pharynx. However, gene expression or function is typically monitored by the use of fluorescent reporters marked with the same fluorophore, presenting a challenge to sort worm populations of interest, particularly at early larval stages. Here, we develop a sorting strategy capable of selecting homozygous mutants carrying a GFP stress reporter from GFP-balanced animals at the second larval stage. Because sorting is not completely error-free, we develop an automated high-throughput image analysis protocol that identifies and discards animals carrying the chromosome balancer. We demonstrate the experimental usefulness of combining sorting of homozygous lethal mutants and automated image analysis in a functional genomic RNA interference (RNAi) screen for genes that genetically interact with mitochondrial prohibitin (PHB). Lack of PHB results in embryonic lethality, while homozygous PHB deletion mutants develop into sterile adults due to maternal contribution and strongly induce the mitochondrial unfolded protein response (UPR mt ). In a chromosome-wide RNAi screen for C. elegans genes having human orthologues, we uncover both known and new PHB genetic interactors affecting the UPR mt and growth. Conclusions: The method presented here 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.European Research Council ERC-2011-StG-281691Ministerio de Economía y Competitividad BFU2012–3550

The importance of long-lived proteins: Not just nuclear anymore

The significance of mitochondrial long-lived proteins (mitoLLPs) to tissue health has remained mysterious for over a decade. In this issue of Developmental Cell, Krishna et al. demonstrate that mitochondrial lifetimes are highly heterogeneous and that mitoLLPs promote respiratory capacity by facilitating supercomplex assembly within the electron transport chain

GSK-3 intestinal activity impacts mitochondrial function and ageing

Resumen del trabajo presentado en EMBO Workshop C. elegans development, cell biology and gene expression and European Worm Meeting, celebrado en Barcelona (España) del 13 al 17 de junio de 2018.Impaired mitochondrial function is a hallmark of ageing and age-related pathologies. The mitochondrial prohibitin complex, composed of PHB-1 and PHB-2, has emerged as a context-dependent modulator of lifespan. While PHB-1 or PHB-2 depletion shortens the lifespan of wild type worms, it dramatically extends lifespan under compromised metabolic conditions, as is the case of daf-2(e1370) mutants. To better understand the function of PHBs in ageing regulation, we performed a phenotype-based RNAi screening for prohibitin regulatory kinases. We identified the Glycogen Synthase Kinase -3 (GSK-3) as a suppressor of the reduced Nile Red phenotype in both phb-2(tm2998) and phb-2(tm2998);daf-2(e1370) mutants. GSK-3 is the worm orthologue of human GSK-3ß, a pleiotropic kinase involved in multiple cellular processes and linked to different diseases such as Alzheimer's, diabetes, cancer or neurodegeneration. Interestingly, GSK-3 depletion decreases the lifespan of wild-type worms and strongly suppresses the long-lived phenotypes of daf-2 and daf-2;phb-2, while it mildly affects phb-2 mutants. We used CRISPR/Cas9 to generate a gsk-3 endogenous gene tagging. GSK-3 is ubiquitously expressed and shows different cellular localization patterns depending on the tissue and prohibitin expression. We combine transcriptomic analysis and metabolic assays to demonstrate a role for GSK-3 as a global metabolic regulator. GSK-3 alters glycogen and fat stores and impacts mitochondrial lipid composition and respiration in a genotype-dependent manner. Moreover, we identify a gsk-3-dependent transcriptional blueprint related to ageing where intestinal specific genes are overrepresented. By SapTrap toolkit, we generated different gsk-3 tissue-specific endogenous knockouts and demonstrate that GSK-3 impact on ageing specifically relies on its intestinal activity

Combined flow cytometry and high-throughput image analysis for the study of essential genes in Caenorhabditis elegans

© Artal-Sanz et al. 2018[Background]: Advances in automated image-based microscopy platforms coupled with high-throughput liquid workflows have facilitated the design of large-scale screens utilising multicellular model organisms such as Caenorhabditis elegans to identify genetic interactions, therapeutic drugs or disease modifiers. However, the analysis of essential genes has lagged behind because lethal or sterile mutations pose a bottleneck for high-throughput approaches, and a systematic way to analyse genetic interactions of essential genes in multicellular organisms has been lacking.[Results]: In C. elegans, non-conditional lethal mutations can be maintained in heterozygosity using chromosome balancers, commonly expressing green fluorescent protein (GFP) in the pharynx. However, gene expression or function is typically monitored by the use of fluorescent reporters marked with the same fluorophore, presenting a challenge to sort worm populations of interest, particularly at early larval stages. Here, we develop a sorting strategy capable of selecting homozygous mutants carrying a GFP stress reporter from GFP-balanced animals at the second larval stage. Because sorting is not completely error-free, we develop an automated high-throughput image analysis protocol that identifies and discards animals carrying the chromosome balancer. We demonstrate the experimental usefulness of combining sorting of homozygous lethal mutants and automated image analysis in a functional genomic RNA interference (RNAi) screen for genes that genetically interact with mitochondrial prohibitin (PHB). Lack of PHB results in embryonic lethality, while homozygous PHB deletion mutants develop into sterile adults due to maternal contribution and strongly induce the mitochondrial unfolded protein response (UPRmt). In a chromosome-wide RNAi screen for C. elegans genes having human orthologues, we uncover both known and new PHB genetic interactors affecting the UPRmt and growth.[Conclusions]: The method presented here 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.This work was mainly funded by grants from the European Research Council (ERC-2011-StG-281691) and the Spanish Ministerio de Economía y Competitividad (BFU2012–35509) to M.A.S and a Marie-Curie Intra-European Fellowship (FP7-PEOPLE-2013-IEF/GA Nr: 627263) to M.A.S. and M.O. The study was also supported by the Deutsche Forschungsgemeinschaft (DFG) (SFB746, SFB850) to R.B. and from the BIOSS Centre for Biological Signalling Studies to R.B and M.A.S