Prohibitin-mediated lifespan and mitochondrial stress implicate SGK-1, insulin/IGF and mTORC2 in C. elegans

This is an open-access article distributed under the terms of the Creative Commons Attribution License.Lifespan regulation by mitochondrial proteins has been well described, however, the mechanism of this regulation is not fully understood. Amongst the mitochondrial proteins profoundly affecting ageing are prohibitins (PHB-1 and PHB-2). Paradoxically, in C. elegans prohibitin depletion shortens the lifespan of wild type animals while dramatically extending that of metabolically compromised animals, such as daf-2-insulin-receptor mutants. Here we show that amongst the three kinases known to act downstream of daf-2, only loss of function of sgk-1 recapitulates the ageing phenotype observed in daf-2 mutants upon prohibitin depletion. Interestingly, signalling through SGK-1 receives input from an additional pathway, parallel to DAF-2, for the prohibitin-mediated lifespan phenotype. We investigated the effect of prohibitin depletion on the mitochondrial unfolded protein response (UPRmt). Remarkably, the lifespan extension upon prohibitin elimination, of both daf-2 and sgk-1 mutants, is accompanied by suppression of the UPRmtinduced by lack of prohibitin. On the contrary, gain of function of SGK-1 results in further shortening of lifespan and a further increase of the UPRmtin prohibitin depleted animals. Moreover, SGK-1 interacts with RICT-1 for the regulation of the UPRmtin a parallel pathway to DAF-2. Interestingly, prohibitin depletion in rict-1 loss of function mutant animals also causes lifespan extension. Finally, we reveal an unprecedented role for mTORC2-SGK-1 in the regulation of mitochodrial homeostasis. Together, these results give further insight into the mechanism of lifespan regulation by mitochondrial function and reveal a cross-talk of mitochondria with two key pathways, Insulin/IGF and mTORC2, for the regulation of ageing and stress response.This work was 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. The study was also supported by grants from the Deutsche Forschungsgemeinschaft DFG (SFB746, SFB850) to R.B. and from BIOSS Centre for Biological Signalling Studies to R.B and M.A.S.Peer Reviewe

IP3 signalling regulates exogenous RNAi in Caenorhabditis elegans.

RNA interference (RNAi) is a widespread and widely exploited phenomenon. Here, we show that changing inositol 1,4,5-trisphosphate (IP3) signalling alters RNAi sensitivity in Caenorhabditis elegans. Reducing IP3 signalling enhances sensitivity to RNAi in a broad range of genes and tissues. Conversely up-regulating IP3 signalling decreases sensitivity. Tissue-specific rescue experiments suggest IP3 functions in the intestine. We also exploit IP3 signalling mutants to further enhance the sensitivity of RNAi hypersensitive strains. These results demonstrate that conserved cell signalling pathways can modify RNAi responses, implying that RNAi responses may be influenced by an animal's physiology or environment.We thank A. Fire, K. Ford, S. Mitani and H. Peterkin for the provision of plasmids and strains. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Other strains were provided by the Mitani Lab through the National Bio‐Resource Project of the MEXT, Japan. We are grateful to J. Ahringer, B. Olofsson and members of the Baylis group for helpful discussions. AIN was funded by Trinity Hall College, Cambridge and the Cambridge European Trust. The work of MDS and RPV‐M was partially funded by a Miguel Servet Grant (CP11/00090) from the Health Research Institute Carlos III, which is partially supported by the European Regional Development Fund. RPV‐M is a Marie Curie fellow (CIG322034). RG was funded by the MRC (G0601106).This is the accepted manuscript. The final version is available from Embo Press at http://embor.embopress.org/content/early/2015/01/21/embr.201439585

Understanding and overcoming the resistance of Plutella xylostella to the Cry1Ac Bacillus thuringiensis toxin

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Energy sensing and mitochondria: investigating prohibitin genetic interactions

Resumen del trabajo presentado al 4th Spanish Worm Meeting, celebrado en Carmona (Sevilla) del 14 al 15 de marzo de 2013.Prohibitins are ubiquitous, evolutionarily strongly conserved proteins that localize in mitochondria. Prohibitin depletion shortens life span in wild type worms while, in contrast, in metabolically challenged animals prohibitin depletion promotes life span. We focus on the interactions of prohibitins with the Insulin/IGF-1-like signaling pathway. Mutations in the insulin receptor daf-2 result in strong lifespan extension and probibitin depletion dramatically enhances this phenotype (Artal-Sanz and Tavernarakis, 2009). In order to understand better this opposing effect of prohibitins on lifespan, we looked at genes involved in insulin signalling downstream of daf-2. Interestingly, the only component we identified in the pathway to interact with prohibitins is the sgk-1 gene. Similar to daf-2, depletion of prohibitins in the absence of sgk-1 extends life span. SGK-1 is a serine/threonine protein kinase that is orthologous to the mammalian serumand glucocorticoid-inducible kinases (SGKs) and it acts in parallel to the AKT kinases to mediate DAF-2 signaling for the control of development, stress response, and longevity (Hertweck et al, 2004). Various sources of evidence (Soukas et al, 2009; Jones et al, 2009) have suggested that sgk-1 acts as a receiver of information coming from the Rictor/TORC2 pathway, which is implicated in sensing of the nutrient status of C. elegans, and this information can be further processed to regulate life span. Data on the interaction of prohibitins with SGK-1 in the regulation of lifespan will be discussed.Peer reviewe

Mechanisms of Resistance to Bacillus thuringiensis in the Diamondback Moth

Plutella xylostella, the diamondback moth, has become a model organism for the study of resistance to Bacillus thuringiensis based insecticides, since it is the only insect that has readily acquired resistance to this agent in the field. A number of different mechanisms have been proposed that result in this resistance phenotype, including the loss of a midgut binding site and the reduced activation of protoxin to toxin. The latter mechanism has been proposed on the basis of resistant insects being more susceptible to toxin than protoxin. We investigated whether a reduction in proteolytic activity, and consequently toxin activity, could be the basis of the resistant phenotype. Our results suggest that there was no qualitative difference in the protease activity between gut extract from susceptible and resistant populations. In this paper we review the different resistant mechanism proposed for this insect and critically analyse the hypothesis that the preferential activity of toxin compared to protoxin is due to a defect in activation

Common, but complex, mode of resistance of Plutella xylostella to Bacillus thuringiensis toxins Cry1Ab and Cry1Ac

A field collected population of Plutella xylostella (SERD4) was selected in the laboratory with Bacillus thuringiensis endotoxins Cry1Ac (Cry1Ac-SEL) and Cry1Ab (Cry1Ab-SEL). Both subpopulations showed similar phenotypes: high resistance to the Cry1A toxins and little cross-resistance to Cry1Ca or Cry1D. A previous analysis of the Cry1Ac-SEL showed incompletely dominant resistance to Cry1Ac with more than one factor, at least one of which was sex influenced. In the present study reciprocal mass crosses between Cry1Ab-SEL and a laboratory susceptible population (ROTH) provided evidence that Cry1Ab resistance was also inherited as incompletely dominant trait with more than one factor, and at least one of the factors was sex influenced. Analysis of single pair mating indicated that Cry1Ab-SEL was still heterogeneous for Cry1Ab resistance genes, showing genes with different degrees of dominance. Binding studies showed a large reduction of specific binding of Cry1Ab and Cry1Ac to midgut membrane vesicles of the Cry1Ab-SEL subpopulation. Cry1Ab-SEL was found to be more susceptible to trypsin-activated Cry1Ab toxin than protoxin, although no defect in toxin activation was found. Present and previous results indicate a common basis of resistance to both Cry1Ab and Cry1Ac in selected subpopulations and suggest that a similar set of resistance genes are responsible for resistance to Cry1Ab and Cry1Ac and are selected whichever toxin was used. The possibility of an incompletely dominant trait of resistant to these toxins should be taken into account when considering refuge resistance management strategies

Deciphering the role of mitochondrial prohibitins as lifespan modulators of sgk-1mutants

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

Prohibitin-Mediated Lifespan and Mitochondrial Stress Implicate SGK-1, Insulin/IGF and mTORC2 in <i>C. elegans</i>

<div><p>Lifespan regulation by mitochondrial proteins has been well described, however, the mechanism of this regulation is not fully understood. Amongst the mitochondrial proteins profoundly affecting ageing are prohibitins (PHB-1 and PHB-2). Paradoxically, in <i>C. elegans</i> prohibitin depletion shortens the lifespan of wild type animals while dramatically extending that of metabolically compromised animals, such as <i>daf-2</i>-insulin-receptor mutants. Here we show that amongst the three kinases known to act downstream of <i>daf-2</i>, only loss of function of <i>sgk-1</i> recapitulates the ageing phenotype observed in <i>daf-2</i> mutants upon prohibitin depletion. Interestingly, signalling through SGK-1 receives input from an additional pathway, parallel to DAF-2, for the prohibitin-mediated lifespan phenotype. We investigated the effect of prohibitin depletion on the mitochondrial unfolded protein response (UPR^mt). Remarkably, the lifespan extension upon prohibitin elimination, of both <i>daf-2</i> and <i>sgk-1</i> mutants, is accompanied by suppression of the UPR^mt induced by lack of prohibitin. On the contrary, gain of function of SGK-1 results in further shortening of lifespan and a further increase of the UPR^mt in prohibitin depleted animals. Moreover, SGK-1 interacts with RICT-1 for the regulation of the UPR^mt in a parallel pathway to DAF-2. Interestingly, prohibitin depletion in <i>rict-1</i> loss of function mutant animals also causes lifespan extension. Finally, we reveal an unprecedented role for mTORC2-SGK-1 in the regulation of mitochodrial homeostasis. Together, these results give further insight into the mechanism of lifespan regulation by mitochondrial function and reveal a cross-talk of mitochondria with two key pathways, Insulin/IGF and mTORC2, for the regulation of ageing and stress response.</p></div

<i>rict-1</i> loss of function suppresses the prohibitin depletion-mediated induction of the UPR^mt in a parallel pathway to <i>daf-2</i>.

<p>Fluorescent microscopy of <i>Phsp-6::gfp</i> animals subjected to control RNAi (empty vector pL4440) or <i>phb-1</i> RNAi (right panel) and graphical representation of the quantification of average pixel intensity under the corresponding conditions (left panel). Worms were imaged at the young adult stage. <i>daf-2(e1370)</i> and <i>rict-1(ft7)</i> loss of function at 20°C suppressed the prohibitin induced UPR^mt as recorded by the mitochondrial chaperone reporter, <i>Phsp-6::gfp</i>. <i>daf-2(e1370); rict-1(ft7)</i> caused an additive further suppression of the UPR^mt, suggesting that <i>daf-2</i> and <i>rict-1</i> are acting in parallel pathways to regulate the induction of the UPR^mt upon prohibitin depletion. *** P value <0.0001, * P value <0.01, n.s. not statistically significant difference. Error bars denote SD. P values were calculated by using the student's t-test.</p

<i>sgk-1</i> and <i>phb-1</i> depletion by RNAi increases mitochondrial mass in the intestine and the body wall muscle.

<p>Analysis of mitochondrial content in <i>Pges-1::gfp^mt</i> and <i>Pmyo-3::gfp^mt</i> animals treated with empty vector pL4440 (control RNAi), <i>sgk-1</i> RNAi, or <i>phb-1</i> RNAi. A. Fluorescent microscopy of <i>Pges-1::gfp^mt</i> animals. Worms were imaged at day 1 of adulthood. B. Graphical representation of the quantification of average pixel intensity under the corresponding conditions. Prohibitin and <i>sgk-1</i> depletion at 20°C increased intestinal mitochondrial mass as recorded by the intestinal mitochondrial reporter <i>Pges-1::gfp^mt</i>. *** P value <0.0001, * P value <0.01. Error bars denote SD. P values were calculated by using the student's t-test. C. Graphical representation of the quantification of green fluorescence signal (GFP normalized to time of flight (TOF)) of <i>Pmyo-3::gfp^mt</i> animals using the COPAS worm sorter. TOF and green fluorescence signal were recorded for each individual adult worm and was summarized by mean + SD. Worms were sorted at day 1 of adulthood. *** P value <0.0001, <i>n</i> = 100–200. Error bars denote SD. P values were calculated by using the student's t-test.</p