Autophagy Regulates Ageing in C. elegans

The role of autophagy in ageing regulation has been suggested based on studies in C. elegans, in which knockdown of the expression of bec-1 (ortholog of the yeast and mammalian autophagy genes ATG6/VPS30 and beclin 1, respectively) shortens the lifespan of the daf-2(e1370) mutant C. elegans. However, Beclin1/ATG6 is also known to be involved in other cellular functions in addition to autophagy. In the current study, we knocked down two other autophagy genes, atg-7 and atg-12, in C. elegans using RNAi. We showed that RNAi shortened the lifespan of both wild type and daf-2 mutant C. elegans, providing strong support for a role of autophagy in ageing regulation.     

Lifespan extension in hypomorphic daf-2 mutants of Caenorhabditis elegans is partially mediated by glutathione transferase CeGSTP2-2

Electrophilic stress caused by lipid peroxidation products such as 4-hydroxynonenal (4-HNE) and/or related compounds may contribute to aging. The major mode of 4-HNE metabolism involves glutathione conjugation catalyzed by specialized glutathione transferases. We have previously shown that glutathione transferase CeGSTP2-2, the product of the Caenorhabditis elegans gst-10 gene, has the ability to conjugate 4-HNE, and that its overexpression extends lifespan of C. elegans. We now demonstrate that the expression level of CeGSTP2-2 correlates highly with lifespan in a series of hypomorphic daf-2 mutants of C. elegans. The overexpression of CeGSTP2-2 in daf-2 is abrogated in daf-16; daf-2 mutants, indicating that expression of the gst-10 gene is modulated by insulin-like growth factor signaling. To determine whether the relationship between CeGSTP2-2 and lifespan is causal, we used RNAi to knock down CeGSTP2-2. Treatment with gst-10-specific dsRNA decreased CeGSTP2-2 protein in wild-type N2 and in daf-2 strains to an approximately equal level. The ability to conjugate 4-HNE was similarly decreased by RNAi, suggesting that the increment of that activity in daf-2 over N2 is due largely to the overexpression of CeGSTP2-2. RNAi-mediated knock-down of CeGSTP2-2 led to an increased susceptibility to 4-HNE, paraquat, and heat shock, and to a shortening of lifespan by 13% in both N2 and daf-2 strains. These results indicate that CeGSTP2-2 significantly contributes to the maintenance of the soma, and that this function is augmented in daf-2 mutants concordantly with other longevity assurance genes, probably via insulin-like growth factor signaling.     

The Age-1 and Daf-2 Genes Function in a Common Pathway to Control the Lifespan of Caenorhabditis Elegans

Recessive mutations in two genes, daf-2 and age-1, extend the lifespan of Caenorhabditis elegans significantly. The daf-2 gene also regulates formation of an alternative developmental state called the dauer. Here we asked whether these two genes function in the same or different lifespan pathways. We found that the longevity of both age-1 and daf-2 mutants requires the activities of the same two genes, daf-16 and daf-18. In addition, the daf-2(e1370); age-1(hx546) double mutant did not live significantly longer than the daf-2 single mutant. We also found that, like daf-2 mutations, the age-1(hx546) mutation affects certain aspects of dauer formation. These findings suggest that age-1 and daf-2 mutations do act in the same lifespan pathway and extend lifespan by triggering similar if not identical processes.     

Differential Function of the Two Atg4 Homologues in the Aggrephagy Pathway in Caenorhabditis elegans

The presence of multiple homologues of the same yeast Atg protein endows an additional layer of complexity on the autophagy pathway in higher eukaryotes. The physiological function of the individual genes, however, remains largely unknown. Here we investigated the role of the two Caenorhabditis elegans homologues of the cysteine protease Atg4 in the pathway responsible for degradation of protein aggregates. Loss of atg-4.1 activity causes defective degradation of a variety of protein aggregates, whereas atg-4.2 mutants remove these substrates normally. LGG-1 precursors accumulate in atg-4.1 mutants, but not atg-4.2 mutants. LGG-1 puncta, formation of which depends on lipidation of LGG-1, are present in atg-4.1 and atg-4.2 single mutants, but are completely absent in atg-4.1; atg-4.2 double mutants. In vitro enzymatic analysis revealed that ATG-4.1 processes LGG-1 precursors about 100-fold more efficiently than ATG-4.2. Expression of a mutant form LGG-1, which mimics the processed precursor, rescues the defective autophagic degradation of protein aggregates in atg-4.1 mutants and, to a lesser extent, in atg-4.1; atg-4.2 double mutants. Our study reveals that ATG-4.1 and ATG-4.2 are functionally redundant yet display differential LGG-1 processing and deconjugating activity in the aggrephagy pathway in C. elegans.     

The miR-58 microRNA family is regulated by insulin signaling and contributes to lifespan regulation in Caenorhabditis elegans

microRNAs regulate diverse biological processes such as development and aging by promoting degradation or inhibiting translation of their target mRNAs. In this study, we have found that the miR-58 family microRNAs regulate lifespan in C.elegans. Intriguingly, members of the miR-58 family affect lifespan differently, sometimes in opposite directions, and have complex genetic interactions. The abundances of the miR-58 family miRNAs are up-regulated in the long-lived daf-2 mutant in a daf-16-dependent manner, indicating that these miRNAs are effectors of insulin signaling in C. elegans. We also found that miR-58 is regulated by insulin signaling and partially required for the lifespan extension mediated by reduced insulin signaling,germline ablation, dietary restriction, and mild mitochondrial dysfunction. We further identified the daf-21, ins-1, and isw-1 mRNAs as endogenous targets of miR-58. Our study shows that miRNAs function in multiple lifespan extension mechanisms,and that the seed sequence is not the dominant factor defining the role of a miRNA in lifespan regulation.     

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

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 (UPR^mt). Remarkably, the lifespan extension upon prohibitin elimination, of both daf-2 and sgk-1 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 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.     

C.elegans中dyn-1基因对寿命和发育的影响

细胞极性对于细胞的多样性起着很重要的作用。发动蛋白是一个大的GTP酶,作用于胞吞作用和肌动蛋白的动力学过程。C.elegans中发动蛋白的同源基因dyn-1起着维持早期细胞极性的功能。我们对C.elegans中dyn-1基因进行了克隆,并构建到表达载体和RNAi载体中。经IPTG诱导表达得到了约90 kDa的DYN-1融合蛋白。同时,利用RNAi方法研究了dyn-1基因沉默后对三种线虫虫株N2、daf-2（e1370）和daf-16（e1038）寿命的影响。C.elegans在喂食dyn-1 RNAi食物后寿命明显缩短,也会导致严重的不育和胚胎致死。     

The two C. elegans ATG-16 homologs have partially redundant functions in the basal autophagy pathway

The presence of multiple homologs of the same yeast ATG genes endows an extra layer of complexity on the autophagic machinery in higher eukaryotes. The physiological function of individual homologs in the autophagy pathway remains poorly understood. Here we characterized the function of the two atg16 homologs, atg-16.1 and atg-16.2, in the autophagy pathway in C. elegans. We showed that atg-16.2 mutants exhibit a stronger autophagic defect than atg-16.1 mutants. atg-16.2; atg-16.1 double mutants display a much more severe defect than either single mutant. ATG-16.1 and ATG-16.2 interact with themselves and each other and also directly associate with ATG-5. atg-16.1 mutant embryos exhibit a wild-type expression and distribution pattern of LGG-1/Atg8, while LGG-1 puncta are markedly fewer in number and weaker in intensity in atg-16.2 mutants. In atg-16.2; atg-16.1 double mutants, the lipidated form of LGG-1 accumulates, but LGG-1 puncta are completely absent. ATG-16.2 ectopically expressed on the plasma membrane provides novel sites of LGG-1 puncta formation. We also demonstrated that the C-terminal WD repeats are dispensable for the role of atg-16.2 in aggrephagy (the degradation of protein aggregates by autophagy). Genetic epistasis analysis placed atg-16.2 upstream of atg-2, epg-6, and atg-18. Our study indicated that C. elegans ATG-16s are involved in specifying LGG-1 puncta formation and the two ATG-16 homologs have partially redundant yet distinct functions in the aggrephagy pathway.     

必需氨基酸延缓秀丽隐杆线虫衰老的研究

利用模式生物秀丽隐杆线虫,考察8种人体必需氨基酸对衰老的影响。首先建立秀丽隐杆线虫寿命模型,以雷帕霉素为阳性对照药,分别考察8种必需氨基酸对线虫生存时间的影响。再用筛选出的氨基酸处理线虫21d,通过秀丽隐杆线虫-绿脓杆菌感染模型,考察氨基酸对线虫的抗感染能力的影响,利用实时荧光定量Real-Time RT-PCR方法检测氨基酸处理线虫后DAF-16/FOXO下游基因和免疫相关基因的表达水平。结果表明8种必需氨基酸中苏氨酸和异亮氨酸既能延长野生型线虫的寿命又能延长daf-16突变型线虫的寿命,同时还能增强秀丽隐杆线虫抗绿脓杆菌感染的能力,并提高免疫相关基因lys-7、clec-67的表达水平,而DAF-16/FOXO下游基因表达没有明显变化。因此苏氨酸和异亮氨酸能延长线虫寿命、提高抗感染能力,且对线虫寿命的延长作用不完全依赖于DAF-16/FOXO转录因子。     

Glyceraldehyde-3-phosphate dehydrogenase mediates anoxia response and survival in Caenorhabditis elegans

Oxygen deprivation has a role in the pathology of many human diseases. Thus it is of interest in understanding the genetic and cellular responses to hypoxia or anoxia in oxygen-deprivation-tolerant organisms such as Caenorhabditis elegans. In C. elegans the DAF-2/DAF-16 pathway, an IGF-1/insulin-like signaling pathway, is involved with dauer formation, longevity, and stress resistance. In this report we compared the response of wild-type and daf-2(e1370) animals to anoxia. Unlike wild-type animals, the daf-2(e1370) animals have an enhanced anoxia-survival phenotype in that they survive long-term anoxia and high-temperature anoxia, do not accumulate significant tissue damage in either of these conditions, and are motile after 24 hr of anoxia. RNA interference was used to screen DAF-16-regulated genes that suppress the daf-2(e1370)-enhanced anoxia-survival phenotype. We identified gpd-2 and gpd-3, two nearly identical genes in an operon that encode the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. We found that not only is the daf-2(e1370)-enhanced anoxia phenotype dependent upon gpd-2 and gpd-3, but also the motility of animals exposed to brief periods of anoxia is prematurely arrested in gpd-2/3(RNAi) and daf-2(e1370);gpd-2/3(RNAi) animals. These data suggest that gpd-2 and gpd-3 may serve a protective role in tissue exposed to oxygen deprivation.     

The WD40 repeat PtdIns(3)P-binding protein EPG-6 regulates progression of omegasomes to autophagosomes

PtdIns(3)P plays critical roles in the autophagy pathway. However, little is known about how PtdIns(3)P effectors act with autophagy proteins in autophagosome formation. Here we identified an essential autophagy gene in C.?elegans, epg-6, which encodes a WD40 repeat-containing protein with PtdIns(3)P-binding activity. EPG-6 directly interacts with ATG-2. epg-6 and atg-2 regulate progression of omegasomes to autophagosomes, and their loss of function?causes accumulation of enlarged early autophagic structures. Another WD40 repeat PtdIns(3)P effector, ATG-18, plays a distinct role in autophagosome formation. We also established the hierarchical relationship of autophagy genes in degradation of?protein aggregates and revealed that the UNC-51/Atg1 complex, EPG-8/Atg14, and binding of lipidated LGG-1 to protein aggregates are required for?omegasome formation. Our study demonstrates that autophagic PtdIns(3)P effectors play distinct roles in autophagosome formation and also provides?a framework for understanding the concerted action of autophagy genes in protein aggregate degradation.     

The nuclear hormone receptor DAF-12 has opposing effects on Caenorhabditis elegans lifespan and regulates genes repressed in multiple long-lived worms

The orphan nuclear hormone receptor gene daf-12 in Caenorhabditis elegans plays a key role in the regulation of development and determination of adult longevity. To understand the effects of daf-12 on aging we characterized the lifespan of loss-of-function and gain-of-function daf-12 alleles that have been identified on the basis of their effects on dauer development. We find that these mutations have opposing effects on longevity and resistance to oxidative and thermal stress which makes daf-12 the first gene with alleles that can extend or shorten lifespan. We find that the shortened lifespan of the loss-of-function mutation is due to accelerated aging in young adulthood rather than an adverse effect of the mutation on development. Microarray analysis of worms carrying the two alleles revealed a relatively small number of genes differentially expressed between the two genotypes. Comparison of the expression profiles with the profiles associated with dauer formation and long-lived daf-2 mutants revealed that while the profiles are largely different, there is significant overlap among the genes down-regulated, but not up-regulated, in all profiles. Several of these genes down-regulated in multiple long-lived worms have known effects on lifespan, and many of the genes belong to a family of poorly characterized genes that are strongly down-regulated in dauers, daf-2 mutants, and long-lived daf-12 mutants. Our results point to daf-12 modulating aging and stress responses in part through the repression of specific genes, and emphasize the role that the repression of genes that curtail maximal lifespan plays in lifespan determination.     

Autophagy is required for dietary restriction-mediated life span extension in C. elegans

Dietary restriction extends life span in diverse species including Caenorhabditis elegans. However, the downstream cellular targets regulated by dietary restriction are largely unknown. Autophagy, an evolutionary conserved lysosomal degradation pathway, is induced under starvation conditions and regulates life span in insulin signaling C. elegans mutants. We now report that two essential autophagy genes (bec-1 and Ce-atg7) are required for the longevity phenotype of the C. elegans dietary restriction mutant (eat-2(ad1113) animals. Thus, we propose that autophagy mediates the effect, not only of insulin signaling, but also of dietary restriction on the regulation of C. elegans life span. Since autophagy and longevity control are highly conserved from C. elegans to mammals, a similar role for autophagy in dietary restriction-mediated life span extension may also exist in mammals.