ASM-3 Acid Sphingomyelinase Functions as a Positive Regulator of the DAF-2/AGE-1 Signaling Pathway and Serves as a Novel Anti-Aging Target

In C. elegans, the highly conserved DAF-2/insulin/insulin-like growth factor 1 receptor signaling (IIS) pathway regulates longevity, metabolism, reproduction and development. In mammals, acid sphingomyelinase (ASM) is an enzyme that hydrolyzes sphingomyelin to produce ceramide. ASM has been implicated in CD95 death receptor signaling under certain stress conditions. However, the involvement of ASM in growth factor receptor signaling under physiological conditions is not known. Here, we report that in vivo ASM functions as a positive regulator of the DAF-2/IIS pathway in C. elegans. We have shown that inactivation of asm-3 extends animal lifespan and promotes dauer arrest, an alternative developmental process. A significant cooperative effect on lifespan is observed between asm-3 deficiency and loss-of-function alleles of the age-1/PI 3-kinase, with the asm-3; age-1 double mutant animals having a mean lifespan 259% greater than that of the wild-type animals. The lifespan extension phenotypes caused by the loss of asm-3 are dependent on the functions of daf-16/FOXO and daf-18/PTEN. We have demonstrated that inactivation of asm-3 causes nuclear translocation of DAF-16::GFP protein, up-regulates endogenous DAF-16 protein levels and activates the downstream targeting genes of DAF-16. Together, our findings reveal a novel role of asm-3 in regulation of lifespan and diapause by modulating IIS pathway. Importantly, we have found that two drugs known to inhibit mammalian ASM activities, desipramine and clomipramine, markedly extend the lifespan of wild-type animals, in a manner similar to that achieved by genetic inactivation of the asm genes. Our studies illustrate a novel strategy of anti-aging by targeting ASM, which may potentially be extended to mammals.     

Daf-16 mediated repression of cytosolic ribosomal protein genes facilitates a hypoxia sensitive to hypoxia resistant transformation in long-lived germline mutants

In C. elegans, germline ablation leads to long life span and stress resistance. It has been reported that mutations that block oogenesis or an upstream step in germline development confer strong resistance to hypoxia. We demonstrate here that the hypoxia resistance of sterile mutants is dependent on developmental stage and age. In just a 12-hour period, sterile animals transform from hypoxia sensitive L4 larvae into hypoxia resistant adults. Since this transformation occurs in animals with no germline, the physiological programs that determine hypoxia sensitivity in germline mutants occur independently of germline signals and instead rely on signals from somatic tissues. Furthermore, we found two distinct mechanisms of hypoxia resistance in germline deficient animals. First, a DAF-16/FoxO independent mechanism that occurs in all hypoxia resistant sterile adults and, second, a DAF-16/FoxO dependent mechanism that confers an added layer of resistance, or “super-resistance”, to animals with no germline as they age past day 1 of adulthood. RNAseq data showed that genes involved in both cytosolic and mitochondrial protein translation are repressed in sterile adults and further repressed only in germline deficient mutants as they age. Importantly, mutation of daf-16 specifically blocked the repression of cytosolic ribosomal protein genes, but not mitochondrial ribosomal protein genes, implicating DAF-16/FoxO mediated repression of cytosolic ribosomal protein genes as a mechanism of hypoxia super-resistance. Consistent with this hypothesis, the hypoxia super-resistance of aging germline deficient adults was also suppressed by dual mutation of ncl-1 and larp-1, two regulators of protein translation and ribosomal protein abundance. These studies provide novel insight into a profound physiological transformation that takes place in germline mutants during development, showing that some of the unique physiological properties of these long-lived animals are derived from developmentally dependent DAF-16/FoxO mediated repression of genes involved in cytosolic protein translation.     

Role of CBP and SATB-1 in Aging,Dietary Restriction,and Insulin-Like Signaling

How dietary restriction (DR) increases lifespan and decreases disease burden are questions of major interest in biomedical research. Here we report that hypothalamic expression of CREB-binding protein (CBP) and CBP-binding partner Special AT-rich sequence binding protein 1 (SATB-1) is highly correlated with lifespan across five strains of mice, and expression of these genes decreases with age and diabetes in mice. Furthermore, in Caenorhabditis elegans, cbp-1 is induced by bacterial dilution DR (bDR) and the daf-2 mutation, and cbp-1 RNAi specifically in adults completely blocks lifespan extension by three distinct protocols of DR, partially blocks lifespan extension by the daf-2 mutation but not of cold, and blocks delay of other age-related pathologies by bDR. Inhibiting the C. elegans ortholog of SATB-1 and CBP-binding partners daf-16 and hsf-1 also attenuates lifespan extension by bDR, but not other protocols of DR. In a transgenic Aβ42 model of Alzheimer''s disease, cbp-1 RNAi prevents protective effects of bDR and accelerates Aβ42-related pathology. Furthermore, consistent with the function of CBP as a histone acetyltransferase, drugs that enhance histone acetylation increase lifespan and reduce Aβ42-related pathology, protective effects completely blocked by cbp-1 RNAi. Other factors implicated in lifespan extension are also CBP-binding partners, suggesting that CBP constitutes a common factor in the modulation of lifespan and disease burden by DR and the insulin/IGF1 signaling pathway.     

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.     

A novel endoplasmic reticulum adaptation is critical for the long-lived Caenorhabditis elegans rpn-10 proteasomal mutant

The loss of proteostasis due to reduced efficiency of protein degradation pathways plays a key role in multiple age-related diseases and is a hallmark of the aging process. Paradoxically, we have previously reported that the Caenorhabditis elegans rpn-10(ok1865) mutant, which lacks the RPN-10/RPN10/PSMD4 subunit of the 19S regulatory particle of the 26S proteasome, exhibits enhanced cytosolic proteostasis, elevated stress resistance and extended lifespan, despite possessing reduced proteasome function. However, the response of this mutant against threats to endoplasmic reticulum (ER) homeostasis and proteostasis was unknown. Here, we find that the rpn-10 mutant is highly ER stress resistant compared to the wildtype. Under unstressed conditions, the ER unfolded protein response (UPR) is activated in the rpn-10 mutant as signified by increased xbp-1 splicing. This primed response appears to alter ER homeostasis through the upregulated expression of genes involved in ER protein quality control (ERQC), including those in the ER-associated protein degradation (ERAD) pathway. Pertinently, we find that ERQC is critical for the rpn-10 mutant longevity. These changes also alter ER proteostasis, as studied using the C. elegans alpha-1 antitrypsin (AAT) deficiency model, which comprises an intestinal ER-localised transgenic reporter of an aggregation-prone form of AAT called ATZ. The rpn-10 mutant shows a significant reduction in the accumulation of the ATZ reporter, thus indicating that its ER proteostasis is augmented. Via a genetic screen for suppressors of decreased ATZ aggregation in the rpn-10 mutant, we then identified ecps-2/H04D03.3, a novel ortholog of the proteasome-associated adaptor and scaffold protein ECM29/ECPAS. We further show that ecps-2 is required for improved ER proteostasis as well as lifespan extension of the rpn-10 mutant. Thus, we propose that ECPS-2-proteasome functional interactions, alongside additional putative molecular processes, contribute to a novel ERQC adaptation which underlies the superior proteostasis and longevity of the rpn-10 mutant.     

The Receptor-Bound Guanylyl Cyclase DAF-11 Is the Mediator of Hydrogen Peroxide-Induced Cgmp Increase in Caenorhabditis elegans

Adenosine 3′, 5′-cyclic monophosphate (cAMP) and guanosine 3′, 5′-cyclic monophosphate (cGMP) are well-studied second messengers that transmit extracellular signals into mammalian cells, with conserved functions in various other species such as Caenorhabditis elegans (C. elegans). cAMP is generated by adenylyl cyclases, and cGMP is generated by guanylyl cyclases, respectively. Studies using C. elegans have revealed additional roles for cGMP signaling in lifespan extension. For example, mutants lacking the function of a specific receptor-bound guanylyl cyclase, DAF-11, have an increased life expectancy. While the daf-11 phenotype has been attributed to reductions in intracellular cGMP concentrations, the actual content of cyclic nucleotides has not been biochemically determined in this system. Similar assumptions were made in studies using phosphodiesterase loss-of-function mutants or using adenylyl cyclase overexpressing mutants. In the present study, cyclic nucleotide regulation in C. elegans was studied by establishing a special nematode protocol for the simultaneous detection and quantitation of cyclic nucleotides. We also examined the influence of reactive oxygen species (ROS) on cyclic nucleotide metabolism and lifespan in C. elegans using highly specific HPLC-coupled tandem mass-spectrometry and behavioral assays. Here, we show that the relation between cGMP and survival is more complex than previously appreciated.     

A steroid hormone that extends the lifespan of Caenorhabditis elegans

Removing the germline of Caenorhabditis elegans extends lifespan. This lifespan extension requires the nuclear receptor DAF-12 and the cytochrome P450 DAF-9, suggesting that a lipophilic hormone is involved. Here we show that C. elegans contains several hormonal steroids that are also present in humans, including pregnenolone (3beta-hydroxy-pregn-5-en-20-one; PREG) and other pregnane and androstane derivatives. We find that PREG can extend the lifespan of C. elegans. Moreover, PREG levels rise when the germline is removed in a daf-9-dependent fashion. PREG extends the lifespan of germline-defective daf-9 mutants dramatically, but has no effect on daf-12 mutants. Thus, germline removal may extend lifespan, at least in part, by stimulating the synthesis of PREG.