Improved resilience and proteostasis mediate longevity upon DAF-2 degradation in old age.

Little is known about the possibility of reversing age-related biological changes when they have already occurred. To explore this, we have characterized the effects of reducing insulin/IGF-1 signaling (IIS) during old age. Reduction of IIS throughout life slows age-related decline in diverse species, most strikingly in the nematode Caenorhabditis elegans. Here we show that even at advanced ages, auxin-induced degradation of DAF-2 in single tissues, including neurons and the intestine, is still able to markedly increase C. elegans lifespan. We describe how reversibility varies among senescent changes. While senescent pathologies that develop in mid-life were not reversed, there was a rejuvenation of the proteostasis network, manifesting as a restoration of the capacity to eliminate otherwise intractable protein aggregates that accumulate with age. Moreover, resistance to several stressors was restored. These results support several new conclusions. (1) Loss of resilience is not solely a consequence of pathologies that develop in earlier life. (2) Restoration of proteostasis and resilience by inhibiting IIS is a plausible cause of the increase in lifespan. And (3), most interestingly, some aspects of the age-related transition from resilience to frailty can be reversed to a certain extent. This raises the possibility that the effect of IIS and related pathways on resilience and frailty during aging in higher animals might possess some degree of reversibility.

Longevity interventions modulate mechanotransduction and extracellular matrix homeostasis in C. elegans.

Dysfunctional extracellular matrices (ECM) contribute to aging and disease. Repairing dysfunctional ECM could potentially prevent age-related pathologies. Interventions promoting longevity also impact ECM gene expression. However, the role of ECM composition changes in healthy aging remains unclear. Here we perform proteomics and in-vivo monitoring to systematically investigate ECM composition (matreotype) during aging in C. elegans revealing three distinct collagen dynamics. Longevity interventions slow age-related collagen stiffening and prolong the expression of collagens that are turned over. These prolonged collagen dynamics are mediated by a mechanical feedback loop of hemidesmosome-containing structures that span from the exoskeletal ECM through the hypodermis, basement membrane ECM, to the muscles, coupling mechanical forces to adjust ECM gene expression and longevity via the transcriptional co-activator YAP-1 across tissues. Our results provide in-vivo evidence that coordinated ECM remodeling through mechanotransduction is required and sufficient to promote longevity, offering potential avenues for interventions targeting ECM dynamics.

In-vivo screening implicates endoribonuclease Regnase-1 in modulating senescence-associated lysosomal changes.

Accumulation of senescent cells accelerates aging and age-related diseases, whereas preventing this accumulation extends the lifespan in mice. A characteristic of senescent cells is increased staining with ß-galactosidase (ß-gal) ex vivo. Here, we describe a progressive accumulation of ß-gal staining in the model organism C. elegans during aging. We show that distinct pharmacological and genetic interventions targeting the mitochondria and the mTORC1 to the nuclear core complex axis, the non-canonical apoptotic, and lysosomal-autophagy pathways slow the age-dependent accumulation of ß-gal. We identify a novel gene, rege-1/Regnase-1/ZC3H12A/MCPIP1, modulating ß-gal staining via the transcription factor ets-4/SPDEF. We demonstrate that knocking down Regnase-1 in human cell culture prevents senescence-associated ß-gal accumulation. Our data provide a screening pipeline to identify genes and drugs modulating senescence-associated lysosomal phenotypes.

Removal of extracellular human amyloid beta aggregates by extracellular proteases in C. elegans.

The amyloid beta (Aß) plaques found in Alzheimer's disease (AD) patients' brains contain collagens and are embedded extracellularly. Several collagens have been proposed to influence Aß aggregate formation, yet their role in clearance is unknown. To investigate the potential role of collagens in forming and clearance of extracellular aggregates in vivo, we created a transgenic Caenorhabditis elegans strain that expresses and secretes human Aß1-42. This secreted Aß forms aggregates in two distinct places within the extracellular matrix. In a screen for extracellular human Aß aggregation regulators, we identified different collagens to ameliorate or potentiate Aß aggregation. We show that a disintegrin and metalloprotease a disintegrin and metalloprotease 2 (ADM-2), an ortholog of ADAM9, reduces the load of extracellular Aß aggregates. ADM-2 is required and sufficient to remove the extracellular Aß aggregates. Thus, we provide in vivo evidence of collagens essential for aggregate formation and metalloprotease participating in extracellular Aß aggregate removal.

DNA damage signals from somatic uterine tissue arrest oogenesis through activated DAF-16.

Germ line integrity is crucial for progeny fitness. Organisms deploy the DNA damage response (DDR) signaling to protect the germ line from genotoxic stress, facilitating the cell-cycle arrest of germ cells and DNA repair or their apoptosis. Cell-autonomous regulation of germ line quality in response to DNA damage is well studied; however, how quality is enforced cell non-autonomously on sensing somatic DNA damage is less known. Using Caenorhabditis elegans, we show that DDR disruption, only in the uterus, when insulin/IGF-1 signaling (IIS) is low, arrests oogenesis in the pachytene stage of meiosis I, in a FOXO/DAF-16 transcription factor-dependent manner. Without FOXO/DAF-16, germ cells of the IIS mutant escape the arrest to produce poor-quality oocytes, showing that the transcription factor imposes strict quality control during low IIS. Activated FOXO/DAF-16 senses DDR perturbations during low IIS to lower ERK/MPK-1 signaling below a threshold to promote germ line arrest. Altogether, we elucidate a new surveillance role for activated FOXO/DAF-16 that ensures optimal germ cell quality and progeny fitness in response to somatic DNA damage.

CRISPR-activated expression of collagen col-120 increases lifespan and heat tolerance.

Transgenic overexpression of collagen col-120 increases the lifespan of C. elegans . However, whether post-developmental enhancement of collagen expression could also increase the lifespan is unknown. Recently, we described a method to induce the expression of a target gene using catalytically dead Cas9 (dCas9)-engineered C. elegans via ingestion of bacteria expressing a pair of promoter-specific single guide RNAs (sgRNA). Here, we cloned col-120 promoter-specific sgRNA oligo pair into L4440-Biobrick-sgRNA and fed these bacteria to dCas9::VP64 transgenic C. elegans . We observed a similar percentage of lifespan extension by post-developmentally dCas9-induced expression of col-120 , as previously reported through transgenic overexpression of col-120 . Consistent with this result is that induction of another previously shown longevity-promoting collagen, col-10 , also increased lifespan. Furthermore, we found an enhanced resilience to heat stress and increased expression of hsp-16.2 upon dCas9-activated col-120 expression. Together, these results provide an orthogonal method to validate longevity by enhancing col-120 expression and point towards a potential role of collagen enhancement in thermotolerance.

Rilmenidine extends lifespan and healthspan in Caenorhabditis elegans via a nischarin I1-imidazoline receptor.

Repurposing drugs capable of extending lifespan and health span has a huge untapped potential in translational geroscience. Here, we searched for known compounds that elicit a similar gene expression signature to caloric restriction and identified rilmenidine, an I1-imidazoline receptor agonist and prescription medication for the treatment of hypertension. We then show that treating Caenorhabditis elegans with rilmenidine at young and older ages increases lifespan. We also demonstrate that the stress-resilience, health span, and lifespan benefits of rilmenidine treatment in C. elegans are mediated by the I1-imidazoline receptor nish-1, implicating this receptor as a potential longevity target. Consistent with the shared caloric-restriction-mimicking gene signature, supplementing rilmenidine to calorically restricted C. elegans, genetic reduction of TORC1 function, or rapamycin treatment did not further increase lifespan. The rilmenidine-induced longevity required the transcription factors FOXO/DAF-16 and NRF1,2,3/SKN-1. Furthermore, we find that autophagy, but not AMPK signaling, was needed for rilmenidine-induced longevity. Moreover, transcriptional changes similar to caloric restriction were observed in liver and kidney tissues in mice treated with rilmenidine. Together, these results reveal a geroprotective and potential caloric restriction mimetic effect by rilmenidine that warrant fresh lines of inquiry into this compound.

Ingestion of single guide RNAs induces gene overexpression and extends lifespan in Caenorhabditis elegans via CRISPR activation.

Inhibition of gene expression in Caenorhabditis elegans, a versatile model organism for studying the genetics of development and aging, is achievable by feeding nematodes with bacteria expressing specific dsRNAs. Overexpression of hypoxia-inducible factor 1 (hif-1) or heat-shock factor 1 (hsf-1) by conventional transgenesis has previously been shown to promote nematodal longevity. However, it is unclear whether other methods of gene overexpression are feasible, particularly with the advent of CRISPR-based techniques. Here, we show that feeding C. elegans engineered to stably express a Cas9-derived synthetic transcription factor with bacteria expressing promoter-specific single guide RNAs (sgRNAs) also allows activation of gene expression. We demonstrate that CRISPR activation via ingested sgRNAs specific for the respective promoter regions of hif-1 or hsf-1 increases gene expression and extends lifespan of C. elegans. Furthermore, and as an in silico resource for future studies aiming to use CRISPR activation in C. elegans, we provide predicted promoter-specific sgRNA target sequences for >13,000 C. elegans genes with experimentally defined transcription start sites. We anticipate that the approach and components described herein will help to facilitate genome-wide gene overexpression studies, for example, to identify modulators of aging or other phenotypes of interest, by enabling induction of transcription by feeding of sgRNA-expressing bacteria to nematodes.

The genetic paradigms of dietary restriction fail to extend life span in cep-1(gk138) mutant of C. elegans p53 due to possible background mutations.

Dietary restriction (DR) increases life span and improves health in most model systems tested, including non-human primates. In C. elegans, as in other models, DR leads to reprogramming of metabolism, improvements in mitochondrial health, large changes in expression of cytoprotective genes and better proteostasis. Understandably, multiple global transcriptional regulators like transcription factors FOXO/DAF-16, FOXA/PHA-4, HSF1/HSF-1 and NRF2/SKN-1 are important for DR longevity. Considering the wide-ranging effects of p53 on organismal biology, we asked whether the C. elegans ortholog, CEP-1 is required for DR-mediated longevity assurance. We employed the widely-used TJ1 strain of cep-1(gk138). We show that cep-1(gk138) suppresses the life span extension of two genetic paradigms of DR, but two non-genetic modes of DR remain unaffected in this strain. We find that two aspects of DR, increased autophagy and up-regulation of the expression of cytoprotective xenobiotic detoxification program (cXDP) genes, are dampened in cep-1(gk138). Importantly, we find that background mutation(s) in the strain may be the actual cause for the phenotypic differences that we observed and cep-1 may not be directly involved in genetic DR-mediated longevity assurance in worms. Identifying these mutation(s) may reveal a novel regulator of longevity required specifically by genetic modes of DR.

Polyunsaturated fatty acids and p38-MAPK link metabolic reprogramming to cytoprotective gene expression during dietary restriction.

The metabolic state of an organism instructs gene expression modalities, leading to changes in complex life history traits, such as longevity. Dietary restriction (DR), which positively affects health and life span across species, leads to metabolic reprogramming that enhances utilisation of fatty acids for energy generation. One direct consequence of this metabolic shift is the upregulation of cytoprotective (CyTP) genes categorized in the Gene Ontology (GO) term of "Xenobiotic Detoxification Program" (XDP). How an organism senses metabolic changes during nutritional stress to alter gene expression programs is less known. Here, using a genetic model of DR, we show that the levels of polyunsaturated fatty acids (PUFAs), especially linoleic acid (LA) and eicosapentaenoic acid (EPA), are increased following DR and these PUFAs are able to activate the CyTP genes. This activation of CyTP genes is mediated by the conserved p38 mitogen-activated protein kinase (p38-MAPK) pathway. Consequently, genes of the PUFA biosynthesis and p38-MAPK pathway are required for multiple paradigms of DR-mediated longevity, suggesting conservation of mechanism. Thus, our study shows that PUFAs and p38-MAPK pathway function downstream of DR to help communicate the metabolic state of an organism to regulate expression of CyTP genes, ensuring extended life span.

A novel gene-diet pair modulates C. elegans aging.

Diet profoundly affects metabolism and incidences of age-related diseases. Animals adapt their physiology to different food-types, modulating complex life-history traits like aging. The molecular mechanisms linking adaptive capacity to diet with aging are less known. We identify FLR-4 kinase as a novel modulator of aging in C. elegans, depending on bacterial diet. FLR-4 functions to prevent differential activation of the p38MAPK pathway in response to diverse food-types, thereby maintaining normal life span. In a kinase-dead flr-4 mutant, E. coli HT115 (K12 strain), but not the standard diet OP50 (B strain), is able to activate p38MAPK, elevate expression of cytoprotective genes through the nuclear hormone receptor NHR-8 and enhance life span. Interestingly, flr-4 and dietary restriction utilize similar pathways for longevity assurance, suggesting cross-talks between cellular modules that respond to diet quality and quantity. Together, our study discovers a new C. elegans gene-diet pair that controls the plasticity of aging.