A dicer-related helicase opposes the age-related pathology from SKN-1 activation in ASI neurons.

Coordination of cellular responses to stress is essential for health across the lifespan. The transcription factor SKN-1 is an essential homeostat that mediates survival in stress-inducing environments and cellular dysfunction, but constitutive activation of SKN-1 drives premature aging thus revealing the importance of turning off cytoprotective pathways. Here, we identify how SKN-1 activation in two ciliated ASI neurons in Caenorhabditis elegans results in an increase in organismal transcriptional capacity that drives pleiotropic outcomes in peripheral tissues. An increase in the expression of established SKN-1 stress response and lipid metabolism gene classes of RNA in the ASI neurons, in addition to the increased expression of several classes of noncoding RNA, define a molecular signature of animals with constitutive SKN-1 activation and diminished healthspan. We reveal neddylation as a unique regulator of the SKN-1 homeostat that mediates SKN-1 abundance within intestinal cells. Moreover, RNAi-independent activity of the dicer-related DExD/H-box helicase, drh-1, in the intestine, can oppose the effects of aberrant SKN-1 transcriptional activation and delays age-dependent decline in health. Taken together, our results uncover a cell nonautonomous circuit to maintain organism-level homeostasis in response to excessive SKN-1 transcriptional activity in the sensory nervous system.

Redirection of SKN-1 abates the negative metabolic outcomes of a perceived pathogen infection.

Early host responses toward pathogens are essential for defense against infection. In Caenorhabditis elegans, the transcription factor, SKN-1, regulates cellular defenses during xenobiotic intoxication and bacterial infection. However, constitutive activation of SKN-1 results in pleiotropic outcomes, including a redistribution of somatic lipids to the germline, which impairs health and shortens lifespan. Here, we show that exposing C. elegans to Pseudomonas aeruginosa similarly drives the rapid depletion of somatic, but not germline, lipid stores. Modulating the epigenetic landscape refines SKN-1 activity away from innate immunity targets, which alleviates negative metabolic outcomes. Similarly, exposure to oxidative stress redirects SKN-1 activity away from pathogen response genes while restoring somatic lipid distribution. In addition, activating p38/MAPK signaling in the absence of pathogens, is sufficient to drive SKN-1-dependent loss of somatic fat. These data define a SKN-1- and p38-dependent axis for coordinating pathogen responses, lipid homeostasis, and survival and identify transcriptional redirection, rather than inactivation, as a mechanism for counteracting the pleiotropic consequences of aberrant transcriptional activity.

Hypodermal responses to protein synthesis inhibition induce systemic developmental arrest and AMPK-dependent survival in Caenorhabditis elegans.

Across organisms, manipulation of biosynthetic capacity arrests development early in life, but can increase health- and lifespan post-developmentally. Here we demonstrate that this developmental arrest is not sickness but rather a regulated survival program responding to reduced cellular performance. We inhibited protein synthesis by reducing ribosome biogenesis (rps-11/RPS11 RNAi), translation initiation (ifg-1/EIF3G mutation and egl-45/EIF3A RNAi), or ribosome progression (cycloheximide treatment), all of which result in a specific arrest at larval stage 2 of C. elegans development. This quiescent state can last for weeks-beyond the normal C. elegans adult lifespan-and is reversible, as animals can resume reproduction and live a normal lifespan once released from the source of protein synthesis inhibition. The arrest state affords resistance to thermal, oxidative, and heavy metal stress exposure. In addition to cell-autonomous responses, reducing biosynthetic capacity only in the hypodermis was sufficient to drive organism-level developmental arrest and stress resistance phenotypes. Among the cell non-autonomous responses to protein synthesis inhibition is reduced pharyngeal pumping that is dependent upon AMPK-mediated signaling. The reduced pharyngeal pumping in response to protein synthesis inhibition is recapitulated by exposure to microbes that generate protein synthesis-inhibiting xenobiotics, which may mechanistically reduce ingestion of pathogen and toxin. These data define the existence of a transient arrest-survival state in response to protein synthesis inhibition and provide an evolutionary foundation for the conserved enhancement of healthy aging observed in post-developmental animals with reduced biosynthetic capacity.

WDR23 regulates NRF2 independently of KEAP1.

Cellular adaptation to stress is essential to ensure organismal survival. NRF2/NFE2L2 is a key determinant of xenobiotic stress responses, and loss of negative regulation by the KEAP1-CUL3 proteasome system is implicated in several chemo- and radiation-resistant cancers. Advantageously using C. elegans alongside human cell culture models, we establish a new WDR23-DDB1-CUL4 regulatory axis for NRF2 activity that operates independently of the canonical KEAP1-CUL3 system. WDR23 binds the DIDLID sequence within the Neh2 domain of NRF2 to regulate its stability; this regulation is not dependent on the KEAP1-binding DLG or ETGE motifs. The C-terminal domain of WDR23 is highly conserved and involved in regulation of NRF2 by the DDB1-CUL4 complex. The addition of WDR23 increases cellular sensitivity to cytotoxic chemotherapeutic drugs and suppresses NRF2 in KEAP1-negative cancer cell lines. Together, our results identify WDR23 as an alternative regulator of NRF2 proteostasis and uncover a cellular pathway that regulates NRF2 activity and capacity for cytoprotection independently of KEAP1.

Omega-3 and -6 fatty acids allocate somatic and germline lipids to ensure fitness during nutrient and oxidative stress in Caenorhabditis elegans.

Animals in nature are continually challenged by periods of feast and famine as resources inevitably fluctuate, and must allocate somatic reserves for reproduction to abate evolutionary pressures. We identify an age-dependent lipid homeostasis pathway in Caenorhabditis elegans that regulates the mobilization of lipids from the soma to the germline, which supports fecundity but at the cost of survival in nutrient-poor and oxidative stress environments. This trade-off is responsive to the levels of dietary carbohydrates and organismal oleic acid and is coupled to activation of the cytoprotective transcription factor SKN-1 in both laboratory-derived and natural isolates of C. elegans. The homeostatic balance of lipid stores between the somatic and germ cells is mediated by arachidonic acid (omega-6) and eicosapentaenoic acid (omega-3) precursors of eicosanoid signaling molecules. Our results describe a mechanism for resource reallocation within intact animals that influences reproductive fitness at the cost of somatic resilience.

A soma-to-germline transformation in long-lived Caenorhabditis elegans mutants.

Unlike the soma, which ages during the lifespan of multicellular organisms, the germ line traces an essentially immortal lineage. Genomic instability in somatic cells increases with age, and this decline in somatic maintenance might be regulated to facilitate resource reallocation towards reproduction at the expense of cellular senescence. Here we show that Caenorhabditis elegans mutants with increased longevity exhibit a soma-to-germline transformation of gene expression programs normally limited to the germ line. Decreased insulin-like signalling causes the somatic misexpression of the germline-limited pie-1 and pgl family of genes in intestinal and ectodermal tissues. The forkhead boxO1A (FOXO) transcription factor DAF-16, the major transcriptional effector of insulin-like signalling, regulates pie-1 expression by directly binding to the pie-1 promoter. The somatic tissues of insulin-like mutants are more germline-like and protected from genotoxic stress. Gene inactivation of components of the cytosolic chaperonin complex that induce increased longevity also causes somatic misexpression of PGL-1. These results indicate that the acquisition of germline characteristics by the somatic cells of C. elegans mutants with increased longevity contributes to their increased health and survival.

Longevity and the long arm of epigenetics: acquired parental marks influence lifespan across several generations.

A recent study reported that longevity in Caenorhabditits elegans can be inherited over several generations. This is probably achieved through the following epigenetic mechanism: inherited demethylated histones at some central loci, such as miRNA, transcription factors or signaling regulators affect the expression of certain genes leading to the longevity phenotype.

Activated SKN-1 alters the aging trajectories of long-lived C. elegans mutants.

In the presence of stressful environments, the SKN-1 cytoprotective transcription factor is activated to induce the expression of gene targets that can restore homeostasis. However, chronic activation of SKN-1 results in diminished health and a reduction of lifespan. Here we demonstrate the necessity of modulating SKN-1 activity to maintain the longevity-promoting effects associated with genetic mutations that impair daf-2/insulin receptor signaling, the eat-2 model of caloric restriction, and glp-1-dependent loss of germ cell proliferation. A hallmark of animals with constitutive SKN-1 activation is the age-dependent loss of somatic lipids and this phenotype is linked to a general reduction in survival in animals harboring the skn-1gf allele, but surprisingly, daf-2lf; skn-1gf double mutant animals do not redistribute somatic lipids which suggests the insulin signaling pathway functions downstream of SKN-1 in the maintenance of lipid distribution. As expected, the eat-2lf allele, which independently activates SKN-1, continues to display somatic lipid depletion in older ages with and without the skn-1gf activating mutation. In contrast, the presence of the skn-1gf allele does not lead to somatic lipid redistribution in glp-1lf animals that lack a proliferating germline. Taken together, these studies support a genetic model where SKN-1 activity is an important regulator of lipid mobilization in response to nutrient availability that fuels the developing germline by engaging the daf-2/insulin receptor pathway.

Disrupting the SKN-1 homeostat: mechanistic insights and phenotypic outcomes.

The mechanisms that govern maintenance of cellular homeostasis are crucial to the lifespan and healthspan of all living systems. As an organism ages, there is a gradual decline in cellular homeostasis that leads to senescence and death. As an organism lives into advanced age, the cells within will attempt to abate age-related decline by enhancing the activity of cellular stress pathways. The regulation of cellular stress responses by transcription factors SKN-1/Nrf2 is a well characterized pathway in which cellular stress, particularly xenobiotic stress, is abated by SKN-1/Nrf2-mediated transcriptional activation of the Phase II detoxification pathway. However, SKN-1/Nrf2 also regulates a multitude of other processes including development, pathogenic stress responses, proteostasis, and lipid metabolism. While this process is typically tightly regulated, constitutive activation of SKN-1/Nrf2 is detrimental to organismal health, this raises interesting questions surrounding the tradeoff between SKN-1/Nrf2 cryoprotection and cellular health and the ability of cells to deactivate stress response pathways post stress. Recent work has determined that transcriptional programs of SKN-1 can be redirected or suppressed to abate negative health outcomes of constitutive activation. Here we will detail the mechanisms by which SKN-1 is controlled, which are important for our understanding of SKN-1/Nrf2 cytoprotection across the lifespan.

Loss of WDR23 proteostasis impacts mitochondrial homeostasis in the mouse brain.

Mitochondrial adaptation is important for stress resistance throughout life. Here we show that WDR23 loss results in an enrichment for genes regulated by nuclear respiratory factor 1 (NRF1), which coordinates mitochondrial biogenesis and respiratory functions, and an increased steady state level of several nuclear coded mitochondrial resident proteins in the brain. Wdr23KO also increases the endogenous levels of insulin degrading enzyme (IDE) and the relaxin-3 peptide (RLN3), both of which have established roles in mediating mitochondrial metabolic and oxidative stress responses. Taken together, these studies reveal an important role for WDR23 as a component of the mitochondrial homeostat in the murine brain.

Hepatic WDR23 proteostasis mediates insulin homeostasis by regulating insulin-degrading enzyme capacity.

Maintaining insulin homeostasis is critical for cellular and organismal metabolism. In the liver, insulin is degraded by the activity of the insulin-degrading enzyme (IDE). Here, we establish a hepatic regulatory axis for IDE through WDR23-proteostasis. Wdr23KO mice have increased IDE expression, reduced circulating insulin, and defective insulin responses. Genetically engineered human cell models lacking WDR23 also increase IDE expression and display dysregulated phosphorylation of insulin signaling cascade proteins, IRS-1, AKT2, MAPK, FoxO, and mTOR, similar to cells treated with insulin, which can be mitigated by chemical inhibition of IDE. Mechanistically, the cytoprotective transcription factor NRF2, a direct target of WDR23-Cul4 proteostasis, mediates the enhanced transcriptional expression of IDE when WDR23 is ablated. Moreover, an analysis of human genetic variation in WDR23 across a large naturally aging human cohort in the US Health and Retirement Study reveals a significant association of WDR23 with altered hemoglobin A1C (HbA1c) levels in older adults, supporting the use of WDR23 as a new molecular determinant of metabolic health in humans.

Oolonghomobisflavans from Camellia sinensis disaggregate tau fibrils across Alzheimer's disease models.

Alzheimer's disease (AD) is a common debilitating neurodegenerative disease with limited treatment options. Amyloid-ß (Aß) and tau fibrils are well-established hallmarks of AD, which can induce oxidative stress, neuronal cell death, and are linked to disease pathology. Here, we describe the effects of Oolonghomobisflavan A (OFA) and Oolonghomobisflavan B (OFB) on tau fibril disaggregation and prionogenic seeding. Transcriptomic analysis of OF-treated animals reveals the induction of a proteostasis-enhancing and health-promoting signature. OFA treatment reduced the burden of Tau protein aggregation in a C. elegans model expressing pathogenic human tau ("hTau-expressing") and promoted Tau disaggregation and inhibited seeding in assays using ex vivo brain-derived paired helical filament tau protein fibrils from Alzheimer's disease brain donors. Correspondingly, treatment with OF improved multiple fitness and aging-related health parameters in the hTau-expressing C. elegans model, including reproductive output, muscle function, and importantly, reversed the shortened lifespan stemming from pathogenic Tau expression. Collectively, this study provides new evidence supporting the neuroprotective effects of OFs and reveal a new therapeutic strategy for targeting AD and other neurodegenerative diseases characterized by tauopathy.

WDR23 mediates NRF2 proteostasis and cytoprotective capacity in the hippocampus.

Pathogenic brain aging and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease are characterized by chronic neuroinflammation and the accumulation of dysfunctional or misfolded proteins that lead to progressive neuronal cell death. Here we demonstrate that a murine model with global loss of the CUL4-DDB1 substrate receptor WDR23 (Wdr23KO) results in changes in multiple age-related hippocampal-dependent behaviors. The behavioral differences observed in Wdr23KO animals accompany the stabilization of the NRF2/NFE2L2 protein, an increase in RNA transcripts regulated by this cytoprotective transcription factor, and an increase in the steady state level of antioxidant defense proteins. Taken together, these findings reveal a role for WDR23-proteostasis in mediating cytoprotective capacity in the hippocampus and reveal the potential for targeting WDR23-NRF2 signaling interactions for development of therapies for neurodegenerative disorders.

SKN-1 isoform-c is essential for C. elegans development.

The transcription factor SKN-1 in Caenorhabditis elegans is a critical regulator of various biological processes, impacting development, diet and immune responses, cellular detoxification, and lipid metabolism; thereby playing a pivotal role in regulating the health and lifespan of the organism. The primary isoforms of SKN-1 ( SKN-1 a, SKN-1 b, and SKN-1 c) exhibit distinct functions resembling mammalian Nrf transcription factors. This study investigates the specific role of the SKN-1 c isoform in development by generating mutants with targeted missense mutations in the skn-1 c and skn-1 a isoforms. The skn-1 c Met1Ala mutants, which replaces a start methionine with alanine, renders SKN-1 c non-functional while preserving other isoforms, produced inviable embryos, requiring a balancer chromosome for proper embryonic development. In contrast, skn-1 a Met1Ala mutants, which replaces the start methionine with alanine for this isoform, displayed normal embryonic development and hatching. Moreover, the data suggest that SKN-1 c plays a crucial role in embryonic development, as strains without maternally deposited SKN-1 c lead to embryos that are developmentally arrested. Together, these findings contribute to our understanding of SKN-1 c's specific role in influencing embryogenesis and development in C. elegans.

Serotonin deficiency from constitutive SKN-1 activation drives pathogen apathy.

When an organism encounters a pathogen, the host innate immune system activates to defend against pathogen colonization and toxic xenobiotics produced. C. elegans employ multiple defense systems to ensure survival when exposed to Pseudomonas aeruginosa including activation of the cytoprotective transcription factor SKN-1/NRF2. Although wildtype C. elegans quickly learn to avoid pathogens, here we describe a peculiar apathy-like behavior towards PA14 in animals with constitutive activation of SKN-1, whereby animals choose not to leave and continue to feed on the pathogen even when a non-pathogenic and healthspan-promoting food option is available. Although lacking the urgency to escape the infectious environment, animals with constitutive SKN-1 activity are not oblivious to the presence of the pathogen and display the typical pathogen-induced intestinal distension and eventual demise. SKN-1 activation, specifically in neurons and intestinal tissues, orchestrates a unique transcriptional program which leads to defects in serotonin signaling that is required from both neurons and non-neuronal tissues. Serotonin depletion from SKN-1 activation limits pathogen defense capacity, drives the pathogen-associated apathy behaviors and induces a synthetic sensitivity to selective serotonin reuptake inhibitors. Taken together, our work reveals new insights into how animals perceive environmental pathogens and subsequently alter behavior and cellular programs to promote survival. KEY POINTS: Identify an apathy-like behavioral response for pathogens resulting from the constitutive activation of the cytoprotective transcription factor SKN-1.Uncover the obligate role for serotonin synthesis in both neuronal and non-neuronal cells for the apathy-like state and ability of serotonin treatment to restore normal behaviors.Characterize the timing and tissue specificity of SKN-1 nuclear localization in neurons and intestinal cells in response to pathogen exposure.Define the unique and context-specific transcriptional signatures of animals with constitutive SKN-1 activation when exposed to pathogenic environments.Reveal necessity for both neuronal and non-neuronal serotonin signaling in host survival from pathogen infection.

Distinct mechanisms of non-autonomous UPRER mediated by GABAergic, glutamatergic, and octopaminergic neurons.

The capacity to deal with stress declines during the aging process, and preservation of cellular stress responses is critical to healthy aging. The unfolded protein response of the endoplasmic reticulum (UPRER) is one such conserved mechanism, which is critical for the maintenance of several major functions of the ER during stress, including protein folding and lipid metabolism. Hyperactivation of the UPRER by overexpression of the major transcription factor, xbp-1s, solely in neurons drives lifespan extension as neurons send a neurotransmitter-based signal to other tissue to activate UPRER in a non-autonomous fashion. Previous work identified serotonergic and dopaminergic neurons in this signaling paradigm. To further expand our understanding of the neural circuitry that underlies the non-autonomous signaling of ER stress, we activated UPRER solely in glutamatergic, octopaminergic, and GABAergic neurons in C. elegans and paired whole-body transcriptomic analysis with functional assays. We found that UPRER-induced signals from glutamatergic neurons increased expression of canonical protein homeostasis pathways and octopaminergic neurons promoted pathogen response pathways, while minor, but statistically significant changes were observed in lipid metabolism-related genes with GABAergic UPRER activation. These findings provide further evidence for the distinct role neuronal subtypes play in driving the diverse response to ER stress.

Different methods of killing bacteria diets differentially influence Caenorhabditis elegans physiology.

Across species, diet plays a critical role in most, if not all life history traits. Caenorhabditis elegans is an important and facile organism for research across modalities, but the use of live bacteria as sources of nutrition can exert pleiotropic outcomes that stem from the action of host-pathogen defenses. Recently, a powerful new approach to readily generate dead and metabolically inactive Escherichia coli was developed that enabled reproducible measures of health across the lifespan. Here we further characterize additional comparisons of developmental and physiological parameters of animals fed either bacteria killed by treatment with ultraviolet (UV) light and bactericidal antibiotics or low-dose paraformaldehyde (PFA). Unlike bacteria killed by UV/Antibiotic treatment, PFA-killed diets resulted in a 25% reduction in body size just prior to adulthood and an overall reduction in stored intracellular lipids. Moreover, a small but reproducible number of animals fed PFA-killed bacteria display age-dependent depletion of somatic lipids, which does not normally occur on live bacteria or bacteria killed by UV/antibiotics. Lastly, animals fed PFA-treated, but not UV-antibiotic treated bacteria display a 10% increase in crawling speed. Taken together, these new data more thoroughly define the physiological impact two methodologies to prepare C. elegans diets that should be considered during experimental design.

Comparative analysis of the molecular and physiological consequences of constitutive SKN-1 activation.

Molecular homeostats play essential roles across all levels of biological organization to ensure a return to normal function after responding to abnormal internal and environmental events. SKN-1 is an evolutionarily conserved cytoprotective transcription factor that is integral for the maintenance of cellular homeostasis upon exposure to a variety of stress conditions. Despite the essentiality of turning on SKN-1/NRF2 in response to exogenous and endogenous stress, animals with chronic activation of SKN-1 display premature loss of health with age, and ultimately, diminished lifespan. Previous genetic models of constitutive SKN-1 activation include gain-of-function alleles of skn-1 and loss-of-function alleles of wdr-23 that impede the turnover of SKN-1 by the ubiquitin proteasome. Here, we define a novel gain-of-function mutation in the xrep-4 locus that results in constitutive activation of SKN-1 in the absence of stress. Although each of these genetic mutations results in continuously unregulated transcriptional output from SKN-1, the physiological consequences of each model on development, stress resistance, reproduction, lipid homeostasis, and lifespan are distinct. Here, we provide a comprehensive assessment of the differential healthspan impacts across multiple models of constitutive SKN-1 activation. Although our results reveal the universal need to reign in the uncontrolled activity of cytoprotective transcription factors, we also define the unique signatures of each model of constitutive SKN-1 activation, which provides innovative solutions for the design of molecular "off-switches" of unregulated transcriptional homeostats.

WDR23 mediates NRF2 proteostasis and cytoprotective capacity in the hippocampus.

Pathogenic brain aging and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease are characterized by chronic neuroinflammation and the accumulation of dysfunctional or misfolded proteins that lead to progressive neuronal cell death. Here we demonstrate that a murine model with global loss of the CUL4-DDB1 substrate receptor WDR23 ( Wdr23KO ) results in changes in multiple age-related hippocampal-dependent behaviors. The behavioral differences observed in Wdr23KO animals accompany the stabilization of the NRF2/NFE2L2 protein, an increase in RNA transcripts regulated by this cytoprotective transcription factor, and an increase in the steady state level of antioxidant defense proteins. Taken together, these findings reveal a role for WDR23-proteostasis in mediating cytoprotective capacity in the hippocampus and reveal the potential for targeting WDR23-NRF2 signaling interactions for development of therapies for neurodegenerative disorders. HIGHLIGHTS: WDR23 regulates NRF2/NFE2L2 stability in the mouse hippocampus Loss of Wdr23 significantly increases the expression of NFE2L2/NRF2 target genes Global loss of WDR23 influences age-related behaviors differentially in males and females.

A dicer-related helicase opposes the age-related pathology from SKN-1 activation in ASI neurons.

Coordination of cellular responses to stress are essential for health across the lifespan. The transcription factor SKN-1 is an essential homeostat that mediates survival in stress-inducing environments and cellular dysfunction, but constitutive activation of SKN-1 drives premature aging thus revealing the importance of turning off cytoprotective pathways. Here we identify how SKN-1 activation in two ciliated ASI neurons in C. elegans results in an increase in organismal transcriptional capacity that drives pleiotropic outcomes in peripheral tissues. An increase in the expression of established SKN-1 stress response and lipid metabolism gene classes of RNA in the ASI neurons, in addition to the increased expression of several classes of non-coding RNA, define a molecular signature of animals with constitutive SKN-1 activation and diminished healthspan. We reveal neddylation as a novel regulator of the SKN-1 homeostat that mediates SKN-1 abundance within intestinal cells. Moreover, RNAi-independent activity of the dicer-related DExD/H-box helicase, drh-1 , in the intestine, can oppose the e2ffects of aberrant SKN-1 transcriptional activation and delays age-dependent decline in health. Taken together, our results uncover a cell non-autonomous circuit to maintain organism-level homeostasis in response to excessive SKN-1 transcriptional activity in the sensory nervous system. SIGNIFICANCE STATEMENT: Unlike activation, an understudied fundamental question across biological systems is how to deactivate a pathway, process, or enzyme after it has been turned on. The irony that the activation of a transcription factor that is meant to be protective can diminish health was first documented by us at the organismal level over a decade ago, but it has long been appreciated that chronic activation of the human ortholog of SKN-1, NRF2, could lead to chemo- and radiation resistance in cancer cells. A colloquial analogy to this biological idea is a sink faucet that has an on valve without a mechanism to shut the water off, which will cause the sink to overflow. Here, we define this off valve.