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Aging and the age-associated decline of the proteome is determined in part through neuronal control of evolutionarily conserved transcriptional effectors, which safeguard homeostasis under fluctuating metabolic and stress conditions by regulating an expansive proteostatic network. We have discovered theCaenorhabditis eleganshomeodomain-interactingproteinkinase (HPK-1) acts as a key transcriptional effector to preserve neuronal integrity, function, and proteostasis during aging. Loss ofhpk-1results in drastic dysregulation in expression of neuronal genes, including genes associated with neuronal aging. During normal aginghpk-1expression increases throughout the nervous system more broadly than any other kinase. Within the aging nervous system,hpk-1is co-expressed with key longevity transcription factors, includingdaf-16(FOXO),hlh-30(TFEB),skn-1(Nrf2), andhif-1, which suggestshpk-1expression mitigates natural age-associated physiological decline. Consistently, pan-neuronal overexpression ofhpk-1extends longevity, preserves proteostasis both within and outside of the nervous system, and improves stress resistance. Neuronal HPK-1 improves proteostasis through kinase activity. HPK-1 functions cell non-autonomously within serotonergic and GABAergic neurons to improve proteostasis in distal tissues by specifically regulating distinct components of the proteostatic network. Increased serotonergic HPK-1 enhances the heat shock response and survival to acute stress. In contrast, GABAergic HPK-1 induces basal autophagy and extends longevity. Our work establisheshpk-1as a key neuronal transcriptional regulator critical for preservation of neuronal function during aging. Further, these data provide novel insight as to how the nervous system partitions acute and chronic adaptive response pathways to delay aging by maintaining organismal homeostasis.

The nervous system systemically coordinates proteostasis to delay organismal aging. However, the neuronal regulatory mechanisms that coordinate cellular anti-aging programs across tissue and cell-types are relatively unknown. In this work, we identify the homeodomain-interacting protein kinase (HPK-1), a transcriptional cofactor, as a novel neuronal component of the proteostatic network: its overexpression produces a paracrine signal to hyper-induce molecular chaperones and a neuroendocrine signal to induce autophagy in peripheral tissues. Neuronal HPK-1 signaling improves proteostasis in distal tissues through neurotransmitters. These pro-longevity modalities are independently regulated within serotonergic and GABAergic neurons, respectively, through distinct adaptive responses, either of which improve proteostasis in a cell non-autonomous manner. Serotonergic HPK-1 activity amplifies the heat shock response and protects the proteome from acute stress, without altering longevity. Conversely, increased GABAergic HPK-1 activity is sufficient to induce autophagy and extend longevity, without altering acute stress survival. Consistently, GABAergic neurons, but not serotonin, is essential for the cell non-autonomous induction of autophagy by neuronal HPK-1. These findings provide novel insight into how the nervous system partitions and coordinates unique adaptive response pathways to delay organismal aging, and reveals a key role for neuronal HPK-1 in regulating the proteostatic network throughout an intact metazoan animal.Significance StatementAging and the age-associated decline of the proteome is determined in part through neuronal control of evolutionarily conserved transcriptional effectors, which safeguard homeostasis under fluctuating metabolic and stress conditions by regulating an expansive proteostatic network in peripheral tissues. How neuronal signaling mechanisms are primed, relayed through an organism, and specific responses are initiated in receiving cell types remain poorly understood. We have discovered that the Caenorhabditis elegans homeodomain-interacting protein kinase (HPK-1) is a novel transcriptional effector that functions within two distinct neuronal cell-types to non-autonomously regulate divergent components of the proteostatic network to enhance stress resistance, improve proteostasis and delay aging.

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington’s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Lifespan is the most straightforward surrogate measure of aging, as it is easily quantifiable. A common approach to measure Caenorhabditis elegans lifespan is to follow a population of animals over time and score viability based on movement. We previously developed an alternative approach, called the Replica Set method, to quantitatively measure lifespan of C. elegans in a high-throughput manner. The replica set method allows a single investigator to screen more treatments or conditions in the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. Unlike traditional approaches, the Replica Set method centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with “traditional” longitudinal methods. The protocols provided here describe both the traditional experimental approach and the Replica Set method, as well as practical considerations for each.

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

An extensive proteostatic network comprised of molecular chaperones and protein clearance mechanisms functions collectively to preserve the integrity and resiliency of the proteome. The efficacy of this network deteriorates during aging, coinciding with many clinical manifestations, including protein aggregation diseases of the nervous system. A decline in proteostasis can be delayed through the activation of cytoprotective transcriptional responses, which are sensitive to environmental stress and internal metabolic and physiological cues. The homeodomain-interacting protein kinase (hipk) family members are conserved transcriptional co-factors that have been implicated in both genotoxic and metabolic stress responses from yeast to mammals. We demonstrate that constitutive expression of the sole Caenorhabditis elegans Hipk homolog, hpk-1, is sufficient to delay aging, preserve proteostasis, and promote stress resistance, while loss of hpk-1 is deleterious to these phenotypes. We show that HPK-1 preserves proteostasis and extends longevity through distinct but complementary genetic pathways defined by the heat shock transcription factor (HSF-1), and the target of rapamycin complex 1 (TORC1). We demonstrate that HPK-1 antagonizes sumoylation of HSF-1, a post-translational modification associated with reduced transcriptional activity in mammals. We show that inhibition of sumoylation by RNAi enhances HSF-1-dependent transcriptional induction of chaperones in response to heat shock. We find that hpk-1 is required for HSF-1 to induce molecular chaperones after thermal stress and enhances hormetic extension of longevity. We also show that HPK-1 is required in conjunction with HSF-1 for maintenance of proteostasis in the absence of thermal stress, protecting against the formation of polyglutamine (Q35::YFP) protein aggregates and associated locomotory toxicity. These functions of HPK-1/HSF-1 undergo rapid down-regulation once animals reach reproductive maturity. We show that HPK-1 fortifies proteostasis and extends longevity by an additional independent mechanism: induction of autophagy. HPK-1 is necessary for induction of autophagosome formation and autophagy gene expression in response to dietary restriction (DR) or inactivation of TORC1. The autophagy-stimulating transcription factors pha-4/FoxA and mxl-2/Mlx, but not hlh-30/TFEB or the nuclear hormone receptor nhr-62, are necessary for extended longevity resulting from HPK-1 overexpression. HPK-1 expression is itself induced by transcriptional mechanisms after nutritional stress, and post-transcriptional mechanisms in response to thermal stress. Collectively our results position HPK-1 at a central regulatory node upstream of the greater proteostatic network, acting at the transcriptional level by promoting protein folding via chaperone expression, and protein turnover via expression of autophagy genes. HPK-1 therefore provides a promising intervention point for pharmacological agents targeting the protein homeostasis system as a means of preserving robust longevity., Author summary Aging is the gradual and progressive decline of vitality. A hallmark of aging is the decay of protective mechanisms that normally preserve the robustness and resiliency of cells and tissues. Proteostasis is the term that applies specifically to those mechanisms that promote stability of the proteome, the collection of polypeptides that cells produce, by a combination of chaperone-assisted folding and degradation of misfolded or extraneous proteins. We have identified hpk-1 (encoding a homeodomain-interacting protein kinase) in the nematode C. elegans as an important transcriptional regulatory component of the proteostasis machinery. HPK-1 promotes proteostasis by linking two distinct mechanisms: first by stimulating chaperone gene expression via the heat shock transcription factor (HSF-1), and second by stimulating autophagy gene expression in opposition to the target of rapamycin (TOR) kinase signaling pathway. HPK-1 therefore provides an attractive target for interventions to preserve physiological resiliency during aging by preserving the overall health of the proteome.

Spns1 (Spinster homolog 1 [Drosophila]) in vertebrates, as well as Spin (Spinster) in Drosophila, is a hypothetical lysosomal H+-carbohydrate transporter, which functions at a late stage of macroautophagy (hereafter autophagy). The Spin/Spns1 defect induces aberrant autolysosome formation that leads to developmental senescence in the embryonic stage and premature aging symptoms in adulthood. However, the molecular mechanism by which loss of Spin/Spns1 leads to the specific pathogenesis remains to be elucidated. Using chemical, genetic and CRISPR/Cas9-mediated genome-editing approaches in zebrafish, we investigated and determined a mechanism that suppresses embryonic senescence as well as autolysosomal impairment mediated by Spns1 deficiency. Unexpectedly, we found that a concurrent disruption of the vacuolar-type H+-ATPase (v-ATPase) subunit gene, atp6v0ca (ATPase, H+ transporting, lysosomal, V0 subunit ca) led to suppression of the senescence induced by the Spns1 defect, whereas the sole loss of Atp6v0ca led to senescent embryos similar to the single spns1 mutation. Moreover, we discovered that the combined stable defect seen in the presence of both the spns1 and atp6v0ca mutant genes still subsequently induced premature autophagosome-lysosome fusion marked by insufficient acidity, while extending developmental life span, compared with the solely mutated spns1 defect. Our data suggest that Spns1 and the v-ATPase orchestrate proper autolysosomal biogenesis with optimal acidification that is critically linked to developmental senescence and survival.