Samuelson, Andrew et al. (2009) International Worm Meeting "Insulin signaling pathway genes facilitating the maintenance of thermotolerance and protein homeostasis."

Ruvkun, Gary, Samuelson, Andrew, Carr, Christopher

[International Worm Meeting, 2009]

The daf-2 insulin-like signaling pathway is the most potent pathway for lifespan extension in C. elegans, converging on the DAF-16 transcription factor to regulate a large number of genes including free radical detoxifying genes and stress resistance genes. During aging, specific protein damage by reactive oxygen species increases exponentially to challenge protein homeostasis. Processes impacting protein homeostasis include protein synthesis, folding, assembly, repair, translocation and degradation. Activation of stress response pathways, with induction of heat shock proteins, is central to the maintenance of protein homeostasis after stress. The ability to maintain protein homeostasis depends on both chaperone-mediated protein folding (i.e. chronic maintenance) and induction of heat shock proteins (i.e. survive acute stress). Additionally, chronic growth at higher temperature shortens lifespan and accelerates aging. Protein homeostasis, as measured through protein aggregation, is controlled by insulin signaling, dependent on HSF-1 and DAF-16. From a comprehensive functional genomic screen we have previously identified 103 genes that are necessary for decreased insulin signaling to extend lifespan. Animals are progeric after these gene inactivations by several independent measures including: premature age pigment accumulation and increased rate of aging, without drastically altering progeny production or an established biomarker for C. elegans aging; the activity ratio, or proportion of life an animal actively responds to stimuli. We sought to identify the progeric gene inactivations that impair the heat shock response or challenge protein homeostasis. We identified the progeric gene inactivations that are necessary for the increased thermotolerance conferred by decreased insulin signaling. Additionally, we tested the progeric gene inactivations for differential lifespan at varying temperature, premature protein aggregation (polyQ), and induction of heat shock proteins. This comprehensive functional analysis identifies the subset of progeric gene inactivations that negatively impact protein homeostasis after acute or chronic stress. Results from this analysis will be presented.

Andrew Samuelson et al. (2007) International Worm Meeting "Functional genomic analysis for gene inactivations that accelerate aging reveals a role for endocytosis-associated mediators."

Andrew Samuelson, Gary Ruvkun, Christopher Carr

[International Worm Meeting, 2007]

Comprehensive RNAi screens for gene inactivations that extend lifespan have revealed that the most potent pathway for lifespan extension in C. elegans is the daf-2 insulin-like signaling pathway. This pathway converges on the DAF-16 transcription factor which regulates a large number of genes. We sought to identify the full range of genes which are necessary for the long lifespan induction in a daf-2 insulin signaling deficient mutant. To this end, we have identified genes whose inactivation shortened the lifespan of long-lived daf-2 mutant animals through a systematic genome-wide RNAi screen of 18,681 clones. The primary screen identified 513 clones (2.6% of the genome) which suppressed the long lifespan of daf-2 mutant animals to some extent. A more stringent lifespan, morphological, and reproductive analysis narrowed the 513 positives to 143 distinct gene inactivations which shorten lifespan and might function in aging. A major complication of any screen for shortened lifespan lies in discriminating aging genes from those that cause sickness upon inactivation. Thus any screen to study aging by shortening lifespan must have a means to distinguish aging from sickness. We classified gene inactivations which only shortened the lifespan of a daf-2 mutant, and those which shortened the lifespan of control as well as a daf-2 mutant. Additionally, analysis for each gene inactivation includes longitudinal scoring for changes in premature accumulation of aging pigments and changes in the expression of the DAF-16 target gene, sod-3. This allowed a classification of those genes that accelerate the aging process versus those that show signs of poor health. Cumulative analysis of these phenotypes has allowed us to separated gene inactivations that accelerate aging from those which simply shorten lifespan and segregate the gene inactivations into refined classes. Highly represented in the gene inactivations that suppress lifespan extension in the daf-2 mutant are genes that encode proteins that mediate endocytotic trafficking of membrane proteins to lysosomes. These gene inactivations act upstream of DAF-16 to suppress sod-3. Inactivation of these genes may allow signaling to DAF-16 in the absence of insulin receptor signals, or may animate other signaling pathways that bypass the need for kinase input to DAF-16.

Andrew Samuelson et al. (2007) International Worm Meeting "Characterization of gene inactivations identified in a functional genomic screen for accelerated aging based on changes in stress resistance."

Fred Ausubel, Andrew Samuelson, Gary Ruvkun, Javier Irazoqui, Jennifer Powell, Christopher Carr

[International Worm Meeting, 2007]

The daf-2 insulin-like signaling pathway is the most potent pathway for lifespan extension in C. elegans, converging on the DAF-16 transcription factor to regulate a large number of genes including free radical detoxifying genes, stress resistance genes, and pathogen resistance genes. We sought to identify the full range of genes which are necessary for the long lifespan induction in a daf-2 insulin signaling deficient mutant and to better understand how lifespan extension relates to increased stress resistance. To this end, we have identified 143 genes whose inactivation shortened the lifespan of long-lived daf-2 mutant animals when inactivated through a systematic genome-wide RNAi screen. Gene inactivations were classified on the basis on whether they only shortened the lifespan of a daf-2 mutant, or whether they shortened the lifespan of control as well as a daf-2 mutant. We accessed whether these gene inactivations resulted in decreased thermotolerance, resistance to oxidative stress, lowered innate immunity, or changed the expression of the DAF-16 target gene, sod-3. Analysis of these phenotypes have separated genes into refined classes. Results from this detailed analysis will be presented.

Andrew Samuelson et al. (2007) International Worm Meeting "Uncoupling daf-2 lifespan extension: pathway specificity, demographic, and movement rate analysis of gene inactivations identified in a functional genomic screen for accelerated aging."

Gary Ruvkun, Christopher Carr, Andrew Samuelson

[International Worm Meeting, 2007]

The daf-2 insulin-like signaling pathway is the most potent pathway for lifespan extension in C. elegans, converging on the DAF-16 transcription factor to regulate a large number of genes. We sought to identify the full range of genes which are necessary for the long lifespan induction in a daf-2 insulin signaling deficient mutant. To this end, we have identified 143 genes whose inactivation shortened the lifespan of long-lived daf-2 mutant animals when inactivated through a systematic genome-wide RNAi screen. We classified gene inactivations which only shortened the lifespan of a daf-2 mutant, and those which shortened the lifespan of wild type as well as a daf-2 mutant. Detailed measurements of lifespan allowed the calculation of the relative mortality rate doubling time (MRDT, proportional to the reciprocal of the rate of aging) and initial mortality rate (IMR) for each gene inactivation. Additionally, analysis for each gene inactivation includes longitudinal scoring for changes in movement rate (activespan), one measure to distinguish genes that accelerate the aging process versus those that show signs of poor health. Analysis of these phenotypes has allowed us to segregate the gene inactivations into refined classes based on whether there is accelerated aging or poor health. For instance, some of the gene inactivations that shorten lifespan in both control and daf-2 mutant animals, such as the previously described heat shock transcriptional regulator hsf-1, accelerated MRDT with only a modest increase in IMR. Results from this detailed analysis will be presented.

Das, Ritika et al. (2015) International Worm Meeting "The homeodomain protein kinase, HPK-1 influences aging and is a central component of the heat shock response ."

Samuelson, Andrew, Das, Ritika, Kim, Richard, Schwartz, Elliot

[International Worm Meeting, 2015]

The homeodomain protein kinase, HPK-1 influences aging and is a central component of the heat shock response Ritika Das1,2, Richard Kim2, Elliot Schwartz3, and Andrew V. Samuelson21.Department of Biology, University of Rochester, Rochester NY2.Department of Biomedical Genetics, University of Rochester Medical Center, Rochester NY3.Program for Neuroscience, University of California, Los Angeles, Los Angeles CAAging, stress resistance, and the maintenance of protein homeostasis are dependent on the activity of the heat shock transcription factor HSF-1, which in turn regulates the expression of cellular chaperones. Yet the underlying signaling mechanisms that activate HSF-1 remain poorly understood. We find that the homeodomain protein kinase, HPK-1, is both necessary and sufficient to alter aging in Caenorhabditis elegans, as measured by changes in lifespan and protein homeostasis. Interestingly, we provide genetic, molecular, and cellular evidence that HPK-1 and HSF-1 function in a common genetic pathway. Furthermore, HPK-1 is induced by thermal stress, implying that HPK-1 is a bona fide heat shock response gene. Consistent with this notion hpk-1 null mutant animals have compromised thermotolerance, which is accompanied with reduced induction of heat shock responsive chaperones hsp-16.2 and hsp-70 after thermal stress. Additionally, hpk-1 mutants fail to the obtain the hormetic benefit conferred by a transient heat stress, which in wild-type animals manifests as increased lifespan, implying that the roles of HPK-1 in the heat shock response, proteostasis, and aging are intimately connected. Collectively, we propose that HPK-1 is an essential component of the heat shock response whose function ultimately influences both protein homeostasis and aging.

Das, Ritika et al. (2011) International Worm Meeting "Role of endocytosis and ER proteostasis in C. elegans longevity."

Stolzenburg, Lindsay, Rath, Sneha, Samuelson, Andrew V., Das, Ritika, Lee, Nam

[International Worm Meeting, 2011]

Insulin signaling is a major pathway controlling lifespan in multiple organisms, such as worms, flies and mammals. The insulin signaling pathway initiated by the activation of the insulin receptor (DAF-2) brings about the repression of the downstream transcription factor DAF-16. DAF-16 is one of the key transcription factors that regulate the expression of genes involved in lifespan as well as other functions such as development, immunity and stress resistance. We have previously conducted a genome wide RNAi screen to identify genes that are essential for enhanced longevity in daf-2 mutant worms {Samuelson, 2007}1. This screen led to the identification of 103 gene inactivations which cause premature aging (the "progeric gene panel", PGP) . Interestingly, 19 genes in the PGP are annotated to be involved in endosomal protein sorting or vesicular trafficking. Similar studies in yeast have also identified genes involved in vacuolar protein sorting to be essential for longevity suggesting a conserved role for proper endosomal function in regulating aging {Fabrizio, 2010}2. However, the mechanism by which endocytosis influences longevity is still not clear. To explore this further, our lab has undertaken a systems level analysis to identify the members of the PGP that are involved in endocytosis, the ER unfolded protein response (ERUPR), and protein homeostasis. To this end, transgenic worms for different endocytosis markers such as the Rab GTPases and GFP fusions to those proteins whose proper localization within the worm depends on functional endocytic machinery have been screened. While the endocytosis machinery is critical to protein degradation and proper protein sorting, ER serves as a primary site for protein synthesis and folding and daf-2 mutant animals exhibit lower levels of the ERUPR marker hsp-4::GFP indicating improved ER homeostasis {Korenblit, 2010}3 . To identify the members of the PGP that influence longevity via the ER stress response pathway, hsp-4::GFP transgenic worms have been screened to identify gene inactivations that either induce the ERUPR in the absence of stress or conversely impair the ERUPR after stress. Lastly, we have identified the members of the PGP that cause the premature collapse of protein homeostasis, as measured by foci formation of Q35::YFP. Highlights of this comprehensive analysis will be presented. References 1. Samuelson et al (2007) Genes Dev 21(22):2976-94 2. Fabrizio et al (2010) PLOS Genet. 6 (7):e1001024 3. Korenblit et al (2010) PNAS 107(21):9730-5.

Wendy Wong et al. (2006) European Worm Meeting "The Integrative Interaction Map for Caenorhabditis elegans"

Wendy Wong, Andrew Fraser

[European Worm Meeting, 2006]

. Wendy Wong and Andrew Fraser. C. elegans is a popular model system for the systematic analysis. However, relatively little is known for its networks of genetic interactions. We hereby construct an integrative interaction map for C. elegans by integrating diverse functional genomics Datasets. We will also present some of the novel tools in querying the interaction map for answering powerful biological hypotheses.

Johnson, David W. et al. (2013) International Worm Meeting "The Max/Mlx transcriptional network influences aging in C. elegans."

Johnson, David W., Farrell, Sara, Samuelson, Andrew V., Llop, Jesse

[International Worm Meeting, 2013]

We have identified a role for the C. elegans Max and Mlx ("Max-like") transcriptional network as a key modulator of aging. Orthologous mammalian transcription factors have been implicated in nutrient sensing and metabolic control, suggesting that this network emerged early in evolution to provide a mechanism coupling nutrient availability to longevity. We hypothesize that the Max-like transcription factors form a conserved regulatory system at the intersection of nutrient sensing and longevity control. Notably, a deletion in the genomic region of one of the human MML-1 homologs is associated with Williams-Beuren Syndrome, which notably causes metabolic dysfunctions, silent diabetes, and symptoms of premature aging. In C. elegans the Max-like transcriptional network consists of two heterodimeric transcription complexes: MXL-1 (Max) dimerizes with MDL-1 (Mad) to repress transcription, while MXL-2 (Mlx) forms a dimer with MML-1 (Mondo) to activate transcription. Our data indicate that the Mlx (MML-1:MXL-2) and Max (MDL-1:MXL-1) dimers have opposing functions in longevity, reflecting their antagonistic roles in transcription. Furthermore, our data show that transcriptional activation via the Mlx complex is required for the full longevity conferred through reduced ILS and DR, and that the extended lifespan observed in the absence of the Max complex requires DAF-16/FoxO and PHA-4/FoxA. Interestingly, examination of published ChIP-Seq data revealed extensive transcriptional regulatory overlap between DAF-16, PHA-4, and MDL-1. Taken together our results suggest that Max-like transcription factors reside at a key nexus between and help to coordinate the transcriptional outputs of ILS and DR.

Lazaro-Pena, Maria et al. (2021) International Worm Meeting "HPK-1 prevents the decline of proteostasis through neuroendocrine control of the proteostatic network"

Diaz-Balzac, Carlos, Samuelson, Andrew, Das, Ritika, Lazaro-Pena, Maria

[International Worm Meeting, 2021]

The progressive decline of cellular proteostasis is a hallmark of normal organismal aging, and is the basis for the onset and progression of a growing number of neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's disease. These diseases share a common characteristic: the accumulation of proteotoxic aggregates that result in cellular dysfunction and death. Proteotoxic disease is opposed by a cellular proteostatic network (PN) of approximately 1500-2000 proteins, which maintain the proteome by balancing rates of protein synthesis, degradation, folding, and sequestration. We have identified the transcriptional cofactor HPK-1 (homeodomain-interacting protein kinase) as an important PN component that preserves proteostasis and extends longevity. We find HPK-1 is primarily expressed in the nervous system during adulthood. Loss of neuronal hpk-1 shortens lifespan, while neuronal overexpression of hpk-1 is sufficient to increase lifespan. Neuronal HPK-1 is responsive to both acute heat shock and chronic nutritional stress, suggesting HPK-1 may act within the nervous system to integrate diverse signals and coordinate adaptive responses within the PN. We investigated whether HPK-1 acts cell autonomously in neurons to mediate stress response and proteostasis, or alternatively functions cell non-autonomously from neurons to regulate these processes in peripheral tissues. Neuronal loss of hpk-1 hastens the collapse of both neuronal and muscle proteostasis. Conversely, neuronal overexpression of hpk-1 delays the progressive decline of both neuronal and muscle proteostasis. Neuronal HPK-1 overexpression produces a paracrine signal to hyper-induce molecular chaperone expression locally and a neuroendocrine signal to induce autophagy in peripheral tissues. To further investigate the non-cell autonomous regulation of neuronal HPK-1, we expressed HPK-1 in different types of neurons and found that overexpression of HPK-1 in serotonergic and GABAergic neurons is sufficient to preserve proteostasis in the muscle. Interestingly, overexpression of HPK-1 in serotonergic neurons, but not in GABAergic neurons, is sufficient to increase heat stress resistance, suggesting HPK-1 exerts a different PN function in these types of neurons to preserve proteostasis. Overexpression of HPK-1 in single types of neurons is not sufficient to increase lifespan, suggesting that its combinatorial roles in neuronal cell-types is required to extend longevity. Collectively, our results position HPK-1 at a central regulatory node acting from the nervous system, upstream of the greater PN, by exerting distinct but complementary roles in different types of neurons.

Farrell, Sara et al. (2011) International Worm Meeting "Insulin signaling pathway genes facilitating thermotolerance and protein homeostasis."

Llop, Jesse, Stolzenburg, Lindsay, Samuelson, Andrew V., Farrell, Sara, Johnson, David

[International Worm Meeting, 2011]

The daf-2 insulin-like signaling pathway is the most potent pathway for lifespan extension in C. elegans, converging on the DAF-16 transcription factor to regulate a large number of genes including free radical detoxifying genes and stress resistance genes. During aging, specific protein damage by reactive oxygen species increases exponentially to challenge protein homeostasis. Activation of stress response pathways, with induction of heat shock proteins, is central to the maintenance of protein homeostasis after stress. The ability to maintain protein homeostasis depends on both chaperone-mediated protein folding and induction of heat shock proteins. Additionally, chronic growth at higher temperature shortens lifespan and accelerates aging, presumably due to increased thermal stress that negatively impacts protein homeostasis. Protein homeostasis, as measured through protein aggregation is controlled by insulin signaling, dependent on HSF-1 and DAF-16. From a comprehensive functional genomic screen we have previously identified 103 genes that are necessary for decreased insulin signaling to extend lifespan. Animals are progeric after these gene inactivations by several independent measures including: premature age pigment accumulation and increased rate of aging, without drastically altering progeny production or an established biomarker for C. elegans aging; the activity ratio, or proportion of life an animal actively responds to stimuli. We sought to identify the progeric gene inactivations that impair the heat shock response or challenge protein homeostasis. The over-arching goal our research is to gain a comprehensive, systems-level understanding of the mechanisms that promote longevity in conditions of reduced daf-2 insulin-like signaling. We identified the progeric gene inactivations that are necessary for the increased intrinsic and acquired thermotolerance conferred by decreased insulin signaling. Additionally, we tested the progeric gene inactivations for differential lifespan at varying temperature, premature collapse of proteostasis, induction of heat shock proteins, and the suppression of hsf-1(o/e) lifespan. This comprehensive functional analysis identifies the subset of progeric gene inactivations that negatively impact protein homeostasis after acute or chronic stress.