Andrew Dillin - US grants

It was recently discovered that reduced ETC signaling in neuronal cells is sufficient to extend the lifespan of C. elegans. It was also found that this effect is dependent upon the activity of an essential component of the mitochondrial stress response or UPRmt. It is not yet understood, however, the fundamental mechanisms by which this life span extension occurs or how the signal is sent and perceived. Moreover, the essential role that the mitochondrion has in cellular homeostasis and energy production suggests that it may act as a reactive sensor of random intrinsic or extrinsic variables capable of influencing an organism's susceptibility to disease. Changes within the mitochondria thus also might be responsible for the emergent properties displayed in such a system in response to stochastic changes, and/or may play a significant role in coordinating the activation of non-mitochondrial stress response pathways. A prediction that genetic modifications will decrease the capacity for stochastic variation in mitochondrial function will ultimately negatively affect the fitness of the organism. Such a hypothesis is in keeping with recent evidence suggesting that deleterious mutations actually decrease the sensitivity of gene expression in response to small environmental changes (a loss of phenotypic robustness). A further hypothesis is it may predict co-variance between the UPRmt and stress response pathways, currently thought to act in distinct regulatory networks, and seek to discover the potential mechanisms by which this co-variance occurs.

Project Summary Mitochondrial dysfunction is a primary consequence of nearly all age-onset neurodegenerative diseases, and can be the consequence of exposure to even mild levels of a wide-range of environmental toxins. Surprisingly, the applicant's lab has uncovered that mitochondrial stress in one cell type can be communicated to a distal cell type that has not undergone mitochondrial stress. Using issue specific promoters in the nematode C. elegans that drive expression of dsRNA of a subunit of complex IV of the ETC in the nervous system, it has been shown that neuronal mitochondrial stress can be communicated to distal cells, such as the intestine. The ability of the nervous system to communicate this mild stress results in increased longevity and survival of the entire animal. The goal of the proposed studies is to identify the source and nature of this signaling mechanism.

DESCRIPTION (provided by applicant): Within invertebrate model organisms such as C. elegans and Drosophila, evidence strongly suggests that tissue-specific manipulations of stress response pathways can affect the aging process of the entire organism. While originating from a single tissue, these manipulations appear capable of propagating synchronous changes to age-related phenotypes across multiple tissues and organs. These compartment-specific stress responses share a role in ensuring maintenance of the proteome, a loss of which would otherwise be catastrophic to the viability of the cell. Because dysfunction in the endoplasmic reticulum (ER) has been associated with a wide-range of age-onset metabolic diseases, including diabetes, obesity, and atherosclerosis, we hypothesized that a restoration of the ER stress response might also have a protective effect on the viability of older animals. We did not know if such a manipulation would affect ER stress response and function cell-autonomously or whether the ER stress response too could be recognized and responded to by distal tissues. Surprisingly, we have discovered that activation of the UPRER in one cell type can also be communicated to a distal cell type that has not undergone ER stress. Using issue specific promoters in the nematode C. elegans that drive expression of spliced version of the UPRER -activating transcription factor XBP-1, we find that neuronal UPR activation can be communicated to distal cells, such as the intestine, resulting in the remote upregulation of ER chaperones. As a consequence, UPRER activation in the nervous system results in increased longevity and stress resistance of the entire animal. This cell non-autonomous response reinforces the idea that in a multi-cellular organism, the sensing of protein folding stress must b conveyed and responded to by the entire organism. The endocrine system is thus an integral and necessary part of multiple conserved cellular stress response pathways. Our data suggest that the UPRER is a cell non-autonomous regulator of age-dependent stress resistance and longevity. We do not yet know the source of this signal, and we do not yet understand the underlying mechanisms of its action. In this proposal, we employ a multi-pronged approach combining genetics, metabolomics, ribosomal profiling, and peptidomics to identify and characterize the signal and its origin. We then use similar techniques to examine the perception of the signal and its consequences in responding tissue. We undertake this research in the hope that novel mechanisms involved in cell non- autonomous UPRER signaling may provide new therapeutic targets for age-onset diseases. We further more hope that such explorations provide valuable insight towards understanding adaptations by which an environmental, extrinsic signal can be sensed and then amplified across the entire animal to coordinate the appropriate onset of reproduction, senescence and/or aging.

DESCRIPTION (provided by applicant): The enclosed proposal presents a revised training program in genetics at UC Berkeley. The program spans four separate administrative units on the Berkeley campus: the Departments of Molecular & Cell Biology (MCB), Plant & Molecular Biology (PMB), Integrative Biology (IB), and the School of Public Health (PH). The program includes 43 training faculty, all prominent researchers in their respective areas of study (10 are members of the National Academy of Sciences). Training faculty represent three major areas of genetics research: developmental genetics, cell & systems-level analysis, and population genetics & evolution. Funds are requested to support the research activities of 15 graduate student trainees (per year) in genetics-oriented laboratories located within the four aforementioned administrative units. Trainees will be appointed by a steering committee composed of prominent faculty from each of the participating administrative units. This committee is also responsible for renewing the appointments of training faculty and appointing new faculty to the training program. In addition, the committee will review the efficacy of the training program by soliciting comments from past and present trainees and faculty. A number of mechanisms have been established to ensure that the proposed genetics training program offers a unique educational experience that is distinct from other programs on the Berkeley campus, such as genomics, cell & molecular biology, and immunology. First, genetics trainees are required to present their current research findings at an annual retreat that is restricted to trainees and their mentors. Second, all trainees will be required to enroll in at least one advanced genetics course, such as MCB 240. Third, all trainees are required to attend the annual Genetics, Genomics, and Development (GGD) retreat regardless of their departmental affiliations. And fourth, all trainees must participate in at least one additional genetics-oriented forum, such as the weekly GGD seminar series or regularly scheduled journal clubs and research meetings focused on topics in genetic analysis. A variety of outreach strategies will be employed to ensure that members of under- represented minorities, women, and disabled or otherwise disadvantaged students are given the opportunity to obtain training in this program and thereby gain access to the wonderful world of genetics.

Mitochondrial dysfunction is a primary consequence of nearly all age-onset neurodegenerative diseases. Across eukaryotic species, however, mild mitochondrial stress can have beneficial effects on the lifespan of organisms. Studies on the roles of mitochondria in the aging process have suggested that reduced mitochondrial function during a critical window of development in the nematode C. elegans is sufficient to extend the lifespan of the organism. Mitochondrial stress during this time results in a massive and persistent restructuring in gene expression patterns, as evidenced by analyses of long-lived mitochondrial mutant animals. This sustained response to an early metabolic stress may allow the organism to adapt its adult metabolism to match predicted states of nutrient availability. Previously, we reported that reduced mitochondrial function specifically in the neurons was sufficient to extend the lifespan of the nematode C. elegans. Mild neuronal mitochondrial stress also caused an upregulation in mitochondrial stress signaling across distal tissues of the organism. We now report evidence for the requirement of a class of metabolic neurotransmitters in the dissemination of perceived mitochondrial stress. We also observe a neuron-specific epigenetic remodeling in response to mitochondrial dysfunction. We hypothesize that, after sensing metabolic stress, neurons transcriptionally remodel their gene expression patterns by activating a class of neuron-specific chromatin modifying enzymes. Transcriptional changes in the neurons then initiate a downstream neuroendocrine signaling event that is capable of activating mitochondrial stress responsive pathways across tissues and organs. This cascade of responses collectively serves to increase the metabolic fitness and lifespan of the organism.

The conserved heat shock transcription factor-1 (HSF-1) is essential to cellular stress resistance and life-span determination. The canonical function of HSF-1 is to regulate a network of genes encoding molecular chaperones that protect proteins from damage caused by extrinsic environmental stress or intrinsic age-related deterioration. In Caenorhabditis elegans, we discovered a modified HSF-1 strain that increased stress resistance and longevity without enhanced chaperone induction. Intriguingly, both modified HSF-1 and wild type HSF-1 were instead capable of increasing expression of an array of actin regulating genes. These data suggest that HSF-1 has a prominent role in actin cytoskeletal integrity. Surpassingly, upregulation of at least one of these actin components was alone sufficient to increase stress resistance and life span. We hypothesize that a loss in actin homeostasis occurs during the aging process, and that this loss is driven by the inability for HSF-1 to normally mount a response to protect actin from stress in aging cells. In this proposal, we will explore how actin homeostasis becomes compromised during normal aging, and whether the activity of HSF-1 will protect the cells from age-onset declines in function. We will use state-of-the-art, in vivo imaging techniques alongside innovative biochemical analyses to monitor changes in actin structure and dynamics both spatial and temporally. We predict that forced expression of hsf-1 in geriatric animals will restore the function of the actin cytoskeleton, protecting the cell from age-onset damage and extending lifespan. We will further explore the possibility that hsf-1 works as a part of a team of additional stress-responsive proteins designed to manage a ?actin cytoskeletal stress response? that be compromised with age, and propose a series of genetic screens to identify other actin-regulatory factors. Finally, we will explore the idea that changes in actin dynamics must be coordinated across tissues and cells, suggesting a role for hsf-1 in the endocrine mediated regulation of actin dynamics. We will leave this work with a newfound understanding of the role of actin homeostasis plays in many of the destructive diseases seen in older individuals.

Cells are repeatedly exposed to various stressors that disrupt protein homeostasis (such as infection, excess nutrients, heat, genetic mutations), resulting in protein misfolding and aggregation. To maintain protein homeostasis (proteostasis), cells have evolved compartment specific stress responses such as the Unfolded Protein Response of the endoplasmic reticulum (UPRER). In times of ER stress when the load of misfolded or unfolded proteins overwhelms the ER, the UPRER is initiated to restore proteostasis. Unfortunately, the ability to mount an effective UPRER is impaired with age, which likely contributes to the accumulation of misfolded proteins - a central molecular hallmark of aging and many degenerative diseases. Our laboratory discovered that ectopic expression of the UPRER transcription factor xbp-1s in neurons is sufficient to prevent age-onset loss of UPRER throughout the organism. Surprisingly, neuronal expression of xbp-1s leads to cell non-autonomous activation of the UPRER in distal, intestinal cells and extends lifespan in C. elegans. Initially this phenomenon was ascribed only to neurons, however recent data from our lab suggests glial cells are more potent cell non-autonomous regulators of ER stress resistance and longevity. Animals lacking a subtype of glial cell are more susceptible to chronic ER stress. Conversely, expressing xbp-1s in glia results in robust ER stress resistance and lifespan extension in a mechanism that is distinct from that initiated by neuronal xbp-1s. Therefore, we hypothesize that glial cells play a central role in coordinating organismal ER stress resistance and longevity. In this proposal, we outline our strategy to pinpoint the origin and identity of the glial cell non-autonomous signal (Aim 1) and to uncover the mechanism by which the signal is perceived in distal tissues (Aim 2). Our approach utilizes techniques which combine the traditional advantages of using C. elegans as a model system (genetic tractability, transparency, short lifespan), with advanced technologies (large particle flow cytometry and tissue-specific ribosomal profiling) to study cell non-autonomous signaling between tissues in the context of aging. Data generated through this proposal will implicate glia as cell non-autonomous regulators of aging and open new avenues for metabolic and neurodegenerative disease therapeutics.

ABSTRACT All cells within our bodies are surrounded by the extracellular matrix (ECM), which consists of a network of proteins and polysaccharides secreted by cells. The ECM is not only critical for physically supporting cell and tissue structures, but is also mediates a large number of receptor signaling on the cell surface that enables proper cellular functions and tissue homeostasis. For example, recent work from our lab have shown that TMEM2, which breaks down a major ECM polysaccharide, hyaluronic acid (HA), can modulate ER stress resistance and immune response in both human cell culture and the C. elegans model system. This highlights the importance of ECM- derived signals in maintaining cell and organismal health. However, the underlying signaling pathways and specific changes of ECM that regulates ER stress and immunity remain unclear. To understand how ECM changes can signal to alter ER stress and immune response, we propose to first use TMEM2 as a model for ECM alteration and elucidate how TMEM2 activity influences ER function and morphology. Next, we will determine whether such ER changes influence immune signaling and identify genetic regulators downstream of TMEM2 that mediates immune signaling in both C. elegans and human cell culture systems. Finally, to uncover how other alterations in ECM regulates ER stress and immune response, we will characterize how expression of TMEM2 along with other known human and C. elegans ECM-modifying proteins alter the ECM network, and determine whether these changes can also modulate ER proteostasis and immune signaling. Taken together, our proposal will reveal ECM changes regulate ER stress and immune signaling, providing insight into how cells and tissues might sense extracellular signals to promote organismal homeostasis.