Peter M. Douglas, Ph.D. - US grants

DESCRIPTION (provided by applicant): The synchronous communication among different cell types within a tissue or organism to achieve normal function is poorly understood. A variety of secreted extracellular signals that are generated in discrete tissues often have the potential to impact homeostasis throughout the entire organism. Occasionally, these signals can be destructive: in numerous age-onset neurodegenerative diseases, for example, dysfunction arises in a defined subset of neurons and subsequently initiates a degenerative cascade in peripheral tissues. Yet, signals from one tissue to another can also be constructive and end in the coordinate protection of the organism. Dr. Douglas has identified a transduction signaling component that originates in the nervous system and can confer protection against stress in distally associated tissues. Preliminary studies show that expression of a constitutively-activated Heat Shock transcription Factor, HSF-1, exclusively in the nervous system of the nematode C. elegans: (1) protects against heat stress; (2) slows aging; and (3) detoxifies model-disease proteins in distal tissues. Heat-protection conferred by secreted HSF-1 signals requires a functional thermo- sensory neural circuit and RNA transporters, suggesting that a putative RNA signal is required for its propagation. In contrast, long-lifespan and proteo-detoxification phenotypes require a distinct signaling pathway for their propagation involving the insulin/IGF-1 transcription factor DAF-16. Thus the site and nature of HSF-1 activation in the nervous system specifies its mode of protection against different stressors. In this study, Dr. Douglas will characterize the generation, propagation and ultimate receipt of these extracellular signaling events in a living, intact organism. In Aim 1, he will identify key neurons which are capable of secreting protective HSF-1 signals and define the participating machinery within those select neurons. In Aim 2, the identity of each divergent signal will be uncovered. In Aim 3, cellular factors will be identified within recipient tissues which are required to recognize the different HSF-1 signals and initiate protective programs. Training in the mentored phase will prepare Dr. Douglas to direct an independent lab using the well developed and tractable genetics, neurobiology, and biochemistry of C. elegans to address new questions of biological importance. Training under this award will include: learning to utilize the C. elegans nervous system to study neural-born extracellular signaling events in the intact animal; mass spectrometry techniques in quantitative proteomics which are necessary to identify pertinent signaling molecules and associated machinery; bioinformatic techniques necessary for small RNA profiling and tissue-specific ribosomal profiling; new investigational methods in the fields of protein homeostasis and aging; and mentorship skills such as teaching and grant writing. These will greatly facilitate Dr. Douglas' transition and success as an independent investigator.

Project Summary/Abstract Aging involves the gradual decay of tissues, organs and organ systems. Early in this process, impermeability or selective permeability of tissues deteriorates and gives rise to increasing organ dysfunction. This phenomenon has been termed barrier dysfunction, yet the molecular mechanisms, which drive tissue ?leakiness? and contribute to organ aging are unclear. To interrogate the underlying mechanism, we examine the aging intestine of the nematode, C. elegans, to determine how resiliency of the intestinal barrier withstands the test of time. Preliminary screens from my lab have linked age regulation by the Heat Shock transcription Factor, HSF-1, with the activity of the intestine-specific actin protein, ACT-5. Although expressed in a small number of cells and comprising less than 2% of total worm actin, ACT-5 plays an essential role in intestinal and organismal aging. Through the proposed five-year research period, we aim to understand how age-related decline in HSF-1 activity contributes to tissue dysfunction and animal aging. In particular, we will characterize the molecular mechanism of age progression in which HSF-1 dysregulation impairs cellular architecture and intestinal physiology. We speculate that age-associated decline in HSF-1 activity impairs specialized actin networks in intestinal epithelium, which ultimately compromise vesicular traffic, cell-cell junctional integrity and tissue barrier maintenance. We have already identified the stress-activated JUN kinase, KGB-1, as a repressed transcriptional target of HSF-1, which catalyzes phosphate addition to the ACT-5 protein within its binding site for the actin filament stabilizing Troponin complex. Accumulation of phosphorylated ACT-5 at serine residue 232 dramatically influences the structural integrity of the apical terminal web and vesicular transport across it. Overall, the proposed research will uncover a phosphorylation-dependent actin relaxation mechanism under HSF-1 control, which facilitates vesicular transport across dense, actin-rich ?roadblocks? while still maintaining their structural rigidity and cellular architecture.

Project Summary/Abstract Lipid signaling plays a critical role in the regulation of organismal physiology and metabolic expenditure. Imbalances in lipid homeostasis can deleteriously impact health and cells within the organism tightly regulate lipid absorption, synthesis and metabolism to accommodate energetic demands and ensure energetic reserves later in life. Cells stockpile energy reserves under ample metabolic resources through SREBP-regulated lipogenesis. Yet, less clear is how cells regulate lipid homeostasis under nutrient depleted conditions and in particular, how cells sense metabolic demand and respond by increasing nutrient absorption. Our examination of several lipid depletion paradigms in C. elegans has identified a highly responsive small G protein, RAB-11.2, which is transcriptionally activated upon defects in the isoprenoid/mevalonate synthesis pathway. Through further investigation, we have discovered a new mechanism linking the nucleocytoplasmic dynamics of the nuclear hormone receptor, NHR-49, with nutrient absorption through RAB-11.2. Through the proposed five-year research period, we aim to define the molecular mechanism by which cells sense and respond to their need for de novo lipid synthesis. Our preliminary data suggests that cells sense their capacity to breakdown lipids through monitoring the availability of a particular prenol lipid synthesized through the isoprenoid pathway, geranylgeranyl pyrophosphate. Under conditions of high homeostatic lipid levels, geranylgeranylation of RAB-11.1 enables it to bind and sequester NHR-49 to cytosolic transport vesicles in a transcriptionally inactive state. Under lipid limited conditions caused by starvation or defective lipolysis/?- oxidation, cells lack the resources to synthesize GGPP through the isoprenoid pathway, which prevents RAB- 11.1 from binding vesicles and disrupts endocytic recycling pathways required for nutrient absorption. Due to the inability of its RAB-11.1 binding partner to associate with vesicles, NHR-49 is release from cytosolic vesicles and translocates to the nucleus where it activates transcription of several metabolic enzymes, nutrient transporters and RAB-11.2 to re-establish nutrient absorption.