Kim A. Caldwell - US grants

[unreadable] DESCRIPTION (provided by applicant): The orchestration of the temporal and spatial events responsible for proper cell division requires a host of proteins acting in concert to ensure that accurate mitotic progression occurs reproducibly. The focal point of this proposal is one such evolutionary conserved protein of unknown function termed NUD-1, a gene product that is essential for embryogenesis in the nematode model organism, C. elegans. Changes in protein levels of NUD-1 result in aberrant cell division, including a distinctive absence of midbody microtubules at anaphase during mitosis and loss of gamma-tubulin at centrosomes. Here we propose to further discern the functional relationship between NUD-1 and gamma-tubulin at the centrosome. Additionally, we will investigate the dependence of other centrosome-associated proteins on NUD-1 activity in vivo. Previous work has established that NudC, the mammalian homolog of NUD-1, is phosphorylated by the polo-like kinase Plk1, and that this interaction is necessary for the proper localization of Plk1 and progression through cytokinesis. It has been further demonstrated that Hsp90, a ubiquitous molecular chaperone, regulates Plk1 during mitosis and is required for Plk1 localization to centrosomes. Importantly, Hsp90 activity is dependent upon a co-chaperone termed p23, a protein that shares significant structural homology with NUD-1/NudC. We propose to investigate the possibility that Plk1-NudC interaction entails a regulatory mechanism involving Hsp90 to facilitate events essential to centrosome function in mitosis. Our approach employs a combination of RNA interference (RNAi), biochemistry, immunocytochemistry and fluorescent microscopy to gain a greater understanding of the molecular mechanisms by which NUD-1 and its partner proteins act in cell division, using the C. elegans one-celled embryo as a model system to investigate the detailed consequences of aberrant gene activity at the centrosome. Relevance to Public Health: The proposed proteins of study (NudC, Hsp90, and Plk1) are significant in various cancers including leukemia, breast, prostate and skin cancer. Additionally, prostate tumor growth can be inhibited by overproduction of NudC in prostate cancer cells in vitro. Elucidating the functional mechanisms mediated by these proteins in cell division provides a better understanding of the cancerous process, thereby contributing to the development of more defined avenues of therapeutic intervention. [unreadable] [unreadable] [unreadable]

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

Protein production, trafficking and degradation are essential for proper cellular function. Perhaps no cells are more sensitive to imbalances in these tightly coordinated processes than neurons. An important cellular compartment, the endoplasmic reticulum (ER), functions to balance levels of neurotransmitter transporters, receptors, and accessory proteins. Thus, deficits in the capacity of the ER to properly handle intracellular stress, such as misfolded proteins, may dramatically affect the activity or survival of neurons and disrupt homeostasis. The focus of this research is an ER-resident protein called torsinA, a member of a family of proteins that appear to have evolved among all multicellular organisms to act as cellular 'gatekeepers' in the prevention of cellular stress. This research uses the well-studied nematode, Caenorhabditis elegans, as a model system whereby the function of torsinA can be more rapidly discerned. Through a combination of behavioral, genomic, and biochemical experiments for evaluating torsinA activity, this study will advance our understanding of how neurons work to prevent stress and maintain homeostatic balance. C. elegans is ideal for this analysis, as this worm has a defined nervous system, a wealth of genetic tools applicable to detailed mechanistic analyses, and a transparent anatomy that enables changes in ER-associated stress to be quantified in living animals using fluorscent biomarkers. Moreover, the ease by which this organism is manipulated is attractive in terms of providing undergraduate students the opportunity to conduct and achieve significant research goals. Outcomes of this CAREER project include mentoring of graduate students, in addition to engaging underrepresented populations of freshmen students in the discovery process through a combination of classroom and mentored-research experiences through an institutionally-supported program, the Alabama Emerging Scholars, aimed at increasing retention of at risk students by early exposure to research.

DESCRIPTION (provided by applicant): Neurodegenerative diseases such as Parkinson disease (PD) comprise a major societal burden with increasing occurrence as our mean population ages. Recent studies have demonstrated that bacteria of distinct Streptomyces species have the capacity to produce a neurotoxic metabolite that causes dopaminergic degeneration in the nematode model organism C. elegans and in human SH-SY5Y neuroblastoma cells. These bacteria are commonly found in soil environments and exposure to their excretions may be a contributor to PD, which is more prevalent in individuals with a rural lifestyle. While exposures to pesticides may be a partially responsible, these alone cannot account for prevalence of PD, even in rural areas. This R15 proposal utilizes C. elegans as a primary assay system for scoring dopamine (DA) neuron degeneration to explore mechanisms involved in Streptomyces-induced neurodegeneration. While chronic exposures to the toxic metabolite cause DA neurodegeneration, experiments will be performed to determine if acute exposure paradigms also result in neurodegeneration (Aim I). Established mutant and transgenic worm strains will be utilized to investigate whether differential degeneration is observed in distinct genetic backgrounds related to PD (Aim II). Cellular pathways associated with neurodegeneration, such as apoptosis, DA metabolism and mitochondrial function will also be assessed following exposure to the metabolite (Aim III). Finally, studies in human neuronal cell cultures will be conducted, both to validate results gleaned from the invertebrate system and to expand upon data demonstrating that the neurotoxic activity of the Streptomyces metabolite extends to these cultures (Aim IV). Taken together, these studies represent an integrated strategy to rapidly discern the significance of this novel environmental factor and its influence on neurodegeneration as it pertains to PD. Included among the broader impacts of this proposal are an understanding of one possible cause of health disparities in rural populations, in addition to training and mentorship of graduate and undergraduate students. PUBLIC HEALTH RELEVANCE: Parkinson's Disease (PD) is the most common movement disorder affecting over 1 million Americans, yet underlying causes of this neurodegenerative disease have largely eluded medical science. This proposal is designed to address an unmet challenge of discerning environmental factors that contribute to PD by investigating the potential for exposures to a toxic compound produced by common soil bacteria to enhance susceptibility to neurodegeneration over the course of aging. Through an integrated training and experimental strategy involving student-centered research, mechanisms underlying neurotoxicity will be explored using a whole animal model system, as well as human cell culture experiments, whereby outcomes include potentially establishing a new paradigm for understanding neurodegeneration that may aid in the development of models to advance diagnostic and therapeutic strategies to combat PD.

Project Summary/Abstract As our population ages, neurodegenerative disorders such as Parkinson disease (PD) comprise a major societal burden. While mechanisms for PD etiology are still emerging, evidence of mitochondrial dysfunction in the pathogenesis of this disease is abundant. Another component of PD pathology is the protein a-synuclein (a-syn); it is found within Lewy Body inclusions, yet causes of cellular toxicity remain unclear. A strategy that mitochondria employ for managing stress is to engage the mitochondrial unfolded protein response (UPRmt), which coordinates nuclear expression of chaperones and proteases that translocate to the mitochondria to handle damaged and/or unfolded proteins. When activated in response to acute stressors, the UPRmt re- establishes homeostasis and promotes cell survival. However, it can become dysregulated when challenged with a long-term genetic stressor such as misfolded a-syn and becomes cytotoxic. Notably, molecular variants of a-syn can interact with TOMM20, an outer mitochondrial membrane protein, and initiate a physical block of mitochondrial protein import. We speculate that the increased UPRmt response observed in a-syn-expressing neurons is a consequence of blocked mitochondrial import. Although attention to a role for mitochondrial quality control in neurodegenerative disease has proven increasingly insightful, there is a pivotal gap that remains to be addressed in demonstrating a direct functional correlation between dysregulated UPRmt activity and neurodegeneration. Importantly, our research illustrates an insidious aspect of mitochondrial signaling in which the UPRmt pathway exacerbates disruption of dopaminergic neurons in vivo, resulting in the neuron loss characteristic of PD. Our approach exploits the expedience of genetic manipulation in Caenorhabditis elegans research, and the rigor with which large, isogenic populations can be scored for neurodegeneration with unprecedented accuracy, at the single-neuron level. We will systematically investigate combinations of transgenic worms co-expressing structural variants of a-syn and transcription factors that activate the UPRmt to discern functional requirements for UPRmt activation with neurodegeneration as the primary endpoint. The studies in Aim 1 will investigate the hypothesis that the a-syn-TOMM20 mitochondrial import block triggers the UPRmt pathway and will explore a role for dopamine in potentially exacerbating the deleterious consequences of this process. As a distinct strategy, Aim 2 will involve the identification of molecular components associated with UPRmt signaling through a forward genetic screening strategy that takes advantage of a strain we have generated that reveals an uncharacterized compensatory mechanism for UPRmt induction. Phenotypic bioassays and genetic screening using C. elegans are routinely conducted by undergraduates in our lab and will serve as an excellent training opportunity for students through this R15 proposal. These studies represent a timely and mechanistic strategy towards defining nuclear-mitochondrial dynamics, specifically with respect to dopaminergic neurodegeneration, with potential to inform a translational path for therapeutic development.