Guy Caldwell - US grants

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Dystonia is estimated to be six times more prevalent than Huntington's Disease, ALS, or Muscular Dystrophy. However, as few as 5% of the over 350,000 persons in North America estimated to be affected have been correctly diagnosed and are under treatment (NIH Budget Office). The most severe early-onset form of this disorder has been linked to a mutation in a human gene named TOR1A that encodes torsinA, a protein that is also localized to inclusions in the brains of Parkinson's patients termed Lewy bodies. While a causative genetic mutation has been identified, the cellular mechanisms of pathogenesis underlying dystonia remain unknown. We are applying the advantages of the model organism, Caenorhabditis elegans, towards a detailed analysis of two specific torsin-related gene products in this nematode. The chromosomal positioning of these genes suggests that they may represent a functionally co-expressed unit and preliminary studies from our laboratory indicate they act neuronally. Phylogenetic analysis of the torsin family indicates these proteins share distant sequence similarity with the functionally diverse AAA+ family of proteins. We have determined that ectopic overexpression of a C. elegans torsin homolog results in a reduction of polyglutamine repeat-induced protein aggregation in a manner similar to that previously reported for molecular chaperones. The suppressive effects of torsin overexpression quantitatively persisted as animals aged. Antibody staining of transgenic animals using antisera specific to TOR-2 indicated this protein was highly localized to sites of protein aggregation. We propose to extend these preliminary studies through a combination of reverse genetic approaches designed to investigate the cellular role of torsin proteins in the nematode. The specific aims of the proposed project include: 1) to determine what phenotypes are associated with C. elegans torsin homologues; 2) to define sites of C. elegans torsin protein function; and 3) to determine potential effectors of torsin activity. These studies will further our understanding of the molecular mechanisms responsible for early-onset torsion dystonia. Moreover, the aberrant protein deposition associated with diverse neurodegenerative disorders like Parkinson's Disease and those caused by polyglutamine expansion such as Huntington's Disease warrants further investigation of any putative neuroprotective effects of torsins. [unreadable] [unreadable]

0237956
Guy A. Caldwell

CAREER: An Integrated Discovery-Based Genomic Approach to Discerning the Role of Nuclear Positioning Genes in Neurodevelopment

The positioning of nuclei within cells is essential for development and is involved in such diverse processes as fertilization, cell division, as well as migration and synaptic activity of neurons. Dr. Caldwell's laboratory has established a link between genes associated with the cellular cytoskeleton and deficits in neuronal activity, specifically related to proteins implicated in nuclear positioning. As the complexity of the human brain renders it intractable to detailed molecular characterization, the proposed studies will utilize the powerful attributes of the nematode model organism Caenorhabditis elegans. This simple animal shares most cellular features commonly associated with human neuronal function (ion channels and neurotransmitters like dopamine and GABA). Dr. Caldwell's group has identified a set of evolutionarily conserved nuclear positioning genes and are performing a comprehensive series of experiments to examine the mechanisms by which genetic factors regulate cytoskeletal establishment of neuronal development and activity.

Results of the research will have an impact beyond developmental neuroscience into areas of cell biology in general. This CAREER proposal provides thoughtful integration of research and educational concepts.
Its focal point is a discovery-based genomics lab course that incorporates Internet-based bioinformatics exercises within an experimental context. The education impacts of these studies include the involvement of minority participants, enhancement of research alliances with liberal arts colleges and other universities, cross-disciplinary projects in bioinformatics, and a novel student-edited interdisciplinary on-line science journal designed to foster scientific literacy.

This award provides partial support for the acquisition of a laser scanning confocal microscope to be housed in the Optical Analysis Center, a multi-user facility in the Department of Biological Sciences at the University of Alabama. The instrument will meet the growing needs of faculty, students and postdocs in the Departments of Biological Sciences, Chemistry, and Chemical Engineering. The equipment will also support three interdisciplinary centers: The Center for Biomolecular Products, The Center for Freshwater Studies, and The Center for Applied Biological Engineering Research. The instrument will replace a 10-year old scanning confocal which is now obsolete, and will allow in-house optical analyses that currently require researchers to travel to the University of Alabama, Birmingham, over 50 miles away. In addition, the instrument will be available for collaboration and training of students and faculty at several two and four-year institutions in western Alabama. The existing microscope will be retained both to support needs for analysis using inverted optics, as a training instrument for an existing graduate course in microscopy, and for research training of undergraduate students.

This award provides funding for the purchase of a new transmission electron microscope and ultramicrotome for the Optical Analysis Facility in the Department of Biological Sciences at the University of Alabama. These instruments will provide state of the art equipment to meet the needs of the rapidly growing research groups in cellular and molecular biology, ecology and molecular systematics in the Department of Biological Sciences, and for faculty in the Department of Chemistry and the Department of Chemical Engineering. The transmission electron microscope will be equipped with a LaB6 gun, eucentric goniometer stage, cold trap anti-contamination device, a sheet film camera and a CCD camera with digital imaging acquisition hardware, computer and software. The ultramicrotome will be equipped with eucentric tilt capability, a touch screen and a high quality binocular system. This instrument will add a microtome with modern features, flexibility, and the capability to produce many sections of consistent thickness required for serial sectioning at the Facility. The microscope and ultramicrotome will be used for a broad range of studies including the cytoskeletal structure and organelle arrangement in wild type and mutant forms of the heterotophic protist Tetrahymena, the cellular management of protein misfolding, the fine structures of chytrid zoospores, the internal structures of sponges and their spicules, the cellular mechanisms that regulate intestinal performance, localization of 1L-myo-inositol 1-phosphate synthase in plants, and imaging hydrogel materials for use as biomaterial deposits for targeted drug delivery.

The Optical Analysis Facility is a major resource for individual and collaborative research efforts in the Departments of Biological Sciences, Chemistry and Chemical Engineering, and three interdisciplinary centers, the Center for Biomolecular Products, the Center for Freshwater Studies and the Center for Green Manufacturing. Research conducted with the equipment will have broad impact in such diverse areas as the lineage of a group fungi that are common parasites on organisms from algae to frogs, factors involved in the cellular dysfunction in the neurological diseases dystonia and Parkinson's disease, and targeted drug delivery. In addition, these instruments will be available for training on an individual basis and in the existing graduate course in microscopical techniques and for demonstrations and tours for classes at the University of Alabama and area students from two-year and four-year colleges, primary and secondary schools. These instruments will replace aging equipment and insure that reliable equipment optimized for biological specimen preparation and observation will be available.

[unreadable] DESCRIPTION (provided by applicant): Parkinson's disease (PD) results from an imbalance in cellular mechanisms designed to cope with environmental stresses to neurons. The two major clinical hallmarks of PD, protein inclusions termed Lewy bodies and dopamine neuron degeneration, are representative of failure in the intracellular management of stress. While genetic forms of PD are rare, these mutations highlight the involvement of pathways that regulate protein folding and oxidative damage in cells. Given the predominance of sporadic PD, environmental sources of toxins may serve as potential risk factors for individuals with specific genetic predispositions. One environmental factor that may contribute to PD is exposure to certain bacteria that produce proteasome inhibitors, such as specific strains of the order Actinomycetales. Here we propose to utilize the nematode roundworm, C. elegans, to mechanistically investigate exposure to bacterial strains implicated in PD. Our lab has previously established this worm model for rapid evaluation of factors influencing both the misfolding of human alpha-synuclein and neuroprotection of dopamine neurons. The aims of our proposal include investigating dopamine neuron degeneration as caused by bacterial exposure in wild-type worms and in genetically defined backgrounds. We will evaluate susceptibility to bacterial exposure in animals defective in worm homologs of known PD genes, in addition to novel PD gene targets obtained from a large-scale RNA interference (RNAi) screen we have performed. Transgenic nematodes containing fluorescent reporter gene constructs will be used to distinguish systemic effects of exposure on various neuronal subtypes, general stress response, and proteasomal inhibition. Differential changes in gene expression in response to bacterial exposure will also be profiled using whole-genome oligonucleotide microarrays to identify potential genes regulated in response to environmental toxins. Relevance to Public Health: The interplay between genetic predisposition and susceptibility to environmental insults lies at the core of PD. Risk factors are best evaluated using systems wherein environmental conditions and genetic differences are strictly controlled. C. elegans, a microscopic worm with precisely 8 dopamine neurons, shares about half of its genes with humans and represents an ideal system to rapidly examine potential sources environmental toxins that may influence development of PD. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Therapeutic advances to treat Parkinson disease (PD) are incumbent upon the identification and mechanistic understanding of cellular factors that influence neuronal survival. We have exploited attributes of the nematode model system, Caenorhabditis elegans, to develop assays that facilitate the screening and isolation of conserved genetic factors that mediate clinical hallmarks of PD, including intracellular aggregation of a-synuclein (a-syn) and dopamine (DA) neuron loss. This R15 application extends prior studies where we have identified neuroprotective targets for translational development and established genetic platforms to evaluate functional modifiers of age-dependent neurodegeneration. Increasing evidence is mounting for a significant role for lysosomal function as a mechanism influencing PD. In this context, a focal point of this application is the ULK2 protein - a human ortholog of the worm unc-51 gene product, a kinase implicated in autophagy, as well as axon elongation and guidance. We previously uncovered a neuroprotective activity for ULK2 in enhancing DA neuron survival in transgenic nematodes. Our data took on greater significance following the report of a human genome-wide association study that also identified a polymorphism in ULK2 as being associated with PD patients. We propose to advance our understanding of ULK2-mediated neuroprotection through a series of structure-function analyses, as well as evaluation of modifiers of ULK2, to mechanistically define the role of ULK2 kinase activity in attenuating neurodegeneration. C. elegans is also among the best-understood animal models in terms of aging mechanisms, with extensive sets of factors implicated in lifespan identified. Thus, as aging represents an unequivocal and defined risk factor for PD, we will use functional genomic screening via RNA interference (RNAi) in C. elegans to knockdown hundreds of genes previously linked to aging-associated pathways to evaluate their distinct contribution to a-syn misfolding and clearance. Preliminary studies in our lab have shown that specific mutations in key components of the daf-2/insulin-like signaling pathway of C. elegans result in substantial effects on DA neuron survival. We have generated a series of transgenic nematode strains to facilitate functional analysis of a-syn modifiers in the context of their potential dependence on this pathway, as well as in autophagy. Transgenic and mutant worms will be generated to examine gene targets from RNAi screening for their impact on DA neuroprotection. This systematic approach provides an unprecedented opportunity to discern age-associated regulators of neurodegeneration that may represent genetic susceptibly markers for PD onset or progression. Collectively, these studies represent an integrated research plan designed to rapidly define the significance of previously uncharacterized factors influencing neurodegeneration. Moreover, our experimental strategy coincides with the specific criteria of the R15 AREA program, as the broader impacts of this application involve extensive undergraduate and graduate student training opportunities in an environment ideally suited to student-centered research. PUBLIC HEALTH RELEVANCE: Over 1 million Americans have been diagnosed with Parkinson's Disease (PD) - the most common movement disorder for which a cure has eluded medical science for decades. This application addresses an unmet challenge of discerning genetic factors that may contribute to this neurodegenerative disease, while accelerating our understanding of underlying cellular mechanisms of PD. In this research, we use a simple animal model system to explore the well-established but poorly understood relationship of aging to PD, in addition to genetic factors that mediate the survival of dopamine-producing neurons. The experimental strategy outlined integrates student-centered research in the context of a systematic approach involving genetic and genomic analysis to uncover mechanisms that facilitate identification of new therapeutic targets with the potential to combat PD.

Project Summary Advances in genomic technologies have accelerated the pace of discovery with respect to genetic variation. The use of model systems is an expeditious route for the assessment of distinctions that render select individuals in a population more resilient to cellular stress and dysfunction. We have developed in vivo assays using the nematode, Caenorhabditis elegans, that facilitate isolation of conserved modulators of dopamine (DA) neurodegeneration and Parkinson?s disease (PD). To discern molecular factors conferring ?resilience?, it is essential to define our experimental goals in practical terms. Here, an inherent capacity of DA neurons to resist progressive degeneration induced by a-synuclein, a protein central to PD, represents an operational definition of resilience. C. elegans facilitates rapid analysis among isogenic populations and highly precise quantitation of neurodegeneration at a single-neuron level in animals challenged by a-synuclein. In Aim I, we will test a hypothesis for the mechanistic nature of dopaminergic resilience that postulates the system by which small RNA transport for epigenetic transmission intersects with regulators of DA transport and endocytosis to provide neuroprotection. A recently identified gene found to contain exonic variation in PD patients, TNK2, encodes a protein that controls dopamine transporter endocytosis. Intriguingly, the sole worm ortholog of TNK2 is SID-3, an endocytic regulator of the SID-1 dsRNA transporter responsible for systemic RNAi. Preliminary data demonstrate that TNK2/SID-3 modulates neurodegeneration. Moreover, we reported the discovery of a neuroprotective small molecule that acts directly on an E3 ligase, Nedd4, responsible for targeting TNK2 for degradation. Using mutant and transgenic analysis, we will dissect the role of Nedd4 in modulating both DA and small RNA transport via TNK2/SID-3. Since we hypothesize resilience is mediated through the activity of select miRNAs, in Aim 2, as a complementary approach, we will evaluate a set of epigenetically-regulated gene targets of a specific miRNA, mir-2 for DA neuroprotection. We have discerned that genomic knockout of mir-2 confers robust protection from a-synuclein-induced DA neurodegeneration in transgenic C. elegans. Therefore, we will conduct a systematic functional analysis of a subset of prioritized candidate gene targets predicted to be regulated by mir-2. Animals will be rigorously examined to quantify functional effects on DA neuron degeneration by conditional knockdown using DA neuron-specific RNAi. We will then counterscreen mir-2 targets to categorize either cell autonomous or non-cell autonomous effects on neuroprotection. We will subsequently coordinate our results with human genomic datasets to identify any single-nucleotide polymorphisms in candidate genes, and functionally evaluate conserved variations. Positives will be advanced for behavioral, lifespan and healthspan analyses. These assays provide excellent training opportunities for undergraduates who routinely conduct such methods in our lab. In all, our strategy is designed as an integrated approach towards expanding new therapeutic targets and mechanisms to halt neurodegeneration.