2003 — 2005 |
Zarnescu, Daniela C |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Characterization of a Novel Fragile X Interacting Gene
DESCRIPTION (provided by applicant): Fragile X syndrome is the most frequent form of inherited mental retardation, affects about 1 in 3,500 males and to date has no cure. Patients have a pleiotropic phenotype that includes mental retardation, facial dismorphia as well as attention deficit and hyperactivity disorder. The disease is caused by mutations in the Fmr1 gene which has a single homolog in Drosophila: dFmr1. To unravel novel players with key roles in the disease mechanism of fragile X syndrome, we recently developed and conducted a genetic screen for dominate modifiers of dFmr1 over-expression in Drosophila. We identified a single major autosomal modifier which we mapped to the lethal (2)giant larvae (I(2)gl)locus). I(2)gl is a component of the cytoskeleton and loss of function mutations lead to neoplastic tumors. On one hand, Lgl bindsmyosin II and interacts genetically with both myosin II and V and on the other hand, FMR protein is involved in transport and translational regulation of target mRNAs. In addition, the latter associates with myosin V to form a common Ribo-Nuclear Particle (RNP). Taken together this data suggest that Lgl and FMR associate physically in a protein complex, perhaps an RNP equiped with molecular motors which enable its travels on the major cellular highways comprised of microtubule and microfilament networks. Specific predictions of this model will be tested in the proposed project: i) I(2)gl phenotypes should overlap with those of dFmrl mutants; ii) 1(2)gl interacts genetically with dFmr1; iii) Lgl and Fmr1 associate in a common protein complex, be it directly, or through an intermediate partner.
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0.966 |
2012 — 2013 |
Zarnescu, Daniela C |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Deciphering Rna Based Mechanisms of Neurodegeneration
DESCRIPTION (provided by applicant): ALS is an adult onset, progressive neurological disorder characterized by selective degeneration and death of motor neurons in the cortex and the spinal cord. In recent years, several RNA binding proteins have been linked to motor neuron disease, including senataxin, angiogenin, TDP-43 and FUS. These findings led to a paradigm shift in the current models for neuronal degeneration mechanisms and suggest that a significant component of ALS may be due to dysregulation of RNA metabolism. At present, TDP-43 has emerged as a common denominator for the majority of ALS cases known to date, however the mechanisms by which it causes neuronal degeneration remain poorly understood. The long-term goals of this research are to decipher the RNA-based mechanisms utilized by TDP-43 in the nervous system and to identify what aspects of RNA metabolism, specific protein partners and RNA targets are dysregulated by TDP-43 mutations linked to ALS. This exploratory proposal aims to test the hypothesis of RNA dysregulation in ALS by focusing on the physical and functional connections between TDP-43 and a repertoire of candidate RNA binding proteins. We will perform the proposed studies in a Drosophila model of ALS based on TDP-43 that we developed in our laboratory and which bears remarkable similarities to the human pathology. With help from collaborators our studies will be extended to human cells and tissue samples obtained from ALS patients. Preliminary data obtained from Drosophila and human cells show that TDP-43 forms a complex with FMRP, an RNA binding proteins with an established role in local translation and implicated in Fragile X syndrome. We also found that TDP-43 colocalizes with PABP in stress granules in motor neurons. In contrast to wild-type TDP-43, the A315T mutant, which has been linked to ALS in human patients, exhibits differential colocalization and genetic interactions with candidate RNA binding proteins including FMRP and PABP. We hypothesize that ALS stems in part from RNA dysregulation and propose to test this through a combination of molecular, electrophysiology, genetic and live imaging approaches. In Aim 1 we will establish the relationship between TDP-43 variants (wild-type and mutant) and several neuronal RNA granule components using colocalization, live trafficking studies, biochemical purifications and cellular fractionations under normal conditions or induced cellular stress. In Aim 2 we will use genetic interaction approaches in conjunction with a battery of phenotypic assays to establish the physiological significance of TDP-43's association with neuronal RNA granule components, including FMRP and established stress granule and P body markers. The proposed experiments will provide insights into what aspects of RNA regulation (e.g., stress granules assembly, composition, translation) are perturbed by disease causing mutations and will identify specific RNA binding proteins that modulate TDP-43's neurotoxicity in vivo. Given our extensive expertise in RNA based neuronal mechanisms and Drosophila genetics as well as the support from a team of expert collaborators we are uniquely positioned to test this novel and exciting hypothesis linking RNA dysregulation to neurodegeneration and to identify novel therapeutic targets for ALS. PUBLIC HEALTH RELEVANCE: The molecular mechanisms underlying motor neuron degeneration in ALS remain largely elusive. This proposal is focused on testing new hypotheses linking ALS to RNA dysregulation. We will employ a combination of molecular, genetic and functional approaches in a Drosophila model of ALS based on the RNA binding protein TDP-43. These experiments will identify specific RNA binding proteins that physically associate with TDP-43, modulate its neurotoxicity in vivo and have the potential to provide novel therapeutic targets for ALS.
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1 |
2015 — 2019 |
Zarnescu, Daniela C |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Rna Dysregulation in Neurodegeneration
? DESCRIPTION (provided by applicant): In recent years, it has become increasingly clear that RNA dysregulation is a critical contributor to the pathophysiology of amyotrophic lateral sclerosis (ALS) and related neurodegenerative diseases. Indeed, several RNA binding proteins have been identified in pathologic aggregates and have also been shown to harbor mutations causative of ALS. Among these, TDP-43, which is linked to the vast majority of ALS cases, has been implicated in several aspects of RNA metabolism including splicing, transport, storage in stress granules (SGs) and translation. These findings suggest an intimate link between TDP-43, RNA SGs, translation and disease. Although recent studies have provided insights into the relationship between TDP-43 and RNA stress granules in ALS, our current knowledge of TDP-43's role in translation, the identity of its mRNA translation targets and their contribution to disease remain poorly understood. The long-term goal of this research is to determine the mechanistic connections between TDP-43 and translation in the nervous system, and to establish the contribution of RNA dysregulation to the pathophysiology of neurodegenerative diseases. We have recently demonstrated that TDP-43 regulates the localization and translation of futsch/MAP1B in Drosophila and defects identified in fly motor neurons are remarkably similar to those found in ALS spinal cords. Using a Drosophila model of ALS based on TDP-43 we also found that FMRP, a well- established translational regulator is neuroprotective by reducing TDP-43 aggregation and restoring the translation of specific mRNA targets. Furthermore, we have identified several new candidate mRNA translation targets including hsc70-4 mRNA, a molecular chaperone that controls synaptic vesicle (SV) trafficking as well as additional candidates implicated in synaptic function. Notably, restoring Hsc70-4 levels in motor neurons by overexpression rescues synaptic vesicle endocytosis defects caused by ALS associated mutant TDP-43. Based on these findings we hypothesize that TDP-43 acts as a translational regulator in motor neurons and that dysregulation of protein synthesis contributes to the pathophysiology of ALS. Our hypothesis will be critically tested in three specific aims. First, we will determine physical and functional interactions between TDP-43 and the translation machinery. Second, we will identify mRNA translation targets of TDP-43 in motor neurons using tagged ribosome affinity purification (TRAP), then will use ribosome footprinting to determine what stage of translation is impacted by TDP-43. Third, we will establish the synaptic defects caused by TDP- 43 and will determine whether restoring candidate targets by overexpression in motor neurons rescues TDP-43 dependent toxicity. Our findings in the Drosophila model will be validated in ALS spinal cords. This research is expected to provide novel insights into TDP-43's function in translation, to identify physiologically significant and disease relevant protein partnrs and mRNA translation targets, which in turn may pinpoint much needed molecular targets and pathways with therapeutic potential for ALS and related neurodegenerative diseases.
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1 |