Michelle Gray, Ph.D. - US grants

The precise regulation of neuronal cell number is essential for proper nervous system function. Mutants exhibiting excess neurons in discreet cell populations are useful for studying the genetic control of neuronal production. Moreover, mutants with extra neurons offer an opportunity to analyze the plasticity of neural circuit formation. We have isolated a mutation in zebrafish, loose ends (loe), that results in supernumerary Mauthner cells. Wild-type zebrafish have a bilateral pair of Mauthner cells on either side of the midline in hindbrain rhombomere 4. In contrast, loe mutants have on average five Mauthner cells on either side of the midline. By cloning the loe gene, we will reveal the identity of the molecule causing this defect. To this end, we will place loe on the genetic map of the zebrafish genome to determine whether it corresponds to a known gene. If a known gene is not revealed, we will positionally clone loe. We will also exploit the fact that loe has an increase in Mauthner cells to ask how the presence of these extra cells affects the formation of the escape response circuit. The escape response circuit involves sensory input onto Mauthner cells and Mauthner cell input onto primary motoneurons. We will characterize both the pre- and post-synaptic contacts in loe mutants to elucidate and how the circuit is organized differently due to the presence of extra Mauthner cells.

DESCRIPTION (provided by applicant): Huntington's disease (HD) is an adult-onset autosomal dominant neurodegenerative disorder characterized clinically by cognitive, psychiatric and motor deficits which progress to severe disability and death. To date there are no affective treatments for HD. The mutated huntingtin (mhtt) protein is widely expressed in neuronal and non-neuronal cells, yet neurodegeneration is highly selective. Understanding the toxicity produced by mhtt and the basis for this selective neurodegeneration are likely to be critical in the design of effective therapies for the disease. The goal of this proposal is to understand the contribution of mhtt expressing astrocytes to neurodegeneration in HD. Astrocytes are critical to the proper function and development of the nervous system and have a newly appreciated role in controlling synaptic activity. Data from cell culture studies show that astrocytes may contribute to HD pathogenesis. However, no in vivo studies have been performed to systematically address this question. Therefore, I will study the contribution of astrocytes to disease pathogenesis in vivo by using conditional mhtt expressing mouse genetic models. Using these models and astrocytes obtained from them I will test three hypotheses: 1) The presence of the mhtt in astrocytes is (1) necessary and sufficient for the development of an HD phenotype;2) the presence of mhtt in astrocytes causes impaired calcium homeostasis and glutamate release from astrocytes;and 3) The presence of mhtt in astrocytes leads to abnormalities in synaptic neurotransmission. Together these studies will help to define the role of mhtt within astrocytes in HD pathogenesis, and may lead to novel therapeutic approaches for treatment of HD. The research plan in this application is designed to allow the applicant to gain additional technical skills and tools for use in studying Huntington's disease pathogenesis, including cell biology and electrophysiology. These new capabilities, together with previous training in generating mouse genetic models of neurodegeneration, will make her a multidisciplinary scientist with the well-rounded set of skills necessary for attaining her long-term career goal of acquiring a tenure track faculty appointment at a major research institution. In addition to the technical skills obtained during this training period, the applicant will further her understanding of neurodegenerative diseases by attending seminars and journal clubs at UAB and outside scientific meetings. She will also take advantage of faculty development seminars and programs that help to teach the required skills and provide the knowledge necessary for a successful career as an independent research scientist including scientific and grant writing skills. Together, the career development component and research plan of this application will provide the candidate with a firm foundation on which to develop an independent laboratory focused on studying neurodegenerative diseases. PUBLIC HEALTH RELEVANCE: Project Narrative Huntington's disease (HD) is a devastating progressive adult-onset neurodegenerative disease. Currently, there is no treatment or a cure for HD. HD is one of the most common familial neurodegenerative disorders, with 30,000 clinically diagnosed HD patients and another 150,000 at risk for HD in the US.

? DESCRIPTION (provided by applicant): Huntington's disease (HD) is an adult-onset autosomal dominant neurodegenerative disorder characterized clinically by cognitive, psychiatric and motor deficits which progress to severe disability and death. To date there are no affective treatments for HD. The mutated huntingtin (mHTT) protein is widely expressed in neuronal and non-neuronal cells, yet neurodegeneration critically affects a few subsets of neuronal cell types in the brain. Understanding the toxicity produced by mhtt in non-neuronal cell types and the basis for this selective neurodegeneration are likely to be critical in the desin of effective therapies for the disease. The goal of this proposal is to expand our knowledge of the contribution of full length-mHTT (fl-mHTT) expressing astrocytes to neurodegeneration in HD. Astrocytes are critical to the proper function and development of the nervous system. They are involved in modulating synaptic activity and neurotransmission. Studies from our laboratory have uncovered increased SNARE-dependent glutamate release from astrocytes in culture taken from the conditional fl-mHTT expressing BACHD mouse model. This is a new found disruption caused by fl-mHTT expression in the astrocytes, which further implicates these cells in HD pathogenesis. Furthermore, we demonstrate that reducing fl-mHTT expression in astrocytes in vivo, contributes to the behavioral and neuropathological phenotypes observed in the conditional BACHD mouse model. The studies proposed here will further our understanding of astrocyte contribution to HD and determine if targeting this cell type for therapeutic intervention is worthwhile. We will use conditional genetic mouse models in these studies and propose three aims: 1) To determine if fl-mHTT expressing astrocytes contribute to HD pathogenesis by increasing glutamate levels in BACHD mice through exocytosis of glutamate; 2) To determine if expression of mutant huntingtin in astrocytes contributes to the abnormal medium spiny neuron physiology observed in BACHD mice; and 3) To determine if decreasing fl-mHTT expression in astrocytes after disease onset in BACHD mice is sufficient to alleviate HD symptoms and whether astrocyte specific expression in a new mouse model is sufficient to cause HD-like phenotypes in mice. Together these studies will help to define the role of fl-mHTT within astrocytes in HD pathogenesis, and may lead to novel therapeutic approaches for treatment of HD.