PD is an age-related, progressive movement disorder characterized by resting tremor, hypokinesia, rigidity and clinical response to dopaminergic therapy. Neuropathologically, PD is defined by the relatively selective loss of dopaminergic neurons in the midbrain and the presence of intraneuronal inclusions known as Lewy bodies. There are several known etiologies of the Parkinsonism syndrome which include infectious, toxic, and other neurodegenerative disorders and """"""""idiopathic"""""""" PD. Focusing on knowledge derived from a rare form of familial PD, the investigator will examine how two recently identified mutations in the alpha-synuclein (alphaS) gene lead to this neurologic condition. The central hypothesis of this research training application is that heterozygous mutations of alphaS initiate the selective degeneration of predominantly dopaminergic neurons in the midbrain of affected patients. These genotypes represent the first clue to the molecular pathogenesis of neuronal dysfunction and cell death in PD. Using biochemical and cell and molecular biological techniques, the investigator plans to study the normal and abnormal function of alphaS in cell culture and examine its effects in vivo as well. The investigator will pursue 4 specific aims, proposing: (1) to characterize the constitutive expression of alphaS in mammalian cells in comparison with human and rodent brain; to that end, the investigator will first express alphaS protein in bacteria and generate high-affinity antibodies to synthetic and bacterially expressed proteins; (2) to systematically explore suitable neural cell models of alphaS overexpression in both human and rodent dopaminergic cell cultures of mesencephalic and non-mesencephalic origin and in rodent oligodendrocytes and will investigate the effects of wild type and mutant human alphaS gene expression in primary neurons and midbrain parenchyma of transgenic mice; (3) to localize alphaS in transfected and primary cells morphologically in order to help determine its function and to search immunocytochemically and biochemically for evidence of insoluble or oligomerized isoforms; and (4) to analyze the phenotypic consequences of mutant alphaS on the metabolism of dopaminergic cell markers (e.g., TH, VMAT-2) and to subject such cells to known mesencephalic neuronal toxins (e.g., MPP+, 6-OH-dopamine). Through these 4 specific aims, the investigator seeks to create dynamic and manipulatable cellular models for alphaS mutation-related dopaminergic neuronal dysfunction. Such cell models should provide important pathogenetic information and should ultimately be useful in screening potential therapeutic compounds.
