Parkinson's disease (PD) is a major public health problem in North America. Although PD has been regarded as having only a minor heritable component, recent discoveries have highlighted the contribution of genetics to this disease, with the description of dominant and recessive inherited forms of PD in several families. A wide variety of mutations in the parkin gene are associated with autosomal recessive forms of PD, especially """"""""juvenile"""""""" onset forms where symptoms start before the fourth decade of life. Inheritance of certain parkin mutations result in greatly reduced parkin protein expression. The function of the parkin protein is unknown but recent results strongly suggest that it is a ubiquitin-protein ligase, or at least a component of a multi subunit ligase. Ubiquitination is a major way in which cells dispose of misfolded or damaged proteins, as well as a way in which the cell regulates levels of specific proteins. A point mutation in a second gene that may affect ubiquitination, UCH-L1, has been reported to be associated with autosomal dominant familial PD. The major pathological hallmark of PD is loss of dopamine containing cells in the substantia nigra (SN). How loss of parkin function causes damage to dopaminergic cells is not yet known, but a prediction of the hypothesis that parkin is an ubiquitin-protein ligase is that parkin is involved in the removal of misfolded and/or potentially toxic proteins from cells. We hypothesize that lack of parkin expression in dopaminergic cells of the SN causes an accumulation of an unidentified protein or set of proteins (protein x) which is/are toxic. The effects of UCH-L1 mutations may be similar, in that these would be predicted to also affect ubiquitination and hence increase the concentration of toxic protein(s) within the cell. The studies in this proposal aim to test this hypothesis in cellular systems which have been carefully chosen to be relevant to dopaminergic cells. Specifically we will perform functional studies of the potential role of parkin and UCH-L1 protein in ubiquitination. We will attempt to understand how mutations in parkin result in altered function of this protein and how it is regulated within the cell. We will also look at the effects of loss of parkin function on cell death and on ubiquitination reactions within the cell. We will also attempt to address the idea that mutations in parkin and UCH-L1 may interact in the same pathway, and examine the balance between use of ubiquitin by reactions catalyzed by parkin, and the production of free ubiquitin via UCH-L1.
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