INHIBITION OF APOPTOSIS IN NEURAL GRAFTS

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Critical to clinical success of transplantation for Parkinson's disease (PD) is the development of methods whereby grafted DA neuron viability and reinnervation of the host striatum are markedly increased. The survival rate of DA neurons in grafts to young rats is 5-10 percent, and is even poorer in grafts to the aged striatum. The focus of this revised KO1 Mentored Research Award application is to pursue previous work by the candidate which demonstrated that striatal target cells can increase the survival of simultaneously grafted DA neurons by supplying diffusible trophic factors. The studies proposed in this application seek to define some of the cellular and molecular mediators of enhanced DA neuron survival, through close collaboration with the mentor's laboratory in the use of cell culture techniques, and apply findings in cell culture to therapeutic intervention in the aged nervous system. Based on suggestions in the literature, it is likely that the striatal oligodendrocyte type 2-astrocyte (0-2A) progenitor cell is one cellular source of striatal-derived trophic activity DA neurons. One goal of this application will be to develop expertise in cell culture techniques and growth factor treatments in culture to selectively enrich striatal 0-2A cells for investigation of their role in neurotrophic effects on DA neuron survival and apoptotic death rates. A second goal of the proposed training will be to develop experience in the neurobiology of aging animals, another focus of the mentor's laboratory. Recent evidence in the mentor's laboratory indicates that morphological and behavioral effects of DA grafts in aged rats with long-term lesions is greatly diminished as compared to grafts in young rats. Information learned from the initial set of culture experiments will be applied to transplantation studies in young and aged rats, in order to determine whether DA neurons co-grafted with 0-2A enriched striatal cells can provide superior morphological and behavioral outcomes in aged subjects. Should these studies indicate that enhanced DA neuron survival is related to the inhibition of apoptosis, then a direct apoptosis inhibition approach also will be studied. Therefore, the final goal of this training proposal will be to gain experience in utilizing viral vectors in cell culture to overexpress bcl-2, an identified "survival" gene, in DA neurons and to examine the effect of this transduction on apoptosis in both cultures and in grafts to young and aged rats. Tissue culture aspects of this study will be learned in the mentor's laboratory, and viral vector will-be provided by Dr. Howard Federoff, Univ. of Rochester School of Medicine. In addition, the candidate will visit the Federoff laboratory learn viral vector transduction techniques. The overall hypothesis of this proposal is that by inhibiting apoptotic cell death of grafted DA neurons, both indirectly by supplying trophic factors and directly by interfering with the genetic death program, it will be possible to increase DA graft viability and subsequent reinnervation of the aged striatum. This training will promote the candidate's development into a fully independent investigator in the fields of neural cell culture, aging in the DA system, apoptosis and neural grafting for PD.