TROPHIC ALTERATIONS IN DOPAMINE NEURON TRANSPLANTATION

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Results form early clinical trials suggest that fetal nigral transplants may offer an exciting new therapeutic strategy for the treatment of Parkinson's disease (PD). However, tissue loss associated with the storage of aborted fetal tissue prior to transplantation, inadequate knowledge about the impact of patient age and/or stage of disease on graft survival, and the lack of a continuous source of tissue for transplantation are among the factors that compromises an otherwise straight forward surgical procedure. Recent work in our laboratory suggests that dopamine (DA) neuron survival can be increased by "preconditioning" DA neurons with trophic molecules during storage. We have also shown that trophic activity in the rat striatum is reduced by aging and increased in direct proportion to the degree of DA denervation. These data suggest that patient age and Parkinsonian stage may have a significant impact on the trophic environment into which grafted tissue is transplanted. In Specific Aim 1 we will therefore systematically evaluate the effect of supplementing storage media with trophic molecules (bFGF, BDNF, GDNF, EGF), antioxidants (U83836E and U74389G), and tissue extracts (striatal, mesencephalic, placental serum). Rat mesencephalon will be stored for up to 5 days under these conditions and then co-cultured with striatal cells. DA neuron survival in these cultures will be evaluated by assessing K+-evoked DA release and tyrosine hydroxylase immunoreactive (THir) cell counts. Optimal storage conditions will be further evaluated by implanting the "enhanced" cells into unilaterally lesioned (6OHDA) rats. Specific Aim 2 will focus on the effects the transplant recipients age and degree of DA denervation has on graft survival. Enhanced cells will be implanted into the striata of 2,9, 17, and 22 month old rats and THir cell counts in the implant site will be compared across groups. Using the intraventricular 6OHDA model, 17 month old rats will be bilaterally denervated 35,50,75, and 90% and transplanted with enhanced cells. THir cell counts will again be compared across groups. Although optimizing fetal tissue storage conditions and identifying the age and degree of DA denervation that maximizes DA neuron survival is likely to have an immediate, positive impact on transplant programs currently under way, these Aims do not address the issues surrounding access to a continuous source of DA neuron enriched tissue. In this regard, we have recently shown that a natural, pluripotent, progenitor cell line can be derived from fetal and adult mesencephalon. Co-culture with freshly harvested mesencephalon converted many of these cells into the DA neuron phenotype whereas known trophic factors did not. Many of these cultures were highly enriched with THir cells. Specific Aim 3 will therefore focus on the future use of the pluripotent progenitor cell as a continuous passage; 2-optimizing and identifying and identifying the crude components of the mesencephalon responsible for its conversion to the DA phenotype; and 3- grafting the progenitor cells in 6OHDA lesioned rats. The successful implementation of the Specific Aims of this proposal addresses several practical aspects of transplantation that could be readily transferred to the clinical setting.