Apoptosis And Excitotoxicity In The Death of Cultured And Grafted Dopaminergic Neurones

Abstract: Over the past two decades, grafting of embryonic nigral tissue has developed into a feasible therapeutic strategy for Parkinson's disease (PD). It has become apparent that the degree of restoration of dopaminergic neurotransmission by the transplanted cells is highly correlated to the symptomatic relief. However, the efficacy of the treatment is still far from optimal: More than 90% of the grafted dopaminergic neurones die during the transplantation procedure and the immediate period (first one to four days) after graft injection. Improvement in the survival of transplanted cells would increase the potential of this therapeutic approach and facilitate a more widespread use. This background provided the motivation of the research described in the present thesis. The focus here was 1), to characterise cell death occurring during nigral transplantation, and, 2), to find and evaluate novel neuroprotective strategies. In the neurone an overload of Ca2+ effects a cascade of events ultimately leading to cell death. Ca2+ entry is mainly mediated either via excitatory amino acid (EAA) receptor activation or via voltage-dependent Ca2+ channels (VDCCs). In the present work, it could be shown that a potent inhibitor of the EAA receptor N-methyl-D-aspartate (NMDA) was able to protect cultured embryonic dopaminergic neurones against stress induced by serum-deprivation. On the other hand, when this compound was applied during mesencephalic grafting, no increase in dopaminergic neurone survival was observed. This indicates that excitotoxicity plays a subordinate role in death of transplanted dopaminergic neurones. In contrast, effective inhibition of VDCCs increased the survival of grafted dopaminergic neurones to 260% compared to control. Bcl-2 is a protein which has proven effective in the prevention of apoptosis in related contexts. Here it could be shown that it protected dopaminergic neurones in cultures subjected to serum-deprivation or staurosporine (STS) insult. However, Bcl-2 overexpression did not increase dopaminergic neurone survival when embryonic ventral mesencephalic tissue from transgenic mice was xenotransplanted into rats. The caspase family of proteases promotes apoptosis and some of its members are closely linked to a Bcl-2-independent pathway. In part of the current thesis, treatment with a caspase inhibitor was found to lead to a remarkable three-to-fourfold increase in the number of surviving implanted dopaminergic neurones, and thus efficiently blocked apoptosis. Activation of poly (ADP-ribose) polymerase (PARP) which is a substrate of caspases has recently been linked to neuronal death. However, its role in the context of apoptosis is still matter of debate. In the final part of the present thesis, it was shown that PARP does not play a role in nigral cell death. The presented research sheds further light on the mechanisms leading to cell death in nigral transplantation and led to the discovery of novel neuroprotective strategies which justify further exploration regarding their relevance for clinical application.