940. Neuroprotective Effects of XIAP Gene Therapy in Models of Neurodegenerative Diseases

Abstract

The pathways and mechanisms leading to cell loss in neurodegerative diseases are still largely unknown. Neuronal cell death is thought to occur via apoptosis and the involvement of several caspases at the late stages of this process is well documented. X-linked inhibitor of apoptosis (XIAP) is a potent inhibitor of caspases 9, 3 and 7 and thus represents an attractive candidate as a potentially therapeutic neuroprotective factor. To test this hypothesis, we examined the effects of XIAP in several in vitro models of Parkinson's and Huntington's disease. Recent reports have documented the pivotal role of the proteasome pathway of protein turnover in the pathogenesis of neurodegenerative disorders and proteasome inhibitors have been employed to model neurodegeneration both in vitro and in vivo. In neuronal cell lines XIAP demonstrated significant protection against apoptosis induced by different proteasome inhibitors. Furthermore, XIAP almost completely prevented cells death induced by overexpression of alpha-synuclein and polyglatamine fusion proteins used to model familial Parkinson's and Huntington's diseases, respectively. To gain insight into the mechanisms of neuroprotective affects of XIAP we performed analysis of deletion and point mutants of this protein. Our experiments revealed that the N-terminal BIR1 domain as well as the C-terminal RING domain are dispensable for anti-apoptotic effects. Deletion or point mutation of the BIR2 domain or the juxtaposed linker region partially abrogated XIAP activity. In contrast, anti-apoptotic effect was lost when a deletion or a point mutation were introduced into the BIR3 domain. Caspases 3 and 7 are believed to be bound by the BIR2 and the linker regions while the caspase 9 is inactivated by the BIR3 domain. Thus, our data suggest that the protective affects of XIAP in these two models of Parkinson's and Huntington's diseases are mostly mediated by the anti-caspase 9 activity. To explore neuroprotective effects of XIAP in vivo we have generated a recombinant AAV (rAAV) vector expressing a stabilized truncated from of XIAP lacking the RING domain (dXIAP). Slowly progressing nigral degeneration triggered by proteasome inhibition is believed to be a close recapitulation of the events that mark sporadic Parkinson's disease in humans. Therefore, for this study we employed a novel PSI rat model of Parkinson's disease which is induced by inhibition of proteasome machinery. While treatment of the animals with PSI resulted in approximately 50% loss of dopaminergic neurons in the substantia nigra on the side injected with a control vector, all the cells were protected on the contralateral side injected with the dXIAP-overexpressing rAAV. Thus, dXIAP delivered by a rAAV provides a long-term protection of nigral neurons against death induced by proteasome inhibition. Furthermore, to our knowledge this is the first demonstration that neuronal loss can be prevented in this novel model of Parkinson's disease. Thus, our studies may provide a foundation for the use of dXIAP for neuroprotection, which can be further exploited for human gene therapy.