Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive degeneration of the dopaminergic nigrostriatal pathway and the abnormal appearance of intracellular inclusions named Lewy bodies (LBs). Over the past few years, the discovery of genes involved in hereditary forms of the disease led to new insights into the pathogenesis of PD. Mutations in the α-synuclein gene have been associated with autosomal dominant PD, and mutations in parkin with autosomal recessive-juvenile parkinsonism (AR-JP). α-synuclein has emerged as a key protein in the pathogenesis of PD, as it appears to be the major structural component of LBs and its accumulation seems to play a prominent role in sporadic PD. To date, genetic PD models based on α-synuclein conventional transgenesis in rodents have been unsuccessful in recapitulating the main features of the human pathology, especially the loss of nigral dopamine neurons. The lack of clear dopaminergic cellular degeneration in these transgenic animal models is likely to be attributed to insufficient expression levels of α-synuclein in the substantia nigra. Injection of viral vectors constitutes an alternative approach for the development of genetic models, as viral vectors allow high gene expression levels in a localized brain region and can be applied in mammalian species other than mice. In the present study, the viral-mediated expression of different α-synuclein forms (normal or mutated) was explored in the substantia nigra of rats. Following the full characterization of this new genetic model, several neuroprotective strategies based on the delivery of potential neuroprotective factors such as the neurotrophic factor GDNF (glial cell line-derived neurotrophic factor), the E3 ligase parkin or the chaperone Hsp104 were evaluated in this α-synuclein rat model. As HIV-1-derived lentiviral vectors can efficiently transduce neurons in the brain, these retroviral vectors were used to overexpress normal or mutated forms of α-synuclein in the substantia nigra of adult rats. In contrast to α-synuclein transgenic mice, the lentiviral-based model developed a progressive and selective loss of nigral dopaminergic neurons associated with a dopaminergic denervation of the striatum in animals expressing either wild-type or mutant forms of human α-synuclein. The neuronal degeneration correlated with the appearance of ?α-synuclein-positive inclusions. This viral-based model thus constitutes an excellent genetic model for testing molecules that can interfere with the neurodegenerative process induced by α-synuclein. Delivery of GDNF, a potent neuroprotective factor for the survival of dopaminergic neurons, is currently the most promising strategy for the treatment of PD. The neuroprotective properties of GDNF were therefore evaluated in the lentiviral-based model expressing mutated human α-synuclein. Although a robust expression of GDNF was observed in the whole nigrostriatal pathway due to retrograde and/or anterograde transport, nigral GDNF delivery did not prevent the α-synuclein-induced neurodegeneration. As loss of function of parkin leads to dopaminergic cell loss in Autosomal Recessive-Juvenile Parkinsonism (AR-JP) patients, parkin may represent a critical survival factor for dopaminergic neurons. The neuroprotective role of the E3 ubiquitin ligase parkin was assessed in the viral-based model overexpressing mutated human α-synuclein. Co-expression of parkin with α-synuclein prevented the nigrostriatal degeneration and increased the number of hyperphosphorylated α-synuclein inclusions. These results suggest that parkin may also play a key role in the formation of aggregates. Abnormal folding and aggregation of α-synuclein is now recognized as a critical issue in the pathology of PD. Chaperones can block or reverse the incorrect folding of misfolded proteins, and increase the clearance of aggregates. To enhance the elimination of aggregated α-synuclein and to understand the importance of inclusions in the α-synuclein pathogenesis, we expressed a protein disaggregase, the yeast chaperone Hsp104, in the lentiviral-based model of PD. This chaperone can facilitate the clearance of misfolded proteins from an aggregate state. Expression of Hsp104 reduced the dopaminergic cell loss induced by lentiviralmediated expression of PD-linked mutated α-synuclein. This neuroprotective effect correlated with a reduction in the formation of hyperphosphorylated α-synuclein inclusions, indicating that enhancing clearance of protein aggregates through the disaggregase activity of Hsp104 may constitute a novel therapeutic strategy for PD and other diseases caused by misfolded protein accumulation. The present thesis demonstrates that lentiviral vectors expressing human α-synuclein constitute a powerful and flexible tool to mimic the neuropathological features of PD. Although not effective in the lentiviral-based model of PD, GDNF may still represent a promising molecule to promote the sprouting of dopaminergic axons. On the contrary, the E3 ligase parkin and the chaperone Hsp104 have demonstrated potent neuroprotective properties against the accumulation of the toxic mutated α-synuclein. Therapeutic approaches aiming at increasing parkin or chaperone levels in the brain may therefore open new perspectives for the treatment of PD.
EPFL2005
