The involvement of alpha-synuclein in Parkinson's disease neurodegeneration is now widely accepted. However, factors and events contributing to the alpha-synuclein toxicity are still being investigated. Among modifications of alpha-synuclein, phosphorylation at Serine 129 has been related to the disease process based on data from postmortem studies and in vitro experiments. In fact, a recent study in transgenic flies suggests that phosphorylation is a necessary event in alpha-synuclein induced dopaminergic neurodegeneration. However, this has never been tested in an in vivo model of alpha-synuclein pathogenesis in mammals.
Our proposal is therefore to translate these data obtained in a lower species (fly) to a mammalian context (rat). For this we will investigate the adequacy of viral vector technology to provide a progressive in vivo model that recapitulates phosphorylation among other Parkinson's characteristics to be used in future Parkinson's disease research studies.
Final Outcome
To elucidate the role of phosphorylation of alpha-synuclein at Serine 129 in the disease process we used a rat PD model based on the local injection of recombinant adeno-associated viral vectors (rAAV). We created various mutations in alpha-synuclein at Ser129, then overexpressed these different types of mutated alpha-synuclein in the midbrain by injecting each rAAV into the substantia nigra. We compared the effect of overexpression in dopaminergic neurons at different time points post-injection.
Our results show that the S129D and S129A variations of alpha-synuclein induced a decrease in dopaminergic cells in the substantia nigra, as well as pathological accumulations of alpha-synuclein, comparable to that seen after the overexpression of non-mutated (wild type) alpha-synuclein. S129D induced faster motor deficit compared to S129A or wild type alpha-synuclein. This observation could be of relevance regarding the role of S129 alpha-synuclein phosphorylation in the neurodegenerative event that takes place prior to cell death in PD.
Based on our in vivo observations at the behavioral level and the work by others in vitro, we hypothesized that alpha-synuclein overexpression leads to abnormal dopaminergic release prior to
cell death. This has resulted in a project focused on the vesicle machinery in this model and we have obtained interesting preliminary data. The project is involving now three
different labs at universities in Denmark, Germany and Australia.
In addition, we are initiating a project where we will perform microdialysis in animals overexpressing alpha-synuclein to elucidate if the accumulation and in turn the presence of increased S129P alpha-synuclein affects dopaminergic neurotransmission.
A third project in progress is dealing with electrophysiological studies in alpha-synuclein transgenic animals, and calcium signaling in synuclein overexpressing cells. These experiments show highly promising preliminary results. The project aims to understand the early events related to alpha-synuclein accumulation and is performed in collaboration with Dr. Kimmo Jensen and his student colleague Kim Boddum (Dept. of Physiology and Biophysics, Aarhus University).