Deciphering the molecular mechanism of Parkinson´s Disease

The ambition of this project is a better understanding of the molecular mechanisms underlying Parkinsons`s disease (PD) in general and the role of a-synuclein and EndoG during cell demise in particular. Death of neurons during PD is linked to cellular age, mitochondrial impairment caused by environmental toxins (e.g. MPTP, rotenone) or genetic mutations, and formation of protein aggregates called Lewy bodies with a-synuclein being the main constituent. Mitochondria and oxidative stress pathways are suggested to be crucial factors determining a- synuclein toxicity. The biochemistry and pathogenicity of a-synuclein has been intensively studied in several model systems like flies, worms, and transgenic mice, implicating a function for a-synuclein in the regulation of synaptic plasticity, vesicular trafficking, and phospholipase D2 activity. In accordance with other models, the yeast system showed a-synuclein to localize to the plasma membrane, to form intracellular inclusions, to function in lipid metabolism and vesicular trafficking, and to inhibit phospholipase D. Our previous work applying chronologically aged yeast (a well established model for ageing of post-mitotic mammalian cells) to mimic age-induced neurodegeneration demonstrates that a-synuclein expression in yeast triggers apoptotic as well as necrotic death. This death is shown to depend on mitochondrial functions. The abrogation of mtDNA resulted in delayed cell death, decreased accumulation of reactive oxygen species (ROS), and reduced apoptotic but not necrotic markers. Our recent data indicates Endonuclease G (EndoG) to be a possible mediator of a-synuclein toxicity (unpublished results). Intriguingly, these data seem to be valid in higher eukaryotes as well, as the knockdown of Drosophila EndoG leads to an inhibition of a-synuclein- mediated apoptosis in a fly model of PD (unpublished data). As this suggests EndoG to be a putative target for a novel PD therapy approach, the identification of inhibitors of EndoG is scheduled in the context of this project. Besides, the influence of EndoG on motor coordination, behaviour, learning capability, and a-synuclein-mediated neuronal death will be analysed in a mouse model for PD. Using yeast clonogenic survival assays combined with flow cytometry- based quantification of apoptotic and necrotic markers, the pathway(s) of a-synuclein-mediated death will be further elucidated. Potential interactors will be identified (using a pulldown assay) and tested for their influence on a-synuclein toxicity and aggregation. Mitochondrial factors implicated in PD like environmental toxins or the proteins parkin, DJ-1, and pink will be further elucidated in respect to survival, ROS-accumulation, apoptotic, necrotic, and autophagic markers, influence on a-synuclein toxicity and aggregation, and their impact on EndoG and other apoptotic and necrotic players. Auspicious results obtained applying the yeast system will be further validated in mammalian cell lines, Drosophila melanogaster, and mice. In general, the intention is to understand the complex interplay between a-synuclein function, toxicity, and aggregation, mitochondrial impairment upon environmental toxins or gene mutations, levels of oxidative phosphorylation, ROS-accumulation, and the pathway of EndoG-mediated cell killing.