4) Mechanism of cell-to-cell propagation of α-Synuclein Pathology in PD

The -synuclein protein, a major constituent of Lewy bodies, the pathological hallmark of PD, has long been implicated in the pathogenesis of PD. Recently, several lines of evidence from a variety of in vivo and in vitro systems point to the tantalizing idea that -synuclein released from one neuron is taken up by neighboring cells, where it “seeds” aggregation of the endogenous -synuclein in the recipient cell, thereby propagating disease-associated aggregates from one cell to another. The first evidence came from the discovery that embryonic dopaminergic (DA) neurons transplanted into PD patients years earlier developed PD pathology with -synuclein containing inclusions most likely as a consequence of factors inherent to the host. These results are congruent with the stereotypical spread of PD pathology in the brain from the lower brainstem to the substantia nigra and eventually to the cortex, proposed by Braak. The observation that non-DA lesions often precede DA degeneration may explain some of the early non-motor PD symptoms such as loss of olfaction and sleep disturbances. Indeed, Lewy bodies have been observed in areas of the peripheral nervous system exposed to the environment such as the olfactory system or the digestive tract, in addition to neurons in the central nervous system. Finally, several studies have demonstrated cell-to-cell transmission of -synuclein in controlled experimental systems including cultured cells and animal models. At present, very little is known about the mechanisms involved or whether inhibition of -synuclein uptake could slow disease progression. In an effort to begin understanding how -synuclein is taken up into cells, we have embarked upon a High-Throughput Screening (HTS) approach funded by Prionet Canada, in collaboration with Dr Robert Screaton at the University of Ottawa and the Center for Drug Research and Development (CDRD) in Vancouver (Fig. 6). Specifically, we are using two complementary approaches: genome-wide siRNA and small-molecule library screening. The goal is to discover new genes and compounds that could modulate -synuclein uptake. We have developed a robust cell-based -synuclein uptake assay that is amenable to HTS. Pilot screens using siRNAs against the ~600 kinases in the human genome and against ~5000 FDA-approved compounds have identified interesting candidates and provide an important proof-of-principle as we embark on the larger scale genetic and small-molecule screens.