Alpha-synuclein amyloid accumulation and its interaction with prion protein

α-Synuclein (α-syn) plays a central role in the pathogenesis of neurodegenerative disorders collectively known as “synucleinopathies” that include Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Understanding the underlying molecular mechanisms of neurodegenerative diseases is indispensably important because of the prevalence of these devastating conditions in the elderly population. Several findings from cell culture and in vivo experiments suggest intercellular transfer of α-syn aggregates. The concept of intracellular α-syn pathology spread was recently extended by the discovery of propagation of α-syn aggregates throughout PD brains. The resulting concept of cell-to-cell propagation of α-syn pathology comprises of its release, uptake, and subsequently seeding of intracellular α-syn aggregation in recipient cells. α-Synuclein (α-syn) plays a central role in the pathogenesis of neurodegenerative disorders collectively known as “synucleinopathies” that include Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Understanding the underlying molecular mechanisms of neurodegenerative diseases is indispensably important because of the prevalence of these devastating conditions in the elderly population. Several findings from cell culture and in vivo experiments suggest intercellular transfer of α-syn aggregates. The concept of intracellular α-syn pathology spread was recently extended by the discovery of propagation of α-syn aggregates throughout PD brains. The resulting concept of cell-to-cell propagation of α-syn pathology comprises of its release, uptake, and subsequently seeding of intracellular α-syn aggregation in recipient cells. In this PhD thesis the methodology used to obtain synthetic mammalian prions has been used to obtain recombinant human and mouse α-syn amyloids in order to characterize whether synthetic material can promote prion-like accumulation of its soluble counterpart in neuronal cell lines in vitro and in wild type (WT) mice in vivo. To address these hypotheses, in the first part of the work the human neuroblastoma SH-SY5Y cell line and WT CD-1 mice were used. A single exposure to human α-syn amyloid fibrils was sufficient to induce aggregation of endogenous α-syn in SH-SY5Y cells. Remarkably, endogenous WT α-syn was sufficient for aggregate formation and overexpression of the protein was not required. Our results provide compelling evidence that endogenous α-syn can accumulate in cell culture after a single exposure to exogenous α-syn short amyloid fibrils. Importantly, using α-syn short amyloid fibrils as seed, endogenous α-syn aggregates and accumulates over several passages in cell culture. We further analyzed this phenomenon in vivo in WT CD-1 mice, where intracerebral inoculation of synthetic α-syn short amyloid fibrils induced the formation of phosphorylated α-syn aggregates in CNS regions distinct from the injection site. After this observation, further studies were performed in order to understand the mechanism of internalization of α-syn amyloids. Indeed, while the function of the cellular prion protein (PrP C) is still under debate, several reports indicate that PrPC interacts with amyloid form proteins, Aβ oligomers and PrP scrapie (PrPSc) fibrils. In order to investigate α-Syn amyloid accumulation and its interaction with prion protein this, we used the same technique as in the first part of our study, working on mouse α-syn protein. Here, we explored the uptake of α-syn amyloids in a neuroblastoma cell line: N2a cells which endogenously express PrPC (N2a), cells knocked out for PrPC (N2a KO), N2a KO where the PrP was re-introduced using transfection protocol (N2a PrPFL), and scrapie infected N2a (ScN2a) cells. Our results show that the uptake of α-syn amyloids is lower in N2a KO compared to control cells (N2a), raising the possibility that PrPmediates the uptake of α-syn amyloids within cytoplasm of N2a cells. Subsequently, biochemical characterization of this putative interaction through the use of two distinct cross-link molecules has been investigated. In the prion replication model, direct interaction between PrPSc and endogenous PrPC is required for the formation of further infectious prions. In fact, molecules that specifically bind cellular PrPC may interrupt the production of prions. To further analyze the effect of the addition of recombinant α-syn amyloids to scrapie infected cells, residual PK-resistant levels of PrP were investigated. ScN2a cells exposed to recombinant α-syn amyloids displayed drastically reduced levels of PK-resistant PrPSc. This observation supports the hypothesis that cell surface PrPC might directly bind α-syn amyloids as previously suggested. Finally, further work was required to validate the importance of this interaction in disease progression in vivo. Thus, stereotaxic injections of α-syn amyloids in substantia nigra pars compacta and striatum in FVB PrPWT and FVB PrPKO mice were performed. In conclusion, our findings suggest a role for PrP C in the regulation of α-syn uptake, thus evidencing a link between the two neurodegeneration associated proteins. Moreover, this study presents new insight on the possible implication of the prion protein in Parkinson’s disease. phenomenon in vivo in WT CD-1 mice, where intracerebral inoculation of synthetic α-syn short amyloid fibrils induced the formation of phosphorylated α-syn aggregates in CNS regions distinct from the injection site.