Induction of de novo α-synuclein fibrillization in a neuronal model for Parkinson's disease

Lewy bodies (LBs) are intraneuronal inclusions consisting primarily of fibrillized human alpha-synuclein (h alpha-Syn) protein, which represent the major pathological hallmark of Parkinson's disease (PD). Although doubling h alpha-Syn expression provokes LB pathology in humans, h alpha-Syn overexpression does not trigger the formation of fibrillar LB-like inclusions in mice. We hypothesized that interactions between exogenous h alpha-Syn and endogenous mouse synuclein homologs could be attenuating ha-Syn fibrillization in mice, and therefore, we systematically assessed ha-Syn aggregation propensity in neurons derived from alpha-Syn-KO, beta-Syn-KO, gamma-Syn-KO, and triple-KO mice lacking expression of all three synuclein homologs. Herein, we show that h alpha-Syn forms hyperphosphorylated (at S129) and ubiquitin-positive LB-like inclusions in primary neurons of alpha-Syn-KO, beta-Syn-KO, and triple-KO mice, as well as in transgenic alpha-Syn-KO mouse brains in vivo. Importantly, correlative light and electron microscopy, immunogold labeling, and thioflavin-S binding established their fibrillar ultrastructure, and fluorescence recovery after photobleaching/photoconversion experiments showed that these inclusions grow in size and incorporate soluble proteins. We further investigated whether the presence of homologous alpha-Syn species would interfere with the seeding and spreading of alpha-Syn pathology. Our results are in line with increasing evidence demonstrating that the spreading of alpha-Syn pathology is most prominent when the injected preformed fibrils and host-expressed alpha-Syn monomers are from the same species. These findings provide insights that will help advance the development of neuronal and in vivo models for understanding mechanisms underlying h alpha-Syn intraneuronal fibrillization and its contribution to PD pathogenesis, and for screening pharmacologic and genetic modulators of alpha-Syn fibrillization in neurons.