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

Abstract

Lewy bodies (LBs) are intraneuronal inclusions consisting primarily of fibrillized human α-synuclein (hα-Syn) protein, which represent the major pathological hallmark of Parkinson's disease (PD). Although doubling hα-Syn expression provokes LB pathology in humans, hα-Syn overexpression does not trigger the formation of fibrillar LB-like inclusions in mice. We hypothesized that interactions between exogenous hα-Syn and endogenous mouse synuclein homologs could be attenuating hα-Syn fibrillization in mice, and therefore, we systematically assessed hα-Syn aggregation propensity in neurons derived from α-Syn-KO, β-Syn-KO, γ-Syn-KO, and triple-KO mice lacking expression of all three synuclein homologs. Herein, we show that hα-Syn forms hyperphosphorylated (at S129) and ubiquitin-positive LB-like inclusions in primary neurons of α-Syn-KO, β-Syn-KO, and triple-KO mice, as well as in transgenic α-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 α-Syn species would interfere with the seeding and spreading of α-Syn pathology. Our results are in line with increasing evidence demonstrating that the spreading of α-Syn pathology is most prominent when the injected preformed fibrils and host-expressed α-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 hα-Syn intraneuronal fibrillization and its contribution to PD pathogenesis, and for screening pharmacologic and genetic modulators of α-Syn fibrillization in neurons.Parkinson's disease | alpha-synuclein | aggregation T he aggregation of proteins into fibrillar structures is a key hallmark of many neurodegenerative disorders. In Parkinson's disease (PD), α-synuclein (α-Syn), a predominantly presynaptic protein involved in the regulation of neurotransmitter release, abnormally fibrilizes and forms intraneuronal inclusions termed "Lewy bodies" (LBs) (1, 2). So far, the mechanisms underlying LB formation remain poorly understood, and the impact of LB presence on neuronal viability remains controversial, in part due to the lack of animal models recapitulating α-Syn fibrillization into LB-like inclusions in the brain.Because patients with familial history of parkinsonism were found carrying either multiplications or point mutations of the α-Syn gene SNCA (2), most animal models of PD have been generated by overexpressing WT human α-Syn (hα-Syn) or mutant forms linked to familial PD (3). Strikingly, although rodent models expressing hα-Syn do not recapitulate the formation of fibrillar LBs within dopaminergic neurons, hα-Syn overexpression in Drosophila led to dramatic neuronal loss accompanied with LB-like structures compri...

β-Synuclein suppresses both the initiation and amplification steps of α-synuclein aggregation via competitive binding to surfaces

“…Although it has been established that β -synuclein can have an inhibitory effect on the aggregation of α -synuclein141516171820, the mechanism by which such inhibition occurs was not previously known but is of very significant biological interest. In earlier work, we have described an experimental and theoretical strategy that has allowed us to dissect the mechanism of aggregation and amyloid formation of α -synuclein in great detail323439.…”

“…Although both proteins are localised at presynaptic terminals and are expressed at similar levels1213, β -synuclein has not been implicated in the etiology of Parkinson’s disease but instead has been observed to inhibit the aggregation of α -synuclein both in vitro 1415 and in vivo 1617181920. Altered expression levels of both α -synuclein and β -synuclein (up-regulated and down-regulated, respectively) have been correlated with disease onset21, and counter-regulating the expression of both proteins decreases α -synuclein aggregation in mammalian cell cultures, suggesting that β -synuclein may act to limit disease progression21.…”

Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson’s Disease in Rodents

“…Recombinant aSyn monomers may be generated in house using protocols such as those described in Volpicelli-Daley, Luk, & Lee (2014) [31], Abdelmotilib et al, 2017 [35], or Fares et al, 2016 [44]. Monomeric aSyn protein specifically-formulated to generate aSyn PFFs may also be purchased from commercial sources (Appendix A).…”

“…Generally, greater homology between the host and species of recombinant aSyn protein will result in greater pathology in vitro and in vivo . This has been demonstrated at the level of seeding recombinant protein as human aSyn PFFs are much more effective at templating amyloid-formation of human aSyn monomers than mouse aSyn monomers when measured by the ThT kinetic seeding assay [44, 51]. Similarly, mouse aSyn PFFs seed amyloid-structure formation in mouse aSyn monomers more effectively than human aSyn monomers [51].…”

“…These forms of aSyn offer the advantage of allowing the sample to undergo the same pretreatment as the aSyn PFFs (heat, shaking, sonication) to control for the effects of these treatments on the protein. Although aggregation-incompetent forms of aSyn have shown promising results as a control in in vitro experiments [44], these proteins have not been tested in vivo . Serum albumin has also been suggested as a control for aSyn PFF experiments as this protein would control for the introduction of a larger protein in the brain and would therefore control for protein size and uptake.…”