Abstract
Recent studies suggest that the cell-to-cell spread of pathological α-synuclein (α-syn) plays important roles in the development of Parkinson’s disease (PD). PD patients who carry α-syn gene mutations often have an earlier onset and more severe clinical symptoms and pathology than sporadic PD cases who carry the wild-type (WT) α-syn gene. However, the molecular mechanism by which α-syn gene mutations promote PD remains unclear. Here, we hypothesized that pathogenic mutations facilitate the intercellular transfer and cytotoxicity of α-syn, favoring an early disease onset and faster progression. We investigated the effects of eight known pathogenic mutations in human α-syn (A18T, A29S, A30P, E46K, H50Q, G51D, A53E, and A53T) on its pathological transmission in terms of secretion, aggregation, intracellular level, cytotoxicity, seeding, and induction of neuroinflammation in SH-SY5Y neuroblastoma cells, cultured rat neurons, and microglia, and the rat substantia nigra pars compacta. We found that 2 of the 8 mutations (H50Q and A53T) significantly increased α-syn secretion while 6 mutations (A18T, A29S, A30P, G51D, A53E, and E46K) tended to enhance it. In vitroα-syn aggregation experiments showed that H50Q promoted while G51D delayed aggregation most strongly. Interestingly, 3 mutations (E46K, H50Q, and G51D) greatly increased the intracellular α-syn level when cultured cells were treated with preformed α-syn fibrils (PFFs) compared with the WT, while the other 5 had no effect. We also demonstrated that H50Q, G51D, and A53T PFFs, but not E46K PFFs, efficiently seeded in vivo and acutely induced neuroinflammation in rat substantia nigra pars compacta. Our data indicate that pathogenic mutations augment the prion-like spread of α-syn at different steps and blockade of this pathogenic propagation may serve as a promising therapeutic intervention for PD.
Highlights
It was thought that α-syn misfolding and intracellular aggregation play a central pathogenic role in synucleinopathies (Conway et al, 1998; Galvin et al, 2001; Beyer, 2006; Uversky, 2007; Tsigelny et al, 2008; Oueslati et al, 2010; Taschenberger et al, 2012)
We found that A30P accelerated α-syn aggregation and increased β-structure content, which agrees with some previous reports (Narhi et al, 1999; FIGURE 1 | Pathogenic mutations increase α-syn secretion in differentiated SH-SY5Y cells. (A) Schematic of pathogenic mutations of α-syn. (B,C) Immunoblots (B) and analysis (C) of cell lysates (n = 7 independent cultures, results of three experiments are shown). (D–F) Immunoblots (n = 4 independent cultures) and ELISA analysis (n = 7 independent cultures) of conditioned medium. (G,H) Normalized MTS levels (G) and mRNA levels quantified by Quantitative Reverse Transcription-PCR (qRT-PCR) (H) (n = 4 independent cultures)
We systematically investigated the modulating effects of pathogenic mutations on the cell-to-cell transmission of α-syn (Table 2) and revealed the following: (1) Pathogenic mutations of α-syn tended to show increased secretion levels overall, with H50Q and A53T enhancing secretion significantly compared to the WT
Summary
It was thought that α-syn misfolding and intracellular aggregation play a central pathogenic role in synucleinopathies (Conway et al, 1998; Galvin et al, 2001; Beyer, 2006; Uversky, 2007; Tsigelny et al, 2008; Oueslati et al, 2010; Taschenberger et al, 2012). Exogenous α-syn aggregates or recombinant preformed α-syn fibrils (PFFs) have been found to enter neurons and act as “seeds” to promote the aggregation of endogenous α-syn, leading to its pathological spread (Volpicelli-Daley et al, 2011; Angot et al, 2012; Luk et al, 2012; Holmqvist et al, 2014; Borland and Vilhardt, 2017; Rutherford et al, 2017; Bieri et al, 2018; Rey et al, 2018; Henderson et al, 2019) This cell-tocell transmission property of α-syn prompted us to investigate whether pathogenic mutations modulate the propagation and cytotoxicity of α-syn and whether these changes correlate with the clinical characteristics. Our results shed light on the understanding of PD pathogenesis and provide insight for intervention
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