Fibril growth and seeding capacity play key roles in α-synuclein-mediated apoptotic cell death

Abstract

The role of extracellular α-synuclein (α-syn) in the initiation and the spreading of neurodegeneration in Parkinson's disease (PD) has been studied extensively over the past 10 years. However, the nature of the α-syn toxic species and the molecular mechanisms by which they may contribute to neuronal cell loss remain controversial. In this study, we show that fully characterized recombinant monomeric, fibrillar or stabilized forms of oligomeric α-syn do not trigger significant cell death when added individually to neuroblastoma cell lines. However, a mixture of preformed fibrils (PFFs) with monomeric α-syn becomes toxic under conditions that promote their growth and amyloid formation. In hippocampal primary neurons and ex vivo hippocampal slice cultures, α-syn PFFs are capable of inducing a moderate toxicity over time that is greatly exacerbated upon promoting fibril growth by addition of monomeric α-syn. The causal relationship between α-syn aggregation and cellular toxicity was further investigated by assessing the effect of inhibiting fibrillization on α-syn-induced cell death. Remarkably, our data show that blocking fibril growth by treatment with known pharmacological inhibitor of α-syn fibrillization (Tolcapone) or replacing monomeric α-syn by monomeric β-synuclein in α-syn mixture composition prevent α-syn-induced toxicity in both neuroblastoma cell lines and hippocampal primary neurons. We demonstrate that exogenously added α-syn fibrils bind to the plasma membrane and serve as nucleation sites for the formation of α-syn fibrils and promote the accumulation and internalization of these aggregates that in turn activate both the extrinsic and intrinsic apoptotic cell death pathways in our cellular models. Our results support the hypothesis that ongoing aggregation and fibrillization of extracellular α-syn play central roles in α-syn extracellular toxicity, and suggest that inhibiting fibril growth and seeding capacity constitute a viable strategy for protecting against α-syn-induced toxicity and slowing the progression of neurodegeneration in PD and other synucleinopathies. 1 Nevertheless, the relationship between α-syn aggregation and neurodegeneration in PD remains elusive. 2A possible role for extracellular α-syn in the pathogenicity of PD emerged from the observation that newly grafted neurons in PD patients exhibit α-syn pathology similar to that of neighboring diseased cells. 3,4 Despite the consensus that α-syn is mainly an intracellular protein, α-syn has been detected in the cerebrospinal fluid under both pathological and healthy conditions. 5 In addition, in vivo rodent models and cellular studies have shown that monomers 6 and aggregated forms 6,7 of α-syn are secreted into the extracellular space via several mechanisms, 7,8 including the nonclassical endoplasmic reticulum/Golgi-independent exocytosis 8 or the exosomal route, 9,10 and are then internalized by neighboring cells. 7This suggests that extracellular α-syn may play a critical role in the spreading of α-syn pathology throughout the brai...

Dynamic structural flexibility of α-synuclein

“…All known mutations associated with familial PD (A30P, E46K, H50Q, G51D, A53E, and A53T) are found in the N-terminal domain (Krüger et al, 2008; Lesage et al, 2013; Pasanen et al, 2014; Polymeropoulos et al, 1997; Proukakis et al, 2013; Zarranz et al, 2004). These mutations, with the exception of G51D, A53E, and A30P, increase the propensity of α-synuclein to form insoluble aggregates and produce morphologically distinct aggregate species (Ghosh et al, 2014; Giasson et al, 1999; Greenbaum et al, 2005; Lesage et al, 2013; Mahul-Mellier et al, 2015; Narhi et al, 1999). Though the precise mechanism by which these mutations promote aggregation has not been conclusively shown, evidence implicate an accelerated formation of oligomers (Conway et al, 2000) likely due to the destabilization of the native N-terminal conformation (Bertoncini et al, 2005a; Burré et al, 2015; Coskuner and Wise-Scira, 2013; Dettmer et al, 2015).…”

Neurotoxicity of pre-incubated alpha-synuclein with neutral nanoliposomes on PC12 and SHSY5Y cell lines

“…Based on the latter, nanoparticles, especially those that possess an ability to accelerate brillation, would be proper candidates to reduce oligomers, and consequently hinder cell death [26]. Furthermore, a recent study has suggested that the trend of brillation and aggregation process plays a crucial role in cell toxicity, and inhibiting bril growth and seeding aptitude may act as a viable strategy to protect cells from degeneration [10]. Finally, according to the existing con icts, more investigations are required to elucidate the mechanism of -Syn pathologic species and its impact on cell death while being treated by nanoparticles.…”

“…The degeneration of dopaminergic neuronal cells has been shown to be connected with the development of -Syn brillation [10]. Hence, there is a considerable interest in inhibiting -Syn bril formation, which can be a virtuous avenue to impede the development of PD [11][12][13].…”

CSF biomarkers in neurodegenerative and vascular dementias

“…Abnormal a-synuclein accumulates in glial (mainly oligodendroglial cells) and in neurons in multiple system atrophy (MSA) (Galvin et al, 2001;Jellinger, 2009;Dickson and Weller, 2011;Love et al, 2015). Oligomeric a-synuclein species are toxic (Winner et al, 2011) and abnormal a-synuclein has also the capacity of seeding thus facilitating disease progression (Angot et al, 2012;Luk et al, 2009;Mahul-Mellier et al, 2015). PD is characterized clinically by motor disturbances known as parkinsonism which are preceded by several years by sleep disorders, depression, loss of olfaction and variegated autonomic symptoms (pre-parkinsonian symptoms of PD) which continue even after the appearance of motor symptoms.…”