A Novel, High-Efficiency Cellular Model of Fibrillar α-Synuclein Inclusions and the Examination of Mutations that Inhibit Amyloid Formation

Parkinson's disease (PD) is the most common movement neurodegenerative disorder and is characterized by a loss of dopaminergic neurons in the substantia nigra pars compacta. Pathological analysis of PD brains reveals intracytoplasmic, perikaryal inclusions, known as Lewy bodies (LBs) in some of the remaining dopaminergic neurons, as well as similar inclusions in neuronal processes, termed Lewy neurites (LNs) (Goedert 2001;von Bohlen Und 2004;Lee and Trojanowski 2006). The major component of LBs and LNs is α-synuclein (α-syn), a 140 amino acid protein that is normally soluble and localized to neuronal synaptic terminals (Goedert 2001;von Bohlen Und 2004;Lee and Trojanowski 2006;Sidhu et al. 2004). A role for α-syn in the pathophysiology of PD is further supported by several missense mutations in α-syn and short chromosomal duplications and triplications that include the α-syn gene, which have been identified in familial forms of PD (Forman et al. 2005;Polymeropoulos et al. 1997). Furthermore, α-syn is the major component of pathological inclusions in other neurodegenerative diseases, including dementia with Lewy bodies (DLB), Lewy body variant of Alzheimer's disease (LBVAD), and multiple systems atrophy (MSA), which are collectively termed α-synucleinopathies (Duda et al. 2000;Lippa et al. 1999;Spillantini et al. 1997;Spillantini et al. 1998;Tu et al. 1998;Goedert 2001;Forman et al. 2005). α-Syn aberrantly polymerizes into 10–15 nm amyloidogenic fibrils that associate to form cellular inclusions, which can contribute to the pathogenesis of PD (Goedert 2001;von Bohlen Und 2004;Lee and Trojanowski 2006). These inclusions can be detected with amyloid binding dyes, such as thioflavin and Congo Red or derivatives (Crystal et al. 2003;Conway et al. 2000). Aggregated α-syn in these inclusions also has several modifications, including ubiquitination (Sampathu et al. 2003) and hyperphosphorylation (Fujiwara et al. 2002;Kahle et al. 2002;Anderson et al. 2006;Nishie et al. 2004;Neumann et al. 2002;Waxman and Giasson 2008). Recombinant α-syn can readily polymerize in vitro into amyloidogenic fibrils, which are structurally similar to pathological inclusions (Conway et al. 1998;Giasson et al. 1999;Hashimoto et al. 1998;Giasson et al. 2001). The middle hydrophobic region of α-syn plays a critical role in amyloid formation (Giasson et al. 2001;Waxman et al. 2009b;Zibaee et al. 2007), such that deletion of several hydrophobic stretches or single-point mutations in this region can prevent polymerization into mature amyloid (Biere et al. 2000;Du et al. 2003;Zibaee et al. 2007;Giasson et al. 2001;Waxman et al. 2009b;Koo et al. 2008;Koo et al. 2009). Such means to prevent the formation of α-syn polymerization are areas of intense investigation. While in vitro studies provide important information regarding the function of α-syn and its role in PD, in vivo systems are required to further the investigation of therapeutics. α-Syn fibril formation is a nucleation-dependent process (Wood et al. 1999), and cellular seeding of amyloidogenic proteins has been previously observed (Ren et al. 2009). The current study presents the development and characterization of a novel, high-efficiency cellular model of α-syn aggregation, employing a combination of cellular seeding of α-syn polymerization and calcium phosphate precipitation. Using this system we examined the effects of single-point mutations as inhibitors of α-syn polymerization. This study provides an ideal model system, with which to investigate such potential inhibitors against α-syn amyloid formation.