Background:A new SNCA mutation, H50Q, has been linked to familial Parkinson disease (PD). Results: The H50Q mutation does not affect the structure, membrane binding, or subcellular localization of ␣-Syn but alters its pathogenic properties. Conclusion: The H50Q mutation increases ␣-Syn aggregation, secretion, and extracellular toxicity. Significance: ␣-Syn mutations contribute to the pathogenesis of PD via multiple mechanisms.

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...

Despite increasing evidence that supports the role of different post-translational modifications (PTMs) in modulating α-synuclein (α-syn) aggregation and toxicity, relatively little is known about the functional consequences of each modification and whether or not these modifications are regulated by each other. This lack of knowledge arises primarily from the current lack of tools and methodologies for the site-specific introduction of PTMs in α-syn. More specifically, the kinases that mediate selective and efficient phosphorylation of C-terminal tyrosine residues of α-syn remain to be identified. Unlike phospho-serine and phospho-threonine residues, which in some cases can be mimicked by serine/threonine → glutamate or aspartate substitutions, there are no natural amino acids that can mimic phosphor-tyrosine. To address these challenges, we developed a general and efficient semisynthetic strategy that enables the site-specific introduction of single or multiple PTMs and the preparation of homogeneously C-terminal modified forms of α-syn in milligram quantities. These advances have allowed us to investigate, for the first time, the effects of selective phosphorylation at Y125 on the structure, aggregation, membrane binding and subcellular localization of α-syn. The development of semisynthetic methods for the site-specific introduction of single or PTMs represents an important advance toward determining the roles of such modifications in α-syn structure, aggregation and functions in heath and disease.

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2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a carcinogenic heterocyclic aromatic amine formed in meat products during cooking. Although the formation of hazardous PhIP metabolites by mammalian enzymes has been extensively reported, research on the putative involvement of the human intestinal microbiota in PhIP metabolism remains scarce. In this study, the in vitro conversion of PhIP into its microbial derivate, 7-hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido[3,2:4,5]imidazo[1,2-a]pyrimidin-5-ium chloride (PhIP-M1), by fecal samples from 18 human volunteers was investigated. High-performance liquid chromatography analysis showed that all human fecal samples transformed PhIP but with efficiencies ranging from 1.8 to 96% after 72 h of incubation. Two PhIP-transforming strains, PhIP-M1-a and PhIP-M1-b, were isolated from human feces and identified by fluorescent amplified fragment length polymorphism and pheS sequence analyses as Enterococcus faecium strains. Some strains from culture collections belonging to the species E. durans, E. avium, E. faecium, and Lactobacillus reuteri were also able to perform this transformation. Yeast extract, special peptone, and meat extract supported PhIP transformation by the enriched E. faecium strains, while tryptone, monomeric sugars, starch, and cellulose did not. Glycerol was identified as a fecal matrix constituent required for PhIP transformation. Abiotic synthesis of PhIP-M1 and quantification of the glycerol metabolite 3-hydroxypropionaldehyde (3-HPA) confirmed that the anaerobic fermentation of glycerol via 3-HPA is the critical bacterial transformation process responsible for the formation of PhIP-M1. Whether it is a detoxification is still a matter of debate, since PhIP-M1 has been shown to be cytotoxic toward Caco-2 cells but is not mutagenic in the Ames assay.Diet is a major risk factor in human cancer (14). Epidemiological studies indicate that the consumption of cooked meat and meat products predisposes individuals to neoplastic disease, particularly of the colon (13). Cooked muscle meats contain potent genotoxic carcinogens belonging to the heterocyclic aromatic amine (HAA) class of chemical compounds (31). Of the 19 heterocyclic amines identified so far, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) is the most mass-abundant heterocyclic amine produced during the cooking of beef, pork, and chicken (15, 40). Experimentally, PhIP is a potent mutagen and genotoxin and has been shown to produce mammary gland, prostate, and colon tumors in rats (23, 39). For humans, less is known about the potential role of PhIP and related heterocyclic amines in tumor development. Several studies have shown that individuals who eat "well-done" meat have an increased risk of breast (52) and colorectal (18) cancers.To determine the potential health risks associated with heterocyclic amines, several dietary studies have been conducted on the metabolism and disposition of these compounds in humans. So far, most investigations have focused on the activation and ...