The H50Q Mutation Enhances α-Synuclein Aggregation, Secretion, and Toxicity

Khalaf, Ossama; Fauvet, Bruno; Oueslati, Abid; Dikiy, Igor; Mahul-Mellier, Anne Laure; Ruggeri, Francesco Simone; Mbefo, Martial K.; Vercruysse, Fangio; Dietler, Giovanni; Lee, Seung-Jae; Eliezer, David; Lashuel, Hilal A.

doi:10.1074/jbc.m114.553297

Cited by 164 publications

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“…This approach has been applied in the present study to characterize the effects of five mutational variants, namely A30P, E46K, H50Q, G51D, and A53T, in α-synuclein that are associated with familial Parkinson's disease (14)(15)(16)(17)(18). Our results show that single-amino acid substitutions in the 140-residue chain of α-synuclein not only increase or decrease the overall rate of aggregation, as noted in earlier studies (22)(23)(24)(25)(26)(27)(28)(29)(30) and as reproduced by us (SI Appendix, Fig. S10), but also dramatically affect the relative importance of individual microscopic steps (Fig.…”

Section: Discussionsupporting

confidence: 49%

“…The effects of these sequence changes on the kinetics of the overall aggregation of α-synuclein have been the subject of intense studies since the discovery of this protein as the main constituent of Lewy bodies (13,(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). The A53T variant is generally considered to accelerate aggregation compared with the WT protein (22), but less agreement exists concerning the effects of the A30P mutation, because this variant has been variously reported to aggregate more slowly (28), more rapidly (23)(24)(25)(26), or with the same rate (22) as the WT protein.…”

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confidence: 99%

“…The A53T variant is generally considered to accelerate aggregation compared with the WT protein (22), but less agreement exists concerning the effects of the A30P mutation, because this variant has been variously reported to aggregate more slowly (28), more rapidly (23)(24)(25)(26), or with the same rate (22) as the WT protein. Moreover, for the E46K (27) and H50Q (29,30) variants, the aggregation process has been reported to be accelerated compared with WT α-synuclein, but the G51D variant has been reported to aggregate more slowly than the WT protein (31). These studies provide key insights into the influence of the mutations on the overall aggregation process, but a more detailed knowledge of how the individual microscopic processes of the aggregation reaction are affected by each of the disease-associated, single-point mutations would be of considerable additional value (32)(33)(34) (Fig.…”

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Mutations associated with familial Parkinson’s disease alter the initiation and amplification steps of α-synuclein aggregation

Flagmeier

Meisl

Vendruscolo

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

271

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Parkinson’s disease is a highly debilitating neurodegenerative condition whose pathological hallmark is the presence in nerve cells of proteinacious deposits, known as Lewy bodies, composed primarily of amyloid fibrils of α-synuclein. Several missense mutations in the gene encoding α-synuclein have been associated with familial variants of Parkinson’s disease and have been shown to affect the kinetics of the aggregation of the protein. Using a combination of experimental and theoretical approaches, we present a systematic in vitro study of the influence of disease-associated single-point mutations on the individual processes involved in α-synuclein aggregation into amyloid fibrils. We find that lipid-induced fibril production and surface catalyzed fibril amplification are the processes most strongly affected by these mutations and show that familial mutations can induce dramatic changes in the crucial processes thought to be associated with the initiation and spreading of the aggregation of α-synuclein.

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Section: Discussionsupporting

confidence: 49%

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confidence: 99%

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confidence: 99%

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Mutations associated with familial Parkinson’s disease alter the initiation and amplification steps of α-synuclein aggregation

Flagmeier

Meisl

Vendruscolo

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

271

315

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“…Several studies have reported that overexpressed hα-Syn in mouse primary neurons exhibits diffuse localization without forming discreet inclusions (8)(9)(10)(11). To assess whether the absence of mα-Syn would affect this distribution, we examined the localization of transiently expressed hα-Syn in primary neurons derived from C57Bl6/J/ola/hsd mice (SNCA…”

Section: Resultsmentioning

confidence: 99%

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

Fares

Maco

Oueslati

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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104

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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...

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“…We recommend a purifi cation protocol that includes a reversed-phase HPLC purifi cation step, in order to ensure complete removal of any enzymes (especially proteases) that may cause problems during the phosphorylation reactions. The expression and purifi cation protocol is described in detail in [ 28 ].…”

Section: Instrumentationmentioning

confidence: 99%

Semisynthesis and Enzymatic Preparation of Post-translationally Modified α-Synuclein

Fauvet

Lashuel

2016

Methods in Molecular Biology

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Posttranslational modifications (PTMs) serve as molecular switches for regulating protein folding, function, and interactome and have been implicated in the misfolding and amyloid formation by several proteins linked to neurodegenerative diseases, including Alzheimer's and Parkinson's disease. Understanding the role of individual PTMs in protein misfolding and aggregation requires the preparation of site-specifically modified proteins, as well as the identification of the enzymes involved in regulating these PTMs. Recently, our group has pioneered the development of enzymatic, synthetic, and semisynthetic strategies that allow site-specific introduction of PTMs at single or multiple sites and generation of modified proteins in milligram quantities. In this chapter, we provide detailed description of enzymatic and semisynthetic strategies for the generation of the phosphorylated α-Synuclein (α-Syn) at S129, (pS129), which has been identified as a pathological hallmark of Parkinson's disease. The semisynthetic method described for generation of α-Syn-pS129 requires expertise with protein chemical ligation, but can be used to incorporate other PTMs (single or multiple) within the α-Syn C-terminus if desired. On the other hand, the in vitro kinase-mediated phosphorylation strategy does not require any special setup and is rather easy to apply, but its application is restricted to the generation of α-Syn_pS129. These methods have the potential to increase the availability of pure and homogenous modified α-Syn reagents, which may be used as standards in numerous applications, including the search for potential biomarkers of synucleinopathies.

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The H50Q Mutation Enhances α-Synuclein Aggregation, Secretion, and Toxicity

Cited by 164 publications

References 61 publications

Mutations associated with familial Parkinson’s disease alter the initiation and amplification steps of α-synuclein aggregation

Mutations associated with familial Parkinson’s disease alter the initiation and amplification steps of α-synuclein aggregation

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

Semisynthesis and Enzymatic Preparation of Post-translationally Modified α-Synuclein

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