The novel Parkinson's disease linked mutation G51D attenuates in vitro aggregation and membrane binding of α-synuclein, and enhances its secretion and nuclear localization in cells

A novel mutation in the α-Synuclein (α-Syn) gene "G51D” was recently identified in two familial cases exhibiting features of Parkinson's disease (PD) and multiple system atrophy (MSA). In this study, we explored the impact of this novel mutation on the aggregation, cellular and biophysical properties of α-Syn, in an attempt to unravel how this mutant contributes to PD/MSA. Our results show that the G51D mutation significantly attenuates α-Syn aggregation in vitro. Moreover, it disrupts local helix formation in the presence of SDS, decreases binding to lipid vesicles C-terminal to the site of mutation and severely inhibits helical folding in the presence of acidic vesicles. When expressed in yeast, α-SynG51D behaves similarly to α-SynA30P, as both exhibit impaired membrane association, form few inclusions and are non-toxic. In contrast, enhanced secreted and nuclear levels of the G51D mutant were observed in mammalian cells, as well as in primary neurons, where α-SynG51D was enriched in the nuclear compartment, was hyper-phosphorylated at S129 and exacerbated α-Syn-induced mitochondrial fragmentation. Finally, post-mortem human brain tissues of α-SynG51D cases were examined, and revealed only partial colocalization with nuclear membrane markers, probably due to post-mortem tissue delay and fixation. These findings suggest that the PD-linked mutations may cause neurodegeneration via different mechanisms, some of which may be independent of α-Syn aggregatio

The novel Parkinson's disease linked mutation G51D attenuates in vitro aggregation and membrane binding of alpha-synuclein, and enhances its secretion and nuclear localization in cells

A novel mutation in the alpha-Synuclein (alpha-Syn) gene "G51D" was recently identified in two familial cases exhibiting features of Parkinson's disease (PD) and multiple system atrophy (MSA). In this study, we explored the impact of this novel mutation on the aggregation, cellular and biophysical properties of alpha-Syn, in an attempt to unravel how this mutant contributes to PD/MSA. Our results show that the G51D mutation significantly attenuates alpha-Syn aggregation in vitro. Moreover, it disrupts local helix formation in the presence of SDS, decreases binding to lipid vesicles C-terminal to the site of mutation and severely inhibits helical folding in the presence of acidic vesicles. When expressed in yeast, alpha-Syn(G51D) behaves similarly to alpha-Syn(A30P), as both exhibit impaired membrane association, form few inclusions and are non-toxic. In contrast, enhanced secreted and nuclear levels of the G51D mutant were observed in mammalian cells, as well as in primary neurons, where alpha-Syn(G51D) was enriched in the nuclear compartment, was hyper-phosphorylated at S129 and exacerbated alpha-Syn-induced mitochondrial fragmentation. Finally, post-mortem human brain tissues of alpha-Syn(G51D) cases were examined, and revealed only partial colocalization with nuclear membrane markers, probably due to post-mortem tissue delay and fixation. These findings suggest that the PD-linked mutations may cause neurodegeneration via different mechanisms, some of which may be independent of alpha-Syn aggregation

The novel Parkinson's disease linked mutation G51D attenuates in vitro aggregation and membrane binding of alpha-synuclein, and enhances its secretion and nuclear localization in cells

A novel mutation in the alpha-Synuclein (alpha-Syn) gene "G51D" was recently identified in two familial cases exhibiting features of Parkinson's disease (PD) and multiple system atrophy (MSA). In this study, we explored the impact of this novel mutation on the aggregation, cellular and biophysical properties of alpha-Syn, in an attempt to unravel how this mutant contributes to PD/MSA. Our results show that the G51D mutation significantly attenuates alpha-Syn aggregation in vitro. Moreover, it disrupts local helix formation in the presence of SDS, decreases binding to lipid vesicles C-terminal to the site of mutation and severely inhibits helical folding in the presence of acidic vesicles. When expressed in yeast, alpha-Syn(G51D) behaves similarly to alpha-Syn(A30P), as both exhibit impaired membrane association, form few inclusions and are non-toxic. In contrast, enhanced secreted and nuclear levels of the G51D mutant were observed in mammalian cells, as well as in primary neurons, where alpha-Syn(G51D) was enriched in the nuclear compartment, was hyper-phosphorylated at S129 and exacerbated alpha-Syn-induced mitochondrial fragmentation. Finally, post-mortem human brain tissues of alpha-Syn(G51D) cases were examined, and revealed only partial colocalization with nuclear membrane markers, probably due to post-mortem tissue delay and fixation. These findings suggest that the PD-linked mutations may cause neurodegeneration via different mechanisms, some of which may be independent of alpha-Syn aggregation

α-Synuclein in central nervous system and from erythrocytes, mammalian cells, and Escherichia coli exists predominantly as disordered monomer

Since the discovery and isolation of α-synuclein (α-syn) from human brains, it has been widely accepted that it exists as an intrinsically disordered monomeric protein. Two recent studies suggested that α-syn produced in Escherichia coli or isolated from mammalian cells and red blood cells exists predominantly as a tetramer that is rich in α-helical structure (Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107-110; Wang, W., Perovic, I., Chittuluru, J., Kaganovich, A., Nguyen, L. T. T., Liao, J., Auclair, J. R., Johnson, D., Landeru, A., Simorellis, A. K., Ju, S., Cookson, M. R., Asturias, F. J., Agar, J. N., Webb, B. N., Kang, C., Ringe, D., Petsko, G. A., Pochapsky, T. C., and Hoang, Q. Q. (2011) Proc. Natl. Acad. Sci. 108, 17797-17802). However, it remains unknown whether or not this putative tetramer is the main physiological form of α-syn in the brain. In this study, we investigated the oligomeric state of α-syn in mouse, rat, and human brains. To assess the conformational and oligomeric state of native α-syn in complex mixtures, we generated α-syn standards of known quaternary structure and conformational properties and compared the behavior of endogenously expressed α-syn to these standards using native and denaturing gel electrophoresis techniques, size-exclusion chromatography, and an oligomer-specific ELISA. Our findings demonstrate that both human and rodent α-syn expressed in the central nervous system exist predominantly as an unfolded monomer. Similar results were observed when human α-syn was expressed in mouse and rat brains as well as mammalian cell lines (HEK293, HeLa, and SH-SY5Y). Furthermore, we show that α-syn expressed in E. coli and purified under denaturing or nondenaturing conditions, whether as a free protein or as a fusion construct with GST, is monomeric and adopts a disordered conformation after GST removal. These results do not rule out the possibility that α-syn becomes structured upon interaction with other proteins and/or biological membranes

The H50Q mutation enhances αα-synuclein aggregation, secretion, and toxicity

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 α-Syn but alters its pathogenic properties. Conclusion: The H50Q mutation increases α-Syn aggregation, secretion, and extracellular toxicity. Significance: α-Syn mutations contribute to the pathogenesis of PD via multiple mechanisms

Phosphorylation of Synucleins by Members of the Polo-like Kinase Family*

Phosphorylation of α-synuclein (α-syn) at Ser-129 is a hallmark of Parkinson disease and related synucleinopathies. However, the identity of the natural kinases and phosphatases responsible for regulating α-syn phosphorylation remain unknown. Here we demonstrate that three closely related members of the human Polo-like kinase (PLK) family (PLK1, PLK2, and PLK3) phosphorylate α-syn and β-syn specifically at Ser-129 and Ser-118, respectively. Unlike other kinases reported to partially phosphorylate α-syn at Ser-129 in vitro, phosphorylation by PLK2 and PLK3 is quantitative (>95% conversion). Only PLK1 and PLK3 phosphorylate β-syn at Ser-118, whereas no phosphorylation of γ-syn was detected by any of the four PLKs (PLK1 to -4). PLK-mediated phosphorylation was greatly reduced in an isolated C-terminal fragment (residues 103–140) of α-syn, suggesting substrate recognition via the N-terminal repeats and/or the non-amyloid component domain of α-syn. PLKs specifically co-localized with phosphorylated Ser-129 (Ser(P)-129) α-syn in various subcellular compartments (cytoplasm, nucleus, and membranes) of mammalian cell lines and primary neurons as well as in α-syn transgenic mice, especially cortical brain areas involved in synaptic plasticity. Furthermore, we report that the levels of PLK2 are significantly increased in brains of Alzheimer disease and Lewy body disease patients. Taken together, these results provide biochemical and in vivo evidence of α-syn and β-syn phosphorylation by specific PLKs. Our results suggest a need for further studies to elucidate the potential role of PLK-syn interactions in the normal biology of these proteins as well as their involvement in the pathogenesis of Parkinson disease and other synucleinopathies