The E46K Mutation in α-Synuclein Increases Amyloid Fibril Formation

Greenbaum, Eric A.; Graves, Charles L.; Mishizen-Eberz, Amanda J.; Lupoli, Michael A.; Lynch, David R.; Englander, S. Walter; Axelsen, Paul H.; Giasson, Benoit I.

doi:10.1074/jbc.m411638200

Cited by 346 publications

(348 citation statements)

References 67 publications

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“…Previous studies have shown that -synuclein mutations (A53T, A30P, E46K) or triplications are associated with some autosomal-dominant PD [3][4][5][6] . Although there is no -synuclein gene mutation identified in idiopathic PD [7,8] , aggregated -synuclein has been found to be the major component of Lewy bodies and Lewy neurites, the pathological hallmarks of PD [9][10][11][12][13][14] .…”

Section: Introductionmentioning

confidence: 99%

RNA interference mediated silencing of α-synuclein in MN9D cells and its effects on cell viability

et al. 2008

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ObjectiveTo silence the expression of -synuclein in MN9D dopaminergic cells using vector mediated RNA interference (RNAi) and examined its effects on cell proliferation and viability. Methods We identified two 19-nucleotide stretches within the coding region of the -synuclein gene and designed three sets of oligonucleotides to generate doublestranded (ds) oligos. The ds oligos were inserted into the pENTR TM /H1/TO vector and transfected into MN9D dopaminergic cells. -Synuclein expression was detected by RT-PCR, real-time PCR, immunocytochemistry staining and Western blot. In addition, we measured cell proliferation using growth curves and cell viability by 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-diphenytetrazoliumromide (MTT). Results The mRNA and protein levels of -synuclein gene were significantly down-regulated in pSH2/ -SYN-transfected cells compared with control MN9D and pSH/CON-transfected MN9D cells, while pSH1/ -SYNtransfected cells showed no significant difference. Silencing -synuclein expression does not affect cell proliferation but may decrease cell viability. Conclusion Our results demonstrated pSH2/ -SYN is an effective small interfering RNA (siRNA) sequence and potent silencing of mouse -synuclein expression in MN9D cells by vector-based RNAi, which provides the tools for studying the normal function of -synuclein and examining its role in Parkinson's disease (PD) pathogenesis.-Synuclein may be important for the viability of MN9D cells, and loss of -synuclein may induce cell injury directly or indirectly.

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Section: Introductionmentioning

confidence: 99%

RNA interference mediated silencing of α-synuclein in MN9D cells and its effects on cell viability

et al. 2008

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“…␣-syn fibrils exhibit properties of amyloid (Spillantini et al, 1998), and ␣-syn assembles into amyloid fibrils in vitro (Han et al, 1995;Giasson et al, 1999), whereas SNCA mutations can accelerate ␣-syn fibrillization (Conway et al, 1998;Greenbaum et al, 2005). Therefore, it is plausible that ␣-syn misfolds and aggregates into amyloid fibrils that are neurotoxic and play a causative role in the pathogenesis of PD.…”

Section: Introductionmentioning

confidence: 99%

α-Synuclein–induced Aggregation of Cytoplasmic Vesicles inSaccharomyces cerevisiae

Soper¹,

Roy²,

Stieber³

et al. 2008

MBoC

146

168

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Aggregated ␣-synuclein (␣-syn) fibrils form Lewy bodies (LBs), the signature lesions of Parkinson's disease (PD) and related synucleinopathies, but the pathogenesis and neurodegenerative effects of LBs remain enigmatic. Recent studies have shown that when overexpressed in Saccharomyces cerevisiae, ␣-syn localizes to plasma membranes and forms cytoplasmic accumulations similar to human ␣-syn inclusions. However, the exact nature, composition, temporal evolution, and underlying mechanisms of yeast ␣-syn accumulations and their relevance to human synucleinopathies are unknown. Here we provide ultrastructural evidence that ␣-syn accumulations are not comprised of LB-like fibrils, but are associated with clusters of vesicles. Live-cell imaging showed ␣-syn initially localized to the plasma membrane and subsequently formed accumulations in association with vesicles. Imaging of truncated and mutant forms of ␣-syn revealed the molecular determinants and vesicular trafficking pathways underlying this pathological process. Because vesicular clustering is also found in LB-containing neurons of PD brains, ␣-syn-mediated vesicular accumulation in yeast represents a model system to study specific aspects of neurodegeneration in PD and related synucleinopathies. INTRODUCTIONParkinson's disease (PD) is the most prevalent neurodegenerative movement disorder and is characterized by bradykinesia, rigidity, postural instability, and resting tremor (Galvin et al., 2001;Forman et al., 2005), as well as by the loss of dopaminergic neurons and presence of neuronal inclusions, known as Lewy bodies (LBs) and Lewy neurites (Forman et al., 2005). The identification of an ␣-syn gene (SNCA) mutation associated with autosomal dominant PD (Polymeropoulos et al., 1997), and the identification of fibrillar ␣-syn as the principal component of LB pathology (Spillantini et al., 1997), indicate that the LBs formed by ␣-syn define classic PD and are implicated in disease pathogenesis. The subsequent identification of additional disease-linked SNCA mutations (Polymeropoulos et al., 1997;Kruger et al., 1998;Zarranz et al., 2004), as well as replications of the SNCA gene (Singleton et al., 2003;Chartier-Harlin et al., 2004), indicate that both mutant ␣-syn and increased expression of wildtype (WT) ␣-syn can cause neurodegeneration.In addition to PD, filamentous ␣-syn inclusions have been detected in other neurodegenerative diseases including the LB variant of Alzheimer's disease, dementia with LBs, neurodegeneration with brain iron accumulation type-1, and multiple system atrophy, which are collectively known as synucleinopathies (Spillantini et al., 1997;Duda et al., 2000). ␣-syn fibrils exhibit properties of amyloid (Spillantini et al., 1998), and ␣-syn assembles into amyloid fibrils in vitro (Han et al., 1995;Giasson et al., 1999), whereas SNCA mutations can accelerate ␣-syn fibrillization (Conway et al., 1998;Greenbaum et al., 2005). Therefore, it is plausible that ␣-syn misfolds and aggregates into amyloid fibrils that are neurotoxic and play a cau...

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“…␣-Synuclein is a major protein component in Lewy bodies and Lewy neurites of sporadic PD (Spillantini et al, 1997(Spillantini et al, , 1998Spillantini and Goedert, 2000;Kahle et al, 2001;Kotzbauer et al, 2001;Forman et al, 2004). Three different mutations in the ␣-synuclein gene (A53T, A30P, and E46K) cause autosomal-dominant hereditary PD (Polymeropoulos et al, 1997;Farrer et al, 1998;Kruger et al, 1998;Zarranz et al, 2004;Greenbaum et al, 2005). Recent reports have shown that ␣-synuclein in Lewy bodies is hyperphosphorylated at S129, the major phosphorylation site of ␣-synuclein Hasegawa et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

-Synuclein Phosphorylation Enhances Eosinophilic Cytoplasmic Inclusion Formation in SH-SY5Y Cells

Smith

Margolis

et al. 2005

Journal of Neuroscience

242

229

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Parkinson's disease (PD) is a neurodegenerative disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. Previous reports have shown that ␣-synuclein deposited in brain tissue from individuals with synucleinopathy is extensively phosphorylated at Ser-129. Here, we investigate the role of phosphorylation of ␣-synuclein in the formation of inclusions involving synphilin-1 and parkin using site-directed mutagenesis to change Ser-129 of ␣-synuclein to alanine (S129A) to abolish phosphorylation at this site. Coexpression of wild-type ␣-synuclein and synphilin-1 in human neuroblastoma SH-SY5Y cells yielded cytoplasmic eosinophilic inclusions with some features resembling Lewy bodies, whereas coexpression of S129A ␣-synuclein and synphlin-1 formed few or no inclusions. Moreover, coexpression of parkin with ␣-synuclein and synphilin-1 formed more ubiquitinated inclusions, but these inclusions decreased with expression of S129A ␣-synuclein instead of wild-type ␣-synuclein. Coimmunoprecipitation assays revealed a decreased interaction of S129A ␣-synuclein with synphilin-1 compared with wild-type ␣-synuclein. Expression of S129A ␣-synuclein instead of wild-type ␣-synuclein also decreased the association of synphilin-1 and parkin and subsequently reduced the parkin-mediated ubiquitination of synphilin-1 and the formation of ubiquitinated inclusions. Treatment of SH-SY5Y cells with H 2 O 2 increased ␣-synuclein phosphorylation and enhanced the formation of inclusions formed by coexpression of ␣-synuclein, synphilin-1, and parkin, whereas treatment with the casein kinase 2 inhibitor 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole had the opposite affect. These results indicate that phosphorylation of ␣-synuclein at S129 may be important for the formation of inclusions in PD and related ␣ synucleinopathies.

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The E46K Mutation in α-Synuclein Increases Amyloid Fibril Formation

Cited by 346 publications

References 67 publications

RNA interference mediated silencing of α-synuclein in MN9D cells and its effects on cell viability

RNA interference mediated silencing of α-synuclein in MN9D cells and its effects on cell viability

α-Synuclein–induced Aggregation of Cytoplasmic Vesicles inSaccharomyces cerevisiae

-Synuclein Phosphorylation Enhances Eosinophilic Cytoplasmic Inclusion Formation in SH-SY5Y Cells

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