Dopamine facilitates α-synuclein oligomerization in human neuroblastoma SH-SY5Y cells

Yamakawa, Kentaro; Izumi, Yasukatsu; Takeuchi, Hiroki; Yamamoto, Norio; Kume, Toshiaki; Akaike, Akinori; Takahashi, Ryōsuke; Shimohama, Shun; Sawada, Hideyuki

doi:10.1016/j.bbrc.2009.11.015

Cited by 38 publications

(33 citation statements)

References 14 publications

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“…Finally, α-syn oligomers were produced by coincubation of monomeric α-syn with excess dopamine. [29][30][31] Under these conditions,~50% of monomeric α-syn was converted to oligomers after 4 days, as assessed by size exclusion chromatography (SEC) (Figure 1h). The purity, heterogeneity and amyloidogenic properties of α-syn oligomers were assessed by SEC, SDS-PAGE electrophoresis, TEM and ThT binding assay (Figures 1h-k).…”

Section: Resultsmentioning

confidence: 99%

“…Interestingly, addition of a mixture of α-syn monomers and dopamine-induced oligomers did not result in any significant cellular toxicity (Figure 2e), consistent with previous reports. 29,30,35 We then investigated which mechanism of cell death was induced by α-syn fibrillization. After 4 days of treatment,~50% of the cells showed caspase 3 activation and DNA fragmentation (Figures 2f and g and Supplementary Figures S1D and E), indicating that α-syn mixture induced apoptosis in neuroblastoma cell lines.…”

Section: Resultsmentioning

confidence: 99%

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Fibril growth and seeding capacity play key roles in α-synuclein-mediated apoptotic cell death

et al. 2015

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Fibril growth and seeding capacity play key roles in α-synuclein-mediated apoptotic cell death

et al. 2015

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“…We first treat asyn-EGFP overexpressing cells and EGFP overexpressing cells as control with dopamine, which is known to lead to the formation of stable αsyn oligomeric species [46]. As reported in Figure S8, SH-SY5Y overexpressing cells treated with dopamine did not show a considerable difference in term of oligomers dimensions.…”

Section: Resultsmentioning

confidence: 99%

Number and Brightness analysis of alpha-synuclein oligomerization and the associated mitochondrial morphology alterations in live cells

Plotegher¹,

Gratton²,

Bubacco³

2014

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background Alpha-synuclein oligomerization is associated to Parkinson's disease etiopathogenesis. The study of alpha-synuclein oligomerization properties in live cell and the definition of their effects on cellular viability are among fields expected to provide the knowledge required to unravel the mechanism(s) of toxicity that lead to the disease. Methods We used Number and Brightness method, which is a method based on fluorescence fluctuation analysis, to monitor alpha-synuclein tagged with EGFP aggregation in living SH-SY5Y cells. The presence of alpha-synuclein oligomers detected with this method was associated with intracellular structure conditions, evaluated by fluorescence confocal imaging. Results Cells overexpressing alpha-synuclein-EGFP present a heterogeneous ensemble of oligomers constituted by less than 10 monomers, when the protein approaches a threshold concentration value of about 90 nM in the cell cytoplasm. We show that the oligomeric species are partially sequestered by lysosomes and that the mitochondria morphology is altered in cells presenting oligomers, suggesting that these mitochondria may be dysfunctional. Conclusions We showed that alpha-synuclein overexpression in SH-SY5Y causes the formation of alpha-synuclein oligomeric species, whose presence is associated with mitochondrial fragmentation and autophagic-lysosomal pathway activation in live cells. General significance The unique capability provided by the Number and Brightness analysis to study alpha-synuclein oligomers distribution and properties, and the study their association to intracellular components in single live cells is important to forward our understanding of the molecular mechanisms Parkinson’s disease and it may be of general significance when applied to the study of other aggregating proteins in cellular models.

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“…Oxidative modification of alpha-syn via dopamine adducts may facilitate aggregation [175]. Similarly, recent data demonstrated that stable overexpression of alpha-syn in SH-SY5Y cells or exposure of cells to dopamine facilitated alpha-syn oligomerization [188]. Dopamine was shown to modulate differently the stability of protofibrils and fibrils composed of wild-type or mutant forms of alpha-syn [189].…”

Section: Protein Aggregation: Alpha-synuclein Oligomerizationmentioning

confidence: 99%

Mitochondrial Dysfunction: The Road to Alpha-Synuclein Oligomerization in PD

Esteves

Arduíno

Silva

et al. 2011

Parkinson's Disease

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While the etiology of Parkinson's disease remains largely elusive, there is accumulating evidence suggesting that mitochondrial dysfunction occurs prior to the onset of symptoms in Parkinson's disease. Mitochondria are remarkably primed to play a vital role in neuronal cell survival since they are key regulators of energy metabolism (as ATP producers), of intracellular calcium homeostasis, of NAD+/NADH ratio, and of endogenous reactive oxygen species production and programmed cell death. In this paper, we focus on mitochondrial dysfunction-mediated alpha-synuclein aggregation. We highlight some of the findings that provide proof of evidence for a mitochondrial metabolism control in Parkinson's disease, namely, mitochondrial regulation of microtubule-dependent cellular traffic and autophagic lysosomal pathway. The knowledge that microtubule alterations may lead to autophagic deficiency and may compromise the cellular degradation mechanisms that culminate in the progressive accumulation of aberrant protein aggregates shields new insights to the way we address Parkinson's disease. In line with this knowledge, an innovative window for new therapeutic strategies aimed to restore microtubule network may be unlocked.

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Dopamine facilitates α-synuclein oligomerization in human neuroblastoma SH-SY5Y cells

Cited by 38 publications

References 14 publications

Fibril growth and seeding capacity play key roles in α-synuclein-mediated apoptotic cell death

Fibril growth and seeding capacity play key roles in α-synuclein-mediated apoptotic cell death

Number and Brightness analysis of alpha-synuclein oligomerization and the associated mitochondrial morphology alterations in live cells

Mitochondrial Dysfunction: The Road to Alpha-Synuclein Oligomerization in PD

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