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ABSTRACT: Most α-synuclein (α-syn) deposited in Lewy bodies, the pathological hallmark of Parkinson's disease (PD), is phosphorylated at serine 129 (Ser129). However, the physiological and pathological roles of this modification are unclear. Here, we investigated the effects of Ser129-phosphorylation on dopamine (DA) uptake in dopaminergic SH-SY5Y cells expressing α-syn. Subcellular fractionation of siRNA-treated cells showed that G-protein-coupled receptor kinase 3 (GRK3), GRK5, GRK6 and casein kinase 2 (CK2) contributed to Ser129-phosphorylation of membrane-associated α-syn, while cytosolic α-syn was phosphorylated exclusively by CK2. Expression of wild-type α-syn increased DA uptake, and this effect was diminished by introducing the S129A mutation into α-syn. However, wild-type and S129A α-syn equally increased the cell surface expression of dopamine transporter (DAT) in SH-SY5Y cells and in non-neuronal HEK293 cells. Additionally, siRNA-mediated knockdown of GRK5 or GRK6 significantly attenuated DA uptake without altering DAT cell surface expression, whereas knockdown of CK2 had no effect on uptake. Taken together, our results demonstrate that membrane-associated α-syn enhances DA uptake capacity of DAT by GRKs-mediated Ser129-phosphorylation, suggesting that α-syn modulates intracellular DA levels with no functional redundancy in Ser129-phosphorylation between GRKs and CK2.
Molecular biology of the cell 04/2013; · 5.98 Impact Factor
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ABSTRACT: Abstract Parkinson's disease is the most common neurodegenerative movement disorder. The motor impairments of Parkinson's disease are caused by the loss of dopaminergic neurons in the substantia nigra and associated with the appearance of fibrillar aggregates of α-synuclein (α-syn) called Lewy bodies. Approximately 90% of α-syn deposited in Lewy bodies is phosphorylated at serine 129 (Ser129). In contrast, only 4% or less of total α-syn is phosphorylated at this residue in the normal brain. This suggests that the accumulation of Ser129-phosphorylated α-syn leads to the formation of Lewy bodies and dopaminergic neurodegeneration in Parkinson's disease. Our laboratory and others have performed experiments using in vivo models of Parkinson's disease to elucidate the role of increased Ser129 phosphorylation in α-syn neurotoxicity. However, there has been a lack of consistency among these models. In this review, we summarize the main findings regarding the relationship between Ser129 phosphorylation and α-syn neurotoxicity, and examine the differences among models. We further discuss the role of Ser129 phosphorylation in α-syn aggregation and the future directions to test the potential of Ser129 phosphorylation as a therapeutic target for slowing the progression of Parkinson's disease.
Reviews in the neurosciences 01/2013; · 2.41 Impact Factor
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ABSTRACT: Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra (SN) and the appearance of fibrillar aggregates of insoluble α-synuclein (α-syn) called Lewy bodies (LBs). Approximately 90% of α-syn deposited in LBs is phosphorylated at serine 129 (Ser129). In contrast, only 4% of total α-syn is phosphorylated in normal brain, suggesting that accumulation of Ser129-phosphorylated α-syn is involved in the pathogenesis of PD. However, the role of Ser129 phosphorylation in α-syn neurotoxicity remains unclear. In this study, we coexpressed familial PD-linked A53T α-syn and G-protein-coupled receptor kinase 6 (GRK6) in the rat SN pars compacta using recombinant adeno-associated virus 2. Coexpression of these proteins yielded abundant Ser129-phosphorylated α-syn and significantly exacerbated degeneration of dopaminergic neurons when compared with coexpression of A53T α-syn and GFP. Immunohistochemical analysis revealed that Ser129-phosphorylated α-syn was preferentially distributed to swollen neurites. However, biochemical analysis showed that the increased expression of Ser129-phosphorylated α-syn did not promote accumulation of detergent-insoluble α-syn. Coexpression of catalytically inactive K215R mutant GRK6 failed to accelerate A53T α-syn-induced degeneration. Furthermore, introducing a phosphorylation-incompetent mutation, S129A, into A53T α-syn did not alter the pace of degeneration, even when GRK6 was coexpressed. Our study demonstrates that authentically Ser129-phosphorylated α-syn accelerates A53T α-syn neurotoxicity without the formation of detergent-insoluble α-syn, and suggests that the degenerative process could be constrained by inhibiting the kinase that phosphorylates α-syn at Ser129.
Journal of Neuroscience 11/2011; 31(46):16884-94. · 7.11 Impact Factor
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ABSTRACT: α-Synuclein (a-Syn) is a major component of fibrillar aggregates in Lewy bodies (LBs), a characteristic hallmark of Parkinson disease. Almost 90% of a-Syn deposited in LBs is phosphorylated at Ser-129. However, the role of Ser-129-phosphorylated a-Syn in the biogenesis of LBs remains unclear. Here, we investigated the metabolism of Ser-129-phosphorylated a-Syn. In SH-SY5Y cells, inhibition of protein phosphatase 2A/1 by okadaic acid, and inhibition of the proteasome pathway by MG132 or lactacystin accumulated Ser-129-phosphorylated a-Syn. However, these inhibitions did not alter the amounts of total a-Syn within the observation time. Inhibition of the autophagy-lysosome pathway by 3-methyladenine or chloroquine accumulated Ser-129-phosphorylated a-Syn in parallel to total a-Syn during longer incubations. Experiments using cycloheximide showed that Ser-129-phosphorylated a-Syn diminished rapidly (t(½) = 54.9 ± 6.4 min), in contrast to the stably expressed total a-Syn. The short half-life of Ser-129-phosphorylated a-Syn was blocked by MG132 to a greater extent than okadaic acid. In rat primary cortical neurons, either MG132, lactacystin, or okadaic acid accumulated Ser-129-phosphorylated a-Syn. Additionally, we did not find that phosphorylated a-Syn was ubiquitinated in the presence of proteasome inhibitors. These data show that Ser-129-phosphorylated a-Syn is targeted to the proteasome pathway in a ubiquitin-independent manner, in addition to undergoing dephosphorylation. The proteasome pathway may play a role in the biogenesis of Ser-129-phosphorylated a-Syn-rich LBs.
Journal of Biological Chemistry 10/2010; 285(52):40732-44. · 4.77 Impact Factor
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Masahiro Sakamoto,
Shigeki Arawaka,
Susumu Hara, Hiroyasu Sato,
Can Cui,
Youhei Machiya,
Shingo Koyama,
Manabu Wada,
Toru Kawanami,
Keiji Kurita,
Takeo Kato
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ABSTRACT: The majority of alpha-synuclein (alphaS) deposited in Lewy bodies, the pathological hallmark of Parkinson's disease (PD), is phosphorylated at serine 129 (Ser129). Ser129 phosphorylation of alphaS has been demonstrated to enhance the alphaS toxicity to dopaminergic neurons in a Drosophila model of PD. Phosphorylation of alphaS at Ser129 seems to play a crucial role in the pathogenesis of PD. Here, we assessed the contribution of ubiquitously expressing members of the G-protein-coupled receptor kinase family (GRK2, GRK3, GRK5, and GRK6) to Ser129 phosphorylation of alphaS in HEK293 cells. To selectively reduce the endogenous expression of each member of the GRK family in cells, we used small interfering RNAs. Knockdown of GRK3 or GRK6 significantly decreased Ser129 phosphorylation of alphaS; however, knockdown of GRK2 or GRK5 did not decrease alphaS phosphorylation. The results indicate that endogenous GRK3 and GRK6, but not GRK2 or GRK5, contribute to Ser129 phosphorylation of alphaS in HEK293 cells.
Biochemical and Biophysical Research Communications 06/2009; 384(3):378-82. · 2.48 Impact Factor
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Hiroki Karube,
Masahiro Sakamoto,
Shigeki Arawaka,
Susumu Hara, Hiroyasu Sato,
Chang-Hong Ren,
Saori Goto,
Shingo Koyama,
Manabu Wada,
Toru Kawanami,
Keiji Kurita,
Takeo Kato
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ABSTRACT: Exposure of alpha-synuclein (alphaS), a major component of Lewy bodies in Parkinson's disease, to polyunsaturated fatty acids (PUFAs) triggers the formation of soluble alphaS oligomers. Here, we demonstrate that PUFA binds recombinant alphaS protein through its N-terminal region (residues 2-60). In HEK293 cells, alphaS mutants lacking the N-terminal region failed to form oligomers in the presence of PUFA. The PUFA-induced alphaS oligomerization was accelerated by C-terminal truncation or Ser129 phosphorylation of alphaS; however, this effect was abolished by deletion of the N-terminus. The results indicate that the N-terminus of alphaS is essential for the PUFA-induced alphaS oligomerization.
FEBS Letters 11/2008; 582(25-26):3693-700. · 3.54 Impact Factor
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ABSTRACT: Chronic kidney disease (CKD) is known as a risk factor for cardiovascular disease. In recent years, several experimental and epidemiological studies have suggested that CKD is associated with endothelial dysfunction; thereby, a CKD state may initiate both large and small vessel damage. The association between renal dysfunction and asymptomatic lacunar infarction was reported in a hospital-based study, whereas the relationship between cerebral small vessel disease (SVD)-related lesions and CKD could not be clarified in a community-based study. We performed a cross-sectional study to determine the relationship between silent cerebral SVD-related lesions and CKD in a total of 625 community-based Japanese elderly. In this study, subjects with lower estimated glomerular filtration rate levels tended to have more lacunar infarcts and higher grades of white matter lesions (WMLs). In addition, the mean grades of WMLs or the mean numbers of lacunar infarction in the subjects with albuminuria were greater than those in subjects without albuminuria. In the logistic regression analysis, the association between the presence of CKD and lacunar infarction or moderate WMLs (Fazekas grades 2 and 3) was statistically significant (odds ratio [OR]: 1.86 and 1.50, respectively). Furthermore, as we performed additional analysis, excluding the subjects with stage 2 hypertension (those with casual blood pressure >or=160/100 mm Hg) or diabetes, CKD remained to be an independent risk for cerebral SVD-related lesions. This is the first study showing the relationship between silent SVD-related brain lesions and the presence of CKD, independently of conventional cardiovascular risk factors, in community-based elderly.
Journal of the Neurological Sciences 07/2008; 272(1-2):36-42. · 2.35 Impact Factor
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ABSTRACT: We report a 54-year-old man (case 1) and a 79-year-old woman (case 2) who presented with encephalopathy of unknown cause. Both patients were on hemodyalysis and took an autumn mashroom, "sugihiratake" (Pleurocybella porrigens), two to three weeks prior to the onset of neurological alterations. The clinical syndrome of those patients was characterized by weakness and involuntary movements of the extremities (cases 1 and 2) or dysarthria (case 1) at the onset of the disease and subsequent intractable focal motor seizures, resulting in generalized status epilepticus or comatose state, six (case 1) or three (case 2) days after the disease onset. Epileptic seizures were gradually improved in both cases. On brain MRI of case 1, no relevant lesions were detectable at the onset day, but, 6 days after onset, T2-high intensity lesions were noted in the subcortical white matter of the insular cortex, claustrum, external capsule, putamen and globus pallidus on both sides. On brain CT scan of case 2, there were no apparent lesions at the onset day, but, 4 days after onset, low density areas were noted bilaterally in the subcortical white matter of the insular cortex. Electroencephalography of the two patients taken on a day of comatose state showed periodic synchronous discharge (PSD), which disappeared when their consciousness levels were improved. As far as we have examined, there was no findings to suggest the cause of the encephalopathy in routine laboratory examinations and various viral antibody studies of the blood and cerebrospinal fluid. The reported patients could constitute a newly recognized disease entity, "sugihiratake" encephalopathy. Our observations suggest that it can be an encephalopathy with subacute progression and affect mainly the basal ganglia. Neuroimaging study and electroencephalographic findings may help the diagnosis, although they may remain unremarkable for several days after onset of the neurological alterations.
Rinsho shinkeigaku = Clinical neurology 03/2005; 45(2):111-6.
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Takeo Kato,
Toru Kawanami,
Hiroshi Shimizu,
Katsuro Kurokawa, Hiroyasu Sato,
Katsuo Nakajima,
Tatsuya Nomoto,
Takehiro Seta,
Tetsumasa Kamei,
Hiide Yoshino, [......],
Minoru Ito,
Shigeru Karasawa,
Hideki Kimura,
Yoshihiro Suzuki,
Noriyuki Degawa,
Asako Tagawa,
Ken Ataka,
Shiori Ando,
Tomoya Omae,
Yukihiro Shikama
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ABSTRACT: In September and October, 2004, an outbreak of encephalopathy of unknown etiology occurred in certain areas of Japan including Yamagata, Akita, and Niigata prefectures. These patients had a history of chronic renal failure, most of them had undergone hemodialysis, and also had a history of eating Sugihiratake (Pleurocybella porrigens), an autumn mushroom without known toxicity. Since clinical details of this type of encephalopathy remain unknown, we analyzed the clinical, radiological and electroencephalographic (EEG) features of ten cases of this encephalopathy in Yamagata prefecture. The summary of the present study is as follows: 1. Ten patients had chronic renal failure, and seven underwent hemodialysis. 2. Each patient had a history of eating Sugihiratake within 2-3 weeks of the onset of neurological symptoms. 3. The onset was subacute; the initial symptoms were tremor, dysarthria, and/or weakness of the extremities, which lasted an average of 4.5 days (ranging from 2 to 11 days), followed by severe consciousness disturbance and intractable seizures, resulting in status epilepticus in 5 patients. Myoclonus was also seen in 4 patients and Babinski reflex in 3. 4. Brain CT and MRI examinations were unremarkable in the early stages of the disease. Three to eight days after onset, however, conspicuous lesions appeared in the areas of the insula and basal ganglia in 6 patients. On MRI, these brain lesions were hyperintense on T2-weighted and FLAIR images, and hypointense on T1-weighted images. 5. EEG examination was performed in 6 patients, all of whom showed abnormal EEG findings. Periodic synchronous discharge (PSD) was seen in 2 patients, spike and wave complex in one patient, and non-specific slow waves in 3. 6. Prognosis was different from case to case. Three patients died at 13, 14, and 29 days after onset. Two patients still showed persistent disturbance of consciousness one month after onset. One patient showed parkinsonism after recovering from consciousness disturbance. Four patients recovered nearly completely around one month after onset In 3 of the 4 recovered patients, renal failure was not severe and they did not need to undergo hemodialysis. This suggests that the degree of renal failure is a key for the prognosis of this type of encephalopathy. The present study suggests that this endemic disease is a newly recognized clinical entity of encephalopathy.
Nō to shinkei = Brain and nerve 01/2005; 56(12):999-1007.
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Nihon Naika Gakkai Zasshi 08/2004; 93(7):1439-41.
