Kaoru Goto

The Role of G-Protein-Coupled Receptor Kinase 5 in Pathogenesis of Sporadic Parkinson's Disease

Sporadic Parkinsons disease (sPD) is a common neurodegenerative disorder, characterized by selective degeneration of dopaminergic neurons in the substantia nigra. Although the pathogenesis of the disease remains undetermined, phosphorylation of α-synuclein and its oligomer formation seem to play a key role. However, the protein kinase(s) involved in the phosphorylation in the pathogenesis of sPD has not been identified. Here, we found that G-protein-coupled receptor kinase 5 (GRK5) accumulated in Lewy bodies and colocalized with α-synuclein in the pathological structures of the brains of sPD patients. In cotransfected cells, GRK5 phosphorylated Ser-129 of α-synuclein at the plasma membrane and induced translocation of phosphorylated α-synuclein to the perikaryal area. GRK5-catalyzed phosphorylation also promoted the formation of soluble oligomers and aggregates of α-synuclein. Genetic association study revealed haplotypic association of the GRK5 gene with susceptibility to sPD. The haplotype contained two functional single-nucleotide polymorphisms, m22.1 and m24, in introns of the GRK5 gene, which bound to YY1 (Yin Yang-1) and CREB-1 (cAMP response element-binding protein 1), respectively, and increased transcriptional activity of the reporter gene. The results suggest that phosphorylation of α-synuclein by GRK5 plays a crucial role in the pathogenesis of sPD.

Nuclear diacylglycerol kinase-zeta is a negative regulator of cell cycle progression in C2C12 mouse myoblasts.

The nucleus contains diacylglycerol kinases (DGKs), i.e., the enzymes that, by converting diacylglycerol (DG) into phosphatidic acid, terminate DG‐dependent events. It has been demonstrated that nuclear DGK‐ζ interferes with cell cycle progression. We previously reported that nuclear DGK‐ζ expression increased during myogenic differentiation, whereas its down‐regulation impaired differentiation. Here, we evaluated the possible involvement of nuclear DGK‐ζ in cell cycle progression of C2C12 myoblasts. Overexpression of a wild‐type DGK‐ζ, which mainly localized to the nucleus (but not of a kinase dead mutant or of a mutant that did not enter the nucleus), blocked the cells in the G1 phase of the cell cycle, as demonstrated by in situ analysis of biotinylated‐16‐dUTP incorporated into newly synthesized DNA and by flow cytometry. In contrast, down‐regulation of endogenous DGK‐ζ by short interfering RNA (siRNA) increased the number of cells in both the S and G2/M phases of the cell cycle. Cell cycle arrest of cells overexpressing wild‐type DGK‐ζ was accompanied by decreased levels of retino‐blastoma protein phosphorylated on Ser‐807/811. Down‐regulation of endogenous DGK‐ζ, using siRNA, prevented the cell cycle block characterizing C2C12 cell myogenic differentiation. Overall, our results identify nuclear DGK‐ζ as a key determinant of cell cycle progression and differentiation of C2C12 cells.—Evan‐gelisti, C., Tazzari, P. L., Riccio, M., Fiume, R., Ho‐zumi, Y., Fala, F., Goto, K., Manzoli, L., Cocco, L., Martelli, A. M. Nuclear diacylglycerol kinase‐ζ is a negative regulator of cell cycle progression in C2C12 mouse myoblasts. FASEB J. 21, 3297–3307 (2007)

The −181 A/C polymorphism in the excitatory amino acid transporter-2 gene promoter affects the personality trait of reward dependence in healthy subjects

There have been some animal and human data suggesting that excitatory amino acid transporter (EAAT)-2, the major subtype of EAAT, is involved in human mental function and behavior. Recently, it has been shown that the -181 A/C polymorphism in the EAAT2 gene promoter affects plasma glutamate concentrations in humans. In the present study, the association of this genetic polymorphism with personality traits was examined in 575 Japanese healthy volunteers. Personality traits were assessed by the Temperament and Character Inventory, and the EAAT2 polymorphism was detected by a PCR-RFLP method. The scores of reward dependence were significantly (p=0.017) lower in the group with the A allele (A/A and A/C) than in that without this allele (C/C). When males and females were analyzed separately, the significant difference between the two genotype groups was observed in females (p=0.021) but not in males. The present study thus suggests that the -181 A/C polymorphism in the EAAT2 gene promoter affects the personality trait of reward dependence in healthy subjects.

Gene expression and localization of diacylglycerol kinase isozymes in the rat spinal cord and dorsal root ganglia

The dorsal root ganglion (DRG) and dorsal horn of the spinal cord are areas through which primary afferent information passes enroute to the brain. Previous studies have reported that, during normal neuronal activity, the regional distribution of a second messenger, diacylglycerol (DG), which is derived from phosphoinositide turnover, is diverse in these areas. However, the way that DG is regulated in these organs remains unknown. The present study was performed to investigate mRNA expression and protein localization of DG kinase (DGK) isozymes, which play a central role in DG metabolism. Gene expression for DGK isozymes was detected with variable regional distributions and intensities in the spinal cord. Among the isozymes, most intense signals were found for DGKζ and DGKι in the DRG. By immunohistochemical analysis, DGKζ immunoreactivity was detected heterogeneously in the nucleus and cytoplasm of small DRG neurons with variable levels of distribution, whereas it was detected exclusively in the cytoplasm of large neurons. On the other hand, DGKι immunoreactivity was distributed solely in the cytoplasm of most of the DRG neurons. Double-immunofluorescent imaging of these isozymes showed that they coexisted in a large population of DRG neurons at distinct subcellular sites, i.e., DGKζ in the nucleus and DGKι in the cytoplasm. Thus, DGK isozymes may have different functional roles at distinct subcellular sites. Furthermore, the heterogeneous subcellular localization of DGKζ between the nucleus and cytoplasm implies the possible translocation of this isozyme in small DRG neurons under various conditions.

No association between the −3081A/T polymorphism in the norepinephrine transporter gene promoter and personality traits in healthy subjects

There have been several data suggesting that norepinephrine neurotransmission is involved in the characterization of personality traits. Recently, the -3081A/T polymorphism in the promoter region of the norepinephrine transporter (NET) gene affecting promoter activity has been reported. In the present study, we studied the association between this NET polymorphism and personality traits in 553 Japanese healthy subjects. Personality traits were assessed by the Temperament and Character Inventory (TCI), and the NET genotypes were identified by a PCR-RFLP method. Multivariate two-factor analysis of covariance with genotype and gender as factors and with age as a covariate showed no association between the NET genotypes and the TCI dimension scores. The present study thus suggests that the -3081A/T polymorphism in the NET gene promoter is not involved in the characterization of personality traits in healthy subjects.