Shigenobu Hayashi

NACP/α-synuclein-positive filamentous inclusions in astrocytes and oligodendrocytes of Parkinson's disease brains

Abstract The precursor of the non-Aβ component of Alzheimer’s disease amyloid (NACP), also called α-synuclein, is a major component of Lewy bodies in Parkinson’s disease (PD) as well as of neuronal and oligodendroglial cytoplasmic inclusions in multiple system atrophy. We previously reported argyrophilic, tau-negative glial inclusions in the midbrains of patients with PD and have now conducted immunocytochemical and ultrastructural examinations. The PD glial inclusions also are immunoreactive for NACP/α-synuclein, but not for β-synuclein, and ultrastructurally are composed of filamentous structures about 25–40 nm in diameter. Double immunolabeling showed that the inclusions were present in both astrocytic and oligodendroglial cells. They were located within the substantia nigra in 13 of 30 patients with PD and outside the nigra in 24. The number of inclusions was correlated with the severity of nigral neuronal loss. These findings indicate that abnormal accumulation of NACP/α-synuclein in glial cells is a pathological feature of PD related to its progression.

Involvement of the cerebral cortex and autonomic ganglia in Machado-Joseph disease

Abstract. Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder caused by expansion of a polyglutamine tract in the disease protein. In this study of brains from four autopsied MJD patients, we have demonstrated immunohistochemically that expanded polyglutamine stretches largely accumulate as inclusions in neuronal nuclei, and less frequently are distributed in the nucleoplasm in a diffuse pattern. These nuclear abnormalities involved many neurons covering a wide range of central and peripheral nervous system regions, including the cerebral cortex, thalamus and autonomic ganglia that have been categorized previously as spared regions by conventional pathological studies. These lesions, newly recognized by polyglutamine immunohistochemistry, may be responsible for the cerebral cortical dysfunctions or autonomic abnormalities pointed out in MJD patients by the recent clinical and neuroradiological studies.

Using a DNA Microarray Method to Examine Gene Expression in Brain from Clozapine-Injected Mice

Abstract: After our 2002 report on the changes of gene expression in the brain from phencyclidine‐treated mouse by using DNA microarray (Toyooka et al., Ann. N.Y. Acad. Sci. 965: 10‐20), we decided to apply this DNA microarray method for the brain of the mouse treated with other drugs. We are now examining the effects of clozapine on the brain function. Clozapine is an atypical antipsychotic drug. Clozapine has affinity for neurotransmitter receptors, including D4 dopamine, serotonin, histamine, and adrenergic receptors. MacGibbon et al. (Mol. Brain Res. 23: 21‐32) reported that clozapine and haloperidol produced a differential pattern of immediate early gene expression in rat caudate‐putamen. Kobayashi et al. (Br. J. Pharmacol. 123: 421‐426) have observed the effects of clozapine on the opioid receptors and G‐protein‐activated inwardly rectifying potassium channel of Xenopus oocytes. Thomas et al. (J. Neurochem. 76: 789‐796) found that clozapine increases apolipoprotein D expression in the mouse brain. Therefore, we are going to apply this DNA microarray method to examine the effects of clozapine on mouse brain function. After we injected clozapine into mice for 20 days, we decapitated the mice. We then used the DNA microarray method to examine the gene expression of mouse brain. We found some changes in the mouse brain treated with clozapine. For example, the intensity of a potassium channel spot decreased, and that of a serotonin receptor spot increased.

Structure and expression of human and rat D2 dopamine receptor genes

D2 dopamine receptor may be related with the pathogenesis of Parkinsons disease and schizophrenia. Furthermore, the antipsychotic drugs have high affinity for D2 dopamine receptor. We carried out the cloning of the genomic DNA for human D2 dopamine receptor and clarified the structure of this gene. Our isolated gene spans about 15 kbp and consists of seven exons interrupted by six introns. However, putative first exon was not yet identified. Spot blot hybridization analysis of cell sorter fractionated human chromosomal DNA with D2 receptor genomic DNA revealed the localization of this gene in the chromosome 11 fraction. We analyzed human genomic DNA by Southern blot hybridization with D2 dopamine receptor genomic DNA as a probe, but so far we could not find RFLP. Northern blot analyses of brain RNA of several animals and rat brain RNA after various treatments were carried out. Developmental changes of D2 dopamine receptor mRNA were observed in the rat brains.

Isolation and structure of the mouse 14-3-3 η chain gene and the distribution of 14-3-3 η mRNA in the mouse brain

Abstract 14-3-3 protein is a brain-specific protein discovered by Moore and Perez, but at present is thought to be a multifunctional protein. To clarify the brain-specific function of the protein, we intend constructing a 14-3-3 η gene knock-out mouse. As the first step of this process, we isolated the mouse 14-3-3 η chain gene and determined its structure. The mouse gene is about 10 kb long and composed of two exons separated by a long intron. The transcription start site was identified and the polyadenylation signals (AATAAA) were found in exon 2 of the mouse gene. In the 5′-upstream sequence, we found several cis elements including a CRE sequence, a TATA box-like sequence, and a C/EBP element. Furthermore, the distribution of 14-3-3 η mRNA in the mouse brain was examined by in situ hybridization histochemistry. The highest signals were found in the Purkinje cells of the cerebellum, the pyramidal cells of the hippocampus and the olfactory bulb neurons of the adult mouse. Neuronal expression of 14-3-3 η in these regions mRNA may generally increase during postnatal brain development. The distribution of protein kinase C γ in the mouse brain was also examined by immunohistochemistry. From the distribution of 14-3-3 η mRNA and protein kinase C γ in the mouse brain, the involvement of these compounds in the induction and maintenance of LTP was discussed.

Immunohistochemical Changes of the Transcription Regulatory Factors in Rat Striatum after Methamphetamine Administration

ABSTRACT: We have found evidence for cyclic adenosine monophosphate (cAMP) response element (CRE) in the 5′‐upstream region of the human 14.3.3 η chain gene during studies on isolation and structure of animal brain 14.3.3 cDNA and the human 14.3.3 h chain gene. cAMP response element‐binding protein (CREB) and phosphorylated CREB (pCREB) may bind to this CRE. Since it was considered that these CREBs may play an important role in molecular mechanisms in the brain of animals treated with methamphetamine, we examined the expression of CREB and pCREB in rat brain after acute and chronic methamphetamine administrations using antibodies against CREB and pCREB. We observed findings for change in the expression of these factors. Our findings should be discussed in relation to the data of other authors.

Gene Expression in the Brain from Fluoxetine‐Injected Mouse Using DNA Microarray

Abstract:  Previously we have examined the effects of phencyclidine and clozapine upon the gene expression in the mouse brain. Recently, fluoxetine (Prozac) has been introduced for the therapeutic purpose as an antidepressant drug. Miledi et al. reported blockage of mouse muscle and neuronal nicotinic acetylcholine receptor by various concentrations of fluoxetine. Furthermore, Kobayashi et al. discovered that fluoxetine inhibits G protein activated inwardly rectifying G protein activated K+ (GIRK) channels using Xenopus oocyte expression assay. From these experiments, we considered that it might be interesting to study the effects of fluoxetine on the gene expression in the mouse brain. After we have injected fluoxetine once a day into mouse for 20 days, we sacrificed mouse by decapitation and extracted RNA from mouse cerebral cortex. We used DNA microarray method for examining the gene expression in the brain. We found the downregulation of many spot signals in the fluoxetine‐treated mouse, for example cholecystockinin and prostaglandin D2 synthase.

The effect of methamphetamine on the mRNA level for 14·3·3 ⥈ chain in the human cultured cells

Abstract14·3·3 protein, a brain-specific protein, is an activator of tyrosine and tryptophan hydroxylases, key enzymes for biosynthesis of dopamine and serotonin. In this article, we describe cloning of cDNA for human brain 14·3·3 ν chain and expression of 14·3·3 ν chain mRNA in some human cultured cells. The cloned cDNA is 1730 bp long and contains 191 bp of a 5′-noncoding region, the complete 738 bp of coding region, and 801 bp of a 3′-noncoding region, containing three polyadenylation signals. This cDNA encoded a polypeptide of 246 amino acids (M, 28,196). Furthermore, usingin situ hybridization histochemistry, the expression of mRNA for this protein was examined in the rat central nervous system.In situ hybridization histochemistry indicated that 14·3·3 ⥈ chain mRNA is detected not only in the monoamine-synthetic neurons, but also in other neurons in the discrete nuclei, which synthesize neither cathecholamine nor serotonin. Northern blot analysis demonstrated that the addition of methamphetamine into the cultured medium increased the mRNA level for 14·3·3 ⥈ chain in U-251 cells, but did not increase that of GFAP.

Pick's disease: selective occurrence of apolipoprotein E-immunoreactive Pick bodies in the limbic system

Abstract We carried out immunohistochemical examination of apolipoprotein E (apoE) in brains from two patients with Pick’s disease. In these cases 1 and 2, the APOE genotypes were ɛ3/4 and ɛ3/3, respectively. In both cases, numerous argyrophilic globular intraneuronal inclusions, Pick bodies (PBs), were distributed widely throughout the brain, and immunohistochemically were occasionally positive for apoE. Interestingly, such apoE-immunoreactive PBs were virtually restricted to neurons in the limbic system; in the dentate gyrus, the proportion of apoE-immunoreactive PBs relative to the total number of argyrophilic PBs was 5.0% in case 1 and 2.7% in case 2, whereas in the frontal and temporal neocortices it was less than 0.1% in both cases. Diffuse cytoplasmic immunoreactivity for apoE was found in only a few limbic system neurons without PBs in both cases. In conclusion, it is considered that apoE may not be positively involved in the process of PB formation and that the preferential distribution of apoE-immunoreactive PBs in the limbic system may reflect the presence of certain regional factors associated with the synthesis or metabolism of apoE in this particular system.

Purification of D2 dopamine receptor by photoaffinity labelling, high-performance liquid chromatography and preparative sodium dodecyl sulphate polyacrylamide gel electrophoresis

[125I]N-azidophenethylspiperone ([125I]azido-NAPS) was used as a photoaffinity ligand for bovine D2 dopamine receptor. On photolysis, [125I]azido-NAPS was covalently incorporated into a major band of 94 kDa in bovine striatal membrane as assessed by autoradiography after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (10% acrylamide gel). The labelled D2 receptor protein from striatal membrane was solubilized and subjected to HPLC using gel filtration (TSK G3000SW) and hydroxyapatite gel (Pentax SH2010C), followed by two steps of preparative SDS-PAGE. The D2 receptor protein could be obtained as a single major polypeptide on SDS-PAGE by either silver staining or autoradiography.