Toshiki Himeda

Effects of age on immunohistochemical changes in the mouse hippocampus.

We investigated the age-related changes in neuronal cell death and synaptophysin of the hippocampal CA1 sector in mice using immunohistochemistry. Microtubule-associated protein 2a, b (MAP2) and synaptophysin immunoreactivity was measured in 2-, 8-, 18-, 42- and 59-week-old mice. MAP2 immunoreactivity was unchanged in the hippocampal CA1 sector up to 42 weeks after birth. In 59-week-old mice, however, a significant decrease in MAP2 immunoreactivity was observed in the hippocampal CA1 sector. Total number of synaptophysin-positive boutons was also unchanged in the hippocampal CA1 sector up to 42 weeks of birth. In 59-week-old mice, however, a significant increase in synaptophysin-positive boutons was observed in the hippocampal CA1 sector. These results demonstrate that dendrites and axons in the hippocampal CA1 neurons are particularly susceptible to ageing processes. In contrast, a marked increase in synaptophysin-positive boutons was found in the hippocampal CA1 sector of aged mice. These findings suggest that increase in synaptophysin-positive boutons may play a role in the maintenance of the structural components in the hippocampal CA1 sector of aged mice although most postsynaptic CA1 pyramidal neurons are generated. Thus, our findings provide further valuable information on age-related neurodegeneration and deficits in hippocampus-dependent memory and synaptic plasticity.

Time dependent alterations of co-localization of S100β and GFAP in the MPTP-treated mice

Summary.S100β is a calcium-binding peptide produced by astrocytes. This protein is expressed at high levels in brain and is known as a marker of brain damage. However, little is known about the role of S100β protein during neuronal damage caused by MPTP. To determine exactly changes of expression of S100β protein in relation to changes of glial cells, we investigated immunohistochemically the expression of S100β protein using MPTP-treated mice. The present study showed that tyrosine hydroxylase (TH) immunoreactivity was decreased in the striatum and substantia nigra from 5 h and 1 day after MPTP treatment, respectively. Thereafter, a severe reduction in TH immunoreactivity was observed in the striatum and substantia nigra 1, 3 and 7 days after MPTP treatment. In our double-labeled immunostaining, the number of S100-positive/GFAP-negative cells decreased from 1 day up to 7 days after MPTP treatment. In contrast, the number of double-labeled S100/GFAP-immnoreactive cells increased from 1 day up to 7 days after MPTP treatment. The number of S100β-positive/GFAP-negative cells also decreased 3 and 7 days after MPTP treatment. In contrast, the number of double-labeled S100β/GFAP-immunoreactive cells increased from 1 day up to 7 days after MPTP treatment. The present study demonstrates that S100β/GFAP-positive cells may play some role in the pathogenesis of MPTP-induced dopaminergic neurodegeneration in the striatum. The present results also suggest the presence of the S100β protein in a subpopulation of GFAP-negative astrocytes in the striatum after MPTP treatment. These results suggest that the modulation of astrocytic activation may offer a novel therapeutic strategy of Parkinson’s disease.

Stoichiometry of subunit e in rat liver mitochondrial H+-ATP synthase and membrane topology of its putative Ca2+-dependent regulatory region

Previous studies have revealed that residues 34-65 of subunit e of mitochondrial H(+)-ATP synthase are homologous with the Ca(2+)-dependent tropomysin-binding region for troponin T and have suggested that subunit e could be involved in the Ca(2+)-dependent regulation of H(+)-ATP synthase activity. In this study, we determined the content of subunit e in H(+)-ATP synthase purified from rat liver mitochondria, and we also investigated the membrane topology of a putative Ca(2+)-dependent regulatory region of subunit e using an antibody against peptide corresponding to residues 34-65 of subunit e. Quantitative immunoblot analysis of subunit e in the purified H(+)-ATP synthase revealed that 1 mol of H(+)-ATP synthase contained 2 mol of subunit e. The ATPase activity of mitoplasts, in which the C-side of F(0) is present on the outer surface of the inner membrane, was significantly stimulated by the addition of the antibody, while the ATPase activity of submitochondrial particles and purified H(+)-ATP synthase was not stimulated. The antibody bound to mitoplasts but not to submitochondrial particles. These results suggest that the putative Ca(2+)-dependent regulatory region of subunit e is exposed on the surface of the C-side of F(0) and that subunit e is involved in the regulation of mitochondrial H(+)-ATP synthase activity probably via its putative Ca(2+)-dependent regulatory region.

Gene Expression of Subunit c(P1), Subunit c(P2), and Oligomycin Sensitivity-conferring Protein May Play a Key Role in Biogenesis of H+-ATP Synthase in Various Rat Tissues

Mammalian H+-ATP synthase is a supramolecule composed of at least 14 subunits that have a constant stoichiometry. Nevertheless the coordinate regulation of the gene expressions of various subunits remains obscure. To clarify the coordinate transcriptional regulatory system of mammalian H+-ATP synthase, we determined the absolute amount of nine species of mRNAs for eight nuclear-encoded subunits of H+-ATP synthase in different tissues of 8-week-old rats by use of the synthetic mRNAs and 32P-labeled DNA probes for each mRNA. Our quantitative analyses of the transcripts of H+-ATP synthase revealed that nine species of the subunits in different tissues of 8-week-old rats were divisible into two groups: a high transcript gene (HTG) group (β-subunit, subunit b, subunit d, subunit e, and Factor 6) and a low transcript gene (LTG) group (subunit c(P1), subunit c(P2), IF1, and oligomycin sensitivity-conferring protein). The transcription step of LTG could constitute a bottleneck in the biogenesis of H+-ATP synthase. Thus, the transcriptional regulatory system of the LTG may play a key role in the biogenesis of mammalian H+-ATP synthase. The HTG were transcribed in a tissue-specific manner that corresponds with energy demand in the tissues. However, there was no tissue specificity in subunit c(P2). Furthermore, the tissue specificity of the transcript of IF1 differed substantially from that of HTG, suggesting that it could be crucial in the protection of mitochondrial membrane under abnormal conditions.

Neuroprotective effect of arundic acid, an astrocyte-modulating agent, in mouse brain against MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) neurotoxicity

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes the damage of dopaminergic neurons as seen in Parkinsons disease. Oxidative stress has been as one of several pathogenic hypotheses for Parkinsons disease. Here we investigated whether arundic acid, an astrocyte-modulating agent, can protect against alterations of nitric oxide synthase (NOS) and superoxide dismutase (SOD) expression on MPTP neurotoxicity in mice, utilizing an immunohistochemistry. For this purpose, anti-tyrosine hydroxylase (TH) antibody, anti-dopamine transporter (DAT) antibody, anti-Cu/Zn-SOD antibody, anti-Mn-SOD antibody, anti-nNOS antibody, anti-eNOS antibody and anti-iNOS antibody were used. The present study showed that the arundic acid had a protective effect against MPTP-induced neuronal damage in the striatum and substantia nigra of mice. The protective effect may be, at least in part, caused by the reductions of the levels of reactive nitrogen (RNS) and oxygen species (ROS) against MPTP neurotoxicity. These results suggest that the pharmacological modulation of astrocyte may offer a novel therapeutic strategy for the treatment of Parkinsons disease. Furthermore, our results provide further evidence that a combination of nNOS inhibitors, iNOS inhibitors and free radical scavengers may be effective in the treatment of neurodegenerative diseases. Thus our present results provide valuable information for the pathogenesis of degeneration of the nigrostriatal dopaminergic neuronal pathway.

Alterations of interneurons of the gerbil hippocampus after transient cerebral ischemia: effect of pitavastatin.

We investigated the immunohistochemical alterations of parvalbumin (PV)-expressing interneurons in the hippocampus after transient cerebral ischemia in gerbils in comparison with neuronal nitric oxide synthase (nNOS)-expressing interneurons. We also examined the effect of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor pitavastatin against the damage of neurons and interneurons in the hippocampus after cerebral ischemia. Severe neuronal damage was observed in the hippocampal CA1 pyramidal neurons 5 and 14 days after ischemia. The PV immunoreactivity was unchanged up to 2 days after ischemia. At 5 and 14 days after ischemia, in contrast, a conspicuous reduction of PV immunoreactivity was observed in interneurons of the hippocampal CA1 sector. Furthermore, a significant decrease of PV immunoreactivity was found in interneurons of the hippocampal CA3 sector. No damage of nNOS-immunopositive interneurons was detected in the gerbil hippocampus up to 1 day after ischemia. Thereafter, a decrease of nNOS immunoreactive interneurons was found in the hippocampal CA1 sector up to 14 days after ischemia. Pitavastatin significantly prevented the neuronal cell loss in the hippocampal CA1 sector 5 days after ischemia. Our immunohistochemical study also showed that pitavastatin prevented significant decrease of PV- and nNOS-positive interneurons in the hippocampus after ischemia. Double-labeled immunostainings showed that PV immunoreactivity was not found in nNOS-immunopositive interneurons of the brain. The present study demonstrates that cerebral ischemia can cause a loss of both PV- and nNOS-immunoreactive interneurons in the hippocampal CA1 sector. Our findings also show that the damage to nNOS-immunopositive interneurons may precede the neuronal cell loss in the hippocampal CA1 sector after ischemia and nNOS-positive interneurons may play some role in the pathogenesis of cerebral ischemic diseases. Furthermore, our present study indicates that pitavastatin can prevent the damage of interneurons in the hippocampus after cerebral ischemia. Thus, our study provides valuable information for the pathogenesis after cerebral ischemia.

Effects of chronic administration with nilvadipine against immunohistochemical changes related to aging in the mouse hippocampus.

We investigated the effect of Ca2+ antagonist nilvadipine on age-related immunohistochemical alterations in ubiquitin and S100β protein of the hippocampal CA1 sector in mice using 8-, 18-, 40-, and 59-week-old mice. No significant changes in the number of neuronal cells were observed in the hippocampal CA1 sector up to 59 weeks after birth. The administration of nilvadipine did not affect the number of the hippocampal CA1 cells of 40-week-old mice. Age-dependent increases in ubiquitin immunoreactivity were observed in the hippocampal CA1 neurons up to 59 weeks after birth. The administration of nilvadipine prevented dose-dependently the increases in the number of ubiquitin-immunoreactive neurons in the hippocampal CA1 sector of 40-week-old mice. S100β immunoreactivity was unchanged in the hippocampal CA1 sector up to 40 weeks after birth. In 59-week-old mice, the level of staining of S100β-immunoreactive cells increased significantly in the hippocampal CA1 sector. The administration of nilvadipine decreased dose-dependently the number of S100β-immunoreactive cells in the hippocampal CA1 sector of 40-week-old mice. The present study demonstrates that age-related increases in ubiquitin system may play a pivotal role in protecting neuronal cell damage during aging. In contrast, our results suggest that expression of S100β protein in the hippocampal CA1 sector may play an exacerbating factor in some neuronal cells damaged by aging. Our results also demonstrate that nilvadipine, a dihydropyridine-type calcium channel blocker, can prevent dose-dependently the increases in the ubiquitin immunoreactive neurons and decrease the number of S100β immunoreactive cells in the hippocampal CA1 neurons of aged mice. These results suggest that nilvadipine may offer a new approach for the treatment of neuronal dysfunction in aged humans.