Toshiyuki Himi

A caspase inhibitor blocks ischaemia-induced delayed neuronal death in the gerbil

Caspases play a critical role in the cell death machinery in various cell types. Here we investigated the involvement of caspases in the delayed neuronal death after transient global forebrain ischaemia in the gerbil. Intrahippocampal injection of benzyloxycarbonyl‐Asp‐CH2‐dichlorobenzene (zD), an irreversible inhibitor of caspases, saved hippocampal CA1 neurones from chromatin condensation and DNA fragmentation at post‐ischaemia day 4, and these neurones maintained normal morphology at day 8 post‐insult. Intrahippocampal injection of interleukin‐1β (IL‐1β) after ischaemic insults did not influence the neuroprotective effect of zD, suggesting that the neuroprotective effect does not depend on the inhibition of mature IL‐1β production. Animals that received zD‐injection showed significant improvement in step‐through and step‐down passive avoidance learning at post‐ischaemia days 4 and 5, suggesting that neural functions were preserved in these animals. At post‐ischaemia day 4, the cleavage of poly(ADP‐ribose)polymerase was observed, and this cleavage was almost completely suppressed in zD‐injected hippocampus, suggesting involvement of caspase‐3 and caspase‐3‐like caspase in the delayed neuronal death. Our findings indicate that caspases play important roles in the delayed neuronal death after transient global forebrain ischaemia in the gerbil, and suggest that ischaemia‐induced brain damage can be blocked by caspase inhibitors.

Role of neuronal glutamate transporter in the cysteine uptake and intracellular glutathione levels in cultured cortical neurons

Summary. Cysteine uptake is the rate-limiting process in glutathione synthesis. Previously we have shown that the inhibitors of excitatory amino acid transporters (EAATs) significantly enhance glutamate toxicity via depletion of intracellular glutathione. In this study we show evidence that the neuronal glutamate transporter EAAT3 is directly enrolled in cysteine uptake in cultured neurons. Neuronal cysteine uptake was dependent on the extracellular sodium, and was suppressed by EAAT inhibitors. Cysteine uptake was suppressed by extracellular glutamate and aspartate, substrates of EAATs, and not by substrates of cysteine transporters. Intracellular glutathione levels were reduced by EAAT inhibitors, and not by inhibitors of cysteine transporters. Knock down of EAAT3 expression using antisense oligonucleotide significantly reduced cysteine uptake, intracellular glutathione level, and neuronal viability against oxidative stress. These facts indicate that EAAT3 functions as a cysteine transporter, and this function seems to be unique and distinct from cysteine transporters that have been reported.

Hepatocyte growth factor protects cultured rat cerebellar granule neurons from apoptosis via the phosphatidylinositol‐3 kinase/Akt pathway

Recent studies suggest that hepatocyte growth factor (HGF) functions as a neurotrophic factor in the central nervous system. In this study, we investigated the neuroprotective effect of HGF and its mechanism of action. We used cultured cerebellar granule neurons that underwent apoptosis when the culture medium was changed from that containing serum with 25 mM K+ to serum‐free medium containing 5 mM K+, and HGF prevented apoptotic cell death. HGF stimulated both mitogen‐activated protein (MAP) kinase and phosphatidylinositol‐3 (PI3)‐kinase activity in cerebellar granule neurons. Two specific inhibitors of PI3‐kinase, wortmannin and LY294002, efficiently blocked this neuroprotective effect of HGF. In contrast, PD98059, a selective inhibitor of MAP kinase kinase (MEK), did not affect the anti‐apoptotic effect of HGF. The downstream signal of PI3‐kinase in this protection was further investigated. HGF‐induced phosphorylation of Akt and pretreatment of the cells with wortmannin completely impaired Akt activation. These results suggest that HGF prevents apoptosis in cerebellar granule neurons via the PI3‐kinase/Akt pathway. J. Neurosci. Res. 59:489–496, 2000

Genetic background conversion ameliorates semi-lethality and permits behavioral analyses in cystathionine β-synthase-deficient mice, an animal model for hyperhomocysteinemia

Cystathionine beta-synthase-deficient mice (Cbs(-/-)) exhibit several pathophysiological features similar to hyperhomocysteinemic patients, including endothelial dysfunction and hepatic steatosis. Heterozygous mutants (Cbs(+/-)) on the C57BL/6J background are extensively analyzed in laboratories worldwide; however, detailed analyses of Cbs(-/-) have been hampered by the fact that they rarely survive past the weaning age probably due to severe hepatic dysfunction. We backcrossed the mutants with four inbred strains (C57BL/6J(Jcl), BALB/cA, C3H/HeJ and DBA/2J) for seven generations, and compared Cbs(-/-) phenotypes among the different genetic backgrounds. Although Cbs(-/-) on all backgrounds were hyperhomocysteinemic/hypermethioninemic and suffered from lipidosis/hepatic steatosis at 2 weeks of age, >30% of C3H/HeJ-Cbs(-/-) survived over 8 weeks whereas none of DBA/2J-Cbs(-/-) survived beyond 5 weeks. At 2 weeks, serum levels of total homocysteine and triglyceride were lowest in C3H/HeJ-Cbs(-/-). Adult C3H/HeJ-Cbs(-/-) survivors showed hyperhomocysteinemia but escaped hypermethioninemia, lipidosis and hepatic steatosis. They appeared normal in general behavioral tests but showed cerebellar malformation and impaired learning ability in the passive avoidance step-through test, and required sufficient dietary supplementation of cyst(e)ine for survival, demonstrating the essential roles of cystathionine beta-synthase in the central nervous system function and cysteine biosynthesis. Our C3H/HeJ-Cbs(-/-) mice could be useful tools for investigating clinical symptoms such as mental retardation and thromboembolism that are found in homocysteinemic patients.

Changed preference for sweet taste in adulthood induced by perinatal exposure to bisphenol A-A probable link to overweight and obesity.

BACKGROUND The preference of obesity has risen dramatically worldwide over the past decades. Some latest reports showed significant increase of obesity in men compared to women. Implication of environmental endocrine disruptors has been focused more and more. Numerous studies in vitro and vivo implied metabolic actions of bisphenol A (BPA), however much less consideration is given to the possibility of BPA exposure-induced change in gender-specific behaviors which result in obesity and overweight. OBJECTIVES To examine whether perinatal exposure to BPA at relative dose to environmental levels can influence sweet preference of male and female rats and consequently lead to alteration in bodyweight. METHODS Rats perinatally exposed to BPA at doses of 0.01, 0.1 and 1.0 mg/L were tested sweet preference for 0.25%, 0.5% saccharin and 15% sucrose by two-bottle choice (water vs. saccharin/sucrose). The food intake, liquid consumption and bodyweight of each rat were monitored daily. At the end of the test, the fat percentage and tail blood pressure were measured. RESULTS Significant sex difference of preference for 0.25% and 0.5% saccharin was shown in control and all BPA-treated groups (p < 0.001, female vs. male). 0.1 and 1.0 mg/L BPA treatment induced the increase of preference for 0.25% saccharin solution in males, but not in females. 0.1 mg/L BPA treatment increased sucrose preference in males at postnatal day (PND) 70 and 140 (p < 0.05 and p < 0.001, compared to control respectively) but decreased sucrose preference in females at PND 140 (p < 0.05, compared to control). The males treated by BPA showed overweight (p < 0.001), high fat percentage (p < 0.001) and tail blood pressure (p < 0.05) than control at PND 140. CONCLUSION Perinatal exposure to a low dose of BPA could increase sweet preference of male rats. Calorie intake may be programmed during early life, leading to changes of body weight depending on the gender. Although further researches concerning the mechanism are required, the results of the present study are particularly important with regards to the more significant increasing prevalence of obesity in men and the environmental endocrine disruptors.

Expression of SLURP‐1, an endogenous α7 nicotinic acetylcholine receptor allosteric ligand, in murine bronchial epithelial cells

Mammalian secreted lymphocyte antigen‐6/urokinase‐type plasminogen activator receptor‐related peptide‐1 (SLURP‐1) is a positive allosteric ligand for α7 nicotinic acetylcholine (ACh) receptors (α7 nAChRs) that potentiates responses to ACh and elicits proapoptotic activity in human keratinocytes. Mutations in the gene encoding SLURP‐1 have been detected in patients with Mal de Meleda, a rare autosomal recessive skin disorder characterized by transgressive palmoplantar keratoderma. On the basis of these findings, SLURP‐1 is postulated to be involved in regulating tumor necrosis factor‐α (TNF‐α) release from keratinocytes and macrophages via α7 nAChR‐mediated pathways. In the present study, we assessed SLURP‐1 expression in lung tissue from C57BL/6J mice to investigate the functions of SLURP‐1 in pulmonary physiology and pathology. Immunohistochemical and in situ hybridization analyses revealed expression of SLURP‐1 protein and mRNA, respectively, exclusively in ciliated bronchial epithelial cells. This was supported by Western blotting showing the presence of the 9.5‐kDa SLURP‐1 protein in whole‐lung tissue and trachea. In addition, high‐affinity choline transporter (CHT1) was detected in apical regions of bronchial epithelial cells and in neurons located in the lamina propria of the bronchus, suggesting that bronchial epithelial cells are able to synthesize both SLURP‐1 and ACh. We also observed direct contact between F4/80‐positive macrophages and bronchial epithelial cells and the presence of invading macrophages in close proximity to CHT1‐positive nerve elements. Collectively, these results suggest that SLURP‐1 contributes to the maintenance of bronchial epithelial cell homeostasis and to the regulation of TNF‐α release from macrophages in bronchial tissue.

Purkinje cells express neuronal nitric oxide synthase after methylmercury administration.

To study the effects of chemical injury on the cerebellar nitric oxide synthase (NOS), we administered methylmercury chloride subcutaneously to mice, 10 mg/kg/day for 9 days. In the methylmercury-treated cerebellum. Purkinje cells were positive both for NADPH-diaphorase and for neuronal NOS. Calcium-dependent NOS activity was increased to 160% of the controls. The present study suggests the ability of Purkinje cells to produce NO through the expression of neuronal NOS.

Neuronal nitric oxide synthase is resistant to ethanol.

To test for a possible role of nitric oxide (NO) in the neurotoxicity of ethanol, we studied the effects of ethanol on the neuronal NO synthase (nNOS) both in vitro and in vivo. Ethanol, up to 200 mM, did not change the NOS activity in the cerebellar homogenate or the production of NO by the cultured cerebellar granule cells. The number of NADPH diaphorase-positive cells in the culture did not change after the exposure to 200 mM ethanol in vitro. The NOS activity in the various brain regions of mice remained similar to the controls after the acute (3 g/kg) and the chronic (33 g/kg/day, 3.5 days) administration of ethanol. N(omega)-nitro-L-arginine, a NOS inhibitor, did not affect the ethanol-withdrawal behavior. These results indicate that nNOS is resistant to ethanol at clinically relevant concentrations and that ethanol affects the NO-operated system in the brain through a pathway other than that of nNOS.

Induction of neuronal nitric oxide synthase by methylmercury in the cerebellum

A free radical, nitric oxide (NO), besides being a messenger molecule in the brain, becomes a neurotoxin if overproduced. We recently reported that methylmercury (MeHg) induces neuronal NO synthase (nNOS) in Purkinje cells. In the present study, we examined the distribution and the mechanism of nNOS induction by MeHg. Subcutaneous administration of MeHg chloride to mice, 10 mg/kg/day for 9 days, increased calcium‐dependent NOS activity to 60% more than the controls only in the cerebellum but not in other brain regions. The Western blots showed a comparable increase in nNOS protein in the cerebellum. A N‐methyl‐D‐aspartate (NMDA) receptor antagonist, MK‐801, did not block, but rather enhanced, the increase in the nNOS activity. Another NMDA antagonist, 3‐(2‐carboxypiperazin‐4‐yl)‐propyl‐1‐phosphonic acid (CPP), did not affect the nNOS activity. The Western blots of protein kinase C (PKC), which is an important cofactor regulating nNOS, did not change after the administration of MeHg. These results show that MeHg induces biologically active nNOS selectively in the cerebellum. The induction is independent of PKC and is not reduced by the blockade of the NMDA receptor. J. Neurosci. Res. 55:352–356, 1999. 

Induction of hepatocyte growth factor (HGF) in rat microglial cells by prostaglandin E2

Hepatocyte growth factor (HGF) is a multifunctional protein that exerts trophic effects on neural cells. HGF is expressed in normal brains and increased after brain injury. Recent studies suggest that neurons and astrocytes are the main producers of HGF in the brain. Here we report that microglia also produce HGF both in vitro and in vivo. Treatment of cultured microglia with prostaglandin E2 (PGE2), one of the major inflammatory mediators in the brain, induced significant production of HGF, and this induction was suppressed by pretreatment with the adenylate cyclase inhibitor SQ22536, suggesting that the induction of HGF by PGE2 in microglia proceeds via a cAMP‐mediated pathway. We further investigated whether microglia also produce HGF in vivo under the pathological condition of cerebral ischemia. We found that HGF expression was increased after permanent occlusion of the middle cerebral artery (MCA), and double immunohistochemical staining revealed that the most of HGF‐positive cells were microglia. PGE2 level was increased 8 hr after start of MCA occlusion, and this enhancement is in parallel with the increase in HGF expression, suggesting that PGE2 not only may induce HGF production in microglia in vitro but may also be an inducer in vivo. J. Neurosci. Res. 62:389–395, 2000.