Atsushi Takaki

PACAP protects hippocampal neurons against apoptosis: Involvement of JNK/SAPK signaling pathway

Abstract: We have demonstrated that the ischemia‐induced apoptosis of neurons in the CA1 region of the rat hippocampus was prevented by either intracerebroventricular or intravenous infusion of pituitary adenylate cyclase‐activating polypeptide (PACAP). However, the molecular mechanisms underlying the anti‐apoptotic effect of PACAP remain to be determined. Within 3–6 h after ischemia, the activities of members of the mitogen‐activated protein (MAP) kinase family, including extracellular signal‐regulated kinase (ERK), Jun N‐terminal kinase (JNK)/stress‐activated protein kinase (SAPK), and p38 were increased in the hippocampus. The ischemic stress had a potent influence on the MAP kinase family, especially on JNK/SAPK. PACAP inhibited the activation of JNK/SAPK after ischemic stress. Secretion of interleukin‐6 (IL‐6) into the cerebrospinal fluid was intensely stimulated after PACAP infusion. IL‐6 inhibited the activation of JNK/SAPK, while it activated ERK. These observations suggest that PACAP and IL‐6 act to inhibit the JNK/SAPK signaling pathway, thereby protecting neurons against apoptosis.

Hydrogen in Drinking Water Reduces Dopaminergic Neuronal Loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine Mouse Model of Parkinson's Disease

It has been shown that molecular hydrogen (H2) acts as a therapeutic antioxidant and suppresses brain injury by buffering the effects of oxidative stress. Chronic oxidative stress causes neurodegenerative diseases such as Parkinsons disease (PD). Here, we show that drinking H2-containing water significantly reduced the loss of dopaminergic neurons in PD model mice using both acute and chronic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The concentration-dependency of H2 showed that H2 as low as 0.08 ppm had almost the same effect as saturated H2 water (1.5 ppm). MPTP-induced accumulation of cellular 8-oxoguanine (8-oxoG), a marker of DNA damage, and 4-hydroxynonenal (4-HNE), a marker of lipid peroxidation were significantly decreased in the nigro-striatal dopaminergic pathway in mice drinking H2-containing water, whereas production of superoxide (O2•−) detected by intravascular injection of dihydroethidium (DHE) was not reduced significantly. Our results indicated that low concentration of H2 in drinking water can reduce oxidative stress in the brain. Thus, drinking H2-containing water may be useful in daily life to prevent or minimize the risk of life style-related oxidative stress and neurodegeneration.

Reduced postischemic apoptosis in the hippocampus of mice deficient in interleukin-1

The cytokine interleukin‐1 (IL‐1) has been implicated in ischemic brain damage, because the IL‐1 receptor antagonist markedly inhibits experimentally induced neuronal loss. However, to date, no studies have demonstrated the involvement of endogenous IL‐1α and IL‐ 1β in neurodegeneration. We report here, for the first time, that mice lacking IL‐1α/β (double knockout) exhibit markedly reduced neuronal loss and apoptotic cell death when exposed to transient cardiac arrest. Furthermore, we show that, despite the reduced neuronal loss, phosphorylation of JNK/SAPK (c‐Jun NH2‐ terminal protein kinase/stress activated protein kinase) and p38 enzymes remain elevated in IL‐1 knockout mice. In contrast, the inducible nitric oxide (iNOS) immunoreactivity after global ischemia was reduced in IL‐1 knockout mice as compared with wild‐type mice. The levels of nitrite (NO2−) and nitrate (NO3−) in the hippocampus of wild‐type mice were increased with time after ischemia‐reperfusion, whereas the increase was significantly inhibited in IL‐1 knockout mice. These observations strongly suggest that endogenous IL‐1 contributes to ischemic brain damage, and this influence may act through the release of nitric oxide by iNOS. J. Comp. Neurol. 448:203–216, 2002.

PACAP Stimulates the Release of Interleukin-6 in Cultured Rat Müller Cells

Abstract:  We have investigated the in vivo effect of PACAP on rat Müller cells that are the predominant glial element in the retina. Müller cells were treated with PACAP38, either alone or in the presence of the PACAP‐selective antagonist, PACAP6–38. Cellular proliferation was determined by measuring the incorporation of bromodeoxyuridine, while interleukin‐6 (IL‐6) levels in the culture medium were examined using a B9 cell bioassay. In cultured rat Müller cells, the expression of PACAP receptor (PAC1‐R) was assessed with immunohistochemistry using a PAC1‐R‐specific antiserum. PACAP stimulated IL‐6 production in Müller cells at a concentration as low as 10−12 M, which was not sufficient to induce cell proliferation. This elevation of IL‐6 production was significantly inhibited by PACAP6–38. These data suggest that Müller cells are one of the target cells for PACAP, stimulating the release of IL‐6, and providing a mechanism whereby PACAP exerts a significant neuroprotective effect in the retina.

Nucleoprotamine diet derived from salmon soft roe protects mouse hippocampal neurons from delayed cell death after transient forebrain ischemia.

The nutritional benefits of nucleoprotamine (NP), the main component of fish soft roe, have been rarely addressed. In the present study, the preventive effect of oral supplements of nucleoprotamine and its derivatives, DNA and protamine (PT), extracted from salmon soft roe, on survival rate and hippocampal cell death induced by transient brain ischemia, was evaluated in mice. Artificially formulated nucleoprotamine-free (NF) diet with/without nucleoprotamine, DNA or protamine was fed orally. One week after commencement of respective diets, animals were subjected to transient brain ischemia, which was performed by common carotid artery (CCA) occlusion for 25 (severe) or 15 min (mild). After severe ischemia, the survival rate of the NF group was lower than that in the group fed standard diet or NP. Morphological changes in the hippocampal CA1 region were estimated 48 h after mild ischemia. The NP and PT groups significantly decreased the neuronal damage compared with the NF group. The number of cell death in the DNA group, however, was affected similar to that of the NF group. Our data suggests that the nucleoprotamine content in salmon soft roe could be a useful nutritional resource for the prevention of cell damage caused by ischemia such as those occurring with cerebral and/or heart infarction.

Suppression of oxidative stress after transient focal ischemia in interleukin-1 knock out mice.

Interleukin-1 (IL-1) contributes to ischemic neurodegeneration. However, the mechanisms regulating action of IL-1 are still poorly understood. In order to clarify this central issue, mice that were gene deficient both IL-1alpha and beta (IL-1 KO) and wild-type mice were subjected to 1 hour transient middle cerebral artery occlusion (tMCAO). The concentration of 8-hydroxy deoxyguanosine (8OHdG) which is considered to be a reliable oxidative DNA damage by superoxide anion, in brain and of total nitric oxide (NO) in plasma were determined by use of HPLC. Twenty-four hours after tMCAO, the ratio of 8OHdG to dG in the ipsilateral hemisphere of wild-type mice were 2.24 x 10(-3) and 4.41 x 10(-3) in the neocortex and striatum, respectively. The concentration of 8OHdG in the ipsilateral hemisphere of the wild-type mice was higher than that of the IL-1 KO mice. The concentration of total NO in the plasma of IL-1 KO mice was also lower than that of the wild-type 24 hours after tMCAO. These results strongly suggest that IL-1 is participated in generating reactive oxygen spices and it aggravates and induces the ischemic neuronal cell death.(183 words).

Electrical stimulation of male monkey's midbrain elicits components of sexual behavior

We electrically stimulated the midbrain of male rhesus monkeys seated in a restraint chair facing the female partners and examined whether sexual behavior could be induced. When the midbrain was stimulated (0.2 ms, 50-500 microA and 50 Hz for 2.5 s), the male monkey touched and held the waist of his partner (latency; 0.9 +/- 0.4 s, mean +/- SD, n = 225), and then mounted her when she responded with presenting her hip toward him. However, this mounting, unlike when the hypothalamus was stimulated, did not lead to thrusting or ejaculation even if the stimulation continued. The sites in the midbrain where the stimulation elicited touching and mounting were the ventral tegmental area, the substantia nigra, the nucleus reticularis mesencephali and the nucleus reticularis pontis oralis et caudalis. Touching and mounting were not elicited when the partner was put away from the male or replaced by submissive male monkeys or humans. The findings suggest that the stimulation-evoked touching and mounting are components of copulatory behavior and that the midbrain structures may be involved in the sexual behavior of male monkeys.

Thermal Stress and Immunity

The rise in deep-body temperature is etiologically categorized into active hyperthermia and passive hyperthermia. A typical example of active hyperthermia is fever. Fever is a centrally regulated rise in core ternperature that is primarily determined by changes in the activity of hypothalamic thermosensitive neurons in response to both pyrogenic cytokines and cryogenic substances derived from the biodefense system. Fever is a phylogenetically old phenomenon observed in invertebrates as well as in vertebrates. Although fever is occasionally harmful, it is likely that fever has evolved as an adaptive host defense response to infection. The febrile temperature exerts detrimental effects on the growth of microorganisms and enhancing effects on T-cellmediated immunity in vitro. Although some functions of the natural immunity (natural killer cell activity and the production of cytokines by macrophages) are suppressed at febrile temperatures, it is presumed that the net effect of febrile temperature on both attacking factors and host defense activities may promote the survival rate of the infected host. Passive hyperthermia as a result of inadequate heat loss in a warm environment, if it is severe, enhances bacterial translocation across the gut mucosal barrier. The gut-derived endotoxin triggers the production of pyrogenic cytokines in the mesenteric lymphoid tissues and liver. Thus, such cytokines activate the immune system on one hand and potentially aggravate environmentally or behaviorally (e.g., exercise) induced hyperthermia by their pyrogenic actions.

Brain-gut-liver-immune axis during non-inflammatory stress in rat

We have demonstrated that acetylcholine (ACh), a classical neurotransmitter, is present in the blood of different species of mammals including humans, and that blood ACh mainly originates from T-lymphocytes. In addition, mRNA expression for various muscarinic (MS) ACh receptors has been reported in lymphocytes. These findings suggest involvement of blood ACh in regulation of interactions between the nervous and immune systems. In this study, we investigated the physiological role of ACh in regulation of the immune systems, by analyzing the effect of bethanechol (BeCh), a selective MS ACh receptor agonist, on intracelIular calcium signals in CEM, a human leukemic T-cell line, used as a model of lymphocytes. BeCh induced a rapid and transient increase of intraceUular calcium ion concentration ([caZ+]i). In addition, an intermittent increase of [C%+]i (calcium oscillation) was observed with time in CEM. However, calcium oscillation via MS ACh receptor stimulation was not observed in the absence of external Ca2+, or abolished in the presence of 1 FM nicardipine, a Ca channel blocker. These results indicate that calcium oscillation may be maintained via Ca2+ entry from receptor-operated Ca channels.

The major source of non-inflammatory stress-induced peripheral IL-6 increase

We have already reported that non-inflammatory stress induced peripheral IL-6 production is, at least partly, mediated by enteric endotoxin (LPS). In the present study, to clarify the major source of peripheral IL-6 induced by non-inflammatory stress, blood samplings were performed via chronically implanted cannulations into the hepatoportal vein (HV), the vena cava inferior (VCI) and/or the vena cava superior (VCS) in the electrical foot shock (FSS) rat model (ImA, 11 times, each one min). The results were as follows; (1) Plasma IL-6 levels in the HV were higher than those in the VCS (3Omin after FSS). (2) The peak of plasma IL-6 in the HV (30min) were earlier than that in the VCS (60 min). (3) Plasma IL-6 levels in the lower portion of the VCI (below the renal branch) were lower than that in the VCS ( 0 and 30min). (4) On the contrary, plasma IL-6 levels in the higher portion of the VCI (nearby the hepatic branch) were not differ from those in the VCS. (5) Both the I-IV and VCS IL-6 increase by FSS were attenuated by in vivo neutralization of the circulating LPS. AU data suggest that (1) LPS is a key mediator to produce the peripheral IL-6 during the non-inflammatory stress, (2) main target sits of the LPS exist in the gut neighboring organs and the liver.