Jun-ichi Kakimura

Activators of peroxisome proliferator-activated receptor-γ (PPARγ) inhibit inducible nitric oxide synthase expression but increase heme oxygenase-1 expression in rat glial cells

Abstract The peroxisome proliferator-activated receptor- γ (PPAR γ ) is activated by 15-deoxy- Δ 12,14 prostaglandin J 2 (15d-PGJ 2 ), anti-diabetic thiazolidinediones and several non-steroidal anti-inflammatory drugs (NSAIDs). In rat glial cells, lipopolysaccharide and interferon- γ (LPS/IFN- γ ) induced expression of both inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1). PPAR γ activators inhibited iNOS expression by LPS and IFN- γ . However, PPAR γ activator alone induced HO-1 expression and further enhanced LPS/IFN- γ -induced HO-1 expression. These results suggest that activation of PPAR γ negatively regulate iNOS expression and positively regulates HO-1 expression in glial cells.

Release and aggregation of cytochrome c and α-synuclein are inhibited by the antiparkinsonian drugs, talipexole and pramipexole

Recently, it has been shown that release of cytochrome c from the mitochondria to the cytosol is required for activation of the caspase-3-dependent cascade in apoptosis, and also for alpha-synuclein aggregation. In the present study, we examined the effects of talipexole and pramipexole on the release of cytochrome c and alpha-synuclein, their aggregations, and activation of caspases. Treatment of human neuroblastoma SH-SY5Y cells with 1-methyl-4-phenylpyridinium (MPP(+), 1 mM) induced the first event, which was the release of cytochrome c from the organellar fraction to the cytosolic fraction, then came the DNA fragmentation, and caused the last event, which was the accumulation of alpha-synuclein protein in the cytosolic fraction. Talipexole and pramipexole at low concentration (0.1-1 mM) significantly inhibited the accumulation of cytochrome c or alpha-synuclein in the cytosolic fraction. These drugs at high concentration (3-10 mM) inhibited in vitro aggregation of cytochrome c by hydrogen peroxide or that of alpha-synuclein by cytochrome c and hydrogen peroxide. In addition, in vitro activation of caspase-3 induced by cytochrome c and/or dATP was also inhibited by drugs at high concentration (5-10 mM). These results suggest that talipexole and pramipexole may have protective effects against the neurodegeneration, which is induced by intracellular accumulation of cytochrome c and alpha-synuclein.

Kainic acid induction of heme oxygenase in vivo and in vitro

Heme oxygenase, catalyses oxidation of the heme molecule in concert with NADPH-cytochrome P450 reductase and then specifically cleaves heme into biliverdin, carbon monoxide, and iron. Biliverdin and its product, bilirubin, are known to be strong antioxidants. Kainic acid is a potent neurotoxin, and induces selective neuronal loss in the rat hippocampus. Kainic acid acts on the kainate receptors, and kainic acid neurotoxicity may be in part mediated by oxidative stress. In this study, we examined whether or not heme oxygenase was activated in kainic acid-induced neurotoxicity. After intracerebroventricular injection of kainic acid, the heme oxygenase-1 protein level was strongly enhanced, although the constitutive heme oxygenase (heme oxygenase-2) protein level was not changed. One day after treatment, the protein level of heme oxygenase-1 reached a maximum and then gradually decreased over a period of three to seven days. In the rat hippocampus, cells expressing heme oxygenase-1 in vivo were predominately microglia and only a few astrocytes. In addition, heme oxygenase-1 immunoreactivity was predominantly co-localized with major histocompatibility complex class II-, and partly co-localized with class I-immunoreactive microglia. In cultured glial cells in vitro, heme oxygenase- protein was expressed in the microglia even with the vehicle treatment, and was strongly induced in astrocytes by kainic acid treatment. These results suggest that ameboid microglia, which express both heme oxygenase-1 and major histocompatibility complex antigens, may play a key role in a delayed episode of kainic acid-induced microglial activation and neurodegeneration.

Possible involvement of both mitochondria- and endoplasmic reticulum-dependent caspase pathways in rotenone-induced apoptosis in human neuroblastoma SH-SY5Y cells.

Recently, it has been shown that rotenone, a specific inhibitor of mitochondrial complex I, is a useful tool in animal models of Parkinsons disease, but the mechanism of rotenone-induced neuronal death is not fully understood. In human neuroblastoma SH-SY5Y cells, rotenone induced the degradation of procaspases-12, -9 and -3, followed by cleavage of poly (adenosine diphosphate-ribose) polymerase, DNA fragmentation and cell death. Pretreatment with phorbol-12-myristate-13-acetate inhibited the rotenone-induced decrease in procaspases-9 and -3, but not that in procaspase-12. In contrast, benzyloxycarbonyl-Val-Ala-Asp(OCH(3))-CH(2)F inhibited the decrease in procaspase-12, but not those in procaspases-9 and -3 in this study. These results suggest that rotenone may induce activation of both mitochondria- and endoplasmic reticulum-dependent caspases in human SH-SY5Y cells.

Increase of Bcl-2 protein in neuronal dendritic processes of cerebral cortex and hippocampus by the antiparkinsonian drugs, talipexole and pramipexole

Treatment of rats for 4 days with the antiparkinsonian drugs, talipexole and pramipexole, markedly increased Bcl-2 immunoreactivity in neuronal dendritic processes in both cerebral cortex and hippocampus, but treatment for 1 day with either of these drugs did not. Repeated administration of talipexole or pramipexole may have neuroprotective effect in neurodegenerative disorders.

Hyperbilirubinemia protects against focal ischemia in rats

Heme oxygenase‐1 (HO1) catalyzes oxidation of the heme molecule in concert with NADPH‐cytochrome P450 reductase following the specific cleavage of heme into carbon monoxide, iron, and biliverdin, which is rapidly metabolized to bilirubin. HO1 is a stress‐inducible protein that protects cells against oxidative injury, but its protective mechanism is not fully understood. The Eizai hyperbilirubinemic rat (EHBR), a mutant strain derived from the Sprague‐Dawley rat (SDR), has a mutation in the gene for the canalicular multispecific organic anion transporter, which results in a phenotype of hyperbilirubinemia, and thus is a model of Dubin‐Johnson syndrome in humans. In this study, we compared EHBR and SDR with regard to neuronal death induced by 2 hr of occlusion of the middle cerebral artery and reperfusion. In EHBR, the area that was immunoreactive for microtubule‐associated protein‐2 was significantly reduced, and the HO1‐immunoreactive area was smaller than that in SDR. These results suggest that bilirubin has essentially a neuroprotective effect against focal ischemia and may participate in HO1‐induced neuroprotection.

Expression of heme oxygenase-1 mediated by non-NMDA and metabotropic receptors in glial cells: possible involvement of reactive oxygen species production and protein kinase C activation

Heme oxygenase (HO) produces biliverdin and bilirubin which are physiological antioxidants and potent scavengers of oxygen radicals. Recently, we found that intracerebroventricular injection of kainic acid (KA) induced inducible HO (HO-1) predominantly in glial cells in the rat hippocampus in vivo. In this study, we examined the mechanism of HO-1 expression induced by agonists for glutamate receptors in cultured glial cells in vitro. The HO-1 protein level was significantly enhanced by several agonists for non-N-methyl-D-aspartate (non-NMDA) receptors and metabotropic glutamate receptors (mGluR) such as KA, quisqualic acid (QA), (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propanoic acid (AMPA), and trans-(+/-)-1-amino-(1S,3R)-cyclopentanedicarboxylic acid (ACPD). Among these agonists, QA had the greatest potency. KA-induced HO-1 expression was inhibited by the non-NMDA antagonist NBQX. In addition, KA induced the marked production of reactive oxygen species (ROS), and KA-induced HO-1 expression was also inhibited by the antioxidants allopurinol and ascorbic acid. ACPD-induced HO-1 expression was inhibited by the mGluR antagonist MCPG and the protein kinase C (PKC) inhibitor calphostin C. These results suggest that induction of HO-1 expression by the activation of non-NMDA receptors is mediated by ROS production, and that expression induced by mGluR activation is mediated by PKC activation in rat glial cells.

α-Synuclein protein is not scavenged in neuronal loss induced by kainic acid or focal ischemia

Alpha-synuclein, a presynaptic protein, is markedly included in Lewy bodies (LB) in Parkinsons and LB diseases. In this study, neuronal loss and the activation of glial cells such as microglia and astrocytes were induced by neurodegenerative insults such as the injection of kainic acid and occlusion of the middle cerebral artery. In contrast, immunoreactivity for alpha-synuclein did not change even at 7 days after these insults. These results suggest that alpha-synuclein protein may be so scarcely scavenged by glial cells that it readily condenses in neurodegenerative regions.

Effects of 15-deoxy-Δ12,14 prostaglandin J2 and interluekin-4 in Toll-like receptor-4-mutant glial cells

Abstract 15-Deoxy-Δ12,14 prostaglandin J2 and interleukin-4 are endogenous anti-inflammatory substances. In this study, we examined the effects of 15-deoxy-Δ12,14 prostaglandin J2 and interleukin-4 in glial cells from the Toll-like receptor-4-mutant (C3H/HeJ) and wild-type (C3H/HeN) mouse brains. The lipopolysaccharide-induced expression of inducible nitric oxide (NO) synthase and cyclooxygenase-2 in the Toll-like receptor-4-mutant glial cells have significantly lower levels (about half and quarter, respectively) than those in the wild-type cells. Treatment with both interleukin-4 (at 10 ng/ml, for 48 h) and 15-deoxy-Δ12,14 prostaglandin J2 (at 3 μM, for 30 min) completely inhibited the lipopolysaccharide-induced expression of inducible NO synthase and cyclooxygenase-2. In contrast, heme oxygenase-1 was induced by 15-deoxy-Δ12,14 prostaglandin J2 alone, but was not changed by interleukin-4 or lipopolysaccharide. The inhibitory protein of nuclear factor-κB was degraded by lipopolysaccharide in both mutant and wild-type glial cells, and this degradation was not inhibited by either 15-deoxy-Δ12,14 prostaglandin J2 or interleukin-4. These results suggest that the response to lipopolysaccharide is partially dependent on Toll-like receptor-4 in mouse glial cells, and that 15-deoxy-Δ12,14 prostaglandin J2 and interleukin-4 differently regulate the expression of inducible NO synthase and cyclooxygenase-2, and heme oxygenase-1.

Kainic acid-induced neuronal loss and glial changes in the hippocampal CA3 of p53-deficient mouse.

We examined kainic acid (KA)-induced neuronal death and changes in glial cells in p53-deficient (p53-/-) and wild-type (p53+/+) mice which were CBA and C57BL/6 background. The p53-/- mouse exhibited a KA-induced loss of CA3 pyramidal neurons similar to that in wild-type mouse. Before neuronal death, c-Jun protein was expressed, phosphorylated and translocated into several nuclei of CA3 pyramidal neurons. In p53-/- mouse, microglial activation was slightly faster and more continuous after 1-7 days than that in p53+/+ mouse. On the other hand, p53-/- astrocytes were relatively resistant to KA cytotoxicity, and marked astrocytosis also occurred after 7 days. These observations suggest that p53-null mutation may influence the activation and proliferation of glial cells rather than neuronal death.