Yuki Kurauchi

Retinoic acid receptor stimulation protects midbrain dopaminergic neurons from inflammatory degeneration via BDNF‐mediated signaling

Functions of retinoic acid receptors (RARs) in adult CNS have been poorly characterized. Here we investigated potential neuroprotective action of tamibarotene (Am80), an RARα/β agonist available for the treatment of acute promyelocytic leukemia, on midbrain dopaminergic neurons. Am80 protected dopaminergic neurons in rat midbrain slice culture from injury mediated by lipopolysaccharide‐activated microglia, without affecting production of nitric oxide, a key mediator of cell injury. The effect of Am80 was mimicked by another RAR agonist, TAC‐101, but not by a retinoid X receptor agonist, HX630, and HX630 did not synergize with Am80. We observed neuronal expression of RARα and RARβ in midbrain slice culture and also found that Am80 increased tissue level of brain‐derived neurotrophic factor (BDNF) mRNA. Exogenous BDNF prevented dopaminergic neurodegeneration, and the neuroprotective effect of Am80 was suppressed by a TrkB inhibitor, K252a, or by anti‐BDNF neutralizing antibody. These results reveal a novel action of RARs mediated by enhancement of BDNF expression. Finally, oral administration of Am80 prevented dopaminergic cell loss in the substantia nigra induced by local injection of lipopolysaccharide in mice, indicating that RARs are a promising target of therapeutics for neurodegenerative disorders.

Caffeic acid phenethyl ester protects nigral dopaminergic neurons via dual mechanisms involving haem oxygenase‐1 and brain‐derived neurotrophic factor

Caffeic acid phenethyl ester (CAPE) is a component of honey bee propolis that can induce expression of haem oxygenase‐1 (HO‐1). Because HO‐1 induction has been suggested to protect dopaminergic neurons in the substantia nigra, we examined the effect of CAPE in experimental models of dopaminergic neurodegeneration.

Midbrain dopaminergic neurons utilize nitric oxide/cyclic GMP signaling to recruit ERK that links retinoic acid receptor stimulation to up-regulation of BDNF

J. Neurochem. (2011) 116, 323–333.

NITRIC OXIDE-CYCLIC GMP SIGNALING PATHWAY LIMITS INFLAMMATORY DEGENERATION OF MIDBRAIN DOPAMINERGIC NEURONS : CELL TYPE-SPECIFIC REGULATION OF HEME OXYGENASE-1 EXPRESSION

Excessive production of nitric oxide (NO) by microglia is at least in part responsible for the pathogenesis of various neurodegenerative disorders including Parkinson disease, but at the same time NO may also play a distinct role as a signaling molecule such as an activator of soluble guanylyl cyclase. Here we investigated potential roles of the NO-soluble guanylyl cyclase-cyclic GMP signaling pathway in the regulation of dopaminergic neurodegeneration. Activation of microglia by interferon-gamma (IFN-gamma) followed by lipopolysaccharide (LPS) caused dopaminergic cell death in rat midbrain slice cultures, which was dependent on NO production. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor, as well as KT5823, an inhibitor of cyclic GMP-dependent protein kinase, exacerbated dopaminergic cell death induced by IFN-gamma/LPS. Conversely, 8-bromo-cyclic GMP attenuated IFN-gamma/LPS cytotoxicity on dopaminergic neurons. Notably, although heme oxygenase-1 (HO-1) was expressed prominently in cells other than dopaminergic neurons in control cultures, robust expression of HO-1 was induced in surviving dopaminergic neurons challenged with IFN-gamma/LPS. ODQ and KT5823 decreased, whereas 8-bromo-cyclic GMP increased, the number of dopaminergic neurons expressing HO-1 after IFN-gamma/LPS challenge, without parallel changes in HO-1 expression in other cell populations. An NO donor 3-(4-morpholinyl)sydnonimine hydrochloride also induced HO-1 expression in dopaminergic neurons, which was abolished by ODQ and augmented by 8-bromo-cyclic GMP. Moreover, IFN-gamma/LPS-induced dopaminergic cell death was augmented by zinc protoporphyrin IX, an HO-1 inhibitor. The NO donor cytotoxicity on dopaminergic neurons was also augmented by ODQ and zinc protoporphyrin IX. These results indicate that the NO-cyclic GMP signaling pathway promotes the induction of HO-1 specifically in dopaminergic neurons, which acts as an endogenous protective system to limit inflammatory degeneration of this cell population.

High fat diet induces specific pathological changes in hypothalamic orexin neurons in mice.

Loss of orexin neurons in the hypothalamus is a prominent feature of narcolepsy and several other neurological conditions. We have recently demonstrated that sleep deprivation stimulates local nitric oxide (NO) production by neuronal NO synthase in the lateral hypothalamus, which leads to selective degeneration of orexin neurons accompanied by formation of orexin-immunoreactive aggregates. Here we analyzed whether lifestyle-related conditions other than sleep deprivation could trigger similar pathological changes in orexin neurons. Four-week-old male C57BL/6 mice were fed with high fat diet (HFD) for 8 weeks. Immunohistochemical analysis revealed that the number of orexin-immunopositive neurons was significantly decreased by HFD intake, whereas the number of melanin-concentrating hormone-immunopositive neurons was unchanged. In addition, HFD promoted formation of intracellular orexin-immunoreactive aggregates in a subset of orexin neurons. We also confirmed that expression of inducible NO synthase (iNOS) in the hypothalamus was upregulated in response to HFD intake. Notably, loss of orexin-immunopositive neurons and formation of orexin-immunoreactive aggregates were not observed in iNOS knockout mice fed with HFD. These results indicate that inappropriate dietary conditions could trigger specific neuropathological events in orexin neurons in an iNOS-dependent manner.

Suppression of CXCL2 upregulation underlies the therapeutic effect of the retinoid Am80 on intracerebral hemorrhage in mice.

We previously demonstrated that a synthetic retinoic acid receptor agonist, Am80, attenuated intracerebral hemorrhage (ICH)‐induced neuropathological changes and neurological dysfunction. Because inflammatory events are among the prominent features of ICH pathology that are affected by Am80, this study investigated the potential involvement of proinflammatory cytokines/chemokines in the effect of Am80 on ICH. ICH induced by collagenase injection into mouse striatum caused prominent upregulation of mRNAs for interleukin (IL)−1β, tumor necrosis factor (TNF)‐α, IL‐6, CXCL1, CXCL2, and CCL3. We found that dexamethasone (DEX) and Am80 differently modulated the increase in expression of these cytokines/chemokines; TNF‐α expression was attenuated only by DEX, whereas CXCL2 expression was attenuated only by Am80. Expression of IL‐1β and IL‐6 was inhibited both by DEX and Am80. Neurological assessments revealed that Am80, but not DEX, significantly alleviated motor dysfunction of mice after ICH. From these results, we suspected that CXCL2 might be critically involved in determining the extent of motor dysfunction. Indeed, magnetic resonance imaging‐based classification of ICH in individual mice revealed that invasion of hematoma into the internal capsule, which has been shown to cause severe neurological disabilities, was associated with higher levels of CXCL2 expression than ICH without internal capsule invasion. Moreover, a CXCR1/2 antagonist reparixin ameliorated neurological deficits after ICH. Overall, suppression of CXCL2 expression may contribute to the beneficial effect of Am80 as a therapeutic agent for ICH, and interruption of CXCL2 signaling may provide a promising target for ICH therapy.

Rapamycin prevents N-methyl-D-aspartate-induced retinal damage through an ERK-dependent mechanism in rats

Recent studies have demonstrated that inhibition of the mammalian target of rapamycin (mTOR) protects against neuronal injury, but the mechanisms underlying this protection are not fully understood. The present study investigates whether rapamycin, an inhibitor of the mTOR pathway, protects against N‐methyl‐D‐aspartate (NMDA)‐induced retinal neurotoxicity and whether the extracellular signal‐regulated kinase (ERK) pathway contributes to this protective effect in rats. Significant cell loss in the ganglion cell layer and a reduction in thickness of the inner plexiform layer were observed 7 days after a single intravitreal injection of NMDA (200 nmol/eye). These NMDA‐induced morphological changes were significantly reduced by rapamycin (20 nmol/eye). The number of terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling‐positive apoptotic cells had increased 6 hr after NMDA injection, an effect that was significantly attenuated by rapamycin. The ERK inhibitor U0126 (1 nmol/eye) almost completely abolished rapamycins inhibition of NMDA‐induced apoptosis. Immunohistochemical studies showed that NMDA caused a time‐dependent increase in levels of the phosphorylated form of the ribosomal protein S6 (pS6), a downstream indicator of mTOR activity. The increased pS6 levels were markedly decreased by rapamycin. Both NMDA and rapamycin increased the level of phosphorylated ERK (pERK) in Müller cells, and coinjection of both agents further increased pERK levels. These results suggest that rapamycin has a neuroprotective effect against NMDA‐induced retinal neurotoxicity and that this effect could be patially mediated by activation of the ERK pathway in retinal Müller cells.

ISO-1, a macrophage migration inhibitory factor antagonist, prevents N-methyl-D-aspartate-induced retinal damage.

Macrophage migration inhibitory factor (MIF) has been shown to play an important role in a variety of inflammatory and immune-mediated diseases. The inflammatory responses contribute to retinal neuronal degeneration. However, the role of MIF in the progression of retinal degeneration has not yet been elucidated. In this study, we determined whether pharmacological inhibition of MIF protects against the retinal damage induced by N-methyl-D-aspartate (NMDA) in rats. Intravitreal injection of NMDA (200 nmol) resulted in (1) cell loss in the ganglion cell layer and reduction in the thickness of the inner plexiform layer, (2) an increase in apoptotic cells, (3) a decrease in parvalbumin-positive amacrine cells, (4) accumulation of leukocytes, and (5) microglia activation. Injection of (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1, 100 nmol), a MIF antagonist, significantly attenuated these NMDA-induced responses. These findings suggest that ISO-1 exerts protective effects against retinal injuries and that MIF may be a target for neuroprotective intervention in retinal diseases associated with glutamate-induced excitotoxicity.

P2X7 receptor antagonists protect against N-methyl-d-aspartic acid-induced neuronal injury in the rat retina

Activation of N-methyl-d-aspartic acid (NMDA) receptors followed by a large Ca(2+) influx is thought to be a mechanism of glaucoma-induced neuronal cell death. It is possible that damage-associated molecular patterns leak from injured cells, such as adenosine triphosphate, causing retinal ganglion cell death in glaucoma. In the present study, we histologically investigated whether antagonists of the P2X7 receptor protected against NMDA-induced retinal injury in the rat in vivo. Under ketamine/xylazine anesthesia, male Sprague-Dawley rats were subjected to intravitreal injection of NMDA. We used A438079 (3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine) and brilliant blue G as P2X7 receptor antagonists. Upon morphometric evaluation 7 days after an intravitreal injection (200 nmol/eye), NMDA-induced cell loss was apparent in the ganglion cell layer. Intravitreal A438079 (50 pmol/eye) simultaneously injected with NMDA and intraperitoneal brilliant blue G (50 mg/kg) administered just before the NMDA injection as well as 24 and 48h after significantly reduced cell loss. In addition, A438079 decreased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells 12h after NMDA injection. P2X7 receptors were immunolocalized in the ganglion cell layer and the inner and outer plexiform layers, whereas the immunopositive P2X7 receptor signal was not detected on the Iba1-positive microglial cells that infiltrated the retina 12h after NMDA injection. The present study shows that stimulation of the P2X7 receptor is involved in NMDA-induced histological damage in the rat retina in vivo. P2X7 receptor antagonists may be effective in preventing retinal diseases caused by glutamate excitotoxicity, such as glaucoma and retinal artery occlusion.

Regulatory Mechanisms of Vitamin D3 on Production of Nitric Oxide and Pro-inflammatory Cytokines in Microglial BV-2 Cells.

Inhibition of pro-inflammatory functions of microglia has been considered a promising strategy to prevent pathogenic events in the central nervous system under neurodegenerative conditions. Here we examined potential inhibitory effects of nuclear receptor ligands on lipopolysaccharide (LPS)-induced inflammatory responses in microglial BV-2 cells. We demonstrate that a vitamin D receptor agonist 1,25-dihydroxyvitamin D3 (VD3) and a retinoid X receptor agonist HX630 affect LPS-induced expression of pro-inflammatory factors. Specifically, both VD3 and HX630 inhibited expression of mRNAs encoding inducible nitric oxide synthase (iNOS) and IL-6, whereas expression of IL-1β mRNA was inhibited only by VD3. The inhibitory effect of VD3 and HX630 on expression of iNOS and IL-6 mRNAs was additive. Effect of VD3 and HX630 was also observed for inhibition of iNOS protein expression and nitric oxide production. Moreover, VD3 and HX630 inhibited LPS-induced activation of extracellular signal-regulated kinase (ERK) and nuclear translocation of nuclear factor κB (NF-κB). PD98059, an inhibitor of ERK kinase, attenuated LPS-induced nuclear translocation of NF-κB and induction of mRNAs for iNOS, IL-1β and IL-6. These results indicate that VD3 can inhibit production of several pro-inflammatory molecules from microglia, and that suppression of ERK activation is at least in part involved in the anti-inflammatory effect of VD3.