Hideya Mizuno

Identification of muscle-specific microRNAs in serum of muscular dystrophy animal models: promising novel blood-based markers for muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder caused by mutations in the dystrophin gene, which encodes a cytoskeletal protein, dystrophin. Creatine kinase (CK) is generally used as a blood-based biomarker for muscular disease including DMD, but it is not always reliable since it is easily affected by stress to the body, such as exercise. Therefore, more reliable biomarkers of muscular dystrophy have long been desired. MicroRNAs (miRNAs) are small, ∼22 nucleotide, noncoding RNAs which play important roles in the regulation of gene expression at the post-transcriptional level. Recently, it has been reported that miRNAs exist in blood. In this study, we hypothesized that the expression levels of specific serum circulating miRNAs may be useful to monitor the pathological progression of muscular diseases, and therefore explored the possibility of these miRNAs as new biomarkers for muscular diseases. To confirm this hypothesis, we quantified the expression levels of miRNAs in serum of the dystrophin-deficient muscular dystrophy mouse model, mdx, and the canine X-linked muscular dystrophy in Japan dog model (CXMDJ), by real-time PCR. We found that the serum levels of several muscle-specific miRNAs (miR-1, miR-133a and miR-206) are increased in both mdx and CXMDJ. Interestingly, unlike CK levels, expression levels of these miRNAs in mdx serum are little influenced by exercise using treadmill. These results suggest that serum miRNAs are useful and reliable biomarkers for muscular dystrophy.

GLUCOCORTICOID ATTENUATES BRAIN-DERIVED NEUROTROPHIC FACTOR-DEPENDENT UPREGULATION OF GLUTAMATE RECEPTORS VIA THE SUPPRESSION OF MICRORNA-132 EXPRESSION

Brain-specific microRNAs (miRs) may be involved in synaptic plasticity through the control of target mRNA translation. Brain-derived neurotrophic factor (BDNF) also contributes to the regulation of synaptic function. However, the possible involvement of miRs in BDNF-regulated synaptic function is poorly understood. Importantly, an increase in glucocorticoid levels and the downregulation of BDNF are supposed to be involved in the pathophysiology of depressive disorders. Previously, we reported that glucocorticoid exposure inhibited BDNF-regulated synaptic function via weakening mitogen-activated protein kinase/extracellular signal-regulated kinase1/2 (MAPK/ERK) and/or phospholipase C-gamma (PLC-gamma) intracellular signaling in cultured neurons [Kumamaru et al (2008) Mol Endocrinol 22:546-558; Numakawa et al (2009) Proc Natl Acad Sci U S A 106:647-652]. Therefore, in this study, we investigate the possible influence of glucocorticoid on BDNF/miRs-stimulated biological responses in cultured cortical neurons. Significant upregulation of miR-132 was caused by BDNF, although miR-9, -124, -128a, -128b, -134, -138, and -16 were intact. Transfection of exogenous ds-miR-132 induced marked upregulation of glutamate receptors (NR2A, NR2B, and GluR1), suggesting that miR-132 has a positive effect on the increase in postsynaptic proteins levels. Consistently, transfection of antisense RNA to inhibit miR-132 function decreased the BDNF-dependent increase in the expression of postsynaptic proteins. U0126, an inhibitor of the MAPK/ERK pathway, suppressed the BDNF-increased miR-132, suggesting that BDNF upregulates miR-132 via the MAPK/ERK1/2 pathway. Interestingly, pretreatment with glucocorticoid (dexamethasone, DEX) reduced BDNF-increased ERK1/2 activation, miR-132 expression, and postsynaptic proteins. We demonstrate that the exposure of neurons to an excess glucocorticoid results in a decrease in the BDNF-dependent neuronal function via suppressing miR-132 expression.

Feeding of Ginkgo biloba extract (GBE) enhances gene expression of hepatic cytochrome P-450 and attenuates the hypotensive effect of nicardipine in rats.

Ginkgo biloba extract (GBE) has been used clinically for improving peripheral vascular diseases in France and Germany and is ingested widely as a herbal medicine in some countries. However, accurate information about its safety as an herbal medicine has not been sufficiently established. To address this issue, we examined the effect of GBE on hepatic drug metabolizing enzymes and their influence on hypotensive drug in rats. Male rats were fed either a control diet or diet containing GBE (0.5% w/w) for 4 weeks. The feeding of a GBE diet did not change the serum transaminase activity, but increased the liver weight and the phospholipid concentration in the liver. In addition, the GBE diet markedly increased the content of cytochrome P-450 (CYP), and the activity of glutathione S-transferase in the liver. Furthermore, the GBE diet markedly induced levels of CYP2B1/2, CYP3A1 and CYP3A2 mRNA in the liver. The levels of CYP1A1, CYP1A2, CYP2E1, CYP2C11 and CYP4A1 were unchanged. The feeding of GBE for 4 weeks significantly reduced the hypotensive effect of nicardipine that was reported to be metabolized by CYP3A2 in rats. These findings suggest that GBE reduces the therapeutic potency of the Ca2+ channel blocker, nicardipine, via enhancement of cytochrome P-450 expression.

Ginkgo biloba extract-induced relaxation of rat aorta is associated with increase in endothelial intracellular calcium level.

Ginkgo biloba extract (GBE) has been used clinically for improving peripheral vascular diseases in France and Germany. In the present study, to clarify the pharmacological properties of vasodilation produced by GBE, we examined the effect of GBE and quercetin, one of the ingredients in GBE, on the thoracic aorta isolated from Wistar rats. GBE produced a dose-dependent relaxation in the aortic ring precontracted with noradrenaline, and the relaxation was abolished by L-N(G)-nitro arginine methyl ester (L-NAME). Quercetin produced a similar relaxation, which was also abolished by L-NAME. We then examined the effects of GBE and quercetin on the intracellular calcium level ([Ca2+]i) of cultured aortic endothelial cells using a fluorescent confocal microscopic imaging system. Both GBE and quercetin produced significant increases in [Ca2+]i in the endothelial cells. The increase in [Ca2+]i by quercetin (10(-6) M) was abolished by removing the extracellular Ca2+, but was not affected by thapsigargin, a calcium pump inhibitor. These findings suggest that a principal ingredient of GBE producing vasodilation is quercetin, which can activate nitric oxide synthesis and release by increasing [Ca2+]i in vascular endothelial cells.

Participation Of Atp In Cell Volume Regulation In The Endothelium After Hypotonic Stress

1. The role of ATP in the regulatory volume decrease (RVD) after hypotonic cell swelling was examined in cultured endothelial cells isolated from the rat caudal artery.

α-Synuclein Transgenic Drosophila As a Model of Parkinson's Disease and Related Synucleinopathies

α-Synuclein (α-Syn) is a major component of protein inclusions known as Lewy bodies, which are hallmarks of synucleinopathies such as Parkinsons disease (PD). The α-Syn gene is one of the familial PD-causing genes and is also associated with an increased risk of sporadic PD. Numerous studies using α-Syn expressing transgenic animals have indicated that α-Syn plays a critical role in the common pathogenesis of synucleinopathies. Drosophila melanogaster has several advantages for modeling human neurodegenerative diseases and is widely used for studying their pathomechanisms and therapies. In fact, Drosophila models expressing α-Syn have already been established and proven to replicate several features of human PD. In this paper, we review the current research on synucleinopathies using α-Syn Drosophila models and, moreover, explore the possibilities of these models for comprehensive genetic analyses and large-scale drug screening towards elucidating the molecular pathogenesis and developing therapies for synucleinopathies.

Pharmacology and Physiology of Perivascular Nerves Regulating Vascular Function

It is generally agreed that the release of norepinephrine (NE) is inhibited by activation of prejunctional purinoceptor. We examined the pharmacological properties of purinoceptors on vascular sympathetic nerve terminals and the source of endogenous adenyl purines. Electrically (1 Hz) evoked NE-release was inhibited by not only P1-agonists but also P2-agonists. Although the inhibition induced by P2-agonists was blocked by P1-antagonists, P2-agonists-induced inhibition was not due to the breakdown to adenosine. Therefore, there may be a new class of purinoceptor that is activated by both P1- and P2-agonists and antagonized by P1-antagonists. Electrical stimulation at 8 Hz but not at 1 Hz evoked the release of adenyl purines such as ATP, ADP, AMP and adenosine, in addition to NE; and the purines-release was blocked by an α1-antagonist. Methoxamine, an α1-agonist, also evoked the release of purines. Electrically (1 Hz)-evoked NE-release was inhibited by methoxamine, and this inhibition was blocked by not only an α1-antagonist but also a P1-antagonist. Therefore, the activation of α1-adrenoceptor appeared to release purines, which in turn inhibited NE-release via prejunctional purinoceptors. From these results, it is suggested that the unique purinoceptor and the endogenous purines released from α1-adrenoceptor-sensitive sources participate in the antidromic transsynaptic modulation of vascular sympathetic neurotransmission.

ATP modulates the release of noradrenaline through two different prejunctional receptors on the adrenergic nerves of rat prostate

1 The effects of adenosine and ATP receptor agonists on the release of endogenous noradrenaline from electrically stimulated (2 Hz, 0.1 msec) rat prostate were examined in order to clarify the pharmacological properties of prejunctional receptors for adenosine and ATP on the adrenergic nerve varicosities in the prostate. Noradrenaline was quantified by HPLC coupled with electrochemical detection techniques. 2 Both adenosine and ATP receptor agonists (1 µmol/L) inhibited noradrenaline release and the relative order of inhibitory effect was N6‐cyclopentyl‐adenosine (CPA) > 5′‐N‐ethylcarboxamidoadenosine > 2‐chloroadenosine > adenosine > 2‐methylthio‐ATP (2mSATP) > AMP > ATP. 3 The adenosine receptor agonist CPA (1 nmol/L–1 µmol/L) and the ATP receptor agonist 2mSATP (100 nmol/L–100 µmol/L) inhibited the stimulation‐induced release of noradrenaline in a concentration‐dependent manner. The concentrations of CPA and 2mSATP that produced 50% inhibition of noradrenaline release were 9.6 nmol/L and 1.4 µmol/L, respectively. 4 1,3‐Dipropyl‐8‐cyclopentylxanthine, an adenosine A1 receptor antagonist, significantly reduced the inhibitory effects of not only CPA, but also 2mSATP. 5 Suramin, an ATP receptor antagonist, significantly reduced the inhibitory effects of 2mSATP, but not those of CPA. 6 Pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid, another ATP receptor antagonist, had no effect on the inhibitory action of either agonist. 7 These results suggest that, in the sympathetic nerve terminals of rat prostate, adenosine and ATP induce inhibition of noradrenaline release via the activation of adenosine A1 and/or xanthine‐sensitive ATP receptors, which play an inhibitory regulatory role in adrenergic neurotransmission in the prostate.

MicroRNA-101 inhibits the expression of Rhes, a striatal-enriched small G-protein, at the post-transcriptional level in vitro

ObjectiveRas homolog enriched in striatum (Rhes) is a small GTP-binding protein that is predominantly localized in the striatal region of the brain. Rhes affects various signaling pathways and plays important roles in Huntington’s disease development caused by striatal anomalies. However, the mechanism underlying the regulation of Rhes expression is not fully understood. We hypothesized that Rhes expression might be regulated by microRNAs (miRNAs), which are small noncoding RNAs that regulate gene expression by interacting with the 3′-untranslated region (3′UTR) of mRNA. This study therefore investigated the interaction between miRNAs and the Rhes mRNA 3′UTR.ResultsThe results of luciferase assay showed that miR-101, the miRNA determined to have the highest possibility of interacting with the Rhes mRNA 3′UTR using DIANA-microT, significantly inhibits luciferase activity, suggesting that miR-101 directly targets the Rhes mRNA 3′UTR. Additionally, Rhes protein levels in cultured cells co-transfected with a plasmid containing the complete Rhes cDNA and miR-101 were significantly downregulated by miR-101 as demonstrated by western blot analysis. These results support our hypothesis that Rhes expression is regulated by miRNA and indicate that miR-101 may be a potent modulator of Rhes expression in striatal neurons.

Bergamottin Promotes Adipocyte Differentiation and Inhibits Tumor Necrosis Factor-α-induced Inflammatory Cytokines Induction in 3T3-L1 Cells

Nowadays, a lot of food ingredients are marketed as dietary supplements for health. Because the effectiveness and mechanisms of these compounds have not been fully characterized, they might have unknown functions. Therefore, we investigated the effect of several food ingredients (Bergamottin, Chrysin, L-Citrulline and β-Carotene) known as health foods on adipocyte differentiation by using 3T3-L1 preadipocytes. In this study, we found that Bergamottin, a furanocoumarin isolated from grapefruit juice, promotes adipocyte differentiation. In addition, Bergamottin increases the expression of adiponectin, an anti-inflammatory adipokine, and peroxisome proliferator activated receptor γ (PPARγ), a nuclear receptor regulating adipocyte differentiation. Furthermore, the anti-inflammatory activity of Bergamottin was demonstrated by its inhibition of the activation of nuclear factor-κB (NF-κB), an inflammatory transcription factor. Stimulation of mature 3T3-L1 adipocytes by tumor necrosis factor-α (TNF-α) decreased the expression of the endogeneous NF-κB inhibitor, IκBα. Treatment with Bergamottin further decreased the TNF-α-induced change in IκBα expression, suggesting that Bergamottin mediated the inhibition of NF-κB activation. In addition, Bergamottin decreased the TNF-α-induced increase in the mRNA levels of pro-inflammatory adipokines, monocyte chemoattractant protein-1 and interleukin-6. Taken together, our results show that Bergamottin treatment could inhibit inflammatory activity through promoting adipocyte differentiation, which in turn suggests that Bergamottin has the potential to minimize the risk factors of metabolic syndrome.