Soichiro Kishi

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.

MicroRNA function and neurotrophin BDNF.

MicroRNAs (miRs), endogenous small RNAs, regulate gene expression through repression of translational activity after binding to target mRNAs. miRs are involved in various cellular processes including differentiation, metabolism, and apoptosis. Furthermore, possible involvement of miRs in neuronal function have been proposed. For example, miR-132 is closely related to neuronal outgrowth while miR-134 plays a role in postsynaptic regulation, suggesting that brain-specific miRs are critical for synaptic plasticity. On the other hand, numerous studies indicate that BDNF (brain-derived neurotrophic factor), one of the neurotrophins, is essential for a variety of neuronal aspects such as cell differentiation, survival, and synaptic plasticity in the central nervous system (CNS). Interestingly, recent studies, including ours, suggest that BDNF exerts its beneficial effects on CNS neurons via up-regulation of miR-132. Here, we present a broad overview of the current knowledge concerning the association between neurotrophins and various miRs.

Prevention of endoplasmic reticulum stress-induced cell death by brain-derived neurotrophic factor in cultured cerebral cortical neurons

Brain-derived neurotrophic factor (BDNF), one of the neurotrophic factors acting in the central nervous system (CNS), prevents ordinary types of neuronal cell death induced by various stimulants. On the other hand, an accumulation of unfolded proteins in the endoplasmic reticulum (ER) leads to ER stress and then induces ER stress-mediated cell death. The ER stress-mediated cell death is distinctive because the caspase-12 activity plays a crucial role in the progression of cell death. We previously showed that nerve growth factor (NGF) attenuated ER stress-mediated cell death in non-neuronal PC12 cells. Here, we report that BDNF suppressed the ER stress-mediated cell death in tunicamycin (Tm)-treated cerebral cortical neurons. An analysis using a specific inhibitor of phosphatidylinositol 3-kinase (PI3-K), LY294002, revealed that BDNF prevented this cell death via the PI3-K signaling pathway. We found that the number of NeuN/TUNEL-double positive cells and the activity of caspase-3 suppressed by BDNF were increased by LY294002. We also discovered that LY294002 diminished the effect of BDNF on the activation of caspase-12, indicating that BDNF prevents ER stress-mediated cell death via a PI3-K-dependent mechanism by suppressing the activation of caspase-12 in cultured CNS neurons.

Nerve growth factor attenuates 2-deoxy-D-glucose-triggered endoplasmic reticulum stress-mediated apoptosis via enhanced expression of GRP78

The glucose analog 2-deoxy-d-glucose (2DG) depletes cells of glucose. Inhibition of glycosylation caused by glucose depletion induces endoplasmic reticulum (ER) stress with subsequent apoptosis. Glucose-regulated protein 78 (GRP78) is a molecular chaperone that acts within the ER. During ER stress, GRP78 expression is induced as part of the unfolded protein response (UPR). We found that nerve growth factor (NGF) prevented 2DG-triggered ER stress-mediated apoptosis, but not the induction of GRP78 expression, in PC12 cells. Surprisingly, GRP78 expression was further up-regulated when NGF was added to 2DG-treated PC12 cells. When a specific inhibitor of phosphatidylinositol 3-kinase (PI3-K), LY294002, was added to 2DG plus NGF-treated cells, both the effects of NGF on 2DG-induced apoptosis and GRP78 expression were significantly diminished. In addition, versipelostatin (VST), a specific inhibitor of GRP78 expression, and small interfering RNA (siRNA) against GRP78 mRNA also decreased both the effects of NGF on 2DG-induced apoptosis and GRP78 expression. RT-PCR and Western blot analyses revealed that enhanced production of nuclear p50 ATF6, but not spliced XBP1, mainly contributed to the NGF-induced enhancement of GRP78 expression in 2DG-treated cells. These results suggest that the NGF-activated PI3-K/Akt signaling pathway plays a protective role against ER stress-mediated apoptosis via enhanced expression of GRP78 in PC12 cells.

NGF-induced phosphatidylinositol 3-kinase signaling pathway prevents thapsigargin-triggered ER stress-mediated apoptosis in PC12 cells

Tunicamycin, an inhibitor of the glycosylation of newly biosynthesized proteins, induces endoplasmic reticulum (ER) stress and subsequent apoptosis, and caspase family proteases are activated during the process of ER stress-mediated apoptosis. In the present study, we showed that thapsigargin (Th), an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA), also induced ER stress-mediated apoptosis, and nerve growth factor (NGF) prevented the apoptosis in PC12 cells. We also found that LY 294002, an inhibitor of phosphatidylinositol 3-kinase (PI 3-K), reduced the survival of cells treated with NGF for 24h in the presence of Th. We discovered that the activities of caspase-3, -9 and -12 were increased time-dependently after the treatment with Th, and NGF suppressed the Th-triggered activation of caspase-3, -9 and -12. LY 294002 diminished the effect of NGF on the inactivation of all these caspases. These results indicate that the NGF-induced PI 3-K signaling pathway prevents Th-triggered ER stress-specific apoptosis via inhibition of caspase-mediated apoptotic signal.

Growth factors stimulate expression of neuronal and glial miR-132.

Brain-specific microRNAs (miRs) and brain-derived neurotrophic factor (BDNF) are both involved in synaptic function. We previously reported that upregulation of miR-132 is involved in BDNF-increased synaptic proteins, including glutamate receptors (NR2A, NR2B, and GluR1) in mature cortical neurons [7]. However, the potential role of other growth factors in miR-132 induction has not been clarified. Here, we examined the effect of growth factors including basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), glial cell line-derived neurotrophic factor (GDNF), and epidermal growth factor (EGF), on expression of miR-132 and glutamate receptors in immature cortical neurons. We found that BDNF and bFGF upregulated levels of miR-132 in cortical cultures, though bFGF failed to increase glutamate receptors such as NR2A, NR2B, and GluR1. IGF-1, GDNF, and EGF did not have a positive influence on miR-132 and glutamate receptors in neuronal cultures. Furthermore, bFGF significantly upregulated miR-132 in cultured astroglial cells, while other growth factors failed to elicit such a response. It is possible that the growth factor-stimulated neuronal and glial action of miR-132 plays a critical role in brain function.

p-Nonylphenol induces endoplasmic reticulum stress-mediated apoptosis in neuronally differentiated PC12 cells.

Endocrine disrupting chemicals (EDCs) induce estrogenic phenotypes in sexual organs and cells by chronic stimulation through binding to estrogen receptors. Although cell death may be induced instead of phenotypic change by EDCs in germ cells, the mechanism of the effect of EDCs in neuronal cells is still obscure. Here we report that p-nonylphenol, one of the EDCs, induced apoptosis with up-regulation of glucose-regulated protein 78 (GRP78) expression and activation of caspase-12, which are involved in endoplasmic reticulum (ER) stress specific phenomena, in NGF-treated neuronally differentiated PC12 cells. Moreover, we observed that p-nonylphenol increased the intracellular Ca(2+) concentration and p-nonylphenol-induced apoptosis was prevented when BAPTA-AM, a membrane-permeable Ca(2+) chelator, was added. Intriguingly, we also discovered that decreased phosphorylation of ERK1/2 was induced by p-nonylphenol in the presence of NGF, whereas p-nonylphenol alone did not induce phosphorylation of ERK1/2. These lines of evidence suggest that p-nonylphenol can induce ER stress-mediated apoptosis via increased intracellular Ca(2+) concentration, and can reduce ERK1/2 phosphorylation to attenuate the cell survival effect of NGF, in neuronally differentiated PC12 cells.

Protective effect of nicotine on tunicamycin-induced apoptosis of PC12h cells.

Nicotine has been reported to have neuroprotective effects. The present study deals with the neuroprotective effect of nicotine on the tunicamycin-induced apoptosis of PC12h cells. Treatment of PC12h cells with tunicamycin causes endoplasmic reticulum stress leading to apoptosis. Nicotine dose-dependently prevented the tunicamycin-induced apoptosis. Hoechst 33258 staining demonstrated the protective effect of nicotine against tunicamycin-induced apoptosis. Treatment with nicotinic acetylcholine receptor (nAChR) and L-type voltage-sensitive calcium channel (L-VSCC) antagonists prevented the nicotine-induced protective effect. A phosphatidylinositol 3-kinase (PI3-K) inhibitor had no influence on the nicotine-induced neuroprotective effect. These results show that the neuroprotective effect of nicotine occurs through nAChRs including the alpha 7 subtype and L-VSCC in PC12h cells and not through the PI3-K/Akt pathway.

Brain-specific microRNA-132 induction by growth factors in cortical neurons

Leukemia inhibitory factor (LIF), a member of the interleukin-6 (IL-6) superfamily of cytokines, contributes to neurogenesis in the cerebral cortex. We previously found a maternal LIF-placental adenocorticotropic hormone (ACTH)-fetal LIF signaling relay pathway (Simamura et al., 2010). In this pathway, maternal LIF stimulates ACTH secretion from the placental trophoblasts into the fetal blood circulation, which in turn induces fetal nucleated red blood cells to secrete LIF. This results in significant increases in neurogenesis in the fetal forebrain. However, the downstream network of LIF action in the fetal cerebrum is unknown. In the present study, we revealed insulin-like growth factor 1 (IGF1), a potent candidate for downstream molecule of LIFACTH-LIF pathway, which is involved in neurogenesis in the fetal forebrain. Slc:Wistar Hannover rats were used. Chronological changes showed that IGF1 concentration in the fetal cerebrospinal fluid (CSF) significantly increased after 16 days post coitus (dpc), following the peaking of LIF in CSF of fetus at 15.5 dpc. The expression of Igf1 in the dorsal cerebrum and the level of IGF1 in CSF of fetuses increased 4 h after injection of recombinant LIF into dams (5 g/kg) at 15.5 dpc intraperitoneally. Moreover, injection of LIF into the fetal cerebral ventricles elevated the expression of Igf1 in the dorsal cerebrum. These results indicate that fetal LIF contributes to neurogenesis in the forebrain via IGF1 signaling downstream to the LIF-ACTH-LIF pathway. Research fund: This work was supported by a KAKENHI (22390216, 21791045), by a Grant for Project Research from the High-Tech Research Center of Kanazawa Medical University (H2009-14, H2010-14) and by grants for promoting research from Kanazawa Medical University (C2010-2-2, S200910, S2009-2, S2010-8).

Interaction between miRNA-132 function and ERK signalings in BDNF-mediated synaptic function

Adult brain vasculature has the blood-brain barrier (BBB) that restricts the movement of molecules between the blood and brain, while the circumventricular organs (CVOs) lack the BBB. The fenestrated microvessels allow neurons and/or glial cells to sense blood ions, pathogenic organisms, toxins, and hormones in sensory circumventricular organs, such as the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO) and area postrema (AP). Moreover, fenestrated microvessels enable neuronal terminals to secrete a variety of peptide hormones into the blood in secretory circumventricular organs, such as the median eminence (ME) and neurohypophysis (NH). In our previous study, we have demonstrated dynamic vascular reconstruction in the adult CVOs. The present study revealed that many VEGF-positive puncta were scattered within the cytoplasm of somatodendrites in the OVLT and SFO, axonal terminals in the ME, and astrocytes in the NH. In the AP, however, VEGF-positive puncta were seen in both astrocytes and neurons of the AP. VEGF-positive puncta were considered as secretory vesicles, since secretory proteins are stored in Glogi-derived vesicles and released extracellularly via exocytosis of the secretory or consecutive pathway. Therefore, it is evident that the CVOs possess sufficient sources of VEGF in neurons and/or astrocytes for angiogenesis. In next experiment, we revealed that the expression of serine-proteases, tissue-type plasminogen activator (tPA) and plasminogen, was prominently higher at neurons in the adult CVOs as compared with adjacent hypothalamic and medulla regions. These data indicate that these extracellular proteases are responsible for rearrangement of neuro-vascular architectures. Research fund: KAKENHI 21500323.