The nSMase2/Smpd3 gene modulates the severity of muscular dystrophy and the emotional stress response in mdx mice.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive, degenerative muscular disorder and cognitive dysfunction caused by mutations in the dystrophin gene. It is characterized by excess inflammatory responses in the muscle and repeated degeneration and regeneration cycles. Neutral sphingomyelinase 2/sphingomyelin phosphodiesterase 3 (nSMase2/Smpd3) hydrolyzes sphingomyelin in lipid rafts. This protein thus modulates inflammatory responses, cell survival or apoptosis pathways, and the secretion of extracellular vesicles in a Ca2+-dependent manner. However, its roles in dystrophic pathology have not yet been clarified. METHODS: To investigate the effects of the loss of nSMase2/Smpd3 on dystrophic muscles and its role in the abnormal behavior observed in DMD patients, we generated mdx mice lacking the nSMase2/Smpd3 gene (mdx:Smpd3 double knockout [DKO] mice). RESULTS: Young mdx:Smpd3 DKO mice exhibited reduced muscular degeneration and decreased inflammation responses, but later on they showed exacerbated muscular necrosis. In addition, the abnormal stress response displayed by mdx mice was improved in the mdx:Smpd3 DKO mice, with the recovery of brain-derived neurotrophic factor (Bdnf) expression in the hippocampus. CONCLUSIONS: nSMase2/Smpd3-modulated lipid raft integrity is a potential therapeutic target for DMD.

Basic fibroblast growth factor increased glucocorticoid receptors in cortical neurons through MAP kinase pathway.

Prolonged and intense stress chronically increases blood concentration of glucocorticoids, which in turn causes downregulation of glucocorticoid receptor (GR) in the central nervous system (CNS). This process has been suggested to be involved in the pathogenesis of major depressive disorder (MDD). Here, we found that basic fibroblast growth factor (bFGF) increased the expression of GR in the rat cerebral cortex and cultured cortical neurons and restored the reduced GR expression caused by glucocorticoid exposure. Among intracellular signaling pathways stimulated by bFGF, extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway was responsible for the upregulation of GR. The bFGF-induced GR was functional as a transcription factor to enhance transcription of a target gene. Because high stress augments bFGF levels in the brain, it is likely that bFGF plays a compensating role for reduced GR expression after stress and thus should be studied as a therapeutic target for the treatment of MDD.

Chronic glucocorticoid exposure suppressed the differentiation and survival of embryonic neural stem/progenitor cells: Possible involvement of ERK and PI3K/Akt signaling in the neuronal differentiation.

Growing evidence suggests that excess glucocorticoids (GCs) exposure during the pregnancy results in behavioral abnormality in offspring. Although research using animal models has demonstrated that systemic GCs treatment impairs development of fetal brain, direct impact of GCs on the phenotype of embryonic neural stem/progenitor cells (eNSPCs) and its mechanism has not been fully understood. Here, we investigated the effect of chronic GCs exposure on cell proliferation, differentiation, and survival of eNSPCs in vitro. Corticosterone (CORT, a murine GC) treatment did not affect the proliferation of eNSPCs. On the other hand, decreased expression of neuronal, synaptic, and astroglial marker proteins were observed when the differentiation of eNSPCs was induced in the presence of CORT. CORT also reduced the survival rate of eNSPCs after the differentiation. Moreover, CORT inhibited extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/Akt (PI3K/Akt) signaling pathways, which were activated during cell differentiation of eNSPCs. Inhibiting these signaling pathways reduced neural differentiation and eNSPCs viability, indicating their essential roles in the eNSPCs differentiation. Furthermore, IGF-I, a potent PI3K/Akt and ERK signaling stimulator, partially restored the adverse effect of CORT on eNSPCs, suggesting a possible involvement of the repression of these intracellular signaling in the GCs-caused eNSPCs impairment.

The inactivation of extracellular signal-regulated kinase by glucagon-like peptide-1 contributes to neuroprotection against oxidative stress.

Glucagon-like peptide-1 (GLP-1), an insulinotropic peptide secreted from enteroendocrine cells, has been known to have a neuroprotective effect. However, it is not fully understood the intracellular mediator of GLP-1 signaling in neuronal cells. In the present study, we examined the change in intracellular signaling of cortical neurons after GLP-1 application and luminal glucose stimulation in vitro and in vivo. GLP-1 receptor was highly expressed in cultured cortical neurons and brain tissues including the prefrontal cortex and hippocampus. The activation of GLP-1 receptor (5min) significantly decreased levels of phosphorylated extracellular signal-regulated kinase (pERK), which is involved in neuronal cell survival and death, in cultured cortical neurons. Oral glucose administration also rapidly reduced pERK levels in the prefrontal cortex, while intraperitoneal glucose injection did not show such an effect. Further, GLP-1 attenuated hydrogen peroxide-induced cell death and hyperactivity of ERK in cultured cortical neurons. It is possible that increased GLP-1 by luminal glucose stimulation affects cortical system including the maintenance of neuronal cell survival.

Self-amplified BDNF transcription is a regulatory system for synaptic maturation in cultured cortical neurons.

Neuronal cell survival and synaptic plasticity are controlled through expression of various neurotrophic factors including brain-derived neurotrophic factor (BDNF). In the present study, we examined the mechanism behind BDNF-induced Bdnf mRNA production and the physiological role of its amplification system using cortical neurons. Exogenous BDNF was applied to the cultured cortical neurons at days in vitro (DIV) 3 and DIV 7 with or without inhibitors for intracellular signaling. Expression levels of total Bdnf and Bdnf variants (exon I, exon IV, and exon VI) were biphasically increased after the BDNF application in different developing stage of neurons. Inhibitor for extracellular signal-regulated kinase, calmodulin dependent protein kinase II, or protein kinase A repressed the BDNF-induced Bdnf mRNA expression. Furthermore, we found that application of TrkB-Fc, which scavenges produced endogenous BDNF, resulted in weakened BDNF/TrkB signaling and decreased expression of postsynaptic proteins, suggesting that newly synthesized BDNF induced by the self-amplification system contributes to the synaptic maturation and function.

Possible role of the dopamine D1 receptor in the sensorimotor gating deficits induced by high-fat diet.

RATIONALE: High-fat diet (HFD) has been recently reported to induce sensorimotor gating deficits, but the underlying mechanisms are not well understood. OBJECTIVE: The purpose of this study is to determine whether HFD induces long-lasting deficits in sensorimotor gating and to examine the involvement of altered dopamine (DA) function. METHODS: C57BL/6J mice were fed HFD for 10 weeks and then normal diet (ND) for 4 weeks. DA D2 receptor (D2R) knockout (KO) mice were also fed HFD for 10 weeks. The mice were evaluated for prepulse inhibition (PPI) of acoustic startle after HFD and the subsequent 4-week ND. We evaluated the effect of SCH23390, a D1 receptor (D1R) antagonist, on PPI and measured protein expression levels of D1R and D2R in the prefrontal cortex (PFC) in HFD mice. The concentrations of monoamines and their metabolites in the cortices of 10-week HFD or ND mice were measured using high performance liquid chromatography. RESULTS: Long-term HFD-induced PPI disruption in WT and D2R KO mice. Even after 4 weeks of subsequent ND, PPI remained to be disrupted. SCH23390 mitigated the PPI disruption. In HFD animals, D1R protein expression in the PFC was significantly decreased, while DA, homovanillic acid, and 3,4-dihydroxyphenylacetic acid levels in the cortex were increased. CONCLUSION: This is the first evidence that HFD can induce long-lasting deficits in sensorimotor gating through alteration of cortical levels of DA and its metabolites. Our data suggest that HFD-induced PPI deficits are related to altered D1R signaling and that D1R antagonists may have therapeutic effects on the deficits.

Glucocorticoid affects dendritic transport of BDNF-containing vesicles.

Brain-derived neurotrophic factor (BDNF) is essential for neuronal survival, differentiation, and functions in the central nervous system (CNS). Because BDNF protein is sorted into secretory vesicles at the trans-Golgi network in the cell body after translation, transport of BDNF-containing vesicles to the secretion sites is an important process for its function. Here we examined the effect of dexamethasone (DEX), a synthetic glucocorticoid, on BDNF-containing vesicle transport and found that DEX decreased the proportion of stationary vesicles and increased velocity of the microtubule-based vesicle transport in dendrites of cortical neurons. Furthermore, DEX increased huntingtin (Htt) protein levels via glucocorticoid receptor (GR) activation, and reduction in the amount of Htt by a specific shRNA reversed the action of DEX on BDNF vesicle transport. Given that Htt protein is a positive regulator for the microtubule-dependent vesicular transport in neurons, our data suggest that glucocorticoid stimulates BDNF vesicle transport through upregulation of Htt protein levels.

IL-1 receptor-antagonist (IL-1Ra) knockout mice show anxiety-like behavior by aging.

Interleukin 1 (IL-1) plays a critical role in stress responses, and its mRNA is induced in the brain by restraint stress. Previously, we reported that IL-1 receptor antagonist (IL-1Ra) knockout (KO) mice, which lacked IL-1Ra molecules that antagonize the IL-1 receptor, showed anti-depression-like behavior via adrenergic modulation at the age of 8 weeks. Here, we report that IL-1Ra KO mice display an anxiety-like phenotype that is induced spontaneously by aging in the elevated plus-maze (EPM) test. This anxiety-like phenotype was improved by the administration of diazepam. The expression of the anxiety-related molecule glucocorticoid receptor (GR) was significantly reduced in 20-week-old but not in 11-week-old IL-1Ra KO mice compared to wild-type (WT) littermates. The expression of the mineralocorticoid receptor (MR) was not altered between IL-1Ra KO mice and WT littermates at either 11 or 20 weeks old. Analysis of monoamine concentration in the hippocampus revealed that tryptophan, the serotonin metabolite 5-hydroxyindole acetic acid (5-HIAA), and the dopamine metabolite homovanillic acid (HVA) were significantly increased in 20-week-old IL-1Ra KO mice compared to littermate WT mice. These findings strongly suggest that the anxiety-like behavior observed in older mice was caused by the complicated alteration of monoamine metabolism and/or GR expression in the hippocampus.

Basic fibroblast growth factor induces miR-134 upregulation in astrocyte for cell maturation.

Evidence suggests that neuronal microRNAs (miRs) contribute to synaptic plasticity, although a role of glial miRs have been unknown. Growth factors including brain-derived neurotrophic factor (BDNF) regulate neuronal functions via upregulation of miRs, while possible influences on expression/function of glial miRs have not been fully understood. Here, we report that basic fibroblast growth factor (bFGF) increased miR-134 expression in astrocyte. The miR-134 was upregulated through stimulating extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling, because inhibitors for each signaling blocked the miR-134 induction by bFGF. We also found upregulation of glial fibrillary acidic protein (astrocyte marker) and decreased extracellular concentration of glutamate after miR-134 overexpression and bFGF application, suggesting that astroglial cell maturation is enhanced by bFGF through induction of miR-134.

Cabergoline, dopamine D2 receptor agonist, prevents neuronal cell death under oxidative stress via reducing excitotoxicity.

Several lines of evidence demonstrate that oxidative stress is involved in the pathogenesis of neurodegenerative diseases, including Parkinson's disease. Potent antioxidants may therefore be effective in the treatment of such diseases. Cabergoline, a dopamine D2 receptor agonist and antiparkinson drug, has been studied using several cell types including mesencephalic neurons, and is recognized as a potent radical scavenger. Here, we examined whether cabergoline exerts neuroprotective effects against oxidative stress through a receptor-mediated mechanism in cultured cortical neurons. We found that neuronal death induced by H2O2 exposure was inhibited by pretreatment with cabergoline, while this protective effect was eliminated in the presence of a dopamine D2 receptor inhibitor, spiperone. Activation of ERK1/2 by H2O2 was suppressed by cabergoline, and an ERK signaling pathway inhibitor, U0126, similarly protected cortical neurons from cell death. This suggested the ERK signaling pathway has a critical role in cabergoline-mediated neuroprotection. Furthermore, increased extracellular levels of glutamate induced by H2O2, which might contribute to ERK activation, were reduced by cabergoline, while inhibitors for NMDA receptor or L-type Ca²âº channel demonstrated a survival effect against H2O2. Interestingly, we found that cabergoline increased expression levels of glutamate transporters such as EAAC1. Taken together, these results suggest that cabergoline has a protective effect on cortical neurons via a receptor-mediated mechanism including repression of ERK1/2 activation and extracellular glutamate accumulation induced by H2O2.

Impairments in brain-derived neurotrophic factor-induced glutamate release in cultured cortical neurons derived from rats with intrauterine growth retardation: possible involvement of suppression of TrkB/phospholipase C-γ activation.

Low birth weight due to intrauterine growth retardation (IUGR) is suggested to be a risk factor for various psychiatric disorders such as schizophrenia. It has been reported that developmental cortical dysfunction and neurocognitive deficits are observed in individuals with IUGR, however, the underlying molecular mechanisms have yet to be elucidated. Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are associated with schizophrenia and play a role in cortical development. We previously demonstrated that BDNF induced glutamate release through activation of the TrkB/phospholipase C-γ (PLC-γ) pathway in developing cultured cortical neurons, and that, using a rat model for IUGR caused by maternal administration of thromboxane A2, cortical levels of TrkB were significantly reduced in IUGR rats at birth. These studies prompted us to hypothesize that TrkB reduction in IUGR cortex led to impairment of BDNF-dependent glutamatergic neurotransmission. In the present study, we found that BDNF-induced glutamate release was strongly impaired in cultured IUGR cortical neurons where TrkB reduction was maintained. Impairment of BDNF-induced glutamate release in IUGR neurons was ameliorated by transfection of human TrkB (hTrkB). Although BDNF-stimulated phosphorylation of TrkB and of PLC-γ was decreased in IUGR neurons, the hTrkB transfection recovered the deficits in their phosphorylation. These results suggest that TrkB reduction causes impairment of BDNF-stimulated glutamatergic function via suppression of TrkB/PLC-γ activation in IUGR cortical neurons. Our findings provide molecular insights into how IUGR links to downregulation of BDNF function in the cortex, which might be involved in the development of IUGR-related diseases such as schizophrenia.

Phencyclidine rapidly decreases neuronal mRNA of brain-derived neurotrophic factor.

Downregulation of brain-derived neurotrophic factor (BDNF), a member of neurotrophin family, has been implicated in psychiatric diseases including schizophrenia. However, detailed mechanisms of its reduction in patients with schizophrenia remain unclear. Here, using cultured cortical neurons, we monitored BDNF mRNA levels following acute application of phencyclidine [PCP; an N-methyl-d-aspartate (NMDA) receptor blocker], which is known to produce schizophrenia-like symptoms. We found that PCP rapidly caused a reduction in total amount of BDNF transcripts without effect on cell viability, while mRNA levels of nerve growth factor was intact. Actinomycin-D (ActD), an RNA synthesis inhibitor, decreased total BDNF mRNA levels similar to PCP, and coapplication of ActD with PCP did not show further reduction in BDNF mRNA compared with solo application of each drug. Among BDNF exons I, IV, and VI, the exon IV, which is positively regulated by neuronal activity, was highly sensitive to PCP. Furthermore, PCP inactivated cAMP response element-binding protein (CREB; a regulator of transcriptional activity of exon IV). The inactivation of CREB was also achieved by an inhibitor for Ca(2+) /calmodulin kinase II (CaMKII), although coapplication with PCP induced no further inhibition on the CREB activity. It is possible that PCP decreases BDNF transcription via blocking the NMDA receptor/CaMKII/CREB signaling.

Phosphatase-mediated intracellular signaling contributes to neuroprotection by flavonoids of Iris tenuifolia.

A variety of flavonoids are suggested to be useful for the treatment of brain-related disorders, including dementia and depression. An investigation on the characteristics of the extracted compounds of Iris tenuifolia Pall. (IT) is of much interest, as this plant has been used as a traditional medicine. In the present study, we examined the effect of total flavonoids obtained from IT on cultured cortical neurons under oxidative-stress and found that pretreatment with IT flavonoids significantly inhibited H 2 O 2-induced cell death in cortical neurons. Such a survival-promoting effect by IT flavonoids was partially blocked by inhibitors for extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase/Akt (PI3K/Akt) cascades, both of which are known as survival-promoting signaling molecules. Furthermore, the phosphorylation of Src homology-2 (SH2) domain-containing phosphatase2 (Shp2) was induced by IT flavonoids, and the protective effect of IT flavonoids was abolished by NSC87877, an inhibitor for Shp2, suggesting the involvement of Shp2-mediated intracellular signaling in flavonoid-dependent neuroprotection.

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.