Emi Kumamaru

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.

Glucocorticoid receptor interaction with TrkB promotes BDNF-triggered PLC-γ signaling for glutamate release via a glutamate transporter

An increase in glucocorticoid levels and down-regulation of BDNF (brain-derived neurotrophic factor) are supposed to be involved in the pathophysiology of depressive disorders. However, possible crosstalk between glucocorticoid- and BDNF-mediated neuronal functions in the CNS has not been elucidated. Here, we examined whether chronic glucocorticoid exposure influences BDNF-triggered intracellular signaling for glutamate release via a glutamate transporter. We found that chronic exposure to dexamethasone (DEX, a synthetic glucocorticoid) suppressed BDNF-induced glutamate release via weakening the activation of the PLC-γ (phospholipase C-γ)/Ca2+ system in cultured cortical neurons. We demonstrated that the GR (glucocorticoid receptor) interacts with receptor tyrosine kinase for BDNF (TrkB). Following DEX treatment, TrkB-GR interaction was reduced due to the decline in GR expression. Corticosterone, a natural glucocorticoid, also reduced TrkB-GR interaction, BDNF-stimulated PLC-γ, and BDNF-triggered glutamate release. Interestingly, BDNF-dependent binding of PLC-γ to TrkB was diminished by DEX. SiRNA transfection to induce a decrease in endogenous GR mimicked the inhibitory action of DEX. Conversely, DEX-inhibited BDNF-activated PLC-γ signaling for glutamate release was recovered by GR overexpression. We propose that TrkB-GR interaction plays a critical role in the BDNF-stimulated PLC-γ pathway, which is required for glutamate release, and the decrease in TrkB-GR interaction caused by chronic exposure to glucocorticoids results in the suppression of BDNF-mediated neurotransmitter release via a glutamate transporter.

Chronic Antidepressants Potentiate via Sigma-1 Receptors the Brain-derived Neurotrophic Factor-induced Signaling for Glutamate Release

Up-regulation of BDNF (brain-derived neurotrophic factor) has been suggested to contribute to the action of antidepressants. However, it is unclear whether chronic treatment with antidepressants may influence acute BDNF signaling in central nervous system neurons. Because BDNF has been shown by us to reinforce excitatory glutamatergic transmission in cultured cortical neurons via the phospholipase-γ (PLC-γ)/inositol 1,4,5-trisphosphate (IP3)/Ca2+ pathway (Numakawa, T., Yamagishi, S., Adachi, N., Matsumoto, T., Yokomaku, D., Yamada, M., and Hatanaka, H. (2002) J. Biol. Chem. 277, 6520-6529), we examined in this study the possible effects of pretreatment with antidepressants on the BDNF signaling through the PLC-γ)/IP3/Ca2+ pathway. Furthermore, because the PLC-γ/IP3/Ca2+ pathway is regulated by sigma-1 receptors (Hayashi, T., and Su, T. P. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 491-496), we examined whether the BDNF signaling is modulated by sigma-1 receptors (Sig-1R). We found that the BDNF-stimulated PLC-γ activation and the ensued increase in intracellular Ca2+ ([Ca2+]i) were potentiated by pretreatment with imipramine or fluvoxamine, so was the BDNF-induced glutamate release. Furthermore, enhancement of the interaction between PLC-γ and TrkB (receptor for BDNF) after imipramine pretreatment was observed. Interestingly, BD1047, a potent Sig-1R antagonist, blocked the imipramine-dependent potentiation on the BDNF-induced PLC-γ activation and glutamate release. In contrast, overexpression of Sig-1R per se, without antidepressant pretreatment, enhances BDNF-induced PLC-γ activation and glutamate release. These results suggest that antidepressant pretreatment selectively enhance the BDNF signaling on the PLC-γ/IP3/Ca2+ pathway via Sig-1R, and that Sig-1R plays an important role in BDNF signaling leading to glutamate release.

Vitamin E protected cultured cortical neurons from oxidative stress-induced cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase

The role of vitamin E in the CNS has not been fully elucidated. In the present study, we found that pre‐treatment with vitamin E analogs including αT (α‐tocopherol), αT3 (α ‐tocotrienol), γT, and γT3 for 24 h prevented the cultured cortical neurons from cell death in oxidative stress stimulated by H2O2, while Trolox, a cell‐permeable analog of αT, did not. The preventive effect of αT was dependent on de novo protein synthesis. Furthermore, we found that αT exposure induced the activation of both the MAP kinase (MAPK) and PI3 kinase (PI3K) pathways and that the αT‐dependent survival effect was blocked by the inhibitors, U0126 (an MAPK pathway inhibitor) or LY294002 (a PI3K pathway inhibitor). Interestingly, the up‐regulation of Bcl‐2 (survival promoting molecule) was induced by αT application. The up‐regulation of Bcl‐2 did not occur in the presence of U0126 or LY294002, suggesting that αT‐up‐regulated Bcl‐2 is mediated by these kinase pathways. These observations suggest that vitamin E analogs play an essential role in neuronal maintenance and survival in the CNS.

Glucocorticoid suppresses BDNF-stimulated MAPK/ERK pathway via inhibiting interaction of Shp2 with TrkB

Grb2 physically interacts with Shp2 by anti bait coimmunoprecipitation (View interaction)

Arc interacts with microtubules/microtubule-associated protein 2 and attenuates microtubule-associated protein 2 immunoreactivity in the dendrites

Arc, activity‐regulated cytoskeleton‐associated gene, is an immediate early gene, and its expression is regulated by a variety of stimuli, such as electric stimulation and methamphetamine. The function of Arc, however, is unknown. To explore this function, we carried out expression experiments by transfecting green fluorescent protein (GFP)‐Arc constructs or by using a protein transduction system in hippocampal cultured neurons. We found that the overexpression of Arc as well as Arc induction by seizure in vivo decreased microtubule‐associated protein 2 (MAP2) staining in the dendrites by immunocytochemistry, although MAP2 content was not changed on Western blot. Furthermore, Arc interacted with newly polymerized microtubules and MAP2, leading to blocking of the epitope of MAP2. The data suggest that Arc increased by synaptic activities would trigger dendritic remodeling by interacting with cytoskeletal proteins.

SA4503, a sigma-1 receptor agonist, prevents cultured cortical neurons from oxidative stress-induced cell death via suppression of MAPK pathway activation and glutamate receptor expression

Many studies suggest that antidepressants act as neuroprotective agents in the central nervous system (CNS), though the underlying mechanism has not been fully elucidated. In the present study, we examined the effect of SA4503, which is a sigma-1 receptor agonist and a novel antidepressant candidate, on oxidative stress-induced cell death in cultured cortical neurons. Exposure of the neurons to H(2)O(2) induced cell death, while pretreatment with SA4503 inhibited neuronal cell death. The SA4503-dependent survival effect was reversed by co-application with BD1047 (an antagonist of sigma-1/2 receptors). Previously we found that H(2)O(2) triggers a series of events including over-activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and intracellular Ca(2+) accumulation via voltage-gated Ca(2+) channels and ionotropic glutamate receptors, resulting in neuronal cell death (Numakawa et al. (2007) [20]). Importantly, we found in this study that SA4503 reduced the activation of the MAPK/ERK pathway and down-regulated the ionotropic glutamate receptor, GluR1. Taking these findings together, it is possible that SA4503 blocks neuronal cell death via repressing activation of the MAPK/ERK pathway and, consequently, expression levels of glutamate receptors.

Phencyclidine-Induced Decrease of Synaptic Connectivity via Inhibition of BDNF Secretion in Cultured Cortical Neurons

Repeated administration of phencyclidine (PCP), a noncompetitive N-methyl-D-aspartate (NMDA) receptor blocker, produces schizophrenia-like behaviors in humans and rodents. Although impairment of synaptic function has been implicated in the effect of PCP, the molecular mechanisms have not yet been elucidated. Considering that brain-derived neurotrophic factor (BDNF) plays an important role in synaptic plasticity, we examined whether exposure to PCP leads to impaired BDNF function in cultured cortical neurons. We found that PCP caused a transient increase in the level of intracellular BDNF within 3 h. Despite the increased intracellular amount of BDNF, activation of Trk receptors and downstream signaling cascades, including MAPK/ERK1/2 and PI3K/Akt pathways, were decreased. The number of synaptic sites and expression of synaptic proteins were decreased 48 h after PCP application without any impact on cell viability. Both electrophysiological and biochemical analyses revealed that PCP diminished glutamatergic neurotransmission. Furthermore, we found that the secretion of BDNF from cortical neurons was suppressed by PCP. We also confirmed that PCP-caused downregulation of Trk signalings and synaptic proteins were restored by exogenous BDNF application. It is possible that impaired secretion of BDNF and subsequent decreases in Trk signaling are responsible for the loss of synaptic connections caused by PCP.

MCI-186 prevents brain tissue from neuronal damage in cerebral infarction through the activation of intracellular signaling

The mechanism by which MCI‐186 (3‐methyl‐1‐phenyl‐2‐prazolin‐5‐one) exerts protective effects during cerebral infarction, other than its function as a radical scavenger, has not been fully elucidated. Here, we found that MCI‐186 stimulates intracellular survival signaling in vivo and in vitro. In a rat infarction model, the infarct area was significantly smaller and the degree of edema was reduced in MCI‐186‐treated animals. In the MCI‐186‐treated rats, the number of single stranded (ss) DNA‐positive damaged cells in the peri‐infarct area was decreased compared with the control, suggesting that MCI‐186 protects cerebral tissues from cell damage. To clarify the mechanisms underlying the effect of MCI‐186, we also examined the survival‐promoting effect of this agent on cultured cortical neurons. In this in vitro system, MCI‐186 blocked serum‐free induced neuronal cell death. Interestingly, an increase in the activation of both Akt (a component of the PI3 kinase pathway) and ERK (a component of the MAP kinase pathway) was observed in the cortical cultures after MCI‐186 exposure. Furthermore, the MCI‐186‐dependent survival effect in vitro was blocked by U0126, an MEK (an upstream of ERK) inhibitor, and also by LY294002, a PI3 kinase inhibitor. We also observed similar increases in the activation of Akt and ERK in the in vivo model, further suggesting that the antiapoptotic role of MCI‐186 is mediated via the PI3 kinase and MAP kinase signaling pathways. We therefore conclude that, in addition to its role as a free radical scavenger, MCI‐186 functions as an antiapoptotic factor by enhancing intracellular survival signaling.

Reticulon3 expression in rat optic and olfactory systems

Reticulon3 (RTN3), which belongs to a reticulon family, is first isolated from the retina, but little is known about its function. We investigated the distribution of RTN3 in rat retina and olfactory bulb by immunohistochemistry. In the retina, Müller cells highly expressed RTN3. The expression level of RTN3 in the optic nerve was high in the embryo, but low in the adult. In the olfactory system, RTN3 was highly expressed in the olfactory nerve both in developmental and adult stages. Further, RTN3 was co-localized with synaptophysin in tubulovesicular structures in the developing axon of cultured cortical neurons. These results suggest that RTN3 may play an important role in the developing axons and also in some glial cells such as Müller cells.