Valproic acid selectively suppresses the formation of inhibitory synapses in cultured cortical neurons.

Valproic acid (VPA) has been used to treat epileptic patients because of its ability to potentiate GABA signaling in the brain. Despite its clinical significance, VPA administration during pregnancy increases the risk of congenital abnormalities, such as neural tube defects and neurodevelopmental disorders including autism. Furthermore, recent studies revealed that early postnatal administration of VPA also leads to neurodevelopmental deficits in rodents. Here, using cultured cortical neurons derived from postnatal day 1 rats, we examined whether exposure to VPA would affect synapse formation. When neurons were exposed to 1mM VPA during early development, expression of the vesicular GABA transporter (VGAT) was selectively reduced, whereas other synaptic markers, including the vesicular glutamate transporters 1 and 2 (VGLUT1 and 2), were not affected. This VPA effect was mediated through inhibition of histone deacetylases (HDACs), since the effects were mostly recapitulated by an HDAC inhibitor, trichostatin A, but not by a VPA derivative, valpromide, which lacks HDAC inhibitor activity. Immunocytochemical analysis demonstrated that VPA exposure resulted in a retardation of axonal growth specific to GABAergic neurons and a decrease in VGAT-positive synapses. Since disturbance of the excitatory and inhibitory (E-I) balance has been implicated as a potential cause of multiple psychiatric disorders, our results may account for one of the cellular mechanisms underlying the pathogenesis of VPA-induced neurodevelopmental impairments.

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.

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

Increased glucocorticoids (GCs) have been implicated in the pathophysiology of depressive disorder. We previously found that dexamethasone (DEX, a synthetic GC) repressed brain-derived neurotrophic factor (BDNF)-induced synaptic proteins via suppressing extracellular signal-regulated protein kinase (ERK) signaling. Here, we investigated the possible involvement of Src homology-2 domain-containing phosphatase2 (Shp2), an ERK signaling mediator. We found that DEX suppressed Shp2 interaction with TrkB, a receptor for BDNF, in cultured cortical neurons. NSC87877, a Shp2 inhibitor, mimicked DEX, and Shp2 overexpression reversed the effect of DEX, suggesting that GCs suppress ERK signaling through inhibiting the interaction of Shp2 with TrkB.

Cyclophosphamide promotes cell survival via activation of intracellular signaling in cultured cortical neurons.

Cyclophosphamide (CP) has been used as an antitumour agent or immunosuppressant clinically, though the potential biological role of CP in the central nervous system (CNS) has not been clarified. In the present study, we found that pretreatment with CP prevented neuronal cell death caused by serum deprivation in cultured cortical neurons. Interestingly, CP stimulated activation of PI3K (phosphatidylinositol 3 kinase) and MAPK/ERK (mitogen-activated protein kinase/extracellular signal-regulated kinase) pathways, which are known as survival-promoting intracellular signalings. Furthermore, CP increased the expression of Bcl2, an anti-apoptotic factor. In the presence of inhibitors for PI3K or MAPK/ERK pathways, the CP-dependent neuronal survival and Bcl-2 up-regulation were both abolished. Importantly, significant increase in BDNF (brain-derived neurotrophic factor) expression was induced by CP application, implying that BDNF up-regulation is involved in the CP effect. We propose that CP has a protective effect on CNS neurons via the activation of intracellular signalings, and up-regulation of Bcl2 and BDNF.

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.

BDNF function and intracellular signaling in neurons.

Brain-derived neurotrophic factor (BDNF) and its receptor, TrkB, are broadly expressed in the developing and adult mammalian brain. BDNF/TrkB-stimulated intracellular signaling is critical for neuronal survival, morphogenesis, and plasticity. It is well known that binding of BDNF to TrkB elicits various intracellular signaling pathways, including mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK), phospholipase Cg (PLCg), and phosphoinositide 3-kinase (PI3K) pathways, and that BDNF exerts biological effects on neurons via activation of similar mechanisms. In addition to TrkB, a low-affinity receptor p75 is also involved in neuronal survival and plasticity. BDNF affects neurons positively or negatively through various intracellular signaling pathways triggered by activation of TrkB or p75. From a clinical standpoint, roles of BDNF have been implicated in the pathophysiology of various brain diseases. The stress-induced steroid hormone, glucocorticoid, and BDNF are putatively associated with the pathophysiology of depression. Recent reports, including our studies, demonstrate possible crosstalk between glucocorticoid- and BDNF/TrkB-mediated signaling. Here, we present a broad overview of the current knowledge concerning BDNF action and associated intracellular signaling as it relates to neuronal protection, synaptic function, and morphological change. Furthermore, understanding the secretion and intracellular dynamics of BDNF proteins is critical as the fate of secreted BDNF may contribute to differences in neuronal response.

Glucocorticoid receptor interaction with TrkB promotes BDNF-triggered PLC-gamma 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-gamma (phospholipase C-gamma)/Ca(2+) 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-gamma, and BDNF-triggered glutamate release. Interestingly, BDNF-dependent binding of PLC-gamma 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-gamma signaling for glutamate release was recovered by GR overexpression. We propose that TrkB-GR interaction plays a critical role in the BDNF-stimulated PLC-gamma 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.

Glucocorticoid prevents brain-derived neurotrophic factor-mediated maturation of synaptic function in developing hippocampal neurons through reduction in the activity of mitogen-activated protein kinase.

An increased level of glucocorticoid may be related to the pathophysiology of depressive disorder. The involvement of brain-derived neurotrophic factor (BDNF) in the antidepressive effect has also been suggested; however, the possible influence of glucocorticoid on the action of BDNF in the developing central nervous system has not been elucidated. In this study, we investigated the effect of glucocorticoid (dexamethasone, DEX) on synaptic maturation and function enhanced by BDNF in early developing hippocampal neurons. In the immature stage, BDNF increased the outgrowth of dendrites and the expression of synaptic proteins including glutamate receptors and presynaptic proteins. Pretreatment with DEX significantly inhibited the BDNF-dependent up-regulation of both dendritic outgrowth and synaptic proteins. In the more mature stage, the BDNF-reinforced postsynaptic Ca(2+) influx was decreased by DEX. BDNF-enhanced presynaptic glutamate release was also suppressed. RU486, a glucocorticoid receptor antagonist, canceled the DEX-dependent blocking effect on the action of BDNF. After down-regulation of glucocorticoid receptor by small interfering RNA application, no inhibitory effect of DEX on the BDNF-increased synaptic proteins was observed. Interestingly, the BDNF-activated MAPK/ERK pathway, which is an essential intracellular signaling pathway for the BDNF-increased synaptic proteins, was reduced by DEX. These results suggest that BDNF-mediated synaptic maturation is disturbed after neurons are exposed to high-level glucocorticoid in their development stage.

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.

Impact of the DISC1 Ser704Cys polymorphism on risk for major depression, brain morphology and ERK signaling.

Disrupted-in-schizophrenia 1 (DISC1), identified in a pedigree with a familial psychosis with the chromosome translocation (1:11), is a putative susceptibility gene for psychoses such as schizophrenia and bipolar disorder. Although there are a number of patients with major depressive disorder (MDD) in the family members with the chromosome translocation, the possible association with MDD has not yet been studied. We therefore performed an association study of the DISC1 gene with MDD and schizophrenia. We found that Cys704 allele of the Ser704Cys single-nucleotide polymorphism (SNP) was associated with an increased risk of developing MDD (P=0.005, odds ratio=1.46) and stronger evidence for association in a multi-marker haplotype analysis containing this SNP (P=0.002). We also explored possible impact of Ser704Cys on brain morphology in healthy volunteers using MR imaging. We found a reduction in gray matter volume in cingulate cortex and a decreased fractional anisotropy in prefrontal white matter of individuals carrying the Cys704 allele compared with Ser/Ser704 subjects. In primary neuronal culture, knockdown of endogenous DISC1 protein by small interfering RNA resulted in the suppression of phosphorylation of ERK and Akt, whose signaling pathways are implicated in MDD. When effects of sDISC1 (Ser704) and cDISC1 (Cys704) proteins were examined separately, phosphorylation of ERK was greater in sDISC1 compared with cDISC1. A possible biological mechanism of MDD might be implicated by these convergent data that Cys704 DISC1 is associated with the lower biological activity on ERK signaling, reduced brain gray matter volume and an increased risk for MDD.

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 alphaT (alpha-tocopherol), alphaT3 (alpha -tocotrienol), gammaT, and gammaT3 for 24 h prevented the cultured cortical neurons from cell death in oxidative stress stimulated by H2O2, while Trolox, a cell-permeable analog of alphaT, did not. The preventive effect of alphaT was dependent on de novo protein synthesis. Furthermore, we found that alphaT exposure induced the activation of both the MAP kinase (MAPK) and PI3 kinase (PI3K) pathways and that the alphaT-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 alphaT application. The up-regulation of Bcl-2 did not occur in the presence of U0126 or LY294002, suggesting that alphaT-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.

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-gamma (PLC-gamma)/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-gamma)/IP3/Ca2+ pathway. Furthermore, because the PLC-gamma/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-gamma 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-gamma 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-gamma activation and glutamate release. In contrast, overexpression of Sig-1R per se, without antidepressant pretreatment, enhances BDNF-induced PLC-gamma activation and glutamate release. These results suggest that antidepressant pretreatment selectively enhance the BDNF signaling on the PLC-gamma/IP3/Ca2+ pathway via Sig-1R, and that Sig-1R plays an important role in BDNF signaling leading to glutamate release.

Characterization of novel Pur alpha-binding proteins in mouse brain.

Pur alpha is an abundant protein in the brain and binds to a (GGN)n sequence, PUR element. It has been shown that Pur alpha not only interacts with single stranded DNA and RNA, but also with various proteins. In the present study, we tried to search for Pur alpha-binding proteins (PurBPs) in mouse brain by the overlay assay with GST-Pur alpha as a ligand. Three PurBPs of 35, 38 and 40 kDa were found mostly in the nuclear extract (N.Ext.) and they were not detected by the pretreatment of N.Ext. with trypsin, but not with RNase or DNase. The three PurBPs disappeared by the addition of ssCRE (single stranded cAMP response element) containing a PUR element, but not by DeltaGGN ssCRE (deletion of the PUR element from the ssCRE). The PurBPs were abundantly expressed in the brain as Pur alpha. We also determined a region in Pur alpha which is required for the association with the PurBPs by using deletion mutants of Pur alpha. These biochemical properties of the PurBPs are different from the reported nuclear Pur alpha-binding proteins such as Sp1 and pRb.

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.

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.