Small noncoding vault RNA modulates synapse formation by amplifying MAPK signaling.

The small noncoding vault RNA (vtRNA) is a component of the vault complex, a ribonucleoprotein complex found in most eukaryotes. Emerging evidence suggests that vtRNAs may be involved in the regulation of a variety of cellular functions when unassociated with the vault complex. Here, we demonstrate a novel role for vtRNA in synaptogenesis. Using an in vitro synapse formation model, we show that murine vtRNA (mvtRNA) promotes synapse formation by modulating the MAPK signaling pathway. mvtRNA is transported to the distal region of neurites as part of the vault complex. Interestingly, mvtRNA is released from the vault complex in the neurite by a mitotic kinase Aurora-A-dependent phosphorylation of MVP, a major protein component of the vault complex. mvtRNA binds to and activates MEK1 and thereby enhances MEK1-mediated ERK activation in neurites. These results suggest the existence of a regulatory mechanism of the MAPK signaling pathway by vtRNAs as a new molecular basis for synapse formation.

The Sox2 promoter-driven CD63-GFP transgenic rat model allows tracking of neural stem cell-derived extracellular vesicles.

Extracellular vesicles (EVs) can modulate microenvironments by transferring biomolecules, including RNAs and proteins derived from releasing cells, to target cells. To understand the molecular mechanisms maintaining the neural stem cell (NSC) niche through EVs, a new transgenic (Tg) rat strain that can release human CD63-GFP-expressing EVs from the NSCs was established. Human CD63-GFP expression was controlled under the rat Sox2 promoter (Sox2/human CD63-GFP), and it was expressed in undifferentiated fetal brains. GFP signals were specifically observed in in vitro cultured NSCs obtained from embryonic brains of the Tg rats. We also demonstrated that embryonic NSC (eNSC)-derived EVs were labelled by human CD63-GFP. Furthermore, when we examined the transfer of EVs, eNSC-derived EVs were found to be incorporated into astrocytes and eNSCs, thus implying an EV-mediated communication between different cell types around NSCs. This new Sox2/human CD63-GFP Tg rat strain should provide resources to analyse the cell-to-cell communication via EVs in NSC microenvironments.

Generation of a novel transgenic rat model for tracing extracellular vesicles in body fluids.

Extracellular vesicles (EVs) play an important role in the transfer of biomolecules between cells. To elucidate the intercellular transfer fate of EVs in vivo, we generated a new transgenic (Tg) rat model using green fluorescent protein (GFP)-tagged human CD63. CD63 protein is highly enriched on EV membranes via trafficking into late endosomes and is often used as an EV marker. The new Tg rat line in which human CD63-GFP is under control of the CAG promoter exhibited high expression of GFP in various body tissues. Exogenous human CD63-GFP was detected on EVs isolated from three body fluids of the Tg rats: blood serum, breast milk and amniotic fluid. In vitro culture allowed transfer of serum-derived CD63-GFP EVs into recipient rat embryonic fibroblasts, where the EVs localized in endocytic organelles. These results suggested that this Tg rat model should provide significant information for understanding the intercellular transfer and/or mother-child transfer of EVs in vivo.

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.

The role of brain-derived neurotrophic factor in comorbid depression: possible linkage with steroid hormones, cytokines, and nutrition.

Increasing evidence demonstrates a connection between growth factor function (including brain-derived neurotrophic factor, BDNF), glucocorticoid levels (one of the steroid hormones), and the pathophysiology of depressive disorders. Because both BDNF and glucocorticoids regulate synaptic function in the central nervous system, their functional interaction is of major concern. Interestingly, alterations in levels of estrogen, another steroid hormone, may play a role in depressive-like behavior in postpartum females with fluctuations of BDNF-related molecules in the brain. BDNF and cytokines, which are protein regulators of inflammation, stimulate multiple intracellular signaling cascades involved in neuropsychiatric illness. Pro-inflammatory cytokines may increase vulnerability to depressive symptoms, such as the increased risk observed in patients with cancer and/or autoimmune diseases. In this review, we discuss the possible relationship between inflammation and depression, in addition to the cross-talk among cytokines, BDNF, and steroids. Further, since nutritional status has been shown to affect critical pathways involved in depression through both BDNF function and the monoamine system, we also review current evidence surrounding diet and supplementation (e.g., flavonoids) on BDNF-mediated brain functions.

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.

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.

Teneurin-4 promotes cellular protrusion formation and neurite outgrowth through focal adhesion kinase signaling.

Teneurin-4 (Ten-4), a transmembrane protein, is highly expressed in the central nervous system; however, its cellular and molecular function in neuronal differentiation remains unknown. In this study, we aimed to elucidate the function of Ten-4 in neurite outgrowth. Ten-4 expression was induced during neurite outgrowth of the neuroblastoma cell line Neuro-2a. Ten-4 protein was localized at the neurite growth cones. Knockdown of Ten-4 expression in Neuro-2a cells decreased the formation of the filopodia-like protrusions and the length of individual neurites. Conversely, overexpression of Ten-4 promoted filopodia-like protrusion formation. In addition, knockdown and overexpression of Ten-4 reduced and elevated the activation of focal adhesion kinase (FAK) and Rho-family small GTPases, Cdc42 and Rac1, key molecules for the membranous protrusion formation downstream of FAK, respectively. Inhibition of the activation of FAK and neural Wiskott-Aldrich syndrome protein (N-WASP), which is a downstream regulator of FAK and Cdc42, blocked protrusion formation by Ten-4 overexpression. Further, Ten-4 colocalized with phosphorylated FAK in the filopodia-like protrusion regions. Together, our findings show that Ten-4 is a novel positive regulator of cellular protrusion formation and neurite outgrowth through the FAK signaling pathway.

Estrogen, predominantly via estrogen receptor α, attenuates postpartum-induced anxiety- and depression-like behaviors in female rats.

Contributions from estrogen receptor (ER) subtypes (ERα and ERß) to postpartum anxiogenic and depressive responses remain unresolved in rats. Using the elevated-plus maze (EPM) and forced swim (FS) tests, we confirmed that primiparous rats exhibited anxiogenic and depressive responses 3 weeks postpartum, improved 5 weeks postpartum (EPM), and recovered at 5 (FS) or 10 weeks postpartum (EPM) compared with diestrus nulliparous females. Immunohistochemistry suggested that these behavioral changes were temporally associated with decreased ERα but not ERß expression in the medial amygdala (MEA). Additionally, ERα expression in the medial preoptic area (MPOA) significantly increased 10 weeks postpartum. Brain-derived neurotrophic factor (BDNF) expression was significantly elevated in the MEA 3 weeks postpartum. BDNF receptor tropomyosin-related kinase expression was significantly elevated in the MEA at 3 and 10 weeks but not at 5 weeks postpartum. The phosphorylation of ERK (pERK)-2 in the MEA, MPOA, and hippocampal CA1 region was significantly elevated 3 and 5 weeks postpartum. The effects of single daily sc injections of the ERα-selective agonist, propyl pyrazoletriol (PPT); ERß-selective agonist, diarylpropionitrile; 17ß-estradiol (E2); and vehicle for 6 days in primiparous rats were assessed. PPT and E2 significantly produced anxiolytic and antidepressant actions in the EPM and FS tests but PPT to a lesser degree than E2 in the EPM test. Diarylpropionitrile affected the EPM test but was not significantly different from vehicle. BDNF expression was significantly increased 3 weeks postpartum by all treatments in the MPOA but not the CA1 and MEA. E2 and PPT treatment significantly increased tropomyosin-related kinase and pERK1/2 expression in the MEA and MPOA and increased pERK1/2 expression in the CA1. The onset of anxiety- and depression-like behaviors in postpartum rats may be partly caused by a complex estrogen-mediated mechanism; nevertheless, changes in the ERα-related system, likely in the MEA, are predominantly involved.

Cortical neurons from intrauterine growth retardation rats exhibit lower response to neurotrophin BDNF.

Intrauterine growth retardation (IUGR) is putatively involved in the pathophysiology of schizophrenia. The animal model of IUGR induced by synthetic thromboxane A2 (TXA2) is useful to clarify the effect of IUGR on pups' brains, however, analysis at the cellular level is still needed. Brain-derived neurotrophic factor (BDNF), which plays a role in neuronal survival and synaptic plasticity in the central nervous system (CNS), may also be associated with schizophrenia. However, the possible relationship between IUGR and BDNF function remains unclear. Here, we examined how IUGR by TXA2 impacts BDNF function by using dissociated cortical neurons. We found that, although BDNF levels in cultured neurons from the cerebral cortex of low birth weight pups with IUGR were unchanged, TrkB (BDNF receptor) was decreased compared with control-rats. BDNF-stimulated MAPK/ERK1/2 and PI3K/Akt pathways, which are downstream intracellular signaling pathways of TrkB, were repressed in IUGR-rat cultures. Expression of glutamate receptors such as GluA1 and GluN2A was also suppressed in IUGR-rat cultures. Furthermore, in IUGR-rat cultures, anti-apoptotic protein Bcl2 was decreased and BDNF failed to prevent neurons from cell death caused by serum-deprivation. Taken together, IUGR resulted in reductions in cell viability and in synaptic function following TrkB down-regulation, which may play a role in schizophrenia-like behaviors.

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.

Functional interactions between steroid hormones and neurotrophin BDNF.

Brain-derived neurotrophic factor (BDNF), a critical neurotrophin, regulates many neuronal aspects including cell differentiation, cell survival, neurotransmission, and synaptic plasticity in the central nervous system (CNS). Though BDNF has two types of receptors, high affinity tropomyosin-related kinase (Trk)B and low affinity p75 receptors, BDNF positively exerts its biological effects on neurons via activation of TrkB and of resultant intracellular signaling cascades including mitogen-activated protein kinase/extracellular signal-regulated protein kinase, phospholipase Cγ, and phosphoinositide 3-kinase pathways. Notably, it is possible that alteration in the expression and/or function of BDNF in the CNS is involved in the pathophysiology of various brain diseases such as stroke, Parkinson's disease, Alzheimer's disease, and mental disorders. On the other hand, glucocorticoids, stress-induced steroid hormones, also putatively contribute to the pathophysiology of depression. Interestingly, in addition to the reduction in BDNF levels due to increased glucocorticoid exposure, current reports demonstrate possible interactions between glucocorticoids and BDNF-mediated neuronal functions. Other steroid hormones, such as estrogen, are involved in not only sexual differentiation in the brain, but also numerous neuronal events including cell survival and synaptic plasticity. Furthermore, it is well known that estrogen plays a role in the pathophysiology of Parkinson's disease, Alzheimer's disease, and mental illness, while serving to regulate BDNF expression and/or function. Here, we present a broad overview of the current knowledge concerning the association between BDNF expression/function and steroid hormones (glucocorticoids and estrogen).

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.

17beta-estradiol protects cortical neurons against oxidative stress-induced cell death through reduction in the activity of mitogen-activated protein kinase and in the accumulation of intracellular calcium.

Although many studies have suggested that estrogen acts as a neuroprotective agent in oxidative stress, the underlying mechanism has not been fully elucidated. In the present study, we examined the effect of 17beta-estradiol (17beta-E2) on H(2)O(2)-induced death signaling in cultured cortical neurons. Exposure of the cortical neurons to H(2)O(2) triggered a series of events, including overactivation of p44/42 MAPK and intracellular Ca(2+) accumulation via voltage-gated Ca(2+) channels and ionotropic glutamate receptors, resulting in apoptotic-like cell death. The MAPK pathway might work as death signaling in our system, because the MAPK pathway inhibitor, U0126, blocked H(2)O(2)-induced MAPK activation, Ca(2+) overload, and cell death. Interestingly, a similar inhibitory effect on H(2)O(2)-triggered MAPK activation, Ca(2+) accumulation, and cell death was observed in cultures incubated with 17beta-E2 for 24 h before exposure to H(2)O(2), suggesting that the protective effect of 17beta-E2 is induced via attenuating overactivation of the MAPK pathway. Furthermore, we found that ionotropic glutamate receptor subunits, including NR2A and GluR2/3, but not NR2B and GluR1, were down-regulated in the 17beta-E2-treated cultures. The down-regulation of these glutamate receptor subunits was also observed after chronic treatment with U0126. Therefore, it is possible that 17beta-E2 down-regulates the expression of the ionotropic glutamate receptors by reducing activity of the MAPK pathway, which might be important for the protective effect of 17beta-E2 against oxidative stress-induced toxicity.

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.

ERK1/2 are involved in low potassium-induced apoptotic signaling downstream of ASK1-p38 MAPK pathway in cultured cerebellar granule neurons.

We have recently reported that the ASK1-p38 MAPK pathway has an important role in the low potassium (LK)-induced apoptosis of cultured cerebellar granule neurons. In the present study, we observed that ERK1/2 were significantly activated 6 h after a change of medium from HK (high potassium) to LK. In addition, U0126, a specific inhibitor of MEKs, remarkably prevented the apoptosis of cultured cerebellar granule neurons. Then, we examined the mechanism underlying the activation of ERK1/2 in the LK-induced apoptotic pathway. The addition of SB203580, an inhibitor of p38 MAPK, suppressed the increase in the phosphorylation of ERK1/2 after the change to LK medium. Furthermore, we found that the expression of a constitutively active mutant of ASK1, an upstream kinase of p38 MAPK, enhanced the phosphorylation of ERK1/2. These results suggest that ERK1/2 play a crucial role in LK-induced apoptosis of cultured cerebellar granule neurons and that the LK-stimulated activation of ERK1/2 is regulated by the ASK1-p38 MAPK pathway.

Evidence of novel neuronal functions of dysbindin, a susceptibility gene for schizophrenia.

Genetic variation in dysbindin (DTNBP1: dystrobrevin-binding protein 1) has recently been shown to be associated with schizophrenia. The dysbindin gene is located at chromosome 6p22.3, one of the most promising susceptibility loci in schizophrenia linkage studies. We attempted to replicate this association in a Japanese sample of 670 patients with schizophrenia and 588 controls. We found a nominally significant association with schizophrenia for four single nucleotide polymorphisms and stronger evidence for association in a multi-marker haplotype analysis (P = 0.00028). We then explored functions of dysbindin protein in primary cortical neuronal culture. Overexpression of dysbindin induced the expression of two pre-synaptic proteins, SNAP25 and synapsin I, and increased extracellular basal glutamate levels and release of glutamate evoked by high potassium. Conversely, knockdown of endogenous dysbindin protein by small interfering RNA (siRNA) resulted in the reduction of pre-synaptic protein expression and glutamate release, suggesting that dysbindin might influence exocytotic glutamate release via upregulation of the molecules in pre-synaptic machinery. The overexpression of dysbindin increased phosphorylation of Akt protein and protected cortical neurons against neuronal death due to serum deprivation and these effects were blocked by LY294002, a phosphatidylinositol 3-kinase (PI3-kinase) inhibitor. SiRNA-mediated silencing of dysbindin protein diminished Akt phosphorylation and facilitated neuronal death induced by serum deprivation, suggesting that dysbindin promotes neuronal viability through PI3-kinase-Akt signaling. Genetic variants associated with impairments of these functions of dysbindin could play an important role in the pathogenesis of schizophrenia.

Neuronal roles of the integrin-associated protein (IAP/CD47) in developing cortical neurons.

Little is known about the role of the integrin-associated protein (IAP, or CD47) in neuronal development and its function in the central nervous system. We investigated neuronal responses in IAP-overexpressing cortical neurons using a virus-gene transfer system. We found that dendritic outgrowth was significantly enhanced in IAP (form 4)-transfected neurons. Furthermore, synaptic proteins including synaptotagmin, syntaxin, synapsin I, and SNAP25 (25-kDa synaptosomal associated protein) were up-regulated. In accordance with this finding, the release of the excitatory transmitter glutamate and the frequencies of Ca2+ oscillations (glutamate-mediated synaptic transmission) were increased. Interestingly, the overexpression of IAP activated mitogen-activated protein kinase (MAPK), and this activation was required for the IAP-dependent biological effects. After down-regulation of the endogenous IAP by small interfering RNA, MAPK activity, synaptic protein levels, and glutamate release decreased. These observations suggest that the IAP plays important roles in dendritic outgrowth and synaptic transmission in developing cortical neurons through the activation of MAPK.

Nerve growth factor-induced glutamate release is via p75 receptor, ceramide, and Ca(2+) from ryanodine receptor in developing cerebellar neurons.

Very little is known about the contribution of a low affinity neurotrophin receptor, p75, to neurotransmitter release. Here we show that nerve growth factor (NGF) induced a rapid release of glutamate and an increase of Ca2+ in cerebellar neurons through a p75-dependent pathway. The NGF-induced release occurred even in the presence of the Trk inhibitor K252a. The release caused by NGF but not brain-derived neurotrophic factor was enhanced in neurons overexpressing p75. Further, after transfection of p75-small interfering RNA, which down-regulated the endogenous p75 expression, the NGF-induced release was inhibited, suggesting that the NGF-induced glutamate release was through p75. We found that the NGF-increased Ca2+ was derived from the ryanodine-sensitive Ca2+ receptor and that the NGF-increased Ca2+ was essential for the NGF-induced glutamate release. Furthermore, scyphostatin, a sphingomyelinase inhibitor, blocked the NGF-dependent Ca2+ increase and glutamate release, suggesting that a ceramide produced by sphingomyelinase was required for the NGF-stimulated Ca2+ increase and glutamate release. This action of NGF only occurred in developing neurons whereas the brain-derived neurotrophic factor-mediated Ca2+ increase and glutamate release was observed at the mature neuronal stage. Thus, we demonstrate that NGF-mediated neurotransmitter release via the p75-dependent pathway has an important role in developing neurons.