Daisaku Yokomaku

Brain-derived neurotrophic factor-induced potentiation of glutamate and GABA release: different dependency on signaling pathways and neuronal activity.

The mechanisms underlying BDNF-modulated neurotransmitter release remain elusive. Here, we found that 24-h exposure of postnatal cortical neurons to BDNF potentiated depolarization-evoked glutamate and GABA release in a protein synthesis-dependent manner. BDNF-potentiated glutamate release occurred through the PLC-gamma and MAPK pathways. The expression of synapsin I, synaptotagmin, and synaptophysin, but not of syntaxin or SNAP25, increased through the PLC-gamma and MAPK pathways. In contrast, BDNF-up-regulated GABA release and GAD65/67 expression depended on MAPK. Furthermore, neuronal activity was necessary for the up-regulation of glutamate release and synapsin I, synaptotagmin, and synaptophysin expression, but not of GABA or GAD65/67. PLC-gamma inhibitor attenuated BDNF-stimulated long-lasting MAPK activation. As BDNF rapidly potentiates glutamatergic transmission through PLC-gamma (J. Biol. Chem. 277, (2002) 6520-6529), PLC-gamma-mediated neuronal activity might sustain MAPK activation, resulting in BDNF-potentiated glutamate release. In conclusion, BDNF potentiates the excitatory and inhibitory system separately, which may be important for the regulation of synaptic plasticity.

Nerve Growth Factor-induced Glutamate Release Is via p75 Receptor, Ceramide, and Ca2+ 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.

Brain-derived neurotrophic factor enhances depolarization-evoked glutamate release in cultured cortical neurons.

Brain‐derived neurotrophic factor (BDNF) has been reported to play an important role in neuronal plasticity. In this study, we examined the effect of BDNF on an activity‐dependent synaptic function in an acute phase. First, we found that short‐term treatment (10 min) with BDNF enhanced depolarization‐evoked glutamate release in cultured cortical neurons. The enhancement diminished gradually according to the length of BDNF treatment. The BDNF‐enhanced release did not require the synthesis of protein and mRNA. Both tetanus toxin and bafilomycin abolished the depolarization‐evoked glutamate release with or without BDNF, indicating that BDNF acted via an exocytotic pathway. Next, we investigated the effect of BDNF on intracellular Ca2+. BDNF potentiated the increase in intracellular Ca2+ induced by depolarization. The Ca2+ was derived from intracellular stores, because thapsigargin completely inhibited the potentiation. Furthermore, both thapsigargin and xestospongin C inhibited the effect of BDNF. These results suggested that the release of Ca2+ from intracellular stores mediated by the IP3 receptor was involved in the BDNF‐enhanced glutamate release. Last, it was revealed that the enhancement of glutamate release by BDNF was dependent on the TrkB‐PLC‐γ pathway. These results clearly demonstrate that short‐term treatment with BDNF enhances an exocytotic pathway by potentiating the accumulation of intracellular Ca2+ through intracellular stores.

Brain‐derived neurotrophic factor triggers a rapid glutamate release through increase of intracellular Ca2+ and Na+ in cultured cerebellar neurons

We reported previously that BDNF induced glutamate release was dependent on intracellular Ca2+ but not extracellular Ca2+ in cerebellar neurons (Numakawa et al., 1999 ). It was revealed that the release was through a non‐exocytotic pathway (Takei et al., 1998 ; Numakawa et al., 1999 ). In the present study, we monitored the dynamics of intracellular Ca2+ and Na+ in cerebellar neurons, and investigated the possibility of reverse transport of glutamate mediated by BDNF. As reported, BDNF increased the intracellular Ca2+ level. We found that the Ca2+ increase induced by BDNF was completely blocked by xestospongin C, an IP3 receptor antagonist, and U‐73122, a PLC‐γ inhibitor. Xestospongin C and U‐73122 also blocked the BDNF‐dependent glutamate release, suggesting that the BDNF‐induced transient increase of Ca2+ through the activation of the PLC‐γ/ IP3 pathway was essential for the glutamate release. We found that BDNF induced a Na+ influx. This was blocked by treatment with TTX. U‐73122 and xestospongin C blocked the BDNF‐induced Na+ influx, suggesting that the Na+influx required the BDNF‐induced Ca2+ increase. Next, we examined the possibility that a co‐transporter of Na+ and glutamate was involved in the BDNF‐induced glutamate release. BDNF‐induced glutamate release was blocked by L‐trans‐pyrollidine‐2,4‐dicalboxylic acid (t‐PDC), a glutamate transporter inhibitor, whereas neither the 4‐aminopyridine (4AP)‐ nor high potassium (HK+)‐induced release was blocked by t‐PDC. In addition, DL‐threo‐β‐benzyloxyaspartate (DL‐TBOA) also blocked the BDNF‐mediated glutamate release, suggesting that reverse transport of glutamate may be involved. All the results therefore suggest that Na+‐dependent reverse transport contributes to BDNF‐mediated transmitter release through the PLC‐γ/IP3‐mediated Ca2+ signaling. J. Neurosci. Res. 66:96–108, 2001.

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, Parkinsons disease, Alzheimers 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 Parkinsons disease, Alzheimers 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).

Basic fibroblast growth factor evokes a rapid glutamate release through activation of the MAPK pathway in cultured cortical neurons

We examined the possibility that basic fibroblast growth factor (bFGF) is involved in synaptic transmissions. We found that bFGF rapidly induced the release of glutamate and an increase in the intracellular Ca2+ concentration through voltage-dependent Ca2+ channels in cultured cerebral cortical neurons. bFGF also evoked a significant influx of Na+. Tetanustoxin inhibited the bFGF-induced glutamate release, revealing that bFGF triggered exocytosis. The mitogen-activated protein kinase (MAPK) pathway was required for these acute effects of bFGF. We also found that pretreatment with bFGF significantly enhanced high K+-elicited glutamate release also in a MAPK activation-dependent manner. Therefore, we propose that bFGF exerts promoting effects on excitatory neuronal transmission via activation of the MAPK pathway.

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.

Expression of CD47/integrin‐associated protein induces death of cultured cerebral cortical neurons

The death and survival of neuronal cells are regulated by various signaling pathways during development of the brain and in neuronal diseases. Previously, we demonstrated that the neuronal adhesion molecule brain immunoglobulin‐like molecule with tyrosine‐based activation motifs/SHP substrate 1 (BIT/SHPS‐1) is involved in brain‐derived neurotrophic factor (BDNF)‐promoted neuronal cell survival. Here, we report the apoptosis‐inducing effect of CD47/integrin‐associated protein (IAP), the heterophilic binding partner of BIT/SHPS‐1, on neuronal cells. We generated a recombinant adenovirus vector expressing a neuronal form of CD47/IAP, and found that the expression of CD47/IAP by infection with CD47/IAP adenovirus induced the death of cultured cerebral cortical neurons. The numbers of TdT‐mediated biotin–dUTP nick‐end labelling (TUNEL)‐positive neurons and of cells displaying apoptotic nuclei increased by expression of CD47/IAP. Neuronal cell death was prevented by the addition of the broad‐spectrum caspase inhibitor Z‐VAD‐fmk. Furthermore, we observed that co‐expression of CD47/IAP with BIT/SHPS‐1 enhanced neuronal cell death, and that BDNF prevented it. These results suggest that CD47/IAP is involved in a novel pathway which regulates caspase‐dependent apoptosis of cultured cerebral cortical neurons. CD47/IAP‐induced death of cultured cortical neurons may be regulated by the interaction of CD47/IAP with BIT/SHPS‐1 and by BDNF.

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.