Tadahiro Numakawa

Nicotine‐induced phosphorylation of extracellular signal‐regulated protein kinase and CREB in PC12h cells

We have investigated mechanisms of nicotine‐induced phosphorylation of extracellular signal‐regulated protein kinase (p42/44 MAP kinase, ERK) and cAMP response element binding protein (CREB) in PC12h cells. Nicotine transiently induced ERK phosphorylation at more than 1u2003µm. The maximal level of nicotine‐induced ERK phosphorylation was lower than that of the membrane depolarization induced and, to a great extent, the nerve growth factor (NGF)‐induced ERK phosphorylation. Nicotinic acetylcholine receptor (nAChR) α7 subunit‐selective inhibitors had no significant effect on nicotine‐induced ERK phosphorylation. l‐Type voltage‐sensitive calcium channel antagonists inhibited nicotine‐induced ERK phosphorylation. Calcium imaging experiments showed that α7‐containing nAChR subtypes were functional at 1u2003µm of nicotine in the nicotine‐induced calcium influx, and non‐α7 nAChRs were prominent in the Ca2+ influx at 50u2003µm of nicotine. An expression of dominant inhibitory Ras inhibited nicotine‐induced ERK phosphorylation. A calmodulin antagonist, a CaM kinase inhibitor, a MAP kinase kinase inhibitor inhibited nicotine‐induced ERK and CREB phosphorylation. The time course of the phosphorylation of CREB induced by nicotine was similar to that of ERK induced by nicotine. These results suggest that non‐α7 nAChRs are involved in nicotine‐induced ERK phosphorylation through CaM kinase and the Ras‐MAP kinase cascade and most of the nicotine‐induced CREB phosphorylation is mediated by the ERK phosphorylation in PC12h cells.

Biological characterization and optical imaging of brain-derived neurotrophic factor-green fluorescent protein suggest an activity-dependent local release of brain-derived neurotrophic factor in neurites of cultured hippocampal neurons.

To visualize the release dynamics of the brain‐derived neurotrophic factor (BDNF) involved in neural plasticity, we constructed a plasmid encoding green fluorescent protein (GFP) fused with BDNF. First, several biological studies confirmed that this fusion protein (BDNF‐GFP) mimics the biological functions and the release kinetics of unfused (native) BDNF. Second, when BDNF‐GFP was expressed in cultured hippocampal neurons, we observed that this protein formed striking clusters in the neurites of mature neurons and colocalized with the PSD‐95 immunoreactivity. Such a clustered BDNF‐GFP rapidly disappeared in response to depolarization with KCl, as revealed by confocal microscopic studies. These data suggest that BDNF is locally and rapidly released at synaptic sites in an activity‐dependent manner. Optical studies using BDNF‐GFP may provide important evidence regarding the participation of BDNF in synaptic plasticity. J. Neurosci. Res. 64:1–10, 2001.

Involvement of phosphatidylinositol-3 kinase in prevention of low K+-induced apoptosis of cerebellar granule neurons

Cerebellar granule neurons obtained from 9-day-old rats die in an apoptotic manner when cultured in serum-free medium containing a low concentration of potassium (5 mM). A high concentration of potassium (26 mM) in the culture medium and BDNF can effectively prevent this apoptosis. The survival effects of high potassium and BDNF were additive, and the effect of high potassium was not blocked by addition of anti-BDNF antibody. These observations indicated that these survival effects were independent. To examine which molecules are involved in the survival pathway induced by BDNF or high K+, we used wortmannin, a specific inhibitor of PI-3 kinase. Wortmannin blocked the survival effects of both BDNF and high K+ on cerebellar granule neurons. Furthermore, in vitro PI-3 kinase assay showed that treatment with BDNF or high K+ induced PI-3 kinase activity, which was diminished by addition of wortmannin. These results indicate that different survival-promoting agents, BDNF and high K+, can prevent apoptosis in cerebellar granule neurons via a common enzyme, PI-3 kinase.

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 (10u2003min) 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.

Production of reactive oxygen species and release of L-glutamate during superoxide anion-induced cell death of cerebellar granule neurons

Abstract: Enhanced production of superoxide anion (O2−) is considered to play a pivotal role in the pathogenesis of CNS neurons. Here, we report that O2− generated by xanthine (XA) + xanthine oxidase (XO) triggered cell death associated with nuclear condensation and DNA fragmentation in cerebellar granule neuron. XA + XO induced significant increases in amounts of intracellular reactive oxygen species (ROS) before initiating loss of cell viability, as determined by measurement of 6‐carboxy‐2′,7′‐dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C‐DCDHF‐DA) for O2− and other ROS and hydroethidine (HEt) specifically for O2− by using fluorescence microscopy and flow cytometry. Catalase, but not superoxide dismutase (SOD), significantly protected granule neurons from the XA + XO‐induced cell death. Catalase effectively reduced C‐DCDHF‐DA but not HEt fluorescence, whereas SOD reduced HEt but not C‐DCDHF‐DA fluorescence, indicating that HEt and C‐DCDHF‐DA fluorescence correlated with O2− and hydrogen peroxide, respectively. The NMDA antagonist MK‐801 prevented the death. XA + XO induced an increase in l‐glutamate release from cerebellar granule neurons. These results indicate that elevation of O2− induces cell death associated with increasing ROS production in cerebellar granule neurons and that XA + XO enhanced release of l‐glutamate.

BDNF potentiates spontaneous Ca2+ oscillations in cultured hippocampal neurons

Brain-derived neurotrophic factor (BDNF) is thought to regulate neuronal plasticity in developing and matured neurons, although the molecular mechanisms are less well characterized. We monitored changes in the intracellular calcium (Ca2+) levels induced by BDNF using a fluorescence Ca2+ indicator (Fluo-3) by means of confocal laser microscopy in rat cultured hippocampal neurons. BDNF acutely potentiated spontaneous Ca2+ oscillations in dendrites and also in the soma of several neurons, although it increased intracellular Ca2+ in only selective proportion of resting neurons without Ca2+ oscillations. The potentiation was observed both in the frequency and the amplitude of Ca2+ oscillations, completely blocked by K-252a, and significantly reduced by 2-aminophosphonovaleric acid. These findings suggest that BDNF increases glutamate release and N-methyl-D-aspartate (NMDA) channel-gated Ca2+ influx via TrkB and regulates the frequency and the amplitude of Ca2+ oscillations. BDNF may have the potential to modulate spontaneous Ca2+ oscillations to regulate neuronal plasticity in developing hippocampal neurons.

Nicotine-induced phosphorylation of Akt through epidermal growth factor receptor and Src in PC12h cells.

Nicotine treatment triggers calcium influx into neuronal cells, which promotes cell survival in a number of neuronal cells. Phosphoinositide (PI) 3‐kinase and downstream PI3‐kinase target Akt have been reported to be important in the calcium‐mediated promotion of survival in a wide variety of cells. We investigated the mechanisms of nicotine‐induced phosphorylation of Akt in PC12h cells, in comparison with nicotine‐induced ERK phosphorylation. Nicotine induced Akt phosphorylation in a dose‐dependent manner. A nicotinic acetylcholine receptor (nAChR) α7 subunit‐selective inhibitor had no significant effect on nicotine‐induced Akt phosphorylation, while a non‐selective nAChR antagonist inhibited the phosphorylation. L‐type voltage‐sensitive calcium channel (VSCC) antagonists, calmodulin antagonist, and Ca2+/calmudulin‐dependent protein kinase (CaM kinase) inhibitor prevented the nicotine‐induced Akt phosphorylation. Three epidermal growth factor receptor (EGFR) inhibitors prevented the nicotine‐induced phosphorylation of both extracellular signal‐regulated protein kinase (p42/44 MAP kinase, ERK) and Akt. In contrast, an inhibitor of the Src family tyrosine kinase prevented the nicotine‐induced Akt phosphorylation but not ERK phosphorylation. These results suggested that nicotine induces the activation of both PI3‐kinase/Akt and ERK pathways via common pathways including non‐α7‐nAChRs, L‐type VSCC, CaM kinase II and EGFR in PC12h cells, but Src family tyrosine kinases only participate in the pathway to activate Akt.

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.

Neurotrophin-elicited short-term glutamate release from cultured cerebellar granule neurons.

Brain-derived neurotrophic factor (BDNF) has been suggested to play an important role in neuronal plasticity. In this study, we investigated the effects of BDNF on short-term transmitter release from cultured CNS neurons. Rapid and transient glutamate and aspartate releases induced by BDNF were observed from cultured cortical, hippocampal, striatal and cerebellar neurons. We furthermore investigated the mechanism of release induced by neurotrophins from cerebellar granule cells, since granule cells represent a large homogeneous glutamatergic population. NGF and NT-3 elicited neurotrophin-induced release of glutamate as well as BDNF from the cerebellar granule neurons. The release was dependent on intracellular Ca(2+) mobilization. Pretreatment with K252a and also TrkB-IgG completely blocked the glutamate and aspartate release elicited by BDNF, but not by NGF. The cerebellar granule neurons expressed trkB and p75 mRNAs at high levels, but not trkA mRNA. These results suggested that while BDNF induced release via TrkB, NGF-elicited release was not mediated by Trks. Furthermore, in the experiment using the styryl dye FM1-43, which selectively labels synaptic vesicles, neither BDNF nor NGF evoked dye loss, suggesting that neurotrophin-induced excitatory amino acid release occurs through a non-exocytotic pathway.

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.