Nobuyuki Takei

Brain-derived neurotrophic factor increases the stimulation-evoked release of glutamate and the levels of exocytosis-associated proteins in cultured cortical neurons from embryonic rats

Abstract: Differentiation and survival of neurons induced by neurotrophins have been widely investigated, but little has been reported about the long‐term effect of brain‐derived neurotrophic factor (BDNF) on synaptic transmission. Among many steps of neurotransmission, one important step is regulated release of transmitters. Therefore, the release of glutamate and GABA from cortical neurons cultured for several days with or without BDNF was measured by an HPLC‐fluorescence method. Although BDNF had little effect on the basal release of glutamate, high K+‐evoked release was greatly increased by BDNF. BDNF also tended to increase evoked release of GABA. Recently, several proteins involved in the step of “regulated release” have been identified. Thus, the effect of BDNF on the levels of these proteins was then investigated. Neurons were cultivated with or without BDNF, collected, and electrophoresed for western blotting. BDNF increased levels of synaptotagmin, synaptobrevin, synaptophysin, and rab3A, which were known as vesicle protein. Levels of syntaxin, SNAP‐25, and β‐SNAP were also increased by BDNF. In addition, the numbers of cored and clear vesicles in nerve terminals or varicosities were also increased by BDNF. These results raise the possibility that BDNF increases regulated release of neurotransmitters through the up‐regulation of secretory mechanisms.

Ca2+ ionophore-induced apoptosis on cultured embryonic rat cortical neurons

The neurotoxicity of ionomycin, a Ca2+ ionophore, was investigated in cultured cortical neurons from embryonic rats. While about 90% of neurons survived 2 h after exposure to ionomycin, the surviving neurons had decreased by about 30 to 40% at 16 h. Both RNA and protein synthesis inhibitors blocked this neurotoxicity. Furthermore, c-Fos immunoreactive neurons increased not only in number but also in the intensity of immunoreactivity. These results suggest that ionomycin-induced neuronal cell death is an active process which requires de novo transcription and translation. In addition, the ultrastructural changes, such as shrinkage of cell body, compaction of nucleus, condensation of chromatin, and membrane blebbing, were observed by electron microscopy. These morphological changes are indexes of apoptosis. Furthermore, DNA fragmentation, a biochemical marker of apoptosis, was also observed. All the results suggest that ionomycin-induced neuronal cell death is apoptotic.

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 INDUCES RAPID AND TRANSIENT RELEASE OF GLUTAMATE THROUGH THE NON-EXOCYTOTIC PATHWAY FROM CORTICAL NEURONS

There is increasing interest in the involvement of neurotrophins in neural transmission and plasticity. Thus, we investigated the effects of brain-derived neurotrophic factor (BDNF) on glutamate release from cortical neurons. Treatment of cultured cortical neurons with BDNF induced rapid and transient release of glutamate. This effect was suggested to be mediated by TrkB activation because K252a inhibited the release of glutamate and BDNF phosphorylated TrkB within 30 s. BDNF-induced glutamate release was observed even when using Ca2+-free assay buffer but was inhibited by BAPTA-AM, a cell-permeable Ca2+ chelator. Therefore, BDNF-induced glutamate release was independent of extracelluar Ca2+ but dependent on intracellular Ca2+. Because normal neurotransmitter release is exocytotic, the involvement of the exocytotic pathway in BDNF-induced glutamate release was examined. As botulinum toxin is known to cleave exocytosis-associated proteins, thereby inhibiting exocytosis, it was applied to neurons prior to the release assay. Although botulinum toxin B cleaved VAMP2 and inhibited Ca2+-triggered glutamate release, it did not inhibit the BDNF-induced release of glutamate. These results strongly suggested that BDNF induces rapid and transient release of glutamate from cortical neurons through a non-exocytotic pathway.

Identification of NAP-22 and GAP-43 (neuromodulin) as major protein components in a Triton insoluble low density fraction of rat brain

NAP-22 is a membrane-localized brain enriched acidic protein having a Ca(2+)-dependent calmodulin binding activity. Further fractionation of the NAP-22 containing membrane showed the localization of NAP-22 in a Triton insoluble fraction of low density. Besides NAP-22, this fraction was found to contain GAP-43 (neuromodulin), trimeric G proteins, and some GPI-anchored proteins such as Thy-1 and N-CAM-120. Presence of some protein tyrosine kinases, such as src and fyn, was also shown.

NGF and BDNF increase the immunoreactivity of vesicular acetylcholine transporter in cultured neurons from the embryonic rat septum.

The expression of vesicular acetylcholine transporter (VAChT), which transports ACh into synaptic vesicles, is coregulated with choline acetyltransferase (ChAT). Therefore, the effects of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) on the levels of VAChT in cultured neurons from the septum of embryonic rats were investigated by immunocytochemistry. NGF and BDNF increased the number of VAChT-immunoreactive neurons by approximately 1.5-fold and enhanced the immunoreactivity in each positive cell. These results suggest that the neurotrophins enhance not only synthesis but also storage of ACh in septal neurons.

BDNF increases the expression of neuropeptide Y mRNA and promotes differentiation/maturation of neuropeptide Y-positive cultured cortical neurons from embryonic and postnatal rats.

The effects of neurotrophic factor on the expression of neuropeptide Y (NPY) mRNA and on morphology of NPY-immunoreactive neurons were investigated. Brain-derived neurotrophic factor (BDNF) increased the expression of NPY mRNA in cultured cortical neurons from both embryonic and postnatal rats. BDNF also increased the number of NPY neurons. Furthermore, multipolar neurites from NPY neurons were observed in cultures treated with BDNF, whereas only monopolar and bipolar neurites were observed in control cultures. These results suggest that BDNF not only increases the expression of NPY mRNA but also promotes the differentiation/maturation of NPY ergic neurons both in number and morphology. NPY expression was strongly increased by neurotrophin-4/5 similarly to BDNF and neurotrophin-3 evoked a slight increase. In contrast, basic fibroblast growth factor, cilliary neurotrophic factor and interferon-gamma had no effect on NPY expression.

Basic fibroblast growth factor inhibited Ca2+ ionophore-induced apoptotic cell death of cultured cortical neurons from embryonic rats

The effect of basic fibroblast growth factor (bFGF) on apoptotic cell death of cultured cortical neurons from embryonic rats induced by ionomycin, a potent Ca2+ ionophore, was investigated. bFGF inhibited Ca2+ ionophore-induced neurotoxicity in a dose-dependent manner. bFGF also reduced the degree of fragmentation of DNA of Ca2+ ionophore-treated neurons. These findings strongly suggest that bFGF inhibited ionomycin-induced neurotoxicity by preventing apoptosis.

Varicosity formation and non-synaptic release of large core vesicles in the co-culture of neuronal and smooth muscle cells

In order to clarify the function and kinetics of varicose swellings in the neurites, we examined the co-culture effects of smooth muscle cells on the varicosity formation in cultured neuronal cells (PC-12 and primary cultured superior cervical ganglion cells). The number and frequency of varicosities of PC-12 cells increased significantly when the cells were grown onto confluently cultured smooth muscle cells. The varicosities moved not only anterogradely but also retrogradely. After stimulation by acetylcholine or high K+, non-synaptic release of core vesicles was demonstrated in the varicosities by electron microscopy. These results indicate that the varicose swellings may function as non-synaptic, flexible nerve terminals.

Interleukin-6 as a Neurotrophic Factor for Promoting Survival of Septal Cholinergic Neurons and Mesencephalic Catecholaminergic Neurons from Postnatal Rats

Neuronal differentiation and survival are supported by several kinds of neurotrophic factors in the central nervous system (CNS) as in the peripheral nervous system (PNS). The well-known nerve frowth factor (NGF), target derived growth factor, is synthesized1–4 and acts on cholinergic neurons5–12 in the CNS; however the action of NGF on other neurons is limited. We have been studying such factors that act on neuronal survival by using primary cultures of postnatal rat brain.