Shinichi Koizumi

Differences in survival‐promoting effects and intracellular signaling properties of BDNF and IGF‐1 in cultured cerebral cortical neurons

Brain‐derived neurotrophic factor (BDNF) and insulin‐like growth factor‐1 (IGF‐1) act on various neurons of the CNS as neurotrophic factors promoting neuronal differentiation and survival. We examined the survival‐promoting effects of BDNF and IGF‐1 on serum deprivation‐induced death in cultured cerebral cortical neurons, and compared the intracellular signaling pathways stimulated by BDNF and IGF‐1 in the neurons. We found that the survival‐promoting effect of BDNF was much weaker than that of IGF‐1 in serum deprivation‐induced death of cultured cortical neurons. We found no differences in the levels of phosphatidylinositol 3‐kinase (PtdIns3‐K) activity or Akt (also called PKB) phosphorylation induced by BDNF and IGF‐1 in the cultured cortical neurons, although many reports suggest that PtdIns3‐K and Akt are involved in survival promotion. In addition, phosphorylation signals of mitogen‐activated protein kinase (MAPK) and cAMP responsive element‐binding protein (CREB), which have also been reported to be involved in survival promotion, were stimulated by BDNF much more potently than by IGF‐1. These results show that there may be, as yet unidentified, intracellular signaling pathways other than the PtdIns3‐K‐Akt, MAPK and CREB signaling, to regulate survival promotion. These unidentified signaling pathways may be responsible for the distinct strengths of the survival‐promoting effects of BDNF and IGF‐1.

BDNF prevents NO mediated glutamate cytotoxicity in cultured cortical neurons.

The effects of brain-derived neurotrophic factor (BDNF) on glutamate-induced cytotoxicity were examined using primary cultures of rat cortical neurons. BDNF induced TrkB tyrosine phosphorylation in rat cultured cortical neurons. The cell viability was significantly reduced when cultures were briefly exposed to glutamate and incubated with normal medium for 24 h. Glutamate cytotoxicity was prevented by MK-801, which is a non-competitive blocker of N-methyl-D-aspartate and N(omega)-nitro-L-arginine, which is a blocker of nitric oxide synthetase. Delayed neurotoxicity was also induced by ionomycin, a calcium ionophore, and nitric oxide (NO) donors such as S-nitrosocysteine (SNOC) and 3-morpholinosydnonimine (SIN-1). Incubating cultures with BDNF for 10 min to 24 h protected cortical neurons against glutamate neurotoxicity. The protective effects of BDNF against glutamate cytotoxicity were dependent on both its concentrations and incubation time. BDNF also prevented the ionomycin-, SNOC-, and SIN-1 induced cytotoxicity. These results indicate that BDNF protects cultured cortical neurons from NMDA receptor-mediated glutamate neurotoxicity by reducing cytotoxic action of NO.

Association of the Src Family Tyrosine Kinase Fyn with TrkB

Abstract: Fyn tyrosine kinase, a member of the Src family, was recently reported to be present in neurons and glia cells. We investigated whether Fyn is involved in the Trk‐dependent signal transduction pathways of neurotrophin. The Fyn‐Src homology domain 2 (SH2) was observed to associate in vitro with the intracellular domain of TrkB (ICD‐TrkB). This association was dependent on the autophosphorylation of ICD‐TrkB. The Fyn‐SH2 domains bound to phosphorylated ICD‐TrkB (plCD‐TrkB) with an affinity similar to the binding of phospholipase Cγ (PLCγ)‐SH2 domains to its autophosphorylation site in TrkB. The Src‐SH2 domains showed substantially lower affinity with plCD‐TrkB, suggesting that the association between Fyn‐SH2 and plCD‐TrkB is not due to non‐specific interactions of SH2 domains with phosphorylated tyrosine residues. This is further supported by the observation that Fyn‐SH2 was able to trap phosphorylated TrkB in cell lysate prepared from primary rat cortical neurons stimulated with brain‐derived neurotrophic factor (BDNF). In contrast, endogenous Fyn was coprecipitated with TrkB from cortical neurons without BDNF stimulation. This basal association showed a threefold increase on BDNF stimulation, probably due to the SH2/phosphotyrosine interaction that was observed in the cell‐free system. All these data suggest the involvement of Fyn in the neurotrophin signal transduction pathways downstream of TrkB.

Acetylcholine Triggers l-Glutamate Exocytosis via Nicotinic Receptors and Inhibits Melatonin Synthesis in Rat Pinealocytes

Rat pinealocytes, melatonin-secreting endocrine cells, contain peripheral glutaminergic systems. l-Glutamate is a negative regulator of melatonin synthesis through a metabotropic receptor-mediated inhibitory cAMP cascade. Previously, we reported that depolarization of pinealocytes by externally added KCl and activation of L-type Ca2+ channels resulted in secretion ofl-glutamate by microvesicle exocytosis. What is unknown is how and what kinds of stimuli trigger glutamate exocytosis under physiological conditions. Here, we report that the nicotinic acetylcholine receptor can trigger glutamate exocytosis from cultured rat pinealocytes. Moreover, acetylcholine or nicotine inhibited norepinephrine-dependent serotonin N-acetyltransferase activity, which results in decreased melatonin synthesis. These activities were blocked by (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine, an antagonist of the metabotropic glutamate receptor. These results suggest that cholinergic stimulation initiates the glutaminergic signaling cascade in pineal glands and that parasympathetic neurons innervating the gland exert negative control over melatonin synthesis by way of the glutaminergic systems.

A Novel Class of Herbicides (Specific Inhibitors of Imidazoleglycerol Phosphate Dehydratase)

A new mode of herbicidal action was established by finding specific inhibitors of imidazoleglycerol phosphate dehydratase, an enzyme of histidine (His) biosynthesis. Three triazole phosphonates inhibited the reaction of the enzyme with Ki values of 40 [plus or minus] 6.5, 10 [plus or minus] 1.6, and 8.5 [plus or minus] 1.4 nM, respectively, and were highly cytotoxic to cultured plant cells. This effect was completely reversed by the addition of His, proving that the cytotoxicity was primarily caused by the inhibition of His biosynthesis. These inhibitors showed wide-spectrum, postemergent herbicidal activity at application rates ranging from 0.05 to 2 kg/ha.

Transient Association of the Phosphotyrosine Phosphatase SHP‐2 with TrkA Is Induced by Nerve Growth Factor

Abstract: Nerve growth factor (NGF) treatment of rat PC12 pheochromocytoma cells results in an increase in the tyrosine phosphorylation of the NGF receptor, TrkA, leading to differentiation to a neuronal phenotype. Dephosphorylation by protein tyrosine phosphatases (PTPases) is thought to play an important role in regulating this signaling pathway. To identify PTPases that are recruited to the activated TrkA receptor, we used an ingel PTPase assay to examine the presence of PTPases in TrkA immunoprecipitates. The Src homology 2 domain containing PTPase SHP‐2 was found to associate transiently with TrkA following receptor activation, reaching a peak after 1 min of NGF treatment and then decreasing rapidly. The association of SHP‐2 with TrkA was accompanied by the tyrosine phosphorylation of SHP‐2 and an association of SHP‐2 with multiple tyrosine‐phosphorylated proteins. In addition, the PTPase activity in SHP‐2 immunoprecipitates increased greater than twofold after 1 min of NGF treatment. This is the first demonstration that the association of SHP‐2 with TrkA is induced by NGF and that this association leads to SHP‐2 activation and tyrosine phosphorylation. We conclude that SHP‐2 plays a significant role in early biochemical events in TrkA‐mediated signal transduction.

Discovery of Orally Bioavailable Cathepsin S Inhibitors for the Reversal of Neuropathic Pain

Cathepsin S inhibitors are well-known to be an attractive target as immunological therapeutic agents. Recently, our gene expression analysis identified that cathepsin S inhibitors could also be effective for neuropathic pain. Herein, we describe the efficacy of selective cathepsin S inhibitors as antihyperalgesics in a model of neuropathic pain in rats after oral administration.

Nerve growth factor induces resistance of PC12 cells to nitric oxide cytotoxicity

Nitric oxide (NO) donors, sodium nitroprusside and NOC 7, caused pheochromocytoma (PC12) cell death in a concentration and time-dependent manner. This cytotoxicity was blocked by the NO trapping agent, oxyhemoglobin. A membrane permeable cGMP analogue had no cytotoxicity in a reasonable concentration. Moreover, the selective inhibitor of cGMP-dependent protein kinase, KT5823, had no effect on NOC 7 cytotoxicity. These results suggest that NO caused PC12 cell death but not through the cGMP pathway. Additionally, this NO-induced PC12 cell death is not accompanied by DNA fragmentation. Nerve growth factor (NGF), which is able to rescue PC12 cells from serum deprivation, failed to protect PC12 cells from NO-induced cell death by acute treatment. However, PC12 cells differentiated by NGF treatment for more than 3 days did not die after NO exposure. The differentiated PC12 cells, but not undifferentiated cells, expressed NO synthase (NOS). NGF-differentiated PC12 cells acquired the resistance to NO, by a mechanism not yet identified, accompanied by the expression of NOS.

Distinct usages of phospholipase Cγ and Shc in intracellular signaling stimulated by neurotrophins

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), members of the neurotrophin family, bind to and activate TrkA, TrkB and TrkC, respectively, members of the Trk receptor tyrosine kinase family, to exert various effects including promotion of differentiation and survival, and regulation of synaptic plasticity in neuronal cells. Many reports have suggested that different neurotrophins show distinct biological functions, although molecular mechanisms by which neurotrophins exert their different functions remain unclear. In the present study, we found distinct usages of phospholipase Cgamma (PLCgamma) and Shc in intracellular signaling stimulated by neurotrophins. BDNF stimulated much stronger interactions of PLCgamma with Trk than NGF and NT-3 in PC12 cells stably expressing TrkB and cultured cerebral cortical neurons, respectively, although BDNF, NGF and NT-3 induced similar levels of tyrosine phosphorylation of Trk. Furthermore, the cultured cortical neurons showed large PLCgamma-dependent increases in intracellular Ca(2+) levels in response to BDNF compared with NT-3. In Shc signaling, NGF, but not BDNF, displayed interactions between Trk and Shc in a phenylarsine oxide (PAO; an inhibitor of tyrosine phosphatase)-dependent manner in TrkB-expressing PC12 cells. These results indicated that neurotrophins stimulate distinct kinds of interactions between Trk and PLCgamma and between Trk and Shc. These differences may lead to the distinct biological functions of neurotrophins.

Purification and Properties of a Monofunctional Imidazoleglycerol-Phosphate Dehydratase from Wheat

Imidazoleglycerol-phosphate dehydratase (EC 4.2.1.19) activity was detected in extracts of several monocotyledonous and dicotyledonous plants using a newly developed assay method. The enzyme was purified 114,000-fold (to apparent homogeneity) from wheat germ by five chromatographic steps. Its native relative molecular weight (Mr) was determined to be 600,000 to 670,000, and it consists of identical subunits of Mr 25,500. In wheat germ, the dehydratase, unlike those of prokaryotic origin, is not associated with histidinol phosphatase activity. The reaction product was identified as imidazoleacetol phosphate (IAP) by comparing it with synthetic IAP as an authentic reference. The Km value for imidazoleglycerol phosphate was 0.36 mM at the optimal pH of 6.6. The enzyme required a reducing agent, such as 2-mercaptoethanol or dithiothreitol, and Mn2+ for maximal activity. 3-Amino-1,2,4-triazole competitively inhibited the activity with a Ki value of 46 [mu]M. The purification of imidazoleglycerol-phosphate dehydratase from wheat germ and histidinol dehydrogenase from cabbage (A. Nagai, A. Scheidegger [1991] Arch Biochem Biophys 284: 127-132) suggests that at least the second half of the histidine biosynthesis in plants is identical to that in microorganisms.