Yoshiko Furukawa

Lipopolysaccharide enhances synthesis of brain‐derived neurotrophic factor in cultured rat microglia

Expression of neurotrophins in pure microglia cultured from embryonic rat brain and the effects of lipopolysaccharide (LPS) on the expression were investigated. In untreated cultures, nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), and neurotrophin (NT)‐4/5 mRNAs were detected by use of reverse transcriptase‐polymerase chain reaction but NT‐3 mRNA was not. LPS stimulation caused a marked increase in BDNF mRNA expression in addition to a slight increment of the NT‐4/5 mRNA level; however, the NGF mRNA level was not affected. LPS also increased BDNF‐like immunoreactivity in cultured microglia, an action consistent with an elevation of BDNF mRNA. These results demonstrate that LPS stimulates synthesis of BDNF and probably NT‐4/5, specific ligands for tyrosine kinase receptor TrkB, suggesting that activated microglia, which appear in the damaged brain, participate in neuronal regeneration via production of such neurotrophins. J. Neurosci. Res. 50:1023–1029, 1997. © 1997 Wiley‐Liss, Inc.

Hydrophobic dipeptide Leu-Ile protects against neuronal death by inducing brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor synthesis

We investigated whether certain hydrophobic dipeptides, Leu‐Ile, Leu‐Pro, and Pro‐Ile, which partially resemble the site on FK506 that binds to immunophilin, could stimulate glial cell line‐derived neurotrophic factor (GDNF) and brain‐derived neurotrophic factor (BDNF) synthesis in cultured neurons and found only Leu‐Ile to be an active dipeptide. Leu‐Ile protected against the death of mesencephalic neurons from wild‐type mice but not from mice lacking the BDNF or GDNF gene. Next, we examined the effects of i.p. or i.c.v. administration of Leu‐Ile on BDNF and GDNF contents. Both types of administration increased the contents of BDNF and GDNF in the striatum of mice. Also, peripheral administration of Leu‐Ile inhibited dopaminergic (DA) denervation caused by unilateral injection of 6‐hydroxydopamine (6‐OHDA) into the striatum of mice. The number of rotations following a methamphetamine challenge was lower in the Leu‐Ile‐treated group than in the nontreated group. Next, we compared the calcineurin activity and immunosuppressant activity of Leu‐Ile with those of FK506. Leu‐Ile was not inhibitory toward calcineurin cellular activity in cultured neuronal cells. Furthermore, Leu‐Ile did not suppress concanavalin A (ConA)‐induced synthesis/secretion of interleukin‐2 by cultured spleen cells, suggesting that the immunosuppressant activity of Leu‐Ile may be negligible when used as a therapeutic tool for neurodegenerative diseases.

Estrogen Stimulates Proliferation and Differentiation of Neural Stem/Progenitor Cells through Different Signal Transduction Pathways

Our previous study indicated that both 17β-estradiol (E2), known to be an endogenous estrogen, and bisphenol A (BPA), known to be a xenoestrogen, could positively influence the proliferation or differentiation of neural stem/progenitor cells (NS/PCs). The aim of the present study was to identify the signal transduction pathways for estrogenic activities promoting proliferation and differentiation of NS/PCs via well known nuclear estrogen receptors (ERs) or putative membrane-associated ERs. NS/PCs were cultured from the telencephalon of 15-day-old rat embryos. In order to confirm the involvement of nuclear ERs for estrogenic activities, their specific antagonist, ICI-182,780, was used. The presence of putative membrane-associated ER was functionally examined as to whether E2 can activate rapid intracellular signaling mechanism. In order to confirm the involvement of membrane-associated ERs for estrogenic activities, a cell-impermeable E2, bovine serum albumin-conjugated E2 (E2-BSA) was used. We showed that E2 could rapidly activate extracellular signal-regulated kinases 1/2 (ERK 1/2), which was not inhibited by ICI-182,780. ICI-182,780 abrogated the stimulatory effect of these estrogens (E2 and BPA) on the proliferation of NS/PCs, but not their effect on the differentiation of the NS/PCs into oligodendroglia. Furthermore, E2-BSA mimicked the activity of differentiation from NS/PCs into oligodendroglia, but not the activity of proliferation. Our study suggests that (1) the estrogen induced proliferation of NS/PCs is mediated via nuclear ERs; (2) the oligodendroglial generation from NS/PCs is likely to be stimulated via putative membrane-associated ERs.

Brain-derived neurotrophic factor alters cell migration of particular progenitors in the developing mouse cerebral cortex

Effects of brain-derived neurotrophic factor (BDNF) on cell migration from the ventricular zone to the cortical plate (CP) in developing mouse cerebral cortex were examined. BDNF (700 ng) was injected into the brain ventricle of 13- or 14-day-old embryos (E13 or E14) after the intraperitoneal administration of 5-bromodeoxyuridine (BrdU) to pregnant mice. BDNF injection at E13 increased the number of BrdU-positive cells migrated into the CP until E15, and caused them to become localized in much deeper layers (V-VI) than expected (IV-V, as in the vehicle-treated mice) by postnatal day 1. However, when the injections were made at E14, BrdU-positive cells predominantly migrated to layers II/III irrespective of BDNF administration. These results demonstrate that BDNF affects particular progenitors at limited stages, and suggest the presence of a Reelin-independent mechanism(s) to regulate cell migration.

Accumulation of nerve growth factor protein at both rostral and caudal stumps in the transected rat spinal cord

Changes in the nerve growth factor (NGF) content in the rat spinal cord during development or after traumatic spinal cord injury were examined by using a two-site enzyme immunoassay (EIA) system and an immunohistochemical technique. From embryonic day (E) 14 to postnatal day (P) 70, the spinal cord contained 200-300 pg NGF/g of wet tissue evenly in all regions tested. After complete spinal cord transection of P49 rats, the NGF level started to increase in the rostral and caudal stumps nearest to the injury site at 2 and 4 days, respectively. The NGF level of the caudal side returned to the original level by 2 weeks, but that of the rostral side remained high even 3 weeks, after the injury. At 4 days after the injury, NGF-like immunoreactivity in both stumps was predominantly localized in the axon-like structures of the white matter and in cells morphologically resembling immune cells. These observations suggest that the NGF was transported within the spinal tracts, and that NGF secreted from immune cells that had invaded into the injured spinal cord had accumulated around the transection site. Increased NGF at the injury site may be advantageous for injured neurons and involved in mechanisms directing to axonal regeneration of the injured spinal cord.

4‐Methylcatechol stimulates phosphorylation of Trk family neurotrophin receptors and MAP kinases in cultured rat cortical neurons

Effects of 4‐methycatechol (4MC), a potent stimulator of nerve growth factor and brain‐derived neurotrophic factor (BDNF) synthesis, on phosphorylation of cellular molecules in cultured rat cortical neurons were examined. 4MC stimulated tyrosine phosphorylation of various proteins of molecular weight from 10–300 kDa including Trks, which are high‐affinity neurotrophin receptors. Moreover, 4MC enhanced the phosphorylation of serine 133 of mitogen‐activated protein kinase (MAPK/ERK) in a dose‐dependent manner. Pretreatment of cultures with PD98059, a selective inhibitor of MAPK kinase (MEK‐1), inhibited 4MC‐induced phosphorylation of ERKs, demonstrating MEK‐1‐mediated activation. Therefore, it seems that 4MC triggered the phosphorylation of Trks, resulting in the activation of the subsequent MAPK/ERK signal cascade, or perhaps the involvement of BDNF action as 4MC can stimulate neuronal BDNF synthesis. The phosphorylation of MAPK/ERK was unaffected, however, in the presence of cycloheximide, a protein synthesis inhibitor, and K252a, a selective inhibitor of Trks, suggesting that the effect of newly synthesized BDNF was negligible on this event, and that primary sites of 4MC actions are not limited only to Trks. These results suggest that 4MC primarily activates multiple signal transduction molecules such as tyrosine kinases, including Trks. A significant increase in the survival rate of cortical neurons in the presence of 10 or 100 nM 4MC supported this idea, because the concentrations were much lower than those for stimulation of BDNF synthesis. Our results strongly suggest that the neurotrophic actions of 4MC found so far are mediated predominantly by direct activation of some intracellular signals including MAPK/ERK rather than by neurotrophin synthesis.

Administration of FGF-2 to embryonic mouse brain induces hydrocephalic brain morphology and aberrant differentiation of neurons in the postnatal cerebral cortex

Fibroblast growth factor‐2 (FGF‐2) was injected into mouse cerebral ventricles at embryonic day (E) 14 in utero and its effects on developing brain morphology and expression of various cell‐ or differentiation‐associated protein markers in the cerebral cortex were examined. High doses of FGF‐2 (200 or 300 ng) caused encephalic alternations such as deformation of the calvarium, enlargement of the ventricular spaces, and thinning of the cerebral cortex. There was no gross abnormality in the alignment of the cerebral neuronal layers, however, both cell number and cell density of the upper layers (II/III) and the lower layers (IV–VI) of the cerebral cortex were increased. Brain‐derived neurotrophic factor (BDNF), tyrosine hydroxylase, nestin, and microtubule‐associated protein 2 were aberrantly or ectopically expressed in the deep areas of the cerebral cortex. A substantial number of these cells coexpressed these antigens. These observations demonstrate that a subpopulation of neurons in the cortical deep layer abnormally differentiated or partly sustained their immature state following a single administration of FGF‐2 at E14. Developmental analysis of localization of BDNF‐positive cells suggested that the abnormality started around P5. Furthermore, cell migration was not affected by FGF‐2 administration. FGF‐2 seems to play predominant roles in the proliferation of neuronal precursors and in neuronal differentiation in the developing mouse cerebral cortex even at relatively late stages of brain neurogenesis. J. Neurosci. Res. 65:228–235, 2001.

Neurotrophins facilitate synthesis of choline acetyltransferase and tyrosine hydroxylase in cultured mouse neural stem cells independently of their neuronal differentiation

Effects of three neurotrophins, i.e., nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3, on the expression of four neurotransmitter-synthesizing enzymes, i.e. choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), dopamine beta hydroxylase (DBH), and glutamate decarboxylase 65 were investigated in cultured mouse neural stem cells. All three neurotrophins enhanced the mRNA expression of ChAT, TH, or DBH of the cells caused to differentiate by the removal of fibroblast growth factor (FGF)-2 from the culture medium, and increased the protein and mRNA levels of ChAT and TH of even the undifferentiated proliferating neural stem cells due to the presence of FGF-2. These results demonstrate that neurotrophins stimulate the synthesis of ChAT and TH of the neural stem cells prior to neuronal differentiation, and suggest that neurotrophins may play roles in the commitment to neuronal cells and choice of specific neurotransmitter phenotypes in early stages of neurogenesis.

Restricted growth and insulin-like growth factor-1 deficiency in mice lacking presenilin-1 in the neural crest cell lineage

Presenilin‐1 (PS1) is a transmembrane protein that is in many cases responsible for the development of early‐onset familial Alzheimers disease. PS1 is essential for neurogenesis, somitogenesis, angiogenesis, and cardiac morphogenesis. We report here that PS1 is also required for maturation and/or maintenance of the pituitary gland. We generated PS1‐conditional knockout (PS1‐cKO) mice by crossing floxed PS1 and Wnt1‐cre mice, in which PS1 was lacking in the neural crest‐derived cell lineage. Although the PS1‐cKO mice exhibited no obvious phenotypic abnormalities for several days after birth, reduced body weight in the mutant was evident by the age of 3–5 weeks. Pituitary weight and serum insulin‐like growth factor (IGF)‐1 level were also reduced in the mutant. Histologic analysis revealed severe atrophy of the cytosol in the anterior and intermediate pituitary lobes of the mutant. Immunohistochemistry did not reveal clear differences in the expression levels of thyroid‐stimulating hormone, adrenocorticotropic hormone, or prolactin in the mutant pituitary. In contrast, growth hormone expression levels were reduced in the anterior lobe of the mutant. PS1 was defective in the posterior lobe, but not the anterior or intermediate lobes, in the mutant pituitary. These findings suggest that PS1 indirectly mediates the development and/or maintenance of the anterior and intermediate lobes in the pituitary gland via actions in other regions, such as the posterior lobe.

Cyclic AMP/protein kinase a signal attenuates Ca2+‐induced fibroblast growth factor‐1 synthesis in rat cortical neurons

Fibroblast growth factor (FGF)‐1 is increased in particular brain regions after birth, suggesting an involvement of some regulatory neuronal circuits. To address the neuronal activity responsible for FGF‐1 synthesis, effects of various neurotransmitter receptor activation on cellular FGF‐1 content were examined using cultured rat cortical neurons. Histamine, glutamate, carbachol, serotonin or γ‐aminobutyric acid (GABA) caused an increase of FGF‐1 content. Because this effect was mimicked by (1) N‐methyl‐D‐aspartate, a glutamatergic agonist; (2) Ca2+ ionophore; (3) depolarization with high concentration of KCl, but was abolished in Ca2+‐free medium, Ca2+ influx was thought to trigger FGF‐1 synthesis. Such Ca2+‐mediated enhancement of FGF‐1 synthesis, however, did not occur in the presence of norepinephrine (NE), but was restored by KT‐5720, an inhibitor of protein kinase A (PKA), suggesting an interplay between Ca2+‐activated and cAMP/PKA signals for neuronal FGF‐1 synthesis. This mechanism was proved to function in vivo by stimulation of FGF‐1 expression in neurons of the cerebral cortex after intracerebral administration of propranolol, an antagonist of adrenergic β receptors. This demonstrates that FGF‐1 synthesis is essentially upregulated by Ca2+ influx through excitatory neuronal activities, but such an effect is abolished by neurotransmission that evokes cAMP/PKA signals. FGF‐1 produced is thought to act on establishment and maintenance of particular neuronal circuits in the brain, which may be one of the ways neurotransmitters regulate brain function.