Elsevier

Neuropharmacology

Volume 42, Issue 7, June 2002, Pages 903-912
Neuropharmacology

Neurotrophins induce BDNF expression through the glutamate receptor pathway in neocortical neurons

https://doi.org/10.1016/S0028-3908(02)00043-6Get rights and content

Abstract

Neurotrophins jointly exert various functions in the nervous system, including neuronal differentiation, survival, and regulation of synaptic plasticity. However, the functional interactions of neurotrophins or mechanisms through which neurotrophins regulate each other are still not clear. In the present study, brain-derived neurotrophic factor (BDNF) mRNA expression is induced by neurotrophin-4/5 (NT-4/5) and by BDNF itself in neocortical neurons. K252a, a specific tyrosine kinase (Trk) inhibitor, completely suppresses BDNF- and NT-4/5-enhanced BDNF mRNA expression. NT-4/5 significantly augments BDNF protein production, which is also reversed by K252a. When neurons are incubated with neurotrophin-3 (NT-3) or nerve growth factor (NGF), there are no significant changes in BDNF mRNA or protein expression. Interestingly, the α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or the N-methyl-D-aspartate (NMDA) receptor blocker AP-5 completely suppresses NT-4/5-enhanced BDNF protein production, while tetrodotoxin (TTX) only suppresses NT-4/5-enhanced BDNF production by 50%. Additionally, the mitogen activated protein (MAP) kinase inhibitor PD98059 enhances BDNF-induced glutamate receptor-1 (GluR1) protein expression, but a phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 strongly reduces BDNF-induced GluR1 protein expression. Taken together, glutamate receptors are important for the regulation of BDNF expression by neurotrophins, and MAP and PI3K kinases differentially modulate AMPA receptor expression in the cortical neurons.

Introduction

The neurotrophins, a family of structurally and functionally related peptide growth factors, include nerve growth factor (NGF), brain-derived growth factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5). Neurotrophins are widely expressed in the developing central and peripheral nervous system (Leibrock et al., 1989, Hohn et al., 1990). In vitro and in vivo experiments suggest that neurotrophins regulate survival of responsive central and peripheral neurons. Among these factors, BDNF has extensively been investigated in the central nervous system (CNS) because of the widespread expression of mRNA for both the factor and its receptor, TrkB (Hofer et al., 1990, Klein et al., 1990. The neurotrophic activity of BDNF is pronounced in various types of CNS neurons (Thoenen, 1995, Lewin and Barde, 1996). Neuronal phenotypic abnormalities in BDNF knockout mice further support the hypothesis that BDNF is essential for the normal development of the brain (Ernfors et al., 1994a, Jones et al., 1994, Marty et al., 1996). It has been suggested that BDNF plays a significant role in brain synaptic plasticity in addition to its classic trophic activities. It can acutely modulate synaptic transmission (Kang and Schuman, 1995, Kang and Schuman, 1996) as well as long-term potentiation (LTP) (Figurov et al., 1996). BDNF also rapidly enhances synaptic current in hippocampal culture (Levine et al., 1995). Hippocampal LTP is impaired in mice lacking the BDNF gene (Korte et al., 1995), but exogenous BDNF can rescue the deficits in basal synaptic transmission and hippocampal LTP (Patterson et al., 1996, Korte et al., 1996).

BDNF is expressed predominantly in neurons of CNS, at least under normal physiologic conditions. Furthermore, the expression of BDNF is regulated by neural activity as demonstrated by seizures (Bengzon et al., 1993, Lauterborn et al., 1995), afferent stimulation, and sensory stimulation (Patterson et al., 1992, Castren et al., 1993, Rocamora et al., 1996). Current evidence suggests that activity-dependent expression of BDNF is primarily mediated by non-NMDA glutamate receptors. The glutamate receptor agonist kainic acid potently induces BDNF in hippocampal neurons, whereas NMDA does not (Zafra et al., 1990). Although these experimental results suggest that neural activity is important for BDNF expression, the regulation of BDNF expression during neuronal development is not fully understood. Neurotrophins influence neural activity (Thoenen, 1995), which implies that neurotrophins may functionally affect each other during neuronal development (Thoenen, 1995). It has been reported that BDNF enhanced NT-3 expression (Lindholm et al., 1994). So, it is possible that BDNF may also be regulated by other neurotrophic factors or even by BDNF itself.

In the present study, we hypothesize that neurotrophins may regulate BDNF during neuronal development. To address this, neurons from rat embryos were prepared and stimulated with various neurotrophic factors. BDNF can be up-regulated by NT-4/5 and by BDNF itself through the glutamate receptor pathway. These findings may lend new insights into the functional interactions of neurotrophins in the CNS.

Section snippets

Reagents

NT-4/5, NT-3 and NGF were purchased from Wako Pure Chemical industries, Ltd (Osaka, Japan). Recombinant BDNF was obtained from Sumitomo Pharmaceuticals Ltd.

Animal procedures

Heterozygous NT-3 mutant mice (Ernfors et al., 1994b) were mated to produce homozygous mutants. The genotypes of the offspring were determined by PCR reaction. Most of the experiments were performed with litters less than 14-days old after birth.

Cell culture

Primary cultures of neocortical neurons were prepared from embryonic rats (Sprague–Dawley).

BDNF up-regulates BDNF mRNA expression in neocortical neurons

In primary cultures of neonatal cortex, whether BDNF regulated its own mRNA expression was first examined. Neurons were prepared from neocortices of rat E18 embryos and grown in serum-free condition for 3 and 7 days, which prevented glial proliferation and suppressed its frequency to less than 5% (Nawa et al., 1993). BDNF (10 and 50 ng/ml) was added daily to the culture. Total cellular RNA was extracted for the detection of BDNF mRNA expression by quantitative RT-PCR. BDNF mRNA expression was

Discussion

BDNF is a neurotrophic factor involved in critical CNS functions including neuronal development and differentiation, as well as synaptic transmission and plasticity (Thoenen, 1995). Due to a central role in these biologic processes, elucidation of the molecular regulation of BDNF gene expression will provide important insight into CNS function. In the present study, we demonstrate that BDNF and NT-4/5 up-regulate BDNF mRNA expression and protein production in neocortical neurons. Neurotrophins

Acknowledgements

We are grateful to Miss Yasuko Kuwabara for taking care of NT-3 deficient mice. We also express our thanks to Dr Jianjun Wang for helpful discussions. This study was supported by a grant from Japanese Society for the Promotion of Sciences (RFTF-96L00203).

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