MEK inhibitors block BDNF-dependent and -independent expression of GABAA receptor subunit mRNAs in cultured mouse cerebellar granule neurons

doi:10.1016/S0165-3806(99)00119-4

Developmental Brain Research

Volume 119, Issue 1, 3 January 2000, Pages 1-10

https://doi.org/10.1016/S0165-3806(99)00119-4 Get rights and content

Abstract

Brain-derived neurotrophic factor (BDNF) can regulate the maturation of developing cerebellar granule neurons. Within 1–2 days of culture, BDNF induces the expression of granule neuron terminal differentiation markers, particularly GABA_A receptor α6 subunit (GABA_Aα6) mRNA. Other trophic factors including insulin-like growth factor, the neurotrophin NT-3, pituitary adenylate cyclase-activating polypeptide (PACAP), and fetal bovine serum failed to induce this early expression. The expression of other GABA_A receptor subunits, including α1 and γ2, was also enhanced by exposure of developing granule neurons to BDNF. This BDNF-dependent expression of GABA_A receptor subunit mRNAs could be effectively blocked by treatment with the mitogen-activated protein kinase kinase (MEK) inhibitors, PD98059 or U0126. In the absence of BDNF, GABA_Aα6 expression occurs but not until 3–4 days of culture. This BDNF-independent expression of GABA_Aα6 was also inhibited by PD98059. Further studies showed that the BDNF-dependent expression GABA_Aα6 could also be reduced by LY294002, an inhibitor of the phosphatidylinositol 3-kinase, or depolarizing concentrations of KCl. These results thus suggest that both BDNF-dependent and -independent expressions of GABA_A receptor subunits require the activation of MEK and the mitogen-activated protein kinase (MAPK) pathway. However, it is also likely that other signaling pathways modulate this maturation process.

Introduction

Brain-derived neurotrophic factor (BDNF) can regulate maturation of CNS neurons. The in vitro terminal differentiation of mouse cerebellar granule neurons can be used as a model for exploring the signaling mechanisms by which BDNF regulates this maturation process 30, 43. In rodents, much of cerebellar granule neuron maturation takes place during postnatal development 1, 2, 26, 40. Granule neurons arise from precursors in the external granule cell layer (EGL). After these precursors exit the cell cycle, they migrate through the developing molecular layer to their adult position in the internal granule layer (IGL). In the IGL, they undergo their final maturation, elaborating dendrites, making synaptic contacts with mossy fiber terminals, and expressing terminal differentiation markers. These differentiation markers include several neurotransmitter receptors subunits including GABA_A receptor subunits, α1, α6, β2, γ2 and δ 4, 24, 26, 27, 50. The expression of GABA_Aα6 is specific for cerebellar granule neurons 24, 26, 27, 50.

Consistent with a role for BDNF in the regulation of granule neuron terminal differentiation and maturation is the observation that expression of the high-affinity BDNF receptor, TrkB, coincides with the beginning of the final granule neuron maturation process [17]. The expression of TrkB first occurs in migrating granule neurons and continues in maturing granule neuron in the IGL. Recent studies of the stargazer (stg) mutant mouse also support this hypothesis. Stg mice have a substantial reduction in BDNF mRNA in the cerebellum and also a reduction in protein level for the maturation marker, GABA_Aα6 38, 48. Studies using cultured granule neuron suggest that BDNF can modulate both neurite extension and the expression of GABA_Aα6 17, 30, 43. In the presence of BDNF, cultured granule neurons express GABA_Aα6 mRNA after 1–2 days of culture. In the absence of added BDNF or in the presence of K252a, an inhibitor of TrkB signaling 5, 37, 47, GABA_Aα6 expression does occur but not until 3–5 days of culture 28, 29, 30. Thus, GABA_Aα6 expression may also be regulated by BDNF-independent mechanisms [35]. BDNF-dependent and independent processes may interact to regulate the final maturation of granule neurons including controlling the appropriate timing and/or level of GABA_Aα6 expression.

A critical issue is to identify the signaling mechanisms by which these processes regulate neuron maturation. The binding of BDNF and other neurotrophins to their cognate receptors results in activation of several downstream signaling pathways including the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3 kinase (PI3-K) pathways [44]. In cultured granule neurons, BDNF can activate both the MAPK pathway and the PI3-K pathway 20, 46. Alternatively, IGF-I, which does not induce the early expression of GABA_Aα6, activates primarily the PI3-K pathway 14, 30. It is possible that BDNF's more substantial activation of the MAPK pathway is responsible for the early induction of GABA_Aα6 expression and maturation of cerebellar granule neurons.

In this report, we present data that suggest that the MAPK pathway may be critical for both BDNF-dependent and -independent regulation of GABA_Aα6 mRNA expression as well as expression of other GABA_A receptor subunit genes. These studies suggest that other signaling pathways may also help regulate the maturation of granule neurons and provide insight into the complex extracellular and intracellular signaling processes that regulate the maturation of developing CNS neurons.

Section snippets

Cerebellar cell culture

Cerebellar granule cell cultures were prepared from postnatal mice. We generated Percoll-purified granule cell cultures using procedures described by Hatten [21]with minor modifications. Cerebella from postnatal day 7 mice were incubated in 1% trypsin (Sigma, St. Louis, MO, type III) and 0.1% DNase I (Boehringer Mannheim, Indianapolis, IN) in calcium- and magnesium-free Tyrode's solution (CMF Tyrode's) for 3 min. We dissociated cells by repeated passage through a small bore pipet in MEM

Results

A minimal culture environment allows cerebellar granule neurons to undergo terminal differentiation 28, 29, 30. We previously observed that purified granule cells, cultured with insulin-like growth factor I (IGF-I) as the only added trophic factor, survive and after 3–5 days of culture express mRNA encoding the GABA_A receptor α6 subunit 28, 29. BDNF added to these cultures, in the presence or absence of IGF-I, accelerated granule neuron maturation with GABA_Aα6 expression occurring 1–2 days

Discussion

The experiments presented in this report suggest that exposing developing cerebellar granule neurons to BDNF can modify the expression of a subset of GABA_A receptor subunit genes that are normally expressed late in granule neuron development. Particularly apparent is an increase in the expression of the GABA_Aα6 subunit RNA, a specific marker of granule neuron terminal differentiation 24, 26, 27, 50. This increased expression may occur at the level of new gene transcription as suggested by

Acknowledgements

We thank D.R. Burt for critical reading of the manuscript and for the gift of the GABA_A receptor subunit cDNAs. We also thank W. Wisden for the gift of the GABA_Aα6/IRES/LacZ promoter construct and A.L. Joyner for the gift of the En-2 cDNA. This work is, in part, supported by an NIH grant (ES/OD08087) to R.F.B.

References (51)

M.M. Berg et al.
K-252a inhibits nerve growth factor-induced trk proto-oncogene tyrosine phosphorylation and kinase activity
J. Biol. Chem.
(1992)
R.F. Bulleit et al.
Methylmercury antagonizes the survival-promoting activity of insulin-like growth factor on developing cerebellar granule neurons
Toxicol. Appl. Pharmacol.
(1998)
P. Chomczynski et al.
Single step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction
Anal. Biochem.
(1987)
A. Cuenda et al.
SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1
FEBS Lett.
(1995)
H. Cui et al.
Inhibition of glycogen synthase kinase 3β activity regulates proliferation of cultured cerebellar granule cells
Dev. Brain Res.
(1998)
S.R. Datta et al.
Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery
Cell
(1997)
M.F. Favata et al.
Identification of a novel inhibitor of mitogen-activated protein kinase kinase
J. Biol. Chem.
(1998)
K.R. Jones et al.
Targeted disruption of the BDNF gene perturbs brain and sensory neuron development but not motor neuron development
Cell
(1994)
K. Kato
Novel GABA_A receptor α subunit is expressed only in cerebellar granule cells
J. Mol. Biol.
(1990)
T. Kubo et al.
Brain-derived neurotrophic factor (BDNF) can prevent apoptosis of rat cerebellar granule neurons in culture
Dev. Brain Res.
(1995)

X. Lin et al.

Cell intrinsic mechanisms regulate mouse cerebellar granule neuron differentiation

Neurosci. Lett.

(1996)

X. Lin et al.

Insulin-like growth factor I (IGF-I) is a critical trophic factor for developing cerebellar granule cells

Dev. Brain Res.

(1997)

X. Lin et al.

Bulleit, BDNF accelerates gene expression in cultured cerebellar granule neurons

Dev. Brain Res.

(1998)

O.H. Lowry et al.

Protein measurement with the folin phenol reagent

J. Biol. Chem.

(1951)

S. Marty et al.

Neurotrophins and activity-dependent plasticity of cortical interneurons

Trends Neurosci.

(1997)

R.M. McKernan et al.

Which GABA_A-receptor subtypes really occur in the brain?

Trends Neurosci.

(1996)

P.M. Schwartz et al.

Abnormal cerebellar development and foliation in BDNF−/− mice reveals a role for neurotrophins in CNS patterning

Neuron

(1997)

F.J. Seil

BDNF and NT-4, but not NT-3, promote development of inhibitory synapses in the absence of neuronal activity

Brain Res.

(1999)

C.L. Thompson et al.

Decreased expression of GABA_A receptor α6 and β3 subunits in stargazer mutant mice: a possible role of brain-derived neurotrophic factor in the regulation of cerebellar GABA_A receptor expression?

Mol. Brain Res.

(1998)

J. Altman

Postnatal development of the cerebellar cortex in the rat: I. The external germinal layer and the transitional molecular layer

J. Comp. Neurol.

(1972)

J. Altman

Postnatal development of the cerebellar cortex in the rat: III. Maturation of the components of the granule layer

J. Comp. Neurol.

(1972)

S. Bahn et al.

Directing gene expression to cerebellar granule cells using γ-aminobutyric acid type A receptor α6 subunit transgene

Proc. Natl. Acad. Sci. U.S.A.

(1997)

C.E. Beattie et al.

Developmental cues modulate GABA_A receptor subunit mRNA expression in cultured cerebellar granule neurons

J. Neurosci.

(1993)

R.J. Cabelli et al.

Inhibition of ocular dominance column formation by infusion of NT-4/5 or BDNF

Science

(1995)

Y. Daaka et al.

Switching of the coupling of the β₂-adrenergic receptor to different G proteins by protein kinase A

Nature

(1997)

Cited by (36)

Influence of BDNF Val66Met polymorphism on excitatory-inhibitory balance and plasticity in human motor cortex
2021, Clinical Neurophysiology
Citation Excerpt :
BDNF has been shown to enhance transmitter release at GABAergic inhibitory terminals (Baldelli et al., 2005, Pattwell et al., 2012) and increase expression and activity of glutamic acid decarboxylase (GAD), a GABA-synthesizing enzyme (Yamada et al., 2002). BDNF also increases the density of inhibitory synapses (Marty et al., 2000) and promotes maturation of GABAergic innervations (Bulleit and Hsieh, 2000). One or several of these mechanisms could account for the influence of BDNF genotype on GABAA and GABAB receptor mediated inhibition.
While previous studies showed that the single nucleotide polymorphism (Val66Met) of brain-derived neurotrophic factor (BDNF) can impact neuroplasticity, the influence of BDNF genotype on cortical circuitry and relationship to neuroplasticity remain relatively unexplored in human.
Using individualised transcranial magnetic stimulation (TMS) parameters, we explored the influence of the BDNF Val66Met polymorphism on excitatory and inhibitory neural circuitry, its relation to I-wave TMS (ITMS) plasticity and effect on the excitatory/inhibitory (E/I) balance in 18 healthy individuals.
Excitatory and inhibitory indexes of neurotransmission were reduced in Met allele carriers. An E/I balance was evident, which was influenced by BDNF with higher E/I ratios in Val/Val homozygotes. Both long-term potentiation (LTP-) and depression (LTD-) like ITMS plasticity were greater in Val/Val homozygotes. LTP- but not LTD-like effects were restored in Met allele carriers by increasing stimulus intensity to compensate for reduced excitatory transmission.
The influence of BDNF genotype may extend beyond neuroplasticity to neurotransmission. The E/I balance was evident in human motor cortex, modulated by BDNF and measurable using TMS. Given the limited sample, these preliminary findings warrant further investigation.
These novel findings suggest a broader role of BDNF genotype on neurocircuitry in human motor cortex.
Negative association between left prefrontal GABA concentration and BDNF serum concentration in young adults
2020, Heliyon
Citation Excerpt :
Some rat and human studies have demonstrated interactions between GABA-ergic neurotransmission and BDNF release. On one hand, BDNF can suppress or presynaptically enhance GABA release through the activation of postsynaptic tropomyosin receptor kinase B (TrkB)-type receptors, which may result in an altered total GABA concentration (Bulleit and Hsieh, 2000; Colino-Oliveira et al., 2016; Kim et al., 2017; Pezet et al., 2002; Tanaka et al., 1997; Wardle and Poo, 2003). Conversely, changes in the balance between excitatory glutamatergic and inhibitory GABAergic neurotransmission has been shown to alter BDNF expression (Saffarpour et al., 2017).
The brain's major inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and the brain-derived neurotrophic factor (BDNF) play important roles in several stress-related disorders. Magnetic resonance spectroscopy (MRS) allows for non-invasive quantification of GABA concentration in the brain. We investigated the relationship between GABA concentration in the left dorsolateral prefrontal cortex (DLPFC) and BDNF concentration in the serum in a community-based sample of young subjects.
For the GABA measurement a single voxel MR spectrum was assessed in the prefrontal lobe (25 × 40 × 30 mm) using the MEGA-PRESS method in 276 subjects. BDNF serum concentrations were assessed with an ELISA kit. For 147 subjects we had both MRS and BDNF serum data, and for 79 subjects we had genotype data on the BDNF rs6265 polymorphism. Depressive psychopathology was assessed using Beck's Depression Inventory (BDI), Montgomery-Asberg Depression Rating Scale (MADRS) and Structured Clinical Interviews for Diagnostic and Statistical Manual of Mental Disorders (SCID) for DSM-IV.
GABA concentration in the left DLPFC was negatively associated with BDNF serum concentration (r = -.264, p = .001). This correlation remained significant if corrected for sex (r = -.264, p = .001). BDNF serum concentration was also positively associated with volumes and surface areas of the left prefrontal cortex (p = .048, p = .005). There were no significant associations or interaction with depressive psychopathology (BDI, MADRS, SCID) or rs6265.
The results of this study suggest that GABA, BDNF and prefrontal brain volumes are interrelated, but do not show a strong association to depressive psychopathology, possibly due to the mild forms of psychiatric conditions present in our community-based sample.
Dopamine D3 receptor-dependent changes in alpha6 GABAA subunit expression in striatum modulate anxiety-like behaviour: Responsiveness and tolerance to diazepam
2015, European Neuropsychopharmacology
Citation Excerpt :
The increase in α6 was already significant after a 3-day treatment (p<0.001) and continued to rise in a time-dependent manner for up to 21 days, reaching a level similar to that of D3R−/− (Fig. 4). Expression of different GABAA subunits may be influenced by BDNF (Bulleit and Hsieh, 2000); furthermore, D3R is critically controlled by BDNF (Guillin et al., 2001). We therefore wondered whether or not changes in α6 GABAA subunit expression were related to mRNA abundance of BDNF and/or D3R in striatum.
Increasing evidence indicates that central dopamine (DA) neurotransmission is involved in pathophysiology of anxiety, in particular the DA receptor subtype 3 (D₃R). We previously reported that D₃R null mice (D₃R^−/−) exhibit low baseline anxiety levels and that acutely administrated diazepam is more effective in D₃R^−/− than in wild type (WT) when tested in the elevated plus maze test (EPM). Here we tested the hypothesis that genetic deletion or pharmacological blockade of D₃R affect GABA_A subunit expression, which in turn modulates anxiety-like behaviour as well as responsiveness and tolerance to diazepam. D₃R^−/− mice exhibited tolerance to diazepam (0.5 mg/kg, i.p.), assessed by EPM, as fast as after 3 day-treatment, performing similarly to untreated D₃R^−/− mice; conversely, WT exhibited tolerance to diazepam after a 14–21 day-treatment. Analysis of GABA_A α6 subunit mRNA expression by qPCR in striatum showed that it was about 15-fold higher in D₃R^−/− than in WT. Diazepam treatment did not modify α6 expression in D₃R^−/−, but progressively increased α6 expression in WT, to the level of untreated D₃R^−/− after 14–21 day-treatment. BDNF mRNA expression in striatum was remarkably (>10-fold) increased after 3 days of diazepam-treatment in both WT and D₃R^−/−; such expression level, however, slowly declined below control levels, by 14–21 days. Following a 7 day-treatment with the selective D₃R antagonist SB277011A, WT exhibited a fast tolerance to diazepam accompanied by a robust increase in α6 subunit expression. In conclusion, genetic deletion or pharmacological blockade of D₃R accelerate the development of tolerance to repeated administrations of diazepam and increase α6 subunit expression, a GABA_A subunit that has been linked to diazepam insensitivity. Modulation of GABA_A receptor by DA transmission may be involved in the mechanisms of anxiety and, if occurring in humans, may have therapeutic relevance following repeated use of drugs targeting D₃R.
Cerebellum volume in high-risk offspring from multiplex alcohol dependence families: Association with allelic variation in GABRA2 and BDNF
2011, Psychiatry Research - Neuroimaging
Offspring from families with multiple cases of alcohol dependence have a greater likelihood of developing alcohol dependence (AD) and related substance use disorders. Greater susceptibility for developing these disorders may be related to structural differences in brain circuits that influence the salience of rewards or modify the efficiency of information processing and AD susceptibility. We examined the cerebellum of 71 adolescent/young adult high-risk (HR) offspring from families with multiple cases of alcohol dependence (multiplex families), and 60 low-risk (LR) controls with no family history of alcohol or drug dependence who were matched for age, gender, socioeconomic status and IQ, with attention given to possible effects of personal use of substances and maternal use during pregnancy. Magnetic resonance images were acquired on a General Electric 1.5-Tesla scanner and manually traced (BRAINS2) blind to clinical information. GABRA2 and BDNF variation were tested for their association with cerebellar volumes. High-risk offspring from multiplex AD families showed greater total volume of the cerebellum and total gray matter (GM), in comparison with LR controls. An interaction between allelic variation in GABRA2 and BDNF genes was associated with GM volumes, suggesting that inherited variation in these genes may promote early developmental differences in neuronal proliferation of the cerebellum.
AMPAR signaling mediating GABA<inf>A</inf>R δ subunit up-regulation in cultured mouse cerebellar granule cells
2010, Neurochemistry International
Depolarization of cerebellar granule cells in culture leads to up-regulation of the GABA_A receptor δ subunit expression. To determine the signaling molecules involved, we examined the effects of protein kinase inhibitors and cyclic AMP-elevating compounds on basal and AMPAR agonist-induced δ mRNA expression in cerebellar granule cells. Treatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 or with pituitary adenylate activating polypeptide increased δ subunit expression by 70%. Selective activation of AMPA receptors with CPW-399 also increased δ mRNA expression (2–4-fold). CPW-399 induction of δ subunit mRNA was reduced by prior treatment with either the MEK1/2 inhibitor U0126 or protein kinase A (PKA) inhibitors KT 5720 and H89. These effects were additive and combined treatment with U0126 and H89 completely prevented induction of δ subunit expression above basal levels. These results suggest that the role of JNK and ERK1/2/PKA on maintainence of δ subunit expression is diammetrically opposite. While JNK activity negatively regulates δ subunit mRNA expression in unstimulated neurons, activity of ERK1/2 and PKA are required for full induction of GABA_A receptor δ subunit expression in response to AMPA receptor stimulation.
AMPA receptors serum-dependently mediate GABA<inf>A</inf> receptor α1 and α6 subunit down-regulation in cultured mouse cerebellar granule cells
2010, Neurochemistry International
Depolarization of cultured mouse cerebellar granule cells with potassium or kainate results in developmentally arrested state that includes down-regulation of GABA_A receptor α1, α6 and β2 subunit expression. These subunits are normally strongly expressed in cerebellar granule cells from second postnatal week throughout the adulthood. In the present study we demonstrate that selective activation of AMPA subtype of glutamate receptors down-regulates α1 and α6 subunit mRNA expression. Removal of AMPA agonist from culture medium restores expression of these subunits indicating reversibility of the down-regulation. In serum-free culture medium AMPA receptor activation did not down-regulate α1 or α6 subunit expression. Furthermore, the down-regulation was strongly attenuated when the cells were cultured in the presence of dialysed fetal calf serum. The results indicate that down-regulation of GABA_A receptor α1 and α6 subunits by AMPA receptor activation is dependent on the presence of low molecular weight compounds present in fetal calf serum. In order to study mouse cerebellar granule cell maturation and/or regulation of GABA_A receptor subunit expression in culture, the experiments should be performed in the absence of fetal calf serum.

View all citing articles on Scopus

View full text

Research reportMEK inhibitors block BDNF-dependent and -independent expression of GABAA receptor subunit mRNAs in cultured mouse cerebellar granule neurons

Abstract

Introduction

Section snippets

Cerebellar cell culture

Results

Discussion

Acknowledgements

J. Biol. Chem.

Toxicol. Appl. Pharmacol.

Anal. Biochem.

FEBS Lett.

Dev. Brain Res.

Cell

J. Biol. Chem.

Cell

J. Mol. Biol.

Dev. Brain Res.

Neurosci. Lett.

Dev. Brain Res.

Dev. Brain Res.

J. Biol. Chem.

Trends Neurosci.

Trends Neurosci.

Neuron

Brain Res.

Mol. Brain Res.

Postnatal development of the cerebellar cortex in the rat: I. The external germinal layer and the transitional molecular layer

J. Comp. Neurol.

Postnatal development of the cerebellar cortex in the rat: III. Maturation of the components of the granule layer

J. Comp. Neurol.

Directing gene expression to cerebellar granule cells using γ-aminobutyric acid type A receptor α6 subunit transgene

Proc. Natl. Acad. Sci. U.S.A.

Developmental cues modulate GABAA receptor subunit mRNA expression in cultured cerebellar granule neurons

J. Neurosci.

Inhibition of ocular dominance column formation by infusion of NT-4/5 or BDNF

Science

Switching of the coupling of the β2-adrenergic receptor to different G proteins by protein kinase A

Nature

Research report
MEK inhibitors block BDNF-dependent and -independent expression of GABA_A receptor subunit mRNAs in cultured mouse cerebellar granule neurons

Developmental cues modulate GABA_A receptor subunit mRNA expression in cultured cerebellar granule neurons

Switching of the coupling of the β₂-adrenergic receptor to different G proteins by protein kinase A