RESUMO
Neuronal activity increases synthesis of brain-derived neurotrophic factor (BDNF) mRNA in vivo and in vitro. We have investigated the pathways through which neuronal activity stimulated by kainic acid regulates BDNF mRNA levels in cultured hippocampal neurons and transgenic mice. Kainic acid induced the transcription of BDNF mRNA without influencing the mRNA stability. Interestingly, the half-life of the 4.2 kb BDNF transcript was much shorter than that of the 1.6 kb transcript (23 +/- 4 min. vs. 132 +/- 30 min). Increase in the BDNF mRNA levels by kainic acid was not blocked by the protein synthesis inhibitor cycloheximide demonstrating that BDNF is regulated as an immediate early gene in hippocampal neurons. Although calmodulin antagonists are known to abolish the effect of kainic acid on BDNF mRNA, this effect was very similar in Ca(+2)-calmodulin-dependent protein kinase II alpha knock-out mice and in wild-type mice. Surprisingly, even high doses of kainic acid failed to increase nerve growth factor (NGF) mRNA in mouse hippocampus although elevation in rat brain has been consistently observed.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Hipocampo/fisiologia , Neurônios/fisiologia , Transcrição Gênica , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/deficiência , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Genes Precoces , Ácido Caínico/farmacologia , Camundongos , Camundongos Knockout , Fatores de Crescimento Neural/genética , RNA Mensageiro/genética , Ratos , Transcrição Gênica/efeitos dos fármacosAssuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica , Glucocorticoides/fisiologia , Fatores de Crescimento Neural/biossíntese , Aldosterona/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Linhagem Celular , Corticosterona/farmacologia , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Nervos Periféricos/efeitos dos fármacos , Nervos Periféricos/fisiologia , Regiões Promotoras Genéticas , TransfecçãoRESUMO
The structure of the mouse neurotrophin-3 (NT-3) gene has been analysed using genomic cloning and the rapid amplification of cDNA ends (RACE) method. The gene consists of two small upstream exons (exons IA and IB) and a larger downstream exon (exon II) that encodes the mature protein. Two classes of NT-3 transcripts, termed transcripts A and B, are generated by alternative splicing of exon IA or exon IB to the common exon II. The NT-3 gene also contains several transcription start sites in both upstream exons, and three different polyadenylation sites in exon II, as shown by RNase protection assays and by RACE, giving rise to multiple NT-3 mRNA variants of slightly different lengths. Cerebellar granule neurons express both classes of NT-3 transcripts, but only transcript B is regulated by tri-iodothyronine (T3) in these neurons. The effect of T3 on NT-3 mRNA is primarily due to transcription enhancement, as shown in nuclear run-on experiments. The levels of NT-3 mRNA are much lower in cultured mouse astrocytes and are undetectable in the human neuroblastoma cell line IMR 32. A TATA box is present in the upstream region of exon IB but not in that of exon IA. Promoter analysis using the chloramphenicol acetyltransferase reporter gene fused to different NT-3 upstream regions showed the presence of two active NT-3 promoters in cerebellar granule neurons. However, in IMR 32 cells, NT-3 promoter activity decreased dramatically with increasing length of the 5' flanking region. This suggests that expression of the NT-3 gene is regulated both by positive influences, such as T3, and by negative silencing elements present in the upstream regions of the NT-3 promoter.
Assuntos
Fatores de Crescimento Neural/genética , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Éxons , Regulação da Expressão Gênica/efeitos dos fármacos , Genes , Camundongos , Dados de Sequência Molecular , Fatores de Crescimento Neural/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Neurotrofina 3 , RNA Mensageiro/metabolismo , Tri-Iodotironina/farmacologiaRESUMO
Neurotrophin-3 (NT-3) is a member of the neurotrophin gene family and is highly expressed in the developing rat cerebellum. Here we show that brain-derived neurotrophic factor (BDNF) increased by approximately 10-fold the NT-3 mRNA levels in cultured cerebellar granule neurons isolated from postnatal rats, whereas nerve growth factor (NGF) and NT-3 itself had no effect. The effect of BDNF was additive to that of triiodothyronine (T3), which also increased NT-3 mRNA in these neurons. The drug K252a inhibited the BDNF-mediated stimulation of NT-3 expression, suggesting an involvement of trkB receptors. Nuclear run-on experiments showed that BDNF enhanced NT-3 transcription, whereas the stability of NT-3 mRNA remained unchanged. The data presented are the first demonstration that one neurotrophin regulates the expression of another and provide evidence that NT-3 production in granule neurons is regulated by both BDNF and T3.
Assuntos
Cerebelo/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/farmacologia , Animais , Fator Neurotrófico Derivado do Encéfalo , Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Neurotrofina 3 , RNA Mensageiro/genética , Ratos , Transcrição Gênica/efeitos dos fármacos , Tri-Iodotironina/farmacologiaRESUMO
Thyroid hormones play an important role in brain development, but the mechanism(s) by which triiodothyronine (T3) mediates neuronal differentiation is poorly understood. Here we demonstrate that T3 regulates the neurotrophic factor, neurotrophin-3 (NT-3), in developing rat cerebellar granule cells both in cell culture and in vivo. In situ hybridization experiments showed that developing Purkinje cells do not express NT-3 mRNA but do express trkC, the putative neuronal receptor for NT-3. Addition of recombinant NT-3 to cerebellar cultures from embryonic rat brain induces hypertrophy and neurite sprouting of Purkinje cells, and upregulates the mRNA encoding the calcium-binding protein, calbindin-28 kD. The present study demonstrates a novel interaction between cerebellar granule neurons and developing Purkinje cells in which NT-3 induced by T3 in the granule cells promotes Purkinje cell differentiation.
