RESUMEN
Reductions in brain-derived neurotrophic factor (BDNF) expression levels have been reported in the brains of patients with neurological disorders such as Alzheimer's disease. Therefore, upregulating BDNF and preventing its decline in the diseased brain could help ameliorate neurological dysfunctions. Accordingly, we sought to discover agents that increase Bdnf expression in neurons. Here, we screened a library of 42 Kampo extracts to identify those with the ability to induce Bdnf expression in cultured cortical neurons. Among the active extracts identified in the screen, we focused on the extract based on the Kampo formula daikenchuto. The extract of daikenchuto in the library used in this study was prepared using the mixture of Zingiberis Rhizoma Processum (ZIN), Zanthoxyli Piperiti Pericarpium (ZAN), and Ginseng Radix (GIN) without Koi. In this study, we defined DKT as the mixture of ZIN, ZAN, and GIN without Koi (DKT extract means the extract prepared from the mixture of ZIN, ZAN, and GIN without Koi). DKT extract significantly increased endogenous Bdnf expression by mediated, at least in part, via Ca2+ signaling involving L-type voltage-dependent Ca2+ channels in cultured cortical neurons. Furthermore, DKT extract significantly improved the survival of cultured cortical neurons and increased neurite complexity in immature neurons. Taken together, our findings suggest that DKT extract induces Bdnf expression and has a neurotrophic effect in neurons. Because BDNF inducers are expected to have therapeutic potential for neurological disorders, re-positioning of Kampo formulations such as daikenchuto may lead to clinical application in diseases associated with reduced BDNF in the brain.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo , Medicina Kampo , Humanos , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Extractos Vegetales/farmacología , Extractos Vegetales/metabolismo , Neuronas , Células CultivadasRESUMEN
Brain-derived neurotrophic factor (BDNF) is a key player in synaptic plasticity, and consequently, learning and memory. Because of its fundamental role in numerous neurological functions in the central nervous system, BDNF has utility as a biomarker and drug target for neurodegenerative and neuropsychiatric disorders. Here, we generated a screening assay to mine inducers of Bdnf transcription in neuronal cells, using primary cultures of cortical cells prepared from a transgenic mouse strain, specifically, Bdnf-Luciferase transgenic (Bdnf-Luc) mice. We identified several active extracts from a library consisting of 120 herbal extracts. In particular, we focused on an active extract prepared from Ginseng Radix (GIN), and found that GIN activated endogenous Bdnf expression via cAMP-response element-binding protein-dependent transcription. Taken together, our current screening assay can be used for validating herbal extracts, food-derived agents, and chemical compounds for their ability to induce Bdnf expression in neurons. This method will be beneficial for screening of candidate drugs for ameliorating symptoms of neurological diseases associated with reduced Bdnf expression in the brain, as well as candidate inhibitors of aging-related cognitive decline.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/genética , Corteza Cerebral/citología , Luciferasas/metabolismo , Tamizaje Masivo , Neuronas/metabolismo , Transcripción Genética , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Dopamina/metabolismo , Ginsenósidos/farmacología , Ratones Transgénicos , Neuronas/efectos de los fármacos , Extractos Vegetales/farmacología , Ratas Sprague-Dawley , Receptores del Ácido Lisofosfatídico/metabolismo , Transducción de Señal , Transcripción Genética/efectos de los fármacosRESUMEN
Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are present in the spinal dorsal horn and dorsal root ganglia, suggesting an important role of PACAP signaling systems in the modulation of spinal nociceptive transmission. Previously, we found that intrathecal injection of PACAP or maxadilan, a selective PACAP type I (PAC1) receptor agonist, induced transient aversive responses followed by a long-lasting mechanical allodynia in mice, suggesting that PACAP-PAC1 receptor systems are involved in chronic pain and that selective PAC1 antagonists may become a new class of analgesics. Although several PAC1 antagonists, such as PACAP 6-38, have been reported, all of them are peptide compounds. In the present study, we identified new small-molecule antagonists of the PAC1 receptor using in silico screening and in vitro/vivo pharmacological assays. The identified small-molecule compounds, named PA-8 and PA-9, dose dependently inhibited the phosphorylation of CREB induced by PACAP in PAC1-, but not VPAC1- or VPAC2-receptor-expressing CHO cells. PA-8 and PA-9 also dose dependently inhibited PACAP-induced cAMP elevation with an IC50 of 2.0 and 5.6 nM, respectively. In vivo pharmacological assays showed that intrathecal injection of these compounds blocked the induction of PACAP-induced aversive responses and mechanical allodynia in mice. In contrast, the compounds when administered alone exerted neither agonistic nor algesic actions in the in vitro/vivo assays. The compounds identified in the present study are new and the first small-molecule antagonists of the PAC1 receptor; they may become seed compounds for developing novel analgesics.
Asunto(s)
Simulación por Computador , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/química , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/antagonistas & inhibidores , Animales , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Evaluación Preclínica de Medicamentos , Hiperalgesia/tratamiento farmacológico , Masculino , Ratones , Simulación del Acoplamiento Molecular , Nocicepción/efectos de los fármacos , Fosforilación/efectos de los fármacos , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/uso terapéutico , Dominios Proteicos , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/química , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismoRESUMEN
Although gene transcription is controlled by neuronal activity, little is known about post-transcriptional regulation in neurons. Using cultured neurons, we found that the half-life of immediate-early gene transcripts is prolonged or shortened by membrane depolarization. Focusing on the activity-dependent stabilization of brain-derived neurotrophic factor (BDNF) mRNA, we constructed a series of plasmids, in which the short 3'-untranslated region (3'-UTR) of the BDNF gene was fused to the firefly luciferase gene, and found that the 3'-UTR prevented destabilization of luciferase mRNA through Ca(2+) signals evoked via depolarization. No such prevention was observed with the simian virus 40 late poly(A) site. The pre-mRNA covering the entire short 3'-UTR, where multiple poly(A) sites including novel ones are located, was stabilized. Deletion analyses of 3'-UTR revealed a core region (about 130 bases long) and a complementary region to be responsible for the prevention, well consistent with the formation of an extended stem-loop RNA structure and the production of poly(A) mRNAs. Thus, the mRNA stability is activity-dependently controlled in neurons and distinct regions of the 3'-UTR of BDNF mRNA are involved in stabilizing mRNA in response to Ca(2+) signals, suggesting a primary role of the RNA secondary structure affecting the availability of poly(A) sites in activity-dependent mRNA stabilization.
Asunto(s)
Regiones no Traducidas 3'/fisiología , Factor Neurotrófico Derivado del Encéfalo/genética , Regulación de la Expresión Génica/genética , ARN Mensajero/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/genética , Células Cultivadas , Corteza Cerebral/citología , Dactinomicina/farmacología , Embrión de Mamíferos , Antagonistas de Aminoácidos Excitadores/farmacología , Genes Inmediatos-Precoces/fisiología , Neuronas , Nicardipino/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Precursores del ARN/genética , Precursores del ARN/metabolismo , Ratas , Ratas Sprague-Dawley , Transfección/métodos , Valina/análogos & derivados , Valina/farmacologíaRESUMEN
In cultures of rat cortical neurons, we found that stimulation of tyrosine receptor kinase B (TrkB) with brain-derived neurotrophic factor (BDNF) induced a biphasic expression of BDNF exon IV-IX mRNA, which became obvious 1-3 h (primary induction) and 24-72 h (delayed induction) after the stimulation, and characterized the delayed induction in relation to the mRNA expression of activity-regulated cytoskeleton-associated protein (Arc). Withdrawal of BDNF from the medium after stimulation for 3 h allowed the delayed induction, which was caused at the transcriptional level and dependent upon the initial contact between exogenously added BDNF and TrkB, the effect of which was time- and dose-dependent. The primary induction was controlled by the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) whereas the secondary induction by the calcium (Ca(2+)) signaling pathway. The enhanced Arc or Zif268 mRNA expression was controlled by activation of the ERK/MAPK pathway, both of which were repressed by blocking the binding of endogenously synthesized BDNF to TrkB. Thus, robust stimulation of TrkB autonomously induces delayed BDNF mRNA expression in an activity-dependent manner in rat cortical neurons, resulting in the stimulation of Arc mRNA expression through endogenously synthesized BDNF, the process being orchestrated by the Ca(2+) and ERK/MAPK signaling pathways.