Regulation of Gene Expression in Striatal Neurons

描述(由申请人提供)： 受体介导的成熟中枢神经系统神经元的基因表达仍然是阐明神经可塑性发展的分子机制的热点。作为基底节的关键结构，纹状体是研究这类基因表达的主要中心部位之一，旨在揭示各种精神疾病(精神和认知障碍)的纹状体机制。代谢性谷氨酸受体(MGluRs)在纹状体投射神经元中密集表达。过去一段时间的研究表明，钙离子连接的I组mGluRs对大鼠纹状体阿片肽基因(前强啡肽和前脑啡肽)的表达起着重要的调节作用。在鉴定I组mGluR信号转导到基因表达的细胞内信号通路的过程中，我们最近发现，I组受体的激活激活了丝裂原激活的蛋白激酶(MAPKs)。因此，作为细胞膜和细胞核之间的信息高速公路，MAPK通路很可能将I组mGluR偶联到基因表达。在这个延续方案中，提出了一系列实验来评估一个重要的假设，即I组mGluRs激活MAPK级联以促进纹状体神经元中阿片类基因的表达。利用多学科的方法，这一假说将在体内和体外的纹状体神经元中得到验证：(1)通过在活体大鼠模型中表征I组mGluR调节的一个主要MAPK亚类，细胞外信号调节激酶1和2(erki/2)的磷酸化，确定I组受体在MAPK调节中的作用；(2)利用亚型选择性激动剂/拮抗剂、反义寡核苷酸和突变小鼠(mGluR1或5l基因敲除)，区分I组两个亚型mGluR1和I mGluR5的相对重要性。(3)在含有选择性抑制剂、反义寡核苷酸和沉默siRNAs的原代培养的纹状体神经元模型中，通过评估钙信号和蛋白激酶(CaMKII、PI3-K、PKC和酪氨酸激酶)的作用，确定I组mGluR信号向MAPK/ERK信号转导的信号通路；(4)通过评估MAPK/ERK两个转录因子靶标Elk-1和CREB在体内和体外对I/ERK敏感的前强啡肽和脑啡肽原基因表达的重要性，确定I组mGluR激活的MAPK/ERK级联信号通路在纹状体基因表达调控中的生理作用。该项目的完成将为成熟中枢神经系统神经元诱导基因表达的受体和信号转导机制提供新的解释。由于可诱导基因表达被认为是神经可塑性发展的重要组成部分，来自该项目的数据最终可以通过靶向ImGluRs和MAPK级联通路来帮助开发新的药物疗法，用于治疗由纹状体功能障碍引起的各种精神疾病。

DESCRIPTION (provided by applicant): Receptor-mediated gene expression in matured CNS neurons remains a hot topic in elucidating molecular mechanisms for the development of neuroplasticity. As a key structure of the basal ganglia, the striatum is among the major central sites for investigating such gene expression aimed to unravel striatal mechanisms underlying a variety of mental illnesses (psychiatric and cognitive disorders). Metabotropic glutamate receptors (mGluRs) are densely expressed in the striatal projection neurons. From studies conducted in the last period, we have established that Ca2+ -Iinked group I mGluRs play a profound role in regulating opioid peptide gene expression (prodynorphin and proenkephalin) in the rat striatum. In the effort of identifying intracellular signaling pathways transducing group I mGluR signals to the gene expression, we recently found that activation of group I receptors activates mitogen-activated protein kinases (MAPKs). As an information superhighway between the surface membrane and the nucleus, the MAPK pathway therefore likely couples group I mGluRs to gene expression. In this continuation proposal, a series of experiments was proposed to evaluate an overarching hypothesis that group I mGluRs activate MAPK cascades to facilitate opioid gene expression in striatal neurons. Using multidisciplinary approaches, this hypothesis will be tested both in vivo and in vitro n striata neurons in four general experiments: (1) define the role of group I receptors in the regulation of MAPKs by characterizing group I I mGluR-regulated phosphorylation of a major MAPK subclass, extracellular signal-regulated kinase 1 and 2 (ERKI/2), in a I well-characterized in vivo rat model, (2) differentiate the relative importance of the two group I subtypes, mGluR1 and I mGluR5, in this event using the subtype-selective agonists/antagonists, antisense oligos and mutant mice (mGluR1 or 5l knockouts) in vivo, (3) identify signaling pathways transducing group I mGluR signals to MAPK/ERK by evaluating roles of Ca 2+ signals and protein kinases (CaMKII, PI3-kinase, PKC, and tyrosine kinases) in our primary striatal neuronal culture model with selective inhibitors, antisense oligos and silencer siRNAs, and (4) define physiological roles of group I mGluRactivated MAPK/ERK cascades in the regulation of striatal gene expression via evaluating the importance of the two transcription factor targets of MAPK/ERK, Elk-1 and CREB, for group I/ERK-sensitive prodynorphin and proenkephalin gene expression in vivo and in vitro. Accomplishment of this project will provide a new interpretation of receptor and signal transduction mechanisms underlying inducible gene expression in matured CNS neurons. Since inducible gene expression is conceived to be an important component of the development of neuroplasticity, data from this project can ultimately contribute to the development of novel pharmacotherapies, by targeting group ImGluRs and MAPK cascades, for the treatment of various mental illnesses resulted from striatal dysfunctions.