Nicotinic Acetylcholine Receptors in Neural Development

神经发育中的烟碱乙酰胆碱受体

• 批准号: 7093071
• 负责人: Rae Nishi
• 金额: $ 33.4万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-15 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7093071
• 关键词: RNA interference; apoptosis; calcium flux; calcium indicator; chick embryo; ciliary ganglion; developmental neurobiology; embryo /fetus toxicology; female; gene induction /repression; glycosylphosphatidylinositols; immunocytochemistry; immunoprecipitation; laboratory mouse; molecular /cellular imaging; neurogenesis; nicotine; nicotinic receptors; nonmammalian vertebrate embryology; protein structure; protein structure function; receptor expression; voltage /patch clamp; western blottings

DESCRIPTION (provided by applicant): In addition to their traditional function of mediating rapid cholinergic transmission, nicotinic acetylcholine receptors (nAChRs) are poised to serve non-traditional functions such as regulating gene transcription through calcium-dependent signaling. Therefore, studying the role that nicotinic signaling plays in guiding the development of the nervous system provides a means for assessing the potential adverse effects of prenatal exposure to drugs such as nicotine. We propose to use the chicken ciliary ganglion as a simple model system in which the role of nicotinic signaling in programmed cell death in vivo can be studied in detail. Programmed cell death normally reduces the total number of neurons in the ciliary ganglion by 50% between embryonic days (E)8 and E14, and embryonic neurons express two functionally distinct populations of nAChRs: homomeric alpha7 containing nAChRs that become extrasynaptically localized by E14, and heteromeric nAChRs containing alpha3, alpha5, Beta2 and Beta4 subunits (alpha3 * nAChRs). Our previous studies suggested that activation of neuronal alpha7 subunit containing nAChRs caused neuronal cell death. We propose that between E6-9, neurons that express a high density of alpha7 nAChRs on their surfaces allow calcium influx that exceeds the set point for survival, thereby inducing apoptosis. After E9, target interactions act to prevent cell death through alpha7 by upregulating an endogenous abtx-like molecule, lynx, that "silences" alpha7 nAChRs. Thus, the central hypothesis of this proposal is that an overabundance of signaling through alpha7 subunit containing nAChRs induces calcium-dependent cell death during normal ciliary ganglion development. To test this hypothesis, the aims of our project are: 1. To determine whether embryonic neurons express more heterogeneity with respect to the levels of alpha7 mRNA prior to cell death and whether neurons expressing elevated alpha7 remain after rescue by MLA and abtx; 2. To determine whether overexpression of alpha7 nAChR subunits causes enhanced levels of intracellular calcium in response to nicotinic stimulation and exacerbates cell death in vivo; 3. To determine whether reducing alpha7 nAChR expression or locally blocking alpha7 nAChRs in ciliary ganglion neurons promotes survival; 4. To test the hypothesis that target- interactions prevent cell death by inducing the expression of lynx, a cell surface molecule with homology to abtx that silences cell surface alpha7 nAChRs. These studies will make a significant contribution towards the detailed understanding of how nicotinic signaling contributes to the development of the nervous system.

描述（由申请人提供）：除了介导快速胆碱能传递的传统功能外，烟碱乙酰胆碱受体（nAChRs）还具有非传统功能，如通过钙依赖性信号调节基因转录。因此，研究尼古丁信号在指导神经系统发育中的作用，为评估产前暴露于尼古丁等药物的潜在不良影响提供了一种手段。我们建议使用鸡睫状神经节作为一个简单的模型系统，在体内可以详细研究尼古丁信号在程序性细胞死亡中的作用。在胚胎期(E)8和E14之间，程序性细胞死亡通常会使睫状神经节中的神经元总数减少50%，并且胚胎神经元表达两种功能不同的nAChRs群体：同源的含有被E14定位的nAChRs的α 7，以及含有α 3、α 5、β 2和β 4亚基的异源的nAChRs （α 3 * nAChRs）。我们之前的研究表明，含有nAChRs的神经元α 7亚基的激活导致神经元细胞死亡。我们认为，在E6-9之间，神经元表面表达高密度的alpha7 nachr，允许钙流入超过生存设定值，从而诱导细胞凋亡。在E9之后，靶标相互作用通过上调内源性abtx样分子lynx来阻止alpha7 nachr的死亡。因此，本研究的中心假设是，在正常的睫状神经节发育过程中，通过含有nAChRs的α 7亚基过量的信号传导诱导钙依赖性细胞死亡。为了验证这一假设，我们项目的目标是：1。确定胚胎神经元在细胞死亡前是否表达更多的α 7 mRNA水平的异质性，以及MLA和abtx拯救后α 7表达升高的神经元是否仍然存在；2. 确定α 7 nAChR亚基的过表达是否会导致细胞内钙水平升高，从而对尼古丁刺激做出反应，并在体内加剧细胞死亡；3. 确定减少睫状神经节神经元中alpha7 nAChR的表达或局部阻断alpha7 nAChR是否能促进存活；4. 为了验证靶标相互作用通过诱导lynx的表达来防止细胞死亡的假设，lynx是一种与abtx同源的细胞表面分子，可以沉默细胞表面的alpha7 nAChRs。这些研究将对详细了解尼古丁信号如何促进神经系统发育做出重大贡献。