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; 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）：除了传统的介导快速胆碱能传播的功能外，烟碱乙酰胆碱受体（NACHR）还准备发挥非传统功能，例如通过钙依赖性信号来调节基因转录。因此，研究烟碱信号在指导神经系统发展中起作用的作用提供了一种方法，可以评估产前暴露于尼古丁等药物的潜在不利影响。我们建议将鸡睫状神经节用作一个简单的模型系统，其中烟碱信号在体内的编程细胞死亡中的作用可以详细研究。通常在胚胎天（e）8和e14之间将睫状神经节神经元的神经元的总数降低50％，而胚胎神经元表达两个在功能上不同的NACHRS：同源α7：含有NACHR的nACHR，含有NACHR，这些NACHR被E14和e14和杂物NACHRA的Alpha3 compla ancha ancha anchra ancha anchra ancha ancha complae2亚基（alpha3 * nachrs）。我们先前的研究表明，含有NACHR的神经元α7亚基的激活导致神经元细胞死亡。我们提出，在E6-9之间，在其表面上表达高密度α7NACHR的神经元使钙的流入超过了生存的设定点，从而诱导了凋亡。在E9之后，目标相互作用作用是通过上调内源性ABTX样分子lynx的“沉默” alpha7 nachrs来防止细胞死亡通过alpha7。因此，该提议的中心假设是通过含有NACHRS的alpha7亚基的信号过多会在正常睫状神经节发育过程中诱导钙依赖性细胞死亡。为了检验这一假设，我们项目的目的是：1。确定胚胎神经元是否在细胞死亡之前就α7mRNA的水平表达了更多的异质性，以及在MLA和ABTX中拯救α7后表达α7的神经元是否保持升高； 2。确定α7NACHR亚基的过表达是否会导致对烟碱刺激的响应增强的细胞内钙水平，并加剧体内细胞死亡； 3。确定减少纤毛神经节神经元中α7NACHR表达或局部阻断α7NACHR是否会促进生存； 4。检验以下假设：靶标相互作用通过诱导Lynx的表达来防止细胞死亡，lynx是与ABTX同源的细胞表面分子，使细胞表面α7NACHRS沉默。这些研究将为详细了解烟碱信号如何促进神经系统的发展做出重大贡献。