MOLECULAR MECHANISMS REGULATING NPY SYNTHESIS IN THE CNS

这是一个申请研究科学家发展奖II级的请求。 神经肽被认为是信息的组成部分 中枢神经系统(CNS)的转移。它们广泛存在，但 离散地、分布和从属于许多生理和 电生理功能。分子生物学的最新进展 使cDNA和编码基因的克隆和测序成为可能 各种神经肽的前体蛋白。这些研究 提供了神经肽基因表达的信息，前体 蛋白质结构和神经肽表达的明显异常 在几种中枢神经系统疾病中。最近发现的一种多肽， 神经肽Y(NPY)是迄今在中枢神经系统中发现的含量最丰富的多肽 并兼有以下生理功能：调节 心血管系统、摄食行为和皮质功能。尽管 神经肽Y的cDNAs和基因的鉴定，鲜为人知 关于神经肽Y生物合成和基因表达的调控 中枢神经系统。拟议的研究将解决NPY合成的调控问题 通过“突触外”和“跨突触”两种机制。“额外的-- “突触”对下丘脑NPY合成的调节将被研究 在设计用来研究分子机制的模型系统中 规范进食行为。拟议的研究将在 体内和体外葡萄糖和胰岛素对下丘脑NPY的调节 正常动物和遗传性肥胖模型中的神经元(Zucker 肥鼠)。探讨神经肽Y的“跨突触”调节 合成，将在体外制备海马和下丘脑切片 以及各种药理制剂的效果，这些药剂 直接调节神经肽Y的表达或调节海马神经元 活动，将被确定。神经肽Y的表达和调控 合成将通过核连续分析、核酸酶保护来监测 前ProNPY信使核糖核酸含量、生物合成标记和 神经肽Y的纯化及放射免疫分析。拟议的研究将 将定义良好的模型系统与各种分子相结合 生物技术，以独特的方式解决问题的若干方面 神经肽Y的表达和合成的调控。此外， 拟议的研究将提供对两个国家之间的相互作用的见解 外周和中枢神经系统调节神经肽的表达，因此是 理解机制多样性的根本重要性 调节行为和中枢神经系统功能。

This is a request for a Research Scientist Development Award Level II. Neuropeptides are recognized as integral components of information transfer in the central nervous system (CNS). They are widely, but discretely, distributed and subserve a number of physiological and electrophysiological functions. Recent progress in molecular biology has allowed the cloning and sequencing of the cDNAs and genes encoding the precursor proteins for a variety of neuropeptides. These studies have provided information on neuropeptide gene expression, precursor protein structure, and apparent abnormalities in neuropeptide expression in several CNS disorders. One recently identiifged peptide, neuropeptide Y (NPY), is the most abundant peptide yet found in the CNS and subserves such physiological functions as regulation of the cardiovascular system, feeding behavior and cortical function. Despite the identification of the cDNA and gene for NPY, little is known regarding the regulation of NPY biosynthesis and gene expression in the CNS. The proposed research will address the regulation of NPY synthesis by both "extra-synaptic" and "Transsynaptic" mechanisms. The "extra- synaptic" modulation of hypothalamic NPY synthesis will be investigated in model systems designed to investigate the molecular mechanisms regulating feeding behavior. The proposed studies will determine in vivo and in vitro how glucose and insulin modulate hypothalamic NPY neurons in normal animals and in a model of genetic obesity (the Zucker fatty rat). To investigate "trans-synaptic" regulation of NPY synthesis, in vitro hippocampal and hypothalamic slices will be prepared and the effects of various pharmacological agents, known to either directly modulate NPY expression or to modulate hippocampal neuronal activity, will be determined. Modulation of NPY expression and synthesis will be monitored by nuclear run-on assay, nuclease protection analysis of preproNPY mRNA content, biosynthetic labeling and purification of NPY and radioimmunoassay. The proposed studies will combine well-defined model systems with a variety of molecular biological techniques to uniquely address several aspects of the regulation of the expression and synthesis of NPY. Moreover, the proposed studies will provide insights into the interactions between the periphery and CNS to modulate neuropeptide expression and, thus, are of fundamental importance to understanding the diversity of mechanisms regulating behavior and CNS function.