Cholinergic and cognitive decline in response to TrkA knockdown using RNAi

描述（由申请人提供）：本申请申请小额拨款（R03），要求支持涉及开发一种方法的研究，该方法将随后用于研究介导认知功能中与年龄相关的损伤发展的神经元机制，以及相关的基底前脑（BF）胆碱能系统调节和完整性的下降。通过酪氨酸激酶受体（TrkA）受体的神经生长因子（NGF）信号的破坏被假设导致BF胆碱能系统的失调，并通过这一主要的皮层输入系统介导认知功能的损害。第一个具体目标是在体外和体内构建和测试病毒介导的RNA干扰（RNAi），以抑制BF胆碱能神经元TrkA受体的表达。最初的实验将包括在RNA聚合酶II启动子的控制下构建表达TrkA microRNA （miRNA）的质粒，并筛选TrkA miRNA序列以抑制大鼠嗜铬细胞瘤细胞中TrkA受体的表达。在体外筛选多种质粒的基础上，我们将构建重组腺相关病毒（AAV）载体，携带表达RNA聚合酶II驱动TrkA miRNA的质粒，用于大鼠BF神经元的体内基因传递。将构建物注入大鼠BF，观察病毒转染BF胆碱能神经元和TrkA抑制情况。由于非胆碱能神经元不表达TrkA受体，因此转染非胆碱能神经元预计仍然无效。将采用几种控制程序来确定胆碱能神经元中潜在的脱靶/非特异性效应。第二系列实验将验证BF TrkA受体的长期沉默导致皮质胆碱能输入释放乙酰胆碱（ACh）的能力衰减的假设。最初的实验将采用胆碱敏感微电极来测定显示TrkA受体表达抑制的大鼠皮层中钾诱发的乙酰胆碱外排。最后的实验将评估持续TrkA受体表达的认知后果。由于注意力表现已被广泛证明表明皮层胆碱能神经传递的状态，TrkA受体表达的持续抑制被假设会导致注意力表现的持久损伤，特别是动物检测注意力触发线索的能力下降。预期的结果将为未来的一般假设的研究奠定基础，即TrkA受体信号的发育中断是BF胆碱能神经元年龄相关失调和相关认知功能障碍的主要变量。此外，作为该项目的一部分开发的方法将潜在地用于旨在沉默体内其他神经元基因产物表达的研究。这项研究计划的预期结果将允许开发一种新方法，可随后用于探索与轻度认知障碍（MCI）和阿尔茨海默病相关的认知功能中年龄相关损伤的分子机制。

DESCRIPTION (provided by applicant): This application for a small grant (R03) requests support for research involving the development of a method to be used subsequently in research on the neuronal mechanisms mediating the development of age-related impairments in cognitive functions and the associated decline in the regulation and integrity of the basal forebrain (BF) cholinergic system. Disruption of nerve growth factor (NGF) signaling via tyrosine kinase receptor (TrkA) receptors has been hypothesized to result in the dysregulation of the BF cholinergic system and in impairments of the cognitive functions mediated via this major cortical input system. The first specific aim focuses on the construction and testing, in vitro and in vivo, of viral-mediated RNA interference (RNAi) to suppress the expression of TrkA receptors by BF cholinergic neurons. The initial experiments will involve the construction of plasmids designed to express TrkA microRNA (miRNA) under the control of a RNA polymerase II promoter, and screening of TrkA miRNA sequences for suppression of TrkA receptor expression in rat pheochromocytoma cells. Based on the screening of several plasmids in vitro, we will construct recombinant adeno-associated virus (AAV) vectors carrying plasmids expressing RNA polymerase II driven TrkA miRNA for in vivo gene delivery in rat BF neurons. Constructs will be infused into the BF of rats and viral transfection of BF cholinergic neurons and TrkA suppression will be evaluated. As non-cholinergic neurons do not express TrkA receptors, transfection of non-cholinergic neurons is expected to remain ineffective. Several control procedures will be employed to determine potential off-target/non-specific effects in cholinergic neurons. A second series of experiments will test the hypothesis that chronic silencing of BF TrkA receptors result in the attenuation of the capacity of cortical cholinergic inputs to release acetylcholine (ACh). Initial experiments will employ choline-sensitive microelectrodes to determine attenuated potassium-evoked ACh efflux in the cortex of rats exhibiting suppression of TrkA receptor expression. The final experiments will assess the cognitive consequences of persistent TrkA receptor expression. As attentional performance has been extensively demonstrated to indicate the status of cortical cholinergic neurotransmission, persistent suppression of TrkA receptor expression is hypothesized to result in enduring impairments in attentional performance, specifically a decrease in the animal's ability to detect attention-triggering cues. The expected results will form the basis for future research on the general hypothesis that developmental disruption of TrkA receptor signaling represents a major variable in the manifestation of age-related dysregulation of BF cholinergic neurons and associated impairments in cognitive functions. Moreover, the methods developed as part of this project will be of potential use for research aiming at silencing the expression of other neuronal gene products in vivo. PUBLIC HEALTH RELEVANCE The expected results of this research proposal will allow the development of a new method that can subsequently be used in exploring molecular mechanisms underlying age-related impairments in cognitive functions associated with mild cognitive impairment (MCI) and Alzheimer's disease.