Cholinergic and cognitive decline in response to TrkA knockdown using RNAi

描述(由申请人提供)：本小额拨款申请(R03)请求支持一项研究，该研究涉及开发一种方法，随后用于研究调节与年龄相关的认知功能损害的神经机制，以及与此相关的基底前脑(BF)胆碱能系统的调节和完整性下降。通过酪氨酸激酶受体(TrkA)受体阻断神经生长因子(NGF)信号被认为会导致BF胆碱能系统的失调，并导致通过这一主要皮质输入系统介导的认知功能受损。第一个特定目的是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。最初的实验将包括构建在RNA聚合酶II启动子控制下表达TrkA microRNA(MiRNA)的质粒，以及筛选TrkA miRNA序列以抑制大鼠嗜铬细胞瘤细胞中TrkA受体的表达。在体外筛选几种载体的基础上，我们将构建携带RNA聚合酶II驱动的TrkA miRNA表达载体的重组腺相关病毒(AAV)，用于大鼠BF神经元的体内基因传递。将构建的载体注入大鼠的BF，并对BF胆碱能神经元的病毒转染和TrkA抑制进行评估。由于非胆碱能神经元不表达TrkA受体，非胆碱能神经元的转基因有望保持无效。将采用几种控制程序来确定胆碱能神经元中潜在的非靶点/非特异性效应。第二系列实验将测试BF TrkA受体的慢性沉默导致皮质胆碱能输入释放乙酰胆碱(ACh)能力减弱的假设。最初的实验将使用胆碱敏感的微电极来确定抑制TrkA受体表达的大鼠大脑皮质中钾诱导的ACh外流减弱。最后的实验将评估持续表达TrkA受体的认知后果。由于注意表现已被广泛证明表明皮质胆碱能神经传递的状态，TrkA受体表达的持续抑制被认为会导致注意表现的持久损害，特别是动物检测注意触发线索的能力下降。这些预期的结果将为未来对TrkA受体信号的发育中断这一普遍假设的研究奠定基础，这一假设是与年龄相关的BF胆碱能神经元调节失调和相关认知功能障碍表现的一个主要变量。此外，作为该项目的一部分开发的方法将对旨在沉默体内其他神经元基因产物表达的研究具有潜在的用途。公共卫生相关性这项研究提案的预期结果将允许开发一种新方法，随后可用于探索与轻度认知障碍(MCI)和阿尔茨海默病相关的与年龄相关的认知功能损害的分子机制。

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.