Effects of Alzheimer's disease on hippocampal alpha7-nAChR protein interactors

描述（由申请人提供）：本项目的目标是阐明的作用？7-烟碱乙酰胆碱受体（nAChR）及其相关蛋白质组（即，本发明的目的是提供一种在阿尔茨海默病（AD）的病理生理学中作为开发更有针对性和更有效的治疗的媒介物的药物组合物（interactome）。我们推测，在体内与海马？7-nAChRs在正常的神经元功能中起着重要的作用，包括那些对记忆至关重要的神经元功能，并且nAChRs的信号机制？7大分子复合物是不利的影响，作为AD的进展，导致蛋白质相互作用组的变化？7-nAChR。使用高通量蛋白质组学技术和死后组织，我们将专注于细胞质蛋白与？7-nAChR，其构成人脑中第二丰富的烟碱受体系统。在目的1，我们将确定蛋白质相互作用的合作伙伴？7-无AD病史的老年受试者（70-75岁）死后海马组织中的nAChR。什么？7-nAChR及其相互作用蛋白将通过配体亲和下拉法（？-银环蛇毒素[Bgtx]-琼脂糖珠）。五百？将M-甲基乌头碱（MLA）加入到对照匀浆中，以选择性地阻断？7-将nAChR与Bgtx-琼脂糖珠结合。结合的蛋白质将用氨甲酰胆碱洗脱，通过SDS-PAGE分级分离，并制备胰蛋白酶解物。将使用最先进的质谱方法确定肽身份，然后将比较实验和对照样品中鉴定的蛋白质，以过滤出以非特异性方式结合的蛋白质。来自至少20个供体的海马组织将被表征，以探测与？7-nAChR。这些结果将作为病理组织研究的正常型基线。在目标2中，我们将确定如何组成？7-晚期AD患者死后海马组织中nAChR相互作用蛋白发生改变。AD样本的蛋白质组数据将进行比较，从目标1的蛋白质组数据，让详细分析AD的蛋白质相互作用的影响？7-nAChR。在目标3中，我们将使用无标记定量质谱法对特定目标1和2中收集的数据进行分析，以确定蛋白质的相对水平如何与蛋白质相互作用？7-nAChR被AD改变。将对目标1和2中收集的质谱数据进行生物信息学重新分析，以量化？7-nAChR相关蛋白。来自混合LTQ-Orbitrap Velos ETD仪器的质谱数据将生成可从中得出鉴定和定量结论的数据。将比较目标1和2的数据，以确定由AD的病理生理机制引起的蛋白质水平的改变。 公共卫生相关性：使用高通量技术和人类死后组织，我们将研究与重要的人脑受体密切相关的细胞蛋白质如何在阿尔茨海默病中受到影响。我们将采用蛋白质组学方法来确定与烟碱α 7受体相关的蛋白质的组成和功能的变化是否与疾病相关。从我们提出的研究结果应该导致更好地了解烟碱α 7受体在阿尔茨海默病中的作用。未来对拟议工作中待鉴定的蛋白质的详细研究可能会导致开发针对这种疾病的更高靶向疗法。

DESCRIPTION (provided by applicant): The goal of this project is to elucidate the role of the ?7- nicotinic acetylcholine receptor (nAChR) and its associated proteome (i.e., interactome) in the pathophysiology of Alzheimer's Disease (AD) as a vehicle towards developing more targeted and efficacious treatments. We hypothesize that the regulatory and signaling proteins closely associated in vivo with hippocampal ?7-nAChRs play an important role in normal neuronal functions including those critical to memory, and that the signaling machinery of the ?7 macromolecular complex is adversely affected as AD progresses resulting in changes in the protein interactome of ?7-nAChRs. Using high-throughput proteomic technologies and post mortem tissue, we will focus on the cytoplasmic proteins associated with the ?7-nAChR, which constitutes the second most abundant nicotinic receptor system in the human brain. In Aim 1, we will determine the protein interacting partners of the ?7-nAChR in post mortem hippocampal tissue of aged subjects (70-75 years) with no history of AD. The ?7-nAChR and its interacting proteins will be isolated from the homogenate by ligand affinity pulldown (?-bungarotoxin[Bgtx]-Sepharose beads). 500 ?M methyllycaconitine (MLA) will be added to control homogenates to block selectively the binding of ?7-nAChR to the Bgtx-Sepharose beads. Bound proteins will be eluted with carbamylcholine, fractionated by SDS-PAGE, and tryptic digests prepared. The peptide identities will be determined using state-of-the-art mass spectrometric methods, and then proteins identified in experimental and control samples will be compared to filter out those that bind in a nonspecific fashion. Hippocampal tissue from at least 20 donors will be characterized in order to probe the population variation among interacting proteins associated with ?7-nAChR. These results will serve as the normotypic baseline for studies of pathological tissue. In Aim 2, we will determine how the composition of ?7-nAChR interacting proteins is altered in the post mortem hippocampal tissue of individuals with late stage AD. The proteomic data from AD samples will be compared to the proteomic data from Aim 1 to allow detailed analysis of the effects of AD on the protein interactors of the ?7-nAChR. In Aim 3, we will use label-free quantitative mass spectrometry on data collected in Specific Aims 1 and 2 to determine how the relative levels of proteins that interact with the ?7-nAChR are altered by AD. The mass spectrometry data collected in Aims 1 and 2 will be re-analyzed bioinformatically to quantify changes in ?7-nAChR associated proteins. The mass spectrometry data from a hybrid LTQ-Orbitrap Velos ETD instrument would generate data from which both identifying and quantitative conclusions could be drawn. The data from Aims 1 and 2 would be compared to determine alterations in protein levels caused by the pathophysiological mechanisms of AD. PUBLIC HEALTH RELEVANCE: Using a high-throughput technology and human post mortem tissue, we will study how cellular proteins found in close working association with an important human brain receptor are affected in Alzheimer's disease. We will pursue a proteomic approach to determine whether changes in the composition and function of the proteins found associated with nicotinic alpha7 receptors can be correlated with the disease. The results from our proposed studies should lead to a better understanding of the role of nicotinic alpha7 receptors in Alzheimer's disease. Future detailed study of the proteins to be identified in the proposed work could lead to the development of more highly targeted therapies for this disease.