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

描述(申请人提供)：该项目的目标是阐明？7-烟碱型乙酰胆碱受体(NAChR)及其相关蛋白质组(即相互作用组)在阿尔茨海默病(AD)病理生理学中的作用，作为开发更有针对性和有效的治疗方法的载体。我们推测，在体内与海马-7-nAChRs密切相关的调节和信号蛋白在正常神经元功能中发挥着重要作用，包括那些对记忆至关重要的功能，并且随着AD的进展，？7-nAChRs的信号机制受到不利的影响，导致？7-nAChRs的蛋白质相互作用体的变化。利用高通量蛋白质组学技术和死后组织，我们将重点研究与？7-nAChR相关的细胞质蛋白质，它构成了人脑中第二丰富的尼古丁受体系统。在目标1中，我们将确定无阿尔茨海默病病史的老年人(70-75岁)死后海马组织中？7-nAChR的蛋白相互作用伙伴。通过配基亲和下拉法(β-银环蛇毒素[BGTX]-琼脂糖珠)从匀浆中分离出β7-nAChR及其相互作用蛋白。加入500M甲基乌头碱(MLA)控制匀浆，选择性地阻断β7-nAChR与BGTX-Sepharose珠的结合。结合的蛋白质将用氨基甲酰胆碱洗脱，用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分离，并制备胰酶消化液。将使用最先进的质谱学方法确定多肽的身份，然后比较在实验和对照样本中识别的蛋白质，以筛选出以非特定方式结合的蛋白质。为了探索与β7-nAChR相关的相互作用蛋白之间的群体差异，将对至少20名捐赠者的海马区组织进行表征。这些结果将作为病理组织研究的正常基线。在目标2中，我们将确定晚期AD患者死后海马组织中？7-nAChR相互作用蛋白的组成是如何改变的。AD样本的蛋白质组数据将与AIM 1的蛋白质组数据进行比较，以详细分析AD对？7-nAChR蛋白质相互作用的影响。在目标3中，我们将对在特定目标1和2中收集的数据进行无标记定量质谱分析，以确定AD如何改变与？7-nAChR相互作用的蛋白质的相对水平。在AIMS 1和2中收集的质谱学数据将被重新进行生物信息学分析，以量化？7-nAChR相关蛋白质的变化。来自混合LTQ-Orbitrap Velos ETD仪器的质谱学数据将产生数据，从中可以得出鉴定和定量结论。AIMS 1和AIMS 2的数据将被比较，以确定AD的病理生理机制导致的蛋白质水平的变化。 公共卫生相关性：利用高通量技术和人类尸检组织，我们将研究与重要的人脑受体密切相关的细胞蛋白在阿尔茨海默病中是如何受到影响的。我们将采用蛋白质组学的方法来确定与尼古丁α7受体相关的蛋白质的组成和功能的变化是否与疾病相关。我们提出的研究结果应该有助于更好地理解尼古丁α7受体在阿尔茨海默病中的作用。未来对拟议工作中确定的蛋白质的详细研究可能会导致开发出更具针对性的治疗这种疾病的方法。

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.