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

阿尔茨海默病对海马 α7-nAChR 蛋白相互作用蛋白的影响

• 批准号: 8114563
• 负责人: Edward Hawrot
• 金额: $ 15.91万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8114563
• 关键词: Address; Adult; Affect; Affinity; Age; Alzheimer' s Disease; Area; Atrophic; Behavior; Behavioral; Binding; Binding Proteins; Biochemistry; Brain; Brain Diseases; Bungarotoxins; Carbamoylcholine; Complex; Cytoplasmic Protein; Data; Data Quality; Dementia; Detergents; Development; Diagnosis; Disease; Elderly; Emotions; Event; Functional disorder; Future; Goals; Healthcare Systems; Hippocampus (Brain); Human; Hybrids; Image; Individual; Label; Lead; Learning; Ligands; Link; Macromolecular Complexes; Mass Spectrum Analysis; Memory; Methods; Molecular; Mus; Neuraxis; Neurons; Nicotinic Receptors; Palliative Care; Pathology; Peptides; Physiological; Play; Population; Prevalence; Proteins; Proteome; Proteomics; Recording of previous events; Relative (related person); Role; SHPS-1 protein; Sampling; Sepharose; Signal Transduction; Staging; System; Technology; Tissues; Universities; Variant; Work; aged; alpha-bungarotoxin receptor; cholinergic; cognitive change; density; high throughput technology; hippocampal atrophy; human RIPK1 protein; in vivo; instrument; mass spectrometer; methyllycaconitine; portion control; protein function; receptor; receptor expression; therapy development

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.

描述（由申请人提供）：这个项目的目标是阐明？7-烟碱乙酰胆碱受体（nAChR）及其相关蛋白质组（即相互作用组）在阿尔茨海默病（AD）病理生理中的作用，作为开发更有针对性和更有效治疗的载体。我们假设体内与海马？7- nachr在正常的神经元功能中起着重要作用，包括那些对记忆至关重要的功能。随着AD的进展，7大分子复合物受到不利影响，导致- 7- nachr蛋白相互作用组发生变化。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，它构成了人脑中第二丰富的尼古丁受体系统。在Aim 1中，我们将确定？无AD病史的老年受试者（70-75岁）死后海马组织中的7-nAChR。的吗?7-nAChR及其相互作用蛋白将通过配体亲和下拉(？金环蛇毒素(Bgtx)琼脂糖珠)。500年?将M甲基莱卡乌碱（MLA）添加到对照匀浆中，以选择性阻断？7-nAChR到Bgtx-Sepharose珠。结合蛋白用氨甲酰胆碱洗脱，SDS-PAGE分离，胰酶消化。肽的特性将使用最先进的质谱方法确定，然后在实验和对照样品中鉴定的蛋白质将进行比较，以过滤掉那些以非特异性方式结合的蛋白质。将对至少20个供体的海马组织进行表征，以探索与- 7-nAChR相关的相互作用蛋白之间的群体差异。这些结果将作为病理组织研究的正型基线。在Aim 2中，我们将确定如何组成？7-nAChR相互作用蛋白在晚期AD患者死后海马组织中发生改变。AD样品的蛋白质组学数据将与Aim 1的蛋白质组学数据进行比较，以便详细分析AD对7-nAChR蛋白质相互作用物的影响。在目标3中，我们将对特异性目标1和2中收集的数据使用无标记定量质谱法，以确定与蛋白质相互作用的相对水平。7-nAChR被AD改变。目标1和目标2中收集的质谱数据将重新进行生物信息学分析，以量化？7-nAChR相关蛋白。混合LTQ-Orbitrap Velos ETD仪器的质谱数据将产生可以得出鉴定和定量结论的数据。Aims 1和Aims 2的数据将被比较，以确定由AD的病理生理机制引起的蛋白水平的改变。