INV OF ASTROCYTE CASPASE ACTV & CD40/CD40L SIGNALING INTERACTIONS IN ALZHEIMER?S

星形胶质细胞天冬氨酸蛋白酶活性的 INV

• 批准号: 7609925
• 负责人: TROY T ROHN
• 金额: $ 6.2万
• 依托单位: UNIVERSITY OF IDAHO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7609925
• 关键词: Address; Adopted; Age; Alzheimer' s Disease; Antibodies; Astrocytes; Autopsy; Biological Models; Brain; Calpain; Calpain I; Caspase; Cell Extracts; Cell Line; Cell model; Cell-Free System; Cells; Circular Dichroism; Cleaved cell; Computer Retrieval of Information on Scientific Projects Database; Condition; Disease; Drug Delivery Systems; Electron Microscopy; Endopeptidases; Ensure; Erythrocytes; Event; Evolution; Funding; Generations; Grant; Human; Immunohistochemistry; In Vitro; Institution; Ischemia; Length; Link; Methods; Microscopy; Molecular Conformation; Molecular Weight; N-terminal; Neuroblastoma; Neurodegenerative Disorders; Neurons; Numbers; Outcome; PC12 Cells; Pathway interactions; Patients; Peptide Hydrolases; Physiological reperfusion; Process; Production; Protein Fragment; Protein Overexpression; Proteins; Proteolytic Processing; Recombinants; Reperfusion Therapy; Research; Research Personnel; Resources; Role; Sampling; Seeds; Signal Transduction; Site; Site-Directed Mutagenesis; Source; Testing; Time; Transgenic Organisms; United States National Institutes of Health; Universities; Western Blotting; abeta accumulation; analytical ultracentrifugation; base; beta pleated sheet; in vitro Assay; in vivo; mind control; molecular mass; mouse model; mutant; research study; tau Proteins

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Aim 1: Examine the a-Syn cleavage product profiles in a cell-free system to identify possible calpain cleavage site(s) within a-Syn. Truncated forms of a-Syn have been identified both in DLB and PD, however, the specific protease(s) involved in this processing is unknown. There are several objectives for this Aim. First, does calpain efficiently cleave a-Syn? Second, if calpain cleaves a-Syn at what specific sites within the protein does this occur? These studies will employ an in vitro approach. Hypothesis: We hypothesize that calpain cleaves a-Syn at specific sites within the protein. Mechanistically, we hypothesize that the cleavage of a-Syn by calpain will generate stable reproducible cleavage products that accumulate over time. Methods: Calpain cleavage of a-Syn will be examined using a cell-free system consisting of human recombinant a-Syn and purified calpain I from erythrocytes. Following cleavage of a-Syn and separation of protein fragments, N-terminal sequencing will be performed to determine exact sites within a-Syn where cleavage occurs. Site-directed mutagenesis of these potential cleavage sites will be performed to verify these potential cleavage sites. Outcome: Several important objectives will be obtained from this Aim. First these experiments will allow us to determine if a-Syn is an efficient substrate for calpain cleavage. Second, the determination of exact sites within a-Syn that are cleaved by calpain will allow us to identify sites that may be amenable to the production of site-directed calpain-cleavage antibodies. Aim 2: Analyze whether calpain cleavage of a-Syn enhances its fibrillization and aggregation in vitro. A critical step in the progression of a-synucleinopathies is the fibrillization and aggregation of a-Syn into LBs. Several recent studies have demonstrated that truncated forms of a-Syn are present in neurodegenerative diseases and are able to promote the aggregation of a-Syn both in vitro and in vivo. In this Aim, we will determine a possible mechanism by which a-Syn aggregation occurs. Hypothesis: We hypothesize that calpain cleavage of a-Syn will enhance its fibrillization and aggregation in vitro. We further hypothesize that calpain cleavage of a-Syn will enhance fibrillization of full-length a-Syn by acting as seeds of nucleation necessary for further fibril assembly. Additionally, we hypothesize that the aggregation of a-Syn following its cleavage by calpain allows it to adopt a beta-sheet conformation similar to what is seen following the aggregation of beta-amyloid (Ab). Methods: To determine if calpain cleavage of a-Syn enhances its fibrillization and aggregation, a number of in vitro assays will be performed including Western blot analysis, analytical ultracentrifugation, circular dichroism microscopy and electron microscopy (EM). These methods will allow us to answer directly whether cleavage of a-Syn leads to its aggregation and fibrillization and whether secondary structural changes accompany this aggregation. Outcome: Because aggregation and fibrillization of a-Syn has been identified as a crucial step in the evolution of LB formation, it is important to not only identify potential proteases involved in processing but whether this processing leads to aggregation. We will determine the mechanisms involved in the proteolytic cleavage of a-Syn and link these events to the aggregation of a-Syn in vitro. Aim 3: Identify the mechanisms underlying the accumulation of truncated a-Syn species in vitro. Recent studies have demonstrated the presence of lower molecular mass a-Syn species in a-Syn aggregates in vivo. In addition, the overexpression of full-length a-Syn in several cell lines leads to the in vitro accumulation of low molecular weight species of a-Syn over time. However, the mechanism that leads to the truncation of full-length a-Syn both in vitro and in vivo is not known. Hypothesis: We hypothesize that calpain is a potential protease that is responsible for the cleavage of a-Syn. More specifically, we hypothesize that overexpression of a-Syn in several cell lines leads to the activation of calpain and the proteolytic processing of a-Syn followed by the accumulation of these cleavage products intracellularly. Methods: We will utilize two different cell lines to address this Aim. SY5Y neuroblastoma cells will be used to artificially activate calpain I and Western blot analysis will be performed to determine if cleavage of a-Syn and whether high-molecular weight species are generated. A second set of cell lines will consist of PC12 cells stably overexpressing either human wild-type a-Syn or mutant A53T a-Syn. PC12 cell lines have been provided to us by Dr. David Rubinsztein (Cambridge University, UK). We will determine if the overexpression of a-Syn leads to the constitutive generation of high-molecular weight a-Syn species. Cell extracts will be examined both for the possibility for calpain activation, and for the presence of low/high molecular weight species of a-Syn by Western blot analysis. Outcome: Experiments proposed in this Aim will allow us to determine the mechanism by which a-Syn is cleaved in vitro, and leads to the generation of truncated, low-molecular weight species of a-Syn. The outcome of these experiments will provide mechanistic support for a role of calpain cleavage of a-Syn, which will help confirm potential in vivo findings (Aim 4, below). Aim 4: Determine whether calpain-cleavage of a-Syn occurs in vivo following application of antibodies by Western blot analysis or immunohistochemistry using postmortem samples from PD or DLB patients. Presently, the protease responsible for the proteolytic processing of a-Syn in a-synucleinopathies is unknown. Because proteolytic fragments of a-Syn have been documented in PD and DLB and these fragments are known to accelerate the evolution of fibril formation, it is critical to identify the mechanisms and pathways involved in a-Syn processing. Hypothesis: The hypothesis to be tested in this Aim is whether or not calpain cleaves a-Syn in vivo. More specifically, we hypothesize that calpain cleavage of a-Syn will be present to a greater extent in both PD and DLB brain sections as compared to age-matched controls. We also hypothesize that in a subset of neurons in DLB and PD, we will find evidence for the co-localization of calpain-cleaved a-Syn together with the caspase-cleavage of tau. This is based in part on previous studies by our lab demonstrating the presence of caspase-cleaved tau within LBs in both PD and LBD postmortem samples. Methods: PD, LBD and control brain samples and extracts will be obtained from the UC Irvine Alzheimers disease research center (ADRC) to be used for immunohistochemical and Western blot analysis. To carry out these experiments, calpain cleavage site-directed antibodies will be utilized. These Abs will be characterized using both in vitro cell model systems and in vivo using an ischemia/reperfusion mouse model to verify recognition to only the calpain-cleavage products (CCPs) of a-Syn following conditions known to activate calpain. In addition, using these Abs, we examine whether calpain cleavage of a-Syn occurs in a transgenic mouse models of PD and AD. To help ensure the successful completion of this aim we also will synthesize additional calpain-cleavage site-directed antibodies to other cleavage sites within a-Syn. Outcome: The outcome of these experiments will determine whether or not a-Syn is cleaved by calpain in PD and DLB. Moreover, these studies may help reveal the pathway leading to LB formation by identifying mechanisms involve in the processing of a-Syn. Finally, these studies may identify new potential drug targets for the treatment of these diseases.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 AIM 1：检查无细胞系统中的A-Syn裂解产物曲线谱，以识别A-Syn中可能的钙蛋白酶裂解位点。 在DLB和PD中都已经鉴定出A-Syn的截断形式，但是，该处理所涉及的特定蛋白酶尚不清楚。 这个目标有几个目标。 首先，钙蛋白酶有效地裂解A-syn吗？ 其次，如果钙蛋白酶在蛋白质内的哪些特定位点裂解A-syn会发生？ 这些研究将采用一种体外方法。 假设：我们假设钙蛋白酶在蛋白质内的特定部位裂解A-syn。 从机械上讲，我们假设Calpain的A-Syn裂解将产生稳定的可重复的切割产物，这些裂解产物会随着时间的流逝而积累。 方法：将使用由人类红细胞的人类重组A-Syn和纯化的Calpain I组成的无细胞系统来检查A-Syn的钙蛋白裂解。裂解A-syn和蛋白质片段的分离后，将进行N末端测序，以确定发生裂解的A-Syn内的精确位点。这些潜在裂解位点的位置定向诱变将进行验证这些潜在的切割位点。 结果：将从这个目标中获得一些重要目标。 首先，这些实验将使我们能够确定A-Syn是否是钙蛋白酶裂解的有效底物。 其次，确定由钙蛋白酶切割的A-Syn中的精确位点将使我们能够识别可能适合生产位置定向的钙蛋白酶裂解抗体的位点。 AIM 2：分析A-Syn的钙蛋白酶裂解是否可以增强其原纤维化和体外聚集。 A-突触核酸进展的关键步骤是A-Syn成纤维化和聚集到LBS中。 最近的一些研究表明，神经退行性疾病中存在截断的A-Syn形式，并且能够在体外和体内促进A-Syn的聚集。在此目标中，我们将确定A-syn聚集的可能机制。 假设：我们假设A-Syn的钙蛋白酶裂解将增强其纤颤和体外聚集。我们进一步假设，A-Syn的钙蛋白酶裂解将通过充当进一步的原纤维组装所需的成核种子来增强全长A-Syn的纤维化。 此外，我们假设A-Syn在其裂解后的聚集使其可以采用与β-淀粉样蛋白（AB）聚集之后看到的β-折叠构象。 方法：为了确定A-syn的钙蛋白酶裂解是否增强其纤维化和聚集，将进行许多体外测定，包括蛋白质印迹分析，分析性超速离心，循环二分性别显微镜和电子显微镜（EM）。 这些方法将使我们能够直接回答A-Syn的裂解是否导致其聚集和纤颤，以及次要结构变化是否伴随着该聚集。 结果：由于A-SYN的聚集和纤颤已被确定为LB形成演变的关键步骤，因此重要的是，不仅重要的是确定涉及加工的潜在蛋白酶，而且是否会导致聚集。 我们将确定A-Syn蛋白水解切割中涉及的机制，并将这些事件与体外A-Syn的聚集联系起来。 目标3：确定体外截短的A-Syn物种积累的基础机制。 最近的研究表明，在体内A-SYN聚集体中存在较低的分子质量A-syn物种。 另外，随着时间的推移，全长A-SYN的过表达导致A-Syn的低分子量物种的体外积累。 然而，尚不清楚导致全长A-Syn截断的机制。 假设：我们假设钙蛋白酶是一种潜在的蛋白酶，负责a-syn的裂解。 更具体地说，我们假设几个细胞系中A-SYN的过表达导致钙蛋白酶的激活和A-Syn的蛋白水解加工，然后在细胞内积累了这些裂解产物。 方法：我们将利用两种不同的单元线来解决此目标。 SY5Y神经母细胞瘤细胞将用于人为地激活Calpain I，并将进行蛋白质印迹分析，以确定是否产生了A-Syn的裂解以及是否产生高分子重量物种。 第二组细胞系将由PC12细胞组成，稳定过表达人类野生型A-Syn或突变体A53T A-Syn。 David Rubinsztein博士（英国剑桥大学）向我们提供了PC12细胞系。 我们将确定A-Syn的过表达是否导致构成生成高分子重量A-Syn物种。 通过蛋白质印迹分析，将检查细胞提取物的可能性，以及是否存在钙蛋白酶激活的可能性，以及是否存在A-Syn的低/高分子量物种。 结果：在此目标中提出的实验将使我们能够确定A-Syn在体外裂解的机制，并导致a-syn的截短的低分子重量物种产生。 这些实验的结果将为A-Syn的钙蛋白酶裂解的作用提供机械支持，这将有助于确认体内的潜在发现（AIM 4，下面）。 AIM 4：确定在使用蛋白质印迹分析或使用PD或DLB患者的后验证样本使用抗体后，在使用抗体后，是否会在体内发生A-Syn的钙蛋白酶裂解。 目前，尚不清楚a-syn蛋白水解加工的蛋白酶是未知的。 由于A-Syn的蛋白水解片段已在PD和DLB中进行了记录，并且已知这些片段可以加速原纤维形成的演化，因此确定与A-Syn加工有关的机制和途径至关重要。 假设：在此目的中要检验的假设是钙蛋白酶在体内是否裂解a-syn。 更具体地说，我们假设与年龄匹配的对照相比，在PD和DLB脑部切片中，A-Syn的Calpain裂解将在更大程度上存在。 我们还假设在DLB和PD中的神经元子集中，我们将找到Calpain-Cheaved A-Syn共定位的证据，以及Tau的caspase-cleavage。 这部分基于我们的实验室的先前研究，该研究证明了在PD和LBD验尸样品中caspase旋转的tau存在。 方法：PD，LBD和对照脑样品和提取物将从UC Irvine Alzheimer疾病研究中心（ADRC）获得，用于免疫组织化学和蛋白质印迹分析。为了进行这些实验，将利用Calpain裂解位置定向的抗体。 这些ABS将使用体外细胞模型系统和体内使用缺血/再灌注小鼠模型来表征这些ABS，以验证A-syn的钙蛋白酶切解产物（CCP）以下遵循已知激活钙蛋白酶的条件的A-Syn。 此外，使用这些ABS，我们检查了A-Syn的钙蛋白酶裂解是否发生在PD和AD的转基因小鼠模型中。 为了帮助确保成功完成此目标，我们还将合成针对A-Syn内其他裂解位点的其他calpain-cleavage定位抗体。 结果：这些实验的结果将确定pd和dlb中的钙蛋白酶是否裂解了a-syn。 此外，这些研究可能有助于揭示导致LB形成的途径，通过识别参与A-SYN处理的机制。 最后，这些研究可能会确定治疗这些疾病的新潜在药物靶标。