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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 目标 1：检查无细胞系统中的 a-Syn 裂解产物谱，以确定 a-Syn 中可能的钙蛋白酶裂解位点。 在 DLB 和 PD 中均已鉴定出 a-Syn 的截短形式，然而，参与此加工的特定蛋白酶尚不清楚。 该目标有几个目标。 首先，钙蛋白酶是否能有效裂解 a-Syn？ 其次，如果钙蛋白酶在蛋白质内的哪些特定位点裂解 a-Syn，则会发生这种情况？ 这些研究将采用体外方法。 假设：我们假设钙蛋白酶在蛋白质内的特定位点切割 a-Syn。 从机制上讲，我们假设钙蛋白酶对 a-Syn 的裂解会产生稳定的、可重复的裂解产物，并随着时间的推移而积累。 方法：使用由人重组 a-Syn 和从红细胞中纯化的钙蛋白酶 I 组成的无细胞系统检查钙蛋白酶对 a-Syn 的裂解。切割 a-Syn 并分离蛋白质片段后，将进行 N 端测序以确定 a-Syn 内发生切割的确切位点。将进行这些潜在切割位点的定点诱变以验证这些潜在切割位点。 结果：从该目标中将实现几个重要目标。 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 a-Syn 内被钙蛋白酶切割的确切位点将使我们能够识别可能适合产生定点钙蛋白酶切割抗体的位点。 目标 2：分析钙蛋白酶对 a-Syn 的裂解是否会增强其体外纤维化和聚集。 α-突触核蛋白病进展的关键步骤是α-Syn 纤维化和聚集成 LB。 最近的几项研究表明，截短形式的 a-Syn 存在于神经退行性疾病中，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 聚合发生的可能机制。 假设：我们假设钙蛋白酶对 a-Syn 的裂解会增强其体外纤维化和聚集。我们进一步假设钙蛋白酶对 a-Syn 的裂解将通过充当进一步原纤维组装所需的成核种子来增强全长 a-Syn 的原纤维化。 此外，我们假设 a-Syn 在被钙蛋白酶裂解后聚集，使其采用与 β-淀粉样蛋白 (Ab) 聚集后所见类似的 β-片层构象。 方法：为了确定钙蛋白酶对 a-Syn 的裂解是否会增强其原纤维化和聚集，将进行许多体外测定，包括蛋白质印迹分析、分析超速离心、圆二色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原纤维化，以及二级结构变化是否伴随着这种聚集。 结果：由于 a-Syn 的聚集和原纤维化已被确定为 LB 形成进化中的关键步骤，因此不仅要确定参与加工的潜在蛋白酶，而且还要确定该加​​工是否会导致聚集。 我们将确定 a-Syn 蛋白水解裂解所涉及的机制，并将这些事件与 a-Syn 体外聚集联系起来。 目标 3：确定截短的 a-Syn 物质在体外积累的机制。 最近的研究表明，体内 a-Syn 聚集体中存在较低分子量的 a-Syn 物质。 此外，全长a-Syn在几种细胞系中的过度表达导致低分子量a-Syn随着时间的推移在体外积累。 然而，导致全长a-Syn在体外和体内截短的机制尚不清楚。 假设：我们假设钙蛋白酶是一种潜在的蛋白酶，负责切割 a-Syn。 更具体地说，我们假设一些细胞系中α-Syn的过度表达导致钙蛋白酶的激活和α-Syn的蛋白水解加工，随后这些裂解产物在细胞内积累。 方法：我们将利用两种不同的细胞系来实现这一目标。 SY5Y 神经母细胞瘤细胞将用于人工激活钙蛋白酶 I，并进行蛋白质印迹分析以确定 a-Syn 是否裂解以及是否产生高分子量物质。 第二组细胞系由稳定过表达人类野生型 a-Syn 或突变型 A53T a-Syn 的 PC12 细胞组成。 PC12 细胞系由 David Rubinsztein 博士（英国剑桥大学）提供给我们。 我们将确定 a-Syn 的过度表达是否会导致高分子量 a-Syn 物种的组成型生成。 将通过蛋白质印迹分析检查细胞提取物是否有钙蛋白酶激活的可能性，以及是否存在低/高分子量α-Syn。 结果：本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，并导致生成截短的、低分子量的 a-Syn。 这些实验的结果将为钙蛋白酶裂解 a-Syn 的作用提供机制支持，这将有助于确认潜在的体内发现（目标 4，见下文）。 目标 4：使用 PD 或 DLB 患者的死后样本，通过蛋白质印迹分析或免疫组织化学确定应用抗体后体内是否发生钙蛋白酶裂解 a-Syn。 目前，在α-突触核蛋白病中负责α-Syn蛋白水解加工的蛋白酶尚不清楚。 由于 a-Syn 的蛋白水解片段已在 PD 和 DLB 中得到记录，并且已知这些片段会加速原纤维形成的进化，因此确定 a-Syn 加工中涉及的机制和途径至关重要。 假设：本目标要检验的假设是钙蛋白酶是否在体内裂解 a-Syn。 更具体地说，我们假设与年龄匹配的对照相比，PD 和 DLB 脑切片中 a-Syn 的钙蛋白酶裂解将更大程度地存在。 我们还假设，在 DLB 和 PD 的神经元子集中，我们将找到钙蛋白酶裂解的 a-Syn 与 tau 的 caspase 裂解共定位的证据。 这部分基于我们实验室之前的研究，该研究表明 PD 和 LBD 死后样本中的 LB 内存在 caspase 裂解的 tau 蛋白。 方法：PD、LBD 和对照脑样本和提取物将从加州大学欧文分校阿尔茨海默病研究中心 (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，将利用钙蛋白酶裂解定点抗体。 这些抗体将使用体外细胞模型系统和体内缺血/再灌注小鼠模型进行表征，以验证在已知激活钙蛋白酶的条件下仅识别 a-Syn 的钙蛋白酶裂解产物 (CCP)。 此外，使用这些 Abs，我们检查了 PD 和 AD 转基因小鼠模型中是否发生钙蛋白酶对 a-Syn 的裂解。 为了帮助确保成功完成这一目标，我们还将针对 a-Syn 内的其他切割位点合成额外的钙蛋白酶切割位点定向抗体。 结果：这些实验的结果将确定 PD 和 DLB 中 a-Syn 是否被钙蛋白酶裂解。 此外，这些研究可能有助于通过确定参与 a-Syn 加工的机制来揭示导致 LB 形成的途径。 最后，这些研究可能会确定治疗这些疾病的新的潜在药物靶点。