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.

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