Formation, Stability, and Structure of Hirano Bodies Using a Live Cell System

使用活细胞系统的平野体的形成、稳定性和结构

• 批准号: 8126431
• 负责人: ANDREW G MASELLI
• 金额: $ 10.03万
• 依托单位: CHICAGO STATE UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8126431
• 关键词: Actin-Binding Protein; Actins; Age; Alzheimer' s Disease; Antibodies; Antigens; Binding; Binding Sites; Biological Assay; Biological Models; Biological Preservation; Brain; Caspase; Cell model; Cell physiology; Cells; Characteristics; Chemicals; Crosslinker; Cytoskeleton; DNA Sequence Rearrangement; Data; Dictyostelium; Dictyostelium discoideum; Event; F-Actin; Fimbrin; Freezing; Funding; Green Fluorescent Proteins; Health; Homologous Gene; Human; Image; Immersion Investigative Technique; Immunoelectron Microscopy; Individual; Kinetics; Lead; Libraries; Life; Light Microscope; Literature; Methodology; Methods; Modeling; Neurodegenerative Disorders; Pathology; Peptide Hydrolases; Phagocytosis; Pinocytosis; Population; Process; Protein Fragment; Protein Isoforms; Proteins; Role; Sepharose; Site; Source; Staging; Structure; System; T-plastin; Technology; Testing; Time; Work; base; cell fixing; cell motility; cell type; comparative; crosslink; light microscopy; plastin; promoter; sample fixation

Neurodegenerative diseases become a major health concern as the population ages. One of the least studied structural manifestations of neurodegenerative disease are Hirano bodies. Hirano bodies are actin based inclusions, which have been identified in the brains of individuals with a broad range of neurodegenerative disorders including Alzheimer's disease. Although the ultrastructure and protein composition of Hirano bodies is well known the cascade of events that leads to Hirano body formation remains unknown. Determining the events that lead to assembly is important to understanding the role of Hirano bodies in pathology. Our hypothesis is that Hirano bodies form as the result of uncontrolled actin cross-linker activity and that this uncontrolled cross-linking, in concert with the cell's natural cross-linking ability, generate a stable Hirano body structure. One of the major reasons that Hirano body formation has not been studied is the lack of a live cell model system. We have shown that the expression of a truncated actin binding protein with uncontrolled actin cross-linking activity can induce the formation of Hirano bodies in the eukaryotic model system Dictyostelium discoideum (Maselli, 2002 ;Maselli, 2003). The model Hirano bodies formed in Dictyostelium have similar characteristics to the Hirano bodies found in the human brain (Maselli, 2002). By expressing a truncated actin binding protein (t-abp) Green Fluorescent Protein (GFP) fusion with an inducible expression system we will be able to observe Hirano body formation in cells. By combining the t-abp probe with a probe for filamentous actin we can observe changes that take place at the earliest stages of Hirano Body formation. Extending our hypothesis, a likely source of t-abp in human cells are proteolytic cleavage fragments of the cells own actin biding proteins. We propose to test our hypothesis by expressing the likely cleavage fragments in cells, and observing the cells for inclusion formation. The ultrastructure of Hirano Bodies is key to understanding both their formation and stability. We will determine the optimal fixation method and used immuno electron microscopy and FIAsH tag technology to correlate our observations from light microscopy to the ultrastructure. Better understanding the cascade of events and the possible triggers for Hirano Body formation will further our understanding of the potential roles of Hirano Bodies in neurodegenerative disease.

随着人口老龄化，神经退行性疾病成为一个主要的健康问题。最少的之一