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.
