Neurodegenerative diseases become a major health concern as the population ages. One of the leaststudied structural manifestations of neurodegenerative disease are Hirano bodies. Hirano bodies are actinbased inclusions, which have been identified in the brains of individuals with a broad range ofneurodegenerative disorders including Alzheimer's disease. Although the ultrastructure and proteincomposition of Hirano bodies is well known the cascade of events that leads to Hirano body formationremains unknown. Determining the events that lead to assembly is important to understanding the role ofHirano bodies in pathology. Our hypothesis is that Hirano bodies form as the result of uncontrolled actincross-linker activity and that this uncontrolled cross-linking, in concert with the cell's natural cross-linkingability, generate a stable Hirano body structure. One of the major reasons that Hirano body formation hasnot been studied is the lack of a live cell model system. We have shown that the expression of a truncatedactin binding protein with uncontrolled actin cross-linking activity can induce the formation of Hirano bodies inthe eukaryotic model system Dictyostelium discoideum (Maselli, 2002 ;Maselli, 2003). The model Hiranobodies 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. Bycombining the t-abp probe with a probe for filamentous actin we can observe changes that take place at theearliest stages of Hirano Body formation. Extending our hypothesis, a likely source of t-abp in human cellsare proteolytic cleavage fragments of the cells own actin biding proteins. We propose to test our hypothesisby expressing the likely cleavage fragments in cells, and observing the cells for inclusion formation. Theultrastructure of Hirano Bodies is key to understanding both their formation and stability. We will determinethe optimal fixation method and used immuno electron microscopy and FIAsH tag technology to correlate ourobservations from light microscopy to the ultrastructure. Better understanding the cascade of events and thepossible triggers for Hirano Body formation will further our understanding of the potential roles of HiranoBodies in neurodegenerative disease.
