Formation of Hirano bodies after inducible expression of a modified form of an actin-cross-linking protein.

Hirano bodies are cytoplasmic inclusions composed mainly of actin and actin-associated proteins. The formation of Hirano bodies during various neurodegenerative disorders, including Alzheimer’s disease and amyotrophic lateral sclerosis, has been reported. Although the underlying molecular mechanisms that lead to the formation of these inclusions in the brain are not known, expression of the C-terminal fragment (CT) (amino acids 124 to 295) from the endogenous 34-kDa actin-binding protein of Dictyostelium discoideum leads to the formation of actin inclusions in vivo. In the current study, we report the development of an inducible expression system to study the early phases of Hirano body formation using an inducible promoter system (rnrB). By fusing the CT to a green fluorescent protein (CT-GFP), we monitored protein expression and localization by fluorescence microscopy, flow cytometry, and Western blot analysis. We observed an increase in the number and size of inclusions formed following induction of the CT-GFP vector system. Time-lapse microscopy studies revealed that the CT-GFP foci associated with the cell cortex and fused to form a single large aggregate. Transmission electron microscopy further demonstrates that these inclusions have a highly ordered ultrastructure, a pathological hallmark of Hirano bodies observed in postmortem brain samples from patients with various neurodegenerative disorders. Collectively, this system provides a method to visualize and characterize the events that surround early actin inclusion formation in a eukaryotic model. Neurodegenerative diseases are characterized pathologically by the formation of protein deposits localized to specific regions of the brain. Notably, protein aggregates derived from the amyloid precursor protein, the microtubule-associated protein tau, and -synuclein have received much attention. However, the intracellular aggregations of actin and actin-binding proteins known as Hirano bodies are less well known. Hirano bodies were first identified in brains affected by Pick’s disease and amyotrophic lateral sclerosis (8, 17). Subsequent studies identified these aggregates in a number of neurodegenerative diseases and other conditions that cause persistent brain injury (7). Although it is clear from this and other observations that the main constituents of Hirano bodies are actin and actinbinding proteins which assemble to form a characteristic ultrastructure (3), little is known about the mechanisms that underlie Hirano body formation. To further understand the spatial and temporal events that surround the formation of these inclusions in vivo, a live cell model that mimics the formation of these structures is necessary. The discovery that Dictyostelium discoideum cells expressing a carboxy-terminal fragment (CT) of the 34-kDa calcium-sensitive actin-binding protein (ABP34) form Hirano bodies in vivo (1, 12, 13) provides a tantalizing clue to a possible mechanism of protein aggregation. Using Dictyostelium as a live cell model system provides the opportunity to control protein expression levels. In this study,