Protein aggregation is associated with the pathology of many diseases, especially neurodegenerative diseases. A variety of structurally polymorphic aggregates or preaggregates including amyloid fibrils is accessible to any aggregating protein. Preaggregates are now believed to be the toxic culprits in pathologies rather than mature aggregates. Although clearly valuable, understanding the mechanism of formation and the structural characteristics of these prefibrillar species is currently lacking. We report here a simple new approach to map the nature of the aggregate core of transient aggregated species directly in the cell. The method is conceptually based on the highly discriminating ability of aggregates to recruit new monomeric species with equivalent molecular structure. Different soluble segments comprising parts of an amyloidogenic protein were transiently pulse-expressed in a tightly controlled, time-dependent manner along with the parent aggregating full-length protein, and their recruitment into the insoluble aggregate was monitored immunochemically. We used this approach to determine the nature of the aggregate core of the metastable aggregate species formed during the course of aggregation of a chimera containing a long polyglutamine repeat tract in a bacterial host. Strikingly, we found that different segments of the full-length protein dominated the aggregate core at different times during the course of aggregation. In its simplicity, the approach is also potentially amenable to screen also for compounds that can reshape the aggregate core and induce the formation of alternative nonamyloidogenic species.Accumulation of macroscopically observable, abnormal protein deposits is the cytological feature of many age-related neurodegenerative diseases, including Alzheimer's, Parkin-son's, and Huntington's diseases (1-3). Although the characteristic pathological aggregates are primarily made up of the fibrillar aggregated form of the disease-associated protein, multiple aggregate morphologies are accessible to each individual amyloidogenic protein. The heterogeneity of aggregate morphologies may arise from (1) variations in the environment and aggregate growth conditions (4-8), (2) different segments of the primary sequence that govern the formation of the aggregate core (8-13), or (3) complex pathways of formation, including † This project was supported by the SysMO (KOSMOBAC), the DFG (project IG 73/4-1, and the Heisenberg award IG 73/1-1) to Z.I., and by the National Institutes of Health (grant GM027616 and a 2006 NIH Director's Pioneer Award to L.M.G.).

Background: Expansion of a polyglutamine repeat in atrophin-1 causes progressive neuronal dysfunction in neurodegenerative dentatorubropallidoluysian atrophy. Results: Nuclear inclusions of expanded atrophin-1 are more dynamic compared with the cytoplasmic aggregates. Conclusion: Structural variations of the aggregate core determine these dynamics. Significance: Probing the molecular properties of the inclusions is crucial for understanding the enhanced cellular toxicity of nuclear aggregates in polyglutamine pathologies.