The ␣-synuclein fibrillation process has been associated with the pathogenesis of several neurodegenerative diseases. Here, we have characterized the cytoplasmic ␣-synuclein aggregates using a fractionation procedure with which different aggregate species can be separated. Overexpression of ␣-synuclein in cells produce two distinct types of aggregates: large juxtanuclear inclusion bodies and small punctate aggregates scattered throughout the cytoplasm. Biochemical fractionation results in an

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... lusion-enriched fraction and two small aggregate fractions. Electron microscopy and thioflavin S reactivity of the fractions show that the juxtanuclear inclusion bodies are filled with amyloidlike ␣-synuclein fibrils, whereas both the small aggregate fractions contain non-fibrillar spherical aggregates with distinct size distributions. These aggregates appear sequentially, with the smallest population appearing the earliest and the fibrillar inclusions the latest. Based on the structural and kinetic properties, we suggest that the small spherical aggregates are the cellular equivalents of the protofibrils. The proteins that co-exist in the Lewy bodies, such as proteasome subunit, ubiquitin, and hsp70 chaperone, are present in the fibrillar inclusions but absent in the protofibrils, suggesting that these proteins may not be directly involved in the early aggregation stage. As predicted in the aggresome model, disruption of microtubules with nocodazole reduced the number of inclusions and increased the size of the protofibrils. Despite the increased size, the protofibrils remained non-fibrillar, suggesting that the deposition of the protofibrils in the juxtanuclear region is important in fibril formation. This study provides evidence that the cellular fibrillation also involves nonfibrillar intermediate species, and the microtubule-dependent inclusion-forming process is required for the protofibril-to-fibril conversion in cells.