Characterization of Cytoplasmic α-Synuclein Aggregates

Abstract

The α-synuclein fibrillation process has been associ-ated with the pathogenesis of several neurodegenera-tive diseases. Here, we have characterized the cytoplas-mic ␣-synuclein aggregates using a fractionation procedure with which different aggregate species can be separated. Overexpression of ␣-synuclein in cells pro-duce two distinct types of aggregates: large jux-tanuclear inclusion bodies and small punctate aggre-gates scattered throughout the cytoplasm. Biochemical fractionation results in an inclusion-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 amyloid-like ␣-synuclein fibrils, whereas both the small aggre-gate fractions contain non-fibrillar spherical aggregates with distinct size distributions. These aggregates ap-pear sequentially, with the smallest population appear-ing the earliest and the fibrillar inclusions the latest. Based on the structural and kinetic properties, we sug-gest that the small spherical aggregates are the cellular equivalents of the protofibrils. The proteins that co-ex-ist in the Lewy bodies, such as proteasome subunit, ubiquitin, and hsp70 chaperone, are present in the fibrillar inclusions but absent in the protofibrils, sug-gesting that these proteins may not be directly involved in the early aggregation stage. As predicted in the ag-gresome model, disruption of microtubules with nocoda-zole 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 non-fibrillar intermediate species, and the microtubule-de-pendent inclusion-forming process is required for the protofibril-to-fibril conversion in cells. A group of human neurodegenerative diseases, such as Par-kinson's disease (PD), 1 dementia with Lewy bodies (LBs), and multiple system atrophy, are characterized by cytoplasmic in-clusion bodies that are mainly composed of ␣-synuclein fibrils (1, 2). Although direct role of fibrils and the inclusion bodies in the disease pathogenesis is the subject of intense debate, an increasing body of evidence suggests that the processes of fi-brillation and inclusion formation are closely related to the disease mechanism. First, two missense mutations (A53T and A30P) that are responsible for the familial PD has been iden-tified in ␣-synuclein gene (3, 4), and the mutant proteins have greater propensity for the self-association and aggregation than the wild type protein (5–7). Indeed, both mutations accel-erated the formation of the pre-fibrillar oligomers in vitro, whereas the fibril formation was slowed by one of the muta-tions (7, 8). Second, transgenic animal models that were gen-erated by the overexpression of human ␣-synuclein developed neuronal cytoplasmic inclusion bodies along with neuronal cell loss and behavioral defects (9 –13). Some of these animals pro-duce fibrillar inclusions (9, 12), but others generate only the granular aggregates (10). In a rat Parkinson's model, estab-lished by a systemic administration of rotenone, nigrostriatal degeneration and the motor symptoms were also accompanied by ␣-synuclein-positive inclusion bodies (14). Therefore, the cellular mechanism of ␣-synuclein aggregation is likely to be linked to at least some aspects of the disease process. Although the process of ␣-synuclein fibril formation has been implicated in the pathogenesis of PD by genetic and biochemical evidence, the exact mechanism by which the processes of ␣-synuclein fibrillation and inclusion body formation contribute to neuro-degeneration is currently unknown. In dilute solution, ␣-synuclein does not have stable structure (15) except for some residual helical structure in the N termi-nus of the protein (16). Fibrillation of ␣-synuclein is a nucleation-dependent process (17) and is initiated by acquiring a partially folded conformation (18), which is subsequently stabilized by self-association (19). Prior to the formation of the fibril, the end product of the process, several non-fibrillar oligomeric aggre-gates, or protofibrils, were identified (20). Earliest and most common protofibrillar species are in a spherical shape with average height of 4.2 nm in case of wild type protein (21). The spherical protofibrils are thought to undergo head-to-tail asso-ciations to form elongated chain-like (22), and ring-like proto-fibrillar species (21). In their search for a potential pathogenic mechanism for ␣-synuclein protofibrils, Lansbury and col-leagues (23, 24) demonstrated that only protofibrillar ␣-synuclein bind tightly and permeabilize synthetic vesicles in a size-selective manner, suggesting membrane disruption via a pore-like mechanism. This hypothesis is supported by the find-ings that the two pathogenic mutations (A53T and A30P) pro-mote the formation of annular pore-like protofibrils (25) and result in an increased permeabilization activity relative to the wild type protein (24). Although some of the basic processes of ␣-synuclein aggregation, including the protofibrils with differ-ent morphologies, have been characterized in vitro, little is known about the fibrillation process or the intermediate pro-tofibrillar species in cells.