||Propagation of pathological α-synuclein by a prion-like mechanism
Intercellular abnormal protein aggregates are a common feature of many neurodegenerative diseases. Misfolded proteins are accumulated in neurons and/or glial cells as a fibriller, phosphorylated and partially ubiquitinated forms. The distribution and spreading of these abnormal proteins in brains of patients are shown to correlate with clinical presentation and disease progression. Recent experimental findings support the concept that structurally changed abnormal proteins convert normal proteins into abnormal forms and spread into neighboring cells in a prion-like manner. This prion-like conversion may account for not only the onset but also the progression of neurodegenerative diseases. α-Synuclein (α-syn) is a 140 amino acid protein that is normally located in presynaptic nerve terminals. In 1997, missense mutation in the α-syn gene was discovered in families of Parkinson’s disease (PD), and subsequent immunohistochemical work with anti-α-syn antibodies revealed that α-syn is the major component of Lewy bodies in PD and dementia with Lewy bodies (DLB), and also glial cytoplasmic inclusions (GCIs) in multiple system atrophy. Purified recombinant α-syn assembles into amyloid-like fibrils that share common properties with the fibrils present in pathological human brains in vitro. Preformed α-syn fibrils and brain extracts from α-synucleinopathies’ patients can work as seeds to induce seed-dependent aggregation in cultured cells. Furthermore, injection of preformed α-syn fibrils into brains of wild-type or transgenic mice overexpressing mutant human α-syn led to development of phosphorylated α-syn pathology several months later. However, it has not been fully elucidated what structures of α-syn molecules (fibrils or oligomers, soluble or insoluble) are the most pathogenic, what sizes of these molecules are the most effective, and what mechanisms underlie the cell-to-cell spreading. In this study, I prepared various kinds of α-syn aggregates or intermediates under various conditions and tested their prion-like properties in vitro, in cells, and in mouse experimental models. I found that only fibril form of α-syn induced seed-dependent aggregation of α-syn in cultured cells and in wild-type mouse brains. I also found that sonication of α-syn fibrils accelerate accumulation of phosphorylated α-syn, indicating that fragmented β-sheet rich fibrous structures of α-syn efficiently induce seed-dependent aggregation and prion-like propagation of pathological α-syn in culture cells and wild-type mouse brains. I also characterized these α-syn species by transmission electron microscopy observations and thioflavin fluorescence assays and found that fibrous structures similar to those observed in α-synucleinopathy brains are formed in α-syn fibril-injected mouse brain and that fibrils containing sarkosyl-insoluble fractions extracted from injected mice functioned as seeds for seed-dependent aggregation of α-syn. These results indicate that fragmented amyloid-like α-syn fibrils less than 50 nm in size are the most effective seeds that triggered prion-like conversion. These results may contribute to understand the molecular mechanisms of neurodegenerative diseases. These cellular and mouse models of α-syn propagation should be useful for screening and evaluation of disease-modifying drugs of α-synucleinopathies.
首都大学東京, 2017-03-25, 博士（理学）