Abstract
The concept that abnormal protein aggregates show prion-like propagation between cells has been considered to explain the onset and progression of many neurodegenerative diseases. Indeed, both synthetic amyloid-like fibrils and pathogenic proteins extracted from patients’ brains induce self-templated amplification and cell-to-cell transmission in vitro and in vivo. However, it is unclear whether exposure to exogenous prion-like proteins can potentially cause these diseases in humans. Here, we investigated in detail the prion-like seeding activities of several kinds of pathogenic α-synuclein (α-syn), including synthetic fibrils and detergent-insoluble fractions extracted from brains of patients with α-synucleinopathies. Exposure to synthetic α-syn fibrils at concentrations above 100 pg/mL caused seeded aggregation of α-syn in SH-SY5Y cells, and seeded aggregation was also observed in C57BL/6 J mice after intracerebral inoculation of at least 0.1 μg/animal. α-Syn aggregates extracted from brains of multiple system atrophy (MSA) patients showed higher seeding activity than those extracted from patients with dementia with Lewy bodies (DLB), and their potency was similar to that of synthetic α-syn fibrils. We also examined the effects of various methods that have been reported to inactivate abnormal prion proteins (PrPSc), including autoclaving at various temperatures, exposure to sodium dodecyl sulfate (SDS), and combined treatments. The combination of autoclaving and 1% SDS substantially reduced the seeding activities of synthetic α-syn fibrils and α-syn aggregates extracted from MSA brains. However, single treatment with 1% SDS or generally used sterilization conditions proved insufficient to prevent accumulation of pathological α-syn. In conclusion, α-syn aggregates derived from MSA patients showed a potent prion-like seeding activity, which could be efficiently reduced by combined use of SDS and autoclaving.
Highlights
Intracellular accumulations of abnormal protein aggregates are common but defining neuropathological features of many neurodegenerative diseases
Seeded aggregation induced by synthetic α-syn fibrils in SH-SY5Y cells First, we investigated the minimum amounts of pathogenic α-syn required to induce seeded aggregation and accumulation of phosphorylated α-syn in SH-SY5Y cells expressing untagged human wild-type (WT) α-syn
Based on protein determination by immunoblotting, the ID50 (50% infectious dose or seeding activity) per 2 μL was calculated to be 102.91 (± 0.51) by the Spearman-Karber method [64]. These results show that exposure of SHSY5Y cells transiently expressing human α-syn to synthetic human α-syn fibrils at a concentration of above 100 pg/mL can induce seeded aggregation of the intracellular α-syn
Summary
Intracellular accumulations of abnormal protein aggregates are common but defining neuropathological features of many neurodegenerative diseases. It has been suggested that the prion-like behavior of abnormal proteins may account for the onset and progression of neurodegenerative diseases [21, 62]. Α-Synucleinopathies, which include Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), are characterized by accumulation of misfolded α-synuclein (α-syn) aggregates in neuronal and/or glial cells, and various pathological phenotypes and clinical symptoms are observed for each disease [22]. In PD and DLB, α-syn pathologies are mainly observed in neurons in the form of Lewy bodies (LBs) and Lewy neurites (LNs) [4, 56], whereas glial cytoplasmic inclusions (GCIs) are seen in oligodendrocytes in MSA [61].
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