Abstract
Synucleinopathies including Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are characterized by pathological accumulation of α-synuclein (α-syn). Amongst the various approaches attempting to tackle the pathological features of synucleinopathies, antibody-based immunotherapy holds much promise. However, the large size of antibodies and corresponding difficulty in crossing the blood-brain barrier has limited development in this area. To overcome this issue, we engineered single-chain variable fragments (scFvs) against fibrillar α-syn, a putative disease-relevant form of α-syn. The purified scFvs showed specific activity towards α-syn fibrils and oligomers in comparison to monomers and recognized intracellular inclusions in human post-mortem brain tissue of Lewy body disease cases, but not aged controls. In vitro studies indicated scFvs inhibit the seeding of α-syn aggregation in a time-dependent manner, decreased α-syn seed-induced toxicity in a cell model of PD, and reduced the production of insoluble α-syn phosphorylated at Ser-129 (pS129-α-syn). These results suggest that our α-syn fibril-specific scFvs recognize α-syn pathology and can inhibit the aggregation of α-syn in vitro and prevent seeding-dependent toxicity. Therefore, the scFvs described here have considerable potential to be utilized towards immunotherapy in synucleinopathies and may also have applications in ante-mortem imaging modalities.
Highlights
Synucleinopathies including Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are characterized by pathological accumulation of α-synuclein (α-syn)
Several lines of evidence indicate that α-syn aggregation is a critical pathogenic event in the natural history of Lewy body diseases. α-Syn is the major protein component of LBs, various point mutations and/or multiplications in the α-syn gene have been described in familial PD, exogenous expression of α-syn in Drosophila and transgenic mice induce the formation of PD-like pathological phenotypes and behavior, and down-regulation of the α-syn protein reduces risk of developing PD6–13
They were capable of inhibiting the seeding of α-syn aggregation in an in vitro assay, whilst reducing the toxicity caused by α-syn seeds in cell model. scFvs blocked the aggregation of α-syn as detected by decreased insoluble α-syn phosphorylated at Ser-129 in another cell model. These results suggest that α-syn pathology-specific scFvs are able to bind α-syn fibrils and oligomers and inhibit the seeding of α-syn aggregation that might lead to the pathogenesis of PD, DLB and MSA
Summary
Synucleinopathies including Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are characterized by pathological accumulation of α-synuclein (α-syn). The large size of antibodies and corresponding difficulty in crossing the blood-brain barrier has limited development in this area To overcome this issue, we engineered single-chain variable fragments (scFvs) against fibrillar α-syn, a putative diseaserelevant form of α-syn. In vitro studies indicated scFvs inhibit the seeding of α-syn aggregation in a time-dependent manner, decreased α-syn seed-induced toxicity in a cell model of PD, and reduced the production of insoluble α-syn phosphorylated at Ser-129 (pS129-α-syn). Synucleinopathies are a group of neurodegenerative disorders comprising Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA)[1]. Already characterized functional monoclonal antibodies can be sequenced and VH and VL sequences responsible for antigen binding can be identified and cloned to synthesize scFv gene
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