Abstract
Recent research implicates soluble aggregated forms of α-synuclein as neurotoxic species with a central role in the pathogenesis of Parkinson's disease and related disorders. The pathway by which α-synuclein aggregates is believed to follow a step-wise pattern, in which dimers and smaller oligomers are initially formed. Here, we used H4 neuroglioma cells expressing α-synuclein fused to hemi:GFP constructs to study the effects of α-synuclein monoclonal antibodies on the early stages of aggregation, as quantified by Bimolecular Fluorescence Complementation assay. Widefield and confocal microscopy revealed that cells treated for 48 h with monoclonal antibodies internalized antibodies to various degrees. C-terminal and oligomer-selective α-synuclein antibodies reduced the extent of α-synuclein dimerization/oligomerization, as indicated by decreased GFP fluorescence signal. Furthermore, ELISA measurements on lysates and conditioned media from antibody treated cells displayed lower α-synuclein levels compared to untreated cells, suggesting increased protein turnover. Taken together, our results propose that extracellular administration of monoclonal antibodies can modify or inhibit early steps in the aggregation process of α-synuclein, thus providing further support for passive immunization against diseases with α-synuclein pathology.
Highlights
Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy are neurodegenerative disorders characterized by the loss of neurons in the brain along with the presence of large intracellular protein inclusions known as Lewy bodies [1,2]
Characterization of mAb49/G Immunization of mice with HNE stabilized a-synuclein oligomers resulted in several monoclonal antibodies, among which mAb49/G was chosen for this study
By inhibition ELISA, the binding of mAb49/G to an HNE stabilized a-synuclein oligomer coated plate was inhibited by addition of serially diluted asynuclein species
Summary
Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy are neurodegenerative disorders characterized by the loss of neurons in the brain along with the presence of large intracellular protein inclusions known as Lewy bodies [1,2]. A-synuclein has a largely unknown function, recent findings suggest it to be involved in neurotransmitter regulation. The aggregation cascade of a-synuclein is believed to begin with the formation of dimers and smaller oligomers before the appearance of larger oligomers or protofibrils [6]. Such soluble pre-aggregated species have been demonstrated to have toxic properties and may play a central role in the pathogenesis [7,8,9,10,11]. The disease associated mutations in the gene encoding for a-synuclein have been found to increase the formation of oligomers/protofibrils, further supporting the pathogenic significance of such species [12,13,14]
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