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.
The small sample volumes and flexibility in choice of liquid matrices using the capillary microsampling technique enable repeated sampling in mouse studies, as well as multi-matrix analysis if needed. Liquid microsampling is well suited for micro- and nano-liter scale immunoassays.
Anti-apoptotic proteins that block death receptor-mediated apoptosis favour tumour evasion of the immune system, leading to enhanced tumour progression. However, it is unclear whether blocking the mitochondrial pathway of apoptosis will protect tumours from immune cell attack. Here, we report that the antiapoptotic protein Bcl-x L , known for its ability to block the mitochondrial pathway of apoptosis, exerted tumour-progressive activity in a murine lymphoma model. Bcl-x L overexpressing tumours exhibited a more aggressive development than control tumours. Surprisingly, Bcl-x L protection of tumours from NK cellmediated attack did not involve protection from NK cell-mediated cytotoxicity. Instead, Bcl-x L -blocked apoptosis resulting from hypoxia and/or nutrient loss associated with the inhibition of angiogenesis caused by NK cell-secreted IFN-c. These results support the notion that NK cells may inhibit tumour growth also by mechanisms other than direct cytotoxicity. Hence, the present results unravel a pathway by which tumours with a block in the mitochondrial pathway of apoptosis can evade the immune system.
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