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O1‐01‐01: CHARACTERIZING PROTEIN AGGREGATES FROM THE HUMAN BRAIN
Author(s) -
De Suman,
Klenerman David
Publication year - 2018
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2018.06.2330
Subject(s) - protein aggregation , vesicle , chemistry , biophysics , membrane , chaperone (clinical) , membrane protein , microbiology and biotechnology , cell membrane , biochemistry , biology , medicine , pathology
Background: The molecular mechanisms that govern protein misfolding and its links with neurodegenerative disease are very complex processes, involving the formation of transiently populated but highly toxic molecular species. Increasing evidence suggest these toxic species are soluble oligomers, a short-lived intermediate of aggregation process. Due to its low abundance and high heterogeneity, the exact mechanism and extent of protein aggregate induced cytotoxicity is still unclear. One of the most consistent pathologies in neurodegenerative disease is small soluble oligomeric aggregate induced membrane disruption followed by unregulated Ca ion influx into the cell. Methods:To characterize this highly heterogeneous oligomers, we have developed a high-throughput biophysical assay by fabricating thousands of nanosized single vesicles which act as an optochemical probe consists of a native-like model system. Small encapsulation of these vesicles (10 L) makes the effective concentrations of encapsulated molecules very high which enables to study protein aggregate induced membrane permeabilization in picomolar concentration. Using this assay we have quantified toxic protein aggregate induced membrane permeabilization by visualizing individual calcium ion influx into hundreds of these individual vesicles in a high throughput manner using highly sensitive total internal reflection fluorescence microscopy (Angewandte Chemie 2017). Results: We have measured and compared the membrane permeabilization ability of recombinant protein aggregates of Ab40/42, a-synuclein, tau. We have also determined if a given chaperone, antibody or nanobody is effective in reducing the protein aggregate inducedmembrane permeabilization and also determinedwhat concentration is needed to prevent this protein aggregate induced membrane permeabilization (Cell Reports 2017). Then using these calibrated assays, we have characterized toxic species present in range of human samples such as cerebrospinal fluids (CSF), brain homogenates and patient derived induced pluripotent stem (iPS) cells. We have also quantified the effect of various antibodies in their ability to counteract the toxicity induced by above mentioned human samples. Conclusions:This ultra-sensitive state-ofthe-art technique provides new insights into the mechanism of protein aggregate induced toxicity in aggregation mixture as well as complex biofluid such as CSF and brain homogenates, allowing us to identify the most toxic aggregate and the most suitable antibodies to prevent aggregate induced damage.