TOPIC · Targeting Cytotoxic Protein Oligomers

TOPIC: Targeting Cytotoxic Protein Oligomers

The overarching aim of TOPIC has been to identify binding partners for oligomeric species involved in neurodegenerative diseases. The original proposal mentioned proteins from four diseases, namely α-synuclein (Parkinson’s), tau (Alzheimer’s), huntingtin (Huntington’s), and amylin (Diabetes Type 2). We have achieved most progress with the first two proteins; huntingtin had to be abandoned due to poor expression and purification yields while amylin proved exceedingly difficult to oligomerize. α-synuclein: Given the importance of obtaining robust samples of aSN oligomers, we devoted considerable effort to the use of physiologically relevant lipid oxidation products, namely docosahexaenoic acid (DHA) and 4-hydroxynonenal (HNE). Both compounds significantly improved oligomer yields, and a biophysical analysis showed similar size and morphology and high b-sheet content, though they differ in terms of species homogeneity and anti-parallel b-sheet content. Our ongoing hydrogen-deuterium exchange mass spectrometry experiments (in collaboration with Daniel Otzen and Assoc. Prof. Thomas Jørgensen, University of Southern Denmark) shows similar regions of protection to the unmodified protein. Both oligomers permeabilize vesicles and we are currently finalizing experiments on their effect on cell signalling and long term potentiation in cells. To identify aSN binding partners, we labeled oligomers with biotin for pull-down experiments. Our oligomers showed puctate distribution in neurons which could be important for toxicity. To improve identification we developed a photoactivated cross-linking method which did not affect neuronal binding and was validated using an aggregate-specific antibody. In the actual pull down we identified 69 cells which constitute a promising start for new targets against oligomer toxicity in cells. We expect one article on the biophysical and functional comparison of different in vitro generated αSOs, including HNE-modified, DHA-modified, and unmodified-αSOs, one article on structural analysis of the oligomers by HDX-MS and one on the binding partners of aSN identified by pull-down experiments. Tau: We established a high-yield purification protocol for recombinant tau and an aggregation protocol where tau aggregation is promoted by heparin. Fluorescently-labeled heparin association to tau oligomers can now be measured by plate readers or scanning protocols. Both filamentous and spherical tau aggregates were observed. Tau oligomers appear to seed aggregation in cell cultures and these oligomers may be used as a well-defined species (as opposed to more heterogeneous fibrils) for future references in cell culture studies. We are finalizing studies to write this up as an article.

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