Moderator of 1 Session
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PRESENCE OF FILAMENTOUS AΒ AND TAU IN AQUEOUSLY SOLUBLE EXTRACTS OF ALZHEIMER DISEASE CORTEX
Abstract
Aims
Aqueously soluble oligomers of the Aβ protein, rather than monomers or insoluble fibrils, are considered the most bioactive form. Although extensive characterization of synthetic oligomers has been performed, little is known about the structure of oligomers that occur in human brain; instead, they are operationally defined as that non-monomeric material present in the supernatant after ultracentrifugation of an aqueously soluble extract.
Methods
Soluble extracts were prepared in TBS using homogenization or soaking, followed by ultracentrifugation in the SW41Ti rotor at 200,000 g. Re-pelleting was performed in a tabletop centrifuge at 20,000 g. Immunogold labeling used D54D2 (rabbit anti-Aβ N-terminus) or various rabbit anti-phosphotau monoclonal antibodies (Cell Signaling Technologies). ELISA utilized m266 and 21F12 after denaturing samples in 5M guanidine hydrochloride.
Results
We found that short filamentous structures could be re-pelleted from soluble extracts of AD but not control cortex in a concentration-dependent manner, and visualized by negative-stain electron microscopy. Immunogold labeling confirmed the presence of Aβ. Re-pelleting of Aβ filaments from soluble post-200,000 g extracts was enhanced by freezing at least overnight and by adding Triton X-100, Tween-20, and digitonin, but not CHAPSO, but was prevented by high ionic strength. Linear time-dependence in re-pelleting suggested the filaments were not reconstituted during re-pelleting and may be present in the initial post-200,000 g aqueous extract. We also observed paired helical filaments re-pelleting from aqueous extracts labeled by anti-phosphotau immunogold.
Conclusions
These results suggest that at least some high molecular weight oligomers of Aβ and tau in putatively soluble fractions are, and/or become, filamentous.