A new biochemical method for ultra-purification of amyloids from Alzheimer's disease brain tissues.

Abstract

The deposition of amyloids is the fundamental feature of several neurodegenerative diseases, including Alzheimer's disease (AD). Amyloid-beta 42 (Aβ42) peptides are the primary constituents of amyloid deposits in the extracellular spaces within AD brains. Over the years, scientists have identified multiple biochemical features and elucidated several structural arrangements of the amyloidic deposits in the brain. Until now, many previous structural studies have been limited by barriers to isolate highly pure amyloid fibrils and plaques from diseased brains. Despite all the technological progress, detailed solid-state NMR structures of naturally occurring Aβ42 amyloid fibrils have yet to be deduced due to the lack in our ability to isolate highly pure material. We recently developed a robust protocol to isolate amyloid threads by using ultrasonication to remove non-specific interacting protein from SDS insoluble fraction of APP-KI brains. This protocol utilizes previously described sucrose gradient ultracentrifugation-based enrichment methods, followed by high shearing energy generated by the advanced Bioruptor Pico Plus sonicator system. We observed a high-efficiency purification of multiple amyloid species from the APP knock-in mice brains. The presence and purity of long fibrils were verified through negative staining electron microscopy and biochemical methods using Aβ42 and fibril-specific antibodies. Immunoblot analyses using different antibodies provide complementary information about the structural and compositional differences among multiple amyloidic formations. The ultrasensitive ELISA values showed enrichment of Aβ42 in a range significantly higher than the previously reported values. Owing to the heterogeneity among the amyloidogenic structures in the extracellular spaces of brain tissues, no clear understanding of the fundamental process of in vivo fibril formation prevails. The lack of biochemical, structural, and clinical correlations further hampers the therapeutic advancement in AD biology. This new method of amyloid purification may help further biochemical and structural characterization of the disease-causing proteinaceous formations in different brain regions.