Diffusible, highly bioactive oligomers represent a critical minority of soluble Aβ in Alzheimer’s disease brain
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Significant data suggest that soluble Aβ oligomers play an important role in Alzheimer’s disease (AD), but there is great confusion over what exactly constitutes an Aβ oligomer and which oligomers are toxic. Most studies have utilized synthetic Aβ peptides, but the relevance of these test tube experiments to the conditions that prevail in AD is uncertain. A few groups have studied Aβ extracted from human brain, but they employed vigorous tissue homogenization which is likely to release insoluble Aβ that was sequestered in plaques during life. Several studies have found such extracts to possess disease-relevant activity and considerable efforts are being made to purify and better understand the forms of Aβ therein. Here, we compared the abundance of Aβ in AD extracts prepared by traditional homogenization versus using a far gentler extraction, and assessed their bioactivity via real-time imaging of iPSC-derived human neurons plus the sensitive functional assay of long-term potentiation. Surprisingly, the amount of Aβ retrieved by gentle extraction constituted only a small portion of that released by traditional homogenization, but this readily diffusible fraction retained all of the Aβ-dependent neurotoxic activity. Thus, the bulk of Aβ extractable from AD brain was innocuous, and only the small portion that was aqueously diffusible caused toxicity. This unexpected finding predicts that generic anti-oligomer therapies, including Aβ antibodies now in trials, may be bound up by the large pool of inactive oligomers, whereas agents that specifically target the small pool of diffusible, bioactive Aβ would be more useful. Furthermore, our results indicate that efforts to purify and target toxic Aβ must employ assays of disease-relevant activity. The approaches described here should enable these efforts, and may assist the study of other disease-associated aggregation-prone proteins.
KeywordsAmyloid β-protein Automated live-cell imaging iPSC-derived human neurons Long-term potentiation Neuritic dystrophy Soluble aggregates
We are grateful to Dr. Frederique Bard for the gift of 3D6, 266, 2G3 and 21F12. We thank Dr. Grant Corbett for assistance preparing iN cells and for Supplementary Fig. 1 and Dr. Tracy Young-Pearse for the gift of virally infected iPSCs. We thank Drs. Dennis Selkoe and Matthew Lavoie for critical appraisal of the revised manuscript.
DMW conceived the idea, directed the research, analyzed data and wrote the manuscript. WH conducted biochemical and IncuCyte experiments, analyzed data, prepared figures and assisted with writing the manuscript. ZW did all electrophysiological recordings, analyzed data and prepared figures. WL cultured iPSC-derived neurons and assisted with biochemical experiments. TTO’M trained WH and conducted initial biochemical experiments. MJ provided critical guidance for the use of iPSC-derived neurons and the IncuCyte. MW and CH provided 2E9 antibody, recombinant APP505–612 and furnished critical guidance on the detection of APP fragments. MF performed postmortem analyses and assessed the disease status of patient brains. All authors critically reviewed the manuscript.
This work was supported by grants to DMW from the National Institutes of Health (AG046275), Bright Focus, and by the Massachusetts Alzheimer’s Disease Research Center (AG05134).
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