Femtomole immunodetection of synthetic and endogenous amyloid-beta oligomers and its application to Alzheimer's disease drug candidate screening.
Alzheimer's disease (AD) is a fatal, progressive dementia for which there is no cure and for which a molecular basis has yet to be established. However, considerable evidence suggests that AD is linked to neurotoxic assemblies of the 42-amino-acid peptide amyloid beta (Abeta). There is now a clear body of evidence that shows this neurotoxicity resides not only in insoluble fibrils of Abeta but also in soluble Abeta ADDLs (Abeta-derived diffusible ligands) and larger protofibrils. Further, anti-Abeta antibodies have been reported to reverse memory failure in human amyloid precursor protein (hAPP)-expressed transgenic mice in a manner that suggests symptom reversal is attributable to targeting of ADDLs. Clearly, a search for drugs targeting the assembly of these soluble Abeta species represents a new and potentially important approach to the treatment of AD. In this work we describe the development of a dot-blot immunoassay to measure ADDL at the femtomole level, its use in defining the time course of ADDL formation, and its use in determining the presence of ADDLs in the hAPP transgenic mouse brain. Discussion of a protocol to screen agents for inhibition of neurotoxic ADDLformation both in vivo and in vitro is also presented. The methods are suitable for screening combinatorial libraries and, importantly, provide the potential for simultaneous information on candidate transport across the blood-brain barrier.
D000544:Alzheimer Disease / Q000188:drug therapy* / Q000378:metabolism; D016229:Amyloid beta-Peptides / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000633:toxicity; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000166:cytology / Q000378:metabolism / Q000503:physiopathology; D003505:Cyclodextrins; D004195:Disease Models, Animal; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D015151:Immunoblotting / Q000379:methods*; D051379:Mice; D008822:Mice, Transgenic; D016716:PC12 Cells; D010446:Peptide Fragments / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000633:toxicity; D011108:Polymers / Q000378:metabolism; D011237:Predictive Value of Tests; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D047392:beta-Cyclodextrins
D016229:Amyloid beta-Peptides; D003505:Cyclodextrins; D010446:Peptide Fragments; D011108:Polymers; D047392:beta-Cyclodextrins; C031215:betadex