Intracellular injection of brain extracts from Alzheimer's disease patients trigger unregulated Ca2+ release from intracellular stores that hinders cellular bioenergetics

Abstract

Strong evidence indicates that amyloid beta (A{beta}) inflicts its toxicity in Alzheimers disease (AD) by promoting uncontrolled elevation of cytosolic Ca2+ in neurons. We have previously shown that synthetic A{beta}42 oligomers stimulate abnormal intracellular Ca2+ release from the endoplasmic reticulum stores, suggesting that a similar mechanism of Ca2+ toxicity may be common to the endogenous A{beta}s oligomers. To investigate this possibility, we use human postmortem brain extracts from control and AD-affected patients and test their ability to trigger Ca2+ fluxes when injected intracellularly into Xenopus oocytes. Immunological characterization of samples from AD patients revealed elevated content of soluble A{beta} oligomers, detected by the conformation-dependent OC-antibody, whereas no immunoreactivity was detected in the normal samples. Intracellular injection of brain extracts from control patients failed to trigger detectable changes in intracellular Ca2+. Conversely, brain extracts from AD patients triggered Ca2+ events consisting of local and global Ca2+ fluorescent transients rising within few seconds after injection and persisting for several seconds. Pre-incubation of brain extracts with the conformation specific OC antibody completely suppressed brain extract ability to trigger cytosolic Ca2+ events. Comparison of the elementary events triggered by brain extracts and synthetic A{beta}42 oligomer showed comparable temporal evolution and amplitudes to events triggered by direct injection of IP3. Moreover, bath application of caffeine reversibly inhibited local and global Ca2+ signals in all the samples confirming the involvement of Ca2+ release from the ER. Analysis of the recorded Ca2+ fluorescence signals by computational modeling allowed quantification of the IP3 and Ca2+ generated by each sample. The model further shows that the abnormal increase of Ca2+ and IP3 may affect mitochondrial bioenergetics. These results, supports the hypothesis that endogenous amyloid oligomer contained in neurons of AD-affected brains may represent the toxic agents responsible for neurons malfunctioning and death, associated with the disruption of neuronal Ca2+ homeostasis.