Operational dissection of β‐amyloid cytopathic effects on cultured neurons

Abstract

Alzheimer disease (AD) affects mainly people over the age of 65 years, suffering from different clinical symptoms such as progressive decline in memory, thinking, language, and learning capacity. The toxic role of β-amyloid peptide (Aβ) has now shifted from insoluble Aβ fibrils to smaller, soluble oligomeric Aβ aggregates. The urgent need for efficient new therapies is high; robust models dissecting the physiopathological aspects of the disease are needed. We present here a model allowing study of four cytopathic effects of Aβ oligomers (AβO): oxidative stress, loss of synapses, disorganization of the neurite network, and cellular death. By generating a solution of AβO and playing on the concentration of and time of exposure to AβO, we have shown that it was possible to reproduce early effects (oxidative stress) and the long-term development of structural alterations (death of neurons). We have shown that 1) all toxic events were linked to AβO according to a specific timing and pathway and 2) AβO were probably the key intermediates in AD pathogenesis. The present model, using Aβ peptide solution containing AβO, reproduced essential neuropathological features of AD; the effects involved were similar whatever the kind of neurons tested (cortical vs. hippocampal). By using a single system, it was possible to embrace all toxic mechanisms at defined times and concentrations, to study each involved pathway, and to study the effects of new molecules on the different neurotoxic pathways responsible for development of AD.

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“…This batch, extensively studied by several techniques contained low and high molecular weight Aβ 1–42 oligomers as assessed by Western blot analyses using the 6E10 antibody. We verified that only preparations containing Aβ 1–42 oligomers were able to induce toxicity in neuronal cultures, while initial peptide preparation that did not contain oligomers was not toxic21, excluding a nonspecific effect of contaminating agents. This allowed us to use non-treated cells as controls without the need for using scrambled peptides as negative controls.…”

“…To test the protective potential of ABC against the effects of toxic soluble oligomeric Aβ 1–42 peptides, we used two models: a primary culture of rat cortical neurons21, and a primary culture of human brain-derived microvascular endothelial cells (HBMEC) that form a tubular network in matrigel22. We used the same batch of Aβ 1–42 peptides described by us previously21.…”

“…We used the same batch of Aβ 1–42 peptides described by us previously21. This batch, extensively studied by several techniques contained low and high molecular weight Aβ 1–42 oligomers as assessed by Western blot analyses using the 6E10 antibody.…”

“…In addition, we note that our Aβ 1–42 oligomeric preparation was performed in media containing phenol red at concentrations (i.e. 44 μM) that did not affect oligomeric formation as assessed by Western blot analyses attesting for the presence of these toxic oligomers21 that were active in our experimental settings. It was also described elsewhere that high concentrations of phenol red should be used over several days to inhibit efficiently Aβ 42 oligomerisation66.…”

“…Rat primary cortical and hippocampal neurons were cultured as described previously21. Briefly, 3 pregnant Wistar female rats of 15 days gestation for the use of cortical neurons or 17 days gestation for hippocampal neurons were killed by cervical dislocation and cortices of foetuses were dissociated.…”