Abstract
Amyloid beta (Abeta) neurotoxicity is believed to play a critical role in the pathogenesis of Alzheimer's disease (AD) mainly because of its deposition in AD brain and its neuronal toxicity. However, there have been discrepancies in Abeta-induced cytotoxicity studies, depending on the assay methods. Comparative analysis of Abeta42-induced in vitro cytotoxicity might be useful to elucidate the etiological role of Abeta in the pathogenesis of AD. In this study, MTT, CCK-8, calcein-AM/EthD-1 assays as well as thorough microscopic examinations were comparatively performed after Abeta42 treatment in a neuronal precursor cells (NT2) and a somatic cells (EcR293). Extensive formation of vacuoles was observed at the very early stage of Abeta42 treatment in both cells. Early observation of Abeta42 toxicity as seen in vacuole formation was also shown in MTT assay, but not in CCK-8 and calcein-AM/EthD-1 assays. In addition, Abeta42 treatment dramatically accelerated MTT formazan exocytosis, implying its effect on the extensive formation of cytoplasmic vacuoles. Abeta42 seems to cause indirect inhibition on the intracellular MTT reduction as well as vacuole formation and exocytosis enhancement. Following the acute cellular dysfunction induced by Abeta42, the prolonged treatment of micromolar concentration of Abeta42 resulted in slight inhibition on redox and esterase activity. The early Abeta42-induced vacuolated morphology and later chronic cytotoxic effect in neuronal cell might be linked to the chronic neurodegeneration caused by the accumulation of Abeta42 in AD patients' brain.
Highlights
Alzheimer’s disease (AD) is characterized by the presence of extracellular amyloid β peptide (Aβ) deposits
With the additional proof by cell counting kit-8 (CCK-8) assay and calcein-AM/ EthD-1 assay, we presented adequate evidence for that, Amyloid β 42 (Aβ42) apparently inhibited the intracellular reduction on MTT without direct inhibition on cellular redox activity
dimethyl sulphoxide (DMSO) treatment did not show any visible vacuoles as expected while Aβ treatment induced vacuole formation as long as cells were treated for longer than 2.5 h
Summary
Alzheimer’s disease (AD) is characterized by the presence of extracellular amyloid β peptide (Aβ) deposits. To be an additional proof for MTT assay for Aβ-induced toxicity, CCK-8 assay can be used as an indicator for the intracellular redox activity and for the cell injury upon treatment with Aβ peptide. Further evidence for evaluating the viability of cells treated with Aβ peptide can be obtained by using calcein-AM assay, which based on readout of the intracellular esterase activity, with mechanism different to that of MTT and CCK-8 assays. With the additional proof by CCK-8 assay and calcein-AM/ EthD-1 assay, we presented adequate evidence for that, Aβ42 apparently inhibited the intracellular reduction on MTT without direct inhibition on cellular redox activity
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