Prevention of Amyloid-Beta Oligomer-Induced Neuronal Death by EGTA, Estradiol, and Endocytosis Inhibitor

Background and objective. Alzheimer’s disease is a progressive neurodegenerative disease that is biochemically characterized by the accumulation of amyloid beta (Aβ) peptides in the brain. The current hypothesis suggests that Aβ oligomers rather than fibrillar aggregates are the most toxic species of Aβ though the mechanisms of their neurotoxicity are unclear. The authors have previously shown that small Aβ_1–42 oligomers at around 1 μM concentration caused rapid (in 24 h) neuronal death in cerebellar granule cell (CGC) cultures. In this study, we aimed to investigate whether protracted (up to 7 days) incubation of CGC cultures with lower submicromolar concentration of various aggregates of Aβ_1–42 had an effect on viability of neurons. In order to get some insight into the mechanism of Aβ-induced cell death, we also sought to determine whether extracellular Ca²⁺ and process of endocytosis contributed to Aβ oligomer-induced neurotoxicity and whether pharmacological interventions into these processes would prevent Aβ oligomer-induced cell death.
Material and Methods. Primary cultures of CGC were treated with various aggregate forms of Aβ_1–42. Cell viability was assessed by fluorescent microscopy using propidium iodide and Hoechst 33342 staining.
Results. Exposure of neurons to 500 nM Aβ_1–42 oligomers for 72–168 h caused extensive neuronal necrosis. Lower concentrations (100–250 nM) were not toxic to cells during 7 days of incubation. Aβ_1–42 monomers and fibrils had no effect on neuronal viability even after 7 days of incubation. Treatment of neurons with EGTA, steroid hormone 17β-estradiol, and methyl-β-cyclodextrin significantly reduced Aβ_1-42 oligomers-induced neuronal death.
Conclusions. The results show that submicromolar concentrations of Aβ_1-42 oligomers were highly toxic to neurons during protracted incubation inducing neuronal necrosis that can be prevented by chelating extracellular Ca2+ with EGTA, inhibiting endocytosis with methyl-β-cyclodextrin, or by estradiol, which may protect against mitochondrial permeability transition pore opening.