DNA Damage Increases Secreted Aβ40 and Aβ42 in Neuronal Progenitor Cells: Relevance to Alzheimer's Disease

Abstract

Background: Recent studies suggest a strong association between neuronal DNA damage, elevated levels of amyloid-β (Aβ), and regions of the brain that degenerate in Alzheimer's disease (AD).

Objective: To investigate the nature of this association, we tested the hypothesis that extensive DNA damage leads to an increase in Aβ40 and Aβ42 generation.

Methods: We utilized an immortalized human neuronal progenitor cell line (NPCs), ReN VM GA2. NPCs or 20 day differentiated neurons were treated with hydrogen peroxide or etoposide and allowed to recover for designated times. Sandwich ELISA was used to assess secreted Aβ40 and Aβ42. Western blotting, immunostaining, and neutral comet assay were used to evaluate the DNA damage response and processes indicative of AD pathology.

Results: We determined that global hydrogen peroxide damage results in increased cellular Aβ40 and Aβ42 secretion 24 h after treatment in ReN GA2 NPCs. Similarly, DNA double strand break (DSB)-specific etoposide damage leads to increased Aβ40 and Aβ42 secretion 2 h and 4 h after treatment in ReN GA2 NPCs. In contrast, etoposide damage does not increase Aβ40 and Aβ42 secretion in post-mitotic ReN GA2 neurons.

Conclusion: These findings provide evidence that in our model, DNA damage is associated with an increase in Aβ secretion in neuronal progenitors, which may contribute to the early stages of neuronal pathology in AD.

Keywords: Alzheimer’s disease; DNA repair; amyloid-β; double strand breaks; etoposide; neurodegenerative disease; oxidative damage.

Fig. 1

Hydrogen peroxide damage increases Aβ₄₀ and Aβ₄₂ secretion in ReN GA2 NPCs, an effect that is mitigated by α-tocopherol treatment. A) Schematic of ReN GA2 NPC experimental design. ReN GA2 NPCs were treated with and without 5μM α-tocopherol prior to treatment with and without 2.5μM hydrogen peroxide for 0.5 h and allowed to recover for 24 h. (Schematic created in BioRender, 2021.) B) Cell viability was determined after indicated treatment and 24 h recovery time. C) Percent secreted Aβ₄₀ and Aβ₄₂ relative to controls after designated treatments and 24 h recovery time. D) Aβ₄₂/Aβ₄₀ ratio calculated from raw values. Error bars represent means±SD of three separate experiments, and the p values were determined using 2-way ANOVA and Tukey’s multiple comparisons test. ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

Fig. 2

Etoposide damage increases Aβ₄₀ and Aβ₄₂ secretion after 2 h and 4 h recovery in ReN GA2 NPCs. A) Treatment schematic for ReN GA2 NPCs treated with and without 10μM etoposide for 6 h and allowed to recover for designated times. (Schematic created in BioRender, 2021.) B) Percent cell viability after 6 h etoposide treatment and designated recovery times. C) Percent secreted Aβ₄₀ and Aβ₄₂ relative to controls after designated treatments and recovery times. D) Aβ₄₂/Aβ₄₀ ratio calculated from raw values. Error bars represent means±SD of three separate experiments, and the p values were determined using 2-way ANOVA and Tukey’s multiple comparisons test. ^*p < 0.05, ^**p < 0.01, ^****p < 0.0001, ns: not significant.

Fig. 3

DSBs increase sAβPPβ, but not total AβPP in ReN GA2 NPCs. A) Western blot of endogenous BACE1 expression in ReN GA2 NPCs and 20 day differentiated neurons treated with and without designated concentrations of etoposide for 6 h and allowed to recover for 2 h. B) Western blot of endogenous AβPP expression in ReN GA2 NPCs treated with and without 10μM etoposide for 6 h and allowed to recover for designated times. C) Western blot of sAβPPβ in ReN GA2 NPCs and 20 day differentiated neurons with and without 10μM of etoposide treatment for 6 h and allowed to recover for 2 h. Error bars represent means±SD of two to three separate experiments, and the p values were determined using two-way ANOVA and Tukey’s multiple comparisons test. ^***p < 0.001, ^****p < 0.0001.

Fig. 4

Etoposide damage does not increase Aβ₄₀ and Aβ₄₂ secretion in differentiated ReN GA2 neurons. A) Treatment schematic for differentiating ReN GA2 neurons and treatment with and without 10μM etoposide for 6 h and designated recovery times. (Schematic created in BioRender, 2021.) B) Immunofluorescent staining of ReN GA2 NPCs and 20-day differentiated neurons with antibodies for anti-MAP2 (neuron) and anti-Ki67 (proliferation marker). C) Percent secreted Aβ₄₀ and Aβ₄₂ relative to controls after 6 h etoposide treatment and designated recovery times. D) Aβ₄₂/Aβ₄₀ ratio calculated from raw values. Error bars represent means±SD of three separate experiments.