The Response to Oxidative DNA Damage in Neurons: Mechanisms and Disease

Abstract

There is a growing body of evidence indicating that the mechanisms that control genome stability are of key importance in the development and function of the nervous system. The major threat for neurons is oxidative DNA damage, which is repaired by the base excision repair (BER) pathway. Functional mutations of enzymes that are involved in the processing of single-strand breaks (SSB) that are generated during BER have been causally associated with syndromes that present important neurological alterations and cognitive decline. In this review, the plasticity of BER during neurogenesis and the importance of an efficient BER for correct brain function will be specifically addressed paying particular attention to the brain region and neuron-selectivity in SSB repair-associated neurological syndromes and age-related neurodegenerative diseases.

Figure 1

Simplified scheme for the short- and long-patch base excision repair pathways.

Figure 2

Cell survival as detected by the MTT assay in neurons from different brain regions treated with increasing H₂O₂ doses for 30 min. Error bars indicate the standard error. ^∗ p < 0.05 and ^∗∗ p < 0.01 versus untreated (NT) by Least Significant Difference (LSD) test. Two-way ANOVA did not show any significant difference in the response to treatment in the neurons derived from the three different brain regions (cell cultures were prepared as previously described) [–139]. In more detail, C57BL wild-type mice were sacrificed at postnatal day 5 (P5) for cerebellar granule cell cultures, whereas at day 17 embryos were collected from pregnant mice for preparation of cortical and hippocampal neurons. Brain regions were dissected and dissociated and cultures were maintained in appropriate media.

Figure 3

H₂O₂-induced DNA damage and repair as detected by γH2AX foci formation in neurons derived from different brain regions. (a) Left: percentage of cells bearing 10 or more γH2AX foci at different times after treatment with 20 μM H₂O₂ for 30 min. One-way ANOVA and post hoc analysis Least Significant Differences (LSD) indicated a significant increase of γH2AX positive cells immediately and 30 minutes after the treatment in all brain regions. Two-way ANOVA did not show any interaction between the treatment and the neuronal cell type. Right: kinetics of γH2AX dephosphorylation following posttreatment times up to 24 hrs. ANOVA test shows any interaction between treatment and the three types of neurons on slopes of focus kinetics. For each time point, at least 100 nuclei were examined. Error bars indicate standard error. ^∗ p < 0.05 versus untreated (NT) by LSD test; ^§ p < 0.05 versus t ₀ by LSD test; ^∗∗ p < 0.01 versus untreated (NT) by LSD test; ^§§ p < 0.01 versus t ₀ by LSD test; ^∗∗∗ p < 0.05 versus untreated (NT) by LSD test. (b) Immunofluorescence of γH2AX (red, Ser 139; Millipore, Billerica, MA, USA) in neurons exposed to H₂O₂ (20 μM, 30 min) with or without 1 hr pretreatment with a specific ATM kinase inhibitor KU55933 (10 μM). Nuclei are stained with DAPI in blue. One representative experiment is shown.

Figure 4

Levels of BER enzymes in neurons derived from cortex (CX), hippocampus (HP), and cerebellum (CV). Immunoblotting was carried out by using antibodies specific for DNA ligase 3 (103 kDa, BD Biosciences Pharmingen, San Diego, CA), FEN1 (43 kDa, Abcam, Atlanta, USA), XRCC1 (85 kDa, Bethyl Laboratories, Inc.), APE1 (35.5 kDa, Santa Cruz Biotechnology, Inc.), and DNA Polβ (38 kDa, Trevigen Inc., Gaithersburg, MD). β-actin (40 kDa, Sigma) and α-tubulin (55 kDa) were used as loading controls. Western blot analysis was conducted on 15 μg of whole cell extracts. One representative experiment is shown.