Neurons efficiently repair glutamate-induced oxidative DNA damage by a process involving CREB-mediated up-regulation of apurinic endonuclease 1

Abstract

Glutamate, the major excitatory neurotransmitter in the brain, activates receptors coupled to membrane depolarization and Ca(2+) influx that mediates functional responses of neurons including processes such as learning and memory. Here we show that reversible nuclear oxidative DNA damage occurs in cerebral cortical neurons in response to transient glutamate receptor activation using non-toxic physiological levels of glutamate. This DNA damage was prevented by intracellular Ca(2+) chelation, the mitochondrial superoxide dismutase mimetic MnTMPyP (Mn-5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine chloride tetrakis(methochloride)), and blockade of the permeability transition pore. The repair of glutamate-induced DNA damage was associated with increased DNA repair activity and increased mRNA and protein levels of apurinic endonuclease 1 (APE1). APE1 knockdown induced accumulation of oxidative DNA damage after glutamate treatment, suggesting that APE1 is a key repair protein for glutamate-induced DNA damage. A cAMP-response element-binding protein (CREB) binding sequence is present in the Ape1 gene (encodes APE1 protein) promoter and treatment of neurons with a Ca(2+)/calmodulin-dependent kinase inhibitor (KN-93) blocked the ability of glutamate to induce CREB phosphorylation and APE1 expression. Selective depletion of CREB using RNA interference prevented glutamate-induced up-regulation of APE1. Thus, glutamate receptor stimulation triggers Ca(2+)- and mitochondrial reactive oxygen species-mediated DNA damage that is then rapidly repaired by a mechanism involving Ca(2+)-induced, CREB-mediated APE1 expression. Our findings reveal a previously unknown ability of neurons to efficiently repair oxidative DNA lesions after transient activation of glutamate receptors.

FIGURE 1.

Glutamate induces reversible oxidative DNA damage in cerebral cortical neurons. A and B, cortical cultures were exposed to 20 μm glutamate for 10 min, then, after 6 or 24 h the cells were harvested and comet assays were performed in the presence of no enzyme, FPG, or T4 endonuclease. Panel A shows representative images of nuclear DNA. First row of panel A is the control group, without glutamate treatment, pre-treated with FPG or T4 endo. The second and third rows of panel A are 6 and 24 h after glutamate treatment, respectively. Panel B shows the results of quantitative measurements of DNA damage (Olive Tail Moment). C, cellular ATP levels at the indicated time points after a 10-min exposure to 20 μm glutamate; values for glutamate-treated neurons were normalized to the value for untreated control cultures. Values for treated cultures are expressed as a percentage of the value for untreated control cultures (100%) (mean ± S.D.; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

FIGURE 2.

Glutamate-induced DNA damage is mediated by Ca²⁺ and mitochondrial superoxide, and requires opening of the mitochondrial permeability transition pore. Cortical cultures were pretreated with vehicle, 10 μm BAPTA-AM, 10 μm MnTMPyP, or 5 μm CsA, and were then exposed to 20 μm glutamate for 10 min. At designated times after glutamate exposure the cells were harvested and comet assays were performed in the presence of no enzyme or FPG. A, images of nuclear DNA from cortical cells that had been exposed to the indicated treatments for 1, 6, or 24 h. B–D, results of quantitative measurements of DNA damage in neurons pretreated with BAPTA-AM (B), MnTMPyP (C), or CsA (D), exposed to glutamate for 10 min, and then incubated for the indicated time periods (in the continued presence of BAPTA-AM, MnTMPyP, or CsA) (mean ± S.D.; ***, p < 0.001).

FIGURE 3.

Incision activity of OGG1 and NEIL1 are not significantly changed after glutamate treatment. The 8-oxoguanine and 5-hydroxycytosine lesions are mainly produced by oxidation and predominately removed by OGG1 and NEIL1, respectively. The results of biochemical and immunoblotting assays for incision activities of OGG1 (A) and NEIL1 (B) (upper panels; *, ³²P-labeled 5′-end; the filled circle marks the 8-OxoG and 5-OHC lesion sites) were not affected by a 10-min pulse glutamate treatment. Values for treated cultures are expressed as a percentage of the value for untreated control cultures (100%).

FIGURE 4.

APE1 DNA incisional activity, and mRNA and protein levels, are elevated in cortical neurons in response to glutamate. Cortical cultures were exposed to glutamate for the indicated time periods and then harvested for analyses of APE1 DNA incision activity (A), APE1 protein levels (B), and APE1 mRNA levels (C). A, an example of an APE1 DNA incision assay (upper; *, ³²P-labeled 5′-end; the filled circle marks the abasic site lesion) and measurements obtained using this assay (graph). B, an example of an APE1 protein immunoblot (upper) and the results of densitometric analysis of blots (graph). C, relative levels of APE1 mRNA as determined by quantitative RT-PCR analysis. Values for treated cultures are expressed as a percentage of the value for untreated control cultures (100%) (mean ± S.D.; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

FIGURE 5.

Glutamate induces CREB phosphorylation in a CaMK-dependent manner. The phosphorylation of CREB was increased at 15 and 30 min after transient exposure to glutamate (A). Glutamate-induced CREB phosphorylation is inhibited in neurons pretreated with the CaMK inhibitor KN-93 (B). Glutamate-induced expression of APE1 is also inhibited by KN-93 (C). Values for treated cultures are expressed as a percentage of the value for untreated control cultures (100%) (mean ± S.D.; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

FIGURE 6.

Depletion of CREB using RNA interference technology abolishes the ability of neurons to repair DNA damaged as a result of glutamate receptor activation. A, Western blot showing that CREB levels are greatly reduced in neurons expressing shRNA directed against the CREB mRNA compared with neurons expressing a scrambled control shRNA. B, images of nuclear DNA from neurons expressing CREB shRNA or scrambled control shRNA at 6 or 24 h after a 10-min exposure to 20 μm glutamate. C, results of quantitative measurements of DNA damage demonstrated that neurons expressing scrambled control shRNA recovered from damage, whereas oxidative lesions in neurons expressing CREB shRNA were not repaired within 24 h (mean ± S.D.; ***, p < 0.001).

FIGURE 7.

Depletion of APE1 using RNA interference technology results in accumulation of glutamate-induced oxidative DNA damage. A, Western blot showing that APE1 levels are greatly reduced in neurons expressing shRNA directed against the APE1 mRNA compared with neurons expressing a scrambled control shRNA. B, images of nuclear DNA from neurons expressing APE1 shRNA or scrambled control shRNA at 6 or 24 h after a 10-min exposure to 20 μm glutamate. C, results of quantitative analysis of DNA damage demonstrated that neurons expressing scrambled control shRNA were recovered from damage, whereas oxidative lesions in neurons expressing APE1 shRNA were not repaired within 24 h (mean ± S.D.; ***, p < 0.001).

FIGURE 8.

Model for the mechanisms of glutamate-induced DNA damage and repair in neurons. Activation of glutamate receptors results in Ca²⁺ influx which, in turn, induces mitochondrial superoxide production and opening of PTP in the mitochondrial membranes. The superoxide or related reactive oxygen species then cause nuclear DNA damage. The DNA damage can be prevented by chelating intracellular Ca²⁺, by removing mitochondrial superoxide and blocking the PTP. Glutamate-induced Ca²⁺ influx also triggers the activation of CaMK and CREB resulting in increased expression of APE1 and repair of the damaged DNA.