Partial loss of the DNA repair scaffolding protein, Xrcc1, results in increased brain damage and reduced recovery from ischemic stroke in mice

Abstract

Oxidative DNA damage is mainly repaired by base excision repair (BER). Previously, our laboratory showed that mice lacking the BER glycosylases 8-oxoguanine glycosylase 1 (Ogg1) or nei endonuclease VIII-like 1 (Neil1) recover more poorly from focal ischemic stroke than wild-type mice. Here, a mouse model was used to investigate whether loss of 1 of the 2 alleles of X-ray repair cross-complementing protein 1 (Xrcc1), which encodes a nonenzymatic scaffold protein required for BER, alters recovery from stroke. Ischemia and reperfusion caused higher brain damage and lower functional recovery in Xrcc1(+/-) mice than in wild-type mice. Additionally, a greater percentage of Xrcc1(+/-) mice died as a result of the stroke. Brain samples from human individuals who died of stroke and individuals who died of non-neurological causes were assayed for various steps of BER. Significant losses of thymine glycol incision, abasic endonuclease incision, and single nucleotide incorporation activities were identified, as well as lower expression of XRCC1 and NEIL1 proteins in stroke brains compared with controls. Together, these results suggest that impaired BER is a risk factor in ischemic brain injury and contributes to its recovery.

Keywords: Base excision repair; Oxidative stress; Stroke; Xrcc1.

Figure 1

Infarct volume, mortality rate, and neurological deficit after middle cerebral artery occlusion/ reperfusion (MCAO/R) in WT and Xrcc1 ^+/− mice. (A) Mouse brain sections stained with 2,3,5-triphenyltetrazolium chloride (TTC) after 48 h of MCAO/R. (B) Quantification of the infarct volume shown in A. The data are presented as a box plot diagram, which represents the minimum to maximum distribution of the average percentage of infarct volume ± SEM. (C) Percentage of death in mice within 48 h of the MCAO/R procedure. (D) Graphic of the assigned neurological deficit score at 24 and 48 h after the MCAO/R procedure. Data are presented as means ± SEM. Statistical analysis was performed by one-way ANOVA followed by Bonferroni’s post-hoc test for pairwise comparisons, *p<0.05, **p<0.01.

Figure 2

Motor dysfunction in Xrcc1 ^+/− and WT mice before and after middle cerebral artery occlusion/ reperfusion (MCAO/R). (A) Average number of falls and (B) average latency of first fall measured during rotarod test. Data indicate average ± SEM, where the sample size is WT, n = 4 and Xrcc1 ^+/− n = 3. A two-way ANOVA followed by Bonferroni’s correction as well as LSD was used to analyze the data. Asterisks denote the various p-values as follows: *, p≤0.024; **p≤0.001; *** p≤0.0001..

Figure 3

Apoptosis in Xrcc1 ^+/− mouse brains after middle cerebral artery occlusion/ reperfusion (MCAO/R). (A) Representative picture of a mouse brain section after TUNEL staining with FITC-labeled dUTP and terminal deoxynucleotidyl transferase enzyme in WT and Xrcc1 ^+/− mouse brains after MCAO/R. (B) Quantification of TUNEL+ nuclei in WT and Xrcc1 ^+/− mice. An average total TUNEL+ cells count was derived from at least six brain section areas using 4 WT and 3 Xrcc1 ^+/− mice is shown. Data are mean ± SEM. *p<0.05, **p<0.01. Data were analyzed by two-tailed Student's t-test. (C) Levels of the full length and cleaved caspase-3 in whole brain lysates of ipsilateral area of the stroked brains. The graph indicates the quantitation of full length caspase 3 and cleaved caspase 3 (sum of p19 + p17) levels shown in the blot normalized to the actin levels.

Figure 4

Expression of Xrcc1 & DNA Ligase III and whole BER activity in Xrcc1 ^+/− mouse brains after middle cerebral artery occlusion/ reperfusion (MCAO/R). (A) A representative immunoblot of Xrcc1 and Ligase III levels in whole brain lysates of contralateral area of the stroked brains. The graph indicates the quantitation of Xrcc1 and ligase III levels shown in the blot normalized to the actin levels. Data were averaged from 4 or 3 mice and at least two independent experiments. (B) Relative dCTP incorporation and ligation into a 91-mer in whole brain lysates of contralateral area of the stroked brains. Data indicate average ± SEM (*p<0.05 using a two-tailed Student's t-test with a sample size of n = 4 for WT and n = 3 for Xrcc1 ^+/−). (C) A representative immunoblot of Xrcc1 and Ligase III levels in whole brain lysates of ipsilateral area of the brains. The graph indicates the quantitation of Xrcc1 and ligase III levels shown in the blot normalized to the actin levels. Data were represented from at least two experiments. (D) Relative dCTP incorporation and ligation into a 91-mer in whole brain lysates of ipsilateral area of the brains. Data indicate average ± SEM.

Figure 5

BER activities in control and stroke human brain samples. (A) Whole tissue lysates from control and stroke postmortem brains were assessed for thymine glycol incision activity using oligonucleotide sequence (Table 2). (B) Whole tissue lysates from control and stroke postmortem brains were assessed for abasic site (tetrahydrofuran [THF]) cleavage using oligonucleotide sequence (Table 2). (C) Whole tissue lysates from control and stroke postmortem brains were assessed for deoxycytidinetriphosphate (dCTP) incorporation and ligation activity using oligonucleotide sequence (Table 2). The data are represented by mean ± SD, n = 9.

Figure 6

XRCC1 expression in control and stroke human brain samples. (A) Representative image of Immunofluorescence staining of XRCC1 in control and stroke human brain samples, n=8 (Insert) image of a single cell is enlarged to show the expression of XRCC1. (B) A representative immunoblot of XRCC1 levels in control and stroke human brain samples. The graph indicates the quantitation of XRCC1 levels shown in the blot normalized to the actin levels. The data are represented by mean ± SD, n = 8.

Figure 7

NEIL1 expression in control and stroke human brain samples. (A) Representative image of Immunofluorescence staining of NEIL1 in control and stroke human brain samples, n=8 (Insert) image of a single cell is enlarged to show the expression of NEIL1. (B) A representative immunoblot of NEIL1 levels in control and stroke human brain samples. The graph indicates the quantitation of NEIL1 levels shown in the blot normalized to the actin levels. The data are represented by mean ± SD, n = 8.