Generation of conditional APE1 knockout mice. (A) Targeting vector strategy. loxP sites were engineered flanking exons IV and V (the third and final coding exons) of the Apex1 gene to create cKO APE1 mice. Additional restriction sites were engineered into the vector to perform restriction enzyme analyses to confirm successful recombination of the loxP sites, including AseI for the 5′ loxP site, and BstEII and BglII for the 3′ loxP site and presence of the Neo gene. The inclusion of the Neo gene was to allow for selection of clones resistant to G418, and was flanked by frt sites to allow for removal from the targeted mouse strain. HSV-TK was included outside of the targeting region to eliminate cells with random integration of the targeting vector, as the expression of this gene will lead to sensitivity to gancylovir allowing these cells to be eliminated. When crossed to transgenic mice expressing the Cre protein in specific cells, the loxP sites recombine in these cells and delete the floxed region of the Apex1 gene knocking out the protein. Open boxes represent noncoding exon regions. Solid arrows represent restriction enzyme cut sites, black arrowheads represent loxP sites, red arrowheads represent Frt sites, and the short red line at the 3′ end represents the external 3′ probe. (B) Southern blot screening of ES clones digested with BstEII led to the identification of a positive clone yielding a band at the predicted size (4.7 kb) when probed with the external 3′ probe (red line in A). Both nontargeted clones (NT) and the targeted clone (T) are shown. (C) The positive clone was amplified and rescreened using BglII to confirm recombination by the 3′ loxP region, yielding a band at the predicted size (6.7 kb) when probed with an external 3′ probe (red line in A). (D) To confirm the presence of the 5′ loxP site, genomic DNA containing the region between exon III and exon IV was amplified and digested with Ase1. The resulting PCR product from the targeted clone (T) produced a single band (lane 3) and was cleaved into two smaller fragments by Ase1 (lane 4, arrows), whereas the PCR product from WT DNA (WT) produced a band (lane 1) that was not cleaved by Ase1 (lane 2) because of the lack of the inserted loxP and Ase1 sequence.

Tamoxifen yields knockout of APE1 protein and inhibition of DNA repair activity in cKO mice. (A) Scheme of breeding to obtain APE1 cKO mice. Homozygous APE1^flox/flox mice were crossed with hemizygous CAGGCre-ER (i.e., CAGGCre-ER; APE1^wt/wt) mice to generate mice, 50% of which were CAGGCre-ER; APE1^flox/wt mice. These mice were bred to APE1^flox/flox mice, yielding offspring 25% of which had the genotype CAGGCre-ER; APE1^flox/flox. (B) Scheme of breeding to obtain APE1 WT mice as age- and gender-matched controls to APE1 cKO mice. Hemizygous CAGGCre-ER mice were crossed with WT C57BL/6J mice (i.e., APE1^wt/wt) to generate mice, 50% of which were CAGGCre-ER; APE1^wt/wt mice. Tan boxes indicate genotypes used for subsequent experiments (APE1 cKO and APE1 WT). (C) PCR genotyping demonstrating generation of heterozygous mice (Left) and subsequent breeding to obtain homozygous mice containing the loxP sites on both alleles (Right). Using primers flanking the 3′ loxP site, the floxed allele runs at a weight of 299 bp, whereas the WT allele has a weight of 217 bp. (D) Successful ablation of APE1 protein expression in APE1 cKO mice treated with tamoxifen for 5 d. Tissue was harvested 3 d after the last tamoxifen injection from the cortex, striatum, and CC from both WT and APE1 cKO mice. Actin immunoblotting served as the loading control. (E) Tamoxifen-induced knockout of APE1 led to loss of DNA excision-repair activity. In vitro excision-repair activity was assessed at 3 d after last tamoxifen injection using lysates derived from the striatum (S) or cortex (C) of WT C57BL/6J mice (Cre⁻; APE1^wt/wt), APE1 WT mice (CAGGCre-ER; APE1^wt/wt), or APE1 cKO (CAGGCre-ER; APE1^flox/flox). The lysates were incubated with a ³²P-labeled 50-bp oligonucleotide substrate containing a synthetic AP site. APE1 activity leads to the generation of a 25-bp product. Lysates derived from the APE1 cKO mouse brain showed marked reduction in the presence of the 25-bp product. As a negative control (Control), the assay done without the protein lysates resulted in absence of the 25-bp product.

Induced knockout of APE1 increases infarct volumes, mortality, and cell death after tFCI. tFCI was induced in tamoxifen-treated CAGGCre-ER; APE1^wt/wt (APE1 WT) and CAGGCre-ER; APE1^flox/flox (APE1 cKO) mice. (A) Cerebral blood flow was assessed by 2D laser-speckle imaging. Representative images show the control, the ischemic area (depicted by the dotted line) during 5 and 30 min of tFCI and after 5, 15, and 45 min of reperfusion following 60 min of tFCI. (Scale bar, 1 mm.) The bar graphs show ischemic areas at 5 and 25 min in both the ischemic core (defined as CBF < 20% of preischemia baseline) and penumbra (P) (CBF = 20–35% of preischemia baseline) during 30 or 60 min of tFCI or 15 min of reperfusion. The results reveal similar CBF changes in APE1 WT and APE1 cKO mice. n = 5 per group. (B and C) Infarct volume was assessed at 48 h after tFCI by TTC (2,3,5-triphenyltetrazolium) staining (the right and left hemispheres are labeled with R and L, respectively) and quantified as described using ImageJ under blinded conditions. Values are means ± SEM, n = 8 per group. **P ≤ 0.01 vs. the matched APE1 WT group. ^##P ≤ 0.01 vs. 30 min WT group. (D) The number of animals that survived (bottom portion of bars) or died (upper red portion of bars) by 28 d after tFCI. Comparisons of animal survival rates were performed by Kaplan–Meier survival analysis. *P ≤ 0.05 vs. the matched APE1 WT group. (E) Histological assessments of cell death were performed at 48 h after tFCI (30 min) using the TUNEL stain for DNA fragmentation and Fluoro-Jade B (Fluo JB) for cell degeneration, respectively. TUNEL⁺ cells were counted using stereology, and Fluoro-Jade B was semiquantified by measuring the fluorescence intensity in the striatum (Str) and cortex (Ctx). Values are mean ± SEM, n = 6 per group. **P ≤ 0.01, ***P ≤ 0.001 vs. the matched APE1 WT group.

APE1 deficiency impairs sensorimotor and cognitive recovery after tFCI. In sham control animals, neither the sensorimotor function tests (rotarod test, corner test) nor the cognitive test (Morris water maze) revealed significant difference between the APE1 WT and APE1 cKO mice. Thus, the “sham group” in A, B, and D refers to the combination of both APE1 WT and APE1 cKO genotypes. (A) Rotarod performance was measured every other day for the first week following 30 min of tFCI. The latency to fall after ischemic injury was normalized to latency before the induction of ischemia. n = 6–8 per group. *P ≤ 0.05; **P ≤ 0.01 vs. the matched APE1 WT group; ^#P ≤ 0.05 vs. the sham group. (B) The corner test was performed up to 28 d following 30 min of tFCI. The number of left turns per 10 trials was recorded and plotted. The sham control group showed no turning bias or asymmetry (average of 5 left turns/10 trials); the postischemic mice showed increased turning toward the body side with unaffected mobility (left, ipsilateral to the ischemic hemisphere). The APE1 WT mice showed complete recovery by 7–10 d, whereas the APE1 cKO mice showed only partial recovery by 28 d. n = 6–8 per group. *P ≤ 0.05; **P ≤ 0.01 vs. the matched APE1 WT group; ^#P ≤ 0.05 vs. the sham group. (C–F) Long-term cognitive functions were assessed by the Morris water maze at 22–27 d after 30 min of tFCI. n = 8–10 per group. Shown are the mean ± SEM *P ≤ 0.05 vs. the matched APE1 WT group; ^##P ≤ 0.01 vs. the sham group. (C) Representative swim path traces (24–26 d after tFCI) of mice attempting to find a hidden platform (top traces, “learning”), or searching for the platform after its removal (bottom traces, “memory”). (D) The escape latency (i.e., time needed to discover the platform) was recorded on days 22–26 after tFCI. (E) Memory was tested at 27 d after tFCI. The percentage of time spent in the target quadrant after the platform was removed was plotted to reflect memory. (F) Swimming speed was assessed at 27 d after tFCI for each genotype, to ensure that the observed differences did not reflect compromised swimming abilities.

APE1 deficiency increases neuronal tissue loss after tFCI. Brains were harvested at 28 d after 30 min tFCI and processed for MAP2 immunostaining. The representative images show MAP2 immunofluorescence in APE1 WT and APE1 cKO brains after tFCI, respectively; the dashed line depicts the MAP2⁻ infarct area on each brain section (30-μm thick). The right and left hemispheres are labeled with R and L, respectively. (Scale bar, 1 mm.) Areas of neuronal tissue loss were measured on six equally-spaced coronal sections within the MCA territory per brain, and subsequently converted into infarct volumes. Data are mean ± SEM, n = 8 per group. **P ≤ 0.01 vs. the matched APE1 WT group.

APE1 deficiency increases the accumulation of ODD after tFCI. (A) AP site frequency was quantitatively measured in cortical and striatal extracts, respectively, from APE1 WT and APE1 cKO mice under sham condition (S) or at 0.5, 2, 8, and 24 h after 30 min of tFCI. Data are expressed as the number of AP site per 10⁶ nucleotides. (B) SSBs were detected immunohistochemically using the PANT assay, and positively labeled cells were counted using stereology at the indicated time points after 30 min of tFCI. Values are means ± SEM, n = 6 per group. *P ≤ 0.05; **P ≤ 0.01 vs. the matched APE1 WT group; ^#P ≤ 0.05; ^##P ≤ 0.01; ^###P ≤ 0.001 vs. the sham group. (C) Levels of phospho-H2AX (marker of genomic damage) were assessed in cortical lysates by Western blotting at 2, 8, and 24 h after tFCI. n = 4 per group. **P ≤ 0.01 vs. the matched APE1 WT group; ^##P ≤ 0.01 vs. the sham group. (D) Double-label immunofluorescent staining for p-H2AX (green) and the neuronal marker NeuN (red) in the cortices of sham controls and at 2 or 24 h after tFCI (a–c, APE1 WT; d–f, APE1 cKO mice). High-magnification images from the boxed regions revealed distinctly punctate patterns of p-H2AX immunofluorescence in postischemic cortical neurons (a′–c′, APE1 WT; d′–f ′, APE1 cKO mice). The frequency of punctate p-H2AX immunofluorescence subsided to control levels at 24 h after tFCI in APE1 WT brain, but remained elevated at 24 h after tFCI in APE1 cKO brains.

APE1 deficiency activates proapoptotic PUMA signaling in postischemic neurons. (A) Representative images demonstrating TUNEL (green, arrows) colabeling with the neuronal marker NeuN (red, arrows) in the postischemic cortex of an APE1 cKO mouse 24 h following 30 min tFCI. DAPI (blue, in merged image) staining was used to identify cell nuclei. (B) Levels of PUMA in total protein extracts and the mitochondrial fraction of cortical tissue were assessed by Western blotting after sham operation or at 2, 8, and 24 h after tFCI. β-Actin and TOM20 (a mitochondria-specific protein) served as the loading controls for whole cell protein extracts and the mitochondrial fraction, respectively. Data are presented as mean± SEM, fold-changes versus sham controls. n = 4 per group. **P ≤ 0.01 vs. the matched APE1 WT group. (C) Triple-label immunofluorescent staining for PUMA (red), TOM20 (green), and DAPI (blue, in merged images only) in the postischemic cortex at 8 h following 30 min of tFCI in APE1 WT (Upper) and APE1 cKO (Lower) mice. PUMA immunofluorescence was increased and was closely associated with TOM20 (arrows) in postischemic cortical neurons of APE1 cKO brains, but not of APE1 WT brains. (D) Close association of intense PUMA immunofluorescence (red, arrows) with TUNEL⁺ staining (green, arrows) in the ischemic cortex 24 h after 30 min of tFCI in an APE1 cKO brain. Cell nuclei were stained with DAPI in the merged image. Two TUNEL⁻ cells were also immunonegative for PUMA (arrowheads).

APE1 deficiency activates proapoptotic PARP1 in postischemic oligodendrocytes. (A) TUNEL staining of brain sections at 48 h following 30 min of tFCI. Ctx, cortex; Str, striatum; CC/EC, corpus callosum and external capsule. Few TUNEL⁺ cells could be detected in these brain regions of the APE1 cKO or APE1 WT mice after sham surgery. (B) The number of TUNEL⁺ cells in the CC/EC region was quantified. n = 6 per group. ***P ≤ 0.001 vs. the matched APE1 WT group; ^#P ≤ 0.05; ^###P ≤ 0.001 vs. the sham group. (C) Colocalization (yellow arrows) of TUNEL (green) with the mature oligodendrocytic marker APC (red) in the CC/EC in sham and 48 h following 30 min of tFCI in APE1 WT and APE1 cKO mice. (D) TUNEL⁺/APC⁺ cells were quantified from randomly selected microscopic fields in the CC/EC. n = 6 per group. ***P ≤ 0.001 vs. the matched APE1 WT group; ^#P ≤ 0.05; ^###P ≤ 0.001 vs. the sham group. (E and F) Western blot analysis of PAR (marker of PARP1 activity, multiple bands) in extracts derived from CC/EC after sham operation (S) or 48 h after ischemia (I). PAR immunoblot results were as fold-changes over sham controls. n = 4 per group. *P ≤ 0.05 vs. the matched APE1 WT group; ^##P ≤ 0.01 vs. the sham group. (G and H) Colocalization (yellow, arrows) of PAR (green) with APC (red) in the CC/EC at 48 h following 30 min of tFCI. PAR⁺/APC⁺ cells were quantified from randomly selected microscopic fields in the CC/EC. n = 6 per group. **P ≤ 0.01 vs. the matched APE1 WT group; ^#P ≤ 0.05; ^###P ≤ 0.001 vs. the sham group.

Astrocytes and microglia are relatively resistant to cell death in the white matter after mild ischemic injury. Double-label staining of TUNEL with the astrocyte marker GFAP (Upper) or the microglial/macrophage marker Iba1 (Lower) in the CC/EC of APE1 WT and APE1 cKO mice, respectively, at 48 h following 30 min tFCI. Few TUNEL⁺ cells are also GFAP⁺ or Iba1⁺, suggesting that, compared with oligodendrocytes, astrocytes and microglia/macrophages in the white matter are less vulnerable to ischemia-induced cell death. Images are representatives of five mice per group with similar results.

APE1 deficiency exacerbates striatal cell death after tFCI and is associated with PARP1 activation. (A) Colocalization (yellow arrows) of TUNEL (green) with the mature oligodendrocytic marker APC (red) in the striatum at 48 h following 30 min of tFCI. Note that the number of TUNEL/APC double-positive cells was greater in APE1 cKO than in APE1 WT postischemic striatum. (B) Colocalization (yellow arrows) of PAR (green) with APC (red) in the striatum at 48 h following 30 min tFCI. PAR/APC double-positive cells were quantified from randomly selected microscopic fields. n = 6 per group. (C) Western blot analysis of PAR in protein extracts derived from the striatum after sham operation (S) or 48 h after ischemia (I). Immunoblot results for PAR were semiquantified. n = 4 per group. ^#P ≤ 0.05; ^##P ≤ 0.01; ^###P ≤ 0.001 vs. sham controls with the same genotype; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 vs. the matched APE1 WT group.

APE1 deficiency elicits long-term loss of white matter integrity after tFCI. (A) Double-label immunofluorescent staining for MBP and dephosphorylated neurofilament protein (SMI-32) in the ipsilesional CC/EC and striatum at 28 d after 30 min of tFCI or sham operation. tFCI decreased MBP labeling and increased SMI-32 labeling in both CC/EC and striatum; APE1 cKO mice were much more affected than APE1 WT mice. The graphs at the bottom illustrate the ratio of SMI-32 to MBP, indicating the degree of demyelination in the CC/EC and striatum. Values are mean ± SEM, n = 6 per group. **P ≤ 0.01; ***P ≤ 0.001 vs. the matched APE1 WT group; ^##P ≤ 0.01; ^###P ≤ 0.001 vs. sham group. (B) Western blot analysis showing multiple MBP bands in protein extracts from the CC/EC or striatum at 28 d after 30 min tFCI or sham operation. The graphs (Right) illustrate fold-changes in MBP protein levels in the CC/EC and striatum after tFCI relative to the sham group. Values were summed for all bands in each lane and are presented as mean ± SEM, n = 4–5 per group. **P ≤ 0.01; ***P ≤ 0.001 vs. the matched APE1 WT group; ^#P ≤ 0.05; ^###P ≤ 0.001 vs. sham group. (C) Functional evaluation of white matter integrity by CAPs at 28 d after 30 min tFCI. The stimulating and recording electrodes were positioned in the CC of coronal brain slices to measure evoked CAPs (a dashed line indicates the approximate location of ischemic lesion). Representative traces of the evoked CAPs in the CC after subthreshold stimuli (labeled as 0), and 1- and 3-mA stimuli are shown. Signal conduction along nerve fibers, as measured by the amplitude of the N1 component of the CAPs in response to increasing stimulus strength (up to 5 mA) were analyzed in sham controls and after tFCI. Data are mean ± SEM, n = 6–8 per group. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 vs. the matched APE1 WT group.

Full-length Western blots of MBP staining. Shown are the original full-length blots from which was generated. Actin immunoblotting served as the loading control. Multiple bands were identified by the anti-MBP antibody, possibly due to the existence of different MBP isoforms in the mouse brains (). Dashed line boxes indicate regions that are presented in .