DNA2 knockout aggravates cerebral ischemia/reperfusion injury by reducing postsynaptic Homer1a

Abstract

DNA2, a multifunctional enzyme with structure-specific nuclease, 5 '-to-3 ' helicase, and DNA-dependent ATPase activities, plays a pivotal role in the cellular response to DNA damage. However, its involvement in cerebral ischemia/reperfusion (I/R) injury remains to be elucidated. This study investigated the involvement of DNA2 in cerebral I/R injury using conditional knockout (cKO) mice ( Nestin-Cre) subjected to middle cerebral artery occlusion (MCAO), an established model of cerebral I/R. Results demonstrated a gradual up-regulation of DNA2 expression, peaking at 72 h post-MCAO. Notably, DNA2 cKO mice exhibited more pronounced brain injury, neurological deficits, and neuronal apoptosis within the penumbra following MCAO. Additionally, DNA2 expression was elevated in an oxygen-glucose deprivation/reoxygenation (OGD/R) cell culture model, and DNA2 knockdown (KD) exacerbated neuronal apoptosis and oxidative stress. Transcriptome analysis of ischemic penumbra tissues via RNA sequencing revealed significant down-regulation of Homer1 in DNA2 cKO mice. Furthermore, in vitro experiments demonstrated that overexpression of Homer1a ameliorated DNA2 KD-induced neuronal apoptosis. Collectively, these findings demonstrate that DNA2 deficiency exacerbates cerebral I/R injury through the down-regulation of Homer1a, highlighting a novel regulatory axis in ischemic neuroprotection.

Keywords: Cerebral I/R; DNA2; Homer1a; Neuronal apoptosis; OGD/R.

Figure 1

Expression pattern of DNA2 in ischemic penumbra following middle cerebral artery occlusion (MCAO) A: Schematic of animal experiments. B: Schematic of ischemic core (red) and penumbra (blue). C: WB images showing levels of DNA2 in ischemic penumbra at indicated time intervals following MCAO. D: Quantification of DNA2 protein levels (n=4), normalized to β-actin, with Sham group set to 1. ^*: P<0.05; ^**: P<0.01; ^***: P<0.001 (one-way ANOVA with Dunnett’s post hoc analysis, F(5,18)=48.15). E: Representative images of IHC staining of DNA2 (red), cell nuclei counterstained with DAPI (blue) in ischemic penumbra from Sham or MCAO groups. Scale bar: 50 μm. F: Quantification of DNA2 fluorescence intensity (n=4). ^***: P<0.001 (Student’s t-tests, t(6)=28.94).

Figure 2

DNA2 deficiency exacerbates neurobehavioral deficits and brain damage in MCAO A: Schematic of DNA2^fl/fl mouse line construction. B: Representative genotyping images of DNA2^fl/fl mice using standard PCR method, with DNA gels showing genotypes of homozygous DNA2^fl/fl (600 bp), WT (500 bp), and Nestin-Cre (900 bp) mice. C: Representative WB images of DNA2 protein levels in cerebral ischemic penumbra. D: Representative brain sections stained with TTC showing infarcts 72 h after MCAO. E: Quantitative analysis of infarct size presented as a percentage of contralateral hemisphere size 3 days after MCAO (n=5). ^***: P<0.001 (Student’s t-tests, t(8)=7.25). F: Neurological deficit scores of mice at 24 h and 72 h after MCAO (n≥6). 24 h: F(2,20)=78.23, 72 h: F(2,20)=48.34. G: Duration of mice on rotarod (n≥6). 24 h: F(2,15)=48.01, 72 h: F(2,15)=38.12. H: Comparison of forelimb grip strength in mice (n≥6). 24 h: F(2,18)=64.68, 72 h: F(2,18)=46.85. I: Time taken by mice to remove tape from their left paw (n≥6). 24 h: F(2,15)=15.22, 72 h: F(2,15)=24.33. ns: Not significant; ^*: P<0.05; ^**: P<0.01; ^***: P<0.001 (one-way ANOVA with Dunnett’s post hoc analysis in F, G, H, and I).

Figure 3

DNA2 conditional knockout (cKO) aggravates MCAO-induced neuronal loss A: Nissl-stained images of neurons in cerebral ischemic penumbra in WT and DNA2 cKO mouse brains. Scale bar: 100 μm. B: Quantitative results of Nissl-positive neurons (n=4). ^***: P<0.001 (one-way ANOVA with Dunnett’s post hoc analysis, F(2,9)=226.4). C: IHC images showing NeuN (green) and TUNEL (red) staining in cerebral ischemic penumbra. Scale bar: 50 μm. D: Quantitative analysis of NeuN-positive cells in cerebral ischemic penumbra (n=4). ^*: P<0.05; ^***: P<0.001 (one-way ANOVA with Dunnett’s post hoc analysis, F(2,9)=117.7). E: Quantitative analysis of TUNEL-positive cells in cerebral ischemic penumbra (n=4). ^***: P<0.001 (one-way ANOVA with Dunnett’s post hoc analysis, F(2,9)=300.5). F: Immunoblotting images showing levels of Bax and Bcl2 at 72 h after MCAO or Sham treatment. G: Quantitative analysis of Bax and Bcl2 protein levels (n=5), normalized to β-actin, with Sham group set to 1. ^**: P<0.01, ^***: P<0.001 (one-way ANOVA with Dunnett’s post hoc analysis, Bax: F(2,12)=317.3, Bcl2: F(2,12)=57.34). H: Representative images of IHC staining of γ-H2AX (red) in ischemic penumbra from Sham or MCAO groups. Scale bar: 100 μm. I: Quantitative analysis of γ-H2AX-positive cells in cerebral ischemic penumbra (n=4). ^**: P<0.01 (Student’s t-tests, t(6)=4.01).

Figure 4

Expression pattern of DNA2 during oxygen-glucose deprivation/reoxygenation (OGD/R) in neuronal cultures A: Schematic of in vitro OGD/R study design. B: Morphological characteristics of HT22 cells at various OGD/R time points. Scale bar: 50 μm. C: Comparison of cell survival ratios under different experimental conditions, determined using CCK-8 assays. D: Representative WB images showing DNA2 levels at various OGD/R time points in HT22 cells. E: Quantitative analysis of DNA2 protein levels, normalized to β-actin, with Ctrl group set to 1. Data are from at least three independent experiments. ^*: P<0.05; ^**: P<0.01 (one-way ANOVA with Dunnett’s post hoc analysis, F(5,12)=19.86).

Figure 5

DNA2 knockdown (KD) enhances apoptosis in HT22 cells subjected to OGD/R A: Representative WB images depicting DNA2 protein levels in HT22 cells. B: Fluorescence images showing ROS levels in HT22 cells transfected with scrambled shRNA (Sc) and DNA2 shRNA (sh-DNA2). Scale bar: 100 μm. C: Quantitative analysis of DHE-positive cells. Data are from at least five independent experiments. ^***: P<0.001 (one-way ANOVA with Dunnett’s post hoc analysis, F(2,12)=217.4). D: WB images showing expression levels of Bax and Bcl2 proteins in HT22 cells post-OGD/R. E: Quantitative analysis of Bax and Bcl2 protein levels, normalized to β-actin, with Sc group set to 1. Data are from at least five independent experiments. ^**: P<0.01; ^***: P<0.001 (one-way ANOVA with Dunnett’s post hoc analysis, Bax: F(2,12)=296.5, Bcl2: F(2,12)=32.55). F, G: Flow cytometry results of annexin V-APC/7-AAD staining, showing percentage of total neuronal apoptosis (sum of Q1 and Q2 quadrants), Q1: Early apoptosis, Q2: Late apoptosis, Q3: Necrosis, Q4: Viable cells. Data are from at least three independent experiments. ^***: P<0.001 (one-way ANOVA with Dunnett’s post hoc analysis, F(2,6)=109.2).

Figure 6

RNA-seq analysis of DNA2 cKO mice compared to WT mice following MCAO A: Venn diagram showing overlap of genes between cKO and WT groups. B: Volcano plot showing differentially expressed genes (DEGs) between DNA2 cKO and WT mice, highlighting significant changes. C: Heatmap of hierarchical cluster analysis for DEGs between cKO and WT mice, with red indicating up-regulated genes and blue indicating down-regulated genes. D: GO analysis of biological processes (BP), cellular components (CC), and molecular functions (MF), identifying key affected categories. E: Chord diagrams of GO enrichment analysis, showing gene names on the left semicircle and their corresponding functions on the right semicircle. F: Analysis of KEGG signaling pathways influenced by DNA2 cKO. Red rectangles highlight key genes or signaling pathways identified by RNA-seq.

Figure 7

DNA2 cKO reduces Homer1a expression at 3 days post-MCAO A: Real-time qPCR analysis of gene expression, selected from RNA-seq data (n=3). ^*: P<0.05; ^**: P<0.01 ^***: P<0.001 (Student’s t-tests, DNA2: t(4)=10.06, Fas: t(4)=5.63, Homer1: t(4)=11.13, Homer1a: t(4)=7.72, Homer1b/c: t(4)=1.18, Gadd45g: t(4)=5.38, Hipk2: t(4)=2.84, Casp12: t(4)=3.80, Icam1: t(4)=22.42, Agt: t(4)=3.17). B: WB analysis of Homer1a protein levels in WT and cKO mice following MCAO. C: Quantitative analysis of Homer1a protein levels (n=5), normalized to β-actin, with WT group set to 1. ^***: P<0.001 (Student’s t-tests, t(8)=17.62). D: IHC images showing Homer1a expression (red) in cerebral ischemic penumbra of WT and cKO mice at 3 days post-MCAO, with nuclei counterstained using DAPI. Scale bar: 50 µm. E: Quantitative analysis of Homer1a-positive cells within ischemic penumbra (n=4). ^***: P<0.001 (Student’s t-tests, t(6)=7.41).

Figure 8

Overexpression (OE) of Homer1a mitigates apoptosis in primary neurons mediated by DNA2 KD in OGD/R A: Representative WB images showing levels of DNA2 and Homer1a at indicated time points during OGD/R in primary neurons. B: WB analysis of pro-apoptotic Bax and anti-apoptotic Bcl2 protein levels in primary neurons following OGD/R treatment. C: Quantitative analysis of Bax and Bcl2 protein levels, normalized to β-actin, with Sc group set to 1. Data are from at least four independent experiments. ^*: P<0.05; ^**: P<0.01; ^***: P<0.001 (one-way ANOVA with Dunnett’s post hoc analysis, Bax: F(3,12)=49.50, Bcl2: F(3,12)=120.5). D: Fluorescence images showing ROS levels in primary neurons from indicated groups. Scale bar: 100 μm. E: Quantitative analysis of ROS-positive cells using DHE probe (red). Data are from at least four independent experiments. ^***: P<0.001 (one-way ANOVA with Dunnett’s post hoc analysis, F(3,12)=259.9).