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. 2018 Sep:18:158-172.
doi: 10.1016/j.redox.2018.06.012. Epub 2018 Jul 7.

Breast cancer susceptibility protein 1 (BRCA1) rescues neurons from cerebral ischemia/reperfusion injury through NRF2-mediated antioxidant pathway

Pengfei Xu  1 , Qian Liu  1 , Yi Xie  1 , Xiaolei Shi  2 , Yunzi Li  1 , Mengna Peng  1 , Hongquan Guo  3 , Rui Sun  4 , Juanji Li  1 , Ye Hong  1 , Xinfeng Liu  5 , Gelin Xu  6
Affiliations

Breast cancer susceptibility protein 1 (BRCA1) rescues neurons from cerebral ischemia/reperfusion injury through NRF2-mediated antioxidant pathway

Pengfei Xu et al. Redox Biol. 2018 Sep.

Erratum in

Abstract

Cellular oxidative stress plays a vital role in the pathological process of neural damage in cerebral ischemia/reperfusion (I/R). The breast cancer susceptibility protein 1 (BRCA1), a tumor suppressor, can modulate cellular antioxidant response and DNA repair. Yet the role of BRCA1 in cerebral I/R injury has not been explored. In this study, we observed that BRCA1 was mainly expressed in neurons and was up-regulated in response to I/R insult. Overexpression of BRCA1 attenuated reactive oxygen species production and lipid peroxidation. Enhanced BRCA1 expression promoted DNA double strand break repair through non-homologous end joining pathway. These effects consequently led to neuronal cell survival and neurological recovery. Mechanically, BRCA1 can interact with the nuclear factor (erythroid-derived 2)-like 2 (NRF2) through BRCA1 C-terminal (BRCT) domain. The cross-talk between BRCT and NRF2 activated the NRF2/Antioxidant Response Element signaling pathway and thus protected injured neurons during cerebral I/R. In conclusion, enhanced BRCA1 after cerebral I/R injury may attenuate or prevent neural damage from I/R via NRF2-mediated antioxidant pathway. The finding may provide a potential therapeutic target against ischemic stroke.

Keywords: BRCA1; DNA damage; Ischemia/Reperfusion; NRF2; Oxidative stress.

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Figures

Fig. 1
Fig. 1
BRCA1 was up-regulated in neurons following ischemic stroke. (A, B) Double immunostaining of BRCA1 with Iba-1 (microglia marker), GFAP (astrocyte marker) and NeuN (neuron marker) were performed in mice brain sections 1 day after reperfusion. Insets show a higher magnification view. Scale bar = 20 µm. (C) Immunoblots and quantitative analysis of BRCA1 in cultured neurons, astrocytes and microglia. (D) Representative immunofluorescence images of BRCA1 co-stained with MAP2 in control and OGD neurons. Insets show a higher magnification view. Scale bar = 20 µm. (E, F) The protein expression pattern and time course of BRCA1 in ischemic penumbra and ischemic core. (D) Immunoblots of BRCA1 in OGD-treated neurons and quantification. All results are presented as mean ± SD; n = 6 for all groups; * P < 0.05, ** P < 0.01, * ** P < 0.001 versus sham mice; #P < 0.05, ##P < 0.01 versus control mice.
Fig. 2
Fig. 2
BRCA1 up-regulation attenuated brain I/R injury. LV-GFP or LV-BRCA1 was injected into the right lateral ventricle and right hippocampus of mice 14 days before MCAO surgery. (A) Brain sections were immunostained for GFP (green) and DAPI (blue). Scale bars = 50 µm. (B) Immunoanalysis and quantification of BRCA1, n = 5. (C) Quantitative real-time PCR analysis of IL-6 and TNF-α, n = 5. (D) Representative image of TTC staining in the indicated groups 1 day after reperfusion. Scale bar = 1 cm. (E, F) Quantitative analysis of infarct volume and edema volume, n = 9. (G) Neurological scores assessment, n = 20. Data are shown as median (range) for neurological scores and mean ± SD for others; † † †P < 0.001 versus control mice; * ** P < 0.001 versus sham mice; ##P < 0.01, ###P < 0.001 versus MCAO mice.
Fig. 3
Fig. 3
Administration of LV-BRCA1 rescued I/R-induced cognitive deficits. (A) Representative tracks of movements of mice for 30 min in an open field box. (B) Cumulative distance traveled per zone. (C-E) Total rearing activities, the time spent in and the frequency entry into the center area during the 30 min session in the OFT. (F) Representative swim path traces of mice in hidden platform test (top traces, “learning”) and probe phase (bottom traces, “memory”). (G) The swim path and (H) escape latency were recorded at day 23–27 after MCAO. (I) Platform crossovers and (J) the percentage of time spent in the target quadrant were recorded at day 28 after MCAO in probe trails. Data are shown as mean ± SD; n = 10 for all groups; ** P < 0.01, * ** P < 0.001 versus sham mice; #P < 0.05, ##P < 0.01, ###P < 0.001 versus MCAO mice.
Fig. 4
Fig. 4
BRCA1 overexpression ameliorated neuronal apoptosis after I/R. (A) Apoptotic neurons were detected by NeuN/TUNEL and TUNEL-AP staining 1 day after reperfusion. Scale bars = 50 µm. (B) Positive apoptotic neurons were calculated using Image J in nine random fields. (C) Western blot and quantitative analysis of p53-dependent proapoptotic proteins in brain extracts of the indicated groups, including p53, Bax, Bcl-2 and Cleaved Caspase-3. n = 6. Data are expressed as mean ± SD; ** P < 0.01, * ** P < 0.001 versus sham group; #P < 0.05, ##P < 0.01 versus MCAO group.
Fig. 5
Fig. 5
BRCA1 overexpression reduced DSBs after I/R. (A) Representative photomicrographs of γH2A.X staining. Scale bar = 50 µm. (B) Statistical analysis of γH2A.X positive cells, n = 9 fields from six mice. (C) Western blot analysis and quantification of nuclear γH2A.X, RAD51, Ku80 and Ku70 in cerebral ischemic penumbra cortex, n = 6. (D) Western blot analysis and quantification of nuclear phosphorylation of DNA-PKcs and DNA-PKcs, n = 6. Data are shown as mean ± SD; * P < 0.05, ** P < 0.001, * ** P < 0.001 versus sham group; #P < 0.01, ###P < 0.001 versus MCAO group.
Fig. 6
Fig. 6
BRCA1 overexpression attenuated oxidative damage post-stroke. (A) Representative images of 8-OHDG and 3-NT staining and (B, C) statistical analysis, n = 9 fields from six mice. Scale bars = 50 µm. (D) ROS production and (E) SOD enzyme activity assay of the indicated groups, n = 6. Lipid peroxidation was evaluated by MDA assay and western blotting. (F) Histograms showing MDA levels in the four groups, n = 6. (G) Western blot analysis and quantification of 4-HNE protein adducts, n = 6. Arrows indicate bands with changed 4-HNE signal at different protein sizes. Data are shown as mean ± SD; * ** P < 0.001 versus sham group; #P < 0.05, ##P < 0.01 versus MCAO group.
Fig. 7
Fig. 7
BRCA1 provoked NRF2-mediated antioxidative pathway. (A) BRCA1 binds to NRF2. The brain lysates were immunoprecipitated with anti-BRCA1 (left) or anti-NRF2 (right) antibodies. Then total lysates and immunoprecipitates were analyzed by immunoblotting with anti-BRCA1 and anti-NRF2. n = 5. (B) Schematic representation of BRCA1 fusion proteins. Interaction was detected between BRCT domians (aa 1591–1784) and NRF2. (C) Western blots showing the protein levels of total, cytosolic and nuclear NRF2, and quantification analysis of total and nuclear NRF2. n = 6. (D) mRNA levels of NRF2 in the indicated groups, n = 6. (E) Dual-luciferase reporter assay of BRCA1 and NRF2 XRE, n = 5. (F) mRNA levels of HO-1, NQO1, GPX4, GCLC, GCLM, SOD1 and SOD2. n = 6. (G) Immunoblots and quantitative analysis of total HO-1, NQO1 and GPX4. n = 6. (H) Schematic diagram of NRF2-mediated antioxidative pathway triggered by BRCA1. Data are expressed as mean ± SD; † † †P < 0.001 versus pc-DNA+ XRE WT group; * P < 0.05, ** P < 0.01, * ** P < 0.001 versus sham group; #P < 0.05, ##P < 0.01, ###P < 0.001 versus MCAO group.
Fig. 8
Fig. 8
LV-BRCA1 treatment facilitated synaptic plasticity after MCAO. (A, B) Transfection efficiency of LV-BRCA1 in hippocampus, n = 5. (C) Western blot and quantitative analysis of PSD95, CaMKII, Synapsin I and synaptophysin, n = 6. (D) Representative micrographs of DCX staining in CA1. (E) Quantification of DCX immunofluorescence density, n = 6. Scale bar = 50 µm. Data are expressed as mean ± SD; * ** P < 0.001 versus sham group; #P < 0.05, ###P < 0.001 versus MCAO group.
Fig. 9
Fig. 9
Schematic depicting neuroprotective roles of BRCA1 towards I/R injury. Excessive ROS production damages macromolecule DNA and lipids. BRCA1 binds directly to NRF2 via its BRCT domain and facilitates NRF2-mediated antioxidant response. Then the downstream ARE genes, including HO-1, NQO1, GCLC, GPX4 and SOD2 are induced, which reduces ROS production and attenuates lipid peroxidation. Meanwhile, BRCA1 promotes DSBs repair through up-regulating Ku70/Ku80 in NHEJ pathway. As a result, neuronal cell death is inhibited.
Fig. S1
Fig. S1
Schematic illustration of the experimental protocol. LV-GFP: lentivirus-GFP; LV-BRCA1: lentivirus-BRCA1; OFT: open field test; MWM: Morris water maze; mNSS: modified Neurological Severity Score.
Fig. S2
Fig. S2
BRCA1co-localized with NeuN positive neurons, but not with Iba1 positive microglia or GFAP positive astrocytes in sham-operated mouse brain. Scale bar = 20 µm.
Fig. S3
Fig. S3
(A, B) mRNA levels of BRCA1 in MCAO mice and OGD/R neurons at indicated times. Data are expressed as mean ± SD; n = 6 for all groups; * P < 0.05, ** P < 0.01, * ** P < 0.001 versus sham group; #P < 0.05, ###P < 0.001 versus control group.
Fig. S4
Fig. S4
BRCA1 overexpression reduced OGD/R-induced neuronal death. Primary neurons were subjected to OGD/R after lentivirus transfection. (A) Representative image of culture neurons infected with LV-BRCA1-GFP. Scale bars = 50 µm. (B) Immunoblots and quantitative analysis of BRCA1 in transfected neurons, n = 5. (C, D) LV-BRCA1 administration reduced OGD/R-induced neuronal death as assessed by PI/Hoechst 33342 and TUNEL-AP staining, n = 6. Scale bar = 50 µm. (E) Cell viability was assayed by CCK-8 kit, n = 6. Data are expressed as mean ± SD; * ** P < 0.001 versus control group; ##P < 0.01 versus OGD/R 24 h group.
Fig. S5
Fig. S5
BRCA1 up-regulation reduced ROS production and DNA damage in vitro. (A) Comet assay of damaged DNA was performed 24 h after reoxygenation. (B) Quantification of the average DNA tail moment, n = 10 fields. (C) Western blot and quantification analysis of nuclear γH2A.X, RAD51, Ku80 and Ku70. n = 6. (D) Representative photographs of DHE fluorescence at 3 h after reoxygenation. Scale bar = 50 µm. (E) Statistical histogram of DHE positive neurons, n = 6. (F) Immunoblots and quantitative analysis of NRF2, HO-1 and NQO1 24 h after oxygenation. n = 6. Data are expressed as mean ± SD; * P < 0.05, ** P < 0.01, * ** P < 0.001 versus control group; §P < 0.05 versus OGD/R 3 h group; #P < 0.05, ##P < 0.01, ###P < 0.001 versus OGD/R 24 h group.
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References

    1. Hankey G.J. Stroke. Lancet. 2017;389(10069):641–654. - PubMed
    1. Manzanero S., Santro T., Arumugam T.V. Neuronal oxidative stress in acute ischemic stroke: sources and contribution to cell injury. Neurochem. Int. 2013;62(5):712–718. - PubMed
    1. Allen C.L., Bayraktutan U. Oxidative stress and its role in the pathogenesis of ischaemic stroke. Int J. Stroke. 2009;4(6):461–470. - PubMed
    1. Radak D., Resanovic I., Isenovic E.R. Link between oxidative stress and acute brain ischemia. Angiology. 2014;65(8):667–676. - PubMed
    1. Dominguez C., Delgado P., Vilches A., Martin-Gallan P., Ribo M., Santamarina E., Molina C., Corbeto N., Rodriguez-Sureda V., Rosell A. Oxidative stress after thrombolysis-induced reperfusion in human stroke. Stroke. 2010;41(4):653–660. - PubMed

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