PARP1 inhibition alleviates injury in ARH3-deficient mice and human cells
Masato Mashimo, Xiangning Bu, Kazumasa Aoyama, Jiro Kato, Hiroko Ishiwata-Endo, Linda A. Stevens, Atsushi Kasamatsu, Lynne A. Wolfe, Camilo Toro, David Adams, Thomas Markello, William A. Gahl, Joel Moss
Masato Mashimo, Xiangning Bu, Kazumasa Aoyama, Jiro Kato, Hiroko Ishiwata-Endo, Linda A. Stevens, Atsushi Kasamatsu, Lynne A. Wolfe, Camilo Toro, David Adams, Thomas Markello, William A. Gahl, Joel Moss
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Research Article Genetics Therapeutics

PARP1 inhibition alleviates injury in ARH3-deficient mice and human cells

Abstract

Poly(ADP-ribosyl)ation refers to the covalent attachment of ADP-ribose to protein, generating branched, long chains of ADP-ribose moieties, known as poly(ADP-ribose) (PAR). Poly(ADP-ribose) polymerase 1 (PARP1) is the main polymerase and acceptor of PAR in response to DNA damage. Excessive intracellular PAR accumulation due to PARP1 activation leads cell death in a pathway known as parthanatos. PAR degradation is mainly controlled by poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose-acceptor hydrolase 3 (ARH3). Our previous results demonstrated that ARH3 confers protection against hydrogen peroxide (H2O2) exposure, by lowering cytosolic and nuclear PAR levels and preventing apoptosis-inducing factor (AIF) nuclear translocation. We identified a family with an ARH3 gene mutation that resulted in a truncated, inactive protein. The 8-year-old proband exhibited a progressive neurodegeneration phenotype. In addition, parthanatos was observed in neurons of the patient’s deceased sibling, and an older sibling exhibited a mild behavioral phenotype. Consistent with the previous findings, the patient’s fibroblasts and ARH3-deficient mice were more sensitive, respectively, to H2O2 stress and cerebral ischemia/reperfusion-induced PAR accumulation and cell death. Further, PARP1 inhibition alleviated cell death and injury resulting from oxidative stress and ischemia/reperfusion. PARP1 inhibitors may attenuate the progression of neurodegeneration in affected patients with ARH3 deficiency.

Authors

Masato Mashimo, Xiangning Bu, Kazumasa Aoyama, Jiro Kato, Hiroko Ishiwata-Endo, Linda A. Stevens, Atsushi Kasamatsu, Lynne A. Wolfe, Camilo Toro, David Adams, Thomas Markello, William A. Gahl, Joel Moss

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Figure 4

Enhanced susceptibility of ARH3–/– mice to cerebral ischemia/reperfusion is associated with increased PAR accumulation and AIF translocation to nuclei.

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Enhanced susceptibility of ARH3–/– mice to cerebral ischemia/reperfusion...
(A) ARH3 expression in whole brains of WT and ARH3–/– mice. Data are representative of experiments performed 3 times with similar results. (B) Infarct area after MCAO. Coronal sections were stained by the Nissl method to evaluate infarct area of WT and ARH3–/– mice subjected to 30-minute ischemia, followed by a 24-hour reperfusion. Infarct area was assessed by four coronal sections. Data are mean ± SEM of values obtained from WT (n = 7) and ARH3–/– (n = 6) mice. ***P <0.001 by 2-tailed Student’s t test. (C) Nissl-labeled neural cells in cerebral cortex in ischemic and control hemispheres of WT and ARH3–/– mice. Scale bar: 50 μm. (D) PAR localization in cerebral cortex. Brain sections were reacted with anti-PAR (green) and anti-NeuN (red) antibodies, and DAPI (blue). Scale bar: 20 μm. Data are representative of experiments performed 3 times with similar results. (E) Percentage of PAR-labeled nuclei of cortical neurons. Data are mean ± SEM of values obtained from 3182 and 1687 cortical neurons in ischemic (Is) and control (Ct) hemispheres of WT mice (n = 5), respectively; and 2626 and 1831 cortical neurons in ischemic and control hemispheres of ARH3–/– mice (n = 5), respectively. ***P < 0.001. (F) Nuclear and cytoplasmic PAR levels in cortex. Data are mean ± SEM of values obtained from WT (n = 5) and ARH3–/– (n = 5) mice. *P < 0.05, **P < 0.01. (G) DNA fragmentation and AIF translocation to nuclei in cerebral cortex. Brain sections were subjected to TUNEL assay (light blue) and reacted with anti-AIF (green) and anti-NeuN (red) antibodies and DAPI (dark blue). Arrowheads indicate nuclei (blue) with DNA fragmentation that are colocalized with AIF. Scale bar: 20 μm. (H) Nuclear localization of AIF. Data are mean ± SEM of values obtained from 666 and 906 cortical neurons in ischemic and control hemispheres of WT mice (n = 3), respectively; and 1804 and 551 cortical neurons in ischemic and control hemispheres of ARH3–/– mice (n = 3), respectively. ***P < 0.001. (I) Percentage of neurons with DNA fragmentation and AIF translocation to nuclei. Data are mean ± SEM of values obtained from 2443 and 1540 cortical neurons in ischemic and control hemispheres of WT mice (n = 5), respectively; and 2575 and 1772 cortical neurons in ischemic and control hemispheres of ARH3–/– mice (n = 5), respectively. **P < 0.01. Statistical tests done in E, F, H, and I were two-way ANOVA with Bonferroni’s post-hoc test.