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 2

PJ34 inhibits H2O2-induced PAR accumulation and AIF-mediated DNA fragmentation in patient fibroblasts.

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PJ34 inhibits H2O2-induced PAR accumulation and AIF-mediated DNA fragmen...
(A) H2O2-induced cytotoxicity in patient fibroblasts (PJ34, 10 μM). Data are mean ± SEM of values obtained from 3 experiments conducted in triplicate. Significant differences were observed with 200 μM. (B) Concentration-dependent effect of PJ34 on H2O2-induced cytotoxicity (H2O2, 300 μM). Data represent mean ± SEM of values obtained from 3 experiments conducted in triplicate and were fitted to a sigmoidal (variable slope) curve. IC50= 158.2 ± 3.21 nM. (C) Effect of PARP activation on NAD+ content. NAD+ concentration (pmol/μg) was measured by HPLC and normalized to protein concentration. Data are mean ± SEM of values obtained from 3 experiments. Significant differences were observed at all time points. (D) Effect of PJ34 on time-dependent PAR localization following exposure to H2O2 (300 μM) in patient fibroblasts. DAPI was used as a nuclear marker. (E) Mean PAR fluorescence intensities in nuclei and cytoplasm following exposure to H2O2. Data are mean ± SEM of values obtained from 30–60 cells from three samples. Significant differences in the nucleus and cytoplasm were observed at 20 min and 40 min, respectively. (F) Effect of PJ34 on H2O2-induced PAR accumulation. These representative data were replicated three times with similar results. (G) Effect of PJ34 on AIF translocation to nuclei and DNA fragmentation. After a 16-hour exposure to 300 μM H2O2 without or with PJ34, patient fibroblasts were subjected to TUNEL assay (light blue), DAPI (blue), and immunolabeling with anti-AIF (green) and anti-Tom20 (marker for mitochondria, red) antibodies. Arrowheads indicate nuclei (blue) with DNA fragmentation that are colocalized with AIF. Scale bar: 20 μm (D and G). (H) Nuclear localization of AIF. Data are mean ± SEM of values obtained from 76–164 cells from 3 samples. ***P < 0.001. (I) Percentage of patient fibroblasts with chromatin condensation and AIF translocation to nuclei. Data are mean ± SEM of values obtained from 97–174 cells in 3 samples. ***P < 0.001. (J) Mean nuclear size assessed by DAPI. Data are mean ± SEM of values obtained from 59–221 cells from 3 samples. **P < 0.01, ***P < 0.001, two-way ANOVA with Bonferroni’s post-hoc test (A, C, E, H–J).