PARP1 inhibition alleviates injury in ARH3-deficient mice and human cells
Masato Mashimo, … , William A. Gahl, Joel Moss
Masato Mashimo, … , William A. Gahl, Joel Moss
Published February 21, 2019
Citation Information: JCI Insight. 2019;4(4):e124519. https://doi.org/10.1172/jci.insight.124519.
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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 1

Truncated ARH3 expressed in patient fibroblasts lacks PAR-degrading activity.

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Truncated ARH3 expressed in patient fibroblasts lacks PAR-degrading acti...
(A) RT-PCR was performed to detect ARH3 mRNA transcript expression in patient fibroblasts (PTs) using ARH3-specific primers, as described in Supplemental Table 1. Plasmid vectors encoding ARH3 WT and truncated ARH3 were used as expression controls. (B) Expression of truncated ARH3 (tARH3), but not full-length ARH3, in patient fibroblasts. Cells were subjected to Western blotting using anti-ARH3 antibodies recognizing the C- (C-ter) and N-terminal regions of ARH3. Recombinant human ARH3 protein (rARH3) was used as a positive control. (C) Expression of truncated ARH3 in the cytoplasm. Purity of nuclear (N), cytoplasmic (C), and mitochondrial (M) fractions was confirmed using protein markers: histone H3 (nucleus), tubulin (cytoplasm), and manganese superoxide dismutase (MnSOD, mitochondria). (D) PAR-degrading activity of ARH3. [14C]-labeled PAR (52,012 cpm, 245 pmol) was incubated with GST-tagged proteins (200 nM) for 60 minutes. As described in Methods, [14C]-ADP-ribose was separated by HPLC using an LC-18T column; radioactivity was measured with a liquid scintillation counter. Data are mean ± SEM of values obtained from 3 samples. ***P < 0.001 by one-way ANOVA with Tukey’s post-hoc test.