Poly(ADP-ribose) glycohydrolase mediates oxidative and excitotoxic neuronal death

Abstract

Excessive activation of poly(ADP-ribose) polymerase 1 (PARP1) leads to NAD(+) depletion and cell death during ischemia and other conditions that generate extensive DNA damage. When activated by DNA strand breaks, PARP1 uses NAD(+) as substrate to form ADP-ribose polymers on specific acceptor proteins. These polymers are in turn rapidly degraded by poly(ADP-ribose) glycohydrolase (PARG), a ubiquitously expressed exo- and endoglycohydrolase. In this study, we examined the role of PARG in the PARP1-mediated cell death pathway. Mouse neuron and astrocyte cultures were exposed to hydrogen peroxide, N-methyl-d-aspartate (NMDA), or the DNA alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Cell death in each condition was markedly reduced by the PARP1 inhibitor benzamide and equally reduced by the PARG inhibitors gallotannin and nobotanin B. The PARP1 inhibitor benzamide and the PARG inhibitor gallotannin both prevented the NAD(+) depletion that otherwise results from PARP1 activation by MNNG or H(2)O(2). However, these agents had opposite effects on protein poly(ADP-ribosyl)ation. Immunostaining for poly(ADP-ribose) on Western blots and neuron cultures showed benzamide to decrease and gallotannin to increase poly(ADP-ribose) accumulation during MNNG exposure. These results suggest that PARG inhibitors do not inhibit PARP1 directly, but instead prevent PARP1-mediated cell death by slowing the turnover of poly(ADP-ribose) and thus slowing NAD(+) consumption. PARG appears to be a necessary component of the PARP-mediated cell death pathway, and PARG inhibitors may have promise as neuroprotective agents.

Figure 1

Effects of PARP and PARG inhibitors on H₂O₂-induced neuronal death. (A) The PARP inhibitor benzamide reduced neuronal death induced by H₂O₂ (200 μM) by more than 80%. (B) The PARG inhibitors nobotanin B and gallotannin prevented neuronal death induced by 1-hr H₂O₂ (100 μM) exposure. Data are mean ± SE; **, P < 0.01; n = 3–7 culture wells per experimental condition. Results are representative of two independent experiments. BNZ, benzamide; NTB, nobotanin B; GT, gallotannin.

Figure 2

Effects of PARP and PARG inhibitors on NMDA-induced neuronal death. (A) The PARP inhibitor benzamide and the PARG inhibitor NTB both reduced NMDA-induced neuronal death. Higher concentrations of these agents had no further effect (not shown). (B) The PARG inhibitor GT similarly reduced NMDA-induced neuronal death. Data are means ± SE; **, P < 0.01; n = 4–7 culture wells per experimental condition. Results are representative of three independent experiments. BNZ, benzamide; NTB, nobotanin B; GT, gallotannin; NMDA, N-methyl-D-aspartate.

Figure 3

Photomicrographs of cortical astrocyte/neuron cocultures treated with NMDA, ± gallotannin, 24 h after drug treatments. Photographs are taken with combined phase contrast and epifluorescence to show propidium iodide staining of dead cells. Neurons have phase bright cell bodies resting above the astrocyte layer. Dead cells are identified by bright propidium iodide fluorescence. Cultures treated with sham washes show only few, scattered dead neurons (Left). Cultures treated with 30 μM NMDA show death of almost all neurons (Center). Cultures treated with NMDA plus 50 μGT show markedly fewer propidium iodide stained (dead) neurons. GT, gallotannin; NMDA, N-methyl-D-aspartate.

Figure 4

PARP-mediated death of astrocytes is also attenuated by PARG inhibitors. (A) The PARP inhibitor benzamide and the PARG inhibitor NTB both reduced astrocyte death after exposure to 300 μM H₂O₂. Higher concentrations of these agents had no further neuroprotective effect (not shown). (B) The PARG inhibitor GT also reduced astrocyte death induced by PARP-activating agents; the oxidant H₂O₂, the DNA alkylating agent MNNG, and the peroxynitrite generator SIN-1. Data are means ± SE; **, P < 0.01; n = 3–9 culture wells per experimental condition. BNZ, benzamide; NTB, nobotanin B; GT, gallotannin; MNNG, N-methyl-N′-nitro-N-nitrosoguanidine; SIN-1, 3morpholinosydnonimine.

Figure 5

Cytoprotective effects of gallotannin (GT) are not attributable to actions as a free radical scavenger. (A) The reduction in H₂O₂-induced astrocyte death afforded by 10 μM GT is significantly greater than that produced by 100-fold higher concentrations of the free radical scavengers N-acetyl cysteine (NAC) or N-tert butyl phenylnitrone (PBN). (B) GT has no effect on H₂O₂-induced inactivation of GAPDH activity in the astrocyte cultures under the same conditions that GT had large effects on astrocyte survival. The 5 mM NAC condition provides a positive control for this experiment. Data are means ± SE; **, P < 0.01; n = 5.

Figure 6

Both the PARP inhibitor BNZ and the PARG inhibitor GT attenuate NAD⁺ depletion in astrocytes exposed to H₂O₂ (A) or MNNG (B). Data are means ± SE; **, P < 0.01; n = 3 cultures wells per experimental condition. Results are representative of three independent experiments.

Figure 7

Effects of BNZ and GT on MNNG-induced poly(ADP-ribose) formation. Western blots were prepared from astrocyte cultures treated with sham washes or 100 μM MNNG for 5 or 10 min. Cultures in each condition were also treated with 5 mM benzamide (BNZ), 50 μM gallotannin (GT), or neither (C). Poly(ADP-ribose) immunostaining shows two prominent bands present at 110 kDa and 89 kDa that probably represent poly(ADP-ribose) on PARP itself. Diffuse staining on proteins of other molecular weights is seen with increasing MNNG incubation periods. This increase in staining is slowed or attenuated in the presence of BNZ, but it is increased in the GT-treated cultures. Numbers in parentheses above each lane indicate the relative optical density of the entire lane between 112 kDa and 210 kDa.

Figure 8

Photomicrographs showing the effects of gallotannin and benzamide on MNNG-induced poly(ADP-ribose) formation in neurons. Astrocyte-neuron cocultures were treated with sham washes (A), 300 μM MNNG for 15 min (B), MNNG plus 3 mM benzamide (C), and MNNG plus 25 μM gallotannin (D).