Poly(ADP-ribose) glycohydrolase as a target for neuroprotective intervention: assessment of currently available pharmacological tools

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Abstract

Poly(ADP-ribose) glycohydrolase (PARG) is being considered as a therapeutic target for the prevention of neurodegeneration. Here, we assessed the pharmacological tools available for target validation. The tannic acid derivative gallotannin inhibited PARG in a cell-free assay but had no detectable effect on PARG function in intact cells. Its cytoprotective actions were associated rather with the radical-scavenging potential of the compound. In astrocytes exposed to high concentrations of the nonoxidative DNA-damaging agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), Poly(ADP-ribose) polymerase (PARP) inhibitors were fully protective, while gallotannin enhanced the damage. The compound N-bis-(3-phenyl-propyl)9-oxo-fluorene-2,7-diamide (GPI 16552), considered a potentially specific PARG inhibitor, had no effect in the different astrocyte death models compared with PARP inhibitors. In an in vitro PARG activity assay, the maximal inhibition that could be achieved with GPI 16552 was only 40% at a drug concentration of 80 μM. We conclude that neither GPI 16552 nor gallotannin are suitable for the evaluation of PARG in cellular death models, and that previous conclusions drawn from the use of these compounds should be interpreted with caution.

Introduction

Poly(ADP-ribosyl)ation and the enzymes involved have attracted large attention as targets for pharmacological intervention under conditions of tissue injury. Poly(ADP-ribose) polymerase-1 (PARP-1, EC 2.4.2.30) is the major nuclear enzyme with poly(ADP-ribosyl)ating activity. The substrate of this enzymatic reaction is β-NAD+, which yields the ADP-ribosyl moiety used for the transfer reaction, and nicotinamide, which is released. The catalytic activity of PARP-1 depends on the presence of DNA single- or double-strand breaks, which are sensed via two zinc fingers in a specific domain of the enzyme. In the activated state, PARP-1 adds chains of poly(ADP-ribose; PAR) consisting of up to 200 ADP-ribosyl units to various nuclear proteins, including histones and PARP-1 itself (Satoh et al., 1994, Pieper et al., 1999). The covalently attached highly negatively charged poly(ADP-ribose) affects protein function and leads to chromatin decondensation, making the DNA strands accessible to DNA repair enzymes (de Murcia et al., 1988). In replicating cells and tissues under mild to moderate genotoxic stress, PARP-1 activity is a survival factor and a guardian of the genome (Ishizuka et al., 2003; for review: Bürkle, 2001). In stark contrast, PARP-1−/− mice show resistance to cerebral or myocardial ischemia, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson syndrome, streptozotocin-induced diabetes, as well as to many forms of inflammation in animal models (Ying et al., 2001, Ha et al., 2002). This suggests a role for PARP-1 in neurodegeneration and tissue destruction. Apparently, excessive activation of PARP-1 under such pathophysiological conditions results in cellular depletion of the NAD+ pool with important implications for energy metabolism (Pieper et al., 1999). Moreover, cellular ATP depletion can occur as a secondary consequence, because NAD+ resynthesis is an energy and adenine nucleotide-demanding process.

Poly(ADP-ribose) glycohydrolase (PARG) negatively regulates the cellular amount of PAR by degrading the PAR chains synthesised by PARP-1 and a number of additional PARP isoforms (Chiarugi, 2002). PARG is an endoexoglycosidase, with a low cellular expression but with a high specific activity, as shown by the fast turnover of poly(ADP-ribose), which has a half life of less than 1 min under conditions of DNA breakage (Alvarez-Gonzalez and Althaus, 1989). Little information is available on the normal cellular function of PARG, (for review, see Davidovic et al., 2001), but it has been suggested that PARG is involved in PARP-1-dependent cell death (Ying et al., 2001). During excessive PARP activation, the simultaneous activity of PARG would result in a vicious NAD+-consuming cycle of synthesis and degradation of PAR. This would result in a complete degradation of NAD+ and of ATP, ultimately converting it to nicotinamide and free monomeric ADP-ribose.

PARP inhibitors like 1,5 dihydroxyisoquinoline and 3-aminobenzamide have been shown to be protective in many models of cell death associated with PARP-1 overactivation (Purnell and Whish, 1980, Banasik et al., 1992, Szabo and Dawson, 1998), and recently, four groups have claimed that PARG inhibitors also are protective in the very same models (Ying and Swanson, 2000, Ying et al., 2001, Hwang et al., 2002, Kim and Koh, 2002, Lu et al., 2003). It was suggested that the mechanisms of action was PARG inhibition preventing the NAD+-consuming vicious cycle, and causing inhibition of PARP-1 due to excessive auto-ADP-ribosylation, which blocks the enzymatic activity of PARP-1 (Zahradka and Ebisuzaki, 1982). These data are mainly based on the use of gallotannin as a putatively selective PARG inhibitor.

In cell-free assays, gallotannin, a complex mixture of tannins purified from oak gall, has been shown to inhibit PARG (Tsai et al., 1992, Aoki et al., 1993), and the compound also showed protection in cell culture models of oxidative stress-induced cell death (Ying and Swanson, 2000, Ying et al., 2001). Another small molecule with putative PARG-inhibitory capacity, N-bis-(3-phenyl-propyl)9-oxo-fluorene-2,7-diamide (GPI-16552), was reported to significantly reduce the infarct volume in a middle cerebral artery occlusion model of cerebral ischemia (Lu et al., 2003). Furthermore, it was postulated that PARG might affect inflammation by inhibition of inducible nitric oxide synthase (iNOS), (Chiesi and Schwaller, 1995). Here, we used astrocyte models to examine putative PARG inhibitors. We used three PARP-1-dependent cell death models plus one inflammation model in primary astrocytes to reinvestigate the effect of GPI-16552 and gallotannin.

Section snippets

Materials

Complete cytokine mix (CCM) contained 10 ng/ml murine tumor necrosis factor-alpha (TNF-α), 10 ng/ml murine interleukin-1β (Sigma-Aldrich, Copenhagen, Denmark), and 5 U/ml recombinant murine interferon gamma (IFN-γ; R&D Systems, Abingdon, UK). Other reagents were S-nitroso-N-acetylpenicillamine (SNAP) and 3-morpholinosydnonimine (SIN-1) purchased from Bie and Berntsen (Rødovre, Denmark). Basic laboratory chemicals and inhibitors were purchased from Sigma, unless stated otherwise. GPI-16552 [N

Models of oxidative and nonoxidative poly(ADP ribose)-mediated cytotoxicity

Three different PARP-1-dependent cell death models were set up in primary cultures of murine fetal astrocytes. DNA strand breaks were triggered either by hydrogen peroxide (through the formation of hydroxyl radicals in the Fenton reaction), by SIN-1 (producing nitric oxide and superoxide, together forming peroxynitrite), or by MNNG (an alkylating agent). All insults were carefully titrated (data not shown), and a concentration inducing near-maximal (80–100%) cell death over a 24-h period was

Discussion

We reviewed the evidence for PARG as a target for tissue protection. Because the outcome of any such evaluation depends strongly on the quality of the compounds used as tools, we undertook a thorough experimental reevaluation. Most evidence on the role of PARG in neuroprotection has been based on the use of gallotannin as a putatively selective inhibitor in cells. We present here evidence confirming that gallotannin is an inhibitor of PARG but also showing that it is not specific, and that it

Acknowledgements

The excellent technical assistance and input of Dr. Christiane Volbracht, Andreas Rassov, and Søren Lund is gratefully acknowledged. We thank Drs. M. Miwa (Tsukuba, Japan) and T. Sugimura (Tokyo, Japan) for 10H hybridoma cells, Dr. Lene Hjorth Alifrangis for the Caco-2 cells, and Dr. Morten Bang Nørdgaard for synthesising GPI-16552.

References (34)

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