JPET Fast Forward. Published on July 6, 2006 as DOI:10.1124/jpet.106.108076
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PARG activity mediates post-traumatic inflammatory reaction after experimental spinal cord trauma*
Salvatore Cuzzocrea, Tiziana Genovese, Emanuela Mazzon, Concetta Crisafulli, Wookee Min, Rosanna Di Paola, Carmelo Muià, Jia-He Li, Emanuela Esposito, Placido Bramanti, Weizhen Xu, Edmond Massuda, Jie Zhang and Zhao-Qi Wang
Department of Clinical and Experimental Medicine and Pharmacology, Torre Biologica, Policlinico Universitario, 98123 Messina, Italy (S.C., T.G., E.M., C.C., R.D.P., C.M.); IRCCS Centro Neurolesi "Bonino-Pulejo" Messina, Italy (S.C., T.G., E.M., P.B.);International Agency for Research on Cancer, 69008 Lyon, France (W.M., J.H.L., Z.Q.W.); Lilium Pharmaceuticals, Cockeysville, MD 21030 USA (E.M); Guilford Pharmaceuticals Inc., Baltimore, MD 21224 USA (W.X., J.Z.): Department of Experimental Pharmacology, University of Naples “Federico II”, Via D. Montesano 49, 80131 Napoli, Italy (E.E)
Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics.
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Running title: PARG inhibitor reduces SCI Number of text pages: 28 Number of tables: 0 Number of figures: 10 Number of references: 40 Number of words in the abstract: 190 Number of words in the introduction: 1069 Number of words in the discussion: 952
Abbreviations: poly (ADP-ribose) glycohydrolase (PARG); spinal cord injury (SCI); reactive oxygen species (ROS); poly(ADP-ribose) polymerase-1 (PARP-1)
Author for correspondence and author who should receive reprint requests: Salvatore Cuzzocrea, PhD, Institute of Pharmacology, School of Medicine, University of Messina, Torre Biologica – Policlinico Universitario Via C. Valeria– Gazzi – 98100 Messina, Italy. Tel.: (39) 090 2213644 Fax.: (39) 090 694951; email:
[email protected]
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ABSTRACT The aim of the present study was to examine the role of poly (ADP-ribose) glycohydrolase (PARG) on the modulation of the inflammatory response and tissue injury associated with neurotrauma. Spinal cord trauma was induced in WT mice by the application of vascular clips (force of 24 g) to the dura via a two-level T6-T7 laminectomy. Spinal cord injury in WT mice resulted in severe trauma characterized by edema, neutrophil infiltration, and cytokine production followed by recruitment of other inflammatory cells, production of a range of inflammation mediators, tissue damage, apoptosis and disease. The genetic disruption of the PARG gene in mice or the pharmacological inhibition of PARG with GPI 16552 (40 mg/kg intraperitoneally bolus), a novel and potent PARG inhibitor, significantly reduced the degree of spinal cord inflammation and tissue injury (histological score), neutrophils infiltration, cytokines production (TNF-α and IL-1β) and apoptosis. In a separate experiment we have clearly demonstrated that PARG inhibition significantly ameliorated the recovery of limb function. Taken together, our results indicate that PARG activity modulates the inflammatory response and tissue injury events associated with spinal cord trauma and participate in target organ damage under these conditions.
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INTRODUCTION Posttraumatic inflammatory reaction may play an important role in the secondary injury processes after spinal cord injury (SCI) (Bartholdi and Schwab, 1995). The cardinal features of inflammation, namely infiltration of inflammatory cells, release of inflammatory mediators, and activation of endothelial cells leading to increased vascular permeability, edema formation, and tissue destruction have been well and extensively characterized in animal SCI models (Popovich et al., 1996; Genovese et al., 2005). The production of reactive oxygen species (ROS) also contribute to the tissue injury observed during posttraumatic inflammatory reaction after SCI (Liu et al., 1997; Xu et al., 2001). Molecularly, ROS also cause DNA damage (Cuzzocrea et al., 1998a,b; Szabò et al., 1998a), which results in the activation of the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1), depletion of NAD+ and ATP and ultimately cell death (Cuzzocrea et al., 1998a; Szabò et al., 1998b). PARP-1 is a member of the PARP enzyme family consisting of PARP-1 and several recently identified novel poly(ADP-ribosylating) enzymes. Despite its function in DNA repair, overactivation of PARP-1 has long been recognized to induce cell death under certain conditions (Berger, 1985). PARP-1 inhibitors and PARP-1 gene disruption can reduce cell death resulted from oxidative stress (Schraufstatter et al., 1986), radiation (Piela-Smith et al., 1992), nitric oxide, peroxynitrite (Szabò et al., 1998c), and other agents that damage DNA (Ha and Snyder, 1999). In vivo, genetic or pharmacological inhibition of PARP-1 reduces ischemic cell death (Szabò et al., 1998a) and prevents neuronal death (Pieper et al., 1999). Poly(ADP-ribose) glycohydrolase (PARG, EC 3.2.1.143) is responsible to degrade poly(ADP-ribose) polymer. Within minutes after its synthesis by PARP-1, the poly(ADP-ribose) is hydrolyzed by PARG to ADP-ribose (Lu et al., 2003). The half life of poly(ADP-ribose) in cells is less than 5 minutes and the NAD level can drop to less than 20% of the normal level within 30 min after severe DNA damage that activates PARP1. PARG cleaves the ribose-ribose bonds of linear and branched portion of polymer, specifically the glycosidic and glycosidic linkages of PAR. The final products of the reaction are mono-ADPribosyl protein and ADP-ribose. ADP-ribose is known to be a weak PARG inhibitor with an
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inhibition of concentration (IC) of 0.1 mM (Slama et al., 1995). Because PARP-1 is inhibited by extensive auto-poly(ADP-ribosyl)ation, PARG inhibitors could thereby indirectly inhibit PARP-1 activity. Previous work has shown that the PARG inhibitor gallotannin can markedly reduce death of astrocytes after oxidative stress (Ying and Swanson, 2000). However, the first generation of PARG inhibitor are often associated with low potency, poor specificity or scarce availability of the compounds, low penetration of the blood–brain barrier, and intercalates into DNA ( Slama et al., 1995). To overcome these limitations, a series of novel small molecule PARG inhibitors with increased potency and reduced molecular weight (Li et al., US Patent ), has been developed as potential therapeutic agents. One such novel PARG inhibitor, N-bis-(3-phenyl-propyl)9-oxofluorene-2,7- diamide, GPI 16552 (IC 5.5 µM, MW 503), a non-tannin small molecule, reduced infarct volume in an in vivo model of brain ischemia/reperfusion injury. The result of neuroprotection by GPI 16552 treatment supports the notion that PARG could potentially be an alternative therapeutic target to regulate the poly(ADP-ribose) pathway. Recently, Patel and colleagues (Patel et al., 2005) have generated mutant mice lacking the 110-kDa isoform of the PARG protein (PARG110KO mice (KO)), which are viable and fertile. However, these KO mice were hypersensitive to genotoxic and endotoxic treatment most likely due to down regulation of PARP-1 automodification activity. On the contrary, recently it has been clearly demonstrated that inflammatory process associated with ischemia and reperfusion (kidney and intestine) is significantly reduced in these KO mice (Cuzzocrea et al., 2005; Patel et al., 2005). Thus, the KO mice represent an useful system to study the function of PARG in rodent model of diseases and validate the pathways that may be target for pharmaceutics application/intervention. To investigate the mechanism that leads to various forms of human SCI, several experimental models have been developed (Beattie et al., 2002). The most commonly used model is the compression model; in this model, injury is induced by applying either a weight or an aneurysm clip to the spinal cord. This model aims to add to that of the contusion model by replicating the persistence of cord compression that is commonly observed in human SCI (Tator, 1995).
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Apoptosis is an important mediator of secondary damage after SCI (Beattie et al., 2002). It incurs its affects through at least two phases: an initial phase, in which apoptosis accompanies necrosis in the degeneration of multiple cell types and a later phase, which is predominantly confined to white matter and involves oligodendrocytes and microglia (Merrill et al., 1993). Generation of free radicals and nitric oxide by activated macrophages has been implicated in causing oligodendrocyte apoptosis (Merrill et al., 1993). Chronologically, apoptosis initially occurs 6 hours post-injury at the lesion center and last for several days associated with the steadily increased number of apoptotic cells in this region . An important intracellular signal transduction pathway that leads to apoptosis after SCI involves activation of the caspases, in particular, caspase-3 (Janicke et al., 1998). This protease is required for DNA fragmentation and morphologic changes associated with apoptotic cell death (Janicke et al., 1998) and PARP-1 is a major substrate of activated caspase-3 (West et al., 2005). However, the role of PARP-1 autopoly(ADP-ribosyl)ation during apoptosis is still unclear and disputed. In this regard, we have recently demonstrated that PARP inhibitors inhibits in vivo apoptotic cell death in spinal cord tissue from mice subjected to trauma (Genovese et al., 2005) suggesting that inhibition of PARP directly protects cells by preventing the activation of the apoptosis pathway. Moreover, it is well known that Bax, a pro-apoptotic gene, plays an important role in developmental cell death (Chittenden et al., 1995) and in CNS injury (Bar-Peled et al., 1999). Similarly, it has been shown that the administration of Bcl-xL fusion protein (Bcl-xL FP), (Bcl-2 is the most expressed antiapoptotic molecule in adult central nervous system) into injured spinal cords significantly increased neuronal survival, suggesting that SCI-induced changes in Bcl-xL contribute considerably to neuronal death (Nesic-Taylor et al., 2005). The objective of this article is to evaluate the molecular pathways of SCI mechanisms and provide information for possible treatments. In the present study, using this experimental model of SCI, we have investigated the role of PARG activation in the pathogenesis of SCI and evaluate the therapeutic effects of GPI 16552.
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METHODS
Animals Wild-type mice (WT) and mice with a targeted disruption of the PARG (Cortes et al., 2004) in 129/Sv/Ola background were kept in the specific pathogen free facility of the International Agency for Research on Cancer, Lyon. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes (D.M. 116192) and with the EEC regulations (O.J. of E.C. L 358/1 12/18/1986).
Experimental designs Mice (male, 8-10 weeks old, 20-22 g) were randomly divided 8 groups: 1)
Sham- (WT) Group. Mice were subjected to identical surgical procedures except for SCI and were maintained under anesthesia for the duration of the experiment.
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SCI-(WT) Group mice. Mice were subjected to SCI.
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Sham-PARG-/- Group. Mice were subjected to identical surgical procedures except for SCI and were maintained under anesthesia for the duration of the experiment.
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SCI-PARG-/- Group. Mice were subjected to SCI.
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Sham- (WT) + GPI 16552 Group; identical to sham-operated mice except for the administration of GPI 16552.
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SCI-(WT) + GPI 16552 Group. Mice were subjected to SCI) and administered GPI 16552 (40 mg/kg, i.p) 30 minutes after SCI .
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Sham-PARG-/- + GPI 16552 Group. Identical to sham-operated mice except for the administration of GPI 16552.
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SCI-PARG-/- + GPI 16552 Group. Mice were subjected to SCI and administered GPI 16552 (40 mg/kg, i.p) 30 minutes after SCI.
At different time points (see Fig. 1) the animals (N=10) mice from each group for each time point were sacrificed in order to evaluate the various parameter as described below. In the experiments investigating the motor score, the animals were treated with GPI 16552 (40 mg/kg, i.p) 30 minutes, 24h after SCI and daily until day 9. The selected dose of 40 mg/kg ip is based on such treatment with GPI 16552 afforded intestinal protection in a mouse model of gut ischemia and reperfusion (Cuzzocrea et al., 2005).
SCI Mice were anaesthetized using chloral hydrate (400mg/kg body weight). A longitudinal incision was made on the midline of the back, exposing the paravertebral muscles. These muscles were dissected away exposing T5-T8 vertebrae. The spinal cord was exposed via a two-level T6-T7 laminectomy and SCI was produced by extradural compression of the spinal cord using an aneurysm clip with a closing force of 24 g. Following surgery, 1.0 cc of saline was administered subcutaneously in order to replace the blood volume lost during the surgery. During recovery from anesthesia, the mice were placed on a warm heating pad and covered with a warm towel. The mice were singly housed in a temperature-controlled room at 27°C for a survival period of 24 hours. Food and water were provided to the mice ad libitum. During this time period, the animals' bladders were manually voided. In all injured groups, the spinal cord was compressed for 1min. Sham injured animals were only subjected to laminectomy.
Measurement of myeloperoxidase activity
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Myeloperoxidase (MPO) activity, which was used as an indicator of polymorphonuclear (PMN) cell infiltration, was measured as previously described at 4 hours and 24 hours after SCI according to precious studies (Carlson et al., 1998).
Annexin -V- evaluation The binding of annexin V-fluorescein isothiocyanate (Ann-V) to externalized phosphatidylserine was used as a measurement of apoptosis in spinal cord tissue section 24 hours after SCI, with an Annexin-V-propidium iodide (PI) apoptosis detection kit (Santa Cruz, DBA Milan Italy) according to the manufacturer's instructions. Briefly, normal viable cells will stain negative for Annexin V FITC and negative for PI. Cells that are induced to undergo apoptosis will stain positive for Annexin V FITC and negative for PI as early as 1 hour after stimulation (Schutte et al., 1998). Both cells in later stages of apoptosis and necrotic cells will stain positive for Annexin V FITC and PI. Sections were washed as before, mounted with 90% glycerol in phosphate saline buffer (PBS), and observed with a LSM 510 Zeiss laser confocal microscope equipped with a 40 X oil objective.
Immunohistochemical localization of tumor necrosis factor α (TNF-α), interleukin 1-β (IL1β), MPO, Bax and Bcl-2 At the 24 hours after SCI, the tissues were fixed in 10% (w/v) PBS-buffered formaldehyde and 8 mm sections were prepared from paraffin embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60% (v/v) methanol for 30 min. The sections were permeablized with 0.1% (w/v) Triton X-100 in PBS for 20 min. Non-specific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin (DBA), respectively. Sections were incubated overnight with anti-TNFα (1:500 in PBS, v/v), anti-IL-1β polyclonal antibody (1:500 in PBS, v/v), anti-MPO (1:1000), antiBax rabbit polyclonal antibody (1:500 in PBS, v/v) or with anti-Bcl-2 polyclonal antibody rat.
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Sections were washed with PBS, and incubated with secondary antibody. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex (DBA). To verify the binding specificity for MPO, TNF-α, IL-1β, Bax, and Bcl-2, some sections were also incubated with only the primary antibody (no secondary) or with only the secondary antibody (no primary). In these situations no positive staining was found in the sections indicating that the immunoreactions were positive in all the experiments carried out. Immunohistochemical photographs (n=5 photos from each samples collected from all mice in each experimental group) were assessed by densitometry using Optilab Graftek software on a Macintosh personal computer.
Terminal Deoxynucleotidyltransferase-Mediated UTP End Labeling (TUNEL) Assay TUNEL assay was conducted by using a TUNEL detection kit according to the manufacturer’s instruction (Apotag, HRP kit DBA, Milan, Italy). Briefly, sections were incubated with 15 µg/ml proteinase K for 15 min at room temperature and then washed with PBS. Endogenous peroxidase was inactivated by 3% H2O2 for 5min at room temperature and then washed with PBS. Sections were immersed in terminal deoxynucleotidyltransferase (TdT) buffer containing deoxynucleotidyl transferase and biotinylated dUTP in TdT buffer, incubated in a humid atmosphere at 37°C for 90 min, and then washed with PBS. The sections were incubated at room temperature for 30 min with anti-horseradish peroxidase-conjugated antibody, and the signals were visualized with diaminobenzidine.
Light microscopy Spinal cord biopsies were taken at 24 hours after the trauma. The biopsies were fixed for 24 hours in para-formaldehyde solution (4% in phosphate buffered saline 0,1M) at room temperature, dehydrated by graded ethanol and embedded in Paraplast (Sherwood Medical, Mahwah, NJ). Tissue sections (thickness 5 µm) were deparaffinized with xylene, stained with haematoxylin/eosin (H&E)
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and Luxol Fast Blue staining (used to assess demyelization) and studied using light microscopy (Dialux 22 Leitz). All the histological studies were performed in a blinded fashion.
Materials Unless stated otherwise, all reagents and compounds used were obtained from Sigma Chemical Company (Sigma, Milan, Italy).
Data analysis. In the experiments involving histology the figures shown are representative of at least three experiments performed on different experimental days. The results were analyzed by one-way ANOVA followed by a Bonferroni's post-hoc test for multiple comparisons. A p-value of less than 0.05 was considered significant.
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RESULTS
PARG inhibition reduces the severity of SCI The severity of the trauma was observed at 24 hours after injury. A significant damage to the spinal cord, at the level of the perilesional area, assessed by the presence of edema as well as alteration of the white matter (Fig. 2B), was observed in SCI- operated WT mice when compared with spinal cord tissue collected from sham-operated mice (Fig. 2A). Notably, a significant protection of the SCI was observed in the tissue collected from KO mice (Fig. 2C) and as well as from WT mice treated with the PARG inhibitor GPI 16552 (40 mg/kg, i.p) (Fig. 2D). Myelin structure was observed by Luxol fast blue staining. In sham animals (Fig. 2E), myelin structure was clearly stained by Luxol fast blue in both lateral and dorsal funiculi of the spinal cord. At 24 hours after the injury in SCI- operated WT mice (Fig. 2F), a significant loss of myelin in lateral and dorsal funiculi was observed. In contrast in KO mice (Fig. 2G) and as well as in WT mice treated with the GPI 16552 (Fig. 2H) the myelin degradation was attenuated in the central part of lateral and dorsal funiculi. In order to evaluate if histological damage to the spinal cord was associated with a loss of motor function, the modified BBB hind limb locomotor rating scale score was evaluated. While motor function was only slightly impaired in sham mice groups, SCI-operated WT mice developed a significant deficits in hind limb movement (Fig. 3). In contrast, a significant amelioration of hind limb motor disturbances was observed in KO mice as well as in WT mice treated with the GPI 16552 (Fig. 3).
Effects of PARG inhibition on neutrophil infiltration The above mentioned histological pattern of SCI appeared to be correlated with the influx of leukocytes into the spinal cord. Therefore, we investigated the role of inhibition of PARG on the neutrophil infiltration by measurement of MPO activity. MPO activity was significantly elevated at 4 hours (Fig. 4A) and 24 hours (Fig. 4B) in SCI-operated WT mice when compared with spinal
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cord tissue collected from sham-operated mice (Fig. 4). Spinal cord MPO activity was significantly attenuated in comparison to those of SCI- operated WT mice group at 4 (Fig. 4A) and 24 hours (Fig. 4B) in KO mice as well as in WT mice treated with the GPI 16552. In addition, tissue sections obtained at 24 hours from SCI-operated WT mice demonstrate positive staining for MPO mainly localized in the infiltrated inflammatory cells in injured area (Fig. 5B, 6). In KO mice (Fig. 5C, 6) as well as in WT mice treated with the GPI 16552 (Fig. 5D, 6), the staining for MPO was visibly and significantly reduced in comparison with the SCI- operated WT mice. There was no staining for MPO in spinal cord tissues obtained from the sham group of mice (Fig. 5A, 6).
PARG inhibition modulates expression of TNF-α and IL-1β . Immunohistological analysis of the spinal cord after SCI was performed to determine whether PARG inhibition may modulate the secondary inflammatory reaction through also the regulation of the secretion of cytokines. A substantial increase of IL-1β (Fig. 6, 7B) and TNF-α (Fig. 6, 7F) formation was found in spinal cord samples collected from SCI- operated WT mice at 24 hours after SCI (Fig. 6). Spinal cord levels of IL-1β and TNF-α were significantly attenuated in KO mice (Fig. 6, 7CG, respectively) and as well as in WT mice treated with the GPI 16552 (Fig. 6 7DH respectively). There was no staining for either IL-1β and TNF-α in spinal cord obtained from the sham group of mice (Fig. 6, 7A,E respectively).
Effect of PARG inhibition on apoptosis in spinal cord tissue after injury To test whether the tissue damage was associated with cell death by apoptosis, we measured Annexin V staining in the spinal cord tissues. Almost no apoptotic cells were detectable in the spinal cord tissue from sham-operated mice (data not shown). However, at 24 hours after SCI, tissues obtained from SCI- operated WT mice demonstrated a marked appearance of positive staining for Annexin-V (Fig. 8A), indicative of cells undergoing apoptosis. In addition, some cells showed a positive intracellular staining to propidium iodide index of cells in the late stage of
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apoptosis (Fig. 8B). On the contrary, spinal cord tissues section from KO mice as well as WT mice treated with the GPI 16552 demonstrated no cells in the earlier stage of apoptosis (Fig. 8DG respectively) and a significantly less presence of cells (positive for propidium iodide) in the later stages of apoptosis (Fig. 8EH respectively). Moreover, in order to confirm whether tissue damage was associated with cell death by apoptosis, we measured also TUNEL-like staining in the perilesional spinal cord tissue. Almost no apoptotic cells were detectable in the spinal cord tissue from sham-operated mice (Fig. 9A). At 24 hours after the trauma tissues obtained from SCIoperated WT mice demonstrated a marked appearance of dark brown (TUNEL-positive) apoptotic cells and intercellular apoptotic fragments (Fig. 9B). In contrast, tissues obtained from KO mice (Fig. 9C) and as well as in WT mice treated with the GPI 16552 (Fig. 9D) contained a smaller number of TUNEL-positive cells.
Effect of PARG inhibition on Bax and Bcl-2 expression Spinal cord tissues were taken at 24 hours after SCI in order to determine the immunohistological staining for Bax and Bcl-2. Sections of spinal cord from sham-operated mice stained negative for Bax (Fig. 6,10A). Spinal cord sections obtained from SCI- operated WT mice exhibited positive staining for Bax (Fig. 6,10B). On the contrary, the degree of positive staining for Bax was significantly reduced in spinal cord tissues collected from KO mice (Fig. 6,10C) as well as from WT mice treated with the GPI 16552 (Fig. 6,10D). In addition, sections of spinal cord from sham-operated mice demonstrated positive staining for Bcl-2 (Fig. 6,10E). Spinal cord sections obtained from SCI- operated WT mice exhibited significantly less staining for Bcl-2 (Fig. 6,10F). The loss of positive staining for Bcl-2 was significantly reduced in spinal cord tissues collected from KO mice (Fig. 6,10GG1) as well as in WT mice treated with the GPI 16552 (Fig. 6,10H).
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DISCUSSION Spinal cord trauma initiates a sequence of events that lead to secondary neuronal cell damage. While the precise mechanisms responsible remain undefined, several studies have implicated ROS in the secondary neuronal damage of SCI (Liu et al., 1997; Xu et al., 2001). ROS and peroxynitrite also cause DNA damage (Cuzzocrea et al., 1998a; Szabò et al., 1998a), which results in the activation of the nuclear enzyme PARP-1. Our recent work clearly demonstrates that the PARP-1 inhibitors significantly reduced the experimental SCI in mice (Genovese et al., 2005).The current study demonstrates that PARG inhibition may be equally effective as PARP-1 inhibition for suppressing the NAD/ATP depletion. Therefore, it also supports the hypothesis that blocking the poly(ADP-ribose) pathway, which can be achieved by either PARG or PARP-1 inhibitors, is the common mechanism for anti-inflammatory effect. Indeed, our data demonstrate that PARG activation plays a major role in SCI, characterized by (1) histological damage (2) motor damage, (3) neutrophil infiltration, (4) cytokine expression (TNF-α and IL-1β), (5) positive for Annexin-V, (6) increased apoptosis (TUNEL staining), (7) induction of Bax and downregulation of Bcl-2. The PARG gene disruption and enzymatic inhibition of PARG reversed the above symptoms. What then is the mechanism by which PARG inhibition protects the spinal cord against injury and dysfunction? Previously, PARG inhibitors have been evaluated in vitro against necrotic cell death. PARG inhibitor, 1,2,3,4,6-o- inhibipenta-b-D-galloylglucose, prevented death of P388D1 macrophage cell line by hydrogen peroxide treatment (Li and Zhang, 2000). Gallotannin and nobotannin reduced the death of the astrocytes and neurons treated with hydrogen peroxide, N-methyl-N9-nitro-Nnitroguanidine or N-methyl-D-aspartate (Ying and Swanson, 2000; Ying et al., 2001). Moreover, Zhang and colleagues (Zhang et al., 1998) have recently demonstrated that the new PARG inhibitor GPI 16552 exerts significant neuroprotection in a cerebral focal ischemia model (Lu et al., 2003). The injured environment during the acute phase of SCI is dominated by the presence of the proinflammatory cytokines tumor necrosis factor α (TNFα), interleukin (IL)-1β and IL-6 (Tyor et al.,
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2002). Direct evidence that TNF-α and IL-1β play a role in the outcome of the damage following injury to the spinal cord has been obtained in animal models in which exogenous local administration of pro-inflammatory cytokines to mice after SCI which may influence these intrinsic processes (Klusman and Schwab, 1997).We confirm in the present study that the model of SCI used here leads to a substantial increase of the levels of TNF-α and IL-1β in the spinal cord. Interestingly, the levels of this pro-inflammatory cytokines are significantly lower in the tissues obtained from KO mice as well as WT mice treated with GPI 16552. These findings, therefore, suggest that inhibition of PARG reduced the activation and the subsequent expression of proinflammatory genes. We demonstrated in the present study that the genetic or pharmacological inhibition of PARG reduces the inflammatory cell infiltration as assessed by the specific granulocyte enzyme myeloperoxidase at different time point. The reduced neutrophil recruitment represents another mechanism for the protective anti-inflammatory effects. Consistent with this notion/hypothesis, Taoka et al. (1997) have demonstrated that activated neutrophils are involved in compression trauma-induced SCI in rats. Neutrophils, recruited into the tissue can contribute to tissue destruction by the production of reactive oxygen metabolites (Tator, 1995; Carlson et al., 1998), granule enzymes, and cytokines that further amplify the inflammatory response. We have identified proapoptotic transcriptional changes, including upregulation of proapoptotic Bax and downregulation of antiapoptotic Bcl-2, by immunohystochemical staining. We report in the present study for the first time that the genetic or pharmacological inhibition of PARG in SCI experimental model documents features of apoptotic cell death after SCI, suggesting that protection from apoptosis may be a prerequisite for regenerative approaches to SCI. In particular, we demonstrated that the genetic or pharmacological inhibition of PARG reduced Bax expression, while on the contrary, Bcl-2 expressed much more in KO mice as well as WT mice treated with GPI 16552. This result suggests that PARG inhibition prevents the lost of the anti-apoptotic way and reduces the pro-apoptotic pathway activation with a mechanism still to be discovered.
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Finally, in the mice spinal cord, two early morphologic changes occur in the myelin sheath after SCI (Zhang et al., 1998). Paranodal myelin breakdown with associated nodal widening and random Wallerian-type degeneration of nerve fibers in the white matter are observed. In this study, the histological examination of the spinal cord from SCI-operated mice revealed that microcystic cavitation and degenerating axons is significantly ameliorated in the SCI-treated KO mice as well as SCI-WT mice treated with GPI 16552 . It is interesting to note that KO mutant mice are hypersensitive to LPS-induced septic shock (Cortes et al., 2004) as well as to post ischemic brain damage (Cozzi et al., 2005). Since PARP-1 can still be activated in KO mutant mice but with a low degree of automodification, DNA damageinduced cell death pathway may become prominent in this model. The involvement of pADPR pathway in pathological conditions is well documented by numerous studies that chemical PARP-1 inhibition and PARP-1 gene deletion (Szabo et al., 1998c;Genovese et al., 2005). The data presented in the present study, together with other reports (Slama et al., 1995; Ying and Swanson, 2000), demonstrate that homeostasis of pADPR modulated by PARP-1/PARG either directly or indirectly regulates the inflammation response. The mechanism of these actions clearly requires further investigation. Further experiments with multiple approaches can be expected to unlock the full potential of intervening the pADPR pathway for therapeutic purposes through PARP-1 and/or PARG inhibition. In conclusion, we have demonstrated for the first time in vivo that genetic and pharmacological inhibition of PARG attenuates SCI. Therefore, our results provide an other important experimental evidence that PARG may be a novel target by therapeutic applications for treating shock and inflammation .
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Acknowledgements: The authors would like to thank Giovanni Pergolizzi and Carmelo La Spada for their excellent technical assistance during this study, Mrs Caterina Cutrona for secretarial assistance and Miss Valentina Malvagni for editorial assistance with the manuscript.
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JPET #108076 Footnotes This study was supported by grant from MURST.
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FIGURE LEGENDS Figure 1: Mice were sacrificed at different time points in order to evaluate the various parameter. n=10 mice from each group for each time point. See Material and methods for further explanations.
Figure 2: PARG inhibition reduces histological alteration of the spinal cord tissue 24 hours after injury. No histological alteration was observed in spinal cord tissues obtained from sham-operated mice (A). Twenty-four hours after the trauma, a significant damage was evident to the spinal cord from SCI- WT treated mice at perilesional level as assessed by the presence of edema as well as an alteration of the white matter (B). Notably, a significant protection of the SCI was observed in the tissue collected from KO (C) as well as from GPI 16552-treated SCI-WT mice (D). In shamoperated animals (E) myelin structure was clearly stained by Luxol fast blue in both lateral and dorsal funiculi of the spinal cord. At 24 hours after the injury in SCI-WT mice (F), a significant loss of myelin was observed. In contrast in the tissue collected from KO mice (G) as well as from GPI 16552-treated SCI-WT mice (H) myelin degradation was attenuated. This figure is representative of at least 3 experiments performed on different experimental days.
Figure 3: Effect of PARG inhibition on hind limb motor disturbance after SCI. The degree of motor disturbance was assessed every day until 10 days after SCI by Basso-Beattie-Bresnahan criteria (Basso et al., 1995). Genetic and pharmacological inhibition of PARG reduces the motor disturbance after SCI. Values shown are mean ± s.e. mean of 10 mice for each group. *P
