NP031112, a thiadiazolidinone compound, prevents inflammation and neurodegeneration under excitotoxic conditions: potential therapeutic role in brain disorders

Abstract

Inflammation and neurodegeneration coexist in many acute damage and chronic CNS disorders (e.g., stroke, Alzheimer's disease, Parkinson's disease). A well characterized animal model of brain damage involves administration of kainic acid, which causes limbic seizure activity and subsequent neuronal death, especially in the CA1 and CA3 pyramidal cells and interneurons in the hilus of the hippocampus. Our previous work demonstrated a potent anti-inflammatory and neuroprotective effect of two thiadiazolidinones compounds, NP00111 (2,4-dibenzyl-[1,2,4]thiadiazolidine-3,5-dione) and NP01138 (2-ethyl-4-phenyl-[1,2,4]thiadiazolidine-3,5-dione), in primary cultures of cortical neurons, astrocytes, and microglia. Here, we show that injection of NP031112, a more potent thiadiazolidinone derivative, into the rat hippocampus dramatically reduces kainic acid-induced inflammation, as measured by edema formation using T2-weighted magnetic resonance imaging and glial activation and has a neuroprotective effect in the damaged areas of the hippocampus. Last, NP031112-induced neuroprotection, both in vitro and in vivo, was substantially attenuated by cotreatment with GW9662 (2-chloro-5-nitrobenzanilide), a known antagonist of the nuclear receptor peroxisome proliferator-activated receptor gamma, suggesting that the effects of NP031112 can be mediated through activation of this receptor. As such, these findings identify NP031112 as a potential therapeutic agent for the treatment of neurodegenerative disorders.

Figures

Figure 1.

Effects of NP031112 on excitotoxic neural injury in vitro. a, Rat primary astrocyte or microglial cultures were treated for 24 h with glutamate (500 μm) in the absence or presence of NP031112 (2.5 μm), and the expression of TNF-α and COX-2 was evaluated by immunofluorescence analysis and confocal microscopy using specific antibodies, as described in Materials and Methods. Some cultures were preincubated 1 h with 30 μm GW9662 before the addition of NP031112 or with 20 mm LiCl before the addition of glutamate. Representative results of three different experiments are shown. Scale bars, 10 μm. Nuclei were counterstained with DAPI (blue). b, Rat primary neuronal cultures were treated for 24 h with glutamate (100 μm) in the absence or presence of NP031112 (2.5 μm). Some cultures were preincubated 1 h with 30 μm GW9662 before the addition of NP031112 or with 20 mm LiCl before the addition of glutamate, and apoptosis was assessed by Annexin-V-FITC staining as described in Materials and Methods. Representative confocal images of three independent experiments are shown. Scale bars, 10 μm. Values in a and b represent the mean ± SD from three different experiments and five independent fields (≥ 50 cells/field) per culture. ***p ≤ 0.001. c, Activation of a PPRE (peroxisome proliferator response element) reporter gene by NP031112. Rat primary astrocytes were transfected with 0.2 μg of the PPRE-tk-luc reporter plasmid, cells were harvested 24 h after treatment with NP031112 at the indicated concentrations, and luciferase activity of cell lysates was determined. Data are expressed relative to the basal values and represent the mean ± SD luciferase activity determined in triplicate in three independent experiments. **p ≤ 0.01; ***p ≤ 0.001, versus control nontreated cultures. GLUT, Glutamate; B, basal; GW, GW9662; NP, NP031112.

Figure 2.

Effect of NP031112 on KA-induced brain edema as detected by MRI. T2-weighted imaging was performed at 7 tesla as described in Materials and Methods at different times after KA injection. a, Representative coronal images of rat brain injected with vehicle (top), KA (middle), and KA plus NP031112 (bottom). Hyperintensity areas in T2-weighted MRIs reveal regions of edema after KA injection. Hyperintense areas were reduced in the NP031112-treated rats compared with the KA-injected rats, demonstrating a decrease in the injured area. No hyperintensity was found in the vehicle-injected rats. Arrows indicate the injection site. An anatomic diagram (Paxinos, 1998) showing (arrow) the precise localization of the microinjection in the rat hippocampus is shown. b, Quantitative analysis of total lesion volumes of KA- and KA plus NP031112-injected rats. The volume of the edemas was significantly lower in NP031112-treated rats, compared with KA-treated group at all of the times studied. Values represent the mean ± SD from five different animals and five independent sections per animal. ***p ≤ 0.001. c, Representative point-resolved spatially spectra acquired from the hippocampal region (white box) with resonances from NAA, lactate (Lac), and creatine (Cr) 9 d after treatment. d, Time course of variation of NAA or lactate values (mean ± SD) in the ipsilateral hemisphere of control-, KA-, and KA plus NP031112-treated animals during 9 d, normalized to the creatine peak. V, Vehicle; NP, NP031112. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001, versus vehicle-injected animals at each time point.

Figure 3.

Neuroprotective effects of NP031112 on KA-induced excitotoxicity. a, Rats were injected with saline, KA, or KA plus NP031112 and killed at different times after injection. Neuronal cell loss was assessed in coronal brain sections, using a Nissl stain. Arrows indicate the injection site. Insets, 400× magnifications of Nissl-stained CA3 cells (boxed areas). No apparent cell loss was observed in control animals. Three days after KA, cell loss was apparent on the CA3 and CA1 subfields of the hippocampus in both KA and KA plus NP031112-injected rats. By day 9, cell loss was more prominent in KA-injected animals, whereas animals treated with NP031112 showed no cell loss. V, Vehicle; NP, NP031112. b, The extent of neuronal damage in the CA3 area of the hippocampus was quantified as described in Materials and Methods. Data were normalized against the mean values given by vehicle-injected rats. Values represent the mean ± SD from five different animals and five independent sections per animal. **p ≤ 0.01; ***p ≤ 0.001, versus vehicle-injected animals at each time point.

Figure 4.

Effect of NP031112 administration on KA-induced glial activation. a–d, GFAP- and OX-42-positive cells in the hippocampus. Rats were injected with saline, KA, or KA plus NP031112 and killed 9 d after injection. Arrows indicate the injection site. Coronal sections (30 μm) were stained with mouse monoclonal antibodies directed against either GFAP (a) or cd11b (OX-42; c) to detect astrocytes or microglial cells, respectively, as described in Materials and Methods. Insets, 400× magnifications of GFAP- and OX-42-stained cells (boxed regions). A significant increase in gliosis is observed after KA injection, which is prevented in the animals treated with NP031112. b, Quantification of the number of reactive astrocytes and their mean immunostaining intensity evaluated in the molecular layer of the hippocampus. Values represent the mean ± SD from five different animals and five independent sections per animal. ***p ≤ 0.001. d, Quantification of the number of reactive microglial cells analyzed in the molecular layer of the hippocampus. Values represent the mean ± SD from five different animals and five independent sections per animal. ***p ≤ 0.001. e, Induction of TNF-α in astrocytes, microglia, and neurons after KA injection is abrogated by NP031112. Coronal sections were double stained with a polyclonal antibody directed against TNF-α and with mouse monoclonal antibodies directed against NeuN, GFAP, or cd11b (OX-42) and examined by confocal microscopy. Photomicrographs showing representative CA3 fields for each group are shown. Scale bars, 10 μm. DAPI was used as a counterstain.

Figure 5.

The PPARγ antagonist GW9662 abolishes NP031112 neuroprotection after KA injection. Rats were injected with saline (V), KA, KA plus NP031112 (NP), or KA plus LiCl and killed 24 h after injection. Some animals were also injected with 0.7 μg GW9662 (GW). Arrows indicate the injection site. a, Neuronal cell loss was assessed in coronal brain sections using a Nissl stain. NP031112 treatment affords robust neuroprotection in the CA3 regions of the hippocampus 24 h after KA injection, whereas LiCl administration only had a small effect on the decrease in CA3 neurons elicited by KA injection. Injection of the PPARγ antagonist GW9662 after NP031112 and KA treatment abolished this neuroprotection. Treatment with the PPARγ antagonist did not affect the number of surviving neurons in any of the other treatment groups. b, Gliosis was assessed in similar brain sections by staining with a mouse monoclonal antibody directed against cd11b (OX-42) to detect microglial cells. Treatment with GW9662 blocked the anti-inflammatory effect of NP031112. c, Quantification of neuronal damage in the CA3 area of the hippocampus. d, Quantification of the number of reactive microglial cells analyzed in the molecular layer of the hippocampus. Values represent the mean ± SD from five different animals and five independent sections per animal. ***p ≤ 0.001 versus vehicle-injected animals; #p ≤ 0.05; ##p ≤ 0.01; ###p ≤ 0.001 versus KA-injected animals.

Figure 6.

Effect of NP031112 administered orally on behavioral activity and neuronal loss after injection of KA. a, b, NP031112 (50 mg/kg) was administered intragastrically 1 h before intraperitoneal injection of KA (10 mg/kg), and the behavioral activity was analyzed. NP031112 has no significant effects on behavioral seizures induced by KA. Each value represents the mean ± SEM from at least nine animals. c, The data correspond to animals that entered SE. Animals were treated as in a, and 72 h after KA injections, animals were killed and coronal hippocampal sections were stained with Nissl. NP031112-treated rats exhibited a diminished loss of neurons in the CA1 and CA3 regions, when compared with KA-injected rats. V, Vehicle; NP, NP031112. Values represent the mean ± SD from five different animals and five independent sections per animal. ***p ≤ 0.001 versus vehicle-injected animals.