Jill Ann Panetta

Transcription Factor Nuclear Factor-Kappa B is Activated in Neurons after Focal Cerebral Ischemia

Nuclear factor-kappa B (NF-kB) is a multisubunit transcription factor that when activated induces the expression of genes encoding acute-phase proteins, cell adhesion molecules, cell surface receptors, and cytokines. NF-kB is composed of a variety of protein subunits of which p50-and p65-kDa (RelA) are the most widely studied. Under resting conditions, these subunits reside in the cytoplasm as an inactive complex bound by inhibitor proteins, IkBα and IkBβ. On activation, IkB is phosphorylated by IkB kinase and ubiquitinated and degraded by the proteasome; simultaneously, the active heterodimer translocates to the nucleus where it can initiate gene transcription. In the periphery, NF-kB is involved in inflammation through stimulation of the production of inflammatory mediators. The role of NF-kB in the brain is unclear. In vitro, NF-kB activation can be either protective or deleterious. The role of NF-kB in ischemic neuronal cell death in vivo was investigated. Adult male rats were subjected to 2 hours of focal ischemia induced by middle cerebral artery occlusion (MCAO). At 2, 6, and 12 hours after reperfusion, the expression and transactivation of NF-kB in ischemic versus nonischemic cortex and striatum were determined by immunocytochemistry and by electrophoretic mobility gel-shift analysis. At all time points studied, p50 and p65 immunoreactivity was found exclusively in the nuclei of cortical and striatal neurons in the ischemic hemisphere. The contralateral nonischemic hemisphere showed no evidence of nuclear NF-kB immunoreactivity. Double immunofluorescence confirmed expression of p50 in nuclei of neurons. Increased NF-kB DNA-binding activity in nuclear extracts prepared from the ischemic hemisphere was further substantiated by electrophoretic mobility gel-shift analysis. Because the activation of NF-kB by many stimuli can be blocked by antioxidants in vitro, the effect of the antioxidant, LY341122, previously shown to be neuroprotective, on NF-kB activation in the MCAO model was evaluated. No significant activation of NF-kB was found by electrophoretic mobility gel-shift analysis in animals treated with LY341122. These results demonstrate that transient focal cerebral ischemia results in activation of NF-kB in neurons and supports previous observations that neuroprotective antioxidants may inhibit neuronal death by preventing the activation of NF-kB.

Cytosolic phospholipase A2 is induced in reactive glia following different forms of neurodegeneration.

Many recent studies have emphasized the deleterious role of inflammation in CNS injury. Increases in free fatty acids, eicosanoids, and products of lipid peroxidation are known to occur in various conditions of acute and chronic CNS injury, including cerebral ischemia, traumatic brain injury, and Alzheimers disease. Although an inflammatory response can be induced by many different means, phospholipases, such as cytosolic phospholipase A2 (cPLA2), may play an important role in the production of inflammatory mediators and in the production of other potential second messengers. cPLA2 hydrolyzes membrane phospholipids and its activity liberates free fatty acids leading directly to the production of eicosanoids. We investigated the cellular localization of cytosolic phospholipase A2 in the CNS following: (1) focal and global cerebral ischemia, (2) facial nerve axotomy, (3) human cases of Alzheimers disease, (4) transgenic mice overexpressing mutant superoxide dismutase, a mouse model of amyotrophic lateral sclerosis, and (5) transgenic mice overexpressing mutant amyloid precursor protein, which exhibits age‐related amyloid deposition characteristic of Alzheimers disease. We show that in every condition evaluated, cytosolic phospholipase A2 is present in reactive glial cells within the precise region of neuron loss. In conditions where neurons did not degenerate or are protected from death, cytosolic phospholipase A2 is not observed. Both astrocytes and microglial cells are immunoreactive for cytosolic phospholipase A2 following injury, with astrocytes being the most consistent cell type expressing cytosolic phospholipase A2. The presence of cytosolic phospholipase A2 does not merely overlap with reactive astroglia, as reactive astrocytes were observed that did not exhibit cytosolic phospholipase A2 immunoreactivity. In most conditions evaluated, inflammatory processes have been postulated to play a pivotal role and may even participate in neuronal cell death. These results suggest that cytosolic phospholipase A2 may prove an attractive therapeutic target for neurodegeneration. GLIA 27:110–128, 1999.

Drug-Induced Neuroprotection From Global Ischemia Is Associated With Prevention of Persistent but Not Transient Activation of Nuclear Factor-κB in Rats

BACKGROUND AND PURPOSE Nuclear factor-kappaB (NF-kappaB) is an oxidative stress responsive transcription factor that is transiently activated in most forebrain neurons in response to transient global ischemia. However, in hippocampal CA1 neurons destined to die, NF-kappaB remains persistently activated. The present study was performed to determine whether an antioxidant (LY231617) that afforded neuroprotection in previous studies had any effect on NF-kappaB activation in hippocampal CA1 neurons after global ischemia. METHODS Rats were subjected to 30 minutes of forebrain ischemia by 4-vessel occlusion (4-VO) and killed at 24 and 72 hours after ischemia. LY231617 was administered orally at a dose of 50 mg/kg 30 minutes before 4-VO and again 4 hours after 4-VO. Neuronal damage was evaluated in sections stained with cresyl violet. Other sections were immunostained with antibodies to NF-kappaB p50 to assess nuclear localization. An electrophoretic mobility shift assay was performed on nuclear extracts from sham- and LY231617-treated rats at 24 and 72 hours after ischemia. RESULTS The administration of LY231617 had a significant protective effect on hippocampal CA1 neurons at 72 hours after ischemia (control group, 16 +/- 7 neurons/mm; treated group, 294 +/- 35 neurons/mm, P<.02) and prevented nuclear translocation of activated NF-kappaB as normally seen at 72 hours after ischemia in untreated controls. In contrast, the untreated controls showed activated NF-kappaB at 72 hours after ischemia. At 24 hours after ischemia, both the control group and the LY231617 group showed intense nuclear localization of NF-kappaB. CONCLUSIONS Activation of NF-kappaB in vitro has been reported to promote proapoptotic as well as antiapoptotic mechanisms, depending on the cell type being investigated. In the present in vivo study, the role of the transient activation of NF-kappaB observed at 24 hours may be responsible for the induction of protective factors in neurons that survive the ischemic insult, whereas the persistent activation of NF-kappaB in hippocampal neurons could be responsible for the induction of proteins that result in CA1 neuronal death.

The antioxidant LY231617 reduces global ischemic neuronal injury in rats.

Background and Purpose In the rat four-vessel occlusion model with 30 minutes of ischemia most agents have failed to be of benefit when given after ischemia. Because postischemia administration is more clinically relevant, we evaluated the antioxidant LY231617 (2,6-bis(1,1-dimethylethyl)-4-[[(1-ethyl)ami-no]methyl]phenol hydrochloride]) when administered after 30 minutes of four-vessel occlusion. Methods Male Wistar rats were subjected to 30 minutes of four-vessel occlusion. LY231617 was either given orally 30 minutes before ischemia or intravenously beginning at 30 minutes after the onset of ischemia. Hippocampal CA1 layer and striatal damage were rated on a scale of 0-3 (0, no damage; 3, >90% cell loss). We also evaluated the ability of LY231617 to prevent iron-dependent lipid peroxidation and to prevent hydrogen peroxide-induced neuronal death of hippocampal neurons in primary culture by exposing cultures to a 50-¨M concentration of hydrogen peroxide for 15 minutes in the presence of LY231617. Results Oral administration of LY231617 reduced both striatal and hippocampal CA1 damage by >75% (p<0.0001). In two separate experiments in which LY231617 was given intravenously beginning 30 minutes after occlusion, hippocampal and striatal damage were reduced by approximately 50% (p<0.03) in the first experiment and by approximately 41% (p<0.002) in the second experiment. Addition of 5 ¨M of LY231617 to primary hippocampal neuronal cultures antagonized the lethal effect of hydrogen peroxide (p<0.05). Iron-dependent lipid peroxidation was also inhibited in a dose-related fashion. Conclusions The significant reduction of ischemia-induced or hydrogen peroxide-induced neuronal damage and inhibition of lipid peroxidation by LY231617 observed in this study suggest that reactive oxygen intermediates play an important role in the events leading to neuronal death after global ischemia/reperfusion.

Effects of LY231617 and angiotensin IV on ischemia-induced deficits in circular water maze and passive avoidance performance in rats

The antioxidant LY231617 has previously been shown to offer significant protection against postischemic cell death in the hippocampus and corpus striatum of rats. The present results extend this observation by demonstrating a concomitant protection against the spatial memory deficits that accompany damage to the hippocampus, as measured by the circular water maze task. These animals were further tested for changes in associative memory by employing a passive avoidance conditioning task. No deficits in passive avoidance conditioning were measured among the 4-vessel occlusion animals treated with LY231617 or vehicle. However, the intracerebroventricular injection of angiotensin IV (Ang IV) immediately prior to foot-shock conditioning improved retention of the conditioned response during the subsequent 2-day period. These results suggest that LY231617 can offer considerable protection against global ischemia-induced cell death in the hippocampus with resulting preservation of spatial memory abilities. In addition, untreated animals that suffered cell losses in the hippocampus remained capable of responding to the facilitory effect of centrally administered Ang IV on a non-spatial memory task. The hypothesized mechanisms of the protection characteristics of LY231617, and the nootropic effect of Ang IV, are discussed.

Synthesis and structure-activity relationships of benzophenones as inhibitors of cathepsin D

Abstract Non peptide inhibitors of cathepsin D, an aspartyl protease that has been implicated in many disease states including Alzheimers disease, were prepared and evaluated. The most potent inhibitor of cathepsin D in this series was found to be (Z)-5-[[4-(4-benzoyl-3-hydroxy-2-propylphenoxy) methylphenyl]methylene]-2-thioxo-4-thiazolidinone ( 3f , IC 50 = 210 nM).

Neuroprotective effects of the antioxidant LY231617 and NO synthase inhibitors in global cerebral ischaemia

Recent studies have shown that the novel antioxidant LY231617 protects against ischaemia-induced neuronal damage in rat models of global cerebral ischaemia. In the present studies we have examined the effects of LY231617 in the gerbil model of global cerebral ischaemia. We also examined the effects of four nitric oxide synthase inhibitors (3-bromo-7-nitroindazole, N(G)-nitro-L-arginine methyl ester, aminoguanidine and S-methylisothiourea sulphate) in this model. LY231617 (50 mg/kg p.o. or 30 mg/kg i.p.) was administered either 30 min prior to occlusion or immediately post-occlusion followed by three further doses at 4, 24 and 48 h after the initial dose. 3-Bromo-7-nitroindazole was administered at 40 mg/kg i.p. immediately after occlusion followed by 20 mg/kg i.p. at 3, 6, 24 and 48 h, N(G)-nitro-L-arginine methyl ester was administered at 10 mg/kg i.p. immediately after occlusion followed by 5 mg/kg i.p. at 3, 6, 24 and 48 h. Aminoguanidine was administered at 80 mg/kg i.p. immediately after occlusion followed by 40 mg/kg i.p. at 3, 6, 24 and 48 h and S-methylisothiourea sulphate was administered at 10 mg/kg i.p. immediately, 3, 6, 24 and 48 h after occlusion. We also examined the effects of aminoguanidine administered at 80 mg/kg i.p. immediately after occlusion followed by 40 mg/kg i.p. at 3, 6, 24, 48, 72 and 96 h and S-methylisothiourea sulphate administered at 10 mg/kg i.p. immediately, 3, 6, 24, 48, 72 and 96 h after occlusion. Control animals were either sham operated or subjected to 5 min bilateral carotid occlusion. Extensive neuronal death was observed in the CA1 layer of the hippocampus in 5-min bilateral carotid artery occluded animals 5 days after surgery. LY231617 provided significant neuroprotection against the ischaemia-induced brain damage when administration was initiated before or after occlusion (P < 0.05). The neuronal NO synthase inhibitors, 3-bromo-7-nitroindazole and a general NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester also provided significant neuroprotection (P < 0.05). In contrast aminoguanidine and S-methylisothiourea sulphate (two inducible NO synthase inhibitors) failed to protect against the ischaemia-induced brain damage. These results indicate that free radicals and nitric oxide are involved in ischaemia-induced brain damage following global cerebral ischaemia. Antioxidants such as LY231617 or neuronal NO synthase inhibitors can prevent the ischaemia-induced neurodegeneration and may be useful as anti-ischaemic agents.

Early treatment with a novel inhibitor of lipid peroxidation (LY341122) improves histopathological outcome after moderate fluid percussion brain injury in rats.

OBJECTIVE Reactive oxygen species are thought to participate in the pathobiology of traumatic brain injury (TBI). This study determined whether treatment with LY341122, a potent inhibitor of lipid peroxidation and an antioxidant, would provide neuroprotection in a rat model of TBI. METHODS To investigate the efficacy of LY341122 in this parasagittal fluid percussion model (1.8-2.1 atm), the rats received oral administration of LY341122 (100 mg/kg) or vehicle 2 hours before and 4 hours after TBI (each group, n = 7). To investigate the therapeutic window for treatment, rats were treated with LY341122 or vehicle for 20 hours by femoral vein infusion starting at 5 minutes, 30 minutes, or 3 hours after TBI (each group, n = 5). Three days after injury, analysis of contusion volumes and the frequency of damaged cortical neurons was conducted. RESULTS Oral administration of LY341122 before and after TBI led to a significant reduction in overall contusion volume (3.28 mm3+/-0.75 mm3 [mean +/- standard error of the mean] versus 1.32 mm3 +/- 0.33 mm3; P < 0.05) and also reduced the frequency of damaged cortical neurons (1191.7 +/- 267.1 versus 474.6 +/- 80.2; P < 0.05). In the second experiment, rats treated with LY341122 at 5 minutes or 30 minutes after TBI also demonstrated a significant reduction (P < 0.05) in contusion volume (1.92 mm3 +/- 0.64 mm3 or 1.59 mm3 +/- 0.50 mm3, respectively) compared with vehicle-treated rats (4.32 mm3 +/- 1.15 mm3). A significant reduction in total cortical necrotic neuron counts was also demonstrated in the 5-minute group (2243.8 +/- 265.3 versus 1457.8 +/- 265.3; P < 0.05). In contrast, histopathological outcome was not significantly improved when treatment was delayed until 3 hours after TBI. CONCLUSION These data reinforce the hypothesis that lipid peroxidation and reactive oxygen species participate in the acute pathogenesis of TBI. Treatment delayed until 3 hours after TBI did not provide significant histopathological protection.

Characterization of LY231617 Protection Against Hydrogen Peroxide Toxicity

Abstract : The compound LY231617 {2,6‐bis(1,1‐dimethylethyl)‐4‐[[(1‐ethyl)amino]methyl]phenol hydrochloride} has been reported to afford significant neuroprotection against hydrogen peroxide (H2O2)‐induced toxicity in vitro and global ischemia in vivo. We now report on further mechanistic studies of H2O2 toxicity and protection by LY231617. Brief exposure to H2O2 (15 min) elicited an oxidative insult comparable with that generated by overnight treatment. H2O2‐mediated cellular degeneration was characterized using lactate dehydrogenase (LDH) release, changes in total glutathione, and a new marker of oxidative stress, 8‐epiprostaglandin F2α (8‐isoprostane). LY231617 attenuated H2O2‐mediated degeneration under a variety of exposure conditions, including a more clinically relevant posttreatment paradigm. Levels of 8‐isoprostane paralleled LDH release under various treatment paradigms of 100 μM H2O2± 5 μM drug. In contrast, despite affording significant protection, LY231617 had modest to no effects on cellular levels of glutathione. Taken together, these results are consistent with a membrane site of action for LY231617 and suggest that the compound affords cytoprotection via its antioxidant properties.

Redox targeting of LY231617, an antioxidant with potential use in the treatment of brain damage

Abstract Several brain-targeting chemical delivery systems (CDS) based on a dihydropyridine ⇌ pyridinium salt-type targetor were synthesized and evaluated for LY231617 (1), a di-tert-butylated phenolic amine antioxidant with potential use in the treatment of brain injuries. The dihydropyridine moiety was chemically attached to the amine (by either amide or various substituted carbamate linkages) or to the phenolic hydroxyl (by carboxylic ester linkage) functionalities of LY231617. In vitro stability and in vivo tissue distribution studies (in the rat) were performed with the novel derivatives. The results indicated that a simple amide-type CDS demonstrated efficient delivery of LY231617-targetor conjugate to the CNS. This derivative which contains the intact pharmacophore might possess intrinsic pharmacological antioxidant activity. Favorable in vitro properties suggested that a substituted carbamate-type CDS might be a better delivery modality for LY231617.