Disease mechanisms revealed by transcription profiling in SOD1-G93A transgenic mouse spinal cord.

Mutations of copper,zinc-superoxide dismutase (cu,zn SOD) are found in patients with a familial form of amyotrophic lateral sclerosis. When expressed in transgenic mice, mutant human cu,zn SOD causes progressive loss of motor neurons with consequent paralysis and death. Expression profiling of gene expression in SOD1-G93A transgenic mouse spinal cords indicates extensive glial activation coincident with the onset of paralysis at 3 months of age. This is followed by activation of genes involved in metal ion regulation (metallothionein-I, metallothionein-III, ferritin-H, and ferritin-L) at 4 months of age just prior to end-stage disease, perhaps as an adaptive response to the mitochondrial destruction caused by the mutant protein. Induction of ferritin-H and -L gene expression may also limit iron catalyzed hydroxyl radical formation and consequent oxidative damage to lipids, proteins, and nucleic acids. Thus, glial activation and adaptive responses to metal ion dysregulation are features of disease in this transgenic model of familial amyotrophic lateral sclerosis.

Protein oxidative damage in a transgenic mouse model of familial amyotrophic lateral sclerosis.

The Gly93-->Ala mutation in the Cu,Zn superoxide dismutase (Cu,Zn-SOD) gene (SOD1) found in some familial amyotrophic lateral sclerosis (FALS) patients has been shown to result in an aberrant increase in hydroxyl radical production by the mutant enzyme that may cause oxidative injury to spinal motor neurons. In the present study, we analyzed the extent of oxidative injury to lumbar and cervical spinal cord proteins in transgenic FALS mice that overexpress the SOD1 mutation [TgN(SOD1-G93A)G1H] in comparison with nontransgenic mice. Total protein oxidation was examined by spectrophotometric measurement of tissue protein carbonyl content by the dinitrophenylhydrazine (DNPH) assay. Four ages were investigated: 30 (pre-motor neuron pathology and clinical disease), 60 (after initiation of pathology, but pre-disease), 100 (approximately 50% loss of motor neurons and function), and 120 (near complete hindlimb paralysis) days. Protein carbonyl content in 30-day-old TgN(SOD1-G93A)G1H mice was twice as high as the level found in age-matched nontransgenic mice. However, at 60 and 100 days of age, the levels were the same. Then, between 100 and 120 days of age, the levels in the TgN(SOD1-G93A)G1H mice increased dramatically (557%) compared with either the nontransgenic mice or transgenic animals that overexpress the wild-type human Cu,Zn-SOD [TgN(SOD1)N29]. The 100-120-day increase in spinal cord protein carbonyl levels was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation and western blot immunoassay, which enabled the identification of heavily oxidized individual proteins using a monoclonal antibody against DNPH-derivatized proteins. One of the more heavily oxidized protein bands (14 kDa) was identified by immunoprecipitation as largely Cu,Zn-SOD. Western blot comparison of the extent of Cu,Zn-SOD protein carbonylation revealed that the level in spinal cord samples from 120-day-old TgN(SOD1-G93A)G1H mice was significantly higher than that found in age-matched nontransgenic or TgN(SOD1)N29 mice. These results suggest that the increased hydroxyl radical production associated with the G93A SOD1 mutation and/or lipid peroxidation-derived radical species (peroxyl or alkoxyl) causes extensive protein oxidative injury and that the Cu,Zn-SOD itself is a key target, which may compromise its antioxidant function.

Anticancer activity of glaucarubinone analogues.

A series of glaucarubinone analogues, obtained from natural sources as well as synthesized by us, were studied both in vitro and in vivo. The focus of the in vitro assessment was to define solid tumor-selective compounds by quantitating differential cytotoxic activity between murine and human solid tumor cells and either murine leukemia or normal cells. Subsequent in vivo studies were aimed at determining the therapeutic efficacy of these analogues against the murine models. Structure-activity analysis consequent to both the in vitro and in vivo studies demonstrated that few changes could be made in the parent glaucarubinone structure (outside of the C-15 position) without abrogating either cytotoxicity or potency. However, significant changes could be made at the C-15 position which modified, either enhanced or diminished, in vitro differential cytotoxicity, potency, human solid tumor selectively, and differential cytotoxicity to a MDR-expressing murine mammary tumor.

Comparative neuroprotective properties of the benzodiazepine receptor full agonist diazepam and the partial agonist PNU-101017 in the gerbil forebrain ischemia model.

Recent studies have demonstrated the neuroprotective properties of the novel imidazoquinoline benzodiazepine receptor partial agonist, PNU-101017, in the gerbil forebrain ischemia model. The compound effectively reduces delayed post-ischemic (5 min bilateral carotid occlusion) hippocampal CA1 neuronal degeneration even when its administration is withheld until 4 h after reperfusion and the effect is unrelated to hypothermia. The purpose of the present study was to determine the comparative abilities of PNU-101017 versus the full agonist diazepam to attenuate post-ischemic CA1 damage. Male gerbils were treated either 30 min before ischemia induction or immediately after reperfusion with an initial dose of PNU-101017 (30 mg/kg i.p.) or diazepam (10 mg/kg i.p.) with a second dose being given at 2 h after reperfusion. Possible hypothermic effects of either compound were prevented by external heating. In vehicle (0.05 N HCl)-treated gerbils, the loss of hippocampal CA1 neurons at 5 days was 85%. PNU-101017 pretreatment reduced the loss to 50% (p<0.05 vs. vehicle) whereas pretreatment with diazepam attenuated damage to only 17% (p<0.001 vs. vehicle). Delaying treatment with PNU-101017 until just after reperfusion still resulted in a reduction in CA1 degeneration statistically that was indistinguishable from that seen with pretreatment. In contrast, diazepam post-treatment did not significantly decrease CA1 neuronal loss. These results suggest that a benzodiazepine receptor partial agonist may have greater neuroprotective practicality than a full agonist for the treatment of global cerebral ischemia. The mechanistic basis for this difference may relate to the partially pro-excitatory neuronal response to endogenous GABA before and after neuronal insult.

Relationship of oxygen radical-induced lipid peroxidative damage to disease onset and progression in a transgenic model of familial ALS.

Transgenic mice that overexpress a mutated human CuZn superoxide dismutase (SOD1) gene (gly93-->ala) found in some patients with familial ALS (FALS) have been shown to develop motor neuron disease, as evidenced by motor neuron loss in the lumbar and cervical spinal regions and a progressive loss of voluntary motor activity. The mutant Cu,Zn SOD exhibits essentially normal dismutase activity, but in addition, generates toxic oxygen radicals as a result of an enhancement of a normally minor peroxidase reaction. In view of the likelihood that the manifestation of motor neuron disease in the FALS transgenic mice involves an oxidative injury mechanism, the present study sought to examine the extent of lipid peroxidative damage in the spinal cords of the TgN(SOD1-G93A)G1H mice over their life span compared to nontransgenic littermates or transgenic mice that overexpress the wild-type human Cu,Zn SOD (TgN(SOD1)N29). Lipid peroxidation was investigated in terms of changes in vitamin E and malondialdehyde (MDA) levels measured by HPLC methods and by MDA-protein adduct immunoreactivity. Four ages were investigated: 30 days (pre-motor neuron pathology and clinical disease); 60 days (after initiation of pathology, but predisease); 100 days (approximately 50% loss of motor neurons and function); and 120 days (near complete hindlimb paralysis). Compared to nontransgenic mice, the TgN(SOD1-G93A)G1H mice showed blunted accumulation of spinal cord vitamin E and higher levels of MDA (P < 0.05 at 30 and 60 days) over the 30-120 day time span. In the TgN(SOD1)N29 mice, levels of MDA at age 120 days were significantly lower than in either the TgN(SOD1-G93A)G1H or nontransgenic mice. MDA-protein adduct immunoreactivity was also significantly increased in the lumbar spinal cord at age 30, 100, and 120 days, and in the cervical cord at 100 and 120 days. The results clearly demonstrate an increase in spinal cord lipid peroxidation in the FALS transgenic model, which precedes the onset of ultrastructural or clinical motor neuron disease. However, the greatest intensity of actual motor neuronal lipid peroxidative injury is associated with the active phase of disease progression. These findings further support a role of oxygen radical-mediated motor neuronal injury in the pathogenesis of FALS and the potential benefits of antioxidant therapy.

Azulenyl nitrones: colorimetric detection of oxyradical end products and neuroprotection in the gerbil transient forebrain ischemia/reperfusion model.

We present analytical and neuroprotective data on a unique spin trapping agent derived from a novel chemical class known as an azulenyl nitrone (AZN). Based on Colorimetric properties, AZN was used to assess the formation of free radicals in a bilateral carotid occlusion (BCO) model in gerbils by monitoring the conversion of the nitrone to the aldehyde in affected tissue. In addition, AZN was tested as a neuroprotectant in this model regarding the preservation of CA1 pyramidal cells of the hippocampus following transient ischemia/reperfusion. AZN was electrochemically oxidized to give the aldehyde using an HPLC system with on line electrochemical oxidation. The oxidation potential associated with a 50% loss of AZN occurred at about 600 mV (half-wave potential versus palladium electrode). The major product detected as AZN oxidation occurred in an aqueous methanolic medium was the corresponding azulenyl aldehyde. Oxidation of AZN was inversely related to the formation of the aldehyde. Based on this test, we considered the in vivo conversion of AZN to aldehyde to be a measurement of oxidative stress in tissue. Results show that 0.3% of hippocampal AZN was converted to aldehyde in animals treated as shams. However, in gerbils subjected to a 7-min ischemic insult plus 7-min reperfusion, the conversion rate was about 3 times higher at 1.0%. In this model, surviving CA1 hippocampal neurons were counted from gerbils that were subjected to 7 mins of BCO followed by 5 days of reperfusion. In sham animals, about 89 cells were counted in a selected field of CA1 neurons. With injury, only 27 cells on average survived (70% loss) and were counted from this selected field. Under similar conditions and AZN treatment, 57 cells survived (36% loss). We conclude, therefore, that the demonstrated neuroprotection occurs because AZN neutralizes radicals which contribute to neuronal damage following ischemia/reperfusion.

Riluzole preserves motor function in a transgenic model of familial amyotrophic lateral sclerosis.

Riluzole was tested in a dose-ranging study for preservation of motor function in a transgenic mouse model of familial ALS. The model is based on expression of mutant human Cu,Zn superoxide dismutase in mouse brain and spinal cord. In contrast with the human ALS trials, in the mouse model, riluzole significantly preserved motor function as assessed by nightly running in a wheel. The effect of riluzole on motor performance was greater earlier in disease than later, suggesting that riluzole may have benefit for "quality-of-life" measures in ALS. Treatment with riluzole was initiated earlier in the transgenic model than in the human ALS trials, which may account for the significantly better outcome.

Neuroprotective properties of the benzodiazepine receptor, partial agonist PNU-101017 in the gerbil forebrain ischemia model.

PNU-101017 is a novel, imidazoquinoline amide and benzodiazepine receptor partial agonist that has high affinity for the GABAA receptor subtypes containing the alpha 1 and alpha 3 or alpha 5 subunits. At each of these receptors, the compound is a partial agonist with approximately 50% of the intrinsic activity of the full agonist diazepam. In view of the previously demonstrated anti-ischemic effects of some GABA agonists, the purpose of this study was to determine the ability of PNU-101017 to salvage selectively vulnerable neuronal populations in the gerbil forebrain ischemia model. In an initial set of experiments, male gerbils were pretreated 30 minutes before ischemia induction (5 minutes) with PNU-101017 (3, 10, or 30 mg/kg intraperitoneally) and again 2 hours after reperfusion. In vehicle (0.05 N HC1)-treated gerbils, the loss of hippocampal CA1 neurons at 5 days was 80%. PNU-101017 was shown to produce a dose-related increase in CA1 neuronal survival; at either 10 or 30 mg/kg, the loss of CA1 neurons was only 21% (P < 0.005 versus vehicle). A second experiment, examined the therapeutic window for PNU-101017 using the dose level of 30 mg/kg intraperitoneally. Administration of the first of two doses (2 hours apart) at the time of reperfusion resulted in an identical decrease in CA1 damage at 5 days to that seen with preischemic treatment (P < 0.003 versus vehicle). Even with a delay of the initial dosing until 4 hours after reperfusion, PNU-101017 reduced CA1 neuronal loss to only 32% (P < 0.01 versus vehicle). In a third experiment in which the duration of the ischemic insult was increased to 10 minutes and the brains were not analyzed until 28 days after ischemia, daily PNU-101017 dosing for the full 28 days still significantly preserved CA1 neurons, although less effectively than in the milder 5 minute-ischemia model. The loss of dopaminergic nigrostriatal neurons was also reduced. The neuroprotective effect of PNU-101017 was not associated with any overt CNS depression and it did not correlate with hypothermia. This benzodiazepine-receptor partial agonist may have potential for the treatment of global cerebral ischemia.

Pyrrolopyrimidines: novel brain-penetrating antioxidants with neuroprotective activity in brain injury and ischemia models.

A novel group of antioxidant compounds, the pyrrolopyrimidines, has been discovered recently. Many of these possess significantly improved oral bioavailability (56-70% in rats), increased efficacy and potency in protecting cultured neurons against iron-induced lipid peroxidative injury and as much as a 5-fold increase in brain uptake compared with the 21-aminosteroid antioxidant compound, tirilazad mesylate (U-74006F), described earlier. They appear to quench lipid peroxidation reactions by electron-donating and/or radical-trapping mechanisms. Several compounds in the series, such as U-101033E and U-104067F, demonstrate greater ability than tirilazad to protect the hippocampal CA1 region in the gerbil transient (5-min) forebrain ischemia model. Delaying treatment until 4 hr after the ischemic insult still results in significant CA1 neuronal protection. U-101033E is still effective in salvaging a portion of the CA1 neuronal population when the ischemic duration is extended to 10 min. In addition, U-101033E has been found to be protective in the context of focal cerebral ischemia, reducing infarct size in the mouse permanent middle cerebral artery occlusion model, in contrast to tirilazad which is minimally effective. These results suggest that antioxidant compounds with improved brain parenchymal penetration are better able to limit certain types of ischemic brain damage than those which are localized in the cerebral microvasculature. However, the activity of U-101033E in improving early post-traumatic recovery in mice subjected to severe concussive head injury is similar to that of tirilazad. Last, the oral bioavailability of many pyrrolopyrimidines suggests that they may be useful for certain chronic neurodegenerative disorders in which lipid peroxidation plays a role.

Neuroprotective effects of the novel brain-penetrating pyrrolopyrimidine antioxidants U-101033E and U-104067F against post-ischemic degeneration of nigrostriatal neurons.

A 10-min period of bilateral carotid occlusion (BCO)-induced forebrain ischemia in gerbils triggers a delayed retrograde degeneration of 35-40% of dopaminergic nigrostriatal (NS) neurons. The mechanism of the NS degeneration is believed to involve oxygen radical formation secondary to a postischemic increase in dopamine turnover (monoamine oxidase, MAO). If the oxygen radical increase is sufficiently severe, lipid peroxidative injury to the striatal NS terminals is followed by retrograde degeneration of the NS cell bodies. In the present study, we examined whether the novel brain-penetrating lipid antioxidant pyrrolopyrimidine, U-101033E, and its aromatized analog, U-104067F, could attenuate dopaminergic neurodegeneration in this model. Male Mongolian gerbils were dosed with U-101033E (1.5, 5, or 15 mg/kg, by mouth, twice daily) or U-104067F (5 or 15 mg/kg, by mouth, twice daily) for 27 days beginning on the day of the 10-min ischemic insult. Preservation of NS neurons was assessed by tyrosine hydroxylase immunohistochemistry at 28 days. In vehicle (40% hydroxypropyl-beta-cyclodextrin)-treated animals, there was a 42% loss of NS neurons. In contrast, gerbils that received 5 or 15 mg/kg U-101033E twice daily had only a 23% or 28% loss of NS neurons, respectively (P < 0.002 vs. vehicle). U-104067F showed little effect at sparing neurons at the 10 mg/kg dose, but did significantly attenuate neuronal loss to only 20% at the 30 mg/kg dose (P < 0.01 vs. vehicle). The results show that both the pyrrolopyrimidines (U-101033E and U-104067F) significantly attenuate the postischemic loss of NS dopaminergic neurons and further support the involvement of a dopamine metabolism-derived, oxygen radical-induced lipid peroxidative mechanism.