Striatal oxidative damage parallels the expression of a neurological phenotype in mice transgenic for the mutation of Huntington’s disease

We examined the degree of oxidative damage to the brain of mice transgenic for the mutation responsible for Huntington’s disease. We found that there is a progressive increase in striatal lipid peroxidation (LP), that parallels the worsening of the neurological phenotype. We consider that these transgenic mice may provide an interesting system to test treatments aimed at protecting cells from damage induced by free radicals.     

Age-dependent changes in nitric oxide synthase activity and protein expression in striata of mice transgenic for the Huntington’s disease mutation

Huntington’s disease (HD) is an autosomal hereditary neurodegenerative disorder caused by an abnormal expansion of the CAG repeats that code for a polyglutamine tract in a novel protein called huntingtin (htt). Both patients and experimental animals exhibit oxidative damage in specific areas of the brain, particularly the striatum. Nitric oxide (NO) is involved in many different physiological processes, and under pathological conditions it may promote oxidative damage through the formation of the highly reactive metabolite peroxynitrite; however, it may also play a role protecting cells from oxidative damage. We previously showed a correlation between the progression of the neurological phenotype and striatal oxidative damage in a line of transgenic mice, R6/1, which expresses a human mutated htt exon 1 with 116 CAG repeats. The purpose of the present work was to explore the participation of NO in the progressive oxidative damage that occurs in the striata of R6/1 mice. We analyzed the role of NO by measuring the activity of nitric oxide synthase (NOS) in the striata of transgenic and control mice at different ages. There was no difference in NOS activity between transgenic and wild-type mice at 11 weeks of age. In contrast, 19-week-old transgenic mice showed a significant increase in NOS activity, compared with same age controls. By 35 weeks of age, there was a decrease in NOS activity in transgenic mice when compared with wild-type controls. NOS protein expression was also determined in 11-, 19- and 35-week-old transgenic mice and wild-type littermates. Our results show increased neuronal NOS expression in 19-week-old transgenic mice, followed by a decreased level in 35-week-old mice, compared with controls, a phenomenon that parallels the changes in NOS enzyme activity. The present results suggest that NO is involved in the process leading to striatal oxidative damage and that it is associated with the onset of the progressive neurological phenotype in mice transgenic for the HD mutation.     

Anticonvulsant valproate reduces seizure-susceptibility in mutant Drosophila

Huntington's disease (HD) is an autosomal hereditary neurodegenerative disorder caused by an abnormal expansion of the CAG repeats that code for a polyglutamine tract in a novel protein called huntingtin (htt). Both patients and experimental animals exhibit oxidative damage in specific areas of the brain, particularly the striatum. Nitric oxide (NO) is involved in many different physiological processes, and under pathological conditions it may promote oxidative damage through the formation of the highly reactive metabolite peroxynitrite; however, it may also play a role protecting cells from oxidative damage. We previously showed a correlation between the progression of the neurological phenotype and striatal oxidative damage in a line of transgenic mice, R6/1, which expresses a human mutated htt exon 1 with 116 CAG repeats. The purpose of the present work was to explore the participation of NO in the progressive oxidative damage that occurs in the striata of R6/1 mice. We analyzed the role of NO by measuring the activity of nitric oxide synthase (NOS) in the striata of transgenic and control mice at different ages. There was no difference in NOS activity between transgenic and wild-type mice at 11 weeks of age. In contrast, 19-week-old transgenic mice showed a significant increase in NOS activity, compared with same age controls. By 35 weeks of age, there was a decrease in NOS activity in transgenic mice when compared with wild-type controls. NOS protein expression was also determined in 11-, 19- and 35-week-old transgenic mice and wild-type littermates. Our results show increased neuronal NOS expression in 19-week-old transgenic mice, followed by a decreased level in 35-week-old mice, compared with controls, a phenomenon that parallels the changes in NOS enzyme activity. The present results suggest that NO is involved in the process leading to striatal oxidative damage and that it is associated with the onset of the progressive neurological phenotype in mice transgenic for the HD mutation.     

Prevention of HIV-1 gp120-induced neuronal damage in the central nervous system of transgenic mice by the NMDA receptor antagonist memantine

To investigate the in vivo role of NMDA receptor stimulation in HIV-1-related CNS neurotoxicity, we evaluated the neuroprotective potential of the NMDA receptor antagonist memantine in transgenic mice which have gp120-induced CNS damage. Brains of mice treated chronically with memantine and of untreated controls were analysed for structural damage by laser scanning confocal microscopy of sections immunolabeled for microtubule-associated protein-2 (MAP-2) and synaptophysin. Qualitative and quantitative analysis of confocal images revealed that memantine treatment substantially decreased neuropathology in gp120 transgenic mice; this included statistically significant improvements in both dendritic and presynaptic terminal density. These results provide in vivo evidence that gp120 can activate neurotoxic pathways that can ultimately result in aberrant NMDA receptor stimulation and neuronal damage in the CNS. They also suggest that clinically tolerated NMDA receptor antagonists may be useful in the prevention of neuronal damage in HIV-1-infected patients.     

Copper-zinc superoxide dismutase prevents the early decrease of apurinic/apyrimidinic endonuclease and subsequent DNA fragmentation after transient focal cerebral ischemia in mice.

BACKGROUND AND PURPOSE: DNA damage and its repair mechanism are thought to be involved in ischemia/reperfusion injury in the brain. We have previously shown that apurinic/apyrimidinic endonuclease (APE/Ref-1), a multifunctional protein in the DNA base excision repair pathway, rapidly decreased after transient focal cerebral ischemia (FCI) before the peak of DNA fragmentation. To further investigate the role of reactive oxygen species in APE/Ref-1 expression in vivo, we examined the expression of APE/Ref-1 and DNA damage after FCI in wild-type and transgenic mice overexpressing copper-zinc superoxide dismutase. METHODS: Transgenic mice overexpressing copper-zinc superoxide dismutase and wild-type littermates were subjected to 60 minutes of transient FCI by intraluminal blockade of the middle cerebral artery. APE/Ref-1 protein expression was analyzed by immunohistochemistry and Western blot analysis. DNA damage was evaluated by gel electrophoresis and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling (TUNEL). RESULTS: A similar level of APE/Ref-1 was detected in the control brains from both groups. APE/Ref-1 was significantly reduced 1 hour after transient FCI in both groups, whereas the transgenic mice had less reduction than that seen in wild-type mice 1 and 4 hours after FCI. DNA laddering was detected 24 hours after FCI and was decreased in transgenic mice. Double staining with APE/Ref-1 and TUNEL showed that the neurons that lost APE/Ref-1 immunoreactivity became TUNEL positive. CONCLUSIONS: These results suggest that reactive oxygen species contribute to the early decrease of APE/Ref-1 and thereby exacerbate DNA fragmentation after transient FCI in mice.     

Oxidative injury to the endoplasmic reticulum in mouse brains after transient focal ischemia

Oxidative damage to the endoplasmic reticulum (ER) could be involved in ischemic neuronal cell death because this organelle is susceptible to reactive oxygen species. Using wild-type mice and copper/zinc-superoxide dismutase (SOD1) transgenic mice, we induced focal cerebral ischemia and compared neuronal degeneration and ER stress, that is, phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) and RNA-dependent protein kinase-like ER eIF2alpha kinase (PERK). We found that neurons with severe and prolonged phosphorylation of eIF2alpha and PERK underwent later degeneration, and that this was partially prevented by SOD1 overexpression. Signals for superoxide production and phospho-PERK were colocalized, which further indicates a pivotal role for superoxide in ER damage. We investigated the molecular mechanisms of oxidative ER stress and found that detachment of glucose-regulated protein 78 from PERK was the key step. We conclude that ER damage is involved in oxidative neuronal injury in the brain after ischemia/reperfusion.     

Immunocytochemical study on the distribution of nitrotyrosine in the brain of the transgenic mice expressing a human Cu/Zn SOD mutation

In the previous study, we reported increased NOS expression in the astrocytes in the spinal cord of the transgenic mice that are used as ALS animal model. In the present study, we performed immunocytochemical studies to investigate the changes of nitrotyrosine-immunoreactivity in the brains of the transgenic mice, and demonstrated in vivo evidence of peroxynitrite-mediated oxidative damage in the pathogenesis of ALS. In the spinal cord of the transgenic mice, immunocytochemistry showed intensely stained nitrotyrosine-IR glial cells with the appearance of astrocytes, but no nitrotyrosine-IR glial cells were observed in the spinal cord of the control mice. In the transgenic mice, nitrotyrosine-IR neurons were observed in the hypoglossal nucleus, lateral reticular nucleus, medullary reticular formation and cerebellar nuclei. Interestingly, nitrotyrosine-IR neurons were observed in the hippocampal formation and septal area of the transgenic mice. In the hippocampus, nitrotyrosine-IR neurons in the CA1 region showed intense staining, and the immunoreactivity was localized mainly in the pyramidal cell layer. Recent studies have shown that antioxidants and selective neuronal NOS inhibitor increase survival in the SOD1 transgenic mouse model of FALS. It is possible that therapy with these agents may slow the neurodegenerative process in human ALS, perhaps through reduction of nitrotyrosine formation.     

Therapeutic efficacy of EGb761 (Gingko biloba extract) in a transgenic mouse model of amyotrophic lateral sclerosis

EGb761 is a standardized extract of green Gingko biloba, which exerts protective effects against mitochondrial damage and oxidative stress. We examined whether oral administration of 0.022% or 0.045% EGb761 in the diet could impart neuroprotective effects in a transgenic mouse model (G93A) of amyotrophic lateral sclerosis (ALS). EGb761 significantly improved motor performance and survival, and protected against a loss of spinal-cord anterior motor horn neurons in male G93A mutant transgenic ALS mice, but not in littermate female mutant transgene mice. While EGb761 extended survival in littermate female G93A mice, significance was not reached. EGb761, however, significantly improved weight loss in both male and female transgenic ALS mice. These findings provide evidence for a gender-specific neuroprotective effect of EGb761 in a transgenic model of ALS and suggest that EGb761 may be a potential effective treatment in patients with ALS.     

N-acetyl-L-cysteine improves survival and preserves motor performance in an animal model of familial amyotrophic lateral sclerosis

Increasing evidence implicates oxidative damage as a major mechanism in the pathogenesis of amyotrophic lateral sclerosis (ALS). We examined the effect of preventative treatment with N-acetyl-L-cysteine (NAC), an agent that reduces free radical damage, in transgenic mice with a superoxide dismutase (SODI) mutation (G93A), used as an animal model of familial ALS. NAC was administered at 1% concentration in the drinking water from 4-5 weeks of age. The treatment caused a significantly prolonged survival and delayed onset of motor impairment in G93A mice treated with NAC compared to control mice. These results provide further evidence for the involvement of free radical damage in the G93A mice, and support the possibility that NAC, an over-the-counter antioxidant, could be explored in clinical trials for ALS.     

Maintenance of susceptibility to neurodegeneration following intrastriatal injections of quinolinic acid in a new transgenic mouse model of Huntington's disease

A transgenic mouse model of Huntington's disease (R6/1 and R6/2 lines) expressing exon 1 of the HD gene with 115-150 CAG repeats resisted striatal damage following injection of quinolinic acid and other neurotoxins. We examined whether excitotoxin resistance characterizes mice with mutant huntingtin transgenes. In a new transgenic mouse with 3 kb of mutant human huntingtin cDNA with 18, 46, or 100 CAG repeats, we found no change in susceptibility to intrastriatal injections of the excitotoxin quinolinic acid, compared to wild-type littermates. The new transgenic mice were injected with the same dose of quinolinic acid (30 nmol) as had been the R6 mice. Our findings highlight the importance of studying pathogenetic mechanisms in different transgenic models of a disease.     

Excitotoxic hippocampal injury is attenuated in thioredoxin transgenic mice.

Thioredoxin is a small, multifunctional protein with a redox-active disulfide/dithiol in the active site. Thioredoxin plays several important biologic roles both in intracellular and extracellular compartments with its redox-regulating and reactive oxygen intermediates scavenging activities. We assayed the seizure response and the excitotoxic hippocampal injury in thioredoxin transgenic and wild-type C57BL/6 mice. Seizure score after kainic acid treatment was significantly lower in thioredoxin transgenic mice. Seven days after kainic acid administration, the damage in the hippocampal CA1 and CA3 regions was significantly attenuated in thioredoxin transgenic mice. Thioredoxin and redox regulation play an important role in excitotoxic brain damage.     

HLA-DR3-DQ2 Mice Do Not Develop Ataxia in the Presence of High Titre Anti-gliadin Antibodies

Recently, it has been suggested that anti-gliadin antibodies (αGAb) may produce “gluten ataxia”, even in the absence of celiac disease enteropathy. αGAb are reportedly present in 12–50 % of patients with sporadic ataxia, but also in 12 % of the general population, such that the importance of αGAb as a cause of sporadic ataxia is not conclusively settled. We aimed to determine whether mice transgenic for HLA-DR3-DQ2 and immunised with gliadin to achieve high titres of αGAb would develop ataxia and/or cerebellar damage. From 6 weeks of age, HLA-DR3-DQ2 transgenic mice were immunised fortnightly with gliadin (n?=?10) or a saline control (n?=?6) in adjuvant. Serum titres were measured by αGAb enzyme-linked immunosorbent assay. At 24 weeks of age, mice were tested for locomotor function using the accelerating rotarod, ledged beam, ink-paw gait, and several neurological severity score subtests. Brains were then collected and processed for immunohistochemistry. Sections were analysed for lymphocytic infiltration, changes in morphology and Purkinje cell (PC) dendritic volume and the number of PCs counted via unbiased stereology. Gliadin-immunised mice developed high αGAb titres while controls did not. There was no statistically significant difference between the gliadin and sham-immunised HLA-DR3-DQ2 mice on any of the tests of motor coordination, in lymphocytic infiltration, PC number or in dendritic volume. High levels of αGAb are not sufficient to produce ataxia or cerebellar damage in HLA-DR3-DQ2 transgenic mice.     

Protection against hypoxic-ischemic injury in transgenic mice overexpressing Kir6.2 channel pore in forebrain

The role of the K-ATP channel pore-forming subunit Kir6.2 on protection from cerebral hypoxic-ischemic injury was assessed in transgenic mice overexpressing normal Kir6.2 or a dominant negative form (AFA) of this subunit in the forebrain. The resulting mice overexpress either the Kir6.2 or the AFA transgene mainly in the cerebral cortex and hippocampus. The Kir6.2 transgenic mice are resistant to hypoxic-ischemic injury showing a decreased region of cortical damage as compared to the dominant negative AFA and the wild-type mice. Moreover, the overexpression of Kir6.2 allowed an important silencing of the neurons present in forebrain regions thus protecting them from ischemic injury. Interestingly, the phenotype observed in Kir6.2 transgenic mice was observed without increased sulfonylurea binding. Taken together, these results indicate that the transgenic overexpression of Kir6.2 in forebrain significantly protects mice from hypoxic-ischemic injury and neuronal damage seen in stroke.     

Mouse model for ablation of proliferating microglia in acute CNS injuries

Activation of microglia, the primary immune effectors of the CNS and proinflammatory signaling, is a hallmark of brain damage. However, it remains controversial whether microglial cells have beneficial or detrimental functions in various neuropathological conditions. We report the generation of transgenic mice that express a mutant form of herpes simplex virus type 1 thymidine kinase (HSV-1 TK(mt-30)) driven by the myeloid-specific CD11b promoter. Using two paradigms of nervous system damage, hypoglossal nerve axotomy, and cortical stab injury, we show that specific ablation of proliferating microglia in CD11b-TK(mt-30) mice can be achieved by administration of ganciclovir. For example, after hypoglossal nerve injury, a 75% reduction in proliferating microglial cells was observed at the site of injury. The CD11b-TK(mt-30) transgenic mouse should provide a valuable tool for studying the role of microglia in CNS damage and repair.     

Increased neuronal damage and apoE immunoreactivity in human apolipoprotein E, E4 isoform-specific, transgenic mice after global cerebral ischaemia

Apolipoprotein E (apoE, protein; APOE, gene) is expressed as three isoforms in humans (E2, E3, E4). The APOE-epsilon4 allele is associated with a poor outcome in patients after head injury of which ischaemic brain damage is a contributor of mortality and morbidity. The aim of the study was to determine whether mice expressing human APOE-epsilon4 displayed more extensive ischaemic neuronal damage 72 h after transient global ischaemia compared with mice which express human APOE-epsilon3. APOE-epsilon3 and -epsilon4 transgenic mice, under the control of a human promoter, were used which express human APOE in neurons and glia. Ischaemic neuronal damage in the CA1 pyramidal cell layer in the APOE-epsilon4 transgenic mice was significantly greater than in the APOE-epsilon3 mice after global ischaemia (36.4+/-8.9%, 18.2+/-7.3%; P<0.05). This was associated with more extensive neuronal apoE immunoreactivity in the CA1 pyramidal cell layer in the APOE-epsilon4 transgenic mice compared with APOE-epsilon3 transgenic mice. In contrast, in the caudate nucleus, there were similar levels of ischaemic neuronal damage in the APOE-epsilon3 and -epsilon4 transgenic mice (39.2 +/-10.1%; 44.6+/-8.4%, P = 0.32). In the caudate, similar numbers of neurons were immunostained for apoE in the APOE-epsilon3 and -epsilon4 transgenic mice. The present study demonstrated that the APOE-epsilon4 allele is associated with an increased vulnerability of a specific brain region to the effects of global ischaemia, which is closely associated with an increase in neuronal apoE. The data extend previous work and are consistent with an association of the APOE-epsilon4 allele with a poor outcome after acute brain injury in humans.     

Elevated Seizure Threshold and Impaired Spatial Learning in Transgenic Mice with Putrescine Overproduction in the Brain

We have studied the role of putrescine by using transgenic mouse lines overexpressing the human ornithine decarboxylase gene in most of their tissues. The aberrant expression of the transgene is most strikingly manifested in the brain, leading to an increase of up to 20-fold in putrescine content. We report that the transgenic mice with grossly elevated putrescine in all brain regions analysed (cortex, striatum, hippocampus and cerebellum) showed a significantly elevated seizure threshold to chemical and electrical stimuli, and impaired performance in spatial learning and memory tests. The view that putrescine may be primarily responsible for these changes was supported by the fact that the concentrations of the major neurotransmitter amino acids, glutamate and GABA in the brain, were not changed in the transgenic animals, and by the finding that a further increase in brain putrescine, achieved by inhibition of the catabolism of l -ornithine, appeared to provide additional protection against electroshock-induced seizures. These results suggest that the commonly observed increase in ornithine decarboxylase activity and the massive increase in brain putrescine in connection with neuron damage is a neuroprotective measure rather than a cause of the damage.     

Methamphetamine toxicity is attenuated in mice that overexpress human manganese superoxide dismutase

We have investigated methamphetamine (MA) toxicity in transgenic mice that overexpress the human form of mitochondrial manganese superoxide dismutase (MnSOD). Our results reveal a significant reduction in the long-term depletion of striatal dopamine and protein oxidation following repeated administration of MA in transgenic vs. non-transgenic littermates. These findings support the notion that ROS contribute to MA-induced brain damage and suggest that mitochondria may play an important role in this form of neurodegeneration.     

Neuroprotective effects of HSP70 overexpression after cerebral ischaemia--an MRI study

Heat shock proteins (HSPs) have been reported to increase cell survival in response to a wide range of cellular challenges. However, the role of HSP70 overexpression is still a matter of debate, with some reports showing protection and others not. In order to resolve these discrepancies and further investigate the action of these proteins in vivo, transgenic mice overexpressing HSP70 have been compared to wild-type mice in a middle cerebral artery occlusion model of permanent cerebral ischaemia. Previously, the effect of HSP70 was assessed histologically postmortem. In this report, magnetic resonance imaging (MRI) was used to assess the mice in vivo after the onset of stroke. The lesion volume, as measured at 24 h using T(2)-weighted MRI, was significantly smaller in HSP70 transgenic mice compared with wild-type mice. The smaller lesion size in HSP70 transgenic mice could not be attributed to differences in vascular anatomy or in cerebral blood flow during occlusion. Additionally, the apparent diffusion coefficient showed different spatial and temporal patterns between the groups, suggesting that the damage within the lesion may be less severe for HSP70 transgenic mice. Thus, we conclude that overexpression of HSP70 reduces the overall lesion size and may also limit the tissue damage within the lesion.     

Partial deficiency of manganese superoxide dismutase exacerbates a transgenic mouse model of amyotrophic lateral sclerosis

The pathogenesis of neuronal cell death as a consequence of mutations in copper/zinc superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis may involve oxidative damage and mitochondrial dysfunction. We examined whether crossing transgenic mice with the G93A SOD1 mutation with transgenic mice with a partial depletion of manganese superoxide dismutase (SOD2) would affect the disease phenotype. Compared with G93A mice alone, the mice with partial deficiency of SOD2 and the G93A SOD1 mutation showed a significant decrease in survival and an exacerbation of motor deficits detected by rotorod testing. There was a significant exacerbation of loss of motor neurons and substantia nigra dopaminergic neurons in the G93A mice with a partial deficiency of SOD2 compared with G93A mice at 110 days. Microvesiculation of large motor neurons was more prominent in the G93A mice with a partial deficiency of SOD2 compared with G93A mice at 90 days. These findings provide further evidence that both oxidative damage and mitochondrial dysfunction may play a role in the pathogenesis of motor neuron death associated with mutations in SOD1.     

Lipoic acid improves survival in transgenic mouse models of Huntington's disease.

There is substantial evidence implicating excitotoxicity and oxidative damage in the pathogenesis of Huntington's disease (HD). We therefore examined whether the antioxidants 2-sulpho-tert-phenyibutyinitrone (S-PBN) and alpha-lipoic acid could exert neuroprotective effects in transgenic mouse models of HD. S-PBN showed no effects on either weight loss or survival in the R6/2 transgenic HD mice. alpha-Lipoic acid produced significant increases in survival in both R6/2 and N171-82Q transgenic mouse models of HD. These findings suggest that alpha-lipoic acid might have beneficial effects in HD patients.