Susan E. Browne

Evidence of increased oxidative damage in both sporadic and familial amyotrophic lateral sclerosis.

Abstract: Some cases of autosomal dominant familial amyotrophic lateral sclerosis (FALS) are associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1), suggesting that oxidative damage may play a role in ALS pathogenesis. To further investigate the biochemical features of FALS and sporadic ALS (SALS), we examined markers of oxidative damage to protein, lipids, and DNA in motor cortex (Brodmann area 4), parietal cortex (Brodmann area 40), and cerebellum from control subjects, FALS patients with and without known SOD mutations, SALS patients, and disease controls (Picks disease, progressive supranuclear palsy, diffuse Lewy body disease). Protein carbonyl and nuclear DNA 8‐hydroxy‐2′‐deoxyguanosine (OH8dG) levels were increased in SALS motor cortex but not in FALS patients. Malondialdehyde levels showed no significant changes. Immunohistochemical studies showed increased neuronal staining for hemeoxygenase‐1, malondialdehyde‐modified protein, and OH8dG in both SALS and FALS spinal cord. These studies therefore provide further evidence that oxidative damage may play a role in the pathogenesis of neuronal degeneration in both SALS and FALS.

Mitochondrial α-Ketoglutarate Dehydrogenase Complex Generates Reactive Oxygen Species

Mitochondria-produced reactive oxygen species (ROS) are thought to contribute to cell death caused by a multitude of pathological conditions. The molecular sites of mitochondrial ROS production are not well established but are generally thought to be located in complex I and complex III of the electron transport chain. We measured H2O2 production, respiration, and NADPH reduction level in rat brain mitochondria oxidizing a variety of respiratory substrates. Under conditions of maximum respiration induced with either ADP or carbonyl cyanide p-trifluoromethoxyphenylhydrazone,α-ketoglutarate supported the highest rate of H2O2 production. In the absence of ADP or in the presence of rotenone, H2O2 production rates correlated with the reduction level of mitochondrial NADPH with various substrates, with the exception of α-ketoglutarate. Isolated mitochondrial α-ketoglutarate dehydrogenase (KGDHC) and pyruvate dehydrogenase (PDHC) complexes produced superoxide and H2O2. NAD+ inhibited ROS production by the isolated enzymes and by permeabilized mitochondria. We also measured H2O2 production by brain mitochondria isolated from heterozygous knock-out mice deficient in dihydrolipoyl dehydrogenase (Dld). Although this enzyme is a part of both KGDHC and PDHC, there was greater impairment of KGDHC activity in Dld-deficient mitochondria. These mitochondria also produced significantly less H2O2 than mitochondria isolated from their littermate wild-type mice. The data strongly indicate that KGDHC is a primary site of ROS production in normally functioning mitochondria.

Oxidative Stress in Huntington's Disease

It has been five years since the elucidation of the genetic mutation underlying the pathogenesis of Huntingtons disease (HD) (97), however the precise mechanism of the selective neuronal death it propagates still remains an enigma. Several different etiological processes may play roles, and strong evidence from studies in both humans and animal models suggests the involvement of energy metabolism dysfunction, excitotoxic processes, and oxidative stress. Importantly, the recent development of transgenic mouse models of HD led to the identification of neuronal intranuclear inclusion bodies in affected brain regions in both mouse models and in HD brain, consisting of protein aggregates containing fragments of mutant huntingtin protein. These observations opened new avenues of investigation into possible huntingtin protein interactions and their putative pathogenetic sequelae. Amongst these studies, findings of elevated levels of oxdative damage products such as malondialdehyde, 8‐hydroxy‐deoxyguanosine, 3‐nitrotyrosine and heme oxygenase in areas of degeneration in HD brain, and of increased free radical production in animal models, indicate the involvement of oxidative stress either as a causative event, or as a secondary constituent of the cell death cascade in the disease. Here we review the evidence for oxidative damage and potential mechanisms of neuronal death in HD.

Inhibition of neuronal nitric oxide synthase by 7-nitroindazole protects against MPTP-induced neurotoxicity in mice.

Abstract: Several studies suggest that nitric oxide (NO•) contributes to cell death following activation of NMDA receptors in cultured cortical, hippocampal, and striatal neurons. In the present study we investigated whether 7‐nitroindazole (7‐NI), a specific neuronal nitric oxide synthase inhibitor, can block dopaminergic neurotoxicity seen in mice after systemic administration of MPTP. 7‐NI dose‐dependently protected against MPTP‐induced dopamine depletions using two different dosing regimens of MPTP that produced varying degrees of dopamine depletion. At 50 mg/kg of 7‐NI there was almost complete protection in both paradigms. Similar effects were seen with MPTP‐induced depletions of both homovanillic acid and 3,4‐dihydroxyphenylacetic acid. 7‐NI had no significant effect on dopamine transport in vitro and on monoamine oxidase B activity both in vitro and in vivo. One mechanism by which NO• is thought to mediate its toxicity is by interacting with superoxide radical to form peroxynitrite (ONOO−), which then may nitrate tyrosine residues. Consistent with this hypothesis, MPTP neurotoxicity in mice resulted in a significant increase in the concentration of 3‐nitrotyrosine, which was attenuated by treatment with 7‐NI. Our results suggest that NO• plays a role in MPTP neurotoxicity, as well as novel therapeutic strategies for Parkinsons disease.

The Energetics of Huntington's Disease*

Huntingtons disease (HD) is a hereditary neurodegenerative disorder that gradually robs sufferers of the ability to control movements and induces psychological and cognitive impairments. This devastating, lethal disease is one of several neurological disorders caused by trinucleotide expansions in affected genes, including spinocerebellar ataxias, dentatorubral-pallidoluysian atrophy, and spinal bulbar muscular atrophy. HD symptoms are associated with region-specific neuronal loss within the central nervous system, but to date the mechanism of this selective cell death remains unknown. Strong evidence from studies in humans and animal models suggests the involvement of energy metabolism defects, which may contribute to excitotoxic processes, oxidative damage, and altered gene regulation. The development of transgenic mouse models expressing the human HD mutation has provided novel opportunities to explore events underlying selective neuronal death in HD, which has hitherto been impossible in humans. Here we discuss how animal models are redefining the role of energy metabolism in HD etiology.

Oxidative damage to mitochondrial DNA in Huntington's disease parietal cortex

Oxidative damage to DNA may play a role in both normal aging and in neurodegenerative diseases. Using a sensitive high-performance liquid chromatography (HPLC) assay, we examined concentrations of 8-hydroxy-2-deoxyguanosine (OH8dG) in mitochondrial DNA (mtDNA) isolated from frontal and parietal cerebral cortex and from cerebellum in 22 Huntingtons disease (HD) patients and 15 age-matched normal controls. A significant increase in OH8dG in mtDNA of parietal cortex was found in HD patients as compared with controls, while there were no significant changes in frontal cortex or cerebellum. The present findings are consistent with regionally specific oxidative damage in HD, which may be a further evidence of a metabolic defect.

Chemotherapy for the Brain: The Antitumor Antibiotic Mithramycin Prolongs Survival in a Mouse Model of Huntington's Disease

Huntingtons disease (HD) is a fully penetrant autosomal-dominant inherited neurological disorder caused by expanded CAG repeats in the Huntingtin gene. Transcriptional dysfunction, excitotoxicity, and oxidative stress have all been proposed to play important roles in the pathogenesis of HD. This study was designed to explore the therapeutic potential of mithramycin, a clinically approved guanosine-cytosine-rich DNA binding antitumor antibiotic. Pharmacological treatment of a transgenic mouse model of HD (R6/2) with mithramycin extended survival by 29.1%, greater than any single agent reported to date. Increased survival was accompanied by improved motor performance and markedly delayed neuropathological sequelae. To identify the functional mechanism for the salubrious effects of mithramycin, we examined transcriptional dysfunction in R6/2 mice. Consistent with transcriptional repression playing a role in the pathogenesis of HD, we found increased methylation of lysine 9 in histone H3, a well established mechanism of gene silencing. Mithramycin treatment prevented the increase in H3 methylation observed in R6/2 mice, suggesting that the enhanced survival and neuroprotection might be attributable to the alleviation of repressed gene expression vital to neuronal function and survival. Because it is Food and Drug Administration-approved, mithramycin is a promising drug for the treatment of HD.

A pivotal role of matrix metalloproteinase-3 activity in dopaminergic neuronal degeneration via microglial activation

Recent studies have demonstrated that activated microglia play an important role in dopamine (DA) neuronal degeneration in Parkinson disease (PD) by generating NADPH‐oxidase (NADPHO)‐derived superoxide. However, the molecular mechanisms that underlie microglial activation in DA cell death are still disputed. We report here that matrix metalloproteinase‐3 (MMP‐3) was newly induced and activated in stressed DA cells, and the active form of MMP‐3 (actMMP‐3) was released into the medium. The released actMMP‐3, as well as catalytically active recombinant MMP‐3 (cMMP‐3) led to microglial activation and superoxide generation in microglia and enhanced DA cell death. cMMP‐3 caused DA cell death in mesencephalic neuron‐glia mixed culture of wild‐type (WT) mice, but this was attenuated in the culture of NADPHO subunit null mice (gp91phox‐/‐), suggesting that NADPHO mediated the cMMP‐3‐induced microglial production of superoxide and DA cell death. Furthermore, in the N‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐injected animal model of PD, nigrostriatal DA neuronal degeneration, microglial activation, and superoxide generation were largely attenuated in MMP‐3‐/‐mice. These results indicate that actMMP‐3 released from stressed DA neurons is responsible for microglial activation and generation of NADPHO‐derived superoxide and eventually enhances nigrostriatal DA neuronal degeneration. Our results could lead to a novel therapeutic approach to PD. Kim, Y. S., Choi, D. H., Block, M. L., Lorenzl, S., Yang, L., Kim, Y. J., Sugama, S., Cho, B. P., Ywang, O., Browne, S. E., Kim, S. Y., Hong, J.‐S., Beal, M. F., Jon, T. H. A pivotal role of matrix metalloproteinase‐3 activity in dopaminergic neuronal degeneration via microglial activation. FASEB J. 21, 179–187 (2007)

Minocycline enhances MPTP toxicity to dopaminergic neurons.

Minocycline has been shown previously to have beneficial effects against ischemia in rats as well as neuroprotective properties against excitotoxic damage in vitro, nigral cell loss via 6‐hydroxydopamine, and to prolong the life‐span of transgenic mouse models of Huntingtons disease (HD) and amyotrophic lateral sclerosis (ALS). We investigated whether minocycline would protect against toxic effects of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), a toxin that selectively destroys nigrostriatal dopaminergic (DA) neurons and produces a clinical state similar to Parkinsons disease (PD) in rodents and primates. We found that although minocycline inhibited microglial activation, it significantly exacerbated MPTP‐induced damage to DA neurons. We present evidence suggesting that this effect may be due to inhibition of DA and 1‐methyl‐4‐phenylpridium (MPP+) uptake into striatal vesicles.

Metabolic dysfunction in familial, but not sporadic, amyotrophic lateral sclerosis

Abstract: Autosomal dominant familial amyotrophic lateral sclerosis (FALS) is associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Previous studies have implicated the involvement of metabolic dysfunction in ALS pathogenesis. To further investigate the biochemical features of FALS and sporadic ALS (SALS), we examined SOD activity and mitochondrial oxidative phosphorylation enzyme activities in motor cortex (Brodmann area 4), parietal cortex (Brodmann area 40), and cerebellum from control subjects, FALS patients with and without known SOD mutations, SALS patients, and disease controls (Picks disease, progressive supranuclear palsy, diffuse Lewy body disease). Cytosolic SOD activity, predominantly Cu/Zn SOD, was decreased ∼50% in all regions in FALS patients with SOD mutations but was not significantly altered in other patient groups. Marked increases in complex I and II–III activities were seen in FALS patients with SOD mutations but not in SALS patients. We also measured electron transport chain enzyme activities in a transgenic mouse model of FALS. Complex I activity was significantly increased in the forebrain of 60‐day‐old G93A transgenic mice overexpressing human mutant SOD1, relative to levels in transgenic wild‐type animals, supporting the hypothesis that the motor neuron disorder associated with SOD1 mutations involves a defect in mitochondrial energy metabolism.