Ole A. Andreassen

Mice Deficient in Cellular Glutathione Peroxidase Show Increased Vulnerability to Malonate, 3-Nitropropionic Acid, and 1-Methyl-4-Phenyl-1,2,5,6-Tetrahydropyridine

Glutathione peroxidase (GSHPx) is a critical intracellular enzyme involved in detoxification of hydrogen peroxide (H2O2) to water. In the present study we examined the susceptibility of mice with a disruption of the glutathione peroxidase gene to the neurotoxic effects of malonate, 3-nitropropionic acid (3-NP), and 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). Glutathione peroxidase knock-out mice showed no evidence of neuropathological or behavioral abnormalities at 2–3 months of age. Intrastriatal injections of malonate resulted in a significant twofold increase in lesion volume in homozygote GSHPx knock-out mice as compared to both heterozygote GSHPx knock-out and wild-type control mice. Malonate-induced increases in conversion of salicylate to 2,3- and 2,5-dihydroxybenzoic acid, an index of hydroxyl radical generation, were greater in homozygote GSHPx knock-out mice as compared with both heterozygote GSHPx knock-out and wild-type control mice. Administration of MPTP resulted in significantly greater depletions of dopamine, 3,4-dihydroxybenzoic acid, and homovanillic acid in GSHPx knock-out mice than those seen in wild-type control mice. Striatal 3-nitrotyrosine (3-NT) concentrations after MPTP were significantly increased in GSHPx knock-out mice as compared with wild-type control mice. Systemic 3-NP administration resulted in significantly greater striatal damage and increases in 3-NT in GSHPx knock-out mice as compared to wild-type control mice. The present results indicate that a knock-out of GSHPx may be adequately compensated under nonstressed conditions, but that after administration of mitochondrial toxins GSHPx plays an important role in detoxifying increases in oxygen radicals.

NEUROPROTECTIVE EFFECTS OF CREATINE IN A TRANSGENIC ANIMAL MODEL OF AMYOTROPHIC LATERAL SCLEROSIS

Mitochondria are particularly vulnerable to oxidative stress, and mitochondrial swelling and vacuolization are among the earliest pathologic features found in two strains of transgenic amyotrophic lateral sclerosis (ALS) mice with SOD1 mutations. Mice with the G93A human SOD1 mutation have altered electron transport enzymes, and expression of the mutant enzyme in vitro results in a loss of mitochondrial membrane potential and elevated cytosolic calcium concentration. Mitochondrial dysfunction may lead to ATP depletion, which may contribute to cell death. If this is true, then buffering intracellular energy levels could exert neuroprotective effects. Creatine kinase and its substrates creatine and phosphocreatine constitute an intricate cellular energy buffering and transport system connecting sites of energy production (mitochondria) with sites of energy consumption, and creatine administration stabilizes the mitochondrial creatine kinase and inhibits opening of the mitochondrial transition pore. We found that oral administration of creatine produced a dose-dependent improvement in motor performance and extended survival in G93A transgenic mice, and it protected mice from loss of both motor neurons and substantia nigra neurons at 120 days of age. Creatine administration protected G93A transgenic mice from increases in biochemical indices of oxidative damage. Therefore, creatine administration may be a new therapeutic strategy for ALS.

Increased oxidative damage to DNA in a transgenic mouse model of Huntington's disease

Mitochondrial dysfunction and oxidative damage may play a role in the pathogenesis of Huntingtons disease (HD). We examined concentrations of 8‐hydroxy‐2‐deoxyguanosine (OH8dG), a well‐established marker of oxidative damage to DNA, in a transgenic mouse model of HD (R6/2). Increased concentrations of OH8dG were found in the urine, plasma and striatal microdialysates of the HD mice. Increased concentrations were also observed in isolated brain DNA at 12 and 14 weeks of age. Immunocytochemistry showed increased OH8dG staining in late stages of the illness. These results suggest that oxidative damage may play a role in the pathogenesis of neuronal degeneration in the R6/2 transgenic mouse model of HD.

Nonlinear decrease over time in N-acetyl aspartate levels in the absence of neuronal loss and increases in glutamine and glucose in transgenic Huntington's disease mice.

Abstract: Mice transgenic for exon I of mutant huntingtin, with 141 CAG repeats, exhibit a profound symptomatology characterized by weight loss, motor disorders, and early death. We performed longitudinal analysis of metabolite levels in these mice using NMR spectroscopy in vivo and in vitro. These mice exhibited a large (53%), nonlinear drop in in vivo N‐acetyl aspartate (NAA) levels over time, commencing at ∼6 weeks of age, coincident with onset of symptoms. These drops in NAA levels occurred in the absence of neuronal death as measured by postmortem Nissl staining and neuronal counting but in the presence of nuclear inclusion bodies. In addition to decreased NAA, these mice showed a large elevation of glucose in the brain (600%) consistent with a diabetic profile and elevations in blood glucose levels both before and after glucose loading. In vitro NMR analysis revealed significant increases in glutamine (100%), taurine (95%) cholines (200%), and scyllo‐inositol (333%) and decreases in glutamate (24%) and succinate (47%). These results lead to two conclusions. NAA is reflective of the health of neurons and thus is a noninvasive marker, with a temporal progression similar to nuclear inclusion bodies and symptoms, of neuronal dysfunction in transgenic mice. Second, the presence of elevated glutamine is evidence of a profound metabolic defect. We present arguments that the elevated glutamine results from a decrease in neuronal‐glial glutamate‐glutamine cycling and a decrease in glutaminase activity.

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 (SOD1) 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.

Mice Deficient in Group IV Cytosolic Phospholipase A2 Are Resistant to MPTP Neurotoxicity

Abstract: Phospholipase A2 (PLA2) enzymes are critical regulators of prostaglandin and leukotriene synthesis, and they may also play an important role in the generation of intracellular free radicals. The group IV cytosolic form of phospholipase A2 (cPLA2) is regulated by changes in intracellular calcium concentration, and the enzyme preferentially acts to release arachidonic acid esterified at the sn‐2 position of phospholipids. We examined the susceptibility of mice carrying a targeted mutation of the cPLA2 gene to 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced neurotoxicity. Mutant mice have no functional cPLA2 activity. Mice that were homozygous for the mutation (cPLA2−/−) were significantly resistant to MPTP‐induced dopamine depletion as compared with littermate control (cPLA2+/+) and heterozygous mice (cPLA2+/−). These findings provide evidence that cPLA2 plays a role in MPTP neurotoxicity and suggest that cPLA2 may play a role in the development of Parkinsons disease in humans.

Huntington's disease of the endocrine pancreas: insulin deficiency and diabetes mellitus due to impaired insulin gene expression.

In a transgenic mouse model of the neurodegenerative disorder Huntingtons disease (HD), age-dependent neurologic defects are accompanied by progressive alterations in glucose tolerance that culminate in the development of diabetes mellitus and insulin deficiency. Pancreatic islets from HD transgenic mice express reduced levels of the pancreatic islet hormones insulin, somatostatin, and glucagon and exhibit intrinsic defects in insulin production. Intranuclear inclusions accumulate with aging in transgenic pancreatic islets, concomitant with the decline in glucose tolerance. HD transgenic mice develop an age-dependent reduction of insulin mRNA expression and diminished expression of key regulators of insulin gene transcription, including the pancreatic homeoprotein PDX-1, E2A proteins, and the coactivators CBP and p300. Disrupted expression of a subset of transcription factors in pancreatic beta cells by a polyglutamine expansion tract in the huntingtin protein selectively impairs insulin gene expression to result in insulin deficiency and diabetes. Selective dysregulation of gene expression in triplet repeat disorders provides a mechanism for pleiotropic cellular dysfunction that restricts the toxicity of ubiquitously expressed proteins to highly specialized subpopulations of cells.

Mice with a partial deficiency of manganese superoxide dismutase show increased vulnerability to the mitochondrial toxins malonate, 3-nitropropionic acid, and MPTP.

There is substantial evidence implicating mitochondrial dysfunction and free radical generation as major mechanisms of neuronal death in neurodegenerative diseases. The major free radical scavenging enzyme in mitochondria is manganese superoxide dismutase (SOD2). In the present study we investigated the susceptibility of mice with a partial deficiency of SOD2 to the neurotoxins 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP), 3-nitropropionic acid (3-NP), and malonate, which are commonly used animal models of Parkinsons and Huntingtons disease. Heterozygous SOD2 knockout (SOD2(+/-)) mice showed no evidence of neuropathological or behavioral abnormalities at 2-4 months of age. Compared to littermate wild-type mice, mice with partial SOD2 deficiency showed increased vulnerability to dopamine depletion after systemic MPTP treatment and significantly larger striatal lesions produced by both 3-NP and malonate. SOD2(+/-) mice also showed an increased production of hydroxyl radicals after malonate injection measured with the salicylate hydroxyl radical trapping method. These results provide further evidence that reactive oxygen species play an important role in the neurotoxicity of MPTP, malonate, and 3-NP. These findings show that a subclinical deficiency in a free radical scavenging enzyme may act in concert with environmental toxins to produce selective neurodegeneration.

Increases in cortical glutamate concentrations in transgenic amyotrophic lateral sclerosis mice are attenuated by creatine supplementation.

Several lines of evidence implicate excitotoxic mechanisms in the pathogenesis of amyotrophic lateral sclerosis (ALS). Transgenic mice with a superoxide dismutase mutation (G93A) have been utilized as an animal model of familial ALS (FALS). We examined the cortical concentrations of glutamate using in vivo microdialysis and in vivo nuclear magnetic resonance (NMR) spectroscopy, and the effect of long‐term creatine supplementation. NMDA‐stimulated and ltrans‐pyrrolidine‐2,4‐dicarboxylate (LTPD)‐induced increases in glutamate were significantly higher in G93A mice compared with littermate wild‐type mice at 115 days of age. At this age, the tissue concentrations of glutamate were also significantly increased as measured with NMR spectroscopy. Creatine significantly increased longevity and motor performance of the G93A mice, and significantly attenuated the increases in glutamate measured with spectroscopy at 75 days of age, but had no effect at 115 days of age. These results are consistent with impaired glutamate transport in G93A transgenic mice. The beneficial effect of creatine may be partially mediated by improved function of the glutamate transporter, which has a high demand for energy and is susceptible to oxidative stress.

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 Huntingtons disease (HD). We therefore examined whether the antioxidants 2-sulpho-tert-phenyibutyinitrone (S-PBN) and α-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. α-Lipoic acid produced significant increases in survival in both R6/2 and N171-82Q transgenic mouse models of HD. These findings suggest that α-lipoic acid might have beneficial effects in HD patients.