Chronic inhibition of superoxide dismutase produces apoptotic death of spinal neurons.

Abstract

Mutations in the gene for Cu/Zn superoxide dismutase (SOD1) have been detected in some families with an autosomal dominant form ofamyotrophic lateral sclerosis; these mutations appear to reduce the activity of this enzyme. To determine whether decreased SOD activity could contribute to motor neuron loss, SODi was inhibited chronically with either antisense oligodeoxynucleotides or diethyldithiocarbamate in spinal cord organotypic cultures. Chronic inhibition of SOD resulted in the apoptotic degeneration of spinal neurons, including motor neurons, over several weeks. Motor neuron loss was markedly potentiated by the inhibition of glutamate transport.In this paradigm, motor neuron toxicity could be entirely prevented by the antioxidant N-acetylcysteine and, to a lesser extent, by the non-N-methyl-D-aspartate glutamate receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-SH-2,3-benzodiazepine hydrochloride. These data support the hypothesis that the loss ofmotor neurons in familial amyotrophic lateral sclerosis could be due to a reduction in SODi activity, possibly potentiated by inefficient glutamate transport.Amyotrophic lateral sclerosis (ALS) is a motor neuron disease characterized clinically by progressive weakness, wasting of muscles, and spasticity due to the slow loss of lower motor neurons in the spinal cord and in the neocortex. Familial ALS represents 5-10o of all cases and is virtually indistinguishable clinically from the more common sporadic form. Mutations in the Cu/Zn superoxide dismutase (SOD1) gene on chromosome 21 have been detected in some families with the autosomal dominant form of familial AML (1, 2). Initial studies indicate that these mutations reduce the activity of SODi (2,3). This protein is a homodimeric metalloenzyme that catalyzes the dismutation of the superoxide anion to oxygen and hydrogen peroxide (4). Excessive levels of oxygen radicals, such as superoxide anion, have been implicated in neuronal injury, either directly or through the formation of more reactive oxygen species such as hydroxyl radicals (5-7). However, it has not been established that a chronic reduction in SODi activity diminishes the viability of spinal motor neurons. To investigate this, we have developed a model of slow toxicity in cultured organotypic spinal cord slices which combines the advantages of long-term survival with partially preserved synaptic connections (8). In this model, SOD activity was inhibited by the use of either antisense oligodeoxynucleotides (ODNs) or metal-chelating agents such as diethyldithiocarbamate (DDC), which has been shown to potentiate oxygen radical-induced toxicity in acute preparations (5). METHODSOrganotypic Spinal Cord Cultures. Eight-day-old neonatal rat pups were decapitated, and the spinal cords were rapidly harvested and cultured (8). Culture medium, including any added pharmacological agent, was changed twice weekly. With this technique, >95% of the explants can be maintained in culture for >3 months with excellent organotypic cellular organization...

Melatonin Prevents Death of Neuroblastoma Cells Exposed to the Alzheimer Amyloid Peptide

“…In line with such an interpretation, melatonin prevented cell death induced by inhibition of superoxide dismutase as well as A␤-induced lipid peroxidation. Inhibition of SOD by DDTC is a well established model of oxidative injury (Omar and Pappolla, 1993) that has been used previously to induce death of spinal cord neurons via an apoptotic pathway (Rothstein et al, 1994). These observations are also in agreement with the oxidative stress hypothesis of AD.…”

Evidence that 4-Hydroxynonenal Mediates Oxidative Stress-Induced Neuronal Apoptosis

“…An increased level of GSH in cells expressing Bcl-2 could account for our results because GSH plays a central role in the cellular metabolism of HNE (Ishikawa et al, 1986;Spitz et al, 1990). In studies of spinal cord organotypic cultures, Rothstein et al (1994) showed that downregulation of Cu/Zn-SOD resulted in neuronal apoptosis, which was inhibited by antioxidants. Troy et al (1996b) reported that downregulation of Cu/Zn-SOD in PC12 cells induces apoptosis, which can be prevented with inhibitors of nitric oxide synthase, suggesting the involvement of peroxynitrite.…”

Production and Actions of Superoxide in the Renal Medulla

“…Doses of DETC chosen for the present study were the doses shown to produce 80% to 100% inhibition of SOD activity in previous studies. 27 The dose of TEMPOL used in our experiments has been reported to induce N G -nitro-L-arginine methyl ester (L-NAME)-blockable reduction of arterial pressure. 14 A single dose of TEMPOL was used because its action duration is relatively long, as has been reported previously.…”