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Josée Roy,1 Sandra Minotti,1 Lichun Dong,2 Denise A. Figlewicz2,3 and Heather D. Durham1
The Journal of Neurosciences, December 1, 1998. 18(23):9673-9684
Presenter: R. Mishra
Presenter's Summary:
Amyotropic lateral sclerosis (ALS) is an adult-onset disease involving loss of motor neurons in the cerebral cortex, brainstem, and spinal cord. Mutations in the gene encoding the enzyme Cu/Zn-superoxide dismutase (SOD-1) has been found in > 10% of the ALS. Several previous studies have supported the importance of excitotoxic mechanisms in motor neuron disease. These neurons have very high levels of glutamatergic input and are extremely vulnerable to excitotoxic cell death. In ALS patients, there is increase in glutamate levels both in serum and CSF, where as glutamate transport is decreased in brain and spinal cord and glutamate receptor activation is increased. Glutamate receptor activation can result in increased intracellular calcium ions, and generation of reactive oxygen species. The results of these studies clearly indicate the role of calcium, in ALS.
Key findings:
1. A general antagonist of AMPA/Kainate receptors (CNQX) and a highly specific antagonist of calcium permeable AMPA receptors (Joro spider toxin) significantly reduced formation of SOD-1 proteinaceous aggregates and prevented death of motor neurons expressing SOD-1 mutants.
2. When Nifedipine, L-type voltage-gated calcium channel blocker was used, a partial protection of motor neurons was also observed implicating role of calcium entry through voltage gated calcium channels.
3. Highly significant neuroprotection was observed when calcium-binding protein, calbindin-28K was co-expressed in SOD-1 mutants.
4. No significant neuroprotection was observed by increasing the intracellular glutathione levels or treatment with the free radical spin trapping reagent, N-tert-butyl-alpha-phenylnitrone. This reagent is commonly used in spin-trapping experiments and can be easily measured by ESR spectroscopy or radio labelling.
In conclusion, the results of these studies provide direct evidence for the calcium entry during neurotransmission, and if cytosolic calcium binding protein is not present in sufficient quantity, then this might lead motor neurons to disease such as amyotropic lateral sclerosis.
General Comments:
Well controlled experiments, describing full details of methods, were carried out in order to test the hypothesis that dysregulation of calcium homostasis plays a major role in the death of motor neurons. There are no obvious flaws in the experiments. The authors clearly indicate the cell death by other mechanisms than simply by formation of free radicals.
Why is this paper important?
In this paper, the authors present evidence in support of a novel mechanism for the cell death in ALS. Lack of calcium binding protein rather than free radicals has been implicated, as both free radical trapping agent and increased glutathione were unable to protect the motor neurons.
Questions Answered:
1. Does the blockade of AMPA/Kainate receptors prevent death of motor neurons?
2. Is the formation of SOD-1 aggregates reduced by ionotropic glutamate receptors?
3. Does the inhibition of L-type voltage-gated calcium channels partially prevent toxicity of mutant SOD-1?
4. Does the co-expression of Calbindin-D28K prevent toxicity of mutant SOD-1 in motor neurons?
Author's Abstract:
Montreal Neurological Institute and Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada H3A 2B4.
[Record supplied by publisher]
Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a subset of familial cases of amyotrophic lateral sclerosis. Using a primary culture model, we have demonstrated that normally nontoxic glutamatergic input, particularly via calcium-permeable AMPA/kainate receptors, is a major factor in the vulnerability of motor neurons to the toxicity of SOD-1 mutants. Wild-type and mutant (G41R, G93A, or N139K) human SOD-1 were expressed in motor neurons of dissociated cultures of murine spinal cord by intranuclear microinjection of plasmid expression vector. Both a general antagonist of AMPA/kainate receptors (CNQX) and a specific antagonist of calcium-permeable AMPA receptors (joro spider toxin) reduced formation of SOD-1 proteinaceous aggregates and prevented death of motor neurons expressing SOD-1 mutants. Partial protection was obtained by treatment with nifedipine, implicating Ca2+ entry through voltage-gated calcium channels as well as glutamate receptors in potentiating the toxicity of mutant SOD-1 in motor neurons. Dramatic neuroprotection was obtained by coexpressing the calcium-binding protein calbindin-D28k but not by increasing intracellular glutathione levels or treatment with the free radical spin trap agent, N-tert-butyl-alpha-phenylnitrone. Thus, generalized oxidative stress could have contributed in only a minor way to death of motor neurons expressing the mutant SOD-1. These studies demonstrated that the toxicity of these mutants is calcium-dependent and provide direct evidence that calcium entry during neurotransmission, coupled with deficiency of cytosolic calcium-binding proteins, is a major factor in the preferential vulnerability of motor neurons to disease.
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