Transgenic mice in the study of ALS: the role of neurofilaments

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurological disorder of multiple etiologies that affects primarily motor neurons in the brain and spinal cord. Abnormal accumulations of neurofilaments (NFs) in motor neurons and a down-regulation of mRNA for the NF light subunit (NF-L) are associated with ALS, but it remains unclear to what extent these NF perturbations contribute to human disease. Transgenic mouse studies demonstrated that overexpression of normal and mutant NF proteins can sometimes provoke a motor neuronopathy characterized by the presence of abnormal NF accumulations resembling those found in ALS. Remarkably, the motor neuronopathy in transgenic mice overexpressing human NF heavy (NF-H) subunits was rescued by the co-expression of a human NF-L transgene at levels that restored a correct stoichiometry of NF-L to NF-H subunits. Transgenic approaches have also been used to investigate the role of NFs in disease caused by Cu/Zn superoxide dismutase (SOD1) mutations, which is responsible for approximately 2% cases of ALS. Studies with transgenic mice expressing low levels of a fusion NF-H/lacZ protein, in which NFs are withheld from the axonal compartment, suggested that axonal NFs are not toxic intermediates required for SOD1-mediated disease. On the contrary, overexpression of human NF-H proteins was found to confer an effective protection against mutant SOD1 toxicity in transgenic mice, a phenomenon that may be due to the ability of NF proteins to chelate calcium. In conclusion, transgenic studies showed that disorganized NFs can sometimes have noxious effects resulting in neuronopathy. However, in the context of motor neuron disease caused by mutant SOD1, there is emerging evidence that NF proteins rather play a protective role.     

Absence of neurofilaments reduces the selective vulnerability of motor neurons and slows disease caused by a familial amyotrophic lateral sclerosis-linked superoxide dismutase 1 mutant

Mutations in superoxide dismutase 1 (SOD1), the only proven cause of amyotrophic lateral sclerosis (ALS), provoke disease through an unidentified toxic property. Neurofilament aggregates are pathologic hallmarks of both sporadic and SOD1-mediated familial ALS. By deleting NF-L, the major neurofilament subunit required for filament assembly, onset and progression of disease caused by familial ALS-linked SOD1 mutant G85R are significantly slowed, while selectivity of mutant-mediated toxicity for motor neurons is reduced. In NF-L-deleted animals, levels of the two remaining neurofilament subunits, NF-M and NF-H, are markedly reduced in axons but are elevated in motor neuron cell bodies. Thus, while neither perikaryal nor axonal neurofilaments are essential for SOD1-mediated disease, the absence of assembled neurofilaments both diminishes selective vulnerability and slows SOD1(G85R) mutant-mediated toxicity to motor neurons.     

Massive mitochondrial degeneration in motor neurons triggers the onset of amyotrophic lateral sclerosis in mice expressing a mutant SOD1

Amyotrophic lateral sclerosis (ALS) involves motor neuron degeneration, skeletal muscle atrophy, paralysis, and death. Mutations in Cu,Zn superoxide dismutase (SOD1) are one cause of the disease. Mice transgenic for mutated SOD1 develop symptoms and pathology similar to those in human ALS. To understand the disease mechanism, we developed a simple behavioral assay for disease progression in mice. Using this assay, we defined four stages of the disease in mice expressing G93A mutant SOD1. By studying mice with defined disease stages, we tied several pathological features into a coherent sequence of events leading to motor neuron death. We show that onset of the disease involves a sharp decline of muscle strength and a transient explosive increase in vacuoles derived from degenerating mitochondria, but little motor neuron death. Most motor neurons do not die until the terminal stage, approximately 9 weeks after disease onset. These results indicate that mutant SOD1 toxicity is mediated by damage to mitochondria in motor neurons, and this damage triggers the functional decline of motor neurons and the clinical onset of ALS. The absence of massive motor neuron death at the early stages of the disease indicates that the majority of motor neurons could be rescued after clinical diagnosis.     

Age-dependent penetrance of disease in a transgenic mouse model of familial amyotrophic lateral sclerosis

The mutation gly93-->ala of Cu,Zn superoxide dismutase (SOD) is found in patients with familial amyotrophic lateral sclerosis and causes motor neuron disease when expressed in transgenic mice. The progression of clinical and pathological disease was studied in a line of mice designated G1H. Clinical disease started at 91 +/- 14 days of age with fine shaking of the limbs, followed by paralysis and death by 136 +/- 7 days of age. Pathological changes begin by 37 days of age with vacuoles derived from swollen mitochondria accumulating in motor neurons. At the onset of clinical disease (90 days), significant death of somatic motor neurons innervating limb muscles has occurred; mice at end-stage disease (136 days) show up to 50% loss of cervical and lumbar motor neurons. However, neither thoracic nor cranial motor neurons show appreciable loss despite vacuolar changes. Autonomic motor neurons also are not affected. Mice that express wild-type human Cu,Zn SOD remain free of disease, indicating that mutations cause neuron loss by a gain-of-function. Thus, the age-dependent penetrance of motor neuron disease in this transgenic model is due to the gradual accumulation of pathological damage in select populations of cholinergic neurons.     

A role for neurofilaments in the pathogenesis of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a late-onset degenerative disease of motor neurons, characterized by abnormal accumulation of neurofilaments (NFs) in perikarya and proximal axons. Two lines of evidence suggest that neurofilament accumulation can play a crucial role in ALS pathogenesis. First, transgenic mouse models overexpressing NF proteins were found to develop motor neuron degeneration and, second, variant alleles of the NF heavy-subunit (NF-H) gene have been found in some human ALS patients. Our axonal transport studies with transgenic mice overexpressing the human NF-H gene, a model of ALS, revealed defects of intracellular transport not only for neurofilament proteins but also for other cytoskeletal proteins and organelles such as mitochondria. Therefore, we propose that neurofilament accumulation in mice causes neurodegeneration by disrupting axonal transport, a mechanism that may account for the pathogenesis of ALS.     

Protein oxidative damage in a transgenic mouse model of familial amyotrophic lateral sclerosis

The Gly93-->Ala mutation in the Cu,Zn superoxide dismutase (Cu,Zn-SOD) gene (SOD1) found in some familial amyotrophic lateral sclerosis (FALS) patients has been shown to result in an aberrant increase in hydroxyl radical production by the mutant enzyme that may cause oxidative injury to spinal motor neurons. In the present study, we analyzed the extent of oxidative injury to lumbar and cervical spinal cord proteins in transgenic FALS mice that overexpress the SOD1 mutation [TgN(SOD1-G93A)G1H] in comparison with nontransgenic mice. Total protein oxidation was examined by spectrophotometric measurement of tissue protein carbonyl content by the dinitrophenylhydrazine (DNPH) assay. Four ages were investigated: 30 (pre-motor neuron pathology and clinical disease), 60 (after initiation of pathology, but pre-disease), 100 (approximately 50% loss of motor neurons and function), and 120 (near complete hindlimb paralysis) days. Protein carbonyl content in 30-day-old TgN(SOD1-G93A)G1H mice was twice as high as the level found in age-matched nontransgenic mice. However, at 60 and 100 days of age, the levels were the same. Then, between 100 and 120 days of age, the levels in the TgN(SOD1-G93A)G1H mice increased dramatically (557%) compared with either the nontransgenic mice or transgenic animals that overexpress the wild-type human Cu,Zn-SOD [TgN(SOD1)N29]. The 100-120-day increase in spinal cord protein carbonyl levels was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation and western blot immunoassay, which enabled the identification of heavily oxidized individual proteins using a monoclonal antibody against DNPH-derivatized proteins. One of the more heavily oxidized protein bands (14 kDa) was identified by immunoprecipitation as largely Cu,Zn-SOD. Western blot comparison of the extent of Cu,Zn-SOD protein carbonylation revealed that the level in spinal cord samples from 120-day-old TgN(SOD1-G93A)G1H mice was significantly higher than that found in age-matched nontransgenic or TgN(SOD1)N29 mice. These results suggest that the increased hydroxyl radical production associated with the G93A SOD1 mutation and/or lipid peroxidation-derived radical species (peroxyl or alkoxyl) causes extensive protein oxidative injury and that the Cu,Zn-SOD itself is a key target, which may compromise its antioxidant function.     

Oxidative stress, mutant SOD1, and neurofilament pathology in transgenic mouse models of human motor neuron disease

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that primarily affects motor neurons in the spinal cord and brain stem. About 10% of all ALS cases are familial (FALS), inherited in an autosomal dominant manner. One fifth of FALS patients carry mutations in the Cu/Zn superoxide dismutase (SOD1) gene, and several lines of transgenic mice have been engineered to express mutant forms of the SOD1 gene that are linked to FALS. Significantly, many of these transgenic lines of mice develop a motor neuron disease (MND) that resembles human FALS. Oxidative stress induced by human SOD1 mutations is believed to play an important role in the pathogenesis of FALS and the FALS-like MND seen in the mutant SOD1 transgenic mice. For example, two lines of these mice showed prominent degeneration of mitochondria and endoplasmic reticulum in spinal cord neurons. Furthermore, recent studies have shown that neurofilament (NF)-rich spheroids. Lewy body-like NF inclusions, altered ubiquitin immunoreactivity, and Golgi fragmentation occur in the spinal cord motoneurons of these mutant SOD1 transgenic mice. Because these lesions recapitulate hallmark abnormalities of human ALS, mutant SOD1 transgenic mice provide a useful model for studies designed to elucidate the pathogenesis of ALS. Furthermore, transgenic mice that overexpress NF proteins also develop a clinical and pathologic phenotype similar to human MND, and polymorphisms in an NF gene have been linked to patients with ALS. Collectively, these observations implicate NF protein abnormalities in the pathogenesis of this disorder. Accordingly, this review summarizes recent insights into mechanisms of motor neuron degeneration in ALS that have emerged from studies of these new animal models of this neurodegenerative disease.     

Aggregation and motor neuron toxicity of an ALS-linked SOD1 mutant independent from wild-type SOD1

Analysis of transgenic mice expressing familial amyotrophic lateral sclerosis (ALS)-linked mutations in the enzyme superoxide dismutase (SOD1) have shown that motor neuron death arises from a mutant-mediated toxic property or properties. In testing the disease mechanism, both elimination and elevation of wild-type SOD1 were found to have no effect on mutant-mediated disease, which demonstrates that the use of SOD mimetics is unlikely to be an effective therapy and raises the question of whether toxicity arises from superoxide-mediated oxidative stress. Aggregates containing SOD1 were common to disease caused by different mutants, implying that coaggregation of an unidentified essential component or components or aberrant catalysis by misfolded mutants underlies a portion of mutant-mediated toxicity.     

Elevated "hydroxyl radical" generation in vivo in an animal model of amyotrophic lateral sclerosis

Mutations in the enzyme copper/zinc superoxide dismutase-1 (SOD1) are associated with familial amyotrophic lateral sclerosis (FALS). The means by which the mutations cause FALS appears to be due to an adverse property of the mutant SOD1 protein that may involve increased generation of free radicals. We used in vivo microdialysis to measure the conversion of 4-hydroxybenzoic acid to 3,4-dihydroxybenzoic acid (3,4-DHBA) as a measure of "hydroxyl radical-like" production in transgenic amyotrophic lateral sclerosis (ALS) mice with the G93A mutation as well as littermate controls. The conversion of 4-hydroxybenzoic acid to 3,4-DHBA was significantly increased in the striatum of transgenic ALS mice at baseline but not in mice overexpressing wild-type human SOD1. Following administration of 3-nitropropionic acid 3,4-DHBA generation was significantly increased as compared with baseline, and the increase in the transgenic ALS mice was significantly greater than those in controls, whereas the increase in mice overexpressing wild-type human SOD1 was significantly attenuated. The present results provide in vivo evidence that expression of mutations in SOD1 can lead to increased generation of "hydroxyl radical-like" activity, which further implicates oxidative damage in the pathogenesis of ALS.     

Antagonistic roles of neurofilament subunits NF-H and NF-M against NF-L in shaping dendritic arborization in spinal motor neurons

Dendrites play important roles in neuronal function. However, the cellular mechanism for the growth and maintenance of dendritic arborization is unclear. Neurofilaments (NFs), a major component of the neuronal cytoskeleton, are composed of three polypeptide subunits, NF-H, NF-M, and NF-L, and are abundant in large dendritic trees. By overexpressing each of the three NF subunits in transgenic mice, we altered subunit composition and found that increasing NF-H and/or NF-M inhibited dendritic arborization, whereas increasing NF-L alleviated this inhibition. Examination of cytoskeletal organization revealed that increasing NF-H and/or NF-M caused NF aggregation and dissociation of the NF network from the microtubule (MT) network. Increasing NF-H or NF-H together with NF-M further reduced NFs from dendrites. However, these changes were reversed by elevating the level of NF-L with either NF-H or NF-M. Thus, NF-L antagonizes NF-H and NF-M in organizing the NF network and maintaining a lower ratio of NF-H and NF-M to NF-L is critical for the growth of complex dendritic trees in motor neurons.     

Up-regulation of protein chaperones preserves viability of cells expressing toxic Cu/Zn-superoxide dismutase mutants associated with amyotrophic lateral sclerosis

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene underlie some familial cases of amyotrophic lateral sclerosis, a neurodegenerative disorder characterized by loss of cortical, brainstem, and spinal motor neurons. We present evidence that SOD-1 mutants alter the activity of molecular chaperones that aid in proper protein folding and targeting of abnormal proteins for degradation. In a cultured cell line (NIH 3T3), resistance to mutant SOD-1 toxicity correlated with increased overall chaperoning activity (measured by the ability of cytosolic extracts to prevent heat denaturation of catalase) as well as with up-regulation of individual chaperones/stress proteins. In transgenic mice expressing human SOD-1 with the G93A mutation, chaperoning activity was decreased in lumbar spinal cord but increased or unchanged in clinically unaffected tissues. Increasing the level of the stress-inducible chaperone 70-kDa heat shock protein by gene transfer reduced formation of mutant SOD-containing proteinaceous aggregates in cultured primary motor neurons expressing G93A SOD-1 and prolonged their survival. We propose that insufficiency of molecular chaperones may be directly involved in loss of motor neurons in this disease.     

Carotenoids and intercellular communication via gap junctions

Long-term cultured murine embryonic yolk sac cells that are capable of forming capillary structures when cultured on base membrane proteins (Matrigel) were successfully transfected with a human growth hormone antagonist (G120R) gene. Cells that stably express relatively high levels of G120R were co-implanted s.c. with Matrigel into BALB/c mice. G120R can be detected in the sera of those implanted mice for more than 14 days at levels from 4 ng/ml to 28 ng/ml. The insulin-like growth factor-1 levels in the sera of those implanted mice were significantly affected by the delivered G120R. One of the physiological effects of G120R delivered by this murine embryonic yolk sac cell-derived mini-organ system is to decrease the growth rate of the implanted mice. This gene delivery system can also be used as an alternative to transgenic animals to study protein function in vivo.     

Small aliphatic dicarboxylic acids inhibit renal uptake of administered mercury

To investigate the role of the neurofilament heavy (NF-H) subunit in neuronal function, we generated mice bearing a targeted disruption of the gene coding for the NF-H subunit. Surprisingly, the lack of NF-H subunits had little effect on axonal calibers and electron microscopy revealed no significant changes in the number and packing density of neurofilaments made up of only the neurofilament light (NF-L) and neurofilament medium (NF-M) subunits. However, our analysis of NF-H knockout mice revealed an approximately 2.4-fold increase of microtubule density in their large ventral root axons. This finding was further corroborated by a corresponding increase in the ratio of assembled tubulin to NF-L protein in insoluble cytoskeletal preparations from the sciatic nerve. Axonal transport studies carried out by the injection of [35S]methionine into spinal cord revealed an increased transport velocity of newly synthesized NF-L and NF-M proteins in motor axons of NF-H knockout mice. When treated with beta,beta'-iminodipropionitrile (IDPN), a neurotoxin that segregates microtubules and retards neurofilament transport, mice heterozygous or homozygous for the NF-H null mutation did not develop neurofilamentous swellings in motor neurons, unlike normal mouse littermates. These results indicate that the NF-H subunit is a key mediator of IDPN-induced axonopathy.     

Susceptibility to hepatotoxicity in transgenic mice that express a dominant-negative human keratin 18 mutant

Keratins 8 and 18 (K8/18) are intermediate filament phosphoglycoproteins that are expressed preferentially in simple-type epithelia. We recently described transgenic mice that express point-mutant human K18 (Ku, N.-O., S. Michie, R.G. Oshima, and M.B. Omary. 1995. J. Cell Biol. 131:1303-1314) and develop chronic hepatitis and hepatocyte fragility in association with hepatocyte keratin filament disruption. Here we show that mutant K18 expressing transgenic mice are highly susceptible to hepatotoxicity after acute administration of acetaminophen (400 mg/Kg) or chronic ingestion of griseofulvin (1.25% wt/wt of diet). The predisposition to hepatotoxicity results directly from the keratin mutation since nontransgenic or transgenic mice that express normal human K18 are more resistant. Hepatotoxicity was manifested by a significant difference in lethality, liver histopathology, and biochemical serum testing. Keratin glycosylation decreased in all griseofulvin-fed mice, whereas keratin phosphorylation increased dramatically preferentially in mice expressing normal K18. The phosphorylation increase in normal K18 after griseofulvin feeding appears to involve sites that are different to those that increase after partial hepatectomy. Our results indicate that hepatocyte intermediate filament disruption renders mice highly susceptible to hepatotoxicity, and raises the possibility that K18 mutations may predispose to drug hepatotoxicity. The dramatic phosphorylation increase in nonmutant keratins could provide survival advantage to hepatocytes.     

Transgenic mice expressing high plasma concentrations of human apolipoprotein B100 and lipoprotein(a)

The B apolipoproteins, apo-B48 and apo-B100, are key structural proteins in those classes of lipoproteins considered to be atherogenic [e.g., chylomicron remnants, beta-VLDL, LDL, oxidized LDL, and Lp(a)]. Here we describe the development of transgenic mice expressing high levels of human apo-B48 and apo-B100. A 79.5-kb human genomic DNA fragment containing the entire human apo-B gene was isolated from a P1 bacteriophage library and microinjected into fertilized mouse eggs. 16 transgenic founders expressing human apo-B were generated, and the animals with the highest expression had plasma apo-B100 levels nearly as high as those of normolipidemic humans (approximately 50 mg/dl). The human apo-B100 in transgenic mouse plasma was present largely in lipoproteins of the LDL class as shown by agarose gel electrophoresis, chromatography on a Superose 6 column, and density gradient ultracentrifugation. When the human apo-B transgenic founders were crossed with transgenic mice expressing human apo(a), the offspring that expressed both transgenes had high plasma levels of human Lp(a). Both the human apo-B and Lp(a) transgenic mice will be valuable resources for studying apo-B metabolism and the role of apo-B and Lp(a) in atherosclerosis.     

T cell epitopes of insulin defined in HLA-DR4 transgenic mice are derived from preproinsulin and proinsulin

Approximately one-half of Caucasians with newly diagnosed insulin-dependent diabetes mellitus (IDDM) have autoantibodies to insulin, and the majority of those express the HLA-DR4 genotype [Ziegler, R., Alper, C. A., Awdeh, Z. L., Castano, L., Brink, S. J., Soeldner, J. S., Jackson, R. A. & Eisenbarth, G. S. (1991) Diabetes 40, 709-714]. However, it has been difficult to demonstrate T cell proliferative responses to human insulin in IDDM patients [Durinovic-Bello, I., Hummel, M. & Ziegler, A. G. (1996) Diabetes 45, 795-800]. We have immunized transgenic mice expressing the susceptible HLA-DR (alpha1*0101,beta1*0401) (hereafter called DRB1*0401) and human CD4 molecules on a murine major histocompatibility complex class II null background, with human preproinsulin (PPI), proinsulin (PI), and insulin and derived large panels of T cell hybridomas to determine the immunogenic epitopes of these proteins. These results show that the prohormones PI or PPI carry the major immunogenic T cell epitope in the DRB1*0401 transgenic mice. The PPI/PI immunodominant epitope LALEGSLQK was localized at the C-peptide/A-chain junction. This T cell epitope PPI/PI LALEGSLQK is unusual because, normally, it is proteolytically destroyed during the maturation of the insulin molecule. Additionally, this T cell epitope is both processed and presented by human DRB1*0401-positive Epstein-Barr virus transformed B cells, and it can also stimulate T cells from the peripheral blood of HLA-DR4-positive patients with type 1 diabetes. These findings may partly explain why susceptibility to type 1 diabetes is associated with HLA-DR4-positive individuals and why T cell responses to the mature insulin protein are rarely detected in IDDM patients.     

Effects of drying methods and additives on structure and function of actin: mechanisms of dehydration-induced damage and its inhibition

Neurofilaments are essential for establishment and maintenance of axonal diameter of large myelinated axons, a property that determines the velocity of electrical signal conduction. One prominent model for how neurofilaments specify axonal growth is that the 660-amino acid, heavily phosphorylated tail domain of neurofilament heavy subunit (NF-H) is responsible for neurofilament-dependent structuring of axoplasm through intra-axonal crossbridging between adjacent neurofilaments or to other axonal structures. To test such a role, homologous recombination was used to generate NF-H-null mice. In peripheral motor and sensory axons, absence of NF-H does not significantly affect the number of neurofilaments or axonal elongation or targeting, but it does affect the efficiency of survival of motor and sensory axons. Loss of NF-H caused only a slight reduction in nearest neighbor spacing of neurofilaments and did not affect neurofilament distribution in either large- or small-diameter motor axons. Since postnatal growth of motor axon caliber continues largely unabated in the absence of NF-H, neither interactions mediated by NF-H nor the extensive phosphorylation of it within myelinated axonal segments are essential features of this growth.     

Overexpression of human glutathione peroxidase protects transgenic mice against focal cerebral ischemia/reperfusion damage

As stroke is a major cause of disability and death in the western world, there is great interest in the basic mechanisms by which ischemia/reperfusion (I/R) causes damage. To this end, extensive research has been carried out which identifies reactive oxygen species (ROS) as key participants in brain damage resultant from I/R. Brain tissue is protected from ROS damage by antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GP). Overexpression of SOD in transgenic mice has already been demonstrated to confer protection against I/R damage in murine stroke models. We are using transgenic mice overexpressing the intracellular form of glutathione peroxidase (GP1) to determine the protective capacity of overexpression of this enzyme on stroke damage. 1 h of focal cerebral ischemia followed by 24 h of reperfusion was induced using the intraliminal suture method. Volume of infarction was reduced by 48% in GP1 mice compared to nontransgenic littermates. Brain edema was reduced by 33%. Behavioral deficits agreed with histologic data. Overexpression of glutathione peroxidase confers significant protection against I/R damage in our stroke model possibly through direct scavenging of ROS or through the influencing of signalling mechanisms which lead to tissue damage.     

Oxidative stress and motor neurone disease

The effects of oxidative stress within post mitotic cells such as neurones may be cumulative, and injury by free radical species is a major potential cause of the age-related deterioration in neuronal function seen in several neurodegenerative diseases. There is strong evidence that oxidative stress plays an important role in the pathogenesis of motor neurone disease (MND). Point mutations in the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1) are found in some pedigrees with the familial form of MND. How mutations in this ubiquitous enzyme cause the relatively selective cell death of specific groups of motor neurones is not clear, although a number of hypotheses have been forwarded. These include (1) the formation of hydroxyl radicals, (2) the catalysis of reactions of the nitrogen centred oxidant species peroxynitrite, (3) toxicity of copper or zinc and (4) protein aggregation. Some experimental support for these different hypotheses has been produced by manipulating cells in culture to express the mutant SOD1 proteins and by generating transgenic mice which over-express mutant SOD1. Observations in these model systems are, in some cases at least, supported by observations made on pathological material from patients with similar SOD1 mutations. Furthermore, there are reports of evidence of free radical mediated damage to neurones in the sporadic form of MND. Several lines of evidence suggest that alterations in the glutamatergic neurotransmitter system may also play a key role in the injury to motor neurones in sporadic MND. There are several important subcellular targets, which may be preferentially impaired within motor neurones, including neurofilament proteins and mitochondria. Future research will need to identify the aspects of the molecular and physiological phenotype of human motor neurones that makes them susceptible to degeneration in MND, and to identify those genetic and environmental factors which combine to cause this disease in individuals and in familial pedigrees.     

Glutamate potentiates the toxicity of mutant Cu/Zn-superoxide dismutase in motor neurons by postsynaptic calcium-dependent mechanisms

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.