Transgenic mouse model for familial amyotrophic lateral sclerosis with superoxide dismutase‐1 mutation

Familial amyotrophic lateral sclerosis (ALS) with mutations in the gene for superoxide dismutase‐1 (SOD1) is clinicopathologically reproduced by transgenic mice expressing mutant forms of SOD1 detectable in familial ALS patients. Motor neuron degeneration associated with SOD1 mutation has been thought to result from a novel neurotoxicity of mutant SOD1, but not from a reduction in activity of this enzyme, based on autosomal dominant transmission of SOD1 mutant familial ALS and its transgenic mouse model, clinical severity of the ALS patients independent to enzyme activity, no ALS‐like disease in SOD1 knockout or wild‐type SOD1‐over‐expressing mice, and clinicopathological severity of mutant SOD1 transgenic mice dependent on transgene copy numbers. Proposed mechanisms of motor neuron de‐generation such as oxidative injury, peroxynitrite toxicity, cytoskeletal disorganization, glutamate excitotoxicity, disrupted calcium homeostasis, SOD1 aggregation, car‐bonyl stress and apoptosis have been discussed. Intracy‐toplasmic vacuoles, indicative of increased oxidative damage to the mitochondria and endoplasmic reticulum, in the neuropil and motor neurons appear in high expressors of mutant SOD1 transgenic mice but not in low expressors of the mice or familial ALS patients, suggesting that overexpression of mutant SOD1 in mice may enhance oxidative stress generation from this enzyme. Thus, transgenic mice carrying small transgene copy numbers of mutant SOD1 would provide a beneficial animal model for SOD1 mutant familial ALS. Such a model would contribute to elucidating the pathomechanism of this disease and establishing new therapeutic agents.     

The neuronal Golgi apparatus is fragmented in transgenic mice expressing a mutant human SOD1, but not in mice expressing the human NF-H gene

Fragmentation of the Golgi apparatus (GA) of motor neurons was first described in sporadic amyotrophic lateral sclerosis (ALS) and later confirmed in transgenic mice expressing the G93A mutation of the gene encoding the enzyme Cu,Zn superoxide dismutase (SOD1(G93A)) found in some cases of familial ALS. In these transgenic mice, however, the fragmentation of the neuronal GA was associated with cytoplasmic and mitochondrial vacuoles not seen in ALS. The present new series of transgenic mice expressing 14-17 trans gene copies of SOD1(G93A), compared to 25 copies in the mice we studied previously, showed consistent fragmentation of the GA of spinal cord motor neurons, axonal swellings, Lewy-like body inclusions in neurons and glia, but none of the cytoplasmic or mitochondrial vacuoles originally reported. Thus, this animal model recapitulates the clinical and most neuropathological findings of sporadic ALS. Neurofilaments (NF) accumulate in axons and, less often, in neuronal perikarya in most cases of sporadic ALS and they have been implicated in its pathogenesis. In order to investigate whether fragmentation of the neuronal GA also occurs in association with accumulation of perikaryal NFs, we studied the organelle in transgenic mice expressing the heavy subunit of human neurofilaments (NF-H) which developed a motor neuronopathy resembling ALS. The neuronal GA of mice expressing NF-H, however, was intact despite massive accumulation of NFs in both perikarya and axons of motor neurons. In contrast, in transgenic mice expressing SOD1(G93A), the GA was fragmented despite the absence of accumulation of perikaryal NFs. These findings suggest that, in transgenic mice with neuronopathies caused by the expression of mutant SOD1(G93A) or the human NF-H, the GA and the perikaryal NFs are independently involved in the pathogenesis. The evidence suggests that the GA plays a central role in the pathogenesis of the vast majority of sporadic ALS and in FALS with SOD1 mutations.     

Immune reactivity in a mouse model of familial ALS correlates with disease progression

OBJECTIVE: The cause of motor neuron death in ALS is incompletely understood. This study aims to define the potential involvement of nonneuronal immune-inflammatory factors in the destruction of motor neurons in mutant superoxide dismutase-1 (SOD1) transgenic mice as a model of ALS. BACKGROUND: The presence of activated microglia, IgG and its receptor for Fc portion (FcgammaRI), and T lymphocytes in the spinal cord of both patients with ALS and experimental animal models of motor neuron disease strongly suggests that immune-inflammatory factors may be actively involved in the disease process. METHODS: The expression of immune-inflammatory factors was followed in both human mutant (G93A) SOD1 transgenic mice and human wild-type SOD1 transgenic mice, at different ages (40, 80, and 120 days). Fixed, frozen, free-floating sections of the lumbar spinal cord were stained with antibodies against CD11b, IgG, FcgammaRI, intercellular adhesion molecule-1 (ICAM-1), CD3, and glial fibrillary acidic protein. RESULTS: The earliest change observed was the upregulation of ICAM-1 in the ventral lumbar spinal cord of 40-day-old mutant SOD1 mice. IgG and FcgammaRI reactivities were detected on motor neurons as early as 40 days and on microglial cells at later stages. Microglial activation was first evident in the ventral horn at 80 days, whereas reactive astrocytes and T cells became most prominent in 120-day-old mutant SOD1 mice. CONCLUSION: The upregulation of proinflammatory factors during early presymptomatic stages as well as the expansion of immune activation as disease progresses in mutant SOD1 transgenic mice suggest that immune-inflammatory mechanisms could contribute to disease progression.     

Familial amyotrophic lateral sclerosis and Cu/Zn superoxide dismutase mutation

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder that involves mainly the motor neuron system. Five to 10 percent of the ALS cases are familial; most others are sporadic. Several mutations in the superoxide dismutase-1 (SOD1) gene have recently been shown to be associated with about 20% of familial ALS patients. The reduced enzyme activity of many mutant SOD1 points to the possibility that a loss-of-function effect of the mutant enzyme is responsible for the pathogenesis of the disease. However, this conflicts with the autosomal dominant inheritance of SOD1 mutation-associated ALS and the normal SOD1 activity in homozygous patients in a SOD1-linked ALS family. Current biochemical investigations have provided evidence that mutant SOD1 may catalyze the peroxynitrite-mediated nitration of protein tyrosine residues, release copper and zinc ions, facilitate apoptosis of neurons and have enhanced peroxidase activity. Immunocytochemical studies demonstrated the presence of intense SOD1 immunoreactivity in Lewy body-like inclusions, which are characteristic features of a certain form of familial ALS with posterior column involvement, in the lower motor neurons of patients in ALS families with different SOD1 mutations. More recently, strains of transgenic mice expressing mutant SOD1 have been established. These mice clinicopathologically develop a motor neuron disease mimicking human ALS with the exception of pronounced intraneuronal vacuolar degeneration. The overexpression of wild-type SOD1 in mice has failed to give rise to the disease. Only one transgene for mutant SOD1 is enough to cause motor neuron degeneration and the severity of clinical course correlates with the transgene copy number. These observations in SOD1-linked familial ALS and its transgenic mouse model suggest a novel neurotoxic function of mutant SOD1.     

Peripherin is not a contributing factor to motor neuron disease in a mouse model of amyotrophic lateral sclerosis caused by mutant superoxide dismutase

Peripherin is a type III intermediate filament protein detected in axonal spheroids associated with amyotrophic lateral sclerosis (ALS). The overexpression of peripherin induces degeneration of spinal motor neurons during aging in transgenic mice and in cultured neuronal cells derived from peripherin transgenic embryos. Here, we investigated whether peripherin is a contributor of pathogenesis in mice overexpressing a mutant superoxide dismutase 1 (SOD1(G37R)) gene linked to familial ALS. This was done by the generation and analysis of SOD1(G37R) mice that either overexpress a peripherin transgene (G37R;TgPer mice) or lack the endogenous peripherin gene (G37R;Per-/- mice). Surprisingly, upregulation or suppression of peripherin expression had no effects on disease onset, mortality, and loss of motor neurons in SOD1(G37R) mice. These results provide compelling evidence that peripherin is not a key contributor of motor neuron degeneration associated with toxicity of mutant SOD1.     

Protective effects of heat shock protein 27 in a model of ALS occur in the early stages of disease progression

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder, characterised by progressive motor neuron degeneration and muscle paralysis. Heat shock proteins (HSPs) have significant cytoprotective properties in several models of neurodegeneration. To investigate the therapeutic potential of heat shock protein 27 (HSP27) in a mouse model of ALS, we conducted an extensive characterisation of transgenic mice generated from a cross between HSP27 overexpressing mice and mice expressing mutant superoxide dismutase (SOD1(G93A)). We report that SOD1(G93A)/HSP27 double transgenic mice showed delayed decline in motor strength, a significant improvement in the number of functional motor units and increased survival of spinal motor neurons compared to SOD1(G93A) single transgenics during the early phase of disease. However, there was no evidence of sustained neuroprotection affecting long-term survival. Marked down-regulation of HSP27 protein occurred during disease progression that was not associated with a reduction in HSP27 mRNA, indicating a translational dysfunction due to the presence of mutant SOD1 protein. This study provides further support for the therapeutic potential of HSPs in ALS and other motor neuron disorders.     

Reduced activity of AMP-activated protein kinase protects against genetic models of motor neuron disease

A growing body of research indicates that amyotrophic lateral sclerosis (ALS) patients and mouse models of ALS exhibit metabolic dysfunction. A subpopulation of ALS patients possesses higher levels of resting energy expenditure and lower fat-free mass compared to healthy controls. Similarly, two mutant copper zinc superoxide dismutase 1 (mSOD1) mouse models of familial ALS possess a hypermetabolic phenotype. The pathophysiological relevance of the bioenergetic defects observed in ALS remains largely elusive. AMP-activated protein kinase (AMPK) is a key sensor of cellular energy status and thus might be activated in various models of ALS. Here, we report that AMPK activity is increased in spinal cord cultures expressing mSOD1, as well as in spinal cord lysates from mSOD1 mice. Reducing AMPK activity either pharmacologically or genetically prevents mSOD1-induced motor neuron death in vitro. To investigate the role of AMPK in vivo, we used Caenorhabditis elegans models of motor neuron disease. C. elegans engineered to express human mSOD1 (G85R) in neurons develops locomotor dysfunction and severe fecundity defects when compared to transgenic worms expressing human wild-type SOD1. Genetic reduction of aak-2, the ortholog of the AMPK α2 catalytic subunit in nematodes, improved locomotor behavior and fecundity in G85R animals. Similar observations were made with nematodes engineered to express mutant tat-activating regulatory (TAR) DNA-binding protein of 43 kDa molecular weight. Altogether, these data suggest that bioenergetic abnormalities are likely to be pathophysiologically relevant to motor neuron disease.     

Inhibition of p38 mitogen activated protein kinase activation and mutant SOD1(G93A)-induced motor neuron death

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the selective loss of motor neurons. Stress activated protein kinases (SAPK) have been suggested to play a role in the pathogenesis of ALS. We studied the relevance of p38 MAPK for motor neuron degeneration in the mutant SOD1 mouse. Increased levels of phospho-p38 MAPK were present in the motor neurons and microglia of the ventral spinal cord. The p38 MAPK-inhibitor, SB203580, completely inhibited mutant SOD1-induced apoptosis of motor neurons and blocked LPS-induced activation of microglia. Semapimod, a p38 MAPK inhibitor suitable for clinical use, prolonged survival of mutant SOD1 mice to a limited extent, but largely protected motor neurons and proximal axons from mutant SOD1-induced degeneration. Our data confirm the abnormal activation of p38 MAPK in mutant SOD1 mice and the involvement of p38 MAPK in mutant SOD1-induced motor neuron death. We demonstrate the effect of p38 MAPK inhibition on survival of mutant SOD1 mice and reveal a dissociation between the effect on survival of motor neurons and that on survival of the animal, the latter likely depending on the integrity of the entire motor axon.     

Compensatory mechanism of motor defect in SOD1 transgenic mice by overactivation of striatal cholinergic neurons.

Expression of a mutant superoxide dismutase 1 (SOD1) gene in transgenic mice induces a gradual degeneration of cholinergic motor neurons in the spinal cord, causing progressive muscle weakness and hindlimb paralysis. Transgenic mice over-expressing the human SOD1 gene containing a Gly-->Ala substitution at position 93 (G93A) were employed to explore the effects of the SOD1 mutation on choline acetyltransferase (ChAT) expression in the striatum, and in the lumbar and cervical spinal cord. These mice showed a progressive loss of their spinal cord motor neurons, and at 130 days of age showed an up-regulation of ChAT mRNA expression in the striatum. On the other hand, ChAT mRNA decreased in cervical and lumbar motor neurons. These findings suggest that cholinergic interneurons in striatum in SOD1 transgenic mice are over-activated in an attempt to compensate for the death of spinal motor neurons.     

Absence of p53: no effect in a transgenic mouse model of familial amyotrophic lateral sclerosis

Familial amyotrophic lateral sclerosis (ALS) has been linked in some families to dominantly inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (Cu-Zn SOD1). Transgenic mice expressing a mutant human Cu-Zn SOD1 (G93A) develop a dominantly inherited adult-onset paralytic disorder that replicates many of the clinical and pathological features of familial ALS. Increased p53 immunoreactivity has been reported in the motor cortex and spinal ventral horns of postmortem tissue from ALS patients. The nuclear phosphoprotein p53 is an important regulator of cellular proliferation, and increasing evidence supports the role of p53 in regulating cellular apoptosis. To assess the role of p53-mediated apoptosis in amyotrophic lateral sclerosis, mice deficient in both p53 alleles (p53-/-) were crossed with transgenic mice expressing the G93A mutant (G93A+), creating novel transgenic knockout mice. The animals (p53 +/+G93A+, p53+/-G93A+, p53-/-G93A+) were examined at regular intervals for cage activity, upper and lower extremity strength, and mortality. At 120 days from birth mice from each genotype were sacrificed, and L2-L3 anterior horn motor neurons were counted. There was no significant difference in time to onset of behavioral decline, mortality, or motor neuron degeneration between the different genotypes. Despite evidence that p53 plays an important role after acute neuronal injury, the current study suggests that p53 is not significantly involved in cell death in the G93A+ transgenic mouse model of familial ALS.     

Progressive spinal axonal degeneration and slowness in ALS2-deficient mice

OBJECTIVE: Homozygous mutation in the ALS2 gene and the resulting loss of the guanine exchange factor activity of the ALS2 protein is causative for autosomal recessive early-onset motor neuron disease that is thought to predominantly affect upper motor neurons. The goal of this study was to elucidate how the motor system is affected by the deletion of ALS2. METHODS: ALS2-deficient mice were generated by gene targeting. Motor function and upper and lower motor neuron pathology were examined in ALS2-deficient mice and in mutant superoxide dismutase 1 (SOD1) mice that develop ALS-like disease from expression of an ALS-linked mutation in SOD1. RESULTS: ALS2-deficient mice demonstrated progressive axonal degeneration in the lateral spinal cord that is also prominent in mutant SOD1 mice. Despite the vulnerability of these spinal axons, lower motor neurons in ALS2-deficient mice were preserved. Behavioral studies demonstrated slowed movement without muscle weakness in ALS2(-/-) mice, consistent with upper motor neuron defects that lead to spasticity in humans. INTERPRETATION: The combined evidence from mice and humans shows that deficiency in ALS2 causes an upper motor neuron disease that in humans closely resembles a severe form of hereditary spastic paralysis, and that is quite distinct from amyotrophic lateral sclerosis.     

Therapeutic benefit of intrathecal injection of insulin-like growth factor-1 in a mouse model of Amyotrophic Lateral Sclerosis

Insulin-like growth factor (IGF)-1 has been shown to have a protective effect on motor neurons both in vitro and in vivo, but has limited efficacy in patients with amyotrophic lateral sclerosis (ALS) when given subcutaneously. To examine the possible effectiveness of IGF-1 in a mouse model of familial ALS, transgenic mice expressing human Cu/Zn superoxide dismutase (SOD1) with a G93A mutation were treated by continuous IGF-1 delivery into the intrathecal space of the lumbar spinal cord. We found that the intrathecal administration of IGF-1 improved motor performance, delayed the onset of clinical disease, and extended survival in the G93A transgenic mice. Furthermore, it increased the expression of phosphorylated Akt and ERK in spinal motor neurons, and partially prevented motor neuron loss in these mice. Taken together, the results suggest that direct administration of IGF-1 into the intrathecal space may have a therapeutic benefit for ALS.     

Coincident thresholds of mutant protein for paralytic disease and protein aggregation caused by restrictively expressed superoxide dismutase cDNA

Familial amyotrophic lateral sclerosis (FALS) has been modeled in transgenic mice by introducing mutated versions of human genomic DNA encompassing the entire gene for Cu,Zn superoxide dismutase (SOD1). In this setting, the transgene is expressed throughout the body and results in mice that faithfully recapitulate many pathological and behavioral aspects of FALS. By contrast, transgenic mice made by introducing recombinant vectors, encoding cDNA genes, that target mutant SOD1 expression to motor neurons, only, or astrocytes, only, do not develop disease. Here, we report that mice transgenic for human SOD1 cDNA with the G37R mutation, driven by the mouse prion promoter, develop motor neuron disease. In this model, expression of the transgene is highest in CNS (both neurons and astrocytes) and muscle. The gene was not expressed in cells of the macrophage lineage. Although the highest expressing hemizygous transgenic mice fail to develop disease by 20 months of age, mice homozygous for the transgene show typical ALS-like phenotypes as early as 7 months of age. Spinal cords and brain stems from homozygous animals with motor neuron disease were found to contain aggregated species of mutant SOD1. The establishment of this SOD1-G37R cDNA transgenic model indicates that expression of mutant SOD1 proteins in the neuromuscular unit is sufficient to cause motor neuron disease. The expression levels required to induce disease coincide with the levels required to induce the formation of SOD1 aggregates.     

Activated p38MAPK is a novel component of the intracellular inclusions found in human amyotrophic lateral sclerosis and mutant SOD1 transgenic mice

Cytoskeletal abnormalities with accumulation of ubiquilated inclusions in the anterior horn cells are a pathological hallmark of both familial and sporadic amyotrophic lateral sclerosis (ALS) and of mouse models for ALS. Phosphorylated neurofilaments besides ubiquitin and dorfin have been identified as one of the major components of the abnormal intracellular perikaryal aggregates. As we recently found that p38 mitogen-activated protein kinase (p38MAPK) colocalized with phosphorylated neurofilaments in spinal motor neurons of SOD1 mutant mice, a model of familial ALS, we investigated whether this kinase also contributed to the inclusions found in ALS patients and SOD1 mutant mice. Intense immunoreactivity for activated p38MAPK was observed in degenerating motor neurons and reactive astrocytes in ALS cases. The intracellular immunostaining for activated p38MAPK appeared in some neurons as filamentous skein-like and ball-like inclusions, with an immunohistochemical pattern identical to that of ubiquitin. Intracellular p38MAPK-positive aggregates containing ubiquitin and neurofilaments were also found in the spinal motor neurons of SOD1 mutant mice. Our observations indicate that activation of p38MAPK might contribute significantly to the pathology of motor neurons in ALS.     

Decreased expression of calretinin in the cerebral cortex and hippocampus of SOD1G93A transgenic mice

In the present study, we investigated the changes of calretinin (CR) expression in the central nervous system of SOD1G93A transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS). In wild-type SOD1 (wtSOD1) transgenic mice, many CR-immunoreactive neurons were found in all cortical regions. In the cerebral cortex of SOD1G93A transgenic mice, the number and staining intensity of CR-positive neurons were decreased. In the hippocampal formation, layer-specific alterations in the staining intensity of CR-immunoreactive neurons were observed in the CA1-3 areas and dentate gyrus. In wtSOD1 transgenic mice, CR-immunoreactive neurons with long processes were found in the stratum oriens and stratum radiatum of CA1-3 areas, and heavily stained band-like molecular layer was prominent in the dentate gyrus. CR immunoreactivity was decreased in each layer of CA1-3 areas and dentate gyrus of SOD1G93A transgenic mice. The first demonstration of decreased immunoreactivity for CR in the cerebral cortex and hippocampus of SOD1G93A transgenic mice may provide insights into the pathogenesis of motor neuron degeneration in human ALS although further quantitative studies are needed.     

Disruption of the structure of the Golgi apparatus and the function of the secretory pathway by mutants G93A and G85R of Cu, Zn superoxide dismutase (SOD1) of familial amyotrophic lateral sclerosis

The Golgi apparatus of motor neurons (GA) is fragmented in sporadic amyotrophic lateral sclerosis (ALS), in familial ALS with SOD1 mutations, and in mice that express SOD1G93A of familial ALS, in which it was detected months before paralysis. In paralyzed transgenic mice expressing SOD1G93A or SOD1G85R, mutant proteins aggregated not only in the cytoplasm of motor neurons, but also in astrocytes and oligodendrocytes. Furthermore, aggregation of the G85R protein damaged astrocytes and was associated with rapidly progressing disease. In order to gain insight into the functional state of the fragmented GA, we examined the effects of S0D1 mutants G93A and G85R in Chinese Hamster Ovary Cells (CHO). In contrast to cells expressing the wt and G93A, the G85R expressers had no SOD1 activity. However, cells expressing both mutants, and to a lesser degree the wt, showed decreased survival, fragmentation of the GA, and dysfunction of the secretory pathway, which was assessed by measuring the amount of cell surface co-expressed CD4, a glycoprotein processed through the GA. The G93A and wt proteins were partially recovered in detergent insoluble fractions; while the recovery of G85R was minimal. Both mutants showed equal reductions of cell survival and function of the secretory pathway, in comparison to the wt and cells expressing mutant alsin, a protein found in rare cases of fALS. These results are consistent with the conclusion that the two SOD1 mutants, by an unknown mechanism, promote the dispersion of the GA and the dysfunction of the secretory pathway. This and other in vitro models of mutant SOD1 toxicity may prove useful in the elucidation of pathogenetic mechanisms.     

A low expressor line of transgenic mice carrying a mutant human Cu,Zn superoxide dismutase (SOD1) gene develops pathological changes that most closely resemble those in human amyotrophic lateral sclerosis

About 15–20% of patients with familial amyotrophic lateral sclerosis (ALS) carry one of several missense mutations in the gene for Cu,Zn superoxide dismutase (SOD1). We have previously reported on an animal model of this disease produced by the transgenic expression of a mutant form of human SOD1 containing a Gly⁹³→Ala amino acid substitution. Several lines of transgenic mice were produced, characterized by a differing tempo and severity of disease that generally correlated with the number of mutant gene copies that these lines expressed. We reported that mice expressing high copy numbers (18–25) developed a disease with a relatively short course and with a pathology mainly characterized by severe vacuolar degeneration of motor neurons and their processes. Lewy-like bodies and swollen axons were also present. The exquisite localization to motor neurons was the feature that made the pathology in these overexpressors germane to the human disease. Severe vacuolar degeneration, however, was considered to be at variance with human ALS, in which similar changes have not been described. In the present study, we have made a temporal characterization of microscopic and immunohistochemical changes in a line of transgenic mice expressing lower copy numbers of the mutant gene. These mice, designated G5/G5, survive more than 400 days and present pathological changes which are virtually identical to those in the human disease. In fact, in these animals, anterior horn cell depletion, atrophy, astrocytosis, and the presence of numerous ubiquitinated Lewy-like bodies and axonal swellings are the main pathological features, while vacuolar pathology is minimal. This study underscores the importance of the level of expression of the mutant enzyme in the resulting clinical and pathological disease, and supports the value of this transgenic model as an excellent tool for investigating both pathogenesis of human ALS and possible therapeutic avenues. Received: 11 September 1996 / Revised, accepted: 18 October 1996