Cytoskeletal abnormalities in amyotrophic lateral sclerosis: beneficial or detrimental effects?

Cytoskeletal abnormalities have been reported in cases of amyotrophic lateral sclerosis (ALS) including abnormal inclusions containing neurofilaments (NFs) and/or peripherin, reduced mRNA levels for the NF light (NF-L) protein and mutations in the NF heavy (NF-H) gene. Recently, transgenic mouse approaches have been used to address whether cytoskeletal changes may contribute to motor neuron disease. Mice lacking one of the three NF subunits are viable and do not develop motor neuron disease. Nonetheless, mice with null mutations for NF-L or for both NF-M and NF-H genes developed severe atrophy of ventral and dorsal root axons. The atrophic process is associated with hind limb paralysis during aging in mice deficient for both NF-M and NF-H proteins. The overexpression in mice of transgenes coding for wild-type or mutant NF proteins can provoke abnormal NF accumulations, axonal atrophy and sometimes motor dysfunction. However, the perikaryal NF accumulations are generally well tolerated by motor neurons and, except for expression of a mutant NF-L transgene, they did not provoke massive motor neuron death. Increasing the levels of perikaryal NF proteins may even confer protection in motor neuron disease caused by ALS-linked mutations in the superoxide dismutase (SOD1). In contrast, the overexpression of wild-type peripherin, a type of IF gene upregulated by inflammatory cytokines, provoked the formation of toxic IF inclusions with the high-molecular-weight NF proteins resulting in the death of motor neurons during aging. These results together with the detection of peripherin inclusions at early stage of disease in mice expressing mutant SOD1 suggest that IF inclusions containing peripherin may play a contributory role in ALS pathogenesis.     

The neuroprotective factor Wlds does not attenuate mutant SOD1-mediated motor neuron disease

Selective degeneration and death of motor neurons in SOD1 mutant-mediated amyotrophic lateral sclerosis (ALS) is accompanied by axonal disorganization and reduced slow axonal transport in the three most frequently used mouse models of mutant SOD1-mediated ALS. To test whether suppression of axonal degeneration (frequently known as Wallerian degeneration) could slow disease development, we took advantage of a spontaneous mouse mutant Wld(s) (Wallerian degeneration slow) in which the programmed axonal degenerative process that is normally activated after axonal injury is significantly delayed. Despite its effectiveness in delaying axonal loss in other neurodegenerative models, the presence of Wld(s) did not slow disease onset, ameliorate mutant motor neuron death, axonal degeneration, or preserve synaptic attachments in mice that develop disease from ALS-linked SOD1 mutants SOD1G37R or SOD1G85R. However, presynaptic endings in both the presence and absence of Wld(s) showed high accumulations of mitochondria and synaptic vesicles, implicating errors of retrograde transport as a consequence of SOD1-mutant damage to axons.     

Targeting of monomer/misfolded SOD1 as a therapeutic strategy for amyotrophic lateral sclerosis

There is increasing evidence that toxicity of mutant superoxide dismutase-1 (SOD1) in amyotrophic lateral sclerosis (ALS) is linked to its propensity to misfold and to aggregate. Immunotargeting of differently folded states of SOD1 has provided therapeutic benefit in mutant SOD1 transgenic mice. The specific region(s) of the SOD1 protein to which these immunization approaches target are, however, unknown. In contrast, we have previously shown, using a specific antibody [SOD1 exposed dimer interface (SEDI) antibody], that the dimer interface of SOD1 is abnormally exposed both in mutant SOD1 transgenic mice and in familial ALS cases associated with mutations in the SOD1 gene (fALS1). Here, we show the beneficial effects of an active immunization strategy using the SEDI antigenic peptide displayed on a branched peptide dendrimer to target monomer/misfolded in SOD1(G37R) and SOD1(G93A) mutant SOD1 transgenic mice. Immunization delayed disease onset and extended disease duration, with survival times increased by an average of 40 d in SOD1(G37R) mice. Importantly, this immunization strategy favored a Th2 immune response, thereby precluding deleterious neuroinflammatory effects. Furthermore, the beneficial effects of immunization correlated with a reduction in accumulation of both monomer/misfolded and oligomeric SOD1 species in the spinal cord, the intended targets of the immunization strategy. Our results support that SOD1 misfolding/aggregation plays a central role in SOD1-linked ALS pathogenesis and identifies monomeric/misfolded SOD1 as a therapeutic target for SOD1-related ALS.     

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.     

Therapeutic immunization with a glatiramer acetate derivative does not alter survival in G93A and G37R SOD1 mouse models of familial ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. The cause of motor neuron degeneration remains largely unknown, and there is no potent treatment. Overexpression of various human mutant superoxide dismutase-1 (SOD1) genes in mice and rats recapitulates some of the clinical and pathological characteristics of sporadic and familial ALS. Glatiramer acetate (GA) is an approved drug for the treatment of multiple sclerosis and neuroprotective properties in some neurodegenerative conditions. A recent report suggested that GA immunization could delay disease progression in some, but not all, G93A SOD1 transgenic mouse models of amyotrophic lateral sclerosis (ALS). Moreover, it has been theorized that derivatives of GA could enhance immunogenicity and positively affect disease outcomes. The purpose of our study was to assess the neuroprotective efficacy of TV-5010, a high molecular weight GA, in three different SOD1 mutant mouse models. We used large numbers of two SOD1 transgenic mouse strains overexpressing the G93A mutation, B6SJL-TgN[SOD1-G93A]1Gur and B6.Cg-Tg(SOD1-G93A)1Gur/J, and the SOD1 mutant mouse overexpressing G37R (line 29). Regardless of the frequency of injections and the dose, treatment with TV-5010 was ineffective at altering either disease onset or survival in both SOD1 G93A mutants used and in the SOD1 G37R transgenic mice; in multiple studies, disease was accelerated. These studies suggest that, at a range of dosing regimens and carrier used, TV-5010 immunization was ineffective in delaying disease in multiple preclinical therapeutic models for ALS. The biological response in animals, and ultimate clinical translation, will ultimately be dependent on careful and appropriate dose, route and carrier paradigms.     

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.     

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.     

Nuclear localization of human SOD1 and mutant SOD1-specific disruption of survival motor neuron protein complex in transgenic amyotrophic lateral sclerosis mice

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disease that causes degeneration of motor neurons and paralysis. Approximately 20% of familial ALS cases have been linked to mutations in the copper/zinc superoxide dismutase (SOD1) gene, but it is unclear how mutations in the protein result in motor neuron degeneration. Transgenic (tg) mice expressing mutated forms of human SOD1 (hSOD1) develop clinical and pathological features similar to those of ALS. We used tg mice expressing hSOD1-G93A, hSOD1-G37R, and hSOD1-wild-type to investigate a new subcellular pathology involving mutant hSOD1 protein prominently localizing to the nuclear compartment and disruption of the architecture of nuclear gems. We developed methods for extracting relatively pure cell nucleus fractions from mouse CNS tissues and demonstrate a low nuclear presence of endogenous SOD1 in mouse brain and spinal cord, but prominent nuclear accumulation of hSOD1-G93A, -G37R, and -wild-type in tg mice. The hSOD1 concentrated in the nuclei of spinal cord cells, particularly motor neurons, at a young age. The survival motor neuron protein (SMN) complex is disrupted in motor neuron nuclei before disease onset in hSOD1-G93A and -G37R mice; age-matched hSOD1-wild-type mice did not show SMN disruption despite a nuclear presence. Our data suggest new mechanisms involving hSOD1 accumulation in the cell nucleus and mutant hSOD1-specific perturbations in SMN localization with disruption of the nuclear SMN complex in ALS mice and suggest an overlap of pathogenic mechanisms with spinal muscular atrophy.     

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.     

Development of a rat model of amyotrophic lateral sclerosis expressing a human SOD1 transgene

Mutations in copper–zinc superoxide dismutase gene (SOD1) have been linked to some familial cases of ALS. We report here that rats that express a human SOD1 transgene with two different ALS‐associated mutations (G93A and H46R) develop striking motor neuron degeneration and paralysis. By comparing the two transgenic rats with different SOD1 mutations, we demonstrate that the time course in these rats was similar to human SOD1‐mediated familial ALS. As in the human disease and transgenic ALS mice, pathological analysis shows selective loss of motor neurons in the spinal cords of these transgenic rats. In addition, typical neuronal Lewy body‐like hyaline inclusions as well as astrocytic hyaline inclusions identical to those in human familial ALS are observed in the spinal cords. The larger size of this rat model as compared with the ALS mice will facilitate studies involving manipulations of spinal fluid (implantation of intrathecal catheters for chronic therapeutic studies; CSF sampling) and spinal cord (e.g., direct administration of viral‐ and cell‐mediated therapies).     

Minocycline slows disease progression in a mouse model of amyotrophic lateral sclerosis

There is currently no effective pharmacological treatment for amyotrophic lateral sclerosis (ALS). Because recent evidence suggests that secondary inflammation and caspase activation may contribute to neurodegeneration in ALS, we tested the effects of minocycline, a second-generation tetracycline with anti-inflammatory properties, in mice expressing a mutant superoxide dismutase (SOD1(G37R)) linked to human ALS. Administration of minocycline into the diet, beginning at late presymptomatic stage (7 or 9 months of age), delayed the onset of motor neuron degeneration, muscle strength decline, and it increased the longevity of SOD1(G37R) mice by approximately 5 weeks for approximately 70% of tested mice. Moreover, less activation of microglia was detected at early symptomatic stage (46 weeks) and at the end stage of disease in the spinal cord of SOD1(G37R) mice treated with minocycline. These results indicate that minocycline, which is clinically well tolerated, may represent a novel and effective drug for treatment of ALS.     

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.     

Dorfin ameliorates phenotypes in a transgenic mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by progressive motor neuron degeneration and leads to death within a few years of diagnosis. One of the pathogenic mechanisms of ALS is proposed to be a dysfunction in the protein quality‐control machinery. Dorfin has been identified as a ubiquitin ligase (E3) that recognizes and ubiquitinates mutant SOD1 proteins, thereby accelerating their degradation and reducing their cellular toxicity. We examined the effects of human Dorfin overexpression in G93A mutant SOD1 transgenic mice, a mouse model of familial ALS. In addition to causing a decrease in the amount of mutant SOD1 protein in the spinal cord, Dorfin overexpression ameliorated neurological phenotypes and motor neuron degeneration. Our results indicate that Dorfin overexpression or the activation or induction of E3 may be a therapeutic avenue for mutant SOD1‐associated ALS. © 2009 Wiley‐Liss, Inc.     

Long term proteasome inhibition does not preferentially afflict motor neurons in organotypical spinal cord cultures.

Ubiquitinated inclusions are a constant feature of amyotrophic lateral sclerosis (ALS). It has been hypothesised that these inclusions reflect overload or failure of the ubiquitin-proteasome system, and that this failure contributes to the degeneration of motor neurons. In the present study we have examined the effect of low concentrations of proteasome inhibitors on protein aggregation and viability of neurons in organotypical spinal cord cultures. We found a dose-dependent degeneration of neurons after a one-week exposure to the proteasome inhibitors lactacystin and epoxomicin. Neuronal degeneration was associated with an increase in poly-ubiquitination, consistent with failure of the ubiquitin-proteasome system. Proteasome inhibition caused degeneration of both motor neurons and interneurons, and no difference in survival between motor neurons and interneurons was observed. Since protein aggregation may particularly play a role in ALS patients with superoxide dismutase 1 (SOD1) mutations, we have compared the effect of proteasome inhibition between spinal cord cultures from non-transgenic and SOD1(G93A) transgenic mice. There was no difference between the viability of motor neurons from transgenic and non-transgenic mice.     

GluR2 deficiency accelerates motor neuron degeneration in a mouse model of amyotrophic lateral sclerosis

AMPA receptor-mediated excitotoxicity has been implicated in the selective degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). Motor neurons in vitro are particularly vulnerable to excessive AMPA receptor stimulation and one of the factors underlying this selective vulnerability is the presence of a large proportion of Ca2+ -permeable (i.e. GluR2-lacking) AMPA receptors. However, the precise role of GluR2-lacking AMPA receptors in motor neuron degeneration remains to be defined. We therefore studied the impact of GluR2 deficiency on motor neuron death in vitro and in vivo. Cultured motor neurons from GluR2-deficient embryos displayed an increased Ca2+ influx through AMPA receptors and an increased vulnerability to AMPA receptor-mediated excitotoxicity. We deleted the GluR2 gene in mutant SOD1G93A mice by crossbreeding them with GluR2 knockout mice. GluR2 deficiency clearly accelerated the motor neuron degeneration and shortened the life span of mutant SOD1G93A mice. These findings indicate that GluR2 plays a pivotal role in the vulnerability of motor neurons in vitro and in vivo, and that therapies that limit Ca2+ entry through AMPA receptors might be beneficial in ALS patients.     

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.     

Lack of changes in the PI3K/AKT survival pathway in the spinal cord motor neurons of a mouse model of familial amyotrophic lateral sclerosis

The vulnerability of motor neurons in transgenic SOD1G93A mice, a model of familial amyotrophic lateral sclerosis (ALS), may depend on the failure of these cells to activate survival mechanisms in response to the toxic mutant SOD1. To test this we investigated whether defects in the PI3K/Akt pathway, a survival signal, and of its neuron-specific activator, Rai, were important for motor neuron degeneration in these mice. No substantial changes were found in the levels of Rai, PI3K(p85) or phosphorylated Akt (P-Akt) in the ventral horn of spinal cord of SOD1G93A mice during disease progression. P-Akt immunoreactivity was the same in degenerating and healthy motor neurons. Rai ablation in SOD1G93A mice slightly accelerated the motor dysfunction without affecting their life span. Thus, motor neurons in SOD1G93A mice do not lose the pro-survival PI3K/Akt signal nor increase it in order to suppress the cell death mechanisms.     

Effect of rosmarinic acid in motor dysfunction and life span in a mouse model of familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a late‐onset progressive neurodegenerative disease affecting motor neurons. About 2% of patients with the disease are associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). The purpose of this study is to assess the effect of rosemary extract and its major constituents, rosmarinic acid (RA) and carnosic acid (CA), in human SOD1 G93A transgenic mice, which are well‐established mouse models for ALS. The present study demonstrates that intraperitoneal administration of rosemary extract or RA from the presymptomatic stage significantly delayed motor dysfunction in paw grip endurance tests, attenuated the degeneration of motor neurons, and extended the life span of ALS model mice. In addition, RA administration significantly improved the clinical score and suppressed body weight loss compared with a vehicle‐treated group. In conclusion, this study provides the first report that rosemary extract and, especially, RA have preventive effects in the mouse model of ALS. © 2009 Wiley‐Liss, Inc.     

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.     

Spinal inhibitory interneuron pathology follows motor neuron degeneration independent of glial mutant superoxide dismutase 1 expression in SOD1-ALS mice

Motor neuron degeneration and skeletal muscle denervation are hallmarks of amyotrophic lateral sclerosis (ALS), but other neuron populations and glial cells are also involved in ALS pathogenesis. We examined changes in inhibitory interneurons in spinal cords of the ALS model low-copy Gurney G93A-SOD1 (G1del) mice and found reduced expression of markers of glycinergic and GABAergic neurons, that is, glycine transporter 2 (GlyT2) and glutamic acid decarboxylase (GAD65/67), specifically in the ventral horns of clinically affected mice. There was also loss of GlyT2 and GAD67 messenger RNA-labeled neurons in the intermediate zone. Ubiquitinated inclusions appeared in interneurons before 20 weeks of age, that is, after their development in motor neurons but before the onset of clinical signs and major motor neuron degeneration, which starts from 25 weeks of age. Because mutant superoxide dismutase 1 (SOD1) in glia might contribute to the pathogenesis, we also examined neuron-specific G93A-SOD1 mice; they also had loss of inhibitory interneuron markers in ventral horns and ubiquitinated interneuron inclusions. These data suggest that, in mutant SOD1-associated ALS, pathological changes may spread from motor neurons to interneuronsin a relatively early phase of the disease, independent of the presence of mutant SOD1 in glia. The degeneration of spinal inhibitory interneurons may in turn facilitate degeneration of motor neurons and contribute to disease progression.