Neuroprotective effect of oxidized galectin-1 in a transgenic mouse model of amyotrophic lateral sclerosis

Abnormal accumulation of neurofilaments in motor neurons is a characteristic pathological finding in amyotrophic lateral sclerosis (ALS). Recently, we revealed that galectin-1, whose oxidized form has axonal regeneration-enhancing activity, accumulates in the neurofilamentous lesions in ALS. To investigate whether oxidized galectin-1 has a beneficial effect on ALS, oxidized recombinant human galectin-1 (rhGAL-1/ox) or physiological saline was injected into the left gastrocnemius muscle of the transgenic mice over-expressing a mutant copper/zinc superoxide dismutase (SOD1) with a substitution of histidine to arginine at position 46 (H46R SOD1). The H46R SOD1 transgenic mice, which represented a new animal model of familial ALS, were subsequently assessed for their disease onset, life span, duration of illness, and motor function. Furthermore, the number of remaining large anterior horn cells of spinal cords was also compared between the two groups. The results showed that administration of rhGAL-1/ox to the mice delayed the onset of their disease and prolonged the life of the mice and the duration of their illness. Motor function, as evaluated by a Rotarod performance, was improved in rhGAL-1/ox-treated mice. Significantly more anterior horn neurons of the lumbar and cervical cords were preserved in the mice injected with rhGAL-1/ox than in those injected with physiological saline. The study suggests that rhGAL-1/ox administration could be a new therapeutic strategy for ALS.     

A dopamine receptor antagonist L-745,870 suppresses microglia activation in spinal cord and mitigates the progression in ALS model mice

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurons in the motor cortex, brainstem, and spinal cord. It has been shown that oxidative stress plays a pivotal role in the progression of this motor neuron loss. We have previously reported that L-745,870, a dopamine D4 receptor antagonist, selectively inhibits oxidative stress-induced cell death in vitro and exerts a potent neuroprotective effect against ischemia-induced neural cell damage in gerbil. To investigate the efficacy of L-745,870 in the treatment of ALS, we here conducted a chronic administration of L-745,870 to transgenic mice expressing a mutated form of human superoxide dismutase gene (SOD1^H46R); a mouse model of familial ALS, and assessed whether the mice benefit from this treatment. The pre-onset administration of L-745,870 significantly delayed the onset of motor deficits, slowed the disease progression, and extended a life span in transgenic mice. These animals showed a delayed loss of anterior horn cells in the spinal cord concomitant with a reduced level of microglial activation at a late symptomatic stage. Further, the post-onset administration of L-745,870 to the SOD1^H46R transgenic mice remarkably slowed the disease progression and extended their life spans. Taken together, our findings in a rodent model of ALS may have implication that L-745,870 is a possible novel therapeutic means to the treatment of ALS.     

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     

DJ-1 Changes in G93A-SOD1 Transgenic Mice: Implications for Oxidative Stress in ALS

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal, neurodegenerative disorder. The causes of ALS are still obscure. Accumulating evidence supports the hypothesis that oxidative stress and mitochondrial dysfunction can be implicated in ALS pathogenesis. DJ-1 plays an important role in the oxidative stress response. The aim of this study was to discover whether there are changes in DJ-1 expression or in DJ-1-oxidized isoforms in an animal model of ALS. We used mutant SOD1^G93A transgenic mice, a commonly used animal model for ALS. Upregulation of DJ-1 mRNA and protein levels were identified in the brains and spinal cords of SOD1^G93A transgenic mice as compared to wild-type controls, evident from an early disease stage. Furthermore, an increase in DJ-1 acidic isoforms was detected, implying that there are more oxidized forms of DJ-1 in the CNS of SOD1^G93A mice. This is the first report of possible involvement of DJ-1 in ALS. Since DJ-1 has a protective role against oxidative stress, it may suggest a possible therapeutic target in ALS.     

Progressive motor neuron impairment in an animal model of familial amyotrophic lateral sclerosis

Mutations of Cu,Zn superoxide dismutase cause an autosomal dominant form of familial amyotrophic lateral sclerosis. An animal model of the disease has been produced by expressing mutant human SOD1 in transgenic mice (G93A). In order to quantify the dysfunction of the motor unit in transgenic mice, electromyographic recordings were performed during the course of the disease. The first alterations in neuromuscular function appeared between P63 and P90. The deficits became even more striking after P100; compound muscle action potentials in the hindlimb decreased by 80% of initial value. Spontaneous fibrillation potentials were measured in more than 50% of transgenic mice. The number of motor units in the gastrocnemius muscle was progressively reduced over time, down to 18% of the control value at P130. Moreover, distal motor latencies increased after P120. These data suggest that the initial dysfunctions of motor unit are related to a severe motor axonal degeneration, which is followed at later periods by myelin alteration. © 1997 John Wiley & Sons, Inc.     

Restricted expression of mutant SOD1 in spinal motor neurons and interneurons induces motor neuron pathology

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective loss of motor neurons (MNs). Approximately 10% of ALS cases are familial (known as FALS), and  20% of FALS cases are caused by mutations in Cu/Zn superoxide dismutase type 1 (SOD1). Mutant (MT) SOD1 induces FALS as a result of a toxicity that remains poorly defined. Several studies suggest that the toxicity involves a non-cell autonomous mechanism. In this study, we generated transgenic mice that had a restricted and repressible expression of MTSOD1 in spinal MNs and interneurons. Although the transgenic mice were not weak, they weighed less than control mice and had pathological and immunohistochemical abnormalities of MNs confined to cells that expressed MTSOD1. These results suggest that MTSOD1-induced MN degeneration is at least partly cell autonomous. Mouse models similar to the one presented here will be valuable for spatially and temporally controlling expression of mutant genes involved in neurodegenerative diseases.     

Magnesium supplementation does not delay disease onset or increase survival in a mouse model of familial ALS

Treatment of amyotrophic lateral sclerosis (ALS) with anti-glutamate agents has had some success, but the search continues for more effective glutamate blockers. Magnesium (Mg) ions inhibit the opening of some glutamate receptors, so we increased dietary Mg in a mouse model of ALS in an attempt to modify the course of the disease. From the age of 6 weeks, mutant superoxide dismutase 1 (SOD1) transgenic mice and wild-type controls had either 0, 21.5 or 43 g/l of Mg pidolate added to their drinking water. Disease onset was measured by tests for coordination and forelimb strength, and survival by standard endpoints. Mg levels in the brain were measured in wild-type mice using mass spectrometry. Mutant SOD1 mice on no added Mg became weak at about 105 days, and survived between 114 and 137 days. No difference in either time of onset of weakness, or survival, was seen in mutant SOD1 mice on different doses of Mg. No increase in wild-type brain Mg was found after supplemental Mg. From these results, it appears that a trial of oral Mg supplementation in human ALS is not warranted.     

Familial amyotrophic lateral sclerosis: a SOD1-unrelated Japanese family of bulbar type with Bunina bodies and ubiquitin-positive skein-like inclusions in lower motor neurons

We describe a new family with adult onset amyotrophic lateral sclerosis (FALS), in which the disease was characterized clinically by relatively rapid progression of bulbar symptoms. Gene analysis of Cu/Zn superoxide dismutase (SOD1) performed in one patient showed no mutations. Autopsy of another patient demonstrated degenerative changes restricted to the upper and lower motor neuron systems; no evident changes were observed in the posterior column, Clarke’s column or spinocerebellar tracts. The presence of Bunina bodies and ubiquitin-positive skein-like inclusions in the lower motor neuron was of considerable interest. Cases of FALS with such pathological features are quite rare in the literature. Identification of the gene responsible for the disease is desirable in order to shed further light on the molecular pathology of not only familial, but also sporadic, ALS.     

Mutant SOD1 from spinal cord of G93A rats is destabilized and binds to inner mitochondrial membrane

Mutations in Cu/Zn superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS). Mechanisms of mutant SOD1 toxicity are unknown, but increased SOD1 activity can boost production of reactive oxygen species (ROS) in the mitochondrial intermembrane space (IMS). Using non-reducing SDS-PAGE we found that in G93A-SOD1 rats the mutant SOD1 was prominently destabilized only in the diseased spinal cord, where this mutant enzyme was also up regulated in the IMS with increased ability to bind the inner membrane of isolated non-transgenic mitoplasts. These mitoplasts increased ROS production when exposed to mutant SOD1 from the spinal cord at the presymptomatic stage. The levels of disulfide-reduced SOD1 peaked at the end stage of the disease, whereas protein disulfide isomerase (PDI), a chaperone capable of rearranging disulfide bonds between cysteine residues of SOD1, was increased prior to the end stage. IMS binding and increased ROS production by destabilized SOD1 may contribute to mitochondrial damage in G93A-SOD1 rats.     

Stabilization of mutant Cu/Zn superoxide dismutase (SOD1) protein by coexpressed wild SOD1 protein accelerates the disease progression in familial amyotrophic lateral sclerosis mice

Transgenic mice carrying familial amyotrophic lateral sclerosis (FALS)-linked mutant Cu/Zn superoxide dismutase (SOD1) genes such as G93A (G93A-mice) and G85R (G85R-mice) genes develop limb paresis. Introduction of human wild type SOD1 (hWT-SOD1) gene, which does not cause motor impairment by itself, into different FALS mice resulted in different effects on their clinical courses, from no effect in G85R-mice to acceleration of disease progression in G93A-mice. However, the molecular mechanism which causes the observed difference, has not been clarified. We hypothesized that the difference might be caused by the stability of mutant SOD1 proteins. Using a combination of mass spectrometry and enzyme-linked immunosorbent assay, we found that the concentration of G93A-SOD1 protein was markedly elevated in tissues of transgenic mice carrying both G93A- and hWT-SOD1 genes (G93A/hWT-mice) compared to that in G93A-mice, and also found that the concentration of G93A-SOD1 protein had a close relation to the disease duration. The concentration of metallothionein-I/II in the spinal cord, reflecting the degree of copper-mediated oxidative stress, was highest in G93A/hWT-mice, second in G93A-mice, and normal in the mice carrying hWT-SOD1 gene. These results indicated that the increase of G93A-SOD1 protein was responsible for the increase of oxidative stress and disease acceleration in G93A/hWT-mice. We speculate that coexpression of hWT-SOD1 protein is deleterious to transgenic mice carrying a stable mutant such as G93A-SOD1, because this mutant protein is stabilized by hWT-SOD1 protein, but not to transgenic mice carrying an unstable mutant such as G85R-SOD1, because this mutant protein is not stabilized by hWT-SOD1.     

Limited effects of glatiramer acetate in the high-copy number hSOD1-G93A mouse model of ALS

In amyotrophic lateral sclerosis (ALS), an involvement of the immune system in the degenerative processes has been shown in both humans and the transgenic SOD1-G93A mice. We previously showed that Glatiramer acetate (also known as copolymer-1; COP-1; Copaxone) improves motor function and extends survival times in an inbred strain of ALS mice probably by interacting with pro-inflammatory T(H) lymphocytes. In the course of this study we tested whether these beneficial effects could be reproduced by repeated vaccination of animals with COP-1 without co-administration of complete Freund's adjuvant. In an outbred strain we could not demonstrate a positive effect of COP-1 on survival times, but found a significant improvement of motor performance during the late stage of disease and a moderate decrease of the production of the inflammatory cytokines interferon-gamma and IL-4 by T lymphocytes isolated from the mice's spleen. In conclusion, the effects of COP-1 in the applied hybrid strain displaying a faster disease progression were less pronounced than in the earlier tested inbred strain of ALS mice.     

铜/锌超氧化物岐化物1突变致肌萎缩侧索硬化一家系分析并文献复习

目的探索一肌萎缩侧索硬化(ALS)家系基因突变位点并进行文献复习。方法对已知常见的ALS致病基因进行检测,进而对国内铜/锌超氧化物岐化物1(SOD1)基因突变型ALS进行文献复习。结果该家系患者平均起病年龄为(37.8±11.6)岁,均以肢体症状起病,平均病程约1.3年,死于呼吸衰竭。该家系SOD1基因4号外显子第305位存在AG突变(D102G)。目前国内报道的SOD1突变基因有26种。起病年龄最早者20岁,最晚者67岁;病程最短者仅1月,最长者达14年。86.4%的患者以肢体症状起病,4.5%以延髓症状起病,7.7%的患者以肢体和延髓症状起病。SOD1基因可表现为完全外显或不完全外显。结论 D102 G为国内首次报道的ALS疾病相关突变。不同SOD1基因突变位点临床症状具有异质性。     

Protein‐bound crotonaldehyde accumulates in the spinal cord of superoxide dismutase‐1 mutation‐associated familial amyotrophic lateral sclerosis and its transgenic mouse model

Growing evidence documents oxidative stress involvement in ALS. We previously demonstrated accumulation of a protein‐bound form of the highly toxic lipid peroxidation product crotonaldehyde (CRA) in the spinal cord of sporadic ALS patients. In the present study, to the determine the role for CRA in the disease processes of superoxide dismutase‐1 (SOD1) mutation‐associated familial ALS (FALS), we performed immunohistochemical and semiquantitative cell count analyses of protein‐bound CRA (P‐CRA) in the spinal cord of SOD1‐mutated FALS and its transgenic mouse model. Immunohistochemical analysis revealed increased P‐CRA immunoreactivity in the spinal cord of the FALS patients and the transgenic mice compared to their respective controls. In the FALS patients, P‐CRA immunoreactivity was localized in almost all of the chromatolytic motor neurons, neurofilamentous conglomerates, spheroids, cordlike swollen axons, reactive astrocytes and microglia, and the surrounding neuropil in the affected areas represented by the anterior horns. In the transgenic mice, P‐CRA immunoreactivity was localized in only a few ventral horn glia in the presymptomatic stage, in almost all of the vacuolated motor neurons and cordlike swollen axons and some of the ventral horn reactive astrocytes and microglia in the onset stage, and in many of the ventral horn reactive astrocytes and microglia in the advanced stage. Cell count analysis on mouse spinal cord sections disclosed a statistically significant increase in the density of P‐CRA‐immunoreactive glia in the ventral horns of the young to old G93A mice compared to the age‐matched control mice. The present results indicate that enhanced CRA formation occurs in motor neurons and reactive glia in the spinal cord of SOD1‐mutated FALS and its transgenic mouse model as well as sporadic ALS, suggesting implications for CRA in the pathomechanism common to these forms of ALS.     

Calcineurin A and calbindin immunoreactivity in the spinal cord of G93A superoxide dismutase transgenic mice

A qualitative immunohistochemical study was performed on calcineurin A- and calbindin-positive neurons in the spinal cord of transgenic mice, an animal model of amyotrophic lateral sclerosis, carrying the G93A mutation of the Cu/Zn-superoxide dismutase gene. The results show that calcineurin A-immunoreactive motoneurons are affected by the neurodegenerative process; in contrast, calbindin-positive cells are selectively spared. The findings suggest that calcineurin plays a role as an accessory factor responsible for selective vulnerability in the neurodegenerative process of amyotrophic lateral sclerosis.     

Methionine-free brain-derived neurotrophic factor in wobbler mouse motor neuron disease: dose-related effects and comparison with the methionyl form

We compared the clinical and pharmacodynamic effects of N-terminal methionine brain-derived neurotrophic factor (met-BDNF) and endogenous met-free BDNF in wobbler mouse motor neuron disease (MND). Met- or met-free BDNF at 5 or 20 mg/kg was subcutaneously injected daily, six times/week for 4 weeks. At 20 mg/kg, grip strength (P<0.05, met-free BDNF; P<0.01, met-BDNF) and running speed (P<0.01 for both groups) improved compared to vehicle. At 5 mg/kg, the beneficial effect was more modest. Plasma BDNF levels after the final injection were dose-dependent and did not differ between BDNF groups. Endogenous met-free BDNF exerts effects similar to met-BDNF in wobbler MND.     

Selective vulnerability of alpha motor neurons in ALS: Relation to autoantibodies toward acetylcholinesterase (AChE) in ALS patients

The degenerative process in amyotrophic lateral sclerosis (ALS) concerns primarily alpha motor neurons in the spinal cord and brain stem, and neurons forming descending pathways to the cord, especially in the pyramidal tract. Some degeneration of large peripheral sensory nerve fibers often occurs too, but preganglionic autonomie neurons and gamma motor neurons are most often spared in the disease. The vulnerability of alpha motor neurons compared to other types of neurons in ALS is discussed in relation to retrograde axoplasmic transport from peripheral blood of foreign noxious macromolecules, interneuronal transport of such molecules, and neuronal surface structure properties relevant to uptake for retrograde axoplasmic transport. Certain differences in these aspects between alpha motor neurons and other neuronal types exist. Some differences concern the neuronal turnover of acetylcholinesterase (AChE), which could be of special interest in view of the recent demonstration of regular occurrence of autoantibodies towards this enzyme in ALS patients.     

Positive effect of transplantation of hNT neurons (NTera 2/D1 cell-line) in a model of familial amyotrophic lateral sclerosis

Transplantation of hNT Neurons derived from the human teratocarcinoma cell-line (NTera2/D1) has been shown to ameliorate motor dysfunction in a number of injury or disease models in which the deficits are fairly localized. However, these cells have not been used before in a model with more extensive neurodegeneration. The aim of this study is to determine the effects of hNT Neuron transplants on motor neuron function in a mouse model of familial amyotrophic lateral sclerosis (FALS) in which there is a substitution of Alanine for Glycine at position 93 of the human SOD1 gene (G93A). Amyotrophic lateral sclerosis is a fatal degenerative motor neuron disease affecting the spinal cord, brainstem, and cortex. This disease clinically manifests as progressive muscular weakness and atrophy, leading to paralysis and death within 3-5 years of diagnosis. The FALS represents 10-13% of all cases. A range of behavioral tests was used to examine spontaneous locomotor activity, coordination, and muscle strength of mice. Long-term (10-11 weeks) transplantation of hNT Neurons into the L(4)-L(5) segments of the ventral horn spinal cord of FALS(G93A) mice at 7 weeks of age (before onset of overt behavioral symptoms of disease) delayed the onset of motor dysfunction for at least 3 weeks. The average lifespan of the transplanted mice was 128 days compared to 106 days for media-injected group. The last mouse in the hNT Neuron transplanted group was euthanized at 135 days of age when it display partial paralysis of the hindlimbs. Immunohistochemical analysis of the implanted spinal cords demonstrated the survival of grafted hNT Neurons and showed many healthy-appearing motor neurons near the implant site. These results suggest that hNT Neuron transplantation may be a promising therapeutic strategy for ALS.     

Ammonium tetrathiomolybdate delays onset, prolongs survival, and slows progression of disease in a mouse model for amyotrophic lateral sclerosis

Mutations in copper/zinc superoxide dismutase (SOD1) cause a form of familial amyotrophic lateral sclerosis (ALS). The pathogenesis of familial ALS may be associated with aberrant copper chemistry through a cysteine residue in mutant SOD1. Ammonium tetrathiomolybdate (TTM) is a copper-chelating drug that is capable of removing a copper ion from copper-thiolate clusters, such as SOD1. We found that TTM exerted therapeutic benefits in a mouse model of familial ALS (SOD1^G93A). TTM treatment significantly delayed disease onset, slowed disease progression and prolonged survival by approximately 20%, 42% and 25%, respectively. TTM also effectively depressed the spinal copper ion level and inhibited lipid peroxidation, with a significant suppression of SOD1 enzymatic activity in SOD1^G93A. These results support the hypothesis that aberrant copper chemistry through a cysteine residue plays a critical role in mutant SOD1 toxicity and that TTM may be a promising therapy for familial ALS with SOD1 mutants.