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.     

Molecular analysis of the superoxide dismutase 1 gene in Spanish patients with sporadic or familial amyotrophic lateral sclerosis

We performed a genetic analysis of the Cu/Zn superoxide dismutase gene (SOD1) in Spanish patients with sporadic or familial amyotrophic lateral sclerosis (ALS). We found mutations in 2 of 11 families (18%) with ALS. In addition, 1 of the 87 sporadic ALS patients studied harbored a mutation in the same gene. We identified G37R in exon 2 of the SOD1 gene in 1 family. Another patient, with sporadic ALS, showed a novel N65S in exon 3. In addition, we found a novel I112M in exon 4 in another family. Our data highlight the genetic heterogeneity of patients with ALS harboring mutations in the SOD1 gene and confirm that families with autosomal dominant inheritance of the trait, regardless of their ethnic background, are more likely to carry mutations in such a gene.     

Neuropathology of familial amyotrophic lateral sclerosis patients with superoxide dismutase 1 gene mutation

More than 60 mutations of the copper/zinc superoxide dismutase 1 (SOD1) gene have been identified. We are aware of 19 reported autopsied cases of familial amyotrophic lateral sclerosis (ALS) linked to these mutations. A review of these cases disclosed remarkable heter-ogenicity of ALS, not only in molecular genetics but also clinicopathologically. However, it is noteworthy that all patients with alanine to valine substitution at codon 4 (A4V) mutation of SOD1 in familial ALS apparently disclose a distinct characteristic phenotype. All these patients manifested a rapid course of progressive muscular atrophy and died less than 1 year after the onset of illness. Microscopic findings were essentially identical in three cases: (i) marked loss of anterior horn neurons and Clarke's nuclei; (ii) the presence of intracytoplasmic Lewy body like hyalin inclusions and cord-like enlargements of the processes in some of the affected neurons. The Lewy body like inclusions were also recognized by antibodies to phos-phorylated neurofilaments protein, ubiquitin, and SOD1. Under electron microscopy, the inclusions consisted of a network of 10 nm neurofilaments intermingled with ill-defined coarse linear structures; (iii) degeneration of spinocerebellar tracts, and middle root zone of the posterior column.     

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.     

Familial amyotrophic lateral sclerosis

The increasing complexity of the pathways implicated in the pathogenesis of familial amyotrophic lateral sclerosis (ALS) has stimulated intensive research in many directions. Genetic analysis of familial ALS has yielded six loci and one disease gene (SOD1), initially suggesting a role for free radicals in the disease process, although the mechanisms through which the mutant exerts toxicity and results in selective motor neuron death remain uncertain. Numerous studies have focused on structural elements of the affected cell, emphasizing the role of neurofilaments and peripherin and their functional disruption in disease. Other topics examined include cellular homeostasis of copper and calcium, particularly in the context of oxidative stress and the processes of protein aggregation, glutamate excitotoxicity, and apoptosis. It has become evident that there is considerable interplay between these mechanisms and, as the role of each is established, a common picture may emerge, enabling the development of more targeted therapies. This study discusses the main areas of investigation and reviews the findings.     

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.     

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.     

Impaired hypoxic sensor Siah‐1, PHD3, and FIH system in spinal motor neurons of an amyotrophic lateral sclerosis mouse model

We recently reported spinal blood flow–metabolism uncoupling in the Cu/Zn‐superoxide dismutase 1 (SOD1)‐transgenic (Tg) mouse model of amyotrophic lateral sclerosis (ALS), suggesting relative hypoxia in the spinal cord. However, the hypoxic stress sensor pathway in ALS has not been well studied. In the present work, we examined the temporal and spatial changes of hypoxic stress sensor proteins (Siah‐1, PHD3, and FIH) following motor neuron (MN) degeneration in the spinal cord of normoxic ALS mice. The expression of Siah‐1 and PHD3 proteins progressively increased in the surrounding glial cells of presymptomatic Tg mice (10 weeks, 10 weeks) compared with the large MN of the anterior horn. In contrast, a significant reduction in Siah‐1 and PHD3 protein expression was evident in end‐stage ALS mice (18 weeks, 18 weeks). Double‐immunofluorescence analysis revealed PHD3 plus Siah‐1 double‐positive cells in the surrounding glia of symptomatic Tg mice (14–18 weeks), with no change in the large MNs. In contrast, FIH protein expression decreased in the surrounding glial cells of Tg mice at end‐stage ALS (18 weeks). The present study suggests a partial loss in the neuroprotective response of spinal MNs in ALS results from a relative hypoxia through the Siah‐1, PHD3, and FIH system under normoxic conditions. This response could be an important mechanism of neurodegeneration in ALS. © 2012 Wiley Periodicals, Inc.     

Coexistence of dominant and recessive familial amyotrophic lateral sclerosis with the D90A Cu,Zn superoxide dismutase mutation within the same country

The Cu,Zn superoxide dismutase (Cu,Zn SOD) mutations described in amyotrophic lateral sclerosis (ALS) have, for the most part, a dominant influence. However, while a few cases with a heterozygous D90A mutation have been described in different countries, D90A has been recently proven to be recessively inherited with a common founder effect in Scandinavia. We screened French ALS families for Cu,Zn SOD mutations. The presence of the D90A allele was found in two index-cases, and their families were subsequently studied. In the first family the ALS patients were homozygotes for D90A, while in the second, all ALS patients were heterozygotes. In both families the disease was found to initially involve the lower limbs with slower progression than in sporadic cases, and frequent atypical signs such as paresthesia and urgency of micturition. We determined the D90A allele frequency in controls (n = 200) and sporadic ALS patients (n = 408). No D90A allele was found. This is the first report of coexistence of dominant and recessive families with the D90A Cu,Zn SOD mutation within the same country.     

Effect of ubiquitin expression on neuropathogenesis in a mouse model of familial amyotrophic lateral sclerosis

The ubiquitin-proteasome system (UPS) is a central component in the cellular defence against potentially toxic protein aggregates. UPS dysfunction is linked to the pathogenesis of both sporadic and inherited neurodegenerative diseases, including dominantly inherited familial amyotrophic lateral sclerosis (fALS). To investigate the role of the UPS in fALS pathogenesis, transgenic mice expressing mutant G9 3A Cu,Zn superoxide dismutase (SOD1) were crossed with transgenic mice expressing epitope tagged, wild-type or dominant-negative mutant ubiquitin (Ub(K48R)). RNase protection assays were used to confirm expression of the Ub transgenes in spinal cord and ubiquitin transgene levels were estimated to account for 9-12% of total ubiquitin. Mice expressing the G9 3A transgene exhibited neurological symptoms and histopathological changes typical of this model irrespective of ubiquitin transgene status. Impaired rotarod performance was observed in all G9 3A transgenics by 7 weeks of age irrespective of ubiquitin genotype. The presence of wild-type or mutant ubiquitin transgenes resulted in a small but significant delay in the onset of clinical symptoms and mild acceleration of disease progression, without influencing overall survival. These data suggest that relatively small changes in ubiquitin expression can influence the development of neurodegenerative disease and are consistent with a neuroprotective role for the UPS.     

Myelin degeneration induced by mutant superoxide dismutase 1 accumulation promotes amyotrophic lateral sclerosis

Myelin is a specialized membrane that wraps around nerve fibers and is essential for normal axonal conduction in neurons. In the central nervous system, oligodendrocytes are responsible for myelin formation. Recent studies have reported pathological abnormalities in oligodendrocytes in human patients with amyotrophic lateral sclerosis (ALS) and a mouse model of ALS expressing the G93A mutation of the human superoxide dismutase 1 (mtSOD1). However, it is unclear whether oligodendrocyte pathology in ALS represents the primary dysfunction induced by mtSOD1 and how mtSOD1 contributes to oligodendrocyte degeneration and ALS pathogenesis. We analyzed GAL4-VP16-UAS transgenic zebrafish selectively expressing mtSOD1 in mature oligodendrocytes. We observed that mtSOD1 directly induced oligodendrocyte degeneration by disrupting the myelin sheath and downregulating monocarboxylate transporter 1 (MCT1), thereby causing spinal motor neuron degeneration. Pathological changes observed in this transgenic zebrafish were similar to the pathology observed in the SOD1^G93A mouse model of ALS, which is characterized by expression of mtSOD1 in all cells. In addition, oligodendrocyte dysfunction induced by mtSOD1 was associated with anxiety-related behavioral abnormalities, learning impairments, and motor defects in the early symptomatic stage. We also found that treatment with potassium channel inhibitors rescued behavioral abnormalities without rescuing MCT1 expression, suggesting that myelin disruption induces behavioral abnormalities independently of MCT1. These results indicate that mtSOD1-induced dysfunction of mature oligodendrocytes is sufficient to induce motor neuron degeneration, thus informing future therapeutic strategies targeted at oligodendrocytes in ALS.     

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.     

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。     

一个肌萎缩侧索硬化家系突变超氧化物歧化酶1相互作用蛋白的酵母双杂交筛选

The present study screened a human fetal brain cDNA library to find the proteins that interact with mutant superoxide dismutase 1 (SOD1) using a yeast two-hybrid system. Using BLAST software, 15 real proteins which interacted with mutant SOD1 were obtained, including 8 known proteins (protein tyrosine-phosphatase non-receptor type 2, TBC1D4, protein kinase family, splicing factor, arginine/serine-rich 2, SRC protein tyrosine kinase Fyn, β-sarcoglycan; glycine receptor α2, micro-tubule associated protein/microtubule affinity-regulating kinase 1, ferritin H chain), and 7 unknown proteins. Results demonstrated interaction of mutant SOD1 with microtubule associated pro-tein/microtubule affinity-regulating kinase 1 and β-sarcoglycan.     

The roles of free radicals in amyotrophic lateral sclerosis

The mutations of the Cu,Zn superoxide dismutase (Cu,Zn-SOD) gene observed in amyotrophic lateral sclerosis (ALS) patients suggest that free radicals play a role in this fatal disease. Free radicals trigger oxidative damage to proteins, membrane lipids, and DNA, thereby destroying neurons. Mutations of the SOD gene may reduce its superoxide dismutase activity, thereby elevating free radical levels. In addition, the mutant SOD protein may function as a peroxidase to oxidize cellular components, and it may also react with peroxynitrite—a product of the reaction between superoxide and nitric oxide—to ultimately form nitrate proteins. The selective degeneration of motor neurons in ALS may be caused by the high level of Cu,Zn-SOD present in and the large number of glutamatergic synapses projecting to these neurons. Free radical-triggered and age-accumulated oxidation may modify the program controlling motor neuron death, thereby initiating apoptosis of motor neurons in young adults.     

The motor cortex and amyotrophic lateral sclerosis

On theoretical grounds, abnormalities of the motor cortex in patients with amyotrophic lateral sclerosis (ALS) could lead to anterograde ("dying-forward") transneuronal degeneration of the anterior horn cells as suggested by Charcot. Conversely, retrograde ("dying-back") degeneration of the corticospinal tracts could affect the motor cortex. Evidence derived from clinical, neuropathological, static, and functional imaging, and physiological studies, favors the occurrence of anterograde degeneration. It is hypothesized that transneuronal degeneration in ALS is an active excitotoxic process in which live but dysfunctional corticomotoneurons, originating in the primary motor cortex, drive the anterior horn cell into metabolic deficit. When this is marked, it will result in more rapid and widespread loss of lower motor neurons. In contrast, slow loss of corticomotoneurons, as occurs in primary lateral sclerosis (PLS), precludes excitotoxic drive and is incompatible with anterograde degeneration. Preservation of slow-conducting non-M1 direct pathways in PLS is not associated with excitotoxicity, and anterior horn cells survive for long periods of time.     

Extensive exercise is not harmful in amyotrophic lateral sclerosis

Whether physical activity increases risk or promotes progression of motor neurone degeneration in amyotrophic lateral sclerosis (ALS) is still debated. Current pathophysiological hypotheses include excitotoxicity, oxidative stress and increased calcium loads as causes of selective degeneration of vulnerable motor neurones. Vigorous exercise might amplify these factors by increasing firing rates at motor neurones. To test this hypothesis, we constrained a transgenic mouse model of ALS overexpressing the mutant human form of the Cu/Zn superoxide dismutase-1 (SOD-1) to a lifetime exercise on motor-driven running wheels for 10 h daily (active group, n = 12). Onset and progression of disease were assessed by grip strength, stride length and tight rope test. Data were compared with SOD-1 mice placed in running wheels set to slow speed (sedentary group, n = 13). Untreated SOD-1 mice were an additional control group (n = 12). We found no differences in disease onset, which was determined by a change-point analysis using an iterative fitting of segmented linear regression models, or in disease progression. However, the running group showed a non-significant 6-day improvement in survival (133.7 +/- 3.2 days) compared with the sedentary group (127.2 +/- 3.2 days) and a 4-day improvement compared with the control group (129.1 +/- 2.5 days). We demonstrate that a lifetime of vigorous exercise does not promote onset or progression of motor degeneration in SOD-1-mediated ALS. Moreover, the results suggest that the level of excitatory input and calcium turnover at motor neurones, both of which should be increased by running activity, do not interfere with the pathophysiology of SOD-1-mediated ALS.     

A novel mutation of SOD-1 (Gly 108 Val) in familial amyotrophic lateral sclerosis

A novel mutation of the SOD-1 gene which encodes the enzyme copper-zinc superoxide dismutase was identified in a family manifesting amyotrophic lateral sclerosis (ALS) in three generations. The mutation is a heterozygote point mutation in exon 4, codon 108 (GGA to GTA), predicting the substitution of valine for glycine. The mutation creates a new restriction site for the endonuclease AccI. The mutation was demonstrated in two affected members of the family, who show features of autosomal dominant inheritance of ALS, but variable age at onset ranging from 48 to 72 years. Over 30 different mutations of SOD-1 have now been identified in families with ALS. The definition of the different mutations causing human disease may allow further investigation of their pathogenicity in transgenic animal models, and also offers insight into the variable phenotypic disease expression both within and between genotypes.