Overexpression of mutant superoxide dismutase 1 causes a motor axonopathy in the zebrafish

The development of small animal models is of major interest to unravel the pathogenesis and treatment of neurodegenerative diseases, especially because of their potential in large-scale chemical and genetic screening. We have investigated the zebrafish as a model to study amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder characterized by the selective loss of motor neurons, caused by mutations in superoxide dismutase 1 (SOD1) in a subset of patients. Overexpression of mutant human SOD1 in zebrafish embryos induced a motor axonopathy that was specific, dose-dependent and found for all mutations studied. Moreover, using this newly established animal model for ALS, we investigated the role of a known modifier in the disease: vascular endothelial growth factor (VEGF). Lowering VEGF induced a more severe phenotype, whereas upregulating VEGF rescued the mutant SOD1 axonopathy. This novel zebrafish model underscores the potential of VEGF for the treatment of ALS and furthermore will permit large-scale genetic and chemical screening to facilitate the identification of new therapeutic targets in motor neuron disease.     

Slowly progressing lower motor neuron disease caused by a novel duplication mutation in exon 1 of the SOD1 gene

Familial amyotrophic lateral sclerosis accounts for about 5% of all cases of the neurodegenerative disorder amyotrophic lateral sclerosis. Genetic mutations in Cu/Zn superoxide dismutase (SOD1) have been associated with one kind of familial amyotrophic lateral sclerosis (ALS1). We identified a novel duplication mutation in exon 1 of the SOD1 gene in a Japanese family whose members had lower motor neuron diseases. The patients showed slow disease progression, with the onset of lower limb muscle weakness and exertional dyspnea. Some patients had mild motor and sensory neuropathy and/or bladder dysfunction, which is further evidence that SOD1 mutation results in a predominantly lower motor neuron phenotype.     

Mice deficient in the Rab5 guanine nucleotide exchange factor ALS2/alsin exhibit age-dependent neurological deficits and altered endosome trafficking

ALS2/alsin is a member of guanine nucleotide exchange factors for the small GTPase Rab5 (Rab5GEFs), which act as modulators in endocytic pathway. Loss-of-function mutations in human ALS2 account for a number of juvenile recessive motor neuron diseases (MNDs). However, the normal physiological role of ALS2 in vivo and the molecular mechanisms underlying motor dysfunction are still unknown. To address these issues, we have generated mice homozygous for disruption of the Als2 gene. The Als2-null mice observed through 21 months of age demonstrated no obvious developmental, reproductive or motor abnormalities. However, immunohistochemical and electrophysiological analyses identified an age-dependent, slowly progressive loss of cerebellar Purkinje cells and disturbance of spinal motor neurons associated with astrocytosis and microglial cell activation, indicating a subclinical dysfunction of motor system in Als2-null mice. Further, quantitative epidermal growth factor (EGF)-uptake analysis identified significantly smaller-sized EGF-positive endosomes in Als2-null fibroblasts, suggesting an alteration of endosome/vesicle trafficking in the cells. Collectively, while loss of ALS2 does not produce a severe disease phenotype in mice, these Als2-null animals should provide a useful model with which to understand the interplay between endosomal dynamics and the long-term viability of large neurons such as Purkinje cells and spinal motor neurons.     

Ataxin-2 intermediate-length polyglutamine expansions in European ALS patients

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disease primarily affecting motor neurons. We recently identified intermediate-length polyglutamine (polyQ) expansions (27-33 Qs) in ataxin 2 as a genetic risk factor for sporadic ALS in North American ALS patients. To extend these findings, we assessed the ataxin 2 polyQ repeat length in 1294 European ALS patients and 679 matched healthy controls. We observed a significant association between polyQ expansions and ALS (>30 Qs; P= 6.2 × 10(-3)). Thus, intermediate-length ataxin 2 polyQ repeat expansions are associated with increased risk for ALS also in the European cohort. The specific polyQ length cutoff, however, appears to vary between different populations, with longer repeat lengths showing a clear association. Our findings support the hypothesis that ataxin 2 plays an important role in predisposing to ALS and that polyQ expansions in ataxin 2 are a significant risk factor for the disease.     

NIPA1 polyalanine repeat expansions are associated with amyotrophic lateral sclerosis

Mutations in NIPA1 cause Hereditary Spastic Paraplegia type 6, a neurodegenerative disease characterized by an (upper) motor neuron phenotype. Deletions of NIPA1 have been associated with a higher susceptibility to amyotrophic lateral sclerosis (ALS). The exact role of genetic variation in NIPA1 in ALS susceptibility and disease course is, however, not known. We sequenced the entire coding sequence of NIPA1 and genotyped a polyalanine repeat located in the first exon of NIPA1. A total of 2292 ALS patients and 2777 controls from three independent European populations were included. We identified two sequence variants that have a potentially damaging effect on NIPA1 protein function. Both variants were identified in ALS patients; no damaging variants were found in controls. Secondly, we found a significant effect of 'long' polyalanine repeat alleles on disease susceptibility: odds ratio = 1.71, P = 1.6 × 10(-4). Our analyses also revealed a significant effect of 'long' alleles on patient survival [hazard ratio (HR) = 1.60, P = 4.2 × 10(-4)] and on the age at onset of symptoms (HR = 1.37, P = 4.6 × 10(-3)). In patients carrying 'long' alleles, median survival was 3 months shorter than patients with 'normal' genotypes and onset of symptoms occurred 3.6 years earlier. Our data show that NIPA1 polyalanine repeat expansions are a common risk factor for ALS and modulate disease course.     

Deficiency in the ALS2 gene does not affect the motor neuron degeneration in SOD1(G93A) transgenic mice

Dysfunction of the ALS2 gene has been linked to one form of juvenile onset autosomal recessive amyotrophic lateral sclerosis (ALS). Previous in vitro studies suggest that over-expression of ALS2 protects cells from mutant Cu/Zn superoxide dismutase (SOD1)-induced cytotoxicity. To test whether ALS2 plays a protective role against mutant SOD1-mediated motor neuron degeneration in vivo, we examined the progression of motor neuron disease in SOD1(G93A) mice on an ALS2 null background. Our data suggest that deficiency in the ALS2 gene does not affect the pathogenesis of SOD1(G93A) mice.     

Pyruvate slows disease progression in a G93A SOD1 mutant transgenic mouse model

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease caused by selective motor neuron death, and currently no effective treatment is available for ALS. In this study, we investigated the neuroprotective effects of pyruvate, which acts as an anti-oxidant and as an energy source. We treated G93A SOD1 transgenic mice with pyruvate (from 70 days of age, i.p., at 1000 mg/kg/week), and found that it prolonged average lifespan by 12.3 days (10.5%), slowed disease progression, and improved motor performance, but did not delay disease onset. Pyruvate treatment was also associated with reduced nitrotyrosine immunoreactivity, gliosis, and increased Bcl-2 expression in the spinal cords of G93A SOD1 transgenic mice. These results suggest that pyruvate treatment may be a potential therapeutic strategy in ALS.     

AIF translocates to the nucleus in the spinal motor neurons in a mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective loss of motor neurons in the brain stem and the spinal cords. One of the causes for the familial ALS has been attributed to the mutations in copper-zinc superoxide dismutase (SOD1). Although the toxic function of the mutant enzyme has not been fully understood, the final cell death pathway has been suggested as caspase-dependent. In the present study, we present evidence that the activation of apoptosis inducing factor (AIF) may play a role to induce motor neuron death during ALS pathogenesis. In the spinal cord of SOD1 G93A transgenic mice, expression of AIF was detected in the motor neurons and astrocytes. The level of AIF expression increased as the disease progressed. In the symptomatic SOD1 G93A transgenic mice, AIF released from the mitochondria and translocated into the nucleus in the motor neurons as evidenced by confocal microscopy and biochemical analysis. These results suggest that AIF may play a role to induce motor neuron death in a mouse model of ALS.     

Vgf is a novel biomarker associated with muscle weakness in amyotrophic lateral sclerosis (ALS), with a potential role in disease pathogenesis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects nerve cells in the brain and the spinal cord. Previous proteomic evidence revealed that the content of certain peptide fragments including Vgf-derived peptide aa 398-411 (Vgf_398-411) of the precursor Vgf protein in the cerebral spinal fluid (CSF) correctly identified patients with ALS from normal and disease controls. Using quantitative ELISA immunoassay we found that the CSF levels of Vgf decreases with muscle weakness in patients with ALS. In SOD1 G93A transgenic mice, loss of full-length Vgf content in CSF, serum and in SMI-32 immunopositive spinal cord motor neurons is noted in asymptomatic animals (approximately 75 days old) and continues to show a progressive decline as animals weaken. In vitro studies show that viral-mediated exogenous Vgf expression in primary mixed spinal cord neuron cultures attenuates excitotoxic injury. Thus, while Vgf may be a reliable biomarker of progression of muscle weakness in patients with ALS, restoration of Vgf expression in spinal cord motor neurons may therapeutically rescue spinal cord motorneurons against excitotoxic injury.     

Drug Screening for ALS Using Patient-Specific Induced Pluripotent Stem Cells

Amyotrophic lateral sclerosis (ALS) is a late-onset, fatal disorder in which the motor neurons degenerate. The discovery of new drugs for treating ALS has been hampered by a lack of access to motor neurons from ALS patients and appropriate disease models. We generate motor neurons from induced pluripotent stem cells (iPSCs) from familial ALS patients, who carry mutations in Tar DNA binding protein-43 (TDP-43). ALS patient-specific iPSC-derived motor neurons formed cytosolic aggregates similar to those seen in postmortem tissue from ALS patients and exhibited shorter neurites as seen in a zebrafish model of ALS. The ALS motor neurons were characterized by increased mutant TDP-43 protein in a detergent-insoluble form bound to a spliceosomal factor SNRPB2. Expression array analyses detected small increases in the expression of genes involved in RNA metabolism and decreases in the expression of genes encoding cytoskeletal proteins. We examined four chemical compounds and found that a histone acetyltransferase inhibitor called anacardic acid rescued the abnormal ALS motor neuron phenotype. These findings suggest that motor neurons generated from ALS patient-derived iPSCs may provide a useful tool for elucidating ALS disease pathogenesis and for screening drug candidates.     

A Mutation that Creates a Pseudoexon in SOD1 Causes Familial ALS

Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease which targets motor neurons of the cortex, brainstem and spinal cord. About 5–10% of all amyotrophic lateral sclerosis cases are familial (FALS), and 15–20% of FALS cases are caused by mutations in the zinc-copper superoxide dismutase gene ( SOD1 ). We identified a large family from France with ten members affected with ALS. Linkage was established to the SOD1 locus on chromosome 21 and genomic and cDNA sequencing was performed for the SOD1 gene. This revealed an activated pseudoexon between exons 4 and 5 that was present in two tested members of the family. Translation of this 43 base pair exon results in the introduction of seven amino acids before a stop codon is present, leading to a prematurely truncated SOD1 protein product of 125 amino acids. Sequencing intron 4 in a patient revealed a heterozygous change 304 bp before exon 5 (c.358 – 304C > G), but only 5 bp after the cryptic exon, thus causing this alternative splice product. This mutation segregated in all affected individuals of the family. This adds an additional genetic mechanism for developing SOD1 -linked ALS and is one which can be more readily targeted by gene therapy.     

Androgens affect muscle,motor neuron,and survival in a mouse model of SOD1-related amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective loss of upper and lower motor neurons and skeletal muscle atrophy. Epidemiologic and experimental evidence suggest the involvement of androgens in ALS pathogenesis, but the mechanism through which androgens modify the ALS phenotype is unknown. Here, we show that androgen ablation by surgical castration extends survival and disease duration of a transgenic mouse model of ALS expressing mutant human SOD1 (hSOD1-G93A). Furthermore, long-term treatment of orchiectomized hSOD1-G93A mice with nandrolone decanoate (ND), an anabolic androgenic steroid, worsened disease manifestations. ND treatment induced muscle fiber hypertrophy but caused motor neuron death. ND negatively affected survival, thereby dissociating skeletal muscle pathology from life span in this ALS mouse model. Interestingly, orchiectomy decreased androgen receptor levels in the spinal cord and muscle, whereas ND treatment had the opposite effect. Notably, stimulation with ND promoted the recruitment of endogenous androgen receptor into biochemical complexes that were insoluble in sodium dodecyl sulfate, a finding consistent with protein aggregation. Overall, our results shed light on the role of androgens as modifiers of ALS pathogenesis via dysregulation of androgen receptor homeostasis.     

Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis

Dominant mutations in superoxide dismutase cause amyotrophic lateral sclerosis (ALS), an adult-onset neurodegenerative disease that is characterized by the loss of motor neurons. Using mice carrying a deletable mutant gene, diminished mutant expression in astrocytes did not affect onset, but delayed microglial activation and sharply slowed later disease progression. These findings demonstrate that mutant astrocytes are viable targets for therapies for slowing the progression of non-cell autonomous killing of motor neurons in ALS.     

Analysis of genome-wide copy number variation in Irish and Dutch ALS populations

Amyotrophic lateral sclerosis (ALS) is an unrelenting neurodegenerative condition characterized by adult-onset loss of motor neurons. Genetic risk factors have been implicated in ALS susceptibility. Copy number variants (CNVs) account for more inter-individual genetic variation than SNPs and have the capacity to alter gene dose and phenotype. We sought to identify the contribution both of commonly polymorphic CNVs and rare ALS-specific CNVs to sporadic ALS (SALS). Using high-density genome-wide data from 408 Irish individuals and 868 Dutch individuals and the QuantiSNP CNV-detection algorithm, we showed that no common CNV locus is significantly associated with ALS risk. However, we identified 39 recurrent CNV loci and 16 replicated ALS-specific gene dose alterations that occur exclusively in patients with ALS and do not occur in more than 11 000 previously identified CNVs in the Database of Genomic Variation. Ataxin genes and the hereditary haemochromatosis locus were implicated along with ENSG00000176605, an uncharacterized gene on chromosome 14. Our data support the hypothesis that multiple rare CNVs may contribute risk for SALS. Future work should seek to profile the contribution of CNVs located in regions not covered on the present SNP platforms.     

Wild-type and A315T mutant TDP-43 exert differential neurotoxicity in a Drosophila model of ALS

The RNA-binding protein TDP-43 has been linked to amyotrophic lateral sclerosis (ALS) both as a causative locus and as a marker of pathology. With several missense mutations being identified within TDP-43, efforts have been directed towards generating animal models of ALS in mouse, zebrafish, Drosophila and worms. Previous loss of function and overexpression studies have shown that alterations in TDP-43 dosage recapitulate hallmark features of ALS pathology, including neuronal loss and locomotor dysfunction. Here we report a direct in vivo comparison between wild-type and A315T mutant TDP-43 overexpression in Drosophila neurons. We found that when expressed at comparable levels, wild-type TDP-43 exerts more severe effects on neuromuscular junction architecture, viability and motor neuron loss compared with the A315T allele. A subset of these differences can be compensated by higher levels of A315T expression, indicating a direct correlation between dosage and neurotoxic phenotypes. Interestingly, larval locomotion is the sole parameter that is more affected by the A315T allele than wild-type TDP-43. RNA interference and genetic interaction experiments indicate that TDP-43 overexpression mimics a loss-of-function phenotype and suggest a dominant-negative effect. Furthermore, we show that neuronal apoptosis does not require the cytoplasmic localization of TDP-43 and that its neurotoxicity is modulated by the proteasome, the HSP70 chaperone and the apoptosis pathway. Taken together, our findings provide novel insights into the phenotypic consequences of the A315T TDP-43 missense mutation and suggest that studies of individual mutations are critical for elucidating the molecular mechanisms of ALS and related neurodegenerative disorders.     

Overexpression of survival motor neuron improves neuromuscular function and motor neuron survival in mutant SOD1 mice

Spinal muscular atrophy results from diminished levels of survival motor neuron (SMN) protein in spinal motor neurons. Low levels of SMN also occur in models of amyotrophic lateral sclerosis (ALS) caused by mutant superoxide dismutase 1 (SOD1) and genetic reduction of SMN levels exacerbates the phenotype of transgenic SOD1^G93A mice. Here, we demonstrate that SMN protein is significantly reduced in the spinal cords of patients with sporadic ALS. To test the potential of SMN as a modifier of ALS, we overexpressed SMN in 2 different strains of SOD1^G93A mice. Neuronal overexpression of SMN significantly preserved locomotor function, rescued motor neurons, and attenuated astrogliosis in spinal cords of SOD1^G93A mice. Despite this, survival was not prolonged, most likely resulting from SMN mislocalization and depletion of gems in motor neurons of symptomatic mice. Our results reveal that SMN upregulation slows locomotor deficit onset and motor neuron loss in this mouse model of ALS. However, disruption of SMN nuclear complexes by high levels of mutant SOD1, even in the presence of SMN overexpression, might limit its survival promoting effects in this specific mouse model. Studies in emerging mouse models of ALS are therefore warranted to further explore the potential of SMN as a modifier of ALS.     

Mutant superoxide dismutase 1 (SOD1), a cause of amyotrophic lateral sclerosis, disrupts the recruitment of SMN, the spinal muscular atrophy protein to nuclear Cajal bodies

Spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS) are among the most common motor neuron diseases to afflict the human population. A deficiency of the survival of motor neuron (SMN) protein causes SMA and is also reported to be an exacerbating factor in the development of ALS. However, pathways linking the two diseases have yet to be defined and it is not clear precisely how the pathology of ALS is aggravated by reduced SMN or whether mutant proteins underlying familial forms of ALS interfere with SMN-related biochemical pathways to exacerbate the neurodegenerative process. In this study, we show that mutant superoxide dismutase-1 (SOD1), a cause of familial ALS, profoundly alters the sub-cellular localization of the SMN protein, preventing the formation of nuclear 'gems' by disrupting the recruitment of the protein to Cajal bodies. Overexpressing the SMN protein in mutant SOD1 mice, a model of familial ALS, alleviates this phenomenon, most likely in a cell-autonomous manner, and significantly mitigates the loss of motor neurons in the spinal cord and in culture dishes. In the mice, the onset of the neuromuscular phenotype is delayed and motor function enhanced, suggestive of a therapeutic benefit for ALS patients treated with agents that augment the SMN protein. Nevertheless, this finding is tempered by an inability to prolong survival, a limitation most likely imposed by the inexorable denervation that characterizes ALS and eventually disrupts the neuromuscular synapses even in the presence of increased SMN.     

Ataxin-2 polyQ expansions in FTLD-ALS spectrum disorders in Flanders-Belgian cohorts

There exists considerable clinical and pathological overlap between frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS), which implies that these 2 neurodegenerative conditions share common pathogenic mechanisms. Recently, intermediate-length (27-33) polyglutamine (polyQ) expansions in ataxin-2 (ATXN2) have been associated with increased risk for ALS, while expansions of > 34 repeats are known to cause spinocerebellar ataxia type 2 (Sca-2). We identified in 72 ALS patients one patient with a 33 polyQ expansion that was absent in 810 control individuals. This allele was also found in one patient with concomitant ALS-Sca-2. In contrast, in a Flanders-Belgian series of 270 FTLD and 22 FTLD-ALS patients, we found no association with intermediate-length polyQ expansions nor did we observe patient-specific long expansions in agreement with the recent observation in a screening of a substantial sized cohort of patients with diverse neurodegenerative brain diseases. Our results provide further support to the notion that ATXN2 associated polyglutamine amplification is specific to the ALS-end of the FTLD-ALS disease spectrum.