Novel mutations in TARDBP (TDP-43) in patients with familial amyotrophic lateral sclerosis

The TAR DNA-binding protein 43 (TDP-43) has been identified as the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U), defining a novel class of neurodegenerative conditions: the TDP-43 proteinopathies. The first pathogenic mutations in the gene encoding TDP-43 (TARDBP) were recently reported in familial and sporadic ALS patients, supporting a direct role for TDP-43 in neurodegeneration. In this study, we report the identification and functional analyses of two novel and one known mutation in TARDBP that we identified as a result of extensive mutation analyses in a cohort of 296 patients with variable neurodegenerative diseases associated with TDP-43 histopathology. Three different heterozygous missense mutations in exon 6 of TARDBP (p.M337V, p.N345K, and p.I383V) were identified in the analysis of 92 familial ALS patients (3.3%), while no mutations were detected in 24 patients with sporadic ALS or 180 patients with other TDP-43-positive neurodegenerative diseases. The presence of p.M337V, p.N345K, and p.I383V was excluded in 825 controls and 652 additional sporadic ALS patients. All three mutations affect highly conserved amino acid residues in the C-terminal part of TDP-43 known to be involved in protein-protein interactions. Biochemical analysis of TDP-43 in ALS patient cell lines revealed a substantial increase in caspase cleaved fragments, including the approximately 25 kDa fragment, compared to control cell lines. Our findings support TARDBP mutations as a cause of ALS. Based on the specific C-terminal location of the mutations and the accumulation of a smaller C-terminal fragment, we speculate that TARDBP mutations may cause a toxic gain of function through novel protein interactions or intracellular accumulation of TDP-43 fragments leading to apoptosis.     

FUS and TARDBP but not SOD1 interact in genetic models of amyotrophic lateral sclerosis

Mutations in the SOD1 and TARDBP genes have been commonly identified in Amyotrophic Lateral Sclerosis (ALS). Recently, mutations in the Fused in sarcoma gene (FUS) were identified in familial (FALS) ALS cases and sporadic (SALS) patients. Similarly to TDP-43 (coded by TARDBP gene), FUS is an RNA binding protein. Using the zebrafish (Danio rerio), we examined the consequences of expressing human wild-type (WT) FUS and three ALS-related mutations, as well as their interactions with TARDBP and SOD1. Knockdown of zebrafish Fus yielded a motor phenotype that could be rescued upon co-expression of wild-type human FUS. In contrast, the two most frequent ALS-related FUS mutations, R521H and R521C, unlike S57Δ, failed to rescue the knockdown phenotype, indicating loss of function. The R521H mutation caused a toxic gain of function when expressed alone, similar to the phenotype observed upon knockdown of zebrafish Fus. This phenotype was not aggravated by co-expression of both mutant human TARDBP (G348C) and FUS (R521H) or by knockdown of both zebrafish Tardbp and Fus, consistent with a common pathogenic mechanism. We also observed that WT FUS rescued the Tardbp knockdown phenotype, but not vice versa, suggesting that TARDBP acts upstream of FUS in this pathway. In addition we observed that WT SOD1 failed to rescue the phenotype observed upon overexpression of mutant TARDBP or FUS or upon knockdown of Tardbp or Fus; similarly, WT TARDBP or FUS also failed to rescue the phenotype induced by mutant SOD1 (G93A). Finally, overexpression of mutant SOD1 exacerbated the motor phenotype caused by overexpression of mutant FUS. Together our results indicate that TARDBP and FUS act in a pathogenic pathway that is independent of SOD1.     

Unique characteristics of the genetics epidemiology of amyotrophic lateral sclerosis in China

Continual discoveries of new genes and unraveling the genetic etiology in amyotrophic lateral sclerosis(ALS) have provided greater insight into the underlying pathogenesis in motor neuron degeneration, as well as facilitating the disease modeling and the testing of targeted therapeutics. While, the genetic etiology accounted for two-thirds of FALS and approximately 11% of SALS in Caucasians. However, the contributions of these causative genes to ALS vary among different populations. Furthermore, the prominent difference between Chinese population and other ethnics remains a source of ongoing debate. We systemically reviewed genetics literature of Chinese ALS populations and updated the mutation frequencies of the main ALS-implicated genes aiming to determine the genetic features of ALS in Chinese population. We also reviewed the associations between ALSimplicated single nucleotide polymorphisms(SNPs) and the risk of ALS in Chinese population. A total of 116 studies were included in this analysis(86 gene mutation study articles and 30 SNPs study articles). The results showed that the overall gene mutation rates of ALS-related causative genes were 55.0% in familial ALS(FALS) and 11.7% in sporadic ALS(SALS) in Chinese population. In Chinese FALS, the highest mutation frequency was found in SOD1 gene(25.6%), followed by FUS(5.8%), TARDBP(5.8%), DCTN1(3.6%) and C9 orf 72(3.5%). In Chinese SALS, the highest mutation frequency was also identified in SOD1 gene(1.6%), followed by ANXA11(1.4%), FUS(1.3%), SQSTM1(1.0%), OPTN(0.9%) and CCNF(0.8%).The associations between several SNPs and risk of ALS were also reported in Chinese population. The genetic features of ALS in Chinese population are significantly different from those in Caucasian population, indicating an association between genetic susceptibility and origin of population. Further explorations are required to understand the gene complexity of ALS, including the contribution of most minor genes and the molecular mechanisms in ALS pathologies.     

Mutation Screening of the <Emphasis Type="Italic">CHCHD10</Emphasis> Gene in Chinese Patients with Amyotrophic Lateral Sclerosis

Mutations in the coiled-coil-helix-coiled-coil-helix domain-containing protein 10 gene (CHCHD10), involved in mitochondrial function, have recently been reported as a causative gene of amyotrophic lateral sclerosis (ALS). The aim of this study was to obtain the mutation prevalence of CHCHD10 and the phenotypes with mutations in Chinese ALS patients. A cohort of 499 ALS patients including 487 sporadic ALS (SALS) and 12 familial ALS (FALS), from the Department of Neurology, West China Hospital of Sichuan University, were screened for mutations of all exons of the CHCHD10 gene by Sanger sequencing. Novel candidate mutations or variants were confirmed by polymerase chain reaction-restriction fragment length polymorphism in 466 healthy individuals. All patients identified with mutations of CHCHD10 gene were screened for mutations of the common ALS causative genes including C9orf72, SOD1, TARDBP, FUS, PFN1, and SQSTM1. Three heterozygous variants, including two missense mutations (c.275A?>?G (p.Y92C) and c.306G?>?C (p.Q102H)) and a synonymous change c.306G?>?A (p.Q102Q), were found in exon 3 of CHCHD10 in three alive SALS individuals (with the longest disease duration of 8.6 years), all of which were not detected in healthy controls. No mutation in CHCHD10 was identified in FALS patients. No mutation was found in the aforementioned common ALS causative genes in the patients who carried CHCHD10 mutations. The mutation frequency of CHCHD10 (0.4 %, 2/487) in a Chinese SALS population suggests CHCHD10 gene mutation appears to be an uncommon cause of ALS in Chinese populations. CHCHD10 mutations are associated with a slow progression and long disease duration.     

Evidence of Increased Oxidative Damage in Both Sporadic and Familial Amyotrophic Lateral Sclerosis

Abstract: Some cases of autosomal dominant familial amyotrophic lateral sclerosis (FALS) are associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1), suggesting that oxidative damage may play a role in ALS pathogenesis. To further investigate the biochemical features of FALS and sporadic ALS (SALS), we examined markers of oxidative damage to protein, lipids, and DNA in motor cortex (Brodmann area 4), parietal cortex (Brodmann area 40), and cerebellum from control subjects, FALS patients with and without known SOD mutations, SALS patients, and disease controls (Pick's disease, progressive supranuclear palsy, diffuse Lewy body disease). Protein carbonyl and nuclear DNA 8-hydroxy-2'-deoxyguanosine (OH⁸dG) levels were increased in SALS motor cortex but not in FALS patients. Malondialdehyde levels showed no significant changes. Immunohistochemical studies showed increased neuronal staining for hemeoxygenase-1, malondialdehyde-modified protein, and OH⁸dG in both SALS and FALS spinal cord. These studies therefore provide further evidence that oxidative damage may play a role in the pathogenesis of neuronal degeneration in both SALS and FALS.     

Superoxide Dismutase Activity, Oxidative Damage, and Mitochondrial Energy Metabolism in Familial and Sporadic Amyotrophic Lateral Sclerosis

The cause of neuronal death in amyotrophic lateral sclerosis (ALS) is unknown. Recently, it was found that some patients with autosomal-dominant familial ALS (FALS) have point mutations in the gene that encodes Cu/Zn superoxide dismutase (SOD1). In this study of postmortem brain tissue, we examined SOD activity and quantified protein carbonyl groups, a marker of oxidative damage, in samples of frontal cortex (Brodmann area 6) from 10 control patients, three FALS patients with known SOD1 mutations (FALS-1), one autosomal-dominant FALS patient with no identifiable SOD1 mutations (FALS-0), and 11 sporadic ALS (SALS) patients. Also, we determined the activities of components of the electron transport chain (complexes I, II-III, and IV) in these samples. The cytosolic SOD activity, which is primarily SOD1 activity, was reduced by 38.8% (p < 0.05) in the FALS-1 patients and not significantly altered in the SALS patients or the FALS-0 patient relative to the control patients. The mitochondrial SOD activity, which is primarily SOD2 activity, was not significantly altered in the FALS-1, FALS-0, or SALS patients. The protein carbonyl content was elevated by 84.8% (p < 0.01) in the SALS patients relative to the control patients. Finally, the complex I activity was increased by 55.3% (p < 0.001) in the FALS-1 patients relative to the control patients. These results from cortical tissue demonstrate that SOD1 activity is reduced and complex I activity is increased in FALS-1 patients and that oxidative damage to proteins is increased in SALS patients.     

Metabolic Dysfunction in Familial, but Not Sporadic, Amyotrophic Lateral Sclerosis

Abstract: Autosomal dominant familial amyotrophic lateral sclerosis (FALS) is associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Previous studies have implicated the involvement of metabolic dysfunction in ALS pathogenesis. To further investigate the biochemical features of FALS and sporadic ALS (SALS), we examined SOD activity and mitochondrial oxidative phosphorylation enzyme activities in motor cortex (Brodmann area 4), parietal cortex (Brodmann area 40), and cerebellum from control subjects, FALS patients with and without known SOD mutations, SALS patients, and disease controls (Pick's disease, progressive supranuclear palsy, diffuse Lewy body disease). Cytosolic SOD activity, predominantly Cu/Zn SOD, was decreased ∼50% in all regions in FALS patients with SOD mutations but was not significantly altered in other patient groups. Marked increases in complex I and II–III activities were seen in FALS patients with SOD mutations but not in SALS patients. We also measured electron transport chain enzyme activities in a transgenic mouse model of FALS. Complex I activity was significantly increased in the forebrain of 60-day-old G93A transgenic mice overexpressing human mutant SOD1, relative to levels in transgenic wild-type animals, supporting the hypothesis that the motor neuron disorder associated with SOD1 mutations involves a defect in mitochondrial energy metabolism.     

Astrocytes from familial and sporadic ALS patients are toxic to motor neurons

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, with astrocytes implicated as contributing substantially to motor neuron death in familial (F)ALS. However, the proposed role of astrocytes in the pathology of ALS derives in part from rodent models of FALS based upon dominant mutations within the superoxide dismutase 1 (SOD1) gene, which account for <2% of all ALS cases. Their role in sporadic (S)ALS, which affects >90% of ALS patients, remains to be established. Using astrocytes generated from postmortem tissue from both FALS and SALS patients, we show that astrocytes derived from both patient groups are similarly toxic to motor neurons. We also demonstrate that SOD1 is a viable target for SALS, as its knockdown significantly attenuates astrocyte-mediated toxicity toward motor neurons. Our data highlight astrocytes as a non-cell autonomous component in SALS and provide an in vitro model system to investigate common disease mechanisms and evaluate potential therapies for SALS and FALS.     

Superoxide Dismutase Concentration and Activity in Familial Amyotrophic Lateral Sclerosis

Abstract: Some cases of autosomal-dominant familial amyotrophic lateral sclerosis (FALS) have been associated with mutations in SOD1 , the gene that encodes Cu/Zn superoxide dismutase (Cu/Zn SOD). We determined the concentrations (µg of Cu/Zn SOD/mg of total protein), specific activities (U/µg of total protein), and apparent turnover numbers (U/µmol of Cu/Zn SOD) of Cu/Zn SOD in erythrocyte lysates from patients with known SOD1 mutations. We also measured the concentrations and activities of Cu/Zn SOD in FALS patients with no identifiable SOD1 mutations, sporadic ALS (SALS) patients, and patients with other neurologic disorders. The concentration and specific activity of Cu/Zn SOD were decreased in all patients with SOD1 mutations, with mean reductions of 51 and 46%, respectively, relative to controls. In contrast, the apparent turnover number of the enzyme was not altered in these patients. For the six mutations studied, there was no correlation between enzyme concentration or specific activity and disease severity, expressed as either duration of disease or age of onset. No significant alterations in the concentration, specific activity, or apparent turnover number of Cu/Zn SOD were detected in the FALS patients with no identifiable SOD1 mutations, SALS patients, or patients with other neurologic disorders. That Cu/Zn SOD concentration and specific activity are equivalently reduced in erythrocytes from patients with SOD1 mutations suggests that mutant Cu/Zn SOD is unstable in these cells. That concentration and specific activity do not correlate with disease severity suggests that an altered, novel function of the enzyme, rather than reduction of its dismutase activity, may be responsible for the pathogenesis of FALS.     

Biotransformation of nitric oxide in the cerebrospinal fluid of amyotrophic lateral sclerosis patients

Recent findings indicate that nitric oxide (NO*) over-production might be an important factor in the pathogenesis of sporadic amyotrophic lateral sclerosis (SALS). We measured significantly higher concentrations of uric acid and thiol group-containing molecules (R-SH groups) in the cerebrospinal fluid (CSF) from SALS patients compared to controls. The above factors, together with a slightly increased free iron concentration found in the CSF, favour conditions necessary for the formation of the dinitrosyl iron complex, capable of NO* bio-transformation. Thus, we performed ex vivo saturation of CSF (from both SALS patients and controls) with NO*. A decrease in the level of R-SH was found. This was more pronounced in the CSF from SALS patients. In the CSF from SALS patients the production of nitrite and hydroxylamine was greater than that observed in the CSF from controls. Moreover, we also found increased Cu,Zn-SOD activity in the CSF from SALS patients (when compared to control subjects) but no activity corresponding to Mn-SOD in any CSF samples. As Cu,Zn-SOD can react with nitroxyl forming NO*, the conditions for a closed, but continuous, loop of NO* biotransformation are present in the CSF of ALS patients.     

A frameshift deletion in peripherin gene associated with amyotrophic lateral sclerosis

Peripherin is a neuronal intermediate filament associated with inclusion bodies in motor neurons of patients with amyotrophic lateral sclerosis (ALS). A possible peripherin involvement in ALS pathogenesis has been suggested based on studies with transgenic mouse overexpressors and with a toxic splicing variant of the mouse peripherin gene. However, the existence of peripherin gene mutations in human ALS has not yet been documented. Therefore, we screened for sequence variants of the peripherin gene (PRPH) in a cohort of ALS patients including familial and sporadic cases. We identified 18 polymorphic variants of PRPH detected in both ALS and age-matched control populations. Two additional PRPH variants were discovered in ALS cases but not in 380 control individuals. One variant consisted of a nucleotide insertion in intron 8 (PRPH(IVS8)(-36insA)), whereas the other one consisted of a 1-bp deletion within exon 1 (PRPH(228delC)), predicting a truncated peripherin species of 85 amino acids. Remarkably, expression of this frameshift peripherin mutant in SW13 cells resulted in disruption of neurofilament network assembly. These results suggest that PRPH mutations may be responsible for a small percentage of ALS, cases and they provide further support of the view that neurofilament disorganization may contribute to pathogenesis.     

A90V TDP-43 variant results in the aberrant localization of TDP-43 in vitro

TAR DNA-binding protein-43 (TDP-43) is a highly conserved, ubiquitously expressed nuclear protein that was recently identified as the disease protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Pathogenic TDP-43 gene (TARDBP) mutations have been identified in familial ALS kindreds, and here we report a TARDBP variant (A90V) in a FTLD/ALS patient with a family history of dementia. Significantly, A90V is located between the bipartite nuclear localization signal sequence of TDP-43 and the in vitro expression of TDP-43-A90V led to its sequestration with endogenous TDP-43 as insoluble cytoplasmic aggregates. Thus, A90V may be a genetic risk factor for FTLD/ALS because it predisposes nuclear TDP-43 to redistribute to the cytoplasm and form pathological aggregates.     

Disease-related changes in the cerebrospinal fluid metabolome in amyotrophic lateral sclerosis detected by GC/TOFMS

Background/Aim

The changes in the cerebrospinal fluid (CSF) metabolome associated with the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) are poorly understood and earlier smaller studies have shown conflicting results. The metabolomic methodology is suitable for screening large cohorts of samples. Global metabolomics can be used for detecting changes of metabolite concentrations in samples of fluids such as CSF.

Methodology

Using gas chromatography coupled to mass spectrometry (GC/TOFMS) and multivariate statistical modeling, we simultaneously studied the metabolome signature of ∼120 small metabolites in the CSF of patients with ALS, stratified according to hereditary disposition and clinical subtypes of ALS in relation to controls.

Principal Findings

The study is the first to report data validated over two sub-sets of ALS vs. control patients for a large set of metabolites analyzed by GC/TOFMS. We find that patients with sporadic amyotrophic lateral sclerosis (SALS) have a heterogeneous metabolite signature in the cerebrospinal fluid, in some patients being almost identical to controls. However, familial amyotrophic lateral sclerosis (FALS) without superoxide dismutase-1 gene (SOD1) mutation is less heterogeneous than SALS. The metabolome of the cerebrospinal fluid of 17 ALS patients with a SOD1 gene mutation was found to form a separate homogeneous group. Analysis of metabolites revealed that glutamate and glutamine were reduced, in particular in patients with a familial predisposition. There are significant differences in the metabolite profile and composition among patients with FALS, SALS and patients carrying a mutation in the SOD1 gene suggesting that the neurodegenerative process in different subtypes of ALS may be partially dissimilar.

Conclusions/Significance

Patients with a genetic predisposition to amyotrophic lateral sclerosis have a more distinct and homogeneous signature than patients with a sporadic disease.     

Effects of cadmium on structure and enzymatic activity of Cu,Zn-SOD and oxidative status in neural cells

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder disease. Ten percent of the ALS patients are congenital (familial ALS), and the other 90% are sporadic ALS (SALS). It has been shown that mutations found in the Cu,Zn-SOD cause 20% of the familial ALS due to its low enzyme activity. We hypothesized that heavy metals may interfere the structure of Cu,Zn-SOD protein to suppress its activity in some of the SALS. In this study, we expressed and characterized the recombinant human Cu,Zn-SOD under various concentrations of Cu(2+), Zn(2+), and Cd(2+). By atomic absorption spectrophotometry, we demonstrated that adding of cadmium significantly increased the content of cadmium ion, but reduced its Zn(2+) content and enzyme activity of the Cu,Zn-SOD protein. The data of circular dichroism spectra demonstrated that the secondary structure of Cu,Zn-SOD/Cd is different from Cu,Zn-SOD, but close to apo-SOD. In addition to the effect of cadmium on Cu,Zn-SOD, cadmium was also shown to induce neural cell apoptosis. To further investigate the mechanism of neural cell apoptosis induced by cadmium, we used proteomics to analyze the altered protein expressions in neural cells treated with cadmium. The altered proteins include cellular structural proteins, stress-related and chaperone proteins, proteins involved in reactive oxygen species (ROS), enzyme proteins, and proteins that mediated cell death and survival signaling. Taken together, in this paper, we demonstrate that cadmium decreases the content of Zn(2+), changes the conformation of Cu,Zn-SOD protein to decrease its enzyme activity, and causes oxidative stress-induced neural cell apoptosis.     

TDP-43 physically interacts with amyotrophic lateral sclerosis-linked mutant CuZn superoxide dismutase

Mutations in the CuZn superoxide dismutase (SOD1) and TAR DNA-binding protein 43 (TDP-43) genes are linked to familial amyotrophic lateral sclerosis, ALS1 and ALS10, respectively. In addition, TDP-43 is a major component protein of the ubiquitinated aggregates observed in sporadic ALS (SALS) patients. However, it remains unclear whether these ALS groups partly have a shared pathogenesis. In the present study, we demonstrate that mutant SOD1, but not wild-type SOD1, interacts with TDP-43 by co-immunoprecipitation assays using cultured cells and G93A mutant SOD1 transgenic mice. The region responsible for this interaction within SOD1 is the dimer interface, namely, the N- and C-terminal regions. Deletion mutants of TDP-43 with or without nuclear localization sequence interacted with mutant SOD1. Cell fractionation assays using cultured cells showed that mutant SOD1 was localized in the cytosolic fraction but not in the nuclear fraction. TDP-43 was detected both in the nuclear and cytosolic fractions, suggesting that mutant SOD1 interacts with TDP-43 in the cytoplasm. Mutant SOD1 overexpression led to an increased amount of mutant SOD1 and, to some extent, its interacting proteins including TDP-43 in the detergent-insoluble fraction. These results indicate that mutant SOD1 could affect the solubility/insolubility of its interacting proteins including TDP-43 through physical interactions. Our findings may contribute to the understanding of links among SALS, ALS1 and ALS10.     

The role of mutant TAR DNA-binding protein 43 in amyotrophic lateral sclerosis and frontotemporal lobar degeneration

TDP-43 (TAR DNA-binding protein 43) has been identified as a key protein of ubiquitinated inclusions in brains of patients with ALS (amyotrophic lateral sclerosis) or FTLD (frontotemporal lobar degeneration), defining a new pathological disease spectrum. Recently, coding mutations have been identified in the TDP-43 gene (TARDBP), which further confirmed the pathogenic nature of the protein. Today, several animal models have been generated to gain more insight into the disease-causing pathways of the FTLD/ALS spectrum. This mini-review summarizes the current status of TDP-43 models, with a focus on mutant TDP-43.     

Tissue-selective regulation of protein homeostasis and unfolded protein response signalling in sporadic ALS

Amyotrophic lateral sclerosis (ALS) is a disorder that affects motor neurons in motor cortex and spinal cord, and the degeneration of both neuronal populations is a critical feature of the disease. Abnormalities in protein homeostasis (proteostasis) are well established in ALS. However, they have been investigated mostly in spinal cord but less so in motor cortex. Herein, we monitored the unfolded protein (UPR) and heat shock response (HSR), two major proteostasis regulatory pathways, in human post-mortem tissue derived from the motor cortex of sporadic ALS (SALS) and compared them to those occurring in spinal cord. Although the UPR was activated in both tissues, specific expression of select UPR target genes, such as PDIs, was observed in motor cortex of SALS cases strongly correlating with oligodendrocyte markers. Moreover, we found that endoplasmic reticulum-associated degradation (ERAD) and HSR genes, which were activated predominately in spinal cord, correlated with the expression of neuronal markers. Our results indicate that proteostasis is strongly and selectively activated in SALS motor cortex and spinal cord where subsets of these genes are associated with specific cell type. This study expands our understanding of convergent molecular mechanisms occurring in motor cortex and spinal cord and highlights cell type–specific contributions.     

TDP-43 physically interacts with amyotrophic lateral sclerosis-linked mutant CuZn superoxide dismutase

Mutations in the CuZn superoxide dismutase (SOD1) and TAR DNA-binding protein 43 (TDP-43) genes are linked to familial amyotrophic lateral sclerosis, ALS1 and ALS10, respectively. In addition, TDP-43 is a major component protein of the ubiquitinated aggregates observed in sporadic ALS (SALS) patients. However, it remains unclear whether these ALS groups partly have a shared pathogenesis. In the present study, we demonstrate that mutant SOD1, but not wild-type SOD1, interacts with TDP-43 by co-immunoprecipitation assays using cultured cells and G93A mutant SOD1 transgenic mice. The region responsible for this interaction within SOD1 is the dimer interface, namely, the N- and C-terminal regions. Deletion mutants of TDP-43 with or without nuclear localization sequence interacted with mutant SOD1. Cell fractionation assays using cultured cells showed that mutant SOD1 was localized in the cytosolic fraction but not in the nuclear fraction. TDP-43 was detected both in the nuclear and cytosolic fractions, suggesting that mutant SOD1 interacts with TDP-43 in the cytoplasm. Mutant SOD1 overexpression led to an increased amount of mutant SOD1 and, to some extent, its interacting proteins including TDP-43 in the detergent-insoluble fraction. These results indicate that mutant SOD1 could affect the solubility/insolubility of its interacting proteins including TDP-43 through physical interactions. Our findings may contribute to the understanding of links among SALS, ALS1 and ALS10.     

Expression of Human A4V Mutant Cu,Zn Superoxide Dismutase in Schizosaccharomyces pombe: Investigations of its Toxic Properties

Cu,Zn superoxide dismutase (SOD1) is an antioxidant enzyme that catalyzes the removal of superoxide radicals generated in various biological oxidations. Amyotrophic lateral sclerosis (ALS) is one of the most common neurodegenerative disorders, occurring in families (FALS) and sporadically (SALS). FALS and SALS are distinguishable genetically but not clinically. More than 100 point mutations in the human SOD 1 gene have been identified that cause FALS. In order to determine the effects of mutant SOD protein, we first cloned wild-type and A4V mutant human SOD1 into Schizosaccharomyces pombe. This study shows viabilities and some antioxidant properties including SOD, catalase, proteasomal activity, and protein carbonyl levels of transformants in SOD1 deleted strain (MN415); and its parental strain (JY741) at different stress conditions. There was no more oxidative damage in the human mutant SOD carrying the transformant strain compared with other strains. These results may help to explain whether ALS progresses as a consequence of cellular oxidative damage.