Molecular Chaperone Mediated Late-Stage Neuroprotection in the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons in the spinal cord, brain stem, and motor cortex. Mutations in superoxide dismutase (SOD1) are associated with familial ALS and lead to SOD1 protein misfolding and aggregation. Here we show that the molecular chaperone, HSJ1 (DNAJB2), mutations in which cause distal hereditary motor neuropathy, can reduce mutant SOD1 aggregation and improve motor neuron survival in mutant SOD1 models of ALS. Overexpression of human HSJ1a (hHSJ1a) in vivo in motor neurons of SOD1^G93A transgenic mice ameliorated disease. In particular, there was a significant improvement in muscle force, increased motor unit number and enhanced motor neuron survival. hHSJ1a was present in a complex with SOD1^G93A and led to reduced SOD1 aggregation at late stages of disease progression. We also observed altered ubiquitin immunoreactivity in the double transgenic animals, suggesting that ubiquitin modification might be important for the observed improvements. In a cell model of SOD1^G93A aggregation, HSJ1a preferentially bound to mutant SOD1, enhanced SOD1 ubiquitylation and reduced SOD1 aggregation in a J-domain and ubiquitin interaction motif (UIM) dependent manner. Collectively, the data suggest that HSJ1a acts on mutant SOD1 through a combination of chaperone, co-chaperone and pro-ubiquitylation activity. These results show that targeting SOD1 protein misfolding and aggregation in vivo can be neuroprotective and suggest that manipulation of DnaJ molecular chaperones might be useful in the treatment of ALS.     

A mutation in dynein rescues axonal transport defects and extends the life span of ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition characterized by motoneuron degeneration and muscle paralysis. Although the precise pathogenesis of ALS remains unclear, mutations in Cu/Zn superoxide dismutase (SOD1) account for approximately 20-25% of familial ALS cases, and transgenic mice overexpressing human mutant SOD1 develop an ALS-like phenotype. Evidence suggests that defects in axonal transport play an important role in neurodegeneration. In Legs at odd angles (Loa) mice, mutations in the motor protein dynein are associated with axonal transport defects and motoneuron degeneration. Here, we show that retrograde axonal transport defects are already present in motoneurons of SOD1(G93A) mice during embryonic development. Surprisingly, crossing SOD1(G93A) mice with Loa/+ mice delays disease progression and significantly increases life span in Loa/SOD1(G93A) mice. Moreover, there is a complete recovery in axonal transport deficits in motoneurons of these mice, which may be responsible for the amelioration of disease. We propose that impaired axonal transport is a prime cause of neuronal death in neurodegenerative disorders such as ALS.     

Transgenic small interfering RNA halts amyotrophic lateral sclerosis in a mouse model

Many autosomal dominant diseases such as familial amyotrophic lateral sclerosis (ALS) with copper/zinc superoxide dismutase (SOD1) mutation may be induced by missense point mutations that result in the production of proteins with toxic properties. Reduction in the encoding of proteins from such mutated genes can therefore be expected to improve the disease phenotype. The duplex of 21-nucleotide RNA, known as small interfering RNA (siRNA), has recently emerged as a powerful gene silencing tool. We made transgenic (Tg) mice with modified siRNA, which had multiple mismatch alternations within the sense strand, to prevent the "shutdown phenomenon" of transgenic siRNA. Consequently, the in vivo knockdown effect of siRNA on SOD1 expression did not diminish over four generations. When we crossed these anti-SOD1 siRNA Tg mice with SOD1G93A Tg mice, a model for ALS, siRNA prevented the development of disease by inhibiting mutant G93A SOD1 production in the central nervous system. Our findings clearly proved the principle that siRNA-mediated gene silencing can stop the development of familial ALS with SOD1 mutation.     

The Legs at odd angles (Loa) Mutation in Cytoplasmic Dynein Ameliorates Mitochondrial Function in SOD1G93A Mouse Model for Motor Neuron Disease

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute ∼10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15–20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1^G93A. In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1^G93A motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1^G93A motor neurons.     

Informatics-assisted protein profiling in a transgenic mouse model of amyotrophic lateral sclerosis

One of the causes of amyotrophic lateral sclerosis (ALS) is due to mutations in Cu,Zn-superoxide dismutase (SOD1). The mutant protein exhibits a toxic gain of function that adversely affects the function of neurons in the spinal cord, brain stem, and motor cortex. A proteomic analysis of protein expression in a widely used mouse model of ALS was undertaken to identify differences in protein expression in the spinal cords of mice expressing a mutant protein with the G93A mutation found in human ALS. Protein profiling was done on soluble and particulate fractions of spinal cord extracts using high throughput two-dimensional liquid chromatography coupled to tandem mass spectrometry. An integrated proteomics-informatics platform was used to identify relevant differences in protein expression based upon the abundance of peptides identified by database searching of mass spectrometry data. Changes in the expression of proteins associated with mitochondria were particularly prevalent in spinal cord proteins from both mutant G93A-SOD1 and wild-type SOD1 transgenic mice. G93A-SOD1 mouse spinal cord also exhibited differences in proteins associated with metabolism, protein kinase regulation, antioxidant activity, and lysosomes. Using gene ontology analysis, we found an overlap of changes in mRNA expression in presymptomatic mice (from microarray analysis) in three different gene categories. These included selected protein kinase signaling systems, ATP-driven ion transport, and neurotransmission. Therefore, alterations in selected cellular processes are detectable before symptomatic onset in ALS mouse models. However, in late stage disease, mRNA expression analysis did not reveal significant changes in mitochondrial gene expression but did reveal concordant changes in lipid metabolism, lysosomes, and the regulation of neurotransmission. Thus, concordance of proteomic and mRNA expression data within multiple categories validates the use of gene ontology analysis to compare different types of "omic" data.     

Different human copper-zinc superoxide dismutase mutants, SOD1G93A and SOD1H46R, exert distinct harmful effects on gross phenotype in mice

Amyotrophic lateral sclerosis (ALS) is a heterogeneous group of fatal neurodegenerative diseases characterized by a selective loss of motor neurons in the brain and spinal cord. Creation of transgenic mice expressing mutant Cu/Zn superoxide dismutase (SOD1), as ALS models, has made an enormous impact on progress of the ALS studies. Recently, it has been recognized that genetic background and gender affect many physiological and pathological phenotypes. However, no systematic studies focusing on such effects using ALS models other than SOD1(G93A) mice have been conducted. To clarify the effects of genetic background and gender on gross phenotypes among different ALS models, we here conducted a comparative analysis of growth curves and lifespans using congenic lines of SOD1(G93A) and SOD1(H46R) mice on two different genetic backgrounds; C57BL/6N (B6) and FVB/N (FVB). Copy number of the transgene and their expression between SOD1(G93A) and SOD1(H46R) lines were comparable. B6 congenic mutant SOD1 transgenic lines irrespective of their mutation and gender differences lived longer than corresponding FVB lines. Notably, the G93A mutation caused severer disease phenotypes than did the H46R mutation, where SOD1(G93A) mice, particularly on a FVB background, showed more extensive body weight loss and earlier death. Gender effect on survival also solely emerged in FVB congenic SOD1(G93A) mice. Conversely, consistent with our previous study using B6 lines, lack of Als2, a murine homolog for the recessive juvenile ALS causative gene, in FVB congenic SOD1(H46R), but not SOD1(G93A), mice resulted in an earlier death, implying a genetic background-independent but mutation-dependent phenotypic modification. These results indicate that SOD1(G93A)- and SOD1(H46R)-mediated toxicity and their associated pathogenic pathways are not identical. Further, distinctive injurious effects resulted from different SOD1 mutations, which are associated with genetic background and/or gender, suggests the presence of several genetic modifiers of disease expression in the mouse genome.     

Vascular endothelial growth factor prevents G93A‐SOD1‐induced motor neuron degeneration

Amyotrophic lateral sclerosis (ALS) is an adult‐onset neurodegenerative disorder characterized by selective loss of motor neurons (MNs). Twenty percent of familial ALS cases are associated with mutations in Cu²⁺/Zn²⁺ superoxide dismutase (SOD1). To specifically understand the cellular mechanisms underlying mutant SOD1 toxicity, we have established an in vitro model of ALS using rat primary MN cultures transfected with an adenoviral vector encoding a mutant SOD1, G93A‐SOD1. Transfected cells undergo axonal degeneration and alterations in biochemical responses characteristic of cell death such as activation of caspase‐3. Vascular endothelial growth factor (VEGF) is an angiogenic and neuroprotective growth factor that can increase axonal outgrowth, block neuronal apoptosis, and promote neurogenesis. Decreased VEGF gene expression in mice results in a phenotype similar to that seen in patients with ALS, thus linking loss of VEGF to the pathogenesis of MN degeneration. Decreased neurotrophic signals prior to and during disease progression may increase MN susceptibility to mutant SOD1‐induced toxicity. In this study, we demonstrate a decrease in VEGF and VEGFR2 levels in the spinal cord of G93A‐SOD1 ALS mice. Furthermore, in isolated MN cultures, VEGF alleviates the effects of G93A‐SOD1 toxicity and neuroprotection involves phosphatidylinositol 3‐kinase/protein kinase B (PI3K/Akt) signaling. Overall, these studies validate the usefulness of VEGF as a potential therapeutic factor for the treatment of ALS and give valuable insight into the responsible signaling pathways and mechanisms involved. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Increased mitochondrial antioxidative activity or decreased oxygen free radical propagation prevent mutant SOD1-mediated motor neuron cell death and increase amyotrophic lateral sclerosis-like transgenic mouse survival

The molecular mechanisms of selective motor neuron degeneration in human amyotrophic lateral sclerosis (ALS) disease remain largely unknown and effective therapies are not currently available. Mitochondrial dysfunction is an early event of motor neuron degeneration in transgenic mice overexpressing mutant superoxide dismutase (SOD)1 gene and mitochondrial abnormality is observed in human ALS patients. In an in vitro cell culture system, we demonstrated that infection of mouse NSC-34 motor neuron-like cells with adenovirus containing mutant G93A-SOD1 gene increased cellular oxidative stress, mitochondrial dysfunction, cytochrome c release and motor neuron cell death. Cells pretreated with highly oxidizable polyunsaturated fatty acid elevated lipid peroxidation and synergistically exacerbated motor neuron-like cell death with mutant G93A-SOD1 but not with wild-type SOD1. Similarly, overexpression of mitochondrial antioxidative genes, MnSOD and GPX4 by stable transfection significantly increased NSC-34 motor neuron-like cell resistance to mutant SOD1. Pre-incubation of cells with spin trapping molecule, 5',5'-dimethylpryrroline-N-oxide (DMPO), prevented mutant SOD1-mediated mitochondrial dysfunction and cell death. Furthermore, treatment of mutant G93A-SOD1 transgenic mice with DMPO significantly delayed paralysis and increased survival. These findings suggest a causal relationship between enhanced oxidative stress and mutant SOD1-mediated motor neuron degeneration, considering that enhanced oxygen free radical production results from the SOD1 structural alterations. Molecular approaches aimed at increasing mitochondrial antioxidative activity or effectively blocking oxidative stress propagation can be potentially useful in the clinical management of human ALS disease.     

Treatment with arimoclomol, a coinducer of heat shock proteins, delays disease progression in ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition in which motoneurons of the spinal cord and motor cortex die, resulting in progressive paralysis. This condition has no cure and results in eventual death, usually within 1-5 years of diagnosis. Although the specific etiology of ALS is unknown, 20% of familial cases of the disease carry mutations in the gene encoding Cu/Zn superoxide dismutase-1 (SOD1). Transgenic mice overexpressing human mutant SOD1 have a phenotype and pathology that are very similar to that seen in human ALS patients. Here we show that treatment with arimoclomol, a coinducer of heat shock proteins (HSPs), significantly delays disease progression in mice expressing a SOD1 mutant in which glycine is substituted with alanine at position 93 (SOD1(G93A)). Arimoclomol-treated SOD1(G93A) mice show marked improvement in hind limb muscle function and motoneuron survival in the later stages of the disease, resulting in a 22% increase in lifespan. Pharmacological activation of the heat shock response may therefore be a successful therapeutic approach to treating ALS, and possibly other neurodegenerative diseases.     

Treatment with Hydrogen-Rich Saline Delays Disease Progression in a Mouse Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is the most frequent adult-onset motor neuron disease, and accumulating evidence indicates that oxidative mechanisms contribute to ALS pathology, but classical antioxidants have not performed well in clinical trials. The aim of this work was to investigate the effect of treatment with hydrogen molecule on the development of disease in mutant SOD1 G93A transgenic mouse model of ALS. Treatment of mutant SOD1 G93A mice with hydrogen-rich saline (HRS, i.p.) significantly delayed disease onset and prolonged survival, and attenuated loss of motor neurons and suppressed microglial and glial activation. Treatment of mutant SOD1 G93A mice with HRS inhibited the release of mitochondrial apoptogenic factors and the subsequent activation of downstream caspase-3. Furthermore, treatment of mutant SOD1 G93A mice with HRS reduced levels of protein carbonyl and 3-nitrotyrosine, and suppressed formation of reactive oxygen species (ROS), peroxynitrite, and malondialdehyde. Treatment of mutant SOD1 G93A mice with HRS preserved mitochondrial function, marked by restored activities of Complex I and IV, reduced mitochondrial ROS formation and enhanced mitochondrial adenosine triphosphate synthesis. In conclusion, hydrogen molecule may be neuroprotective against ALS, possibly through abating oxidative and nitrosative stress and preserving mitochondrial function.     

Integrative role of cPLA with COX-2 and the effect of non-steriodal anti-inflammatory drugs in a transgenic mouse model of amyotrophic lateral sclerosis

Cyclooxygenase-2 (COX-2) is a key molecule in the inflammatory pathway in amyotrophic lateral sclerosis (ALS). Cytosolic phospholipase A (cPLA2) is an important enzyme providing substrate for cyclooxygenases. We therefore examined cPLA2 expression in human ALS and mutant Cu/Zn superoxide dismutase (SOD1) transgenic mice and its relation to COX-2. Immunohistochemistry and real-time RT-PCR revealed elevated cPLA2 protein and its mRNA levels in the lumbar spinal cord of mutant SOD1 mice. COX-2 immunoreactivity was increased in lumbar spinal cord sections from both familial ALS (FALS) and sporadic ALS (SALS) as compared to controls, and cPLA2 immunoreactivity was increased in a patient with FALS. Oral administration of the non-selective cyclooxygenase (COX) inhibitor, sulindac, extended the survival (by 10%) of G93A SOD1 mice as compared to littermate controls. Sulindac, as well as the selective COX-2 inhibitors, rofecoxib and celecoxib reduced cPLA2 immunoreactivity in the lumbar spinal cord of G93A transgenic mice. Sulindac treatment preserved motor neurons, and reduced microglial activation and astrocytosis, in the spinal cord of G93A SOD1 transgenic mice. These results suggest that cPLA2 plays an important role in supplying arachidonic acid to the COX-2 driven inflammatory pathway in ALS associated with SOD1 mutations.     

Changes in the Spinal Cord Proteome of an Amyotrophic Lateral Sclerosis Murine Model Determined by Differential In-gel Electrophoresis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by loss of motor neurons resulting in progressive paralysis. To date, more than 140 different mutations in the gene encoding CuZn-superoxide dismutase (SOD1) have been associated with ALS. Several transgenic murine models exist in which various mutant SOD1s are expressed. We used DIGE to analyze the changes in the spinal cord proteome induced by expression of the unstable SOD1 truncation mutant G127insTGGG (G127X) in mice. Unlike mutants used in most other models, G127X lacks SOD activity and is present at low levels, thus reducing the risk of overexpression artifacts. The mice were analyzed at their peak body weights just before onset of symptoms. Variable importance plot analysis showed that 420 of 1,800 detected protein spots contributed significantly to the differences between the groups. By MALDI-TOF MS analysis, 54 differentially regulated proteins were identified. One spot was found to be a covalently linked mutant SOD1 dimer, apparently analogous to SOD1-immunoreactive bands migrating at double the molecular weight of SOD1 monomers previously detected in humans and mice carrying mutant SOD1s and in sporadic ALS cases. Analyses of affected functional pathways and the subcellular representation of alterations suggest that the toxicity exerted by mutant SODs induces oxidative stress and affects mitochondria, cellular assembly/organization, and protein degradation.Amyotrophic lateral sclerosis (ALS)¹ is a devastating neurodegenerative disease characterized by loss of motor neurons in the motor cortex, the brainstem, and the spinal cord. This results in progressive muscular atrophy, and the patients usually succumb to respiratory failure within a few years. About 10% of ALS cases are familial (1), and in some patients the disease is linked to mutations in the CuZn-superoxide dismutase (SOD1) gene (2). SOD1 is a ubiquitously expressed antioxidant enzyme. Overall about 6% of all cases with ALS show SOD1 mutations, and more than 140 such mutations have been identified (3). The mutations confer a cytotoxic gain of function of unknown character to the enzyme (4, 5). ALS caused by mutant SOD1s shows the same spectrum of disease phenotypes as is seen in sporadic cases lacking such mutations. This suggests that the pathogenesis of ALS induced by SOD1 mutations should show significant similarities with that in sporadic disease, e.g. similar pathogenic protein alterations.ALS has been modeled in mice via transgenic overexpression of mutant SOD1s (5–8). To cause disease within the short lifespan of a mouse, the mutant SOD1s have to be expressed at rates around 25-fold higher than the rate of expression of the endogenous murine enzyme (9). Mostly structurally stable mutants have been used, resulting in up to 10-fold increases in SOD activity and 20-fold increases in SOD1 protein levels in the CNS that may cause overexpression artifacts. Overloading of mitochondria with mutant SOD1s and vacuolization have been observed in such models (10).To explore ALS pathogenesis, we studied changes in the proteome of spinal cords of SOD1 transgenic mice using DIGE. To reduce the risk of overexpression artifacts, we used mice that express the unstable human SOD1 (hSOD1) truncation mutant G127insTGGG (G127X) (7). These mice develop an aggressive form of the disease, which is of short duration, despite the fact that the mutant lacks SOD activity and the fact that the level of G127X hSOD1 protein is less than half that of the endogenous murine SOD1. The mice were studied at their peak body weights just before development of paralytic symptoms. Here we present the identity of and discuss the possible significance of 53 proteins found to be differentially regulated in ALS transgenic mice.     

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.     

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.     

An RNAi strategy for treatment of amyotrophic lateral sclerosis caused by mutant Cu,Zn superoxide dismutase

Amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) is a neurodegenerative disease characterized by motor neuron degeneration, paralysis and death. One cause of this disease is mutations in the Cu,Zn superoxide dismutase (SOD1) gene. As mutant SOD1 acquires a toxic property that kills motor neurons, by reducing the mutant protein the disease progression may be slowed or prevented. While mutant SOD1 is toxic, the wild-type SOD1 is indispensable for motor neuron health. Therefore, the ideal therapeutic strategy would be to inhibit selectively the mutant protein expression. Previously we have demonstrated that RNA interference (RNAi) can selectively inhibit some mutant SOD1 expression. However, more than 100 SOD1 mutants can cause ALS and all mutants cannot be inhibited selectively by RNAi. To overcome this obstacle, we have designed a replacement RNAi strategy. Using this strategy, all mutants and wild-type genes are inhibited by RNAi. The wild-type SOD1 function is then replaced by designed wild-type SOD1 genes that are resistant to the RNAi. Here we demonstrate the concept of this strategy.     

Gene Expression Profiles of APP and BACE1 in Tg SOD1G93A Cortical Cells

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease defined by motor neuron loss. Transgenic mouse model (Tg SOD1G93A) shows pathological features that closely mimic those seen in ALS patients. An hypothetic link between AD and ALS was suggested by finding an higher amount of amyloid precursor protein (APP) in the spinal cord anterior horn neurons, and of Aβ peptides in ALS patients skin. In this work, we have investigated the expression of some genes involved in Alzheimer’s disease, as APP, β- and γ-secretase, in an animal model of ALS, to understand some possible common molecular mechanisms between these two pathologies. For gene expression analysis, we carried out a quantitative RT-PCR in ALS mice and in transgenic mice over-expressing human wild-type SOD1 (Tg hSOD1). We found that APP and BACE1 mRNA levels were increased 1.5-fold in cortical cells of Tg SOD1G93A mice respect to Tg hSOD1, whereas the expression of γ-secretase genes, as PSEN1, PSEN2, Nicastrin, and APH1a, showed no statistical differences between wild-type and ALS mice. Biochemical analysis carried out by immunostaining and western blotting, did not show any significant modulation of the protein expression compared to the genes, suggesting the existence of post-translational mechanisms that modify protein levels.     

Transduction of familial amyotrophic lateral sclerosis-related mutant PEP-1-SOD proteins into neuronal cells

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the selective death of motor neurons. Mutations in the SOD1 gene are responsible for a familial form of ALS (FALS). Although many studies suggest that mutant SOD1 proteins are cytotoxic, the mechanism is not fully understood. To investigate the role of mutant SOD1 in FALS, human SOD1 genes were fused with a PEP-1 peptide in a bacterial expression vector to produce in-frame PEP-1-SOD fusion proteins (wild type and mutants). The expressed and purified PEP-1-SOD fusion proteins were efficiently transduced into neuronal cells. Neurones harboring the A4V, G93A, G85R, and D90A mutants of PEP-1-SOD were more vulnerable to oxidative stress induced by paraquat than those harboring wild-type proteins. Moreover, neurones harboring the mutant SOD proteins had lower heat shock protein (Hsp) expression levels than those harboring wild-type SOD. The effects of the transduced SOD1 fusion proteins may provide an explanation for the association of SOD1 with FALS, and Hsps could be candidate agents for the treatment of ALS.     

Mutant SOD1-induced neuronal toxicity is mediated by increased mitochondrial superoxide levels

Amyotrophic lateral sclerosis (ALS), the most common motor neuron disease in adults, is characterized by the selective degeneration and death of motor neurons leading to progressive paralysis and eventually death. Approximately 20% of familial ALS cases are associated with mutations in SOD1, the gene encoding Cu/Zn-superoxide dismutase (CuZnSOD). Previously, we reported that overexpression of the mitochondrial antioxidant manganese superoxide dismutase (MnSOD or SOD2) attenuates cytotoxicity induced by expression of the G37R-SOD1 mutant in a human neuroblastoma cell culture model of ALS. In the present study, we extended these earlier findings using several different SOD1 mutants (G93C, G85R, and I113T). Additionally, we tested the hypothesis that mutant SOD1 increases mitochondrial-produced superoxide (O(2) (*)) levels and that SOD2 overexpression protects neurons from mutant SOD1-induced toxicity by reducing O(2) (*) levels in mitochondria. In the present study, we demonstrate that SOD2 overexpression markedly attenuates the neuronal toxicity induced by adenovirus-mediated expression of all four SOD1 mutants (G37R, G93C, G85R, or I113T) tested. Utilizing the mitochondrial-targeted O(2) (*)-sensitive fluorogenic probe MitoSOX Red, we observed a significant increase in mitochondrial O(2) (*) levels in neural cells expressing mutant SOD1. These elevated O(2) (*) levels in mitochondria were significantly diminished by the overexpression of SOD2. These data suggest that mitochondrial-produced O(2) (*) radicals play a critical role in mutant SOD1-mediated neuronal toxicity and implicate mitochondrial-produced free radicals as potential therapeutic targets in ALS.