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Purpose: We characterized the response to the extremely low frequency magnetic field (ELF-MF) in an in vitro model of familial Amyotrophic Lateral Sclerosis (fALS), carrying two mutant variants of the superoxide dismutase 1 (SOD1) gene. Materials and methods: SH-SY5Y human neuroblastoma cells, stably over-expressing the wild type, the G93A or the H46R mutant SOD1 cDNA, were exposed to either the ELF-MF (50 Hz, 1 mT) or the sham control field, up to 72 h. Analysis of (i) viability, proliferation and apoptosis, (ii) reactive oxygen species generation, and (iii) assessment of the iron metabolism, were carried out in all clones in response to the MF exposure. Results: We report that 50-Hz MF exposure induces: (i) no change in proliferation and viability; (ii) no modulation of the intracellular superoxide and H2O2 levels; (iii) a significant deregulation in the expression of iron-related genes IRP1, MFRN1 and TfR1, this evidence being exclusive for the SOD1G93A clone and associated with a slight (p =.0512) difference in the total iron content. Conclusions: 50-Hz MF affects iron homeostasis in the in vitro SOD1G93A ALS model. [ABSTRACT FROM AUTHOR]

Abstract Current understanding of the pathogenesis of the familial form of amyotrophic lateral sclerosis has been aided by the study of transgenic mice that over-express mutated forms of the human CuZn-superoxide dismutase (SOD1) gene. While mutant SOD1 in motor neurons determines disease onset, other non-cell autonomous factors are critical for disease progression, and altered energy metabolism has been implicated as a contributing factor. Since most energy expended by laboratory mice is utilized to defend body temperature (T b), we analyzed thermoregulation in transgenic mice carrying the G93A mutation of the human SOD1 gene, using implantable temperature data loggers to continuously record T b for up to 85 days. At room (22 °C) ambient temperature, G93A mice exhibited a diminished amplitude of the daily T b rhythm compared to C57BL/6J controls, secondary to decreased T b values during the dark (behaviorally active) phase of the light-dark cycle. The defect arose at 85–99 days of age, around the age of symptom onset (as assessed by grip strength), well before observable weakness and weight loss, and could not be accounted for by decreased levels of locomotor activity or food consumption. Housing under thermoneutral (29 °C) ambient temperature partially rescued the defect, but age-dependently (only in animals >100 days of age), suggesting that the deficit in older mice was due in part to inadequate thermogenesis by "peripheral" thermogenic organs as the disease progressed. In younger mice, we found that cold-induced thermogenesis and energy expenditure were intact, hinting that an initial "central" defect might localize to the subparaventricular zone, involving neural output pathways from the circadian clock in the hypothalamic suprachiasmatic nucleus to forebrain thermoregulatory circuitry. Highlights • G93A SOD1 mice exhibit a decreased amplitude of their daily body temperature rhythm. • The defect is not due to decreased locomotor activity and arises prior to weight loss. • Housing under thermoneutrality implicates both "central" and "peripheral" mechanisms. • The "central" deficit may localize to neural output pathways from the circadian clock. • The "peripheral" deficit is superimposed later, as thermogenic organs begin to fail. [ABSTRACT FROM AUTHOR]

Here we report a gain in function for mutant (mt) superoxide dismutase I (SOD1), a cause of familial amyotrophic lateral sclerosis (FALS), wherein small soluble oligomers of mtSOD1 acquire a membrane toxicity. Phosphatidylglycerol (PG) lipid domains are selectively targeted, which could result in membrane damage or “toxic channels” becoming active in the bilayer. This PG-selective SOD1-mediated membrane toxicity is largely reversible in vitro by a widely-available FDA-approved surfactant and membrane-stabilizer P188. Treatment of G93ASOD1 transgenic mice with P188 significantly delayed symptoms onset, extended survival and decreased motoneuron death. The use of P188 or an analogue, which targets mtSOD1 misfolding-induced membrane toxicity, may provide a new direction for ALS treatment. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by degeneration and loss of motor neurons in the spinal cord, brainstem and motor cortex. Up to 10% of ALS cases are inherited (familial, fALS) and associated with mutations, frequently in the superoxide dismutase 1 (SOD1) gene. Rodent transgenic models of ALS are often used to elucidate a complex pathogenesis of this disease. Of importance, both ALS patients and animals carrying mutated human SOD1 gene show symptoms of oxidative stress and iron metabolism misregulation. The aim of our study was to characterize changes in iron metabolism in one of the most commonly used models of ALS - transgenic mice overexpressing human mutated SOD1G93A gene. We analyzed the expression of iron-related genes in asymptomatic, 2-month-old and symptomatic, 4-month-old SOD1G93A mice. In parallel, respective age-matched mice overexpressing human non-mutated SOD1 transgene and control mice were analyzed. We demonstrate that the overexpression of both SOD1 and SOD1G93A genes account for a substantial increase in SOD1 protein levels and activity in selected tissues and that not all the changes in iron metabolism genes expression are specific for the overexpression of the mutated form of SOD1. [ABSTRACT FROM AUTHOR]

Background: In vivo animal models of familial amyotrophic lateral sclerosis (fALS) are widely used to delineate the potential role that genetic mutations play in the neurodegenerative process. While these models are extensively used for establishing the safety and efficacy of putative therapeutics during pre-clinical development, effective clinical translation of pharmacological interventions has been largely unsuccessful. Results: In this report we compare a recent cohort of G37R (line 29) mice generated from mating wild-type females with transgenic males obtained commercially to a previous set of offspring produced with transgenic male breeders from a colony established at a local collaborator's facility. Commercially derived progeny presented with a tightly clustered genomic signature for the mutant human superoxide dismutase1 transgene (hSOD1) locus, and exhibited a greater than two-fold reduction in the number of transgene copies present in the genome compared to offspring derived locally. Decrease in transgene levels corresponded with delayed ALS progression and a significant increase in overall lifespan (146%). Conclusions: These results highlight some key challenges inherent to the use of G37R (line 29) animals in pre-clinical studies for the development of ALS therapeutics. Without stringent assessment of mutant SOD1 copy number/protein levels, heterogeneity of transgene levels within cohorts may influence the behavioural and pathological presentation of disease and thus calls to question the validity of any detected therapeutic effects. Nuanced changes in mutant SOD1 copy number that currently remain unreported may undermine research endeavours, delay efforts for clinical translation, and compromise the rigor of animal studies by limiting reproducibility amongst research groups. [ABSTRACT FROM AUTHOR]

Abstract: This study examined whether activity is a contributing factor to motor terminal degeneration in mice that overexpress the G93A mutation of the SOD1 enzyme found in humans with inherited motor neuron disease. Previously, we showed that overload of muscles accomplished by synergist denervation accelerated motor terminal degeneration in dogs with hereditary canine spinal muscular atrophy (HCSMA). In the present study, we found that SOD1 plantaris muscles overloaded for 2months showed no differences of neuromuscular junction innervation status when compared with normally loaded, contralateral plantaris muscles. Complete elimination of motor terminal activity using blockade of sciatic nerve conduction with tetrodotoxin cuffs for 1month also produced no change of plantaris innervation status. To assess possible effects of activity on motor terminal function, we examined the synaptic properties of SOD1 soleus neuromuscular junctions at a time when significant denervation of close synergists had occurred as a result of natural disease progression. When examined in glucose media, SOD1 soleus synaptic properties were similar to wildtype. When glycolysis was inhibited and ATP production limited to mitochondria, however, blocking of evoked synaptic transmission occurred and a large increase in the frequency of spontaneous mEPCs was observed. Similar effects were observed at neuromuscular junctions in muscle from dogs with inherited motor neuron disease (HCSMA), although significant defects of synaptic transmission exist at these neuromuscular junctions when examined in glucose media, as reported previously. These results suggest that glycolysis compensates for mitochondrial dysfunction at motor terminals of SOD1 mice and HCSMA dogs. This compensatory mechanism may help to support resting and activity-related metabolism in the presence of dysfunctional mitochondria and prolong the survival of SOD1 motor terminals. [Copyright &y& Elsevier]

Abstract: Loss of astrocytic glutamate transport capacity in ALS spinal cord supports an excitotoxic contribution to motor neuron (MN) damage in the disease, and dominant gain of function mutations in Cu/Zn superoxide dismutase (SOD1) cause certain familial forms of ALS. We have used organotypic slice cultures from wild type and G93A SOD1 mutant rat spinal cords to examine interactions between excitotoxicity and the presence of mutant SOD1 in the induction of MN degeneration. Slice cultures were prepared from 1week old pups, and after an additional week in vitro, some were exposed to either a low level (30μM) of the glutamate uptake inhibitor, trans-pyrrolidine-2,4-dicarboxylic acid (PDC) for 3weeks, or a higher level (50μM) for 48h, followed by histochemical labeling to assess MN injury. In wild type animals these exposures caused relatively little MN degeneration. Similarly, little MN degeneration was seen in slices from SOD1 mutant animals that were not exposed to PDC. However, addition of PDC to SOD1 mutant slices resulted in substantial MN injury, which was markedly attenuated by a Ca2+ permeable AMPA-type (Ca-AMPA) glutamate channel blocker, or by a nitric oxide synthase antagonist. These observations illustrate the utility of the organotypic culture model for the investigation of intracellular interactions underlying MN degeneration in ALS, and support the hypothesis that activation of Ca-AMPA channels on MNs provides a metabolic burden that synergizes with deleterious effects of mutant SOD1 in the induction of MN injury. [Copyright &y& Elsevier]

Abstract: Mutant superoxide dismutase 1 (mSOD1) is often found as aggregates at the outer-membrane of mitochondria in motor neurons of various mouse models and familial amyotrophic lateral sclerosis (f-ALS) patients. It has been postulated that disruption of mitochondrial function by physical association of misfolded mSOD1 aggregates may actually be the trigger for initiation of degeneration of motor neurons in ALS. However, it was not clear if the same mechanism is involved in muscle degeneration and mitochondrial dysfunction in skeletal muscles of ALS. Recent study from our laboratory show that two skeletal muscle proteins, namely creatine kinase (CK) and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) undergo major conformational and functional changes in the f-ALS mouse model of ALS (G93A). In this paper, we report two intriguing observations which are as follows:(i) G93A protein does not form aggregates in skeletal muscle at any stages of disease process probably due to high chymotrypsin-like activity of proteasome and thus G93A protein aggregates have no direct effects on progressive loss of muscle mass and global changes in protein conformation in ALS, and (ii) the soluble G93A protein does not have direct effects on mitochondrial dysfunction as determined by quantifying the release of reactive oxygen species (ROS) in skeletal muscle mitochondria; instead, the proteins affected by G93A possibly affect mitochondrial ROS release. These data strongly suggest for the first time that unlike in motor neurons, the soluble and aggregation states of the G93A protein do not have direct effects on protein misfolding and mitochondrial dysfunction in skeletal muscle during ALS. [Copyright &y& Elsevier]

Abstract: Defect of metallothionein-III (MT-III) has been reported to be a contributor to the progression of amyotrophic lateral sclerosis (ALS). We explored the expression and effects of MT-III on the motor neurons of spinal cords of ALS model mice (G93A Cu/Zn superoxide dismutase (SOD-1) mutant-transgenic (Tg) mice) using a retrograde viral delivery system. Once-weekly injection of the adenovirus encoding LacZ or MT-III gene was started at the age of 20 weeks, which was the mean age of ALS onset. Gene expression was detected in the motor neurons of the lumbar spinal cord. At 160 days of age (14 days after injection), the mean numbers of Nissl-stained α neurons were 15.42±5.32, 16.50±1.35, and 24.75±4.01 in 5-μm sections of the lumbar hemispinal cord from the untreated group, LacZ group, and MT-III group, respectively. The mean durations of illness were 15.20±5.30 days, 10.33±4.27 days, and 25.71±7.67 days in the untreated group, LacZ group, and MT-III group, respectively. The mean life spans were 163.20±7.72 days, 159.50±3.27 days, and 178.14±12.97 days in the untreated group, LacZ group, and MT-III group, respectively. We demonstrated that MT-III prevents the loss of motor neurons of ALS model mice and prolongs the life span, even when the administration is started at the time of onset. [Copyright &y& Elsevier]

mutant form of the ubiquitous copper/zinc superoxide dismutase (SOD1) protein has been found in some patients with amyotrophic lateral sclerosis (ALS). We monitored oxidative stress in an animal model of ALS, the SOD mouse, which develops a disease similar to ALS with an accelerated course. The aim of this work was to show that ALS damages several organs and tissues, from an oxidative stress point of view. We measured lipid and protein oxidative damage in different tissue homogenates of SOD mice. The biomarkers that we analyzed were malondialdehyde + 4-hydroxyalkenal (MDA + 4-HDA) and carbonyls, respectively. The spinal cord and brain of SOD mice showed increased lipid peroxidation after 100 or 130 days compared to age-matched littermate controls. The CNS was most affected, but lipid peroxidation was also detected in the skeletal muscle and liver on day 130. No changes were observed in protein carbonylation in the homogenates. Our results are consistent with a multisystem etiology of ALS and suggest that oxidative stress may play a primary role in ALS pathogenesis. Thus, oxidative stress represents a potential biomarker that might be useful in developing new therapeutic strategies for ALS. [ABSTRACT FROM AUTHOR]

Abstract: Amyotrophic Lateral Sclerosis (ALS) is a devastating adult-onset motor neuron disorder with marginal therapeutic options. The disease is characterized by progressive degeneration of motor neurons in spinal cord and motor cortex. Transgenic mice carrying the G93A mutation of the superoxide dismutase 1 (SOD1) gene develop a neurodegenerative disease closely mimicking human ALS. Several methods are currently used to record disease onset and progression of the animals in preclinical studies. For the interpretation of these preclinical trials, it is important to assess neurological function as sensitively as possible. In the present study, five different parameters (rotarod performance, weight, footprint analysis for both step length and runtime and the general condition of the mice scored from 1 to 5) were compared with respect to their significance to detect symptom onset and to monitor disease progression in transgenic G93A ALS mice. The rotarod and footprint analyses were performed weekly while the weight was recorded up to three times a week at later time points. General condition was assessed daily. First deficits were detected by rotarod testing and step length analyses. General condition score and weight showed first changes two weeks later. For preclinical testing of novel drug treatments rotarod and footprint analysis for step length therefore seem to be the most effective methods to detect symptom onset and potential treatment induced improvements. [Copyright &y& Elsevier]

Abstract: Therapeutic success in the superoxide dismutase (SOD1) mouse model of amyotrophic lateral sclerosis (ALS) has not translated into effective therapy for human ALS, calling into question the utility of such preclinical data for identifying therapeutic agents that are worthy of further study in humans. This random effects meta-analysis of treatment trials in the superoxide dismutase (SOD1) mouse was undertaken in order to explore possible reasons for this failure of translational research and to identify potential pharmacological interventions that might be used in either a preventative or therapeutic trial in familial ALS. Among studies in which treatment was initiated presymptomatically, the weighted mean differences (WMDs) comparing the active treatment to control treated animals were 12 days (onset), 13 days (survival) and 5 days (survival interval). Among studies in which treatment was initiated at the time of symptom onset, the WMDs were 15 days (survival) and 8 days (survival interval). Subgroup analysis suggests that drugs such as minocycline and Cox-2 inhibitors with an anti-inflammatory mechanism of action, and anti-oxidative agents such as creatine or the manganese porphyrin AEOL-10150, appear to be the most promising for preventative and therapeutic trials respectively in patients with familial ALS. These conclusions should be tempered by the methodological limitations of the relevant literature. [Copyright &y& Elsevier]

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease which results from selective loss of upper and lower motor neurons. Mouse models of ALS, such as one carrying the G93A mutant of the human Cu-Zn superoxide dismutase gene [SOD1(G93A)], develop motor neuron pathology and clinical symptoms similar to those observed in ALS patients. There is compelling evidence that both direct and indirect glutamate toxicity contribute to the pathogenesis of motor neuron degeneration. However, the therapeutic effect of various glutamate receptor antagonists has not been clearly demonstrated. Memantine is a noncompetitive N-methyl- d-aspartate (NMDA) receptor antagonist. It has been shown to protect neurons against NMDA- or glutamate-induced toxicity in vitro and in animal models of neurodegenerative diseases. In the current study, we have examined the therapeutic efficacy of memantine in an ALS mouse model carrying a high copy number of SOD1(G93A). Memantine treatment significantly delayed the disease progression and increased the life span of SOD1(G93A) mice, from 121.4 ± 5.5 to 129.7 ± 4.5 days ( P = 0.032) . Furthermore, NMDA receptor subunits were reliably detected in the spinal cord of SOD1(G93A) mice and their expression levels were similar to those in the wild-type littermate control. Therefore, the neuroprotective effect of memantine in SOD1(G93A) mice is most probably due to the inhibition of spinal cord NMDA receptors. In view of the long-term usage of memantine for dementia patients, with excellent tolerance and safety, these data suggest that memantine may be used in ALS patients alone or in combination with other therapies to prolong survival. [ABSTRACT FROM AUTHOR]