Novel behavioural characteristics of the superoxide dismutase 1 G93A (SOD1G93A) mouse model of amyotrophic lateral sclerosis include sex‐dependent phenotypes

Amyotrophic lateral sclerosis (ALS) involves the rapid degeneration of upper and lower motor neurons leading to weakening and paralysis of voluntary movements. Mutations in copper‐zinc superoxide dismutase 1 (SOD1) are a known genetic cause of ALS, and the SOD1 ^G93A mouse has been used extensively to investigate molecular mechanisms in ALS. In recent years, evidence suggests that ALS and frontotemporal dementia form a spectrum disorder ranging from motor to cognitive dysfunctions. Thus, we tested male and female SOD1 ^G93A mice for the first time before the onset of debilitating motor impairments in behavioural domains relevant to both ALS and frontotemporal dementia. SOD1 ^G93A males displayed reduced locomotion, exploration and increased anxiety‐like behaviours compared with control males. Intermediate‐term spatial memory was impaired in SOD1 ^G93A females, whereas long‐term spatial memory deficits as well as lower acoustic startle response, and prepulse inhibition were identified in SOD1 ^G93A mice of both sexes compared with respective controls. Interestingly, SOD1 ^G93A males exhibited an increased conditioned cue freezing response. Nosing behaviours were also elevated in both male and female SOD1 ^G93A when assessed in social paradigms. In conclusion, SOD1 ^G93A mice exhibit a variety of sex‐specific behavioural deficits beyond motor impairments supporting the notion of an ALS‐frontotemporal spectrum disorder. Thus, SOD1 ^G93A mice may represent a useful model to test the efficacy of therapeutic interventions on clinical symptoms in addition to declining motor abilities.     

Progressive loss of a glial potassium channel (KCNJ10) in the spinal cord of the SOD1 (G93A) transgenic mouse model of amyotrophic lateral sclerosis

Transgenic mice expressing the superoxide dismutase G93A mutation (SOD1(G93A)) were used to investigate the role of glial inwardly rectifying K(+) (Kir)4.1 channels, which buffer extracellular K(+) increases in response to neuronal excitation. A progressive decrease in Kir4.1 immunoreactivity was observed predominantly in the ventral horn of SOD1(G93A) mutants. Immunoblotting of spinal cord extracts mirrored these changes by showing a loss of Kir4.1 channels from presymptomatic stages onwards. Kir4.1 channels were found to be expressed in the spinal cord grey matter, targetting astrocytes and clustering around capillaries, supporting their role in clearance of extracellular K(+). To understand the functional implications of extracellular K(+) increases, we challenged the NSC34 motor neurone cell line with increasing extracellular K(+) concentrations. Exposure to high extracellular K(+) induced progressive motor neurone cell death. We suggest that loss of Kir4.1 impairs perineural K(+) homeostasis and may contribute to motor neurone degeneration in SOD1(G93A) mutants by K(+) excitotoxic mechanisms.     

Temporal patterns of cytokine and apoptosis-related gene expression in spinal cords of the G93A-SOD1 mouse model of amyotrophic lateral sclerosis

Familial amyotrophic lateral sclerosis (FALS) is often caused by gain-of-function mutations in Cu,Zn-superoxide dismutase (SOD1). Multiprobe ribonuclease protection assays (RPAs) were used to investigate expression of 36 different cytokines and apoptosis-related genes in spinal cords of mice that ubiquitously express human SOD1 bearing a glycine (r) alanine substitution at residue 93 (G93A-SOD1). Mice were studied at late presymptomatic stage (80 days), and at 120 days when the animals experience severe hindlimb paralysis and accumulation of oxidatively modified proteins. Spinal cord tissue from G93A-SOD1 mice expressed a selective subset of macrophage-typical cytokines (monokines) including interleukin (IL)1alpha, IL1beta and IL1RA at 80 days increasing by 120 days. Contrastingly, T-cell derived cytokines (lymphokines) including IL2, IL3 and IL4 were detected at low levels in non-transgenic mice but these were not elevated in G93A-SOD1 mice even at 120 days. Apoptosis-related genes were generally unaffected at 80 days but multiple caspases and death receptor components were up-regulated at 120 days; the only exceptions being FADD and the tumor necrosis factor (TNF)alpha receptor p55 which was up-regulated at 80 days and increased further at 120 days. These data indicate that in the G93A-SOD1 mouse: (i) cytokine expression changes precede bulk protein oxidation and apoptosis gene expression; (ii) lymphocyte contributions to cytokine expression in FALS are likely minor; and (iii) TNFalpha and its receptors may link inflammation to apoptosis in ALS.     

Differential expression of inflammation- and apoptosis-related genes in spinal cords of a mutant SOD1 transgenic mouse model of familial amyotrophic lateral sclerosis

Familial amyotrophic lateral sclerosis (FALS)-linked mutations in copper-zinc superoxide dismutase (SOD1) cause motor neuron death through one or more acquired toxic properties. We analyzed the molecular mechanism underlying motor neuron degeneration in the transgenic mouse model expressing the SOD1 gene with G93A mutation. Using cDNA microarray, the differentially expressed genes were identified in the spinal cords of G93A mice, 30 being elevated and seven decreased. cDNA microarray analysis to monitor gene expression during neurodegeneration revealed an up-regulation of genes related to an inflammatory process, such as the tumor necrosis factor-alpha (TNF-alpha) gene, resulting from glial cell activation, together with the change in apoptosis-related gene expression, such as caspase-1. The increased expression of the inflammation- and apoptosis-related genes occurred at 11 weeks of age in the presymptomatic stage prior to motor neuron death. These results suggest a mechanism of neurodegeneration that includes an inflammatory response as an important component. Thus, ALS has paralleled other neurodegenerative disorders, such as Alzheimer's and prion diseases, in which the inflammatory process is believed to participate directly in neuronal death.     

Structural consequences of the familial amyotrophic lateral sclerosis SOD1 mutant His46Arg

The His46Arg (H46R) mutant of human copper-zinc superoxide dismutase (SOD1) is associated with an unusual, slowly progressing form of familial amyotrophic lateral sclerosis (FALS). Here we describe in detail the crystal structures of pathogenic H46R SOD1 in the Zn-loaded (Zn-H46R) and metal-free (apo-H46R) forms. The Zn-H46R structure demonstrates a novel zinc coordination that involves only three of the usual four liganding residues, His 63, His 80, and Asp 83 together with a water molecule. In addition, the Asp 124 "secondary bridge" between the copper- and zinc-binding sites is disrupted, and the "electrostatic loop" and "zinc loop" elements are largely disordered. The apo-H46R structure exhibits partial disorder in the electrostatic and zinc loop elements in three of the four dimers in the asymmetric unit, while the fourth has ordered loops due to crystal packing interactions. In both structures, nonnative SOD1-SOD1 interactions lead to the formation of higher-order filamentous arrays. The disordered loop elements may increase the likelihood of protein aggregation in vivo, either with other H46R molecules or with other critical cellular components. Importantly, the binding of zinc is not sufficient to prevent the formation of nonnative interactions between pathogenic H46R molecules. The increased tendency to aggregate, even in the presence of Zn, arising from the loss of the secondary bridge is consistent with the observation of an increased abundance of hyaline inclusions in spinal motor neurons and supporting cells in H46R SOD1 transgenic rats.     

Over-expression of Hsp27 does not influence disease in the mutant SOD1(G93A) mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a chronic, adult-onset neurodegenerative disorder characterized by the selective loss of upper and lower motor neurons, resulting in severe atrophy of muscles and death. Although the exact pathogenic mechanism of mutant superoxide dismutase 1 (SOD1) causing familial ALS is still elusive, toxic protein aggregation leading to insufficiency of chaperones is one of the main hypotheses. In this study, we investigated the effect of over-expressing one of these chaperones, heat shock protein 27 (Hsp27), in ALS. Mice over-expressing the human, mutant SOD1^G93A were crossed with mice that ubiquitously over-expressed human Hsp27. Even though the single transgenic hHsp27 mice showed protection against spinal cord ischemia, the double transgenic SOD1^G93A/hHsp27 mice did not live longer, and did not show a significant delay in the onset of disease compared to their SOD1^G93A littermates. There was no protective effect of hHsp27 over-expression on the motor neurons and on the mutant SOD1 aggregates in the double transgenic SOD1^G93A/hHsp27 mice. In conclusion, despite the protective action against acute motor neuron injury, Hsp27 alone is not sufficient to protect against the chronic motor neuron injury due to the presence of mutant SOD1.     

Comparative study of behavioural tests in the SOD1G93A mouse model of amyotrophic lateral sclerosis

In preclinical trials, a sensitive functional test is required to detect changes in the motor behaviour of the SOD1G93A mouse model of amyotrophic lateral sclerosis (ALS). We evaluated changes in body weight and motor impairment in behavioural tests, such as the rotarod, the hanging-wire test and the treadmill, of transgenic and wild type mice. We found differences in detection of the onset of symptoms and progression of the disease between the different tests assessed. Moreover, the data showed significant gender differences in the motor behaviour of this mouse model. The rotarod and the hanging-wire test were more sensitive to detect early motor impairment. Moreover, the results suggested that the rotarod and hanging-wire became the most accurate tests rather than treadmill to characterise the ALS disease phenotype.     

A copper chaperone–mimetic polytherapy for SOD1-associated amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons progressively and rapidly degenerate, eventually leading to death. The first protein found to contain ALS-associated mutations was copper/zinc superoxide dismutase 1 (SOD1), which is conformationally stable when it contains its metal ligands and has formed its native intramolecular disulfide. Mutations in SOD1 reduce protein folding stability via disruption of metal binding and/or disulfide formation, resulting in misfolding, aggregation, and ultimately cellular toxicity. A great deal of effort has focused on preventing the misfolding and aggregation of SOD1 as a potential therapy for ALS; however, the results have been mixed. Here, we utilize a small-molecule polytherapy of diacetylbis(N(4)-methylthiosemicarbazonato)copper(II) (CuATSM) and ebselen to mimic the metal delivery and disulfide bond promoting activity of the cellular chaperone of SOD1, the “copper chaperone for SOD1.” Using microscopy with automated image analysis, we find that polytherapy using CuATSM and ebselen is highly effective and acts in synergy to reduce inclusion formation in a cell model of SOD1 aggregation for multiple ALS-associated mutants. Polytherapy reduces mutant SOD1-associated cell death, as measured by live-cell microscopy. Measuring dismutase activity via zymography and immunoblotting for disulfide formation showed that polytherapy promoted more effective maturation of transfected SOD1 variants beyond either compound alone. Our data suggest that a polytherapy of CuATSM and ebselen may merit more study as an effective method of treating SOD1-associated ALS.     

Cell-permeable peptide antioxidants as a novel therapeutic approach in a mouse model of amyotrophic lateral sclerosis

Reactive oxygen species (ROS) play a major role in the pathogenesis of neurodegenerative diseases. They are important contributors to necrotic and apoptotic cell death. A major proportion of cellular ROS is generated at the inner mitochondrial membrane by the respiratory chain. In the present study, we investigated a novel peptide antioxidant (SS-31) targeted to the inner mitochondrial membrane for its therapeutic effects both in vitro and in vivo in the G93A mouse model of amyotrophic lateral sclerosis (ALS). SS-31 protected against cell death induced by hydrogen peroxide in vitro in neuronal cells stably transfected with either wild-type or mutant Cu/Zn superoxide dismutase (SOD1). Daily intraperitoneal injections of SS-31 (5 mg/kg), starting at 30 days of age, led to a significant improvement in survival and motor performance. In comparison with vehicle-treated G93A mice, SS-31-treated mice showed a decreased cell loss and a decrease in immunostaining for markers of oxidative stress in the lumbar spinal cord. This further enhances the concept that pharmacological modification of oxidative stress is a therapeutic option for the treatment of ALS.     

Changes in skeletal muscle iron metabolism outpace amyotrophic lateral sclerosis onset in transgenic rats bearing the G93A hmSOD1 gene mutation

Abstract

Objective. Recently, iron and the adaptor protein “p66Shc” have been shown to play an important role in the development of amyotrophic lateral sclerosis (ALS) in rats. We hypothesized that changes in muscle p66Shc activity and iron metabolism would appear before visible symptoms of the disease occurred. Methods. In the present study, we used transgenic rats bearing the G93A hmSOD1 gene mutation and their non-transgenic littermates to test this hypothesis. We examined muscle p66Shc phosphorylation and iron metabolism in relation to oxidative stress in animals at three disease stages: asymptomatic (ALS I), disease onset (ALS II), and end-stage disease (ALS III). Results. Significant changes in iron metabolism and markers of lipid and protein oxidation were detected in ALS I animals, which manifested as decreased levels of ferritin H and ferroportin 1 (Fpn1) and increased levels of ferritin L levels. Muscles of ALS I rats possessed increased levels of p66Shc phosphorylated at Ser³⁶ compared with muscles of control rats. During disease progression, level of ferritin H significantly increased and was accompanied by iron accumulation. Conclusions. This study showed that multiple mechanisms may underlie iron accumulation in muscles of ALS transgenic rats, which include changes in blood hepcidin and muscle Fpn1 and increased level of muscle ferritin H. These data suggest that impaired iron metabolism is not a result of changes in motor activity.     

Proteomic analysis of spinal cord of presymptomatic amyotrophic lateral sclerosis G93A SOD1 mouse

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, whose primary mechanisms or causes are still not defined and for which no effective treatment is available. We have recently reported that before disease onset the level of tyrosine nitrated proteins is increased in the G93A SOD1 transgenic mouse model of ALS. In the present investigation, we carried out a proteomic analysis of spinal cord extracts from G93A SOD1 mice at the presymptomatic stage of the disease to further unravel primary events in the pathogenesis and tentatively screen for potential pharmacological targets. Using a robust two-dimensional gel electrophoresis-based proteomic approach, we detected a number of proteins differentially represented in presymptomatic mice in comparison with controls. Alterations of these proteins correlate with mitochondrial dysfunction, aggregation, and stress response. Moreover, we found a variation in the isoform pattern of cyclophilin A, a molecular chaperone that protects cells from the oxidative stress.     

Oxidative inactivation of calcineurin by Cu,Zn superoxide dismutase G93A, a mutant typical of familial amyotrophic lateral sclerosis

Calcineurin is a serine/threonine phosphatase involved in a wide range of cellular responses to calcium mobilizing signals. Previous evidence supports the notion of the existence of a redox regulation of this enzyme, which might be relevant for neurodegenerative processes, where an imbalance between generation and removal of reactive oxygen species could occur. In a recent work, we have observed that calcineurin activity is depressed in two models for familial amyotrophic lateral sclerosis (FALS) associated with mutations of the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1), namely in neuroblastoma cells expressing either SOD1 mutant G93A or mutant H46R and in brain areas from G93A transgenic mice. In this work we report that while wild-type SOD1 has a protective effect, calcineurin is oxidatively inactivated by mutant SOD1s in vitro; this inactivation is mediated by reactive oxygen species and can be reverted by addition of reducing agents. Furthermore, we show that calcineurin is sensitive to oxidation only when it is in an 'open', calcium-activated conformation, and that G93A-SOD1 must have its redox-active copper site available to substrates in order to exert its pro-oxidant properties on calcineurin. These findings demonstrate that both wild-type and mutant SOD1s can interfere directly with calcineurin activity and further support the possibility of a relevant role for calcineurin-regulated biochemical pathways in the pathogenesis of FALS.     

MTOR-independent,autophagic enhancer trehalose prolongs motor neuron survival and ameliorates the autophagic flux defect in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder caused by selective motor neuron degeneration. Abnormal protein aggregation and impaired protein degradation pathways may contribute to the disease pathogenesis. Although it has been reported that autophagy is altered in patients and animal model of ALS, little is known about the role of autophagy in motor neuron degeneration in this disease. Our previous study shows that rapamycin, an MTOR-dependent autophagic activator, accelerates disease progression in the SOD1^G93A mouse model of ALS. In the present report, we have assessed the role of the MTOR-independent autophagic pathway in ALS by determining the effect of the MTOR-independent autophagic inducer trehalose on disease onset and progression, and on motor neuron degeneration in SOD1^G93A mice. We have found that trehalose significantly delays disease onset prolongs life span, and reduces motor neuron loss in the spinal cord of SOD1^G93A mice. Most importantly, we have documented that trehalose decreases SOD1 and SQSTM1/p62 aggregation, reduces ubiquitinated protein accumulation, and improves autophagic flux in the motor neurons of SOD1^G93A mice. Moreover, we have demonstrated that trehalose can reduce skeletal muscle denervation, protect mitochondria, and inhibit the proapoptotic pathway in SOD1^G93A mice. Collectively, our study indicated that the MTOR-independent autophagic inducer trehalose is neuroprotective in the ALS model and autophagosome-lysosome fusion is a possible therapeutic target for the treatment of ALS.     

MTOR-independent,autophagic enhancer trehalose prolongs motor neuron survival and ameliorates the autophagic flux defect in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder caused by selective motor neuron degeneration. Abnormal protein aggregation and impaired protein degradation pathways may contribute to the disease pathogenesis. Although it has been reported that autophagy is altered in patients and animal model of ALS, little is known about the role of autophagy in motor neuron degeneration in this disease. Our previous study shows that rapamycin, an MTOR-dependent autophagic activator, accelerates disease progression in the SOD1^G93A mouse model of ALS. In the present report, we have assessed the role of the MTOR-independent autophagic pathway in ALS by determining the effect of the MTOR-independent autophagic inducer trehalose on disease onset and progression, and on motor neuron degeneration in SOD1^G93A mice. We have found that trehalose significantly delays disease onset prolongs life span, and reduces motor neuron loss in the spinal cord of SOD1^G93A mice. Most importantly, we have documented that trehalose decreases SOD1 and SQSTM1/p62 aggregation, reduces ubiquitinated protein accumulation, and improves autophagic flux in the motor neurons of SOD1^G93A mice. Moreover, we have demonstrated that trehalose can reduce skeletal muscle denervation, protect mitochondria, and inhibit the proapoptotic pathway in SOD1^G93A mice. Collectively, our study indicated that the MTOR-independent autophagic inducer trehalose is neuroprotective in the ALS model and autophagosome-lysosome fusion is a possible therapeutic target for the treatment of ALS.     

Focal dysfunction of the proteasome: a pathogenic factor in a mouse model of amyotrophic lateral sclerosis

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a familial form of amyotrophic lateral sclerosis (fALS). The present study demonstrated impaired proteasomal function in the lumbar spinal cord of transgenic mice expressing human SOD-1 with the ALS-causing mutation G93A (SOD-1(G93A)) compared to non-transgenic littermates (LM) and SOD-1(WT) transgenic mice. Chymotrypsin-like activity was decreased as early as 45 days of age. By 75 days, chymotrypsin-, trypsin-, and caspase-like activities of the proteasome were impaired, at about 50% of control activity in lumbar spinal cord, but unchanged in thoracic spinal cord and liver. Both total and specific activities of the proteasome were reduced to a similar extent, indicating that a change in proteasome function, rather than a decrease in proteasome levels, had occurred. Similar decreases of total and specific activities of the proteasome were observed in NIH 3T3 cell lines expressing fALS mutants SOD-1(G93A) and SOD-1(G41S), but not in SOD-1(WT) controls. Although overall levels of proteasome were maintained in spinal cord of SOD-1(G93A) transgenic mice, the level of 20S proteasome was substantially reduced in lumbar spinal motor neurons relative to the surrounding neuropil. It is concluded that impairment of the proteasome is an early event and contributes to ALS pathogenesis.     

Dorfin prevents cell death by reducing mitochondrial localizing mutant superoxide dismutase 1 in a neuronal cell model of familial amyotrophic lateral sclerosis

Abstract Dorfin is a RING-finger type ubiquitin ligase for mutant superoxide dismutase 1 (SOD1) that enhances its degradation. Mutant SOD1s cause familial amyotrophic lateral sclerosis (FALS) through the gain of unelucidated toxic properties. We previously showed that the accumulation of mutant SOD1 in the mitochondria triggered the release of cytochrome c, followed by the activation of the caspase cascade and induction of neuronal cell death. In the present study, therefore, we investigated whether Dorfin can modulate the level of mutant SOD1 in the mitochondria and subsequent caspase activation. We showed that Dorfin significantly reduced the amount of mutant SOD1 in the mitochondria, the release of cytochrome c and the activation of the following caspase cascade, thereby preventing eventual neuronal cell death in a neuronal cell model of FALS. These results suggest that reducing the accumulation of mutant SOD1 in the mitochondria may be a new therapeutic strategy for mutant SOD1-associated FALS, and that Dorfin may play a significant role in this.     

Dysregulation of intracellular copper trafficking pathway in a mouse model of mutant copper/zinc superoxide dismutase-linked familial amyotrophic lateral sclerosis

Mutations in copper/zinc superoxide dismutase (SOD1) are responsible for 20% of familial amyotrophic lateral sclerosis through a gain-of-toxic function. We have recently shown that ammonium tetrathiomolybdate, an intracellular copper-chelating reagent, has an excellent therapeutic benefit in a mouse model for amyotrophic lateral sclerosis. This finding suggests that mutant SOD1 might disrupt intracellular copper homeostasis. In this study, we investigated the effects of mutant SOD1 on the components of the copper trafficking pathway, which regulate intracellular copper homeostasis. We found that mutant, but not wild-type, SOD1 shifts intracellular copper homeostasis toward copper accumulation in the spinal cord during disease progression: copper influx increases, copper chaperones are up-regulated, and copper efflux decreases. This dysregulation was observed within spinal motor neurons and was proportionally associated with an age-dependent increase in spinal copper ion levels. We also found that a subset of the copper trafficking pathway constituents co-aggregated with mutant SOD1. These results indicate that the nature of mutant SOD1 toxicity might involve the dysregulation of the copper trafficking pathway, resulting in the disruption of intracellular copper homeostasis.     

Arylsulfanyl pyrazolones block mutant SOD1-G93A aggregation. Potential application for the treatment of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an orphan neurodegenerative disease currently without a cure. Mutations in copper/zinc superoxide dismutase 1 (SOD1) have been implicated in the pathophysiology of this disease. Using a high-throughput screening assay expressing mutant G93A SOD1, two bioactive chemical hit compounds (1 and 2), identified as arylsulfanyl pyrazolones, were identified. The structural optimization of this scaffold led to the generation of a more potent analogue (19) with an EC₅₀ of 170 nM. To determine the suitability of this class of compounds for further optimization, 1 was subjected to a battery of pharmacokinetic assays; most of the properties of 1 were good for a screening hit, except it had a relatively rapid clearance and short microsomal half-life stability. Compound 2 was found to be blood-brain barrier penetrating with a brain/plasma ratio = 0.19. The optimization of this class of compounds could produce novel therapeutic candidates for ALS patients.