Respiratory impairment in a mouse model of amyotrophic lateral sclerosis.

Amyothrophic lateral sclerosis (ALS) is a progressive, lethal neuromuscular disease that is associated with the degeneration of cortical and spinal motoneurons, leading to atrophy of limb, axial, and respiratory muscles. Patients with ALS invariably develop respiratory muscle weakness and most die from pulmonary complications. Overexpression of superoxide dismutase 1 (SOD1) gene mutations in mice recapitulates several of the clinical and pathological characteristics of ALS and is therefore a valuable tool to study this disease. The present study is intended to evaluate an age-dependent progression of respiratory complications in SOD1(G93A) mutant mice. In each animal, baseline measurements of breathing pattern [i.e., breathing frequency and tidal volume (VT)], minute ventilation (VE), and metabolism (i.e., oxygen consumption and carbon dioxide production) were repeatedly sampled at variable time points between 10 and 20 wk of age with the use of whole-body plethysmographic chambers. To further characterize the neurodegeneration of breathing, VE was also measured during 5-min challenges of hypercapnia (5% CO(2)) and hypoxia (10% O(2)). At baseline, breathing characteristics and metabolism remained relatively unchanged from 10 to 14 wk of age. From 14 to 18 wk of age, there were significant (P < 0.05) increases in baseline VT, VE, and the ventilatory equivalent (VE/oxygen consumption). After 18 wk of age, there was a rapid decline in VE due to significant (P < 0.05) reductions in both breathing frequency and VT. Whereas little change in hypoxic VE responses occurred between 10 and 18 wk, hypercapnic VE responses were significantly (P < 0.05) elevated at 18 wk due to an augmented VT response. Like baseline breathing characteristics, hypercapnic VE responses also declined rapidly after 18 wk of age. The phenotypic profile of SOD1(G93A) mutant mice was apparently unique because similar changes in respiration and metabolism were not observed in SOD1 controls. The present results outline the magnitude and time course of respiratory complications in SOD1(G93A) mutant mice as the progression of disease occurs in this mouse model of ALS.     

Transgenic mouse models of amyotrophic lateral sclerosis

The discovery of missense mutations in the gene coding for the Cu/Zn superoxide dismutase 1 (SOD1) in subsets of familial cases was rapidly followed by the generation of transgenic mice expressing various forms of SOD1 mutants. The mice overexpressing high levels of mutant SOD1 mRNAs do develop motor neuron disease but unraveling the mechanisms of pathogenesis has been very challenging. Studies with mouse lines suggest that the toxicity of mutant SOD1 is unrelated to copper-mediated catalysis but rather to propensity of a subfraction of mutant SOD1 proteins to form misfolded protein species and aggregates. However, the mechanism of toxicity of SOD1 mutants remains to be elucidated. Involvement of cytoskeletal components in ALS pathogenesis is supported by several mouse models of motor neuron disease with neurofilament abnormalities and with genetic defects in microtubule-based transport. Here, we describe how transgenic mouse models have been used for understanding pathogenic pathways of motor neuron disease and for pre-clinical drug testing.     

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.     

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.     

Abnormal exocytotic release of glutamate in a mouse model of amyotrophic lateral sclerosis

Glutamate-mediated excitotoxicity plays a major role in the degeneration of motor neurons in amyotrophic lateral sclerosis and reduced astrocytary glutamate transport, which in turn increases the synaptic availability of the amino acid neurotransmitter, was suggested as a cause. Alternatively, here we report our studies on the exocytotic release of glutamate as a possible source of excessive glutamate transmission. The basal glutamate efflux from spinal cord nerve terminals of mice-expressing human soluble superoxide dismutase (SOD1) with the G93A mutation [SOD1/G93A(+)], a transgenic model of amyotrophic lateral sclerosis, was elevated when compared with transgenic mice expressing the wild-type human SOD1 or to non-transgenic controls. Exposure to 15 mM KCl or 0.3 μM ionomycin provoked Ca(2+)-dependent glutamate release that was dramatically increased in late symptomatic and in pre-symptomatic SOD1/G93A(+) mice. Increased Ca(2+) levels were detected in SOD1/G93A(+) mouse spinal cord nerve terminals, accompanied by increased activation of Ca(2+)/calmodulin-dependent kinase II and increased phosphorylation of synapsin I. In line with these findings, release experiments suggested that the glutamate release augmentation involves the readily releasable pool of vesicles and a greater capability of these vesicles to fuse upon stimulation in SOD1/G93A(+) mice.     

Early functional deficit and microglial disturbances in a mouse model of amyotrophic lateral sclerosis

Background

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by selective motoneurons degeneration. There is today no clear-cut pathogenesis sequence nor any treatment. However growing evidences are in favor of the involvement, besides neurons, of several partners such as glia and muscles. To better characterize the time course of pathological events in an animal model that recapitulates human ALS symptoms, we investigated functional and cellular characteristics of hSOD1^G93A mice.

Methods and Findings

We have evaluated locomotor function of hSOD1^G93A mice through dynamic walking patterns and spontaneous motor activity analysis. We detected early functional deficits that redefine symptoms onset at 60 days of age, i.e. 20 days earlier than previously described. Moreover, sequential combination of these approaches allows monitoring of motor activity up to disease end stage. To tentatively correlate early functional deficit with cellular alterations we have used flow cytometry and immunohistochemistry approaches to characterize neuromuscular junctions, astrocytes and microglia. We show that (1) decrease in neuromuscular junction''s number correlates with motor impairment, (2) astrocytes number is not altered at pre- and early-symptomatic ages but intraspinal repartition is modified at symptoms onset, and (3) microglia modifications precede disease onset. At pre-symptomatic age, we show a decrease in microglia number whereas at onset of the disease two distinct microglia sub-populations emerge.

Conclusions

In conclusion, precise motor analysis updates the onset of the disease in hSOD1^G93A mice and allows locomotor monitoring until the end stage of the disease. Early functional deficits coincide with alterations of neuromuscular junctions. Importantly, we identify different sets of changes in microglia before disease onset as well as at early-symptomatic stage. This finding not only brings a new sequence of cellular events in the natural history of the disease, but it may also provide clues in the search for biomarkers of the disease, and potential therapeutic targets.     

Bax and Bcl-2 interaction in a transgenic mouse model of familial amyotrophic lateral sclerosis

It has been proposed that mutations in copper/zinc-superoxide dismutase (SOD1), the only proven cause of amyotrophic lateral sclerosis (ALS), induce the disease by a toxic property that promotes apoptosis. Consistent with this, we have demonstrated that overexpression of Bcl-2, a protein that inhibits apoptosis, attenuates neurodegeneration produced by the familial ALS-linked SOD1 mutant G93A (mSOD1). Herein, we assessed the status of key members of the Bcl-2 family in the spinal cord of transgenic mSOD1 mice at different stages of the disease. In asymptomatic transgenic mSOD1 mice, expression of Bcl-2, Bcl-XL, Bad, and Bax does not differ from that in nontransgenic mice. In contrast, in symptomatic mice, expression of Bcl-2 and Bcl-XL, which inhibit apoptosis, is reduced, whereas expression of Bad and Bax, which stimulate apoptosis, is increased. These alterations are specific to affected brain regions and are caused by the mutant and not by the normal SOD1 enzyme. Relevant to the neuroprotective effects of Bcl-2 in transgenic mSOD1 mice, overexpression of Bcl-2 increases the formation of Bcl-2:Bax heterodimers, which abolish the Bax proapoptotic property. This study demonstrates significant alterations in the expression of key members of the Bcl-2 family associated with mSOD1 deleterious effects. That these changes contribute to the neurodegenerative process in this model of ALS is supported by our observations in double transgenic mSOD1/Bcl-2 mice in which the pernicious increase of Bax is tempered by an increase in formation of Bcl-2:Bax heterodimers. Based on these findings, it may be concluded that Bcl-2 family members appear as invaluable targets for the development of new neuroprotective therapies in ALS.     

Trace elements, age, and sex in amyotrophic lateral sclerosis disease

The statistical tests analysis of variance, analysis of covariance, correlation coefficient, Kolmogorov-Smirnov test,t-test, and Tukey test were applied to copper, magnesium, managenese, and zinc content in serum (S) and in cerebrospinal fluid (CSF) of controls and of a sporadic form of Amyotrophic Lateral Sclerosis (ALS) disease. This is carried out in order to evaluate statistically the possible relationships among the trace elements when ALS patients and controls are considered as independent groups, within sex groups and within age decades of both patients and control classes. A statistically significant difference between older controls (age >40) and ALS patients (age>40) for copper in CSF, copper in S, manganese in S, and zinc in CSF was found. Statistically significant correlation coefficients within the different classes formed for this study were observed. Within this pool, a correlation of patient group can differ statistically from the corresponding one of controls and vice versa. Thus, this correlation could be characteristic of the group from which is extracted, e.g., the correlation between copper in S and zinc in S, which is characteristic of ALS patients when considered as an independent group as well as members of the male patient class.     

Glutamate release and uptake processes are altered in a new mouse model of amyotrophic lateral sclerosis

In this paper, we showed that in the cortex of mice expressing an abberant form of FUS protein that model amyotrophic lateral sclerosis (ALS), the processes of KCl-induced and basal [³H]glutamate release and uptake are altered at the presymptomatic stage as compared to the non-transgenic littermates. The change in these three parameters in transgenic animals causes excitotoxicity, which, in turn, may lead to massive loss of motor neurons and the onset of ALS symptoms.     

Early abnormalities in transgenic mouse models of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative and fatal human disorder characterized by progressive loss of motor neurons. Transgenic mouse models of ALS are very useful to study the initial mechanisms underlying this neurodegenerative disease. We will focus here on the earlier abnormalities observed in superoxide dismutase 1 (SOD1) mutant mice. Several hypotheses have been advanced to explain the selective loss of motor neurons such as apoptosis, neurofilament disorganisation, oxidative stress, mitochondrial dysfunction, astrogliosis and excitotoxicity. Although disease onset appears at adulthood, recent studies have detected abnormalities during embryonic and postnatal maturation in animal models of ALS. We reported that SOD1(G85R) mutant mice exhibit specific delays in acquiring sensory-motor skills during the first week after birth. In addition, physiological measurements on in vitro spinal cord preparations reveal defects in evoking rhythmic activity with N-methyl-DL-aspartate and serotonin at lumbar, but not sacral roots. This is potentially significant, as functions involving sacral roots are spared at late stages of the disease. Moreover, electrical properties of SOD1 lumbar motoneurons are altered as early as the second postnatal week when mice begin to walk. Alterations concern the input resistance and the gain of SOD1 motoneurons which are lower than in control motoneurons. Whether or not the early changes in discharge firing are responsible for the uncoupling between motor axon terminals and muscles is still an open question. A link between these early electrical abnormalities and the late degeneration of motoneurons is proposed in this short review. Our data suggest that ALS, as other neurodegenerative diseases, could be a consequence of an abnormal development of neurons and network properties. We hypothesize that the SOD1 mutation could induce early changes during the period of maturation of motor systems and that compensatory mechanisms-linked to developmental spinal plasticity-might explain the late onset of the disease.     

One universal common endpoint in mouse models of amyotrophic lateral sclerosis

There is no consensus among research laboratories around the world on the criteria that define endpoint in studies involving rodent models of amyotrophic lateral sclerosis (ALS). Data from 4 nutrition intervention studies using 162 G93A mice, a model of ALS, were analyzed to determine if differences exist between the following endpoint criteria: CS 4 (functional paralysis of both hindlimbs), CS 4+ (CS 4 in addition to the earliest age of body weight loss, body condition deterioration or righting reflex), and CS 5 (CS 4 plus righting reflex >20 s). The age (d; mean ± SD) at which mice reached endpoint was recorded as the unit of measurement. Mice reached CS 4 at 123.9±10.3 d, CS 4+ at 126.6±9.8 d and CS 5 at 127.6±9.8 d, all significantly different from each other (P<0.001). There was a significant positive correlation between CS 4 and CS 5 (r = 0.95, P<0.001), CS 4 and CS 4+ (r = 0.96, P<0.001), and CS 4+ and CS 5 (r = 0.98, P<0.001), with the Bland-Altman plot showing an acceptable bias between all endpoints. Logrank tests showed that mice reached CS 4 24% and 34% faster than CS 4+ (P = 0.046) and CS 5 (P = 0.006), respectively. Adopting CS 4 as endpoint would spare a mouse an average of 4 days (P<0.001) from further neuromuscular disability and poor quality of life compared to CS 5. Alternatively, CS 5 provides information regarding proprioception and severe motor neuron death, both could be important parameters in establishing the efficacy of specific treatments. Converging ethics and discovery, would adopting CS 4 as endpoint compromise the acquisition of insight about the effects of interventions in animal models of ALS?     

Mitochondrial electron transport chain complex dysfunction in a transgenic mouse model for amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease that causes degeneration of motoneurons. Mutation of Cu,Zn superoxide dismutase (SOD1) is one cause for this disease. In mice, expression of mutant protein causes motoneuron degeneration and paralysis resembling the human disease. Morphological change, indicative of mitochondrial damage, occurs at early stages of the disease. To determine whether mitochondrial function changes during the course of disease progression, enzyme activities of mitochondrial electron transport chain in spinal cords from mice at different disease stages were measured using three different methods: spectrophotometric assay, in situ histochemical enzyme assay, and blue native gel electrophoresis combined with in-gel histochemical reaction. The enzyme activities were decreased in the spinal cord, particularly in the ventral horn, beginning at early disease stages. This decrease persisted throughout the course of disease progression. This decrease was not detected in the spinal cords of non-transgenic animals, of mice expressing the wild-type protein, and in cerebellum and dorsal horn of the spinal cords from mice expressing mutant protein. These results demonstrate a functional defect in mitochondria in the ventral horn region and support the view that mitochondrial damage plays a role in mutant SOD1-induced motoneuron degeneration pathway.     

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.     

In vivo optical imaging of motor neuron autophagy in a mouse model of amyotrophic lateral sclerosis

Autophagy is involved in the pathological process of motor neuron death in amyotrophic lateral sclerosis (ALS). We have generated a novel double transgenic (DTg) mouse line by mating a green fluorescent protein (GFP)-fused microtubule-associated protein 1 light chain 3 (LC3) transgenic (LC3-Tg) mouse and a G93A mutant human Cu/Zn superoxide dismutase (mSOD1) transgenic (mSOD1-Tg) mouse. In vivo imaging of autophagy with these novel DTg mice was conducted at 10 (presymptomatic), 17 (early symptomatic) and 19 (late symptomatic) weeks of age. Fluorescence imaging analysis revealed a strong fluorescent signal in vivo over the T₃-S₁ level at 17 and 19 weeks of age only in the DTg mice. Ex vivo autophagy imaging of spinal cord sections (20 μm) also showed a progressive increase of the fluorescence signal from 17 to 19 weeks in DTg mice in the anterior horn at the L4-5 level, and the fluorescence signals were clearly observed in the gray matter of the spinal cord with a progressive increase of the signal and decreases in large motor neurons. Protein gel blot analysis revealed maximum LC3-I and LC3-II expressions at 19 weeks, consistent with the results from the in vivo autophagy imaging experiment. This method could also be applied as a unique tool for clarifying the role of autophagy, and to monitor the pathologic processes involving autophagy not only in ALS, but also other neurological diseases.     

In vivo optical imaging of motor neuron autophagy in a mouse model of amyotrophic lateral sclerosis

Autophagy is involved in the pathological process of motor neuron death in amyotrophic lateral sclerosis (ALS). We have generated a novel double transgenic (DTg) mouse line by mating a green fluorescent protein (GFP)-fused microtubule-associated protein 1 light chain 3 (LC3) transgenic (LC3-Tg) mouse and a G93A mutant human Cu/Zn superoxide dismutase (mSOD1) transgenic (mSOD1-Tg) mouse. In vivo imaging of autophagy with these novel DTg mice was conducted at 10 (presymptomatic), 17 (early symptomatic) and 19 (late symptomatic) weeks of age. Fluorescence imaging analysis revealed a strong fluorescent signal in vivo over the T?-S? level at 17 and 19 weeks of age only in the DTg mice. Ex vivo autophagy imaging of spinal cord sections (20 μm) also showed a progressive increase of the fluorescence signal from 17 to 19 weeks in DTg mice in the anterior horn at the L?-? level, and the fluorescence signals were clearly observed in the gray matter of the spinal cord with a progressive increase of the signal and decreases in large motor neurons. Protein gel blot analysis revealed maximum LC3-I and LC3-II expressions at 19 weeks, consistent with the results from the in vivo autophagy imaging experiment. This method could also be applied as a unique tool for clarifying the role of autophagy, and to monitor the pathologic processes involving autophagy not only in ALS, but also other neurological diseases.     

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.     

Innate immunity in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition in which motor neurons are selectively targeted. Although the underlying cause remains unclear, evidence suggests a role for innate immunity in disease pathogenesis. Neuroinflammation in areas of motor neuron loss is evident in presymptomatic mouse models of ALS and in human patients. Efforts aimed at attenuating the inflammatory response in ALS animal models have delayed symptom onset and extended survival. Seemingly conversely, attempts to sensitize cells of the innate immune system and modulate their phenotype have also shown efficacy. Effectors of innate immunity in the CNS appear to have ambivalent potential to promote either repair or injury. Because ALS is a syndromic disease in which glutamate excitotoxicity, altered cytoskeletal protein metabolism, oxidative injury, mitochondrial dysfunction and neuroinflammation all contribute to motor neuron degeneration, targeting inflammation via modulation of microglial function therefore holds significant potential as one aspect of therapeutic intervention and could provide insight into the exclusive vulnerability of motor neurons.     

Mitochondrial involvement in amyotrophic lateral sclerosis

The causes of motor neuron death in amyotrophic lateral sclerosis (ALS) are so far unknown. The involvement of mitochondria in the disease was initially suggested by ultrastructural studies. More recently these observations have been supported by studies of mitochondrial function in ALS. Alterations in the activity of complexes which make up the mitochondrial electron transport chain have been recorded as well as mutations in the mitochondrial genome. The calcium buffering function of the mitochondria may also be affected in the disease. This review will discuss how mitochondrial dysfunction could be of relevance in ALS and the evidence that an alteration of mitochondrial function is a feature of the disease. The way in which the involvement of mitochondria fits with other aetiological hypotheses for ALS will also be discussed.