Mitochondria in amyotrophic lateral sclerosis: a trigger and a target

Strong evidence shows that mitochondrial dysfunction is involved in amyotrophic lateral sclerosis (ALS), but despite the fact that mitochondria play a central role in excitotoxicity, oxidative stress and apoptosis, the intimate underlying mechanism linking mitochondrial defects to motor neuron degeneration in ALS still remains elusive. Morphological and functional abnormalities occur in mitochondria in ALS patients and related animal models, although their exact nature and extent are controversial. Recent studies postulate that the mislocalization in mitochondria of mutant forms of copper-zinc superoxide dismutase (SOD1), the only well-documented cause of familial ALS, may account for the toxic gain of function of the enzyme, and hence induce motor neuron death. On the other hand, mitochondrial dysfunction in ALS does not seem to be restricted only to motor neurons as it is also present in other tissues, particularly the skeletal muscle. The presence of this 'systemic' defect in energy metabolism associated with the disease is supported in skeletal muscle tissue by impaired mitochondrial respiration and overexpression of uncoupling protein 3. In addition, the lifespan of transgenic mutant SOD1 mice is increased by a highly energetic diet compensating both the metabolic defect and the motorneuronal function. In this review, we will focus on the mitochondrial dysfunction linked to ALS and the cause-and-effect relationships between mitochondria and the pathological mechanisms thought to be involved in the disease.     

The neuronal Golgi apparatus is fragmented in transgenic mice expressing a mutant human SOD1, but not in mice expressing the human NF-H gene

Fragmentation of the Golgi apparatus (GA) of motor neurons was first described in sporadic amyotrophic lateral sclerosis (ALS) and later confirmed in transgenic mice expressing the G93A mutation of the gene encoding the enzyme Cu,Zn superoxide dismutase (SOD1(G93A)) found in some cases of familial ALS. In these transgenic mice, however, the fragmentation of the neuronal GA was associated with cytoplasmic and mitochondrial vacuoles not seen in ALS. The present new series of transgenic mice expressing 14-17 trans gene copies of SOD1(G93A), compared to 25 copies in the mice we studied previously, showed consistent fragmentation of the GA of spinal cord motor neurons, axonal swellings, Lewy-like body inclusions in neurons and glia, but none of the cytoplasmic or mitochondrial vacuoles originally reported. Thus, this animal model recapitulates the clinical and most neuropathological findings of sporadic ALS. Neurofilaments (NF) accumulate in axons and, less often, in neuronal perikarya in most cases of sporadic ALS and they have been implicated in its pathogenesis. In order to investigate whether fragmentation of the neuronal GA also occurs in association with accumulation of perikaryal NFs, we studied the organelle in transgenic mice expressing the heavy subunit of human neurofilaments (NF-H) which developed a motor neuronopathy resembling ALS. The neuronal GA of mice expressing NF-H, however, was intact despite massive accumulation of NFs in both perikarya and axons of motor neurons. In contrast, in transgenic mice expressing SOD1(G93A), the GA was fragmented despite the absence of accumulation of perikaryal NFs. These findings suggest that, in transgenic mice with neuronopathies caused by the expression of mutant SOD1(G93A) or the human NF-H, the GA and the perikaryal NFs are independently involved in the pathogenesis. The evidence suggests that the GA plays a central role in the pathogenesis of the vast majority of sporadic ALS and in FALS with SOD1 mutations.     

In vivo pathogenic role of mutant SOD1 localized in the mitochondrial intermembrane space

Mutations in Cu,Zn superoxide dismutase (SOD1) are associated with familial amyotrophic lateral sclerosis (ALS). Mutant SOD1 causes a complex array of pathological events, through toxic gain of function mechanisms, leading to selective motor neuron degeneration. Mitochondrial dysfunction is among the well established toxic effects of mutant SOD1, but its mechanisms are just starting to be elucidated. A portion of mutant SOD1 is localized in mitochondria, where it accumulates mostly on the outer membrane and inside the intermembrane space (IMS). Evidence in cultured cells suggests that mutant SOD1 in the IMS causes mitochondrial dysfunction and compromises cell viability. Therefore, to test its pathogenic role in vivo we generated transgenic mice expressing G93A mutant or wild-type (WT) human SOD1 targeted selectively to the mitochondrial IMS (mito-SOD1). We show that mito-SOD1 is correctly localized in the IMS, where it oligomerizes and acquires enzymatic activity. Mito-G93ASOD1 mice, but not mito-WTSOD1 mice, develop a progressive disease characterized by body weight loss, muscle weakness, brain atrophy, and motor impairment, which is more severe in females. These symptoms are associated with reduced spinal motor neuron counts and impaired mitochondrial bioenergetics, characterized by decreased cytochrome oxidase activity and defective calcium handling. However, there is no evidence of muscle denervation, a cardinal pathological feature of ALS. Together, our findings indicate that mutant SOD1 in the mitochondrial IMS causes mitochondrial dysfunction and neurodegeneration, but per se it is not sufficient to cause a full-fledged ALS phenotype, which requires the participation of mutant SOD1 localized in other cellular compartments.     

Mitochondrial dynamics and bioenergetic dysfunction is associated with synaptic alterations in mutant SOD1 motor neurons

Mutations in Cu,Zn superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis (FALS), a rapidly fatal motor neuron disease. Mutant SOD1 has pleiotropic toxic effects on motor neurons, among which mitochondrial dysfunction has been proposed as one of the contributing factors in motor neuron demise. Mitochondria are highly dynamic in neurons; they are constantly reshaped by fusion and move along neurites to localize at sites of high-energy utilization, such as synapses. The finding of abnormal mitochondria accumulation in neuromuscular junctions, where the SOD1-FALS degenerative process is though to initiate, suggests that impaired mitochondrial dynamics in motor neurons may be involved in pathogenesis. We addressed this hypothesis by live imaging microscopy of photo-switchable fluorescent mitoDendra in transgenic rat motor neurons expressing mutant or wild-type human SOD1. We demonstrate that mutant SOD1 motor neurons have impaired mitochondrial fusion in axons and cell bodies. Mitochondria also display selective impairment of retrograde axonal transport, with reduced frequency and velocity of movements. Fusion and transport defects are associated with smaller mitochondrial size, decreased mitochondrial density, and defective mitochondrial membrane potential. Furthermore, mislocalization of mitochondria at synapses among motor neurons, in vitro, correlates with abnormal synaptic number, structure, and function. Dynamics abnormalities are specific to mutant SOD1 motor neuron mitochondria, since they are absent in wild-type SOD1 motor neurons, they do not involve other organelles, and they are not found in cortical neurons. Together, these results suggest that impaired mitochondrial dynamics may contribute to the selective degeneration of motor neurons in SOD1-FALS.     

DJ-1 Changes in G93A-SOD1 Transgenic Mice: Implications for Oxidative Stress in ALS

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal, neurodegenerative disorder. The causes of ALS are still obscure. Accumulating evidence supports the hypothesis that oxidative stress and mitochondrial dysfunction can be implicated in ALS pathogenesis. DJ-1 plays an important role in the oxidative stress response. The aim of this study was to discover whether there are changes in DJ-1 expression or in DJ-1-oxidized isoforms in an animal model of ALS. We used mutant SOD1^G93A transgenic mice, a commonly used animal model for ALS. Upregulation of DJ-1 mRNA and protein levels were identified in the brains and spinal cords of SOD1^G93A transgenic mice as compared to wild-type controls, evident from an early disease stage. Furthermore, an increase in DJ-1 acidic isoforms was detected, implying that there are more oxidized forms of DJ-1 in the CNS of SOD1^G93A mice. This is the first report of possible involvement of DJ-1 in ALS. Since DJ-1 has a protective role against oxidative stress, it may suggest a possible therapeutic target in ALS.     

The mitochondrial permeability transition pore in motor neurons: Involvement in the pathobiology of ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons (MNs) that causes paralysis. Some forms of ALS are inherited, caused by mutations in the superoxide dismutase-1 (SOD1) gene. The mechanisms of human mutant SOD1 (mSOD1) toxicity to MNs are unresolved. Mitochondria in MNs might be key sites for ALS pathogenesis, but cause–effect relationships between mSOD1 and mitochondriopathy need further study. We used transgenic mSOD1 mice to test the hypothesis that the mitochondrial permeability transition pore (mPTP) is involved in the MN degeneration of ALS. Components of the multi-protein mPTP are expressed highly in mouse MNs, including the voltage-dependent anion channel, adenine nucleotide translocator (ANT), and cyclophilin D (CyPD), and are present in mitochondria marked by manganese SOD. MNs in pre-symptomatic mSOD1-G93A mice form swollen megamitochondria with CyPD immunoreactivity. Early disease is associated with mitochondrial cristae remodeling and matrix vesiculation in ventral horn neuron dendrites. MN cell bodies accumulate mitochondria derived from the distal axons projecting to skeletal muscle. Incipient disease in spinal cord is associated with increased oxidative and nitrative stress, indicated by protein carbonyls and nitration of CyPD and ANT. Reducing the levels of CyPD by genetic ablation significantly delays disease onset and extends the lifespan of G93A-mSOD1 mice expressing high and low levels of mutant protein in a gender-dependent pattern. These results demonstrate that mitochondria have causal roles in the disease mechanisms in MNs in ALS mice. This work defines a new mitochondrial mechanism for MN degeneration in ALS.     

Familial amyotrophic lateral sclerosis and Cu/Zn superoxide dismutase mutation

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder that involves mainly the motor neuron system. Five to 10 percent of the ALS cases are familial; most others are sporadic. Several mutations in the superoxide dismutase-1 (SOD1) gene have recently been shown to be associated with about 20% of familial ALS patients. The reduced enzyme activity of many mutant SOD1 points to the possibility that a loss-of-function effect of the mutant enzyme is responsible for the pathogenesis of the disease. However, this conflicts with the autosomal dominant inheritance of SOD1 mutation-associated ALS and the normal SOD1 activity in homozygous patients in a SOD1-linked ALS family. Current biochemical investigations have provided evidence that mutant SOD1 may catalyze the peroxynitrite-mediated nitration of protein tyrosine residues, release copper and zinc ions, facilitate apoptosis of neurons and have enhanced peroxidase activity. Immunocytochemical studies demonstrated the presence of intense SOD1 immunoreactivity in Lewy body-like inclusions, which are characteristic features of a certain form of familial ALS with posterior column involvement, in the lower motor neurons of patients in ALS families with different SOD1 mutations. More recently, strains of transgenic mice expressing mutant SOD1 have been established. These mice clinicopathologically develop a motor neuron disease mimicking human ALS with the exception of pronounced intraneuronal vacuolar degeneration. The overexpression of wild-type SOD1 in mice has failed to give rise to the disease. Only one transgene for mutant SOD1 is enough to cause motor neuron degeneration and the severity of clinical course correlates with the transgene copy number. These observations in SOD1-linked familial ALS and its transgenic mouse model suggest a novel neurotoxic function of mutant SOD1.     

Neuroprotective effect of activity-dependent neurotrophic factor against toxicity from familial amyotrophic lateral sclerosis-linked mutant SOD1 in vitro and in vivo

Amyotrophic lateral sclerosis (ALS) is the most common fatal motor neuron disease, affecting mostly middle-aged people. There are no curative therapies for ALS. Several lines of evidence have supported the notion that the proapoptotic property of familial ALS (FALS)-linked mutant Cu/Zn-superoxide dismutase-1 (SOD1) genes may play an important role in the pathogenesis of some FALS cases. Here we found that activity-dependent neurotrophic factor (ADNF), a neurotrophic factor originally identified to have the anti-Alzheimer's disease (AD) activity, protected against neuronal cell death caused by FALS-linked A4T-, G85R- and G93R-SOD1 in a dose-responsive fashion. Notably, ADNF-mediated complete suppression of SOD1 mutant-induced neuronal cell death occurs at concentrations as low as 100 fM. ADNF maintains the neuroprotective activity even at concentrations of more than 1 nM. This is in clear contrast to the previous finding that ADNF loses its protective activity against neurotoxicity induced by AD-relevant insults, including some familial AD genes and amyloid beta peptide at concentrations of more than 1 nM. Characterization of the neuroprotective activity of ADNF against cell death caused by SOD1 mutants revealed that CaMKIV and certain tyrosine kinases are involved in ADNF-mediated neuroprotection. Moreover, in vivo studies showed that intracerebroventricularly administered ADNF significantly improved motor performance of G93A-SOD1 transgenic mice, a widely used model of FALS, although survival was extended only marginally. Thus, the neuroprotective activity of ADNF provides a novel insight into the development of curative drugs for ALS.     

Protein‐bound crotonaldehyde accumulates in the spinal cord of superoxide dismutase‐1 mutation‐associated familial amyotrophic lateral sclerosis and its transgenic mouse model

Growing evidence documents oxidative stress involvement in ALS. We previously demonstrated accumulation of a protein‐bound form of the highly toxic lipid peroxidation product crotonaldehyde (CRA) in the spinal cord of sporadic ALS patients. In the present study, to the determine the role for CRA in the disease processes of superoxide dismutase‐1 (SOD1) mutation‐associated familial ALS (FALS), we performed immunohistochemical and semiquantitative cell count analyses of protein‐bound CRA (P‐CRA) in the spinal cord of SOD1‐mutated FALS and its transgenic mouse model. Immunohistochemical analysis revealed increased P‐CRA immunoreactivity in the spinal cord of the FALS patients and the transgenic mice compared to their respective controls. In the FALS patients, P‐CRA immunoreactivity was localized in almost all of the chromatolytic motor neurons, neurofilamentous conglomerates, spheroids, cordlike swollen axons, reactive astrocytes and microglia, and the surrounding neuropil in the affected areas represented by the anterior horns. In the transgenic mice, P‐CRA immunoreactivity was localized in only a few ventral horn glia in the presymptomatic stage, in almost all of the vacuolated motor neurons and cordlike swollen axons and some of the ventral horn reactive astrocytes and microglia in the onset stage, and in many of the ventral horn reactive astrocytes and microglia in the advanced stage. Cell count analysis on mouse spinal cord sections disclosed a statistically significant increase in the density of P‐CRA‐immunoreactive glia in the ventral horns of the young to old G93A mice compared to the age‐matched control mice. The present results indicate that enhanced CRA formation occurs in motor neurons and reactive glia in the spinal cord of SOD1‐mutated FALS and its transgenic mouse model as well as sporadic ALS, suggesting implications for CRA in the pathomechanism common to these forms of ALS.     

Restricted expression of mutant SOD1 in spinal motor neurons and interneurons induces motor neuron pathology

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective loss of motor neurons (MNs). Approximately 10% of ALS cases are familial (known as FALS), and  20% of FALS cases are caused by mutations in Cu/Zn superoxide dismutase type 1 (SOD1). Mutant (MT) SOD1 induces FALS as a result of a toxicity that remains poorly defined. Several studies suggest that the toxicity involves a non-cell autonomous mechanism. In this study, we generated transgenic mice that had a restricted and repressible expression of MTSOD1 in spinal MNs and interneurons. Although the transgenic mice were not weak, they weighed less than control mice and had pathological and immunohistochemical abnormalities of MNs confined to cells that expressed MTSOD1. These results suggest that MTSOD1-induced MN degeneration is at least partly cell autonomous. Mouse models similar to the one presented here will be valuable for spatially and temporally controlling expression of mutant genes involved in neurodegenerative diseases.     

Transgenic mouse model for familial amyotrophic lateral sclerosis with superoxide dismutase‐1 mutation

Familial amyotrophic lateral sclerosis (ALS) with mutations in the gene for superoxide dismutase‐1 (SOD1) is clinicopathologically reproduced by transgenic mice expressing mutant forms of SOD1 detectable in familial ALS patients. Motor neuron degeneration associated with SOD1 mutation has been thought to result from a novel neurotoxicity of mutant SOD1, but not from a reduction in activity of this enzyme, based on autosomal dominant transmission of SOD1 mutant familial ALS and its transgenic mouse model, clinical severity of the ALS patients independent to enzyme activity, no ALS‐like disease in SOD1 knockout or wild‐type SOD1‐over‐expressing mice, and clinicopathological severity of mutant SOD1 transgenic mice dependent on transgene copy numbers. Proposed mechanisms of motor neuron de‐generation such as oxidative injury, peroxynitrite toxicity, cytoskeletal disorganization, glutamate excitotoxicity, disrupted calcium homeostasis, SOD1 aggregation, car‐bonyl stress and apoptosis have been discussed. Intracy‐toplasmic vacuoles, indicative of increased oxidative damage to the mitochondria and endoplasmic reticulum, in the neuropil and motor neurons appear in high expressors of mutant SOD1 transgenic mice but not in low expressors of the mice or familial ALS patients, suggesting that overexpression of mutant SOD1 in mice may enhance oxidative stress generation from this enzyme. Thus, transgenic mice carrying small transgene copy numbers of mutant SOD1 would provide a beneficial animal model for SOD1 mutant familial ALS. Such a model would contribute to elucidating the pathomechanism of this disease and establishing new therapeutic agents.     

Mouse motor neuron disease caused by truncated SOD1 with or without C-terminal modification

Mutation of Cu/Zn superoxide dismutase (SOD1) contributes to a portion of the cases of familial amyotrophic lateral sclerosis (FALS). We previously reported on a FALS family whose members had a mutant form of SOD1 characterized by a 2-base pair (bp) deletion at codon 126 of the SOD1 gene. To investigate the cellular consequences of this mutation, we produced transgenic mice that expressed normal and mutated copies of human SOD1: wild-type SOD1 (W), wild-type SOD1 with a FLAG epitope at C-terminal (WF), mutated SOD1 with the 2-bp deletion (D), and SOD1 with the 2-bp deletion with FLAG (DF). The mice heterozygotic for the human mutated SOD1 (D and DF) showed distinct ALS-like motor symptoms, whereas the mice heterozygotic for the normal SOD1 (W and WF) mice did not. Homozygotes of D and DF lines showed the ALS symptoms at an earlier age and died earlier than the heterozygotes. By Northern blot analysis, the mRNAs for all human SOD1s were confirmed in these lines. All the human SOD1 proteins, except the D mutant, were detectable by immunoblot. The D protein was only confirmed when it was concentrated by immunoprecipitation. Neuropathologically, loss of spinal motor neurons and reactive gliosis were common features in the symptomatic lines. The remaining motor neurons in these mice also exhibited eosinophilic inclusions. The biochemical and pathological characteristics of these mice are quite similar to those of human FALS patients with same mutation. This intriguing model will provide an important source of information of the pathogenesis of FALS.     

The efficacy of trientine or ascorbate alone compared to that of the combined treatment with these two agents in familial amyotrophic lateral sclerosis model mice

One of the hypotheses regarding the pathogenesis of familial ALS (FALS) is a copper-mediated oxidative toxicity derived from the mutant Cu, Zn-superoxide dismutase (SOD1). We have previously demonstrated the efficacy of the combined treatment with a copper chelator (trientine) and an antioxidant (ascorbate) on the disease expression of the FALS-linked mutated SOD1 transgenic mice. Here, we investigated the efficacy of trientine or ascorbate alone on FALS mice when administered before or after the onset of the disease. The mice with a high dose of trientine or ascorbate administered before the onset survived significantly longer than the control. In the combined treatment with a high dose of trientine and ascorbate initiated before the onset, survival lengthened and the motor function of the mice remained more significantly than the control. None of the treatments affected the mean age of the onset, and none of the agents administered after the onset prolonged survival. These findings suggest that better outcomes may be expected by the administration of these agents at the preonset stage of the disease, and the combination of the agents acting on different sites might be useful in preserving the motor performance in FALS.     

Mice lacking cytosolic copper/zinc superoxide dismutase display a distinctive motor axonopathy.

OBJECTIVE: To characterize the motor neuron dysfunction in two models by performing physiologic and morphometric studies. BACKGROUND: Mutations in the gene encoding cytosolic superoxide dismutase 1 (SOD1) account for 25% of familial ALS (FALS). Transgenes with these mutations produce a pattern of lower motor neuron degeneration similar to that seen in patients with FALS. In contrast, mice lacking SOD1 develop subtle motor symptoms by approximately 6 months of age. METHODS: Physiologic measurements, including motor conduction and motor unit estimation, were analyzed in normal mice, mice bearing the human transgene for FALS (mFALS mice), and knockout mice deficient in SOD1 (SOD1-KO). In addition, morphometric analysis was performed on the spinal cords of SOD1-KO and normal mice. RESULTS: In mFALS mice, the motor unit number in the distal hind limb declined before behavioral abnormalities appeared, and motor unit size increased. Compound motor action potential amplitude and distal motor latency remained normal until later in the disease. In SOD1-KO mice, motor unit numbers were reduced early but declined slowly with age. In contrast with the mFALS mice, SOD1-KO mice demonstrated only a modest increase in motor unit size. Morphometric analysis of the spinal cords from normal and SOD1-KO mice showed no significant differences in the number and size of motor neurons. CONCLUSIONS: The physiologic abnormalities in mFALS mice resemble those in human ALS. SOD1-deficient mice exhibit a qualitatively different pattern of motor unit remodeling that suggests that axonal sprouting and reinnervation of denervated muscle fibers are functionally impaired in the absence of SOD1.     

SOD1 and cognitive dysfunction in familial amyotrophic lateral sclerosis

Background   Sporadic Amyotrophic Lateral Sclerosis (sALS) is associated with frontotemporal dementia (ALS-FTD) or milder deficits of cognitive (predominantly executive) dysfunction (ALSCi) in some patients. Some forms of familial ALS (FALS) have a family history of FTD, ALS-FTD, or both, but there have been few reports of ALS-FTD in FALS patients with mutations of the gene superoxide dismutase-1 (SOD1 FALS). The aim of this study was to test the hypothesis that ALSCi may be found in non-SOD1 FALS, but that SOD1 FALS patients would show little or no evidence of cognitive change. Methods   A neuropsychological test battery was administered to 41 SALS patients, 35 control participants, 7 FALS patients with a SOD1 mutation (SOD1 FALS) and 10 FALS patients without a SOD1 mutation (non-SOD1 FALS). Results   Relative to control participants, non-SOD1 FALS patients had impaired performance on written verbal fluency and confrontation naming, and reported higher levels of executive behavioural problems. These deficits were absent in SOD1 FALS patients. SALS patients performed poorer than controls only on the Graded Naming Test. All ALS groups had higher levels of behavioural apathy and emotional lability than were found in control participants. Cognitive domains of memory, receptive language, and visuospatial perception were spared. Groups were matched for age, gender, premorbid full-scale IQ, anxiety and depression. Discussion   Individuals with SOD1 gene mutations are less likely to have significant cognitive changes compared to non-SOD1 FALS patients. Cognitive abnormalities in ALS are heterogeneous and may reflect underlying genetic variations rather than a simple spectrum of extra-motor involvement.     

Immunocytochemical study on the distribution of nitrotyrosine in the brain of the transgenic mice expressing a human Cu/Zn SOD mutation

In the previous study, we reported increased NOS expression in the astrocytes in the spinal cord of the transgenic mice that are used as ALS animal model. In the present study, we performed immunocytochemical studies to investigate the changes of nitrotyrosine-immunoreactivity in the brains of the transgenic mice, and demonstrated in vivo evidence of peroxynitrite-mediated oxidative damage in the pathogenesis of ALS. In the spinal cord of the transgenic mice, immunocytochemistry showed intensely stained nitrotyrosine-IR glial cells with the appearance of astrocytes, but no nitrotyrosine-IR glial cells were observed in the spinal cord of the control mice. In the transgenic mice, nitrotyrosine-IR neurons were observed in the hypoglossal nucleus, lateral reticular nucleus, medullary reticular formation and cerebellar nuclei. Interestingly, nitrotyrosine-IR neurons were observed in the hippocampal formation and septal area of the transgenic mice. In the hippocampus, nitrotyrosine-IR neurons in the CA1 region showed intense staining, and the immunoreactivity was localized mainly in the pyramidal cell layer. Recent studies have shown that antioxidants and selective neuronal NOS inhibitor increase survival in the SOD1 transgenic mouse model of FALS. It is possible that therapy with these agents may slow the neurodegenerative process in human ALS, perhaps through reduction of nitrotyrosine formation.     

Metallothionein expression is altered in a transgenic murine model of familial amyotrophic lateral sclerosis

Missense mutations in the gene encoding copper zinc superoxide dismutase (SOD1) have been found to cause one form of familial amyotrophic lateral sclerosis (FALS). Although the exact mechanism of disease is unknown, abnormalities in the ability of mutant SOD1 to bind zinc or copper ions may be crucial in the pathogenesis of disease. Because members of the metallothionein (MT) family of zinc and copper binding proteins function as important cellular regulators of metal ion bioavailability in the central nervous system, we used in situ hybridization and immunohistochemistry to study the expression pattern of these molecules in a transgenic mouse model of familial ALS. In adult wild-type mouse spinal cord, expression of MT-I and MT-II is restricted to ependymal cells and a subset of astrocytes located in white matter tracts, while MT-III synthesis is limited to neurons within gray matter. Compared to wild-type littermates, transgenic mice carrying the G93A SOD1 mutation demonstrate markedly increased expression of MT-I and MT-II within astrocytes in both white and gray matter as weakness develops. MT-III synthesis in neurons is also greatly upregulated as G93A SOD1 animals age, with glial cell expression of MT-III evident by later stages of the disease. Changes in MT expression occur before the onset of motor deficits or significant motor neuron pathology in G93A SOD1 mice and remarkably extend beyond ventral horn populations of neurons and glia. These results are consistent with the hypothesis that metallothioneins may serve an early and important protective function in FALS.     

Selective changes in mitochondria respiratory properties in oxidative or glycolytic muscle fibers isolated from G93AhumanSOD1 transgenic mice

Cases of familial amyotrophic lateral sclerosis (FALS) are associated with mutations in cytosolic copper, zinc superoxide dismutase (SOD1). Total SOD activity and functional mitochondrial properties were studied in muscles and nervous tissues of control and transgenic mice mimicking the disease. It was found that total SOD activity was lower in nervous tissues than in muscles in both transgenic and control mice. In addition SOD activity increased during progression of disease in muscle but not in nervous tissue of transgenic mice. Maximal oxygen consumption and apparent Km for ADP were decreased in mitochondria from transgenic soleus (an oxidative muscle). However there was no difference between control and transgenic mice in respiratory parameters of mitochondria in the EDL muscle (a glycolytic muscle). These findings indicate that oxidative stress due to SOD1 mutations could alter energy metabolism in FALS mice, thereby affecting primarily oxidative muscle of the limbs, independently of motoneuron loss.     

ALS pathogenesis: Recent insights from genetics and mouse models

For the vast majority of cases of amyotrophic lateral sclerosis (ALS) the etiology remains unknown. After the discovery of missense mutations in the gene coding for the Cu/Zn superoxide dismutase 1 (SOD1) in subsets of familial ALS, several transgenic mouse lines have been generated with various forms of SOD1 mutants overexpressed at different levels. Studies with these mice yielded complex results with multiple targets of damage in disease including mitochondria, proteasomes, and secretory pathways. Many unexpected discoveries were made. For instance, the toxicity of mutant SOD1 seems unrelated to copper-mediated catalysis but rather to formation of misfolded SOD1 species and aggregates. Transgenic studies revealed a potential role of wtSOD1 in exacerbating mutant SOD1-mediated disease. Another key finding came from chimeric mouse studies and from Cre-lox mediated gene deletion experiments which have highlighted the importance of non-neuronal cells in the disease progression. 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. Recently, the generation of new animal models of ALS has been made possible with the discovery of ALS-linked mutations in other genes encoding for alsin, dynactin, senataxin, VAPB, TDP-43 and FUS. Following the discovery of mutations in the TARDBP gene linked to ALS, there have been some reports of transgenic mice with high level overexpression of WT or mutant forms of TDP-43 under strong gene promoters. However, these TDP-43 transgenic mice do not exhibit all pathological features the human ALS disease. Here, we will describe these new TDP-43 transgenic mice and discuss their validity as animal models of human ALS.     

L-carnitine suppresses the onset of neuromuscular degeneration and increases the life span of mice with familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal disease caused by progressive degeneration of motor neurons in the spinal cord and motor cortex. Although the etiology of ALS remains unknown, a mutation of the gene encoding Cu,Zn-superoxide dismutase (SOD1) has been reported in 20% of familial cases of ALS (FALS). Transgenic mice that overexpress a mutated human SOD1 exhibit a phenotype and pathology similar to those observed in patients with FALS. Mitochondrial abnormality has been reported in patients with ALS and in animal models of FALS. We recently reported that L-carnitine, an essential cofactor for the beta-oxidation of long-chain fatty acids, effectively inhibits various types of mitochondrial injury and apoptosis both in vitro and in vivo. The present study demonstrates that oral administration of L-carnitine prior to disease onset significantly delayed the onset of signs of disease (log-rank P=0.0008), delayed deterioration of motor activity, and extended life span (log-rank P=0.0001) in transgenic mice carrying a human SOD1 gene with a G93A mutation (Tg). More importantly, subcutaneous injection of L-carnitine increased the life span of Tg mice (46% increase in male, 60% increase in female) even when given after the appearance of signs of disease.