Early-Stage Treatment with Withaferin A Reduces Levels of Misfolded Superoxide Dismutase 1 and Extends Lifespan in a Mouse Model of Amyotrophic Lateral Sclerosis

Approximately 20 % of cases of familial amyotrophic lateral sclerosis (ALS) are caused by mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Recent studies have shown that Withaferin A (WA), an inhibitor of nuclear factor-kappa B activity, was efficient in reducing disease phenotype in a TAR DNA binding protein 43 transgenic mouse model of ALS. These findings led us to test WA in mice from 2 transgenic lines expressing different ALS-linked SOD1 mutations, SOD1^G93A and SOD1^G37R. Intraperitoneal administration of WA at a dosage of 4 mg/kg of body weight was initiated from postnatal day 40 until end stage in SOD1^G93A mice, and from 9 months until end stage in SOD1^G37R mice. The beneficial effects of WA in the SOD1^G93A mice model were accompanied by an alleviation of neuroinflammation, a decrease in levels of misfolded SOD1 species in the spinal cord, and a reduction in loss of motor neurons resulting in delayed disease progression and mortality. Interestingly, WA treatment triggered robust induction of heat shock protein 25 (a mouse ortholog of heat shock protein 27), which may explain the reduced level of misfolded SOD1 species in the spinal cord of SOD1^G93A mice and the decrease of neuronal injury responses, as revealed by real-time imaging of biophotonic SOD1^G93A mice expressing a luciferase transgene under the control of the growth-associated protein 43 promoter. These results suggest that WA may represent a potential lead compound for drug development aiming to treat ALS.

Electronic supplementary material

The online version of this article (doi:10.1007/s13311-014-0311-0) contains supplementary material, which is available to authorized users.Key Words: ALS, Neuroinflammation, Withaferin A, SOD1^G93A, SOD1^G37R     

Therapeutic benefit of intrathecal injection of insulin-like growth factor-1 in a mouse model of Amyotrophic Lateral Sclerosis

Insulin-like growth factor (IGF)-1 has been shown to have a protective effect on motor neurons both in vitro and in vivo, but has limited efficacy in patients with amyotrophic lateral sclerosis (ALS) when given subcutaneously. To examine the possible effectiveness of IGF-1 in a mouse model of familial ALS, transgenic mice expressing human Cu/Zn superoxide dismutase (SOD1) with a G93A mutation were treated by continuous IGF-1 delivery into the intrathecal space of the lumbar spinal cord. We found that the intrathecal administration of IGF-1 improved motor performance, delayed the onset of clinical disease, and extended survival in the G93A transgenic mice. Furthermore, it increased the expression of phosphorylated Akt and ERK in spinal motor neurons, and partially prevented motor neuron loss in these mice. Taken together, the results suggest that direct administration of IGF-1 into the intrathecal space may have a therapeutic benefit for ALS.     

Treatment with edaravone, initiated at symptom onset, slows motor decline and decreases SOD1 deposition in ALS mice

Edaravone is a free-radical scavenger, an agent being widely used for cerebral ischemia in Japan. To evaluate its efficacy for possible treatment of amyotrophic lateral sclerosis (ALS), we performed a randomized blind trial in ALS model mice. After identification of the clinical onset in each female G93A mutant SOD1 transgenic mouse, we intraperitoneally administered multiple doses of edaravone to the mice and observed their motor symptoms. We also counted the number of lumbar motoneurons, determined the 3-nitrotyrosine/tyrosine ratio, and evaluated the abnormal SOD1 aggregation in the spinal cord at the 10th day after the edaravone injection. Edaravone significantly slowed the motor decline of the transgenic mice. The remaining motoneurons were significantly preserved in the higher-dose edaravone-administered group, and the 3-nitrotyrosine/tyrosine ratios were reduced dose-dependently. Intriguingly, the area of abnormal SOD1 deposition in the spinal cord was significantly decreased in the higher-dose edaravone-administered group. Our results indicate that edaravone was effective to slow symptom progression and motor neuron degeneration in the ALS model mice. These favorable actions might be attributable to the yet unidentified mechanism responsible for reducing the deposition of mutant SOD1.     

Heme Oxygenase in the Experimental ALS Mouse

Heme oxygenase-1 (HO-1) is a stress protein inducible in some cells by oxidative stress. The status of heme oxygenase was investigated in a transgenic mouse model of amyotrophic lateral sclerosis (ALS) since oxidative mechanisms are postulated in neuronal injury. Three ALS mice [(SOD1-G93A)1Gur] and three controls [(SOD-1)2Gur] were obtained from The Jackson Laboratory. Behavioral differences suggestive of neurodegeneration in ALS mice developed at 4–5 months of age. All mice were killed at 7–8 months of age. Tissue vacuolation, cell loss, and the presence of GFAP⁺cells were noted in the spinal cords of ALS mice. Spinal cord motor neurons in both control and ALS mice stained positive for heme oxygenase-2 (HO-2). While not precluding the presence of low levels of HO-1 neither immunohistochemical staining nor Western blot analysis provided evidence for significant HO-1 induction in degenerating spinal cord.     

Relationship of oxygen radical-induced lipid peroxidative damage to disease onset and progression in a transgenic model of familial ALS

Transgenic mice that overexpress a mutated human CuZn superoxide dismutase (SOD1) gene (gly⁹³→ala) found in some patients with familial ALS (FALS) have been shown to develop motor neuron disease, as evidenced by motor neuron loss in the lumbar and cervical spinal regions and a progressive loss of voluntary motor activity. The mutant Cu,Zn SOD exhibits essentially normal dismutase activity, but in addition, generates toxic oxygen radicals as a result of an enhancement of a normally minor peroxidase reaction. In view of the likelihood that the manifestation of motor neuron disease in the FALS transgenic mice involves an oxidative injury mechanism, the present study sought to examine the extent of lipid peroxidative damage in the spinal cords of the TgN(SOD1-G93A)G1H mice over their life span compared to nontransgenic littermates or transgenic mice that overexpress the wild-type human Cu,Zn SOD (TgN(SOD1)N29). Lipid peroxidation was investigated in terms of changes in vitamin E and malondialdehyde (MDA) levels measured by HPLC methods and by MDA-protein adduct immunoreactivity. Four ages were investigated: 30 days (pre-motor neuron pathology and clinical disease); 60 days (after initiation of pathology, but predisease); 100 days (approximately 50% loss of motor neurons and function); and 120 days (near complete hindlimb paralysis). Compared to nontransgenic mice, the TgN(SOD1-G93A)G1H mice showed blunted accumulation of spinal cord vitamin E and higher levels of MDA (P < 0.05 at 30 and 60 days) over the 30–120 day time span. In the TgN(SOD1)N29 mice, levels of MDA at age 120 days were significantly lower than in either the TgN(SOD1-G93A)G1H or nontransgenic mice. MDA-protein adduct immunoreactivity was also significantly increased in the lumbar spinal cord at age 30, 100, and 120 days, and in the cervical cord at 100 and 120 days. The results clearly demonstrate an increase in spinal cord lipid peroxidation in the FALS transgenic model, which precedes the onset of ultrastructural or clinical motor neuron disease. However, the greatest intensity of actual motor neuronal lipid peroxidative injury is associated with the active phase of disease progression. These findings further support a role of oxygen radical-mediated motor neuronal injury in the pathogenesis of FALS and the potential benefits of antioxidant therapy. J. Neurosci. Res. 53:66–77, 1998. © 1998 Wiley-Liss, Inc.     

Immune reactivity in a mouse model of familial ALS correlates with disease progression

OBJECTIVE: The cause of motor neuron death in ALS is incompletely understood. This study aims to define the potential involvement of nonneuronal immune-inflammatory factors in the destruction of motor neurons in mutant superoxide dismutase-1 (SOD1) transgenic mice as a model of ALS. BACKGROUND: The presence of activated microglia, IgG and its receptor for Fc portion (FcgammaRI), and T lymphocytes in the spinal cord of both patients with ALS and experimental animal models of motor neuron disease strongly suggests that immune-inflammatory factors may be actively involved in the disease process. METHODS: The expression of immune-inflammatory factors was followed in both human mutant (G93A) SOD1 transgenic mice and human wild-type SOD1 transgenic mice, at different ages (40, 80, and 120 days). Fixed, frozen, free-floating sections of the lumbar spinal cord were stained with antibodies against CD11b, IgG, FcgammaRI, intercellular adhesion molecule-1 (ICAM-1), CD3, and glial fibrillary acidic protein. RESULTS: The earliest change observed was the upregulation of ICAM-1 in the ventral lumbar spinal cord of 40-day-old mutant SOD1 mice. IgG and FcgammaRI reactivities were detected on motor neurons as early as 40 days and on microglial cells at later stages. Microglial activation was first evident in the ventral horn at 80 days, whereas reactive astrocytes and T cells became most prominent in 120-day-old mutant SOD1 mice. CONCLUSION: The upregulation of proinflammatory factors during early presymptomatic stages as well as the expansion of immune activation as disease progresses in mutant SOD1 transgenic mice suggest that immune-inflammatory mechanisms could contribute to disease progression.     

Acute glial activation by stab injuries does not lead to overt damage or motor neuron degeneration in the G93A mutant SOD1 rat model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease where motor neurons within the brain and spinal cord are lost, leading to paralysis and death. Recently, a correlation between head trauma and the incidence of ALS has been reported. Furthermore, new invasive neurosurgical studies are being planned which involve inserting needles directly to the spinal cord. We therefore tested whether acute trauma to the spinal cord via a knife wound injury would lead to accelerated disease progression in rodent models of ALS (SOD1^G93A rats). A longitudinal stab injury using a small knife was performed within the lumbar spinal cord region of presymptomatic SOD1^G93A rats. Host glial activation was detected in the lumbar area surrounding a micro-knife lesion at 2 weeks after surgery in both wild type and SOD1^G93A animals. However, there was no sign of motor neuron loss in the injured spinal cord of any animal and normal motor function was maintained in the ipsilateral limb. These results indicate that motor neurons in presymptomatic G93A animals are not affected by an invasive puncture wound injury involving reactive astrocytes. Furthermore, acute trauma alone does not accelerate disease onset or progression in this ALS model which is important for future strategies of gene and cell therapies directly targeting the spinal cord of ALS patients.     

Development of a rat model of amyotrophic lateral sclerosis expressing a human SOD1 transgene

Mutations in copper–zinc superoxide dismutase gene (SOD1) have been linked to some familial cases of ALS. We report here that rats that express a human SOD1 transgene with two different ALS‐associated mutations (G93A and H46R) develop striking motor neuron degeneration and paralysis. By comparing the two transgenic rats with different SOD1 mutations, we demonstrate that the time course in these rats was similar to human SOD1‐mediated familial ALS. As in the human disease and transgenic ALS mice, pathological analysis shows selective loss of motor neurons in the spinal cords of these transgenic rats. In addition, typical neuronal Lewy body‐like hyaline inclusions as well as astrocytic hyaline inclusions identical to those in human familial ALS are observed in the spinal cords. The larger size of this rat model as compared with the ALS mice will facilitate studies involving manipulations of spinal fluid (implantation of intrathecal catheters for chronic therapeutic studies; CSF sampling) and spinal cord (e.g., direct administration of viral‐ and cell‐mediated therapies).     

Amyotrophic lateral sclerosis: recent insights from transgenic animal models with SOD1 mutations]

Mutations in Cu/Zn superoxide dismutase (SOD1) have been linked to some familial cases of amyotrophic lateral sclerosis (ALS). In order to reproduce the different degree of toxicity to the mutant protein by mutations, we generated new transgenic mice with two mutations from which the progression of the disease in human family is rapid (L84V) or extremely slow (H46R). By comparing the two transgenic mice with different SOD1 mutations, we demonstrate that the time course and the first symptoms in these mice were likely to human SOD1-mediated familial ALS. In addition, we report here that rats that express a human SOD1 transgene with two different ALS-associated mutations (G93A and H46R) develop striking motor neuron degeneration and paralysis. The larger size of this rat model as compared with the ALS mice will facilitate studies involving manipulations of spinal fluid (implantation of intrathecal catheters for chronic therapeutic studies; CSF sampling) and spinal cord (e.g., direct administration of viral- and cell-mediated therapies). Using this rat model we showed that intrathecal administration of the hepatocyte growth factor attenuates motoneuron death and prolongs the duration of the disease of transgenic rats.     

The relationship between Bayesian motor unit number estimation and histological measurements of motor neurons in wild-type and SOD1(G93A) mice

Objective

To assess the relationship between Bayesian MUNE and histological motor neuron counts in wild-type mice and in an animal model of ALS.

Methods

We performed Bayesian MUNE paired with histological counts of motor neurons in the lumbar spinal cord of wild-type mice and transgenic SOD1^G93A mice that show progressive weakness over time. We evaluated the number of acetylcholine endplates that were innervated by a presynaptic nerve.

Results

In wild-type mice, the motor unit number in the gastrocnemius muscle estimated by Bayesian MUNE was approximately half the number of motor neurons in the region of the spinal cord that contains the cell bodies of the motor neurons supplying the hindlimb crural flexor muscles. In SOD1^G93A mice, motor neuron numbers declined over time. This was associated with motor endplate denervation at the end-stage of disease.

Conclusion

The number of motor neurons in the spinal cord of wild-type mice is proportional to the number of motor units estimated by Bayesian MUNE. In SOD1^G93A mice, there is a lower number of estimated motor units compared to the number of spinal cord motor neurons at the end-stage of disease, and this is associated with disruption of the neuromuscular junction.

Significance

Our finding that the Bayesian MUNE method gives estimates of motor unit numbers that are proportional to the numbers of motor neurons in the spinal cord supports the clinical use of Bayesian MUNE in monitoring motor unit loss in ALS patients.     

Relationship between neuropathology and disease progression in the SOD1(G93A) ALS mouse

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of upper and lower motor neurons. However, recent reports suggest an active role of non-neuronal cells in the pathogenesis of the disease. Here, we examined quantitatively the temporal development of neuropathologic features in the brain and spinal cord of a mouse model of ALS (SOD1^G93A). Four phases of the disease were studied in both male and female SOD1^G93A mice: presymptomatic (PRE-SYM), symptomatic (SYM), endstage (ES) and moribund (MB). Compared to their control littermates, SOD1^G93A mice showed an increase in astrogliosis in the motor cortex, spinal cord and motor trigeminal nucleus in the SYM phase that worsened progressively in ES and MB animals. Associated with this increase in astrogliosis was a concomitant increase in motor neuron cell death in the spinal cord and motor trigeminal nucleus in both ES and MB mice, as well as in the ventrolateral thalamus in MB animals. In contrast, microglial activation was significantly increased in all the same regions but only when the mice were in the MB phase. These results suggest that astrogliosis preceded or occurred concurrently with neuronal degeneration whereas prominent microgliosis was evident later (MB stage), after significant motor neuron degeneration had occurred. Hence, our findings support a role for astrocytes in modulating the progression of non-cell autonomous degeneration of motor neurons, with microglia playing a role in clearing degenerating neurons.     

Progressive spinal axonal degeneration and slowness in ALS2-deficient mice

OBJECTIVE: Homozygous mutation in the ALS2 gene and the resulting loss of the guanine exchange factor activity of the ALS2 protein is causative for autosomal recessive early-onset motor neuron disease that is thought to predominantly affect upper motor neurons. The goal of this study was to elucidate how the motor system is affected by the deletion of ALS2. METHODS: ALS2-deficient mice were generated by gene targeting. Motor function and upper and lower motor neuron pathology were examined in ALS2-deficient mice and in mutant superoxide dismutase 1 (SOD1) mice that develop ALS-like disease from expression of an ALS-linked mutation in SOD1. RESULTS: ALS2-deficient mice demonstrated progressive axonal degeneration in the lateral spinal cord that is also prominent in mutant SOD1 mice. Despite the vulnerability of these spinal axons, lower motor neurons in ALS2-deficient mice were preserved. Behavioral studies demonstrated slowed movement without muscle weakness in ALS2(-/-) mice, consistent with upper motor neuron defects that lead to spasticity in humans. INTERPRETATION: The combined evidence from mice and humans shows that deficiency in ALS2 causes an upper motor neuron disease that in humans closely resembles a severe form of hereditary spastic paralysis, and that is quite distinct from amyotrophic lateral sclerosis.     

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.     

Celastrol blocks neuronal cell death and extends life in transgenic mouse model of amyotrophic lateral sclerosis

There is substantial evidence that both inflammation and oxidative damage contribute to the pathogenesis of motor neuron degeneration in the G93A SOD1 transgenic mouse model of amyotrophic lateral sclerosis (ALS). Celastrol is a natural product from Southern China, which exerts potent anti-inflammatory and antioxidative effects. It also acts potently to increase expression of heat shock proteins including HSP70. We administered it in the diet to G93A SOD1 mice starting at 30 days of age. Celastrol treatment significantly improved weight loss, motor performance and delayed the onset of ALS. Survival of celastrol-treated G93A mice increased by 9.4% and 13% for 2 mg/kg/day and 8 mg/kg/day doses, respectively. Cell counts of lumbar spinal cord neurons confirmed a protective effect, i.e. 30% increase in neuronal number in the lumbar spinal cords of celastrol-treated animals. Celastrol treatment reduced TNF-alpha, iNOS, CD40, and GFAP immunoreactivity in the lumbar spinal cord sections of celastrol-treated G93A mice compared to untreated G93A mice. TNF-alpha immunoreactivity co-localized with SMI-32 (neuronal marker) and GFAP (astrocyte marker). HSP70 immunoreactivity was increased in lumbar spinal cord neurons of celastrol-treated G93A mice. Celastrol has been widely used in treating inflammatory diseases in man, and is well tolerated; therefore, it may be a promising therapeutic candidate for the treatment of human ALS.     

Persistent activation of p38 mitogen-activated protein kinase in a mouse model of familial amyotrophic lateral sclerosis correlates with disease progression

The p38 mitogen-activated protein kinase (p38MAPK) is activated via phosphorylation in neurones and glial cells by a variety of stimuli including oxidative stress, excitotoxicity, and inflammatory cytokines. Activated p38MAPK can in turn induce phosphorylation of cytoskeletal proteins and activation of cytokines and nitric oxide, thus contributing to neurodegeneration. We investigated the expression and distribution of p38MAPK in the spinal cord of transgenic mice expressing a superoxide dismutase 1 mutation (SOD1G93A), a model of familial amyotrophic lateral sclerosis (ALS). Accumulation of p38MAPK was found by immunoblotting in the spinal cord of G93A mice during the progression of disease, but no changes were detected in its mRNA levels. Immunostaining for phosphorylated p38MAPK in lumbar spinal cord sections of SOD1G93A mice at the presymptomatic and early stages of disease showed an increased labeling in motor neurones that colocalized with phosphorylated neurofilaments in vacuolized perikarya and neurites, as detected by confocal microscopy. As the disease progressed, activated p38MAPK also accumulated in hypertrophic astrocytes and reactive microglia, as demonstrated by colocalization with GFAP and CD11b immunostaining, respectively. These data suggest that activation of p38MAPK in motor neurons and then in reactive glial cells may contribute, respectively, to the development and progression of motor neuron pathology in SOD1G93A mice.     

Neurochemical changes in the spinal cord in degenerative motor neuron diseases

Human amyotrophic lateral sclerosis (ALS), a typical motor neuron disease, is characterized pathologically by selective degenerative loss of motoneurons in the CNS. We have demonstrated significant reductions of neurotransmitter-related factors, such as acetylcholine-(ACh)-synthesizing enzyme activity and glutamate and aspartate contents in the ALS, compared to the non-ALS spinal cord obtained at autopsy. We have also shown considerable reductions in activities of cytochrome-c oxidase (CO), an enzyme contributing to aerobic energy production, and transglutaminase (TG), a Ca²⁺-dependent marker enzyme for tissue degeneration, in the ALS spinal cord. We found marked increases in fragmented glial fibrillary acidic protein (GFAP), a filamentous protein specifically associated with reactive astrocytes, in the ALS spinal cord relative to non-ALS tissue. These biochemical results corresponded well to pathomor-phological neuronal degenerative loss and reactive proliferation of astroglial components in the ALS spinal cord tissue. However, these results only indicate the final pathological and biochemical outcomes of ALS, and it is difficult to follow up cause and process in the ALS spinal cord during progression of the disease. Therefore, we used an animal model closely resembling human ALS, motor neuron degeneration (Mnd) mutant mice, a subline of C57BL/6 that shows late-onset progressive degeneration of lower motor neurons with paralytic gait beginning around 6.5 mo of age, to follow the biochemical and pathological alterations during postnatal development. We detected significant decreases in CO activity during early development and in activity of superoxide dismutase (SOD), an antioxidant enzyme, in later stages inMnd mutant spinal cord tissue. TG activity in theMnd spinal cord showed gradual increases during early development reaching a maximum at 5 mo, and then tending to decrease thereafter. Amounts of fragmented GFAPs increased continuously during postnatal development inMnd spinal cord. These biochemical changes were observed prior to the appearance of clinical motor dysfunctions in theMnd mutant mice. Such biochemical analyses using appropriate animal models will be useful for inferring the origin and progression of human ALS.     

Fatigability of spinal reflex transmission in a mouse model (SOD1(G93A) ) of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motor neurons. To analyze the progressive motor deficits during the course of this disease, we investigated fatigability and ability of recovery of spinal motor neurons by testing monosynaptic reflex transmission with increasing stimulus frequencies in the lumbar spinal cord of the SOD1(G93A) mouse model for ALS in a comparison with wild-type (WT) mice. Monosynaptic reflexes in WT and SOD1(G93A) mice without behavioral deficits showed no difference with respect to their resistance to increasing stimulus frequencies. During the progression of motor deficits in SOD1(G93A) mice, the vulnerability of monosynaptic reflexes to higher frequencies increased, the required time for reflex recovery was extended, and recovery was often incomplete. Fatigability and demand for recovery of spinal motor neurons in SOD1(G93A) mice rose with increasing motor deficits. This supports the assumption that impairment of the energy supply may contribute to the pathogenesis of ALS.