Differential gene expression in the axotomized facial motor nucleus of presymptomatic SOD1 mice

Previously, we compared molecular profiles of one population of wild-type (WT) mouse facial motoneurons (FMNs) surviving with FMNs undergoing significant cell death after axotomy. Regardless of their ultimate fate, injured FMNs respond with a vigorous pro-survival/regenerative molecular response. In contrast, the neuropil surrounding the two different injured FMN populations contained distinct molecular differences that support a causative role for glial and/or immune-derived molecules in directing contrasting responses of the same cell types to the same injury. In the current investigation, we utilized the facial nerve axotomy model and a presymptomatic amyotrophic lateral sclerosis (ALS) mouse (SOD1) model to experimentally mimic the axonal die-back process observed in ALS pathogenesis without the confounding variable of disease onset. Presymptomatic SOD1 mice had a significant decrease in FMN survival compared with WT, which suggests an increased susceptibility to axotomy. Laser microdissection was used to accurately collect uninjured and axotomized facial motor nuclei of WT and presymptomatic SOD1 mice for mRNA expression pattern analyses of pro-survival/pro-regeneration genes, neuropil-specific genes, and genes involved in or responsive to the interaction of FMNs and non-neuronal cells. Axotomized presymptomatic SOD1 FMNs displayed a dynamic pro-survival/regenerative response to axotomy, similar to WT, despite increased cell death. However, significant differences were revealed when the axotomy-induced gene expression response of presymptomatic SOD1 neuropil was compared with WT. We propose that the increased susceptibility of presymptomatic SOD1 FMNs to axotomy-induced cell death and, by extrapolation, disease progression, is not intrinsic to the motoneuron, but rather involves a dysregulated response by non-neuronal cells in the surrounding neuropil.     

Identification of B6SJL mSOD1G93A mouse subgroups with different disease progression rates

Disease progression rates among patients with amyotrophic lateral sclerosis (ALS) vary greatly. Although the majority of affected individuals survive 3–5 years following diagnosis, some subgroups experience a more rapidly progressing form, surviving less than 1 year, and other subgroups experience slowly progressing forms, surviving nearly 50 years. Genetic heterogeneity and environmental factors pose significant barriers in investigating patient progression rates. Similar to the case for humans, variation in survival within the mSOD1 mouse has been well documented, but different progression rates have not been investigated. The present study identifies two subgroups of B6SJL mSOD1^G93A mice with different disease progression rates, a fast progression group (FPG) and slow progression group, as evidenced by differences in the rate of motor function decline. In addition, increased disease‐associated gene expression within the FPG facial motor nucleus confirmed the presence of a more severe phenotype. We hypothesize that a more severe disease phenotype could be the result of 1) an earlier onset of axonal disconnection with a consistent degeneration rate or 2) a more severe or accelerated degenerative process. We performed a facial nerve transection axotomy in both mSOD1 subgroups prior to disease onset as a method to standardize the axonal disconnection. Instead of leading to comparable gene expression in both subgroups, this standardization did not eliminate the severe phenotype in the FPG facial nucleus, suggesting that the FPG phenotype is the result of a more severe or accelerated degenerative process. We theorize that these mSOD1 subgroups are representative of the rapid and slow disease phenotypes often experienced in ALS. J. Comp. Neurol. 523:2752–2768, 2015. © 2015 Wiley Periodicals, Inc.     

Increased ER stress during motor neuron degeneration in a transgenic mouse model of amyotrophic lateral sclerosis

Abstract

The endoplasmic reticulum (ER), which plays important roles in apoptosis, is susceptible to oxidative stress. ER stress is also thought to be involved in the pathogenesis of neurodegenerative diseases. In this study, we investigated whether ER stress is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) using the anterior part of the lumbar spinal cord of transgenic mice carrying a mutation (G93A) in the superoxide dismutase 1 (SOD1) gene. Western blot and immunohistochemical analyses demonstrated that the expressions of p-PERK and p-eIF2α were increased in the microsome fraction (P3) of the lumbar spinal cord at the pre-symptomatic age of 12 weeks (12W), while the expression of activated caspase-12 was increased in the cytoplasmic fraction (S3) of the lumbar spinal cord at both the pre-symptomatic age of 12W and the late symptomatic age of 20W. In contrast, GRP78 did not show any increases in the microsome fraction (P3) of the lumbar spinal cord at either the pre-symptomatic or symptomatic ages. Thus, the present results strongly suggest that the balance between anti- and pro-apoptotic proteins related to ER stress is impaired from the pre-symptomatic stage in this ALS mouse model, and that this imbalance may be related to the pathogenesis of motor neuron degeneration in ALS.     

Dorfin ameliorates phenotypes in a transgenic mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by progressive motor neuron degeneration and leads to death within a few years of diagnosis. One of the pathogenic mechanisms of ALS is proposed to be a dysfunction in the protein quality‐control machinery. Dorfin has been identified as a ubiquitin ligase (E3) that recognizes and ubiquitinates mutant SOD1 proteins, thereby accelerating their degradation and reducing their cellular toxicity. We examined the effects of human Dorfin overexpression in G93A mutant SOD1 transgenic mice, a mouse model of familial ALS. In addition to causing a decrease in the amount of mutant SOD1 protein in the spinal cord, Dorfin overexpression ameliorated neurological phenotypes and motor neuron degeneration. Our results indicate that Dorfin overexpression or the activation or induction of E3 may be a therapeutic avenue for mutant SOD1‐associated ALS. © 2009 Wiley‐Liss, Inc.     

Motor neuron degeneration in amyotrophic lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death

The mechanisms of human mutant superoxide dismutase-1 (mSOD1) toxicity to motor neurons (MNs) are unresolved. We show that MNs in G93A-mSOD1 transgenic mice undergo slow degeneration lacking similarity to apoptosis structurally and biochemically. It is characterized by somal and mitochondrial swelling and formation of DNA single-strand breaks prior to double-strand breaks occurring in nuclear and mitochondrial DNA. p53 and p73 are activated in degenerating MNs, but without nuclear import. The MN death is independent of activation of caspases-1, -3, and -8 or apoptosis-inducing factor within MNs, with a blockade of apoptosis possibly mediated by Aven up-regulation. MN swelling is associated with compromised Na,K-ATPase activity and aggregation. mSOD1 mouse MNs accumulate mitochondria from the axon terminals and generate higher levels of superoxide, nitric oxide, and peroxynitrite than MNs in control mice. Nitrated and aggregated cytochrome c oxidase subunit-I and alpha-synuclein as well as nitrated SOD2 accumulate in mSOD1 mouse spinal cord. Mitochondria in mSOD1 mouse MNs accumulate NADPH diaphorase and inducible nitric oxide synthase (iNOS)-like immunoreactivity, and iNOS gene deletion extends significantly the life span of G93A-mSOD1 mice. Prior to MN loss, spinal interneurons degenerate. These results identify novel mechanisms for mitochondriopathy and MN degeneration in amyotrophic lateral sclerosis (ALS) mice involving blockade of apoptosis, accumulation of MN mitochondria with enhanced toxic potential from distal terminals, NOS localization in MN mitochondria and peroxynitrite damage, and early degeneration of alpha-synuclein(+) interneurons. The data support roles for oxidative stress, protein nitration and aggregation, and excitotoxicity as participants in the process of MN degeneration caused by mSOD1.     

Sex-specific behavioural effects of environmental enrichment in a transgenic mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterised by motor neuron degeneration, muscle wasting and paralysis. While twin studies support a role for both genetic and environmental factors in ALS, the nature of environmental modifiers is unknown. We therefore compared onset and progression of disease symptoms in female and male transgenic ALS mice (expressing the human SOD1^G93A gene mutation) and their wild-type littermates, housed in environmentally enriched versus standard conditions. Environmental enrichment significantly improved motor performance, as measured using the accelerating rotarod, in particular for female mice. This enhanced motor coordination was observed for both SOD1^G93A and wild-type mice, suggesting this effect is independent of genotype. Female SOD1^G93A mice housed with environmental enrichment were found to reach overt end-stage disease sooner than their standard-housed littermates. However, male SOD1^G93A mice did not show significantly accelerated disease progression. This evidence for environmental modulation of ALS pathogenesis in transgenic mice provides insights into activity-dependent aspects of the disease process, and may help identify molecular targets for pharmacological modulators as future therapeutics.     

Stem cell factor‐activated bone marrow ameliorates amyotrophic lateral sclerosis by promoting protective microglial migration

Amyotrophic lateral sclerosis (ALS) is a progressive disease associated with motor neuron death. Several experimental treatments, including cell therapy using hematopoietic or neuronal stem cells, have been tested in ALS animal models, but therapeutic benefits have been modest. Here we used a new therapeutic strategy, bone marrow transplantation (BMT) with stem cell factor (SCF)‐ or FMS‐like tyrosine kinase 3 (flt3)‐activated bone marrow (BM) cells for the treatment of hSOD1(G93A) transgenic mice. Motor function and survival showed greater improvement in the SCF group than in the group receiving BM cells that had not been activated (BMT alone group), although no improvement was shown in the flt3 group. In addition, larger numbers of BM‐derived cells that expressed the microglia marker Iba1 migrated to the spinal cords of recipient mice compared with the BMT‐alone group. Moreover, after SCF activation, but not flt3 activation or no activation, the migrating microglia expressed glutamate transporter‐1 (GLT‐1). In spinal cords in the SCF group, inflammatory cytokines tumor necrosis factor‐α and interleukin‐1β were suppressed and the neuroprotective molecule insulin‐like growth factor‐1 increased relative to nontreatment hSOD1(G93A) transgenic mice. Therefore, SCF activation changed the character of the migrating donor BM cells, which resulted in neuroprotective effects. These studies have identified SCF‐activated BM cells as a potential new therapeutic agent for the treatment of ALS. © 2014 Wiley Periodicals, Inc.     

Early decrease of the immunophilin FKBP 52 in the spinal cord of a transgenic model for amyotrophic lateral sclerosis

Intrathecal administration of 5-HT(2) receptor agonists produces an anti-allodynic effect in a rat model of neuropathic pain. Several non-serotonergic neurotransmitters have been implicated these anti-nociceptive effects. In the present study, intrathecal pre-treatment with the muscarinic receptor antagonist atropine (10 and 30 microg) and pirenzepine (10 microg) reversed the anti-allodynic effect of the 5-HT(2) receptor agonist alpha-methyl-5-hydroxytryptamine, unlike various other antagonists. Thus, muscarinic receptors may be involved in the anti-allodynic action of intrathecally injected 5-HT(2) receptor agonist.     

Enhanced oxidative stress and the treatment by edaravone in mice model of amyotrophic lateral sclerosis

Oxidative stress is associated with the degeneration of both motor neurons and skeletal muscles in amyotrophic lateral sclerosis (ALS). A free radical scavenger edaravone has been proven as a therapeutic drug for ALS patients, but the neuroprotective mechanism for the oxidative stress of ALS has not been fully investigated. In this study, we investigated oxidative stress in ALS model mice bearing both oxidative stress sensor nuclear erythroid 2-related factor 2 (Nrf2) and G93A-human Cu/Zn superoxide dismutase (Nrf2/G93A) treated by edaravone. In vivo Nrf2 imaging analysis showed the accelerated oxidative stress both in spinal motor neurons and lower limb muscles of Nrf2/G93A mice according to disease progression in addition to the enhancement of serum oxidative stress marker dROMS. These were significantly alleviated by edaravone treatment accompanied by clinical improvements (rotarod test). The present study suggests that in vivo optical imaging of Nrf2 is useful for detecting oxidative stress in ALS, and edaravone alleviates the degeneration of both motor neurons and muscles related to oxidative stress in ALS patients.     

Strong P2X4 purinergic receptor-like immunoreactivity is selectively associated with degenerating neurons in transgenic rodent models of amyotrophic lateral sclerosis

The distribution of the P2X family of ATP receptors was analyzed in a rat model for amyotrophic lateral sclerosis (ALS) expressing mutated human superoxide dismutase (mSOD1(G93A)). We showed that strong P2X(4) immunoreactivity was selectively associated with degenerating motoneurons (MNs) in spinal cord ventral horn. Degenerating P2X(4)-positive MNs did not display apoptotic features such as chromatin condensation, positive TUNEL reaction, or active caspase 3 immunostaining. In contrast, these neurons showed other signs of abnormality, such as loss of the neuronal marker NeuN and recruitment of microglial cells with neuronophagic activity. Similar changes were observed in MNs from the cerebral cortex and brainstem in mSOD1(G93A) in both rat and mice. In addition, P2X(4) immunostaining demonstrated the existence of neuronal degeneration in the locus coeruleus, reticular formation, and Purkinje cells of the cerebellar cortex. It is suggested that abnormal trafficking and proteolytic processing of the P2X(4) receptor protein may underlie these changes.     

TDP‐43 is consistently co‐localized with ubiquitinated inclusions in sporadic and Guam amyotrophic lateral sclerosis but not in familial amyotrophic lateral sclerosis with and without SOD1 mutations

The transactive response (TAR) DNA binding protein 43 (TDP‐43) has been recently implicated as a major component of ubiquitinated inclusions in amyotrophic lateral sclerosis (ALS, motor neuron disease: MND) and ALS‐related disorders. In this study, we examined abnormal TDP‐43 pathology in 13 sporadic ALS (SALS), six familial ALS (FALS) with and without Cu/Zn superoxide dismutase (SOD1) mutations (SOD1‐FALS and non‐SOD1‐FALS), Guam ALS, two frontotemporal lobar degeneration with MND/ALS (FTLD‐MND/ALS), one FTLD with ubiquitin‐only‐immunoreactive inclusions (FTLD‐U) and two progressive supranuclear palsy (PSP). Sections from the spinal cord were processed for immunohistochemistry using antibodies against TDP‐43, ubiquitin, p62, cystatin C, phosphorylated tau protein (P‐tau; AT8), α‐synuclein and phosphorylated neurofilament protein (P‐NF). In 12 out of 13 SALS and both Guam ALS cases ubiquitin and p62‐immunoreactive (IR) neuronal inclusions co‐localized with TDP‐43. In three out of four SOD1‐FALS and one of two non‐SOD1‐FALS cases, TDP‐43‐IR inclusions were absent despite the presence of p62 and/or ubiquitin‐IR inclusions. However, a single TDP‐43‐IR neuronal inclusion co‐localized with p62 and ubiquitin in one SOD1‐FALS (His48Gln) case. Except for one neuron in a Guam case, all TDP‐43‐IR neuronal inclusions were negative for P‐tau (AT8). TDP‐43‐IR glial inclusions and neurites were also demonstrated. The TDP‐43 is a consistent component of the ubiquitinated inclusions in SALS and Guam ALS, but TDP‐43‐IR inclusions are absent or scarce in SOD1‐FALS.     

Expression of N19S-SOD1, an SOD1 mutant found in sporadic amyotrophic lateral sclerosis patients, induces low-grade motoneuronal toxicity

Amyotrophic lateral sclerosis (ALS) is the most common fatal motor neuron disease. It has been generally accepted that the proapoptotic property of the familial ALS (FALS)-linked mutant SOD1 genes plays an important role in the pathogenesis of some FALS cases. We found here that expression of N19S-SOD1, a novel SOD1 mutant originally found in a sporadic ALS patient, induces lower grade death in NSC34 cells than FALS-linked mutant SOD1. In agreement, intracytoplasmic aggregate formation and SOD1 polymerization are less prominently induced by ectopic expression of N19S-SOD1 than FALS-linked mutant SOD1. We further found that additional cell stresses, such as inhibition of proteasomal activity or up-regulation of intracellular oxidative stress, enhance N19S-SOD1-induced aggregate formation and polymerization of N19S-SOD1. Such analysis of the intracellular polymerization and the ubiquitination of N19S-SOD1 have further suggested that it is recognized as a misfolded protein, like FALS-linked mutant SOD1, whereas wild-type SOD1 is not. Altogether, it is speculated that the N19S mutation of SOD1 in cooperation with associated cell stresses contributes to the onset of ALS as a risk factor.     

Progressive motor unit loss in the G93A mouse model of amyotrophic lateral sclerosis is unaffected by gender

We examined whether there are gender differences in the progressive loss of functional motor units in SOD1^G93A transgenic mice. Isometric muscle and motor unit twitch contractions were recorded in fast‐ and slow‐twitch muscles in response to stimulation of the sciatic nerve. Using a modified motor unit number estimation technique (ITS‐MUNE), we found that motor unit numbers declined rapidly from 40 to 90 days of age during the asymptomatic phase of ALS in fast‐ but not slow‐twitch hindlimb muscles of both male and female mice. There was a corresponding decline in twitch and tetanic contractile forces of the fast‐twitch muscles. Gender did not affect the progressive loss of motor units and associated decline in force production. We conclude that gender does not alter progressive, muscle‐specific motor unit loss in ALS, even though gender does influence disease onset. Muscle Nerve 39: 318–327, 2009     

A novel SOD1 gene mutation in a Korean family with amyotrophic lateral sclerosis

BACKGROUND: Superoxide dismutase 1 (SOD1) gene mutations are responsible for approximately 20% of all familial amyotrophic lateral sclerosis (ALS) cases. However, these cases, especially those with SOD1 gene mutations, have not been reported in Korea. OBJECTIVES: The SOD1 gene in Korean family with ALS was screened for potential mutations and the clinical data was collected. MATERIALS AND METHODS: The clinical histories and neurological findings of the family members were obtained. Genomic DNA was isolated from the leukocytes of whole blood samples and the coding region of the SOD1 gene was analyzed by PCR and sequencing. RESULTS: The family with ALS showed a novel missense mutation in the SOD1 gene, which was heterozygous for the mutation, GGC to GTT, causing the substitution of valine for glycine at codon 10 (Gly10Val) in exon 1. Clinically, the patients exhibited early onset and rapid disease progression. CONCLUSIONS: Familial ALS with a novel Gly10Val mutation in the SOD1 gene showed severe clinical features. The mutation lies in a region involved in a dimer contact in the three-dimensional structure of the SOD1 protein. This study expands the number of ALS-associated SOD1 gene mutations.     

Spinal anterior horn has the capacity to self‐regenerate in amyotrophic lateral sclerosis model mice

The exact host environment necessary for neural regeneration in amyotrophic lateral sclerosis (ALS) has not yet been fully elucidated. We first focused on the extracellular matrix proteins in ALS model mice during development of the disease and then attempted to examine whether regeneration occurs in the ALS spinal cord under regenerative conditions. A progressive increase in γ1 laminin (a promoter of regeneration) and a progressive decrease in semaphorin3A (Sema3A; an inhibitor of regeneration) were observed, mainly in the neuropil of the spinal anterior horn from 15 to 18 weeks, when astrocytes began to express both γ1 laminin and Sema3A. On the other hand, a progressive increase in growth‐associated protein 43 (GAP43; synaptic regeneration site) and a progressive decrease in synaptotagmin1 (actual synaptic bouton) were observed in the same areas of the spinal anterior horn from 15 to 18 weeks. Thus, the present data suggest that, although the spinal anterior horn in ALS models loses motor neurons, it initially possesses the capacity to self‐regenerate but displays a progressive loss of ability to regenerate new effective synapses. © 2009 Wiley‐Liss, Inc.     

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.     

Matrix metalloproteinase-9 regulates TNF-alpha and FasL expression in neuronal, glial cells and its absence extends life in a transgenic mouse model of amyotrophic lateral sclerosis

Whether increased levels of matrix metalloproteinases (MMPs) correspond to a role in the pathogenesis of amyotrophic lateral sclerosis (ALS) needs to be determined and it is actively being pursued. Here we present evidence suggesting that MMP-9 contributes to the motor neuron cell death in ALS. We examined the role of MMP-9 in a mouse model of familial ALS and found that lack of MMP-9 increased survival (31%) in G93A SOD1 mice. Also, MMP-9 deficiency in G93A mice significantly attenuated neuronal loss, and reduced neuronal TNF-alpha and FasL immunoreactivities in the lumbar spinal cord. These findings suggest that MMP-9 is an important player in the pathogenesis of ALS. Our data suggest that the mechanism for MMP-9 neurotoxicity in ALS may be by upregulating neuronal TNF-alpha and FasL expression and activation. This study provides new mechanism and suggests that MMP inhibitors may offer a new therapeutic strategy for ALS.     

Locomotor analysis identifies early compensatory changes during disease progression and subgroup classification in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a motoneuron degenerative disease that is challenging to diagnose and presents with considerable variability in survival. Early identification and enhanced understanding of symp-tomatic patterns could aid in diagnosis and provide an avenue for monitoring disease progression. Use of the mSOD1G93A mouse model provides control of the confounding environmental factors and genetic heteroge-neity seen in amyotrophic lateral sclerosis patients, while investigating underlying disease-induced changes. In the present study, we performed a longitudinal behavioral assessment paradigm and identified an early hindlimb symptom, resembling the common gait abnormality foot drop, along with an accompanying fore-limb compensatory mechanism in the mSOD1G93A mouse. Following these initial changes, mSOD1 mice displayed a temporary hindlimb compensatory mechanism resembling an exaggerated steppage gait. As the disease progressed, these compensatory mechanisms were not sufficient to sustain fundamental locomotor parameters and more severe deficits appeared. We next applied these initial findings to investigate the inher-ent variability in B6SJL mSOD1G93A survival. We identified four behavioral variables that, when combined in a cluster analysis, identified two subpopulations with different disease progression rates: a fast progression group and a slow progression group. This behavioral assessment paradigm, with its analytical approaches, provides a method for monitoring disease progression and detecting mSOD1 subgroups with different dis-ease severities. This affords researchers an opportunity to search for genetic modifiers or other factors that likely enhance or slow disease progression. Such factors are possible therapeutic targets with the potential to slow disease progression and provide insight into the underlying pathology and disease mechanisms.