Endocannabinoids accumulate in spinal cord of SOD1 G93A transgenic mice

Approximately 2% of amyotrophic lateral sclerosis (ALS) cases are caused by mutations in the super oxide dismutase 1 (SOD1) gene and transgenic mice for these mutations recapitulate many features of this devastating neurodegenerative disease. Here we show that the amount of anandamide (AEA) and 2-arachidonoylglycerol (2-AG), two endocannabinoids that have neuroprotective properties, increase in spinal cord of SOD1(G93A) transgenic mice. This increase occurs in the lumbar section of spinal cords, the first section to undergo neurodegeneration, and is significant before overt motor impairment. Our results show that chronic neurodegeneration induced by a genetic mutation increases endocannabinoid production possibly as part of an endogenous defense mechanism.     

Transgenic SOD1 G93A mice develop reduced GLT-1 in spinal cord without alterations in cerebrospinal fluid glutamate levels

Glutamate-induced excitotoxicity is suggested to play a central role in the development of amyotrophic lateral sclerosis (ALS), although it is still unclear whether it represents a primary cause in the cascade leading to motor neurone death. We used western blotting, immunocytochemistry and in situ hybridization to examine the expression of GLT-1 in transgenic mice carrying a mutated (G93A) human copper-zinc superoxide dismutase (TgSOD1 G93A), which closely mimic the features of ALS. We observed a progressive decrease in the immunoreactivity of the glial glutamate transporter (GLT-1) in the ventral, but not in the dorsal, horn of lumbar spinal cord. This effect was specifically found in 14- and 18-week-old mice that had motor function impairment, motor neurone loss and reactive astrocytosis. No changes in GLT-1 were observed at 8 weeks of age, before the appearance of clinical symptoms. Decreases in GLT-1 were accompanied by increased glial fibrillary acidic protein (GFAP) levels and no change in the levels of GLAST, another glial glutamate transporter. The glutamate concentration in the cerebrospinal fluid (CSF) of TgSOD1 G93A mice was not modified at any of the time points examined, compared with age-matched controls. These findings indicate that the loss of GLT-1 protein in ALS mice selectively occurs in the areas affected by neurodegeneration and reactive astrocytosis and it is not associated with increases of glutamate levels in CSF. The lack of changes in GLT-1 at the presymptomatic stage suggests that glial glutamate transporter reduction is not a primary event leading to motor neurone loss.     

Presymptomatic and symptomatic ALS SOD1(G93A) mice differ in adenosine A1 and A2A receptor-mediated tonic modulation of neuromuscular transmission

Amyotrophic lateral sclerosis (ALS) is a disease leading to neuromuscular transmission impairment. A_2A adenosine receptor (A2AR) function changes with disease stage, but the role of the A₁ receptors (A1Rs) is unknown and may have a functional cross-talk with A2AR. The role of A1R in the SOD1(G93A) mouse model of ALS in presymptomatic (4–6 weeks old) and symptomatic (12–14 weeks old) phases was investigated by recording endplate potentials (EPPs), miniature endplate potentials (MEPPs), and quantal content (q.c.) of EPPs, from Mg²⁺ paralyzed hemidiaphragm preparations. In presymptomatic mice, the A1R agonist, N⁶-cyclopentyladenosine (CPA) (50 nM), decreased mean EPP amplitude, MEPP frequency, and q.c. of EPPs, an effect quantitatively similar to that in age-matched wild-type (WT) mice. However, coactivation of A2AR with CGS 21680 (5 nM) prevented the effects of CPA in WT mice but not in presymptomatic SOD1(G93A) mice, suggestive of A1R/A2AR cross-talk disruption in this phase of ALS. DPCPX (50 nM) impaired CGS 21680 facilitatory action on neuromuscular transmission in WT but not in presymptomatic mice. In symptomatic animals, CPA only inhibited transmission if added in the presence of adenosine deaminase (ADA, 1 U/mL). ADA and DPCPX enhanced more transmission in symptomatic mice than in age-matched WT mice, suggestive of increase in extracellular adenosine during the symptomatic phase of ALS. The data documents that at the neuromuscular junction of presymptomatic SOD1(G93A) mice, there is a loss of A1R-A2AR functional cross-talk, while in symptomatic mice there is increased A1R tonic activation, and that with disease progression, changes in A1R-mediated adenosine modulation may act as aggravating factors during the symptomatic phase of ALS.     

Prevention of thrombin‐induced motoneuron degeneration with different neurotrophic factors in highly enriched cultures

Previous reports have shown that neuronal and glial cells express functionally active thrombin receptors. The thrombin receptor (PAR‐1), a member of a growing family of protease activated receptors (PARs), requires cleavage of the extracellular amino‐terminus domain by thrombin to induce signal transduction. Studies from our laboratory have shown that PAR‐1 activation following the addition of thrombin or a synthetic thrombin receptor activating peptide (TRAP) induces motoneuron cell death both in vitro and in vivo. In addition to increasing motoneuron cell death, PAR‐1 activation leads to decreases in the mean neurite length and side branching in highly enriched motoneuron cultures. It has been suggested that motoneuron survival depends on access to sufficient target‐derived neurotrophic factors through axonal branching and synaptic contacts. However, whether the thrombin‐induced effects on motoneurons can be prevented by neurotrophic factors is still unknown. Using highly enriched avian motoneuron cultures, we show here that alone, soluble chick skeletal muscle extracts (CMX), brain‐derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and glial cell line–derived neurotrophic factor (GDNF) significantly increased motoneuron survival compared to controls, whereas nerve growth factor (NGF) did not have a significant effect on motoneuron survival. Furthermore, cotreatment with muscle‐derived agents (i.e., CMX, BDNF, GDNF) significantly prevented the death of motoneurons induced by α‐thrombin. Yet, non–muscle‐derived agents (CNTF and NGF) had little or no significant effect in reversing thrombin‐induced motoneuron death. CMX and CNTF significantly increased the mean length of neurites, whereas NGF, BDNF, and GDNF failed to enhance neurite outgrowth compared to controls. Furthermore, CMX and CNTF significantly prevented thrombin‐induced inhibition of neurite outgrowth, whereas BDNF and GDNF only partially reversed thrombin‐induced inhibition of neurite outgrowth. These findings show differential effects of neurotrophic factors on thrombin‐induced motoneuron degeneration and suggest specific overlaps between the trophic and stress pathways activated by some neurotrophic agents and thrombin, respectively. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 571–580, 1999     

Decreased GLT-1 and increased SOD1 and HO-1 expression in astrocytes contribute to lumbar spinal cord vulnerability of SOD1-G93A transgenic mice

The SOD1-G93A transgenic mouse is a widely used ALS model, but the death of lower motor neurons is the hallmark. Here, we show that the SOD1-G93A transgene and HO-1 are preferentially over-expressed in the lumbar spinal cord, particularly in the activated astrocytes of the transgenic mice. We also show down-regulation of GLT-1 in spite of the proliferating astrocytes. However, GLT-1, SOD1-G93A transgene and HO-1 expression were not obviously changed in the motor cortex. Our data link spinal cord vulnerability to relatively decreased expression of GLT-1, and high expression of the transgene and HO-1 in astrocytes in SOD1-G93A transgenic mice.     

Paradoxical roles of serine racemase and D-serine in the G93A mSOD1 mouse model of amyotrophic lateral sclerosis

D-serine is an endogenous neurotransmitter that binds to the NMDA receptor, thereby increasing the affinity for glutamate, and the potential for excitotoxicity. The primary source of D-serine in vivo is enzymatic racemization by serine racemase (SR). Regulation of D-serine in vivo is poorly understood, but is thought to involve a combination of controlled production, synaptic reuptake by transporters, and intracellular degradation by D-amino acid oxidase (DAO). However, SR itself possesses a well-characterized eliminase activity, which effectively degrades D-serine as well. D-serine is increased two-fold in spinal cords of G93A Cu,Zn-superoxide dismutase (SOD1) mice--the standard model of amyotrophic lateral sclerosis (ALS). ALS mice with SR disruption show earlier symptom onset, but survive longer (progression phase is slowed), in an SR-dependent manner. Paradoxically, administration of D-serine to ALS mice dramatically lowers cord levels of D-serine, leading to changes in the onset and survival very similar to SR deletion. D-serine treatment also increases cord levels of the alanine-serine-cysteine transporter 1 (Asc-1). Although the mechanism by which SOD1 mutations increases D-serine is not known, these results strongly suggest that SR and D-serine are fundamentally involved in both the pre-symptomatic and progression phases of disease, and offer a direct link between mutant SOD1 and a glial-derived toxic mediator.     

Death receptor 6 (DR6) antagonist antibody is neuroprotective in the mouse SOD1G93A model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the death of motor neurons, axon degeneration, and denervation of neuromuscular junctions (NMJ). Here we show that death receptor 6 (DR6) levels are elevated in spinal cords from post-mortem samples of human ALS and from SOD1^G93A transgenic mice, and DR6 promotes motor neuron death through activation of the caspase 3 signaling pathway. Blocking DR6 with antagonist antibody 5D10 promotes motor neuron survival in vitro via activation of Akt phosphorylation and inhibition of the caspase 3 signaling pathway, after growth factor withdrawal, sodium arsenite treatment or co-culture with SOD1^G93A astrocytes. Treatment of SOD1^G93A mice at an asymptomatic stage starting on the age of 42 days with 5D10 protects NMJ from denervation, decreases gliosis, increases survival of motor neurons and CC1⁺ oligodendrocytes in spinal cord, decreases phosphorylated neurofilament heavy chain (pNfH) levels in serum, and promotes motor functional improvement assessed by increased grip strength. The combined data provide clear evidence for neuroprotective effects of 5D10. Blocking DR6 function represents a new approach for the treatment of neurodegenerative disorders involving motor neuron death and axon degeneration, such as ALS.     

Characterizing the multiple roles of FGF-2 in SOD1G93A ALS mice in vivo and in vitro

We have previously shown that knockout of fibroblast growth factor-2 (FGF-2) and potential compensatory effects of other growth factors result in amelioration of disease symptoms in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). ALS is a rapidly progressive neurological disorder leading to degeneration of cortical, brain stem, and spinal motor neurons followed by subsequent denervation and muscle wasting. Mutations in the superoxide dismutase 1 (SOD1) gene are responsible for approximately 20% of familial ALS cases and SOD1 mutant mice still are among the models best mimicking clinical and neuropathological characteristics of ALS. The aim of the present study was a thorough characterization of FGF-2 and other growth factors and signaling effectors in vivo in the SOD1^G93A mouse model. We observed tissue-specific opposing gene regulation of FGF-2 and overall dysregulation of other growth factors, which in the gastrocnemius muscle was associated with reduced downstream extracellular-signal-regulated kinases (ERK) and protein kinase B (AKT) activation. To further investigate whether the effects of FGF-2 on motor neuron death are mediated by glial cells, astrocytes lacking FGF-2 were cocultured together with mutant SOD1 ^G93A motor neurons. FGF-2 had an impact on motor neuron maturation indicating that astrocytic FGF-2 affects motor neurons at a developmental stage. Moreover, neuronal gene expression patterns showed FGF-2- and SOD1 ^G93A-dependent changes in ciliary neurotrophic factor, glial-cell-line-derived neurotrophic factor, and ERK2, implying a potential involvement in ALS pathogenesis before the onset of clinical symptoms.     

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

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

Castration reduces motoneuron soma size but not dendritic length in the spinal nucleus of the bulbocavernosus of wild‐type and BCL‐2 overexpressing mice

Motoneurons in the spinal nucleus of the bulbocavernosus (SNB) and their target muscles, bulbocavernosus and levator ani (BC/LA), constitute an androgen‐sensitive neuromuscular system. Testosterone regulates SNB soma size, SNB dendritic length, and BC/LA muscle mass in adult male rats. Recent evidence indicates that the cell death‐regulatory protein, Bcl‐2, may also play a role in adult neural plasticity. The present study examined whether gonadal hormones and/or the Bcl‐2 protein influence the morphology of the SNB neuromuscular system in adult B6D2F1 mice. In Experiment 1, adult wild‐type and Bcl‐2 overexpressing males were castrated and implanted with silastic capsules containing testosterone or left blank. Six weeks after castration, cholera toxin‐horseradish peroxidase was injected into the BC muscle to label SNB dendrites. Animals were killed 48 h later, and BC/LA muscle mass, SNB soma size, and SNB dendritic arbors were examined. In Experiment 2, wild‐type and Bcl‐2 overexpressing males were castrated or sham castrated, implanted with testosterone‐filled or blank capsules, and examined 12 weeks later. In both experiments, BC/LA muscle mass and SNB soma size were significantly reduced in castrates receiving blank capsules. Surprisingly, however, there was no effect of hormone manipulation on any of several measures of dendritic length. Thus, the dendritic morphology of SNB motoneurons appears to be relatively insensitive to circulating androgen levels in B6D2F1 mice. Bcl‐2 overexpression did not influence BC/LA muscle mass, SNB soma size, or SNB dendritic length, indicating that the morphology of this neuromuscular system and the response to castration are not altered by forced expression of the Bcl‐2 protein. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 403–412, 2002     

Gene Expression Profiles of APP and BACE1 in Tg SOD1G93A Cortical Cells

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease defined by motor neuron loss. Transgenic mouse model (Tg SOD1G93A) shows pathological features that closely mimic those seen in ALS patients. An hypothetic link between AD and ALS was suggested by finding an higher amount of amyloid precursor protein (APP) in the spinal cord anterior horn neurons, and of Aβ peptides in ALS patients skin. In this work, we have investigated the expression of some genes involved in Alzheimer’s disease, as APP, β- and γ-secretase, in an animal model of ALS, to understand some possible common molecular mechanisms between these two pathologies. For gene expression analysis, we carried out a quantitative RT-PCR in ALS mice and in transgenic mice over-expressing human wild-type SOD1 (Tg hSOD1). We found that APP and BACE1 mRNA levels were increased 1.5-fold in cortical cells of Tg SOD1G93A mice respect to Tg hSOD1, whereas the expression of γ-secretase genes, as PSEN1, PSEN2, Nicastrin, and APH1a, showed no statistical differences between wild-type and ALS mice. Biochemical analysis carried out by immunostaining and western blotting, did not show any significant modulation of the protein expression compared to the genes, suggesting the existence of post-translational mechanisms that modify protein levels.     

Immunohistochemical localization of insulin-like growth factor binding protein 2 in the central nervous system of SOD1<Superscript>G93A</Superscript> transgenic mice

In the present study, we performed immunohistochemical studies to investigate the changes of insulin-like growth factor binding protein 2 (IGFBP2) in the central nervous system of SOD1^G93A mutant transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS). Decreased immunoreactivity for IGFBP2 was observed in the cerebral cortex, hippocampus and brainstem of SOD1^G93A transgenic mice. In the cerebral cortex, the number of IGFBP2-positive cells was decreased in the somatomotor area, somatosensory area, auditory area, visual area, entorhinal area, piriform area and prefrontal area. In the hippocampal formation, IGFBP2 immunoreactivity was significantly decreased in the CA1-3 areas and the dentate gyrus. In the brainstem, few IGFBP2-immunoreactive cells were observed in the medullary and pontine reticular formation, vestibular nucleus, trigeminal motor nucleus, facial nucleus, hypoglossal nucleus and raphe nucleus. In the spinal cord, IGFBP2 immunoreactivity was not significantly decreased in SOD1^G93A transgenic mice. This study showing decreased IGFBP2 in different brain regions of SOD1^G93A transgenic mice may provide clues for understanding differential susceptibility of neural structures in ALS. S. E. Sim and Y. H. Chung have contributed equally to this work.     

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

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

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

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

Abnormal medial prefrontal cortex connectivity and defective fear extinction in the presymptomatic G93A SOD1 mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a fatal progressive neuropathy associated with the degeneration of spinal and brainstem motor neurons. Although ALS is essentially considered as a lower motor neuron disease, prefrontal cortex atrophy underlying executive function deficits have been extensively reported in ALS patients. Here, we examine whether prefrontal cortex neuronal abnormalities and related cognitive impairments are present in presymptomatic G93A Cu/Zn superoxide dismutase mice, a mouse model for familial ALS. Structural characteristics of prelimbic/infralimbic (PL/IL) medial prefrontal cortex (mPFC) neurons were studied in 3-month-old G93A and wild-type mice with the Golgi–Cox method, while mPFC-related cognitive operations were assessed using the conditioned fear extinction paradigm. Sholl analysis performed on the dendritic material showed a reduction in dendrite length and branch nodes on basal dendrites of PL/IL neurons in G93A mice. Spine density was also decreased on basal dendrite segments of branch order five. Consistent with the altered morphology of PL/IL cortical regions, G93A mice showed impaired extinction of conditioned fear. Our findings indicate that abnormal prefrontal cortex connectivity and function are appreciable before the onset of motor disturbances in this model.     

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

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

Cross‐talk between IGF‐1 and estrogen receptors attenuates intracellular changes in ventral spinal cord 4.1 motoneuron cells because of interferon‐gamma exposure

Insulin‐like growth factor‐1 (IGF‐1) is a neuroprotective growth factor that promotes neuronal survival by inhibition of apoptosis. To examine whether IGF‐1 exerts cytoprotective effects against extracellular inflammatory stimulation, ventral spinal cord 4.1 (VSC4.1) motoneuron cells were treated with interferon‐gamma (IFN‐γ). Our data demonstrated apoptotic changes, increased calpain:calpastatin and Bax:Bcl‐2 ratios, and expression of apoptosis‐related proteases (caspase‐3 and ‐12) in motoneurons rendered by IFN‐γ in a dose‐dependent manner. Post‐treatment with IGF‐1 attenuated these changes. In addition, IGF‐1 treatment of motoneurons exposed to IFN‐γ decreased expression of inflammatory markers (cyclooxygenase‐2 and nuclear factor‐kappa B:inhibitor of kappa B ratio). Furthermore, IGF‐1 attenuated the loss of expression of IGF‐1 receptors (IGF‐1Rα and IGF‐1Rβ) and estrogen receptors (ERα and ERβ) induced by IFN‐γ. To determine whether the protective effects of IGF‐1 are associated with ERs, ERs antagonist ICI and selective siRNA targeted against ERα and ERβ were used in VSC4.1 motoneurons. Distinctive morphological changes were observed following siRNA knockdown of ERα and ERβ. In particular, apoptotic cell death assessed by TUNEL assay was enhanced in both ERα and ERβ‐silenced VSC4.1 motoneurons following IFN‐γ and IGF‐1 exposure. These results suggest that IGF‐1 protects motoneurons from inflammatory insult by a mechanism involving pivotal interactions with ERα and ERβ.

     

Screening the expression characteristics of several miRNAs in G93A‐SOD1 transgenic mouse: altered expression of miRNA‐124 is associated with astrocyte differentiation by targeting Sox2 and Sox9

MicroRNA s (miRNA s) are suspected to be a contributing factor in amyotrophic lateral sclerosis (ALS ). Here, we assess the altered expression of miRNA s and the effects of miR‐124 in astrocytic differentiation in neural stem cells of ALS transgenic mice. Differentially expressed miRNA ‐positive cells (including miR‐124, miR‐181a, miR‐22, miR‐26b, miR‐34a, miR‐146a, miR‐219, miR‐21, miR‐200a, and miR‐320) were detected by in situ hybridization and qRT ‐PCR in the spinal cord and the brainstem. Our results demonstrated that miR‐124 was down‐regulated in the spinal cord and brainstem. In vitro , miR‐124 was down‐regulated in neural stem cells and up‐regulated in differentiated neural stem cells in G93A‐ superoxide dismutase 1 (SOD 1 ) mice compared with WT mice by qRT ‐PCR . Meanwhile, Sox2 and Sox9 protein levels showed converse change with miR‐124 in vivo and vitro . After over‐expression or knockdown of miR‐124 in motor neuron‐like hybrid (NSC 34) cells of mouse, Sox2 and Sox9 proteins were noticeably down‐regulated or up‐regulated, whereas Sox2 and Sox9 mRNA s remained virtually unchanged. Moreover, immunofluorescence results indicated that the number of double‐positive cells of Sox2/glial fibrillary acidic protein (GFAP) and Sox9/glial fibrillary acidic protein (GFAP) was higher in G93A‐SOD 1 mice compared with WT mice. We also found that many Sox2‐ and Sox9‐positive cells were nestin positive in G93A‐SOD 1 mice, but not in WT mice. Furthermore, differentiated neural stem cells from G93A‐SOD 1 mice generated a greater proportion of astrocytes and lower proportion of neurons than those from WT mice. MiR‐124 may play an important role in astrocytic differentiation by targeting Sox2 and Sox9 in ALS transgenic mice.

Cover Image for this issue: doi: 10.1111/jnc.14171 .