Decreased signalling of EphA4 improves functional performance and motor neuron survival in the SOD1G93A ALS mouse model.
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ABSTRACT:
SUBMITTER: Zhao J
PROVIDER: S-EPMC6065374 | biostudies-literature | 2018 Jul
REPOSITORIES: biostudies-literature
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Decreased signalling of EphA4 improves functional performance and motor neuron survival in the SOD1<sup>G93A</sup> ALS mouse model.
Zhao J J Cooper L T LT Boyd A W AW Bartlett P F PF
Scientific reports 20180730 1
Amyotrophic lateral sclerosis (ALS) is an untreatable, progressive, neurodegenerative disease specifically affecting motor neurons. Recently, the tyrosine kinase receptor EphA4 was directly implicated in ALS disease progression. We report that a long-lived mutated form of the EphA4 antagonist EphA4-Fc (mutEphA4-Fc), which blocks EphA4 binding to its ligands and inhibits its function, significantly improved functional performance in SOD1<sup>G93A</sup> ALS model mice, as assessed by rotarod and h ...[more]
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Project description:ALS (amyotrophic lateral sclerosis), the most common motor neuron disease, causes muscle denervation and rapidly fatal paralysis. While motor neurons are the most affected cells in ALS, studies on the pathophysiology of the disease have highlighted the importance of non-cell autonomous mechanisms, which implicate astrocytes and other glial cells. In ALS, subsets of reactive astrocytes lose their physiological functions and become toxic for motor neurons, thereby contributing to disease pathogenesis. Evidence of astrocyte contribution to disease pathogenesis are well established in cellular and animal models of familial ALS linked to mutant SOD1, where astrocytes promote motor neuron cell death. The mechanism underlying astrocytes reactivity in conditions of CNS injury have been shown to involve the MTOR pathway. However, the role of this conserved metabolic signaling pathway, and the potential therapeutic effects of its modulation, have not been investigated in ALS astrocytes. Here, we show elevated activation of the MTOR pathway in human-derived astrocytes harboring mutant SOD1, which results in inhibition of macroautophagy/autophagy, increased cell proliferation, and enhanced astrocyte reactivity. We demonstrate that MTOR pathway activation in mutant SOD1 astrocytes is due to post-transcriptional upregulation of the IGF1R (insulin like growth factor 1 receptor), an upstream positive modulator of the MTOR pathway. Importantly, inhibition of the IGF1R-MTOR pathway decreases cell proliferation and reactivity of mutant SOD1 astrocytes, and attenuates their toxicity to motor neurons. These results suggest that modulation of astrocytic IGF1R-MTOR pathway could be a viable therapeutic strategy in SOD1 ALS and potentially other neurological diseases.Abbreviations: ACM: astrocyte conditioned medium; AKT: AKT serine/threonine kinase; ALS: amyotrophic lateral sclerosis; BrdU: thymidine analog 5-bromo-2'-deoxyuridine; CNS: central nervous system; EIF4EBP1/4EBP1: eukaryotic translation initiation factor 4E binding protein 1; GFAP: glial fibrillary acidic protein; IGF1R: insulin like growth factor 1 receptor; INSR: insulin receptor; iPSA: iPSC-derived astrocytes; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta;MTOR: mechanistic target of rapamycin kinase; NES: nestin; PPK1: 3-phosphoinositide dependent protein kinase 1; PI: propidium iodide; PPP: picropodophyllotoxin; PTEN: phosphatase and tensin homolog; S100B/S100β: S100 calcium binding protein B; SLC1A3/ EAAT1: solute carrier family 1 member 3; SMI-32: antibody to nonphosphorylated NEFH; SOD1: superoxide dismutase 1; TUBB3: tubulin beta 3 class III; ULK1: unc-51 like autophagy activating kinase 1.
| S-EPMC8726657 | biostudies-literature
Project description:BackgroundOxidative stress and reactive oxygen species (ROS) are important in the pathogenesis of amyotrophic lateral sclerosis (ALS). Hypochlorous acid (HOCl) is a powerful oxidant of the reactive oxygen species (ROS) family. HOCl's role in the progress of ALS remains unclear due to the lack of an effective HOCl detection method. Cumulative evidence supports oxidative damage incurred by mutant hSOD1 contributing to motor neuron death; however, whether HOCl as well as its catalytic enzyme myeloperoxidase (MPO) function in the cell death of SOD1G93A ALS remains elusive.MethodsThe hSOD1WT and hSOD1G93A NSC-34 cell and SOD1G93A ALS mouse models were employed. With a novel fluorescent HOCl probe, HKOCl-3, we detected the expressions of HOCl and its catalytic enzyme, MPO, in the above models in vitro and in vivo. The regulation of MPO/HOCl by hSOD1G93A mutation and cell deaths by MPO/HOCl were also assayed, including apoptosis, ferroptosis, and autophagy.ResultsOur results showed that hSOD1G93A mutation promoted the activation of the MPO/HOCl pathway in SOD1G93A ALS cell models. The activation of MPO/HOCl pathways facilitated apoptosis and ferroptosis through increasing the Bax/Bcl-2 ratio and expression of caspase-3 or inhibiting the expressions of GPX4 and NQO1 and thus leading to irreversible lipid peroxidation. Overexpressed FSP1, a glutathione-independent suppressor, could ameliorate ferroptosis. In vivo, we demonstrated that the activation of the MPO/HOCl pathway occurred differently in motor neurons of the motor cortices, brain stems, and spinal cords in male and female SOD1G93A transgenic mice. In addition, inhibiting MPO improved the motor performance of SOD1G93A transgenic mice, as demonstrated by the rotarod test.ConclusionsWe concluded that aggregation of mutant hSOD1 proteins contributed to activation of the MPO/HOCl pathway, triggering apoptosis and ferroptosis in motor neuronal deaths and exerting impaired motor performance.
| S-EPMC8845144 | biostudies-literature
Project description:Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by upper and lower motor neuron (MN) degeneration. Astrocytes surrounding MNs are known to modulate ALS progression. When cocultured with astrocytes overexpressing the ALS-linked mutant Cu/Zn superoxide dismutase (SOD1G93A) or when cultured with conditioned medium from SOD1G93A astrocytes, MN survival is reduced. The exact mechanism of this neurotoxic effect is unknown. Astrocytes secrete extracellular vesicles (EVs) that transport protein, mRNA, and microRNA species from one cell to another. The size and protein markers characteristic of exosomes were observed in the EVs obtained from cultured astrocytes, indicating their abundance in exosomes. Here, we analyzed the microRNA content of the exosomes derived from SOD1G93A astrocytes and evaluated their role in MN survival. Purified MNs exposed to SOD1G93A astrocyte-derived exosomes showed reduced survival and neurite length compared to those exposed to exosomes derived from non-transgenic (non-Tg) astrocytes. Analysis of the miRNA content of the exosomes revealed that miR-155-5p and miR-582-3p are differentially expressed in SOD1G93A exosomes compared with exosomes from non-Tg astrocytes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicates that miR-155-5p and miR-582-3p predicted targets are enriched in the neurotrophin signaling pathway. Importantly, when levels of miR-155-5p were reduced by incubation with a specific antagomir, SOD1G93A exosomes did not affect MN survival or neurite length. These results demonstrate that SOD1G93A-derived exosomes are sufficient to induce MN death, and miRNA-155-5p contributes to this effect. miRNA-155-5p may offer a new therapeutic target to modulate disease progression in ALS.
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| S-EPMC3996124 | biostudies-literature
Project description:While the death of motor neuron is a pathological hallmark of amyotrophic lateral sclerosis (ALS), defects in other cell types or organs may also actively contribute to ALS disease progression. ALS patients experience progressive skeletal muscle wasting that may not only exacerbate neuronal degeneration, but likely has a significant impact on bone function. In our previous published study, we have discovered severe bone loss in an ALS mouse model with overexpression of ALS-associated mutation SOD1G93A (G93A). Here we further provide a mechanistic understanding of the bone loss in ALS animal and cellular models. Combining mitochondrial fluorescent indicators and confocal live cell imaging, we discovered abnormalities in mitochondrial network and dynamics in primary osteocytes derived from the same ALS mouse model G93A. Those mitochondrial defects occur in ALS mice after the onset of neuromuscular symptoms, indicating that mitochondria in bone cells respond to muscle atrophy during ALS disease progression. To examine whether ALS mutation has a direct contribution to mitochondrial dysfunction independent of muscle atrophy, we evaluated mitochondrial morphology and motility in cultured osteocytes (MLO-Y4) with overexpression of mitochondrial targeted SOD1G93A. Compared with osteocytes overexpressing the wild type SOD1 as a control, the SOD1G93A osteocytes showed similar defects in mitochondrial network and dynamic as that of the primary osteocytes derived from the ALS mouse model. In addition, we further discovered that overexpression of SOD1G93A enhanced the expression level of dynamin-related protein 1 (Drp1), a key protein promoting mitochondrial fission activity, and reduced the expression level of optic atrophy protein 1 (OPA1), a key protein related to mitochondrial fusion. A specific mitochondrial fission inhibitor (Mdivi-1) partially reversed the effect of SOD1G93A on mitochondrial network and dynamics, indicating that SOD1G93A likely promotes mitochondrial fission, but suppresses the fusion activity. Our data provide the first evidence that mitochondria show abnormality in osteocytes derived from an ALS mouse model. The accumulation of mutant SOD1G93A protein inside mitochondria directly causes dysfunction in mitochondrial dynamics in cultured MLO-Y4 osteocytes. In addition, the ALS mutation SOD1G93A-mediated dysfunction in mitochondrial dynamics is associated with an enhanced apoptosis in osteocytes, which could be a potential mechanism underlying the bone loss during ALS progression.
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Project description:Increasing evidence indicates an excitatory/inhibitory imbalance may have a critical role in the pathogenesis of amyotrophic lateral sclerosis (ALS). Impaired inhibitory circuitry is consistently reported in the motor cortex of both familial and sporadic patients, closely associated with cortical hyperexcitability and ALS onset. Inhibitory network dysfunction is presumably mediated by intra-cortical inhibitory interneurons, however, the exact cell types responsible are yet to be identified. In this study we demonstrate dynamic changes in the number of calretinin- (CR) and neuropeptide Y-expressing (NPY) interneurons in the motor cortex of the familial hSOD1G93A ALS mouse model, suggesting their potential involvement in motor neuron circuitry defects. We show that the density of NPY-populations is significantly decreased by ~17% at symptom onset (8 weeks), and by end-stage disease (20 weeks) is significantly increased by ~30%. Conversely, the density of CR-populations is progressively reduced during later symptomatic stages (~31%) to end-stage (~36%), while CR-expressing interneurons also show alteration of neurite branching patterns at symptom onset. We conclude that a differential capacity for interneurons exists in the ALS motor cortex, which may not be a static phenomenon, but involves early dynamic changes throughout disease, implicating specific inhibitory circuitry.
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| S-EPMC2744197 | biostudies-literature
Project description:Non-cell autonomous processes involving astrocytes have been shown to contribute to motor neuron degeneration in amyotrophic lateral sclerosis. Mutant superoxide dismutase 1 (SOD1G93A) expression in astrocytes is selectively toxic to motor neurons in co-culture, even when mutant protein is expressed only in astrocytes and not in neurons. To examine metabolic changes in astrocyte-spinal neuron co-cultures, we carried out metabolomic analysis by 1H NMR spectroscopy of media from astrocyte-spinal neuron co-cultures and astrocyte-only cultures. We observed increased glucose uptake with SOD1G93A expression in all co-cultures, but while co-cultures with only SOD1G93A neurons had lower extracellular lactate, those with only SOD1G93A astrocytes exhibited the reverse. Reduced branched-chain amino acid uptake and increased accumulation of 3-methyl-2-oxovalerate were observed in co-culture with only SOD1G93A neurons while glutamate was reduced in all co-cultures expressing SOD1G93A. The shifts in these coupled processes suggest a potential block in glutamate processing that may impact motor neuron survival. We also observed metabolic alterations which may relate to oxidative stress responses. Overall, the different metabolite changes observed with the two SOD1G93A cell types highlight the role of the astrocyte-motor neuron interaction in the resulting metabolic phenotype, requiring further examination of altered met abolic pathways and their impact on motor neuron survival.
| S-EPMC5428359 | biostudies-literature
Project description:Motor neurons become hyperexcitable during progression of amyotrophic lateral sclerosis (ALS). This abnormal firing behavior has been explained by changes in their membrane properties, but more recently it has been suggested that changes in premotor circuits may also contribute to this abnormal activity. The specific circuits that may be altered during development of ALS have not been investigated. Here we examined the Renshaw cell recurrent circuit that exerts inhibitory feedback control on motor neuron firing. Using two markers for Renshaw cells (calbindin and cholinergic nicotinic receptor subunit alpha2 [Chrna2]), two general markers for motor neurons (NeuN and vesicular acethylcholine transporter [VAChT]), and two markers for fast motor neurons (Chondrolectin and calcitonin-related polypeptide alpha [Calca]), we analyzed the survival and connectivity of these cells during disease progression in the Sod1(G93A) mouse model. Most calbindin-immunoreactive (IR) Renshaw cells survive to end stage but downregulate postsynaptic Chrna2 in presymptomatic animals. In motor neurons, some markers are downregulated early (NeuN, VAChT, Chondrolectin) and others at end stage (Calca). Early downregulation of presynaptic VAChT and Chrna2 was correlated with disconnection from Renshaw cells as well as major structural abnormalities of motor axon synapses inside the spinal cord. Renshaw cell synapses on motor neurons underwent more complex changes, including transitional sprouting preferentially over remaining NeuN-IR motor neurons. We conclude that the loss of presynaptic motor axon input on Renshaw cells occurs at early stages of ALS and disconnects the recurrent inhibitory circuit, presumably resulting in diminished control of motor neuron firing. J. Comp. Neurol. 521:1449-1469, 2013. © 2012 Wiley Periodicals, Inc.
| S-EPMC3604165 | biostudies-literature
Project description:Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by death of motor neurons in the brain and spinal cord. However, so far, there is no effective treatment for ALS. Methods: In this study, R13, a prodrug of 7,8-dihydroxyflavone, selectively activating tyrosine kinase receptor B (TrkB) signaling pathway, was administered prophylactically to 40-day old SOD1G93A mice for 90 days. The motor performance was investigated by rotarod test, climbing-pole test, grip strength test and hanging endurance test. Afterwards, the spinal cord and medulla oblongata of 130-day old mice were harvested, and the proteomics revealed the effect of R13 on mouse protein expression profile. Astrocytes and microglial proliferation were assessed by immunohistochemical analysis. The number of motor neurons in the spinal cord is determined by Nissl staining. The effect of R13 on gastrocnemius morphology was assessed by HE staining. The effect of R13 on the survival rate was accomplished with worms stably expressing G93A SOD1. Results: Behavioral tests showed that R13 significantly attenuated abnormal motor performance of SOD1G93A mice. R13 reduced the advance of spinal motor neuron pathology and gastrocnemius muscle atrophy. The proliferation of microglia and astrocytes was reduced by R13 treatment. Mitochondriomics analysis revealed that R13 modified the mitochondrial protein expression profiles in the medulla oblongata and spinal cord of SOD1G93A mice, particularly promoting the expression of proteins related to oxidative phosphorylation (OXPHOS). Further study found that R13 activated AMPK/PGC-1α/Nrf1/Tfam, promoted mitochondrial biogenesis and ameliorated mitochondrial dysfunction. Lastly, R13 prolonged the survival rate of worms stably expressing G93A SOD1. Conclusions: These findings suggest oral R13 treatment slowed the advance of motor system disease in a reliable animal model of ALS, supporting that R13 might be useful for treating ALS.
| S-EPMC8210609 | biostudies-literature