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Altered Metabolic Profiles Associate with Toxicity in SOD1G93A Astrocyte-Neuron Co-Cultures.

ABSTRACT: 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.

SUBMITTER: Valbuena GN

PROVIDER: S-EPMC5428359 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Altered Metabolic Profiles Associate with Toxicity in SOD1G93A Astrocyte-Neuron Co-Cultures.

Valbuena Gabriel N GN Tortarolo Massimo M Bendotti Caterina C Cantoni Lavinia L Keun Hector C HC

Scientific reports 20170303 1

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 f ...[more]

PMID: 28246392

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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.

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.

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Altered Metabolic Profiles Associate with Toxicity in SOD1G93A Astrocyte-Neuron Co-Cultures.

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Altered Metabolic Profiles Associate with Toxicity in SOD1<sup>G93A</sup> Astrocyte-Neuron Co-Cultures.

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