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Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning
Published on: February 17, 2016
Glial metabotropic glutamate receptor-4 increases maturation and survival of oligodendrocytes
Simona Federica Spampinato1, Sara Merlo1, Mariangela Chisari1
1Department of Biomedical and Biotechnological Sciences, Section of Pharmacology, University of Catania Catania, Italy.
Abstract:
Group III metabotropic glutamate (mGlu) receptors mediate important neuroprotective and anti-inflammatory effects. Stimulation of mGlu4 receptor reduces neuroinflammation in a mouse model of experimental autoimmune encephalomyelitis (EAE) whereas mGlu4 knockout mice display exacerbated EAE clinical scores. We now show that mGlu4 receptors are expressed in oligodendrocytes, astrocytes and microglia in culture. Oligodendrocytes express mGlu4 receptors only at early stages of maturation (O4 positive), but not when more differentiated (myelin basic protein, MBP positive). Treatment of immature oligodendrocytes with the mGlu4 receptor agonist L-2-Amino-4-phosphonobutyrate (L-AP4; 50 μM for 48 h) accelerates differentiation with enhanced branching and earlier appearance of MBP staining. Oligodendrocyte death induced by exposure to 1 mM kainic acid for 24 h is significantly reduced by a 30-min pretreatment with L-AP4 (50 μM), an effect observed only in the presence of astrocytes, mimicked by the specific mGlu4 receptor positive allosteric modulator N-Phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) (30 μM) and prevented by pretreatment with the mGlu4 receptor antagonist, cyclopropyl-4-phosphonophenylglycine (CPPG) (100 μM). In astrocytes, mGlu4 receptor is the most expressed among group III mGlu receptors, as by Quantitative real time PCR (QRT-PCR), and its silencing prevents protective effects. Protection is also observed when conditioned medium (CM) from L-AP4-pretreated astrocytes is transferred to oligodendrocytes challenged with kainic acid. Transforming growth factor β (TGF-β) mediates the increased oligodendrocyte survival as the effect of L-AP4 is mimicked by addition of 10 ng/ml TGF-β and prevented by incubation with a neutralizing anti-TGF-β antibody. In contrast, despite the expression of mGlu4 receptor in resting and activated microglia, CM from L-AP4-stimulated microglia does not modify kainate-induced oligodendrocyte toxicity. Our results suggest that mGlu4 receptors expressed in astrocytes mediate enhanced survival of oligodendrocytes under conditions of excitotoxicity.
Insights
Group III metabotropic glutamate 4 (mGlu4) receptors protect oligodendrocytes from death. Astrocytes mediate this protection via transforming growth factor beta (TGF-β), highlighting mGlu4
Area of Science:
- Neuroscience
- Cell Biology
- Neuroinflammation
Background:
- Group III metabotropic glutamate (mGlu) receptors are known for neuroprotective and anti-inflammatory roles.
- mGlu4 receptor stimulation reduces neuroinflammation in experimental autoimmune encephalomyelitis (EAE) models.
- mGlu4 receptor knockout mice exhibit worsened EAE.
- mGlu4 receptors are present in oligodendrocytes, astrocytes, and microglia.
Purpose of the Study:
- To investigate the role of mGlu4 receptors in oligodendrocyte survival and differentiation.
- To determine the cellular mechanisms underlying mGlu4 receptor-mediated neuroprotection.
Main Methods:
- Quantitative real-time PCR (QRT-PCR) to assess mGlu4 receptor expression in astrocytes.
- Treatment of primary oligodendrocyte cultures with mGlu4 receptor agonist (L-AP4) and positive allosteric modulator (PHCCC).
- Induction of oligodendrocyte death using kainic acid and assessment of protection with L-AP4 and antagonist (CPPG).
- Analysis of conditioned medium from treated astrocytes and microglia transferred to oligodendrocytes.
- Investigating the role of transforming growth factor beta (TGF-β) in mediating protective effects.
Main Results:
- mGlu4 receptors are expressed in immature (O4 positive) but not mature (MBP positive) oligodendrocytes.
- L-AP4 treatment accelerates oligodendrocyte differentiation and enhances myelin basic protein (MBP) expression.
- L-AP4 pretreatment significantly reduces kainic acid-induced oligodendrocyte death, but only in the presence of astrocytes.
- Astrocytes mediate protection through TGF-β, as shown by mimicking effects with TGF-β and blocking with anti-TGF-β antibodies.
- Conditioned medium from L-AP4-stimulated astrocytes protects oligodendrocytes, while that from microglia does not.
- mGlu4 receptor silencing in astrocytes abolishes the protective effect.
Conclusions:
- mGlu4 receptors on astrocytes play a crucial role in protecting oligodendrocytes from excitotoxicity.
- Astrocyte-mediated protection involves the release of TGF-β.
- Targeting mGlu4 receptors in astrocytes may offer a therapeutic strategy for demyelinating diseases.
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