mGLU3 metabotropic glutamate receptors modulate the differentiation of SVZ-derived neural stem cells towards the astrocytic lineage.

Neural stem cells (NSCs) isolated from the subventricular zone (SVZ) of postnatal mice, and cultured as neurospheres, expressed functional mGlu3 receptors. Following mitogen withdrawal and plating onto poly-ornitine-coated dishes, cells dissociated from the neurospheres differentiated into GFAP(+) astrocytes (about 85%), and a small percentage of beta-III tubulin(+)-neurons and O1(+)-oligodendrocytes. Activation of mGlu3 receptors with LY379268 (100 nM, applied every other day), during the differentiation period, impaired astrocyte differentiation, favoring the maintenance in culture of proliferating progenitors co-expressing GFAP with the immature markers, Sox1 and nestin. Co-treatment with the preferential mGlu2/3 receptor antagonist, LY341495 (100 nM), reversed this effect. We examined whether mGlu3 receptors could modulate the canonical signaling pathway activated by bone morphogenic proteins (BMPs), which are known to promote astrocyte differentiation of SVZ/NSCs. An acute challenge of cells isolated from the neurospheres with BMP4 (100 ng/mL) led to phosphorylation and nuclear translocation of the transcription factors, Smads. This effect was largely attenuated by the mGlu2/3 receptor agonist, LY379268. The interaction of mGlu3 and BMP4 receptors was mediated by the activation of the mitogen-activated protein kinase (MAPK) pathway. Accordingly, LY379268 failed to affect BMP receptor signaling when combined with the MAPK kinase inhibitor, UO-126 (30 muM). These data raise the intriguing possibility that glutamate regulates differentiation of SVZ/NSCs by activating mGlu3 receptors.

Type-3 metabotropic glutamate receptors negatively modulate bone morphogenetic protein receptor signaling and support the tumourigenic potential of glioma-initiating cells.

Targeted-therapies enhancing differentiation of glioma-initiating cells (GICs) are potential innovative approaches to the treatment of malignant gliomas. These cells support tumour growth and recurrence and are resistant to radiotherapy and chemotherapy. We have found that GICs express mGlu3 metabotropic glutamate receptors. Activation of these receptors sustained the undifferentiated state of GICs in culture by negatively modulating the action of bone morphogenetic proteins, which physiologically signal through the phosphorylation of the transcription factors, Smads. The cross-talk between mGlu3 receptors and BMP receptors was mediated by the activation of the mitogen-activated protein kinase pathway. Remarkably, pharmacological blockade of mGlu3 receptors stimulated the differentiation of cultured GICs into astrocytes, an effect that appeared to be long lasting, independent of the growth conditions, and irreversible. In in vivo experiments, a 3-month treatment with the brain-permeant mGlu receptor antagonist, LY341495 limited the growth of infiltrating brain tumours originating from GICs implanted into the brain parenchyma of nude mice. While clusters of tumour cells were consistently found in the brain of control mice, they were virtually absent in a large proportion of mice treated with LY341495. These findings pave the way to a new non-cytotoxic treatment of malignant gliomas based on the use of mGlu3 receptor antagonists.

Metabotropic glutamate receptors regulate differentiation of embryonic stem cells into GABAergic neurons.

Mouse embryonic stem (ES) cells were stimulated to differentiate either as adherent monolayer cultures in DMEM/F12 supplemented with N2/B27, or as floating embryoid bodies (EBs) exposed to 1 microM retinoic acid (RA) for 4 days, starting from 4 DIV, and subsequently re-plated in DMEM/F12 medium. Adherent monolayer cultures of ES cells expressed mGlu5 receptors throughout the entire differentiation period. Selective pharmacological blockade of mGlu5 receptors with methyl-6-(phenylethynyl)-pyridine (MPEP) (1 microM, added once a day) accelerated the appearance of the neuronal marker, beta-tubulin. In addition, treatment with MPEP increased the number of cells expressing glutamate decarboxylase-65/67 (GAD(65/67)), a marker of GABAergic neurons. In floating EBs, mGlu5 receptors are progressively replaced by mGlu4 receptors. The orthosteric mGlu4/6/7/8 receptor agonist, L-2-amino-4-phosphonobutanoate (L-AP4), or the selective mGlu4 receptor enhancer, PHCCC,--both combined with RA at concentrations of 30 microM--increased the expression of both beta-tubulin and GAD(65/67), inducing the appearance of fully differentiated neurons that released GABA in response to membrane depolarization. We conclude that mGlu receptor subtypes regulate neuronal differentiation of ES cells in a context-dependent manner, and that subtype-selective ligands of these receptors might be used for the optimization of in vitro protocols aimed at producing GABAergic neurons from ES cells.