Selective activation of metabotropic glutamate receptor 7 induces inhibition of cellular proliferation and promotes astrocyte differentiation of ventral mesencephalon human neural stem/progenitor cells

Abstract

Expression of group III metabotropic glutamate receptors (mGluR) was established by RT-PCR and immunocytochemistry on a cultured clonal human neural stem/progenitor cell (hNSPC) line derived from fetal ventral mesencephalon (VM). Selective activation of these receptors by the group III mGluR agonist L-(+)-2-amino-4-phosphonobutyric acid (L-AP4) prevented increases in cAMP levels following forskolin stimulation, suggesting these receptors are coupled to their canonical G-protein coupled signal transduction pathway. Tonic exposure of undifferentiated cultures to L-AP4 resulted in a decrease in cellular metabolism and proliferation in the absence of toxicity, as measured by MTT and LDH assays, in a dose-dependent manner. This was confirmed by a reduction in BrdU incorporation into nuclear DNA, suggestive of an anti-proliferative effect of L-AP4. This effect was rescued by co-addition of the broad-spectrum group III mGluR competitive antagonist (RS)-a-cyclopropyl-4-phosphonophenylglycine (CPPG), demonstrating a receptor-mediated mechanism, but not mimicked by application of the cell permeable cAMP analogue dibutyrl cAMP (db-cAMP). The potency of these effects of L-AP4 indicates that this is an mGlu7 subtype-mediated effect. Tonic exposure of undifferentiated cultures to the mGlu7 selective allosteric agonist N,N'-bis(diphenylmethyl)-1,2-ethanediamine dihydrochloride (AMN082), but not the mGlu4 selective allosteric agonist (±)-cis-2-(3,5-dicholorphenylcarbamoyl)cyclohexanecarboxylic acid (VU0155041), or the mGlu8 selective agonist (S)-3,4-dicarboxyphenylglycine ((S)-3,4-DCPG) resulted in an identical anti-proliferative effect to L-AP4, confirming the involvement of the mGlu7 subtype. In differentiating cultures, tonic exposure to L-AP4 or AMN082 resulted in a significant shift towards an astrocyte cell fate. The mGlu7 receptor therefore provides a new opportunity to influence the proliferation and differentiation of ventral mesencephalon-derived hNSPC.