Syddansk Universitet Activation of Group II Metabotropic Glutamate Receptors Increases Proliferation but does not Influence Neuronal Differentiation of a Human Neural Stem Cell Line

The multiple functions of glutamate include regulation of neural development and stem cells. While the importance of the ionotropic glutamate receptors is well established, less is known about the role of metabotropic glutamate receptors (mGluRs). A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. In this study, we examined the effects of pharmacological activation and inhibition of mGluR2/3 on proliferation, differentiation and viability of a human neural stem cell line. Immunofluorescence staining revealed the presence of mGluR2/3 receptors on both proliferating and differentiating stem cells, including cells differentiated into β-tubulin III-positive immature neurons and glial fibrillary acidic protein-positive astrocytes. Stimulation of mGluR2/3 receptors during cell propagation using the agonist (2S,2’R,3’R)-2-(2’,3’-dicarboxycyclopropyl) glycine (DCG-IV) increased total cell numbers significantly (60% compared to untreated controls). This effect could be inhibited by the specific antagonist (2S)-2-Amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495). The antagonist alone had no effect. No significant decrease in cell death was found following mGluR2/3 stimulation, suggesting that the observed elevation in cell number was not related to cell viability. Subsequent differentiation of the cells resulted in a slight decrease in beta-tubulin III-positive neurons (5.2% to 3.2% of total cells) for DCG-IV pre-treated cultures. Treatment with DCG-IV and LY342495 during cell differentiation alone had no such effect. Western blot analysis revealed that the active, dimeric form of mGluR2/3 was mainly present on the proliferating cells, which may explain our findings. The present study emphasises the importance of glutamate and mGluRs on regulation of human neural stem cells and suggests a significant role of mGluR2/3 during cell proliferation. Glutamate is the major excitatory transmitter in the central nervous system (1,2) exerting its effect by acting on ionotropic (iGluRs) and metabotropic glutamate receptors (mGluRs) (3). MGluRs are divided into three groups based on sequence homology, agonist pharmacology and signal transduction pathways. Group I receptors (mGluR1,5) are positively coupled to phospholipase C, A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. whereas group II (mGluR2,3) and group III (mGluR4,6,7,8) receptors are negatively coupled to the adenylate cyclase (4–6). Several studies suggest that mGluRs play a significant role in the regulation of neural development. MGluRs are expressed on neuronal precursor cells during embryonic development in both rodents and humans (7–10). Activation of mGluR3 and mGluR5 promote proliferation and survival of neural stem cells (NSCs) derived from mouse embryonic forebrain (11). NSCs derived from the developing human forebrain express group I mGluRs and respond to low-dose glutamate treatment with increased proliferation and decreased cell death (12). In addition, mGluRs appear to affect postnatal neurogenesis. In the adult murine brain, mGluRs can be found in the neurogenic zones (13–15), and in vitro studies of NSCs derived from these zones have revealed that receptor manipulation influence proliferation, survival and differentiation of NSCs (11,13,14,16). The group II mGluRs are mainly present on presynaptic membranes on both glutamatergic and GABAergic neurons as well as on glial cells (17). Varying results have been reported regarding the expression of group II mGluRs during development. In one study, neural precursors from mice have been shown to express mGluR2 mRNA (18), whereas in other studies it could not be detected (11,14). More is known about mGluR3, which is expressed by NSCs in the developing and adult rodent forebrain, where it promotes survival and proliferation (11,13,16). However, little is known about the presence and functional importance of group II mGluRs on human NSCs. In one study, adult human glial precursor cells were shown to express mGluR3 and mGluR5a (19). In the present study, we investigated the expression of group II metabotropic glutamate receptors (mGluR2/3) on human forebrain-derived NSCs and whether pharmacological activation of these receptors would influence cell proliferation and differentiation.