A Phenotypic Small-Molecule Screen Identifies an Orphan Ligand-Receptor Pair that Regulates Neural Stem Cell Differentiation

Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
Chemistry & Biology (Impact Factor: 6.65). 10/2007; 14(9):1019-30. DOI: 10.1016/j.chembiol.2007.07.016
Source: PubMed


High-throughput identification of small molecules that selectively modulate molecular, cellular, or systems-level properties of the mammalian brain is a significant challenge. Here we report the chemical genetic identification of the orphan ligand phosphoserine (P-Ser) as an enhancer of neurogenesis. P-Ser inhibits neural stem cell/progenitor proliferation and self-renewal, enhances neurogenic fate commitment, and improves neuronal survival. We further demonstrate that the effects of P-Ser are mediated by the group III metabotropic glutamate receptor 4 (mGluR4). siRNA-mediated knockdown of mGluR4 abolished the effects of P-Ser and increased neurosphere proliferation, at least in part through upregulation of mTOR pathway activity. We also found that P-Ser increases neurogenesis in human embryonic stem cell-derived neural progenitors. This work highlights the tremendous potential of developing effective small-molecule drugs for use in regenerative medicine or transplantation therapy.

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    • "However, drug-screening assays do not require that each neurosphere derived from a single stem or progenitor cell. A low plating cell density of 5000 cells/mL or 1000 cells/mL is sufficient to measure the self-renewal of neural stem cells [33, 34]. "
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    ABSTRACT: Adult neural stem cells (NSCs) persist throughout life to replace mature cells that are lost during turnover, disease, or injury. The investigation of NSC creates novel treatments for central nervous system (CNS) injuries and neurodegenerative disorders. The plasticity and reparative potential of NSC are regulated by different factors, which are critical for neurological regenerative medicine research. We investigated the effects of Psoralen, which is the mature fruit of Psoralea corylifolia L., on NSC behaviors and the underlying mechanisms. The self-renewal and proliferation of NSC were examined. We detected neuron- and/or astrocyte-specific markers using immunofluorescence and Western blotting, which could evaluate NSC differentiation. Psoralen treatment significantly inhibited neurosphere formation in a dose-dependent manner. Psoralen treatment increased the expression of the astrocyte-specific marker but decreased neuron-specific marker expression. These results suggested that Psoralen was a differentiation inducer in astrocyte. Differential gene expression following Psoralen treatment was screened using DNA microarray and confirmed by quantitative real-time PCR. Our microarray study demonstrated that Psoralen could effectively regulate the specific gene expression profile of NSC. The genes involved in the classification of cellular differentiation, proliferation, and metabolism, the transcription factors belonging to Ets family, and the hedgehog pathway may be closely related to the regulation.
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    • "protein-coupled receptor (GPCR) family are divided into three major subtypes: group I (mGluR1 and mGluR 5), group II (mGluR2 and mGluR 3) and group III (mGluR4, mGluR6, mGluR7 and mGluR8) based on their amino acid sequence homology, signal transduction pathway and pharmacology (Conn and Pin, 1997; Schoepp et al., 1999). Specifically, of the eight known mGluR subtypes, expression of group I mGluR (mGluR1 and 5), group II mGluR (mGluR3) and group III mGluR (mGluR4 and 8) have been reported in cultured NSPCs from various brain regions (Canudas et al., 2004; Di Giorgi Gerevini et al., 2004, 2005; Suzuki et al., 2006; Saxe et al., 2007; Gandhi et al., 2008; Nakamichi et al., 2008). Of these, selective activation of mGluR3 and 5 supports proliferation and survival of NSPCs isolated from the subventricular zone (SVZ) of embryonic mouse forebrain (Di Giorgi Gerevini et al., 2004, 2005) and the neocortex (Gandhi et al., 2008), whilst a similar role has also recently been proposed for group II mGluR (Brazel et al., 2005). "
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    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.
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    • "As a result, several steroids that promoted hES cell differentiation were identified, and the antihypertensive drug pinacidil was shown to affect hES cell survival. Phenotypic screens can be advantageous because they can be carried out in cells by examining multiple markers and functional changes (for example, cell morphology and behavior) using automated high-content imaging technologies in a high-throughput manner 1, 4, 36, 37. "
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    ABSTRACT: Embryonic stem (ES) cells and their differentiated progeny offer tremendous potential for regenerative medicine, even in the field of drug discovery. There is an urgent need for clinically relevant assays that make use of ES cells because of their rich biological utility. Attention has been focused on small molecules that allow the precise manipulation of cells in vitro, which could allow researchers to obtain homogeneous cell types for cell-based therapies and discover drugs for stimulating the regeneration of endogenous cells. Such therapeutics can act on target cells or their niches in vivo to promote cell survival, proliferation, differentiation, and homing. In the present paper, we reviewed the use of ES cell models for high-throughput/content drug screening and toxicity assessment. In addition, we examined the role of stem cells in large pharmaceutical companies' R&D and discussed a novel subject, nicheology, in stem cell-related research fields.
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