GABAB receptors role in cell migration and positioning within the ventromedial nucleus of the hypothalamus.

The ventromedial (VMN) and arcuate (ARC) nuclei of the hypothalamus are bilateral nuclear groups at the base of the hypothalamus that are organized through the aggregation of neurons born along the third ventricle that migrate laterally. During development, GABAergic neurons and fibers surround the forming (or primordial) VMN while neurons containing GABA receptors are found within the boundaries of the emerging nucleus. To investigate the role that GABAB receptors play in establishing the VMN, Thy-1 yellow fluorescent protein (YFP) mice were utilized for live video microscopy studies. The Thy-1 promoter drives YFP expression in regions of the hypothalamus during development. Administration of the GABAB receptor antagonist saclofen and the GABAA receptor antagonist bicuculline selectively increased the rate of VMN cell movement in slices placed in vitro at embryonic day 14, when cells that form both the ARC and VMN are migrating away from the proliferative zone surrounding the third ventricle. To further test the role of GABAB receptors in VMN development, GABAB receptor knockout mice were used to examine changes in the positions of phenotypically identified cells within the VMN. Cells containing immunoreactive estrogen receptors (ER) alpha were located in the ventrolateral quadrant of the wild type VMN. In GABABR1 knockout mice, these ERalpha positive neurons were located in more dorsal positions at postnatal day (P) 0 and P4. We conclude that GABA alters cell migration and its effect on final cell positioning may lead to changes in the circuitry and connections within specific nuclei of the developing hypothalamus.

Edg8/S1P5: an oligodendroglial receptor with dual function on process retraction and cell survival.

Endothelial differentiation gene (Edg) proteins are G-protein-coupled receptors activated by lysophospholipid mediators: sphingosine-1-phosphate (S1P) or lysophosphatidic acid. We show that in the CNS, expression of Edg8/S1P5, a high-affinity S1P receptor, is restricted to oligodendrocytes and expressed throughout development from the immature stages to the mature myelin-forming cell. S1P activation of Edg8/S1P5 on O4-positive pre-oligodendrocytes induced process retraction via a Rho kinase/collapsin response-mediated protein signaling pathway, whereas no retraction was elicited by S1P on these cells derived from Edg8/S1P5-deficient mice. Edg8/S1P5-mediated process retraction was restricted to immature cells and was no longer observed at later developmental stages. In contrast, S1P activation promoted the survival of mature oligodendrocytes but not of pre-oligodendrocytes. The S1P-induced survival of mature oligodendrocytes was mediated through a pertussis toxin-sensitive, Akt-dependent pathway. Our data demonstrate that Edg8/S1P5 activation on oligodendroglial cells modulates two distinct functional pathways mediating either process retraction or cell survival and that these effects depend on the developmental stage of the cell.

Modulation of hippocampal excitability by 5-HT4 receptor agonists persists in a transgenic model of Alzheimer's disease.

5-HT(4) receptors are widely distributed in both peripheral and central nervous systems where they couple, via a G-protein, to the activation of adenylate cyclase. In the brain, the highest 5-HT(4) receptor densities are found in the limbic system, including the hippocampus and frontal cortex. It has been suggested that activation of these receptors may be of therapeutic benefit in diseases that produce cognitive deficits such as Alzheimer's disease (AD). Previous electrophysiological studies have shown that the 5-HT(4) agonist, Zacopride, can increase population spike amplitude recorded in region CA1 of rat hippocampal slices in a cyclic AMP (cAMP)/cAMP-dependent protein kinase A-dependent manner. We report here that the 5-HT(4) agonist, Prucalopride, and the 5-HT(4) partial agonist, SL65.0155, produce a similar effect in rat hippocampal slices and that the specific 5-HT(4) antagonist, GR113808, blocks these effects. To investigate the potential use of 5-HT(4) agonists in the treatment of AD, Prucalopride was applied to hippocampal slices from a transgenic mouse line that overexpresses the Abeta peptide. Despite the deficit in synaptic transmission present in these mice, the percentage increase of the CA1 population spike induced by Prucalopride was the same as that observed in wild-type mice. These data support 5-HT(4) receptors as a target for cognitive enhancement and suggest that a partial agonist would be sufficient to produce benefits, while reducing potential peripheral side effects. In addition, we show that 5-HT(4) receptors remain functional in the presence of excess Abeta peptide and may therefore be a useful target in AD.

Distribution of a GABAB-like receptor protein in the rat central nervous system.

Using a homology-based bioinformatics approach we have identified the human and rodent orthologues of a novel putative seven transmembrane G protein coupled receptor, termed GABA(BL). The amino acid sequence homology of these cDNAs compared to GABA(B1) and GABA(B2) led us to postulate that GABA(BL) may be a putative novel GABA(B) receptor subunit. We have developed a rabbit polyclonal antisera specific to the GABA(BL) protein and assessed the distribution of GABA(BL) in the rat CNS by immunohistochemistry. Protein expression was particularly dense in regions previously shown to contain known GABA(B) receptor subunits. Dense immunoreactivity was observed in the cortex, major subfields of the hippocampus and the dentate gyrus. GABA(BL) labelling was very conspicuous in the cerebellum, both in the granule cell layer and in Purkinje cells, and was also observed in the substantia gelatinosa and ventral horn motor neurons of the spinal cord. GABA(BL) immunoreactivity was also noted in a subset of parvalbumin positive hippocampal interneurons. Our data suggest a widespread distribution of GABA(BL) throughout the rat CNS.

Molecular cloning and characterisation of a novel GABAB-related G-protein coupled receptor.

Using a homology-based bioinformatics approach we have analysed human genomic sequence and identified the human and rodent orthologues of a novel putative seven transmembrane G protein coupled receptor, termed GABA(BL). The amino acid sequence homology of these cDNAs compared to GABA(B1) and GABA(B2) led us to postulate that GABA(BL) was a putative novel GABA(B) receptor subunit. The C-terminal sequence of GABA(BL) contained a putative coiled-coil domain, di-leucine and several RXR(R) ER retention motifs, all of which have been shown to be critical in GABA(B) receptor subunit function. In addition, the distribution of GABA(BL) in the central nervous system was reminiscent of that of the other known GABA(B) subunits. However, we were unable to detect receptor function in response to any GABA(B) ligands when GABA(BL) was expressed in isolation or in the presence of either GABA(B1) or GABA(B2). Therefore, if GABA(BL) is indeed a GABA(B) receptor subunit, its partner is a potentially novel receptor subunit or chaperone protein which has yet to be identified.

Cyclic AMP-dependent protein kinase phosphorylation facilitates GABA(B) receptor-effector coupling.

GABA (gamma-aminobutyric acid)(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Here we show that the functional coupling of GABA(B)R1/GABA(B)R2 receptors to inwardly rectifying K(+) channels rapidly desensitizes. This effect is alleviated after direct phosphorylation of a single serine residue (Ser892) in the cytoplasmic tail of GABA(B)R2 by cyclic AMP (cAMP)-dependent protein kinase (PKA). Basal phosphorylation of this residue is evident in rat brain membranes and in cultured neurons. Phosphorylation of Ser892 is modulated positively by pathways that elevate cAMP concentration, such as those involving forskolin and beta-adrenergic receptors. GABA(B) receptor agonists reduce receptor phosphorylation, which is consistent with PKA functioning in the control of GABA(B)-activated currents. Mechanistically, phosphorylation of Ser892 specifically enhances the membrane stability of GABA(B) receptors. We conclude that signaling pathways that activate PKA may have profound effects on GABA(B) receptor-mediated synaptic inhibition. These results also challenge the accepted view that phosphorylation is a universal negative modulator of G protein-coupled receptors.

GABA(B) receptors: from monogamy to promiscuity.

The aim of this review is firstly to describe the current understanding of the diverse physiology and pharmacology of GABA(B) receptors in vivo. We will then focus on recent advances made, since the identification of the GABA(B) receptor subunit genes, in our knowledge of the molecular nature of the receptor, and the recently discovered molecular determinants of functions such as ligand binding, trafficking and signalling. We will conclude with a summary of the GABA(B) receptor-interacting proteins that have been described thus far, and discuss how these may, at least in part, account for the paradox of varied receptor pharmacology in the potential context of a single heterodimeric GABA(B) receptor.

Comparative immunohistochemical localisation of GABA(B1a), GABA(B1b) and GABA(B2) subunits in rat brain, spinal cord and dorsal root ganglion.

GABA(B) receptors are G-protein-coupled receptors mediating the slow onset and prolonged synaptic actions of GABA in the CNS. The recent cloning of two genes, GABA(B1) and GABA(B2), has revealed a novel requirement for GABA(B) receptor signalling. Studies have demonstrated that the two receptor subunits associate as a GABA(B1)/GABA(B2) heterodimer to form a functional GABA(B) receptor. In this study we have developed polyclonal antisera specific to two splice variants of the GABA(B1) subunit, GABA(B1a) and GABA(B1b), as well as an antiserum to the GABA(B2) subunit. Using affinity-purified antibodies derived from these antisera we have mapped out the distribution profile of each subunit in rat brain, spinal cord and dorsal root ganglion. In brain the highest areas of GABA(B1a), GABA(B1b) and GABA(B2) subunit expression were found in neocortex, hippocampus, thalamus, cerebellum and habenula. In spinal cord, GABA(B1) and GABA(B2) subunits were expressed in the superficial layers of the dorsal horn, as well as in motor neurones in the deeper layers of the ventral horn. GABA(B) receptor subunit immunoreactivity in dorsal root ganglion suggested that expression of GABA(B1b) was restricted to the large diameter neurones, in contrast to GABA(B1a) and GABA(B2) subunits which were expressed in both large and small diameter neurones. Although expression levels of GABA(B1) and GABA(B2) subunits varied we found no areas in which GABA(B1) was expressed in the absence of GABA(B2). This suggests that most, if not all, GABA(B1) immunoreactivity may represent functional GABA(B) receptors. Although our data are in general agreement with functional studies, some discrepancies in GABA(B1) subunit expression occurred with respect to other immunohistochemical studies. Overall our data suggest that GABA(B) receptors are widely expressed throughout the brain and spinal cord, and that GABA(B1a) and GABA(B1b) subunits can associate with GABA(B2) to form both pre- and post-synaptic receptors.

GABA(B2) is essential for g-protein coupling of the GABA(B) receptor heterodimer.

GABA(B) receptors are unique among G-protein-coupled receptors (GPCRs) in their requirement for heterodimerization between two homologous subunits, GABA(B1) and GABA(B2), for functional expression. Whereas GABA(B1) is capable of binding receptor agonists and antagonists, the role of each GABA(B) subunit in receptor signaling is unknown. Here we identified amino acid residues within the second intracellular domain of GABA(B2) that are critical for the coupling of GABA(B) receptor heterodimers to their downstream effector systems. Our results provide strong evidence for a functional role of the GABA(B2) subunit in G-protein coupling of the GABA(B) receptor heterodimer. In addition, they provide evidence for a novel "sequential" GPCR signaling mechanism in which ligand binding to one heterodimer subunit can induce signal transduction through the second partner of a heteromeric complex.

Control of progenitor cell number by mitogen supply and demand.

BACKGROUND: Much is known about how cell proliferation is controlled at the single cell level, but much less about the control of cell numbers in developing populations. Cell number might be determined by an intracellular division limiter or, alternatively, by the availability of mitogens or other factors outside the cell. We investigated the relative importance of intracellular and extracellular controls for one well-defined population of neural precursor cells, namely the glial progenitors that give rise to oligodendrocytes in the mouse spinal cord. RESULTS: We found by cumulative BrdU labeling in vivo that the progenitor cell division cycle slows down markedly as their numbers increase during embryogenesis. When cultured in saturating PDGF, the main mitogen for these cells, their cell cycle accelerated and was independent of their prior rate of division in vivo. This shows that mitogens are limiting in vivo, and suggests that division normally slows down because the PDGF concentration declines. In PDGF-transgenic mice, cell number was proportional to the PDGF supply and apparently unsaturable; at ten times the normal rate of supply, cell number was still increasing but the animals were no longer viable. CONCLUSIONS: Progenitor cell proliferation in the embryo is limited by environmental factors, not a cell-intrinsic mechanism. The linear relationship between PDGF supply and final cell number strongly suggests that cells deplete the mitogenic activity in their environment at a rate proportional to the total number of cells. The cells might simply consume the available PDGF or they might secrete autocrine inhibitors, or both.

Association of GABA(B) receptors and members of the 14-3-3 family of signaling proteins.

Two GABA(B) receptors, GABA(B)R1 and GABA(B)R2, have been cloned recently. Unlike other G protein-coupled receptors, the formation of a heterodimer between GABA(B)R1 and GABA(B)R2 is required for functional expression. We have used the yeast two hybrid system to identify proteins that interact with the C-terminus of GABA(B)R1. We report a direct association between GABA(B) receptors and two members of the 14-3-3 protein family, 14-3-3eta and 14-3-3zeta. We demonstrate that the C-terminus of GABA(B)R1 associates with 14-3-3zeta in rat brain preparations and tissue cultured cells, that they codistribute after rat brain fractionation, colocalize in neurons, and that the binding site overlaps partially with the coiled-coil domain of GABA(B)R1. Furthermore we show a reduced interaction between the C-terminal domains of GABA(B)R1 and GABA(B)R2 in the presence of 14-3-3. The results strongly suggest that GABA(B)R1 and 14-3-3 associate in the nervous system and begin to reveal the signaling complexities of the GABA(B)R1/GABA(B)R2 receptor heterodimer.

The C-terminal domains of the GABA(b) receptor subunits mediate intracellular trafficking but are not required for receptor signaling.

GABA(B) receptors are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. These receptors are heterodimers assembled from GABA(B1) and GABA(B2) subunits, neither of which is capable of producing functional GABA(B) receptors on homomeric expression. GABA(B1,) although able to bind GABA, is retained within the endoplasmic reticulum (ER) when expressed alone. In contrast, GABA(B2) is able to access the cell surface when expressed alone but does not couple efficiently to the appropriate effector systems or produce any detectable GABA-binding sites. In the present study, we have constructed chimeric and truncated GABA(B1) and GABA(B2) subunits to explore further GABA(B) receptor signaling and assembly. Removal of the entire C-terminal intracellular domain of GABA(B1) results in plasma membrane expression without the production of a functional GABA(B) receptor. However, coexpression of this truncated GABA(B1) subunit with either GABA(B2) or a truncated GABA(B2) subunit in which the C terminal has also been removed is capable of functional signaling via G-proteins. In contrast, transferring the entire C-terminal tail of GABA(B1) to GABA(B2) leads to the ER retention of the GABA(B2) subunit when expressed alone. These results indicate that the C terminal of GABA(B1) mediates the ER retention of this protein and that neither of the C-terminal tails of GABA(B1) or GABA(B2) is an absolute requirement for functional coupling of heteromeric receptors. Furthermore although GABA(B1) is capable of producing GABA-binding sites, GABA(B2) is of central importance in the functional coupling of heteromeric GABA(B) receptors to G-proteins and the subsequent activation of effector systems.

Platelet-derived growth factor is constitutively secreted from neuronal cell bodies but not from axons.

Neurons synthesise and secrete many growth and survival factors but it is not usually clear whether they are released locally at the cell body or further afield from axons or axon terminals. Without this information, we cannot predict the site(s) of action or the biological functions of many neuron-derived factors. For example, can neuronal platelet-derived growth factor (PDGF) be secreted from axons and reach glial cells in nerve-fibre (white-matter) tracts? To address this question, we expressed PDGF-A in retinal ganglion neurons in transgenic mice and tested for release of PDGF from cell bodies in the retina and from axons in the optic nerve. In both the retina and optic nerve, there are glial cells that express PDGF receptor alpha (PDGFR alpha) [1] and divide in response to PDGF [2-5], so we could detect functional PDGF indirectly through the mitogenic response of glia at both locations. Expressing PDGF-A in neurons under the control of the neuron-specific enolase promoter (NSE-PDGF-A) resulted in a striking hyperplasia of retinal astrocytes, demonstrating that PDGF is secreted from the cell bodies of neurons in the retina [4]. In contrast, glial proliferation in the optic nerve was unaffected, indicating that PDGF is not released from axons. When PDGF was expressed directly in the optic nerve under the control of an astrocyte-specific promoter (GFAP-PDGF-A), oligodendrocyte progenitors hyperproliferated, resulting in a hypertrophic optic nerve. We conclude that PDGF is constitutively secreted from neuronal cell bodies in vivo, but not from axons in white-matter tracts.

The expression of GABA(B1) and GABA(B2) receptor subunits in the cNS differs from that in peripheral tissues.

GABA(B) receptors are G-protein-coupled receptors that mediate the slow and prolonged synaptic actions of GABA in the CNS via the modulation of ion channels. Unusually, GABA(B) receptors form functional heterodimers composed of GABA(B1) and GABA(B2) subunits. The GABA(B1) subunit is essential for ligand binding, whereas the GABA(B2) subunit is essential for functional expression of the receptor dimer at the cell surface. We have used real-time reverse transcriptase-polymerase chain reaction to analyse expression levels of these subunits, and their associated splice variants, in the CNS and peripheral tissues of human and rat. GABA(B1) subunit splice variants were expressed throughout the CNS and peripheral tissues, whereas surprisingly GABA(B2) subunit splice variants were neural specific. Using novel antisera specific to individual GABA(B) receptor subunits, we have confirmed these findings at the protein level. Analysis by immunoblotting demonstrated the presence of the GABA(B1) subunit, but not the GABA(B2) subunit, in uterus and spleen. Furthermore, we have shown the first immunocytochemical analysis of the GABA(B2) subunit in the brain and spinal cord using a GABA(B2)-specific antibody. We have, therefore, identified areas of non-overlap between GABA(B1) and GABA(B2) subunit expression in tissues known to contain functional GABA(B) receptors. Such areas are of interest as they may well contain novel GABA(B) receptor subunit isoforms, expression of which would enable the GABA(B1) subunit to reach the cell surface and form functional GABA(B) receptors.

Molecular cloning and characterization of two novel retinoic acid-inducible orphan G-protein-coupled receptors (GPRC5B and GPRC5C).

Using homology searching of public databases with a metabotropic glutamate receptor sequence from Caenorhabditis elegans, two novel protein sequences (named RAIG-2 (HGMW-approved symbol GPRC5B) and RAIG-3 (HGMW-approved symbol GPRC5C) were identified containing seven putative transmembrane domains characteristic of G-protein-coupled receptors (GPCRs). RAIG-2 and RAIG-3 encode open reading frames of 403 and 442 amino acid polypeptides, respectively, and show 58% similarity to the recently identified retinoic acid-inducible gene-1 (RAIG-1, HGMW-approved symbol RAI3). Analysis of the three protein sequences places them within the type 3 GPCR family, which includes metabotropic glutamate receptors, GABA(B) receptors, calcium-sensing receptors, and pheromone receptors. However, in contrast to other type 3 GPCRs, RAIG-1, RAIG-2, and RAIG-3 have only short N-terminal domains. RAIG-2 and RAIG-3 cDNA sequences were cloned into the mammalian expression vector pcDNA3 with c-myc or HA epitope tags inserted at their N-termini, respectively. Transient transfection experiments in HEK239T cells using these constructs demonstrated RAIG-2 and RAIG-3 expression at the cell surface. Distribution profiles of mRNA expression obtained by semiquantitative Taq-Man PCR analysis showed RAIG-2 to be predominantly expressed in human brain areas and RAIG-3 to be predominantly expressed in peripheral tissues. In addition, expression of RAIG-2 and RAIG-3 mRNA was increased following treatment with all-trans-retinoic acid in a manner similar to that previously described for RAIG-1. Finally, RAIG-2 was mapped to chromosome 16p12 (D16S405-D16S3045) and RAIG-3 to chromosome 17q25 (D17S1352-D17S785). These results suggest that RAIG-1, RAIG-2, and RAIG-3 represent a novel family of retinoic acid-inducible receptors, most closely related to the type 3 GPCR subfamily, and provide further evidence for a linkage between retinoic acid and G-protein-coupled receptor signal transduction pathways.

Defective oligodendrocyte development and severe hypomyelination in PDGF-A knockout mice.

There is a class of oligodendrocyte progenitors, called O-2A progenitors, that is characterized by expression of platelet-derived growth factor &agr;-receptors (PDGFR(&agr;)). It is not known whether all oligodendrocytes are derived from these PDGFRalpha-progenitors or whether a subset(s) of oligodendrocytes develops from a different, PDGFR alpha-negative lineage(s). We investigated the relationship between PDGF and oligodendrogenesis by examining mice that lack either PDGF-A or PDGF-B. PDGF-A null mice had many fewer PDGFR alpha-progenitors than either wild-type or PDGF-B null mice, demonstrating that proliferation of these cells relies heavily (though not exclusively) on PDGF-AA homodimers. PDGF-A-deficient mice also had reduced numbers of oligodendrocytes and a dysmyelinating phenotype (tremor). Not all parts of the central nervous system (CNS) were equally affected in the knockout. For example, there were profound reductions in the numbers of PDGFR alpha-progenitors and oligodendrocytes in the spinal cord and cerebellum, but less severe reductions of both cell types in the medulla. This correlation suggests a close link between PDGFRalpha-progenitors and oligodendrogenesis in most or all parts of the CNS. We also provide evidence that myelin proteolipid protein (PLP/DM-20)-positive cells in the late embryonic brainstem are non-dividing cells, presumably immature oligodendrocytes, and not proliferating precursors.

Oligodendrocyte population dynamics and the role of PDGF in vivo.

Oligodendrocyte progenitors originate near the floor plate of the spinal cord, then proliferate and migrate throughout the cord before giving rise to oligodendrocytes. Progenitor cell proliferation stops before birth because the cell cycle slows down, linked to an increase in differentiation and death. Experiments with transgenic mice show that platelet-derived growth factor (PDGF) drives progenitor cell division and suggest that slowing of and exit from the cycle reflects a decline in PDGF signaling. Overexpressing PDGF induces hyperproliferation of progenitor cells and excessive, ectopic production of oligodendrocytes. However, the superfluous oligodendrocytes die at an immature stage of differentiation, leaving a normal complement of myelin-forming cells. Therefore, cell survival controls override proliferation controls for determining the final number and distribution of mature oligodendrocytes.

PDGF mediates a neuron-astrocyte interaction in the developing retina.

Astrocytes invade the developing retina from the optic nerve head, over the axons of retinal ganglion cells (RGCs). RGCs express the platelet-derived growth factor A-chain (PDGF-A) and retinal astrocytes the PDGF alpha-receptor (PDGFR alpha), suggesting that PDGF mediates a paracrine interaction between these cells. To test this, we inhibited PDGF signaling in the eye with a neutralizing anti-PDGFR alpha antibody or a soluble extracellular fragment of PDGFR alpha. These treatments inhibited development of the astrocyte network. We also generated transgenic mice that overexpress PDGF-A in RGCs. This resulted in hyperproliferation of astrocytes, which in turn induced excessive vasculogenesis. Thus, PDGF appears to be a link in the chain of cell-cell interactions responsible for matching numbers of neurons, astrocytes, and blood vessels during retinal development.