Transgenic approaches to study oligodendrocyte development and function Expression of functional neurotransmitter receptors and ionic channels in glia indicates that glial cells are also responsive elements of the mammalian central nervous system. Receptors and channels may have an important physiological role not only in neurons, but also in non-neuronal cells. Our objective is to understand the functional properties and the associated signal transduction pathways of glial neurotransmitter receptors, and their physiological role in the mature and developing brain. In previous work, we focused on glutamatergic and beta-adrenergic receptors (GluRs and b-ARs, respectively) in oligodendrocyte progenitors (OPs), and demonstrated that activation of these receptors inhibits OP proliferation, but has opposite effects on cell differentiation. Both neurotransmitters cause G1-arrest in OP cell cycle. Glutamate inhibits, whereas norepinephrine stimulates OP differentiation. We have studied cell cycle progression, arrest and withdrawal in OP cells, focusing our attention on the G1 phase and G1/S transition. Not only were proliferating OPs found to display higher protein levels of cyclin E and D, and cyclin-dependent kinases (cdk) 2,4 and 6 than post-mitotic oligodendrocytes, but the kinase activities of both cyclin D-cdk4/6 and cyclin E-cdk2 complexes were also higher in dividing OPs. This was associated to a significant decrease in the formation of the cyclin E-cdk2 and cyclin D-cdk4/cyclin D-cdk6 complexes in differentiated oligodendrocytes that had withdrawn from the cell cycle. Cell cycle arrest in G1 induced by glutamatergic and b-adrenergic receptor activation, or cell depolarization, however, did not modify cyclin E and cdk2 protein expression, compared to proliferating OPs. Instead, these agents caused a strong and selective decrease in cdk2 activity and an impairment of cyclin E-cdk2 complex formation. Although cyclin D protein levels were higher than in proliferating cells, cyclin D-associated kinase activity was not modified in G1-arrested OPs. In order to establish an animal model to study oligodendrocyte development and physiology in situ, we generated a transgenic mouse expressing the green fluorescent protein (GFP) under the control of the 2?-3?-cyclic nucleotide 3?-phosphodiesterase (CNP) promoter. GFP+ cells could be easily visualized in live and fixed brain tissue throughout the entire mouse postnatal development. The spatiotemporal appearance of GFP+ cells in mouse embryos was consistent with the previously described origin of oligodendrocytes. Immunohistochemical analysis in brain tissue using different neural markers demonstrated that GFP expression was restricted to cells of the oligodendrocyte lineage. These cells included oligodendrocyte progenitors (OPs) and oligodendrocytes at distinct developmental stages. GFP+ OPs gave rise to differentiated oligodendrocytes in culture. Fluorescence-activated cell sorting was used to obtain a 100% pure population of GFP+ oligodendrocyte lineage cells. Electrophysiological patch clamp recordings of GFP+ cells in situ demonstrated that OP cells displayed large outward tetraethylammonium (TEA)-sensitive K+-currents and very small inward currents, whereas mature oligodendrocytes were characterized by expression of large inward currents. In tissue slice cultures, the proliferation rate of GFP+ cells in developing white matter decreased with the age of the animals between postnatal day 2 and 20. Proliferation of GFP+ cells in tissue slices was strongly inhibited by TEA. Our findings indicate that oligodendrocyte development and physiology can be studied in live tissue of CNP-GFP transgenic mice, and that these mice represent a source of pure GFP+ oligodendrocyte lineage cells throughout development. Proliferation and differentiation of oligodendroglial cells are tightly linked biological processes that control myelination in the central nervous system. We analyzed expression of cdk2 and its partner cyclin E in vivo in cells purified from transgenic mice selectively expressing the green fluorescent protein in the oligodendroglial lineage. Cdk2 and cyclin E levels decreased during postnatal maturation, consistent with the time-course of oligodendrocyte progenitor (OP) proliferation and differentiation in vivo. In order to establish a causal link between cyclin E/cdk2 activity and OP cell proliferation, we used an in vitro transgenic approach, and selectively targeted cdk2 in OP cells by overexpressing wild-type (wt) or dominant-negative (Dn) versions of this gene. Dn-cdk2 overexpression inhibited mitogen-induced OP cell proliferation, whereas overexpression of wt-cdk2 prevented reversible cell cycle arrest associated with the activation of glutamatergic and b-adrenergic receptors, and with K+ channel blockade. Thus, cdk2 activity plays a pivotal function in OP cell cycle decisions occurring at G1/S checkpoint either in a pro- or anti-mitotic environment. Dn-cdk2- or wt-cdk2-mediated regulation of G1/S transition, per se, did not influence OP cell differentiation. Therefore, molecular mechanisms associated with initiation of OP differentiation are independent from cyclinE/cdk2 checkpoint and from the number of cell cycles that occur prior to the onset of differentiation. Kainate receptor genes and their regulation in the brain Kainate receptors represent a distinct molecular and pharmacological subtype of glutamate receptors. Two kainate receptor gene sub-families encode the subunits GluR5, 6 and 7, and KA1 and 2. When expressed in Xenopus oocytes or heterologous mammalian cells, only GluR5-GluR7 form functional homomeric. KA1 and KA2 are inactive as homomeric ion channels, but associate with members of the GluR5-7 family to form functional heteroligomeric kainate-preferring receptors with biophysical properties and agonist selectivity distinct from homomeric GluR5-GluR7. Functional homomeric and heteroligomeric kainate-preferring receptors are expressed in cultured neurons and glia, and kainate receptor synaptic currents have been found in various areas of the CNS. The GRIK5 gene encodes the kainate receptor subunit KA2. Since KA2 associates with other kainate receptor subunits, it follows that different functional receptor subtypes can be formed as a result of a stringent qualitative and quantitative control of GRIK5 expression. To gain an understanding of how kainate receptor subunit expression is regulated, we have previously cloned and characterized the structure of the GRIK5 gene, including its 5'untranslated region. We have also identified an intronic element of this gene, which displays functional features of a silencer. The GRIK5 promoter is TATA-less and is GC-rich, with multiple consensus initiator sequences. Transgenic mouse lines carrying 4kb of the GRIK5 5? flanking sequence showed lacZ reporter expression predominantly in the nervous system. Reporter assays in central glial (CG-4) and non-neural cells with up to 2kb of 5? flanking sequences indicated that a 1200 bp fragment could sustain neural cell-specific GRIK5 promoter activity. Transcriptional activity was associated with the formation of a TFIID-containing complex on an initiator sequence located 1100 bp upstream from the first intron. In transfection studies, deletion of exonic sequences downstream of the promoter resulted in reporter gene activity that was no longer neural-specific. When a 77bp sequence from the deleted fragment was placed downstream of the GRIK5 promoter, it completely silenced reporter expression in NIH3T3 fibroblasts, while marginally attenuating reporter activity in CG-4 cells. Analysis of the 77bp sequence revealed a functional SP1 binding site and a sequence resembling a neuron-restrictive silencer element. The latter sequence, however, did not display cell-specific binding of REST-like proteins. Our studie
| Gallo, V; Ghiani, C A (2000) Glutamate receptors in glia: new cells, new inputs and new functions. Trends Pharmacol Sci 21:252-8 |
| Ghiani, C A; Yuan, X; Eisen, A M et al. (1999) Voltage-activated K+ channels and membrane depolarization regulate accumulation of the cyclin-dependent kinase inhibitors p27(Kip1) and p21(CIP1) in glial progenitor cells. J Neurosci 19:5380-92 |
