Macroglial cell development in embryonic rat brain: studies using monoclonal antibodies, fluorescence activated cell sorting, and cell culture

Astrocytes, ependymal cells, and oligodendrocytes have been shown to develop on the same schedule in dissociated cell cultures of early embryonic rat brain as in vivo. Subsequent studies showed that there are two major types of astrocyte (type-1 and type-2), which, in cultures of perinatal optic nerve, develop as two distinct lineages. In such cultures, type-2 astrocytes and oligodendrocytes develop from the same, bipotential, (O-2A) progenitor cells, which differentiate into type-2 astrocytes in 10% fetal calf serum (FCS) and into oligodendrocytes in less than or equal to 0.5% FCS. In light of these findings, we now have extended our studies on macroglial cell development in rat brain and show the following: (i) The first astrocytes to develop have a type-1 phenotype, while astrocytes with a type-2 phenotype do not develop until almost 2 weeks later, just as in the optic nerve. (ii) Most importantly, type-2 astrocytes, like the other macroglial cells, develop on the same schedule in cultures of early embryonic (less than or equal to E15) brain as they do in vivo. (iii) By contrast, both oligodendrocytes and type-2 astrocytes develop prematurely in cultures of E17 brain, and FCS influences this development in the same way it does in perinatal optic nerve cultures. (iv) Type-2 astrocyte precursors are labeled by the A2B5 monoclonal antibody, as shown previously for oligodendrocyte precursors in brain and for O-2A progenitor cells in optic nerve. Taken together with our previous findings, these results suggest that oligodendrocytes and type-2 astrocytes in brain develop from bipotential O-2A progenitor cells, whose choice of developmental pathway and timing of differentiation depend on mechanisms that operate independently of brain morphogenesis.     

A role for platelet-derived growth factor in normal gliogenesis in the central nervous system

The bipotential progenitor cells (O-2A progenitors) that produce oligodendrocytes and type-2 astrocytes in the developing rat optic nerve are induced to proliferate in culture by type-1 astrocytes. Here, we show that the astrocyte-derived mitogen is platelet-derived growth factor (PDGF). PDGF is a potent mitogen for O-2A progenitor cells in vitro. Mitogenic activity in astrocyte-conditioned medium comigrates with PDGF on a size-exclusion column, competes with PDGF for receptors, and is neutralized by antibodies to PDGF. PDGF dimers can be immunoprecipitated from astrocyte-conditioned medium, and mRNA encoding PDGF is present in rat brain throughout gliogenesis. We propose that astrocyte-derived PDGF is crucial for the control of myelination in the developing central nervous system.     

Type-2 astrocyte development in rat brain cultures is initiated by a CNTF-like protein produced by type-1 astrocytes

O-2A progenitor cells are bipotential glial precursors that give rise to both oligodendrocytes and type-2 astrocytes on a precise schedule in the rat CNS. Studies in culture suggest that oligodendrocyte differentiation occurs constitutively, while type-2 astrocyte differentiation requires an exogenous inducer such as fetal calf serum. Here we describe a rat brain cell culture system in which type-2 astrocytes develop on schedule in the absence of exogenous inducers. Coincident with type-2-astrocyte development, the cultures produce an approximately 20 kd type-2-astrocyte-inducing factor(s). Purified cultures of type-1 astrocytes can produce a similar factor(s). Under conditions where they produce type-2-astrocyte-inducing factor(s), both brain and type-1 astrocyte cultures produce a factor(s) with ciliary neurotrophic (CNTF)-like activity. Purified CNTF, like the inducers from brain and type-1 astrocyte cultures, prematurely induces type-2 astrocyte differentiation in brain cultures. These findings suggest that type-2 astrocyte development is initiated by a CNTF-like protein produced by type-1 astrocytes.     

PDGF receptors on cells of the oligodendrocyte-type-2 astrocyte (O-2A) cell lineage

It has been shown previously that cultures of rat optic nerve contain three types of macroglial cells--oligodendrocytes and two types of astrocytes. Type-1 astrocytes develop from their own precursor cells beginning before birth, while oligodendrocytes and type-2 astrocytes develop postnatally from a common bipotential precursor called the O-2A progenitor cell. Proliferating O-2A progenitor cells give rise to postmitotic oligodendrocytes beginning around birth, and to type-2 astrocytes beginning in the second postnatal week. Studies in vitro have suggested that platelet-derived growth factor (PDGF), secreted by type-1 astrocytes, plays an important part in timing oligodendrocyte development: PDGF seems to keep O-2A progenitor cells proliferating until an intrinsic clock in the progenitor cells initiates the process leading to oligodendrocyte differentiation. The clock apparently determines when a progenitor cell becomes unresponsive to PDGF, at which point the cell stops dividing and, as a consequence, automatically differentiates into an oligodendrocyte. Here we have used radiolabelled PDGF to show that O-2A progenitor cells have PDGF receptors, suggesting that these cells respond directly to PDGF. The receptors resemble the type A PDGF receptor previously described on human fibroblasts and are initially retained when progenitor cells stop dividing and develop in vitro into oligodendrocytes. The latter finding indicates that receptor loss is not the reason that progenitor cells initially become mitotically unresponsive to PDGF.     

An inducer protein may control the timing of fate switching in a bipotential glial progenitor cell in rat optic nerve

In rat optic nerve, oligodendrocytes and type-2 astrocytes develop from a common (O-2A) progenitor cell. The first oligodendrocytes differentiate at birth, while the first type-2 astrocytes differentiate in the second postnatal week. We previously showed that the timing of oligodendrocyte differentiation depends on an intrinsic clock in the O-2A progenitor cell. Here we provide evidence that the timing of type-2 astrocyte differentiation, by contrast, may depend on an inducing protein that appears late in the developing nerve. We show that extracts of 3- to 4-week-old, but not 1-week-old, rat optic nerve contain a protein (apparent Mr approximately 25,000) that induces O-2A progenitor cells in culture to express glial fibrillary acidic protein (GFAP), an astrocyte-specific marker in the rat central nervous system.     

Localization, characterization and function of pituitary adenylate cyclase-activating polypeptide during brain development

Neural development is controlled by region-specific factors that regulate cell proliferation, migration and differentiation. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that exerts a wide range of effects on different cell types in the brain as early as the fetal stage. Here we review current knowledge concerning several aspects of PACAP expression in embryonic and neonatal neural tissue: (i) the distribution of PACAP and PACAP receptors mRNA in the developing brain; (ii) the characteristic generation of neurons, astrocytes and oligodendrocytes in brain areas where the PACAP receptor is expressed and (iii) the role of PACAP as a regulator of neural development, inducing differentiation and proliferation in association with other trophic factors or signal transduction molecules.     

A quantitative immunohistochemical study of macroglial cell development in the rat optic nerve: in vivo evidence for two distinct astrocyte lineages

We have shown previously that three antibodies--anti-galactocerebroside (GC), anti-glial fibrillary acidic protein (GFAP), and the A2B5 monoclonal antibody--can be used to help distinguish three classes of glial cells in the rat optic nerve: oligodendrocytes are GC+, GFAP-, almost all type-1 astrocytes are A2B5-, GFAP+, and almost all type-2 astrocytes are A2B5+, GFAP+. In the present study we have used these antibodies to examine the timing and sequence of the development of the three types of glial cells in vivo. We show that type-1 astrocytes first appear at embryonic Day 16 (E16), oligodendrocytes at birth (E21), and type-2 astrocytes between postnatal Days 7 and 10 (P7-10). Moreover, we demonstrate quantitatively that astrocytes in the optic nerve develop in two waves, with more than 95% of type-1 astrocytes developing before P15 and more than 95% of type-2 astrocytes developing after P15. Finally, we provide indirect evidence that type-2 astrocytes do not develop from type-1 astrocytes in vivo, supporting previous direct evidence that the two types of astrocytes develop from two serologically distinct precursor cells in vitro.     

Pathways of dehydroepiandrosterone formation in rat brain glia

In peripheral steroidogenic tissues, dehydroepiandrosterone (D) is formed from pregnenolone (P) by the microsomal cytochrome P450c17 enzyme. Although some steroidogenic P450s have been found in brain tissue, no enzyme has been shown to possess P450c17 activity. We recently demonstrated the presence of an alternative, Fe(2+)-dependent pathway responsible for D formation from alternative precursors in rat glioma cells. We and others could not find P450c17 mRNA and protein in rat brain, but demonstrate herein the presence of Fe(2+)-dependent alternative pathway for D formation in rat brain cortex microsomes. Using primary cultures of differentiating rat glial cells, we observed that P450c17 mRNA and protein were present in O-2A oligodendrocyte precursors and mature oligodendrocytes. In the presence of P, O-2A and mature oligodendrocytes formed D. Addition of Fe(2+) together with submaximal concentrations of P increased D formation by these cells. Treatment of oligodendrocytes with the P450c17 inhibitor SU 10603 in the presence or absence of P failed to inhibit D production. These data suggest that D formation in oligodendrocytes occurs independently of the P450c17 protein present in the cells. In isolated type I astrocytes we did not find neither P450c17 mRNA nor protein. These cells responded to Fe(2+) by producing D and addition of P together with Fe(2+) further increased D synthesis. SU 10603 failed to inhibit D formation by astrocytes. Taken together these results suggest that in differentiating rat brain oligodendrocytes and astrocytes D is formed via a P450c17-independent and oxidative stress-dependent alternative pathway.     

神经干细胞向少突胶质前体细胞的定向分化诱导

本研究采用神经胶质瘤细胞株(B104 neuroblatoma cells,B104 cells)培养上清(B104CM)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF),将冷冻复苏的大鼠胚胎脊髓神经干细胞(neural stem cells,NSCs)定向诱导为少突胶质前体细胞(oligodendrocyte precusor cells,OPCs)。形态学和免疫组化的结果显示,诱导后95％以上的细胞具有双极或多极突起的典型OPCs形态,并表达A285和血小板源生长因子受体-α(platelet derived growth factor receptor-α,PDGFR-α等0PCs标志,所有PDGFR-α阳性的OPCs均不表达β-Tublin Ⅲ,其中仅少量细胞表达胶质原纤维酸性蛋白(glia fibrillary acidic protein,GFAP)。在B104CM和bFGF共存的培养条件下,悬浮培养的OPCs可大量增殖形成少突胶质细胞球,该细胞球可通过传代继续扩增,且扩增的OPCs仍能维持其特有的形态和自我增殖的特性。撤去bFGF和B104CM后,OPCs能进一步分化为成熟的少突胶质细胞(oligodendrocytes,OLs)或Ⅱ型星形胶质细胞。实验表明,诱导NSCs产生的OPCs在形态、增殖以及分化格局等方面均与已报道的存在于胚胎脑区的O-2A前体细胞相类似。该培养系统可为实验性细胞移植的研究提供丰富的细胞来源。     

Expression of mRNAs of multidrug resistance proteins (Mrps) in cultured rat astrocytes,oligodendrocytes, microglial cells and neurones

Multidrug resistance proteins (Mrps) are ATP-driven export pumps that mediate the export of organic anions from cells. So far only little information is available on expression and physiological functions of Mrps in brain. The expression of mRNAs of six Mrp paralogs in rat brain, as well as in rat cultures enriched for neurones, astrocytes, oligodendrocytes and microglial cells, was studied by qualitative and semiquantitative RT-PCR analysis. In adult rat brain as well as in neural cell cultures the mRNAs coding for Mrp1, Mrp3, Mrp4 and Mrp5 were detected. Semiquantitative analysis revealed that the mRNAs coding for Mrp1 and Mrp5 were more abundant in the four cell culture types than mRNAs of the other Mrps. mRNAs coding for Mrp3 and Mrp4 were found at significant levels in cultured astrocytes and microglial cells, whereas cultures of neurones and oligodendrocytes contained only marginal quantities of these mRNAs. Putative physiological functions of Mrps in brain cells are discussed.     

A novel member of the leucine-rich repeat superfamily induced in rat astrocytes by beta-amyloid.

beta-Amyloid (Abeta) deposition and senile plaque-associated astrocytes are common neuropathological features of Alzheimer's disease (AD). Although the molecular mechanisms by which Abeta contributes to the progression of neuropathologic changes have not been established entirely, there is little doubt that the association of Abeta with astrocytes, the predominant cell type in brain, has significant influence on exacerbation of the disease. In an effort to identify key molecules involved in AD, we investigated Abeta-responsive genes using rat astrocytes. In this study, we identified a novel Abeta-induced rat gene, designated as Lib, encoding a type I transmembrane protein with an extracellular domain that contains fifteen leucine-rich repeats (LRRs). Human counterpart of rat Lib is located on chromosome 3q29 and human Lib mRNA found in particularly placenta. Lib mRNA levels in rat C6 astrocytoma cells can be increased by pro-inflammatory cytokines and the rat Lib-transfected cells express Lib protein on the cell surfaces. Lib appears to be a member of the LRR superfamily which is involved in cell-cell and/or -extracellular matrix interactions including adhesion or target recognition in neuroinflammatory states.     

Differential Localization of Glutathione-S-Transferase Yp and Yb Subunits in Oligodendrocytes and Astrocytes of Rat Brain

Glutathione-S-transferase Yb subunits were recently identified in rat brain and localized to astrocytes, ependymal cells lining the ventricles, subventricular zone cells, and tanycytes. Another isoform, Yp (pi family), was detected in rat brain by immunoblotting, and its mRNA was detected by Northern hybridizations. Double immunofluorescence localized Yb and Yp in different glial cells. The strongly Yp-positive cells were identified as oligodendrocytes by virtue of their arrangement in rows in white-matter tracts, colocalization in strongly carbonic anhydrase-positive cells, and association with myelinated tracts in the corpus striatum. Ependymal cells in the choroid plexus and ventricular lining were also strongly Yp positive, whereas Yb was not detected in the choroid plexus. The occurrence of Yp at low levels in astrocytes was indicated after immunostaining by a sensitive peroxidase-antiperoxidase method, which revealed weak staining of those cells in the molecular layer of the cortex. The data suggest that Yb and Yp subunits are primarily localized to astrocytes and oligodendrocytes, respectively, and that both are absent from neurons. The glutathione-S-transferase in oligodendrocytes may participate in the removal of toxins from the vicinity of the myelin sheath. The finding of glutathione-S-transferases in ependymal cells and astrocytes in the brain also suggests that this enzyme could be a first line of defense against toxic substances.     

Astrocyte Sodium Signalling and Panglial Spread of Sodium Signals in Brain White Matter

In brain grey matter, excitatory synaptic transmission activates glutamate uptake into astrocytes, inducing sodium signals which propagate into neighboring astrocytes through gap junctions. These sodium signals have been suggested to serve an important role in neuro-metabolic coupling. So far, it is unknown if astrocytes in white matter—that is in brain regions devoid of synapses—are also able to undergo such intra- and intercellular sodium signalling. In the present study, we have addressed this question by performing quantitative sodium imaging in acute tissue slices of mouse corpus callosum. Focal application of glutamate induced sodium transients in SR101-positive astrocytes. These were largely unaltered in the presence of ionotropic glutamate receptors blockers, but strongly dampened upon pharmacological inhibition of glutamate uptake. Sodium signals induced in individual astrocytes readily spread into neighboring SR101-positive cells with peak amplitudes decaying monoexponentially with distance from the stimulated cell. In addition, spread of sodium was largely unaltered during pharmacological inhibition of purinergic and glutamate receptors, indicating gap junction-mediated, passive diffusion of sodium between astrocytes. Using cell-type-specific, transgenic reporter mice, we found that sodium signals also propagated, albeit less effectively, from astrocytes to neighboring oligodendrocytes and NG2 cells. Again, panglial spread was unaltered with purinergic and glutamate receptors blocked. Taken together, our results demonstrate that activation of sodium-dependent glutamate transporters induces sodium signals in white matter astrocytes, which spread within the astrocyte syncytium. In addition, we found a panglial passage of sodium signals from astrocytes to NG2 cells and oligodendrocytes, indicating functional coupling between these macroglial cells in white matter.