Bernhard Reuss

Fibroblast growth factors and their receptors in the central nervous system

Fibroblast growth factors (FGFs) and their receptors constitute an elaborate signaling system that participates in many developmental and repair processes of virtually all mammalian tissues. Among the 23 FGF members, ten have been identified in the brain. Four FGF receptors (FGFRs), receptor tyrosine kinases, are known so far. Ligand binding of these receptors greatly depends on the presence of heparan sulfate proteoglycans, which act as low affinity FGFRs. Ligand binding specificity of FGFRs depends on the third extracellular Ig-like domain, which is subject to alternative splicing. Activation of FGFRs triggers several intracellular signaling cascades. These include phosphorylation of src and PLCγ leading finally to activation of PKC, as well as activation of Crk and Shc. SNT/FRS2 serves as an alternative link of FGFRs to the activation of PKC and, in addition, activates the Ras signaling cascade. In the CNS, FGFs are widely expressed; FGF-2 is predominantly synthesized by astrocytes, whereas other FGF family members, e.g., FGF-5, FGF-8, and FGF-9, are primarily synthesized by neurons. During CNS development FGFs play important roles in neurogenesis, axon growth, and differentiation. In addition, FGFs are major determinants of neuronal survival both during development and during adulthood. Adult neurogenesis depends greatly on FGF-2. Finally, FGF-1 and FGF-2 seem to be involved in the regulation of synaptic plasticity and processes attributed to learning and memory.

Fibroblast growth factor 2 (FGF-2) differentially regulates connexin (cx) 43 expression and function in astroglial cells from distinct brain regions

Fibroblast growth factor (FGF)‐2 is a peptide growth factor that promotes the generation, differentiation, and survival of neurons and glial cells. In the CNS, astroglial cells are coupled in a region‐specific manner by gap junctions consisting of connexin 43 (cx43). In the present study we have investigated effects of FGF‐2 and of other growth factors on the expression and function of cx43 in astroglial cells cultured from telencephalic cortex, striatum, and mesencephalon of newborn rats. Confluent cultures were maintained for two days in low serum, and then exposed to FGF‐2 (10 ng/ml) for 48 h. FGF‐2 caused a reduction of cx43‐protein, ‐mRNA, and intercellular communication revealed by dye spreading. These changes occurred in cortical and striatal cells, but not in mesencephalic astroglial cells. Effects of FGF‐2 were time‐ and concentration‐dependent, with a minimal effective dose of 1 ng/ml FGF‐2, and an onset of effects after 6 h of incubation. The reduction of coupling by FGF‐2 was transient, since in cortical and striatal cultures coupling recovered to control levels 48 h after removal of the growth factor. Like FGF‐2, transforming growth factor‐β3 (TGF‐β3) decreased coupling of cortical and striatal, but not mesencephalic astroglial cells. Astroglial cells from all brain regions showed a slight FGF‐mediated increase in 5‐bromo‐2′‐desoxy‐uridine (BrdU) incorporation, which was abolished upon co‐treatment with TGF‐β3. However, TGF‐β3 did not interfere with the repression of cx43‐function by FGF‐2. Epidermal growth factor (EGF) that has been demonstrated to influence coupling in other cell types had no effect on dye spreading but significantly increased BrdU incorporation. Our results reveal a novel function of FGF‐2 on cultured astroglial cells which may be relevant to the regulation of astroglial cell connectivity in vivo. GLIA 22:19–30, 1998.

Expression and developmental regulation of gap junction connexins cx26, cx32, cx43 and cx45 in the rat midbrain-floor.

Connexins (cx) constitute a family of transmembrane proteins that form gap junction channels allowing metabolic and electrical coupling of cellular networks. Initial studies on the expression of cx in the developing brain have suggested that cx may undergo dynamic changes and may possibly be implicated in synchronizing development and differentiation of neural progenitor cells and young neurons. We have investigated expression of cx26, cx32, cx43, and cx45 in the midbrain floor, where nigrostriatal dopaminergic neurons originate and differentiate. This neuron population is of major importance in regulating motor‐functions. Semiquantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) revealed low levels of cx26‐mRNA in the midbrain floor at E12, which gradually increased during pre‐ and postnatal development, reaching a maximum in the adult. Cx32‐mRNA‐levels reached a first peak at E16, and showed highest levels in adulthood. Cx43 was highly expressed at E12, decreased until E18, and subsequently increased again until adulthood. Cx45 mRNA was prominent at all developmental ages, but slightly decreased after the first postnatal week. Double‐labeling for the dopaminergic neuronal marker tyrosine hydroxylase (TH), and cx‐immunoreactivities (ir) evaluated by quantitative confocal laser microscopy revealed both distinct and similar developmental patterns for the individual cx investigated. Cx26 was highest at E14, decreased towards birth, and subsequently increased again reaching about 50% of the E14 level in the adult. Cx32‐ir peaked at E16 and dropped to low levels after birth. Cx43‐ir was highest at E12, decreased sharply at E14, reached its lowest levels at birth, but modestly increased again afterwards. Cx45‐ir showed a biphasic pattern, with two prominent peaks at E12 and E18, followed by a massive postnatal decrease. Taken together, our results reveal that expression and ir of cx in the midbrain floor and dopaminergic neurons, respectively, follow cx‐type specific patterns that temporally coincide with important steps of midbrain morphogenesis, as e.g. progenitor cell formation and migration (E12), early differentiation (E14‐16), target encounter (E16‐18) and postnatal functional maturation of the nigrostriatal system.

TRANSFORMING GROWTH FACTOR-BETA MEDIATES THE NEUROTROPHIC EFFECT OF FIBROBLAST GROWTH FACTOR-2 ON MIDBRAIN DOPAMINERGIC NEURONS

Fibroblast growth factor (FGF)‐2 is an established neurotrophic factor for dopaminergic (DAergic) neurons in the ventral midbrain. Its survival and differentiation‐promoting effects on DAergic neurons in vitro and in vivo are crucially dependent on the presence, numerical expansion and maturation of astroglial cells. We show now that transforming growth factor (TGF)‐β, an established trophic factor for DAergic neurons and product of astroglial cells, mediates the trophic effect of FGF‐2 on DAergic neurons cultured from the embryonic rat midbrain floor. Antibodies to TGF‐β that neutralize the isoforms ‐β1, ‐β2 and ‐β3 abolish the trophic effect of FGF‐2. FGF‐2 increases TGF‐β3 mRNA and amounts of biologically active TGF‐β determined in a mink lung epithelial cell assay in a time‐dependent manner. FGF‐2 also induces levels of active TGF‐β in neonatal rat astrocytes cultured from midbrain, striatum and cortex. We conclude that TGF‐β is required for mediating the survival promoting effect of FGF‐2 on DAergic and, possibly, cortical and striatal neurons grown in the presence of glial cells.

Fibroblast growth factors-5 and -9 distinctly regulate expression and function of the gap junction protein connexin43 in cultured astroglial cells from different brain regions.

Astroglial cells contribute to neuronal maintenance and function in the normal and diseased brain. Gap junctions formed predominantly by connexin43 (cx43) provide important pathways to coordinate astroglial responses. We have previously shown that fibroblast growth factor (FGF)‐2, which occurs ubiquitously in the CNS, downregulates gap junction communication in cortical and striatal, but not in mesencephalic astroglial cells in vitro (Reuss et al. Glia 22:19–30, 1998). Other members of the FGF family expressed in the CNS include FGF‐5 and FGF‐9. We show that both FGF‐5 and FGF‐9, like FGF‐2, downregulate astroglial gap junctions and functional coupling. However, their effects are strikingly different from different brain regions, with regard to astroglial cells. FGF‐5 specifically affects mesencephalic astroglial cells without changing coupling of cortical and striatal astroglia, while FGF‐9 reduces gap junctional coupling in astroglia from all three brain regions. Both cx43 mRNA and protein levels as well as functional coupling assessed by dye spreading are affected. To clarify whether brain region‐specific effects of FGFs on astroglial coupling are due to differential expression of FGF receptors (FGFR), we monitored expression of the four known FGFR mRNAs in astroglial cultures by RT‐PCR. Irrespective of their regional origin, astroglial cells express mRNAs for FGFR‐2 and FGFR‐3. In summary, our results provide evidence for an important role of FGF‐2, ‐5, and –9 in a distinct, CNS region‐specific regulation mechanism of astroglial gap junction communication. The molecular basis underlying the regionally distinct responsiveness of astrocytes to different FGFs may be sought beyond distinct FGFR expression. GLIA 30:231–241, 2000.

Survival and differentiation of dopaminergic mesencephalic neurons are promoted by dopamine-mediated induction of FGF-2 in striatal astroglial cells.

Survival of dopaminergic (DAergic) midbrain neurons during development and after lesioning depends, in part, on the presence of astroglia-derived growth factors, as, e.g., fibroblast growth factor (FGF)-2. Astrocytes express DA receptors in a brain-region-specific manner. We show here that DA (10(-3) to 10(-6) mol/liter) applied continuously for 12 h or as a 10-min pulse significantly upregulates FGF-2 immunoreactivity quantified by Western blot and densitometry in astrocytes cultured from two target areas of DAergic neurons, striatum and cortex, but not in mesencephalic astroglia. Semiquantitative competitive RT-PCR confirmed the increase in FGF-2 on the mRNA level. The effects were specific in that glutamate, which can also activate receptors on astroglial cells, did not influence FGF-2 synthesis. In addition to the DA-mediated increase in FGF-2 synthesis the capability of conditioned medium (CM) from DA-stimulated striatal and cortical astrocytes to promote survival and process formation of cultured rat DAergic neurons was significantly enhanced. These effects could be fully blocked by preincubation of the CM with an FGF-2-specific polyclonal antiserum. Our results suggest that DA released from DAergic axon terminals in target regions of DAergic neurons and astroglial FGF-2 production are interdependent in that DA triggers synthesis of FGF-2, which, in turn enhances survival and differentiation of DAergic neurons.

GDNF‐related factor persephin is widely distributed throughout the nervous system

Persephin (PSP) is the most recently discovered member of the GDNF family of neurotrophic factors. We have used an RT‐PCR approach to start addressing the putative functional significance of PSP by determining sites of its synthesis in the neonatal rat brain. Generally, two transcripts were found. Sequence analysis of the transcripts identifies an 88 bp intronic sequence. Neural tissues analysed included cortex, hippocampus, striatum, diencephalon, mesencephalon, cerebellum, hindbrain and spinal cord as well as superior cervical, dorsal root ganglia, adrenal gland, and PC12 pheochromocytoma cells. As non‐neuronal tissues, sciatic nerve, optic nerve, primary astroglial, oligodendroglial, O2A progenitor, and glioma cells (C6, B49) were also included. All tissues/cells except oligodendrocytes and O2A progenitor cells were strongly positive for PSP mRNA. To test the hypothesis of whether PSP might act as a target‐derived factor, as suggested for GDNF, the motoneuron–muscle axis has been analysed. PSP is synthesized in skeletal muscle and, to a higher extent, in the spinal cord. Moreover, PSP is synthesized in purified embryonic motoneurons. Together, these data do not support a role for PSP as a typical target‐derived neurotrophic factor for motoneurons. We conclude that PSP is synthesized throughout the nervous system and that it is presumably of both astroglial and neuronal origin, in contrast to GDNF and neurturin, which seem to be predominantly of neuronal origin. J. Neurosci. Res. 53:494–501, 1998.

Atypical Neuroleptic Drugs Downregulate Dopamine Sensitivity in Rat Cortical and Striatal Astrocytes

Psychotic symptoms in different neuropsychiatric disorders are treated by neuroleptic drugs. Neuroleptics are known to block dopamine (DA) neurotransmission, however, cell types mediating their actions have not been determined. Recently, astrocytes have been demonstrated to express D1- and D2-DA receptors, whose activation leads to transient increases in intracellular calcium concentration. We show here that DA-sensitivity of cortical and striatal rat astroglial cultures, as monitored by calcium imaging, is reduced by a 12-h exposure to the atypical antipsychotic agents Clozapine (>1 nmol/liter), Olanzapine (>100 nmol/liter), and Risperidone (>1 nmol/liter), but not by classical neuroleptics Haloperidol and Sulpiride. These effects could not be reverted by the receptor-specific antagonists SCH23390, Sulpiride, L745 870, Ergotamine, and Propranolol. In addition, RT-PCR and Western blot analyses concerning the effects of Clozapine, Olanzapine, and Risperidone on DA receptor expression in cortical and striatal astroglial cells revealed no alterations in mRNAs and immunoreactive protein of D1- and D2-DA receptor subtypes. These results provide the first evidence that atypical but not classical neuroleptic drugs reduce astroglial DA-sensitivity, a mechanism that may be important for a better understanding of differences in effects and side effects between atypical and classical neuroleptic drugs.

Regionally distinct regulation of astroglial neurotransmitter receptors by fibroblast growth factor-2.

Fibroblast growth factor (FGF)-2 is an abundant astroglial cytokine. We have previously shown that FGF-2 downregulates gap junctions in primary astroglial cultures (B. Reuss et al., 1998, Glia 22, 19-30). We demonstrate now that FGF-2 induces astroglial dopamine (DA) sensitivity and D1 dopamine-receptor (D1DR) antigen and message in cortical and striatal astroglial cultures. On the functional level 10 micromol/L DA triggered transient increases in astroglial [Ca(2+)](i). In gap-junction-coupled cells, no FGF-2-dependent changes in proportions of DA-responsive cells were observable. However, uncoupling with octanol or 18alpha-glycirrhetinic acid isolated the smaller population of astrocytes intrinsically sensitive to DA which was significantly increased by FGF-2 in cortical and striatal cultures. Administration of DR-specific substances revealed that FGF-2 upregulated D1DR. These results indicate that downregulation of astroglial gap junctions by FGF-2 is accompanied by an upregulation of D1DR and DA sensitivity, adding a new aspect to the role of FGF-2 in the regulation of brain functions.

Regulation of gap junction communication by growth factors from non‐neural cells to astroglia: A brief review

Astrocytes are coupled by gap junctions, which are composed of connexins, channel‐forming proteins. Connexin43 is the predominant connexin of astrocytes. Gap junctions assemble astrocytes into functional syncytia permitting exchange of small molecules including metabolites, catabolites, and second messenger molecules. Thus, gap junctions of astroglial cells serve the maintenance of extra‐ and intra‐cellular homeostasis in the brain and eventually ascertain neuronal functions. Alterations in astroglial cell coupling can disturb this equilibrium resulting in neuronal dysfunction and death. Growth factors are an important class of substances that can influence coupling in non‐neural cells. Several groups have recently carried the analyses of gap junction regulation by cytokines to the level of neural cells. This review summarizes the recent progress in this field and outlines directions of future research. GLIA 24:32–38, 1998.