Internalization and processing of basic fibroblast growth factor by neurons and astrocytes.

The fate of iodinated basic fibroblast growth factor (FGF) after its binding to cultured astrocytes and hippocampal neurons was studied. Autoradiography after light and electron microscopy establishes that, if cells are returned to 37 degrees C, the 125I-basic FGF bound internalizes into vesicles in the cytoplasm, localizes to the perinuclear cytoplasm, and is translocated to chromatin structures of the nucleus. The radiolabeled protein is long-lived, a finding confirmed by biochemical analyses. Polyacrylamide gel electrophoresis and autoradiography of both hippocampal neurons and astrocyte extracts reveal that these cells internalize 125I-basic FGF and then metabolize it to three major heparin-binding peptides with molecular weights of 15.5, 9, and 4 kDa. These peptides are initially detected 16 hr after binding to neurons and 4 hr after binding to astrocytes but are still detectable 48 and 16 hr, respectively, after initial binding (though present at lower levels). Immunoprecipitation with sequence-specific antisera to basic FGF reveals that the 15.5-kDa fragment is generated by cleavage at the carboxyl terminus, that the 9-kDa peptide contains the sequences between residues 30 and 87, and the 4-kDa peptide is a C-terminus fragment containing the sequence of basic FGF(106-120) but without basic FGF(139-146) immunoreactivity. The internalization of basic FGF is required for this processing; the treatment of cells with trypsin and 2 M NaCl at different times after binding can only prevent the metabolism of basic FGF if it is performed immediately after binding. Similarly, WGA, which inhibits basic FGF binding to its high-affinity receptor, prevents the metabolism of basic FGF. The possible significance of a metabolic pathway that is responsible for the processing of basic FGF after its internalization by cells in the CNS is discussed in light of its potential function as a neurotrophic factor.     

Selective enhancement by basic fibroblast growth factor of NMDA receptor-mediated increase of intracellular Ca2+ concentration in hippocampal neurons.

The short-term effect of bFGF on intracellular Ca2+ concentration ([Ca2+]i) of hippocampal neurons was investigated using dissociated cell cultures. Changes in [Ca2+]i were measured by microfluorometrically monitoring the fluorescence intensities from individual neurons loaded with fura-2. Perfusion of bFGF (20 ng/ml) alone did not affect the basal level of [Ca2+]i in hippocampal neurons, but clearly enhanced the [Ca2+]i increase induced by NMDA. Quisqualate or KCl-induced [Ca2+]i increase was not influenced by bFGF. These results suggest that bFGF selectively enhances the NMDA receptor-mediated response in hippocampal neurons.     

Distinct signaling pathways involved in multiple effects of basic fibroblast growth factor on cultured rat hippocampal neurons

We investigated possible involvement of voltage-dependent Ca(2+) channels (VDCCs) and several intracellular signaling mechanisms in multiple actions of basic fibroblast growth factor (bFGF), such as survival promotion, induction of calbindin D(28k) expression as well as acceleration of neuritic branch formation of cultured rat hippocampal neurons. Immunocytochemical staining with anti-gamma-aminobutyric acid (GABA) antibody showed that the promotion of neuron survival by bFGF in high cell-density cultures were exerted exclusively on GABA-negative neurons. Nicardipine (5 microM) attenuated the effect of bFGF on neuronal survival and formation of neurite branches, suggesting that the activity of L-type VDCCs is required for these effects. In contrast, stimulation of calbindin expression by bFGF was not attenuated by nicardipine. A phospholipase C inhibitor U73122 (1 microM) prevented the effect of bFGF on neurite branch formation, but not on neuronal survival or calbindin expression. On the other hand, chronic application of phorbol-12-myristate-13-acetate (1 microM) inhibited the effect of bFGF on neuronal survival, without inhibiting the other bFGF actions. Forskolin (100 microM) attenuated the effect of bFGF on neuronal survival and neurite branch formation, indicating that cyclic AMP plays negative regulatory roles in these actions of bFGF. Taken together, these results suggest that multiple biological actions of bFGF on hippocampal neurons are exerted through, and modulated by, distinct signaling pathways.     

The effect of basic fibroblast growth factor on glutamate-injured neuroarchitecture and arachidonic acid release in adult hippocampal neurons

During development in culture, basic fibroblast growth factor (bFGF) protected immature primary hippocampal neurons against glutamate-induced neurotoxicity. We investigated the effects of bFGF on mature, differentiated rat hippocampal neurons cultured for 10–12 days after an 8-min exposure to 500 μM glutamate. Seven days post-injury, hippocampal cells demonstrated severe reductions in cellular viability and axonal and dendritic outgrowth, which were accompanied by a marked increase in [³H]arachidonic acid (ARA) release from prelabelled neurons. bFGF applied post-injury attenuated cell death and cytoarchitectural destruction at all concentrations used (500 pg/ml, 1, 10, 20 ng/ml). However, neurite elongation and branching processes were only significantly protected by 10 ng/ml bFGF. [³H]ARA release decreased in a dose-related fashion within a concentration range of 1–10 ng/ml bFGF. 20 ng/ml bFGF was not superior to 10 ng/ml bFGF. Therefore, bFGF's neurotropic actions appear to be concentration-dependent. Our data suggest that bFGF applied post-injury may have a neuroprotective potential for mature, differentiated, completely polarized hippocampal neurons.     

Selective enhancement by basic fibroblast growth factor of NMDA receptor-mediated increase of intracellular Ca concentration in hippocampal neurons

The short-term effect of bFGF on intracellular Ca²⁺ concentration ([Ca²⁺]_i) of hippocampal neurons was investigated using dissociated cell cultures. Changes in [Ca²⁺]_i were measured by microfluorometrically monitoring the fluorescence intesities from indivudual neurons loaded with fura-2. Perfusion of bFGF (20 ng/ml) alone did not affect the basal level of [Ca²⁺]_i in hippocampal neurons, but clearly enhanced the [Ca²⁺]_i increase induced by NMDA. Quisqualate or KCl-induced [Ca²⁺]_i increase was not influenced by bFGF. These results suggest that bFGF selectively enhances the NMDA receptor-mediated response in hippocampal neurons.     

Basic fibroblast growth factor modulates synaptic transmission in cultured rat hippocampal neurons

Basic fibroblast growth factor (bFGF) is one of the effective growth factors that protect neurons against excitotoxic/ischemic injury and promote neuronal survival. In the present study, we examined the acute modulative effect of bFGF on synaptic transmission by monitoring spontaneous intracellular Ca²⁺ ([Ca²⁺]i]) oscillation, the amplitudes of which reflect excitatory and inhibitory inputs. The hippocampal cells from embryonic day 18 rats were cultured for 11–14 days, and changes in [Ca²⁺]i of single neurons were measured by a microfluometrical technique with fura-2. The amplitude of spontaneous oscillation was decreased by 10 ng/ml bFGF, but not by nerve growth factor (10–1000 ng/ml). Acidic FGF (1000 ng/ml) had a weaker depressant effect. The effect of bFGF was counteracted by suramin. bFGF did not affect the increase in [Ca²⁺]i evoked by glutamate agonists, NMDA or kainate, indicating that glutamate receptors are not involved in the mechanism. This is supported by similar results that kainate-evoked current was not affected by bFGF. On the other hand, bicuculline masked the effect of bFGF on the Ca²⁺ oscillation. But GABA-evoked current was slightly decreased by bFGF. These results suggest the possible role of bFGF in modulating GABAergic rather than glutamatergic neurotransmission.     

Regulation of brain-derived neurotrophic factor and nerve growth factor mRNA in primary cultures of hippocampal neurons and astrocytes.

Brain-derived neurotrophic factor (BDNF) and NGF are both expressed by neurons in the hippocampus. In previous studies, it has been demonstrated that both BDNF and NGF mRNA levels are regulated by neuronal activity. Upregulation is predominantly regulated by the glutamate (NMDA and non-NMDA receptors); downregulation, predominantly by the GABA system (Zafra et al., 1990, 1991). In neuronal cultures of the rat hippocampus, potassium depolarization and kainic acid-mediated increases in BDNF and NGF mRNA were eliminated in a dose-dependent manner by the calcium channel blocker nifedipine. Conversely, calcium ionophores (Bay-K8644 and ionomycin) augmented BDNF and NGF mRNA levels by a calmodulin-mediated mechanism. In view of the fact that many potential modulators (conventional transmitters and neuropeptides) of neuronal and astrocytic BDNF and NGF mRNA synthesis may act via the adenylate cyclase system, we studied the effect of forskolin, an activator of adenylate cyclase. Indeed, forskolin enhanced the effects of calcium ionophores and kainic acid on BDNF and NGF mRNA levels. Cytokines, such as interleukin-1 and transforming growth factor-beta 1, which have previously been shown to increase NGF mRNA markedly in astrocytes, were without effect on neuronal BDNF and NGF mRNA levels. In contrast to neuronal cultures, where the regulation of BDNF and NGF mRNA was generally very similar, the regulation in astrocytes was distinctly different. All the cytokines that produce a marked increase in NGF mRNA were without effect on astrocyte BDNF mRNA levels, which under basic conditions were below the detection limit. However, norepinephrine produced a marked elevation of BDNF mRNA in astrocytes, an effect that was further enhanced by glutamate receptor agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical localization of basic fibroblast growth factor in choroid plexus of the rat

Abstract

In the present study, we report that an intense bFGF-immunoreactivity has been detected in the choroid plexus of the brain ventricles of adult rats. These results suggest that epithelial choroid plexus cells may be the source of the cerebrospinal fluid bFGF. [Neurol Res 1994; 16: 310-312]     

Increased expression and nuclear accumulation of basic fibroblast growth factor in primary cultured astrocytes following ischemic-like insults.

Basic fibroblast growth factor (bFGF) is a biological active polypeptide with potent trophic effects on neurons, glia and endothelial cells. In the present study, we examined the temporal expression profile of bFGF protein in cultured cortical astrocytes under ischemic-like conditions such as serum-free, glucose-free or glutamate application. A peak increase of bFGF level was observed at 24 h after the initiation of insults. A striking increase in the bFGF immunoreactivity and a moderate increase in the fibroblast growth factor receptor-1 (FGFR-1) immunoreactivity were also found in the astrocytes treated with serum- or glucose-deprivation or glutamate. The increased bFGF immunoreactivity and FGFR-1 immunoreactivity were mainly accumulated in the nuclei of astrocytes. The results suggest that the expression of bFGF and FGFR-1 in the astrocytes, especially in the nuclear interior, can be up-regulated under ischemic-like conditions and that the up-regulation of bFGF and FGFR-1 may play an important role in the maintenance and repair of the central nervous system (CNS) after ischemia.     

The binding of basic fibroblast growth factor to ischaernic neurons in the rat

Transient occlusion of the right middle cerebral artery for 15 min produced a small ischaemic lesion in the dorsal portion of the right striatum in rats as seen on days 3, 7 and 14 post-operatively. The lesions consisted mainly of reactive astrocytes and 'ischaemic neuron's with chromatin-condensed (pyknotic) nuclei and homogenously eosinophilic cytoplasm. The incubation of tissue sections with basic fibroblast growth factor (bFGF) followed by anti-bFGF, or with biotinylated bFGF without anti-bFGF, labelled virtually all ischaemic neurons, indicating that bFGF had bound to the latter. The pretreatment of sections with heparitinase prevented the binding of bFGF to these cells, suggesting that the chemical substrate for the bFGF binding was heparan sulphate. In light of the findings that many normal-looking neurons were observed in the corresponding portion of the right striatum in most rats on post-operative days 28 and 90, the appearance of bFGF-binding sites in ischaemic neurons may contribute to the repair process of injured neurons.     

Differential expression of fibronectin, tenascin-C and NCAMs in cultured hippocampal astrocytes activated by kainate, bacterial lipopolysaccharide or basic fibroblast growth factor

Different reports demonstrated that reactive glial cells express increased amounts of adhesion and matrix molecules. Despite a wealth of information on the expression of these molecules during development and after lesion, very little is known of how this expression is regulated. In the present report we used Western blots and immunocytochemistry to investigate the expression of neural cell adhesion molecule (NCAM), fibronectin and tenascin-C in cultured astrocytes from rat hippocampus. The effects of three different extracellular signals were analyzed: the glutamatergic receptor agonist kainic acid, the basic fibroblast growth factor (bFGF) and the bacterial lipopolysaccharide. Each treatment had a specific pattern of glial activation and differentially modified the expression of these proteins. Treatment of astrocytes with kainic acid resulted in an increase of tenascin-C, a decrease of fibronectin and a shift of NCAMs isoforms: NCAM 140 and PSA-NCAM (polysialic acid-rich NCAMs) were increased while NCAM 120 was decreased. bFGF increased fibronectin, tenascin-C and NCAM 120, while decreasing PSA-NCAM. Finally, the treatment of astrocytes with lipopolysaccharide induced a significant increase of fibronectin, tenascin-C and NCAM 120 but did not modify the expression of NCAM 140 and PSA-NCAM. These data suggest different mechanisms for modulation of cell surface interactions. They suggest that glial activation by bFGF and lipopolysaccharide are associated with an increase of the adhesive properties, while kainate action is rather associated with a decrease of the adhesiveness of astrocytes.     

Epidermal growth factor exerts neuronotrophic effects on dopaminergic and GABAergic CNS neurons: comparison with basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) have been described to exert neuronotrophic effects on central nervous system neurons in culture. To study the selectivity of trophic actions of these growth factors, neurotransmitter-identified populations of embryonic rat mesencephalon were used. At 20 days in vitro, EGF (3 ng/ml) promoted survival and neurite outgrowth from these neurons. The neuritogenic effect of bFGF (3 ng/ml) was, however, more robust. Quantitative analysis with the neurofilament monoclonal antibody RR97 and ELISA confirmed the differential response, bFGF being 2-2.5 times more effective at all concentrations tested (ED100: 3-10 ng/ml for both EGF and bFGF). At 10 days in vitro, EGF displayed no trophic activity--even at 30 ng/ml. Treatment of mesencephalic cultures with EGF (3 ng/ml) for 20 days stimulated [3H]dopamine and [14C]GABA uptakes about 4-fold. While bFGF (3 ng/ml) also stimulated GABA uptake some 4-fold, dopamine uptake was increased almost 20-fold. Thus, EGF is also capable of enhancing the transmitter traits of selected central neuronal populations; however, the actions of bFGF appear to preferentially address dopaminergic cells.     

Epidermal growth factor and basic fibroblast growth factor: effects on an overlapping population of neocortical neurons in vitro

Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) have trophic effects on rat neocortical neurons in vitro. Concentration-response studies reveal that EGF maximally stimulates neuronal survival and process outgrowth at approximately 10 ng/ml, while the maximal effect of bFGF is seen at 10-30 ng/ml. Treatment with maximal concentrations of bFGF results in cultures containing a greater number of neurons with long processes, as well as greater branching of processes, than does treatment with EGF. When EGF and bFGF are added together to cultures the effects are not additive. In addition, bFGF is capable of supporting the survival of neurons previously treated with EGF. These findings indicate that EGF and bFGF affect a largely overlapping population of neocortical neurons, but that bFGF may be a more effective trophic agent for these cells.     

Neuroprotective effect of chronic infusion of basic fibroblast growth factor on seizure-associated hippocampal damage

Basic fibroblast growth factor (bFGF) has been shown to have neuroprotective effects in animal models of ischemia. To determine whether bFGF is protective against seizure-induced brain damage, we administered bFGF through osmotic pumps prior to, and after treatment with kainic acid (KA). Recombinant bFGF, CS23, a modified human bFGF, was infused into the lateral ventricles in rats for 2 days before and 5 days after the injection of KA. Control rats received equal volumes of phosphated saline over the same period of time. Infusion of 5 μg/ml of bFGF (0.5 μ1/h) did not modify the latency and duration of seizures induced by intraperitoneal injections of KA. However, bFGF prevented cell loss in the hippocampus in 80% of the rats. In control rats, cell loss in the hippocampus was found in all rats. These results indicate that bFGF has a substantial neuropropective effect.     

Overexpression of nerve growth factor and basic fibroblast growth factor in AIDS dementia complex.

Although neurotrophic factors are currently considered as treatment for neurodegenerative diseases, little is still known about their presence in the central nervous system under pathological conditions. We investigated the expression of the neurotrophic molecules NGF, bFGF, BDNF and IGF-1 in brain tissue of patients suffering from AIDS dementia complex. In contrast to IGF-1 and BDNF, NGF and bFGF mRNA levels were significantly elevated. Strong NGF immunoreactivity was found in perivascular areas and was colocalized with infiltrating macrophages, whereas intense bFGF staining was found in cells with characteristic astrocytic morphology. These data suggest that the induction of NGF and bFGF alone appears to be insufficient as a compensatory mechanism to prevent ADC.     

Effects of basic fibroblast growth factor on survival and choline acetyltransferase development of spinal cord neurons.

To investigate the biological role of basic fibroblast growth factor (bFGF) for the development of the spinal cord we studied the in vitro and in vivo effects of this protein on survival and choline acetyltransferase (ChAT)-activity of embryonic chick and rat spinal cord neurons. In vitro, bFGF (ED50 1-2.8 ng/ml) supported the survival of embryonic neurons from the ventral part of the rat spinal cord (ventral spinal cord, vsc), including motoneurons. Addition of bFGF (100 ng/ml) increased the ChAT-activity in embryonic chick vsc cultures to 150% as compared to untreated cultures (100%). The effect of bFGF was dose-dependent. In vivo-application of bFGF resulted in a similar increase of ChAT-activity in chick spinal cord. Since bFGF stimulates the ChAT-activity of spinal cord neurons in vivo and in vitro we therefore conclude that this protein may have a physiological function for the transmitter development of cholinergic spinal cord neurons.     

Oestrogens,via transforming growth factor alpha,modulate basic fibroblast growth factor synthesis in hypothalamic astrocytes: in vitro observations

The data presented here show that, in cultures of type 1 astrocytes obtained from the hypothalamus of neonatal female rat, 17beta-oestradiol is able to increase both the mRNA and the protein levels of basic fibroblast growth factor (bFGF). In particular, after 24 h of exposure to 17beta-oestradiol (10(-9) and 10(-10) m), an increase of messenger levels of bFGF appears in hypothalamic type 1 astrocytes. Similarly, an induction of bFGF protein is also evident at this time of exposure. The effect on the mRNA and protein levels of bFGF is blocked by the presence in the medium of an antibody raised against the transforming growth factor alpha (TGFalpha) receptor. This observation indicates that, TGFalpha, whose synthesis is modulated by oestrogens in hypothalamic astrocytes and which is able to increase, both the mRNA and the protein levels of bFGF in our experimental model, may act as the mediator of the oestrogenic induction of bFGF. Hypothalamic astrocytes, together with hypothalamic neurones synthesizing and secreting luteinizing hormone-releasing hormone (LHRH), form the LHRH network in conjunction with other neuronal systems. Gonadal steroids in general, and oestrogens in particular, play an important role in the control of the activity of this network. In addition, bFGF and TGFalpha, two growth factors released from astrocytes, are able to influence the activity of LHRH neurones. The present observations suggest that oestrogens may also act on LHRH neurones in an indirect fashion (i.e. by modulating the expression of bFGF and TGFalpha in glial cells).     

外源性碱性成纤维细胞生长因子对缺血大鼠脑内源性碱性成纤维细胞生长因子表达的影响

目的 研究外源性碱性成纤维细胞生长因子(bFGF)缩小局灶性脑缺血梗死灶的机制。方法 用免疫组化ABC法检测在局灶性脑缺血模型上给予生理盐水或bFGF后早期生长反应蛋白-1(Egr-1)，bFGF，碱性成纤维细胞生长因子受体(bFGFR)的动态表达。结果 给药组在3h～3d各时间段梗死灶均有不同程度的缩小。对照组和给药组Egr-1表达均表现为3～6h的增强过程，但给药组更强于对照组。对照组12h见有bFGF表达增强，而bFGFR表达3h到达高峰，6h起下降，12h时bFGFR的表达已恢复至正常水平(出现了配体和受体表达时相上不匹配)。给药组bFGF表达提前且增强，3h即见有bFGF表达增强，6h时出现第一峰，从而与bFGFR 3～6h的表达增强过程相吻合。结论 外源性bFGF能缩小梗死灶，该神经保护作用是通过Egr-1蛋白高表达使内源性bFGF的表达增高且提前，从而与bFGFR的表达增强过程重叠而实现的。