Journal of Neuroscience Research 30:493-497 (1991)
Epidermal Growth Factor Exerts Neuronotrophic Effects on Dopaminergic and GABAergic CNS Neurons: Comparison With Basic Fibroblast Growth Factor G. Ferrari, G. Toffano, and S.D. Skaper Fidia Research Laboratories, Abano Terme (PD), Italy
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, neurotransmitteridentified 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 RT97 and ELISA confirmed the differential response, bFGF being 2-2.5 times more effective at all concentrations tested (ED,,,: 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. Key words: epidermal growth factor, trophic action, neurons, dopamine, GABA, mesencephalon INTRODUCTION Neuronotrophic factors, a class of polypeptide growth factors that acts on neurons of the peripheral and central nervous systems, maintain cell vitality and stimulate neurite outgrowth (Varon et al., 1988). Nerve growth factor (NGF), a trophic protein with a well-defined role in the peripheral nervous system (Levi-Montalcini, 1987), has, more recently, been identified within the central nervous system (CNS) and has been proposed to act as a trophic factor for particular subsets of cholinergic neurons (Barde, 1989; Dreyfus, 1989). 0 1991 Wiley-Liss, Inc.
The primary neuronotrophic factor for the majority of CNS neurons remains unknown. Additional trophic activities for various types of CNS neurons have been described, but extensive characterization has been lacking. However, two homogeneous proteins, namely basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), have been reported to enhance survival and neurite extension of CNS neurons in vitro (Ferrari et al., 1989; Hatten et al., 1988; Morrison et al., 1986, 1987, 1988; Unsicker et a]., 1987; Walicke, 1988; Walicke et al., 1986). Epidermal growth factor, a potent polypeptide mitogen originally purified from the male submaxillary gland (Cohen, 1962), has been identified in the mammalian CNS (Fallon et al., 1984; however, see Probstmeier and Schachner, 1986), along with its mRNA (Lazar et al., 1988) and receptor (Adamson and Meek, 1984; Werner et al., 1988; Nieto-Sampedro et al., 1988; Chabot et al., 1988). A nonmitogenic function for EGF in this tissue is supported by the demonstration that EGF enhances survival and neurite outgrowth of postnatal rat striatal, cortical, and cerebellar neurons in primary culture (Knusel et al., 1990; Morrison et al., 1987, 1988). Because of the relative heterogeneity of neuronal cell types obtained from CNS tissue taken from even limited anatomical areas, it is important to define the responses of biochemically identifiable (e.g., in terms of neurotransmitter traits) CNS neurons to the trophic actions of EGF. In this report, we show that EGF promotes survival and stimulates fiber outgrowth from rat mesencephalicderived dopaminergic and GAB Aergic neurons in primary culture.
Received October 3, 1990; revised February 11, 1991; accepted February 11, 1991. Address reprint requests to G. Ferrari, Fidia Research Laboratories, Via Ponte della Fabbrica 3/A, 35031 Abano Terme (PD), Italy.
494
Ferrari et al.
MATERIALS AND METHODS Materials Eagle's basal medium and Ham's F12 medium were obtained from Flow (Milan, Italy). Bovine pancreatic insulin, human transferrin, putrescine, progesterone, soybean trypsin inhibitor, and DNase were from Sigma (St. Louis, MO). Basic fibroblast growth factor was obtained from the FGF Company (San Francisco, CA) and EGF from Bethesda Research Laboratories (Gaithersburg, MD). Fetal calf serum (FCS) was obtained from Seromed (Berlin, FRG), and affinity-purified horseradish peroxidase-conjugated goat antimouse immunoglobulins from Vector. Mesencephalic Cell Cultures Rostra1 mesencephalic tegmentum was dissected from 14-day-old Sprague-Dawley rat embryos (Charles River, Como, Italy), dissociated, and cells cultured as previously described (Ferrari et al., 1989). Polyornithine-coated wells were seeded with 3 X lo5 cells per 22-mm well (12-well cluster) or 4 x lo4 cells per 6-mm well (96-well cluster). Where indicated, EGF or bFGF was added 4 hr after cell plating. No further additions or changes of medium were made. Dopaminergic and GABAergic neurons each represented about 1% of the initially plated neurons.
RT97 (1:1,000 dilution of ascites fluid in 10% FCSPBS) was then applied for 60 min at 25"C, washed three times with PBS containing 10% FCS, and then incubated with affinity-purified horseradish peroxidase-conjugated goat antimouse immunoglobulin (HRP-RAM, 1:1000 dilution) for a further 60 min. After three washes each with PBS and then with distilled water, wells were incubated with 50 p1 of 0.2% (w/v) O-phenylenediamine (OPD) and 0.02% (v/v) H20, in citrate-phosphate buffer, pH 5.0. The conversion of OPD to its oxidized product was stopped after 30 rnin by addition of 50 pl of 4.5 M H,S04. Chromogen formation was determined by measuring optical density at 492 nm with a titertek Multiskan@,and blanking against the appropriate control incubation mixture.
RESULTS
The addition of EGF (3 ng/ml) to dissociated cultures of mesencephalic neurons markedly enhanced cell survival and neurite outgrowth (Fig. 1A,B). Cultures treated with EGF for 20 days exhibited numerous cells with long, branched processes that stained positively for neurofilament protein. Cells immunopositive for GFAP were virtually nonexistent (less than 0.1 % of total cells present), suggesting that EGF does not exert its effects indirectly via astroglial cell proliferation. The fetal age of tissue used would not be expected to favor the growth Neurotransmitter Uptake of glial cells. Note, however, the more extensive neurite High-affinity uptake of [3H]dopamine and y-[ 14C]- field when these same mesencephalic cells are cultured aminobutyric acid was performed as described (Ferrari et for 20 days with 3 ng/ml of bFGF (Fig. 1C). al., 1989). The apparent differential abilities of EGF and bFGF in inducing neurite outgrowth at the microscopic Indirect Immunohistochemical Staining level were quantified using the neurofilament monoStaining of neurofilament and glial fibrillary acidic clonal antibody RT97 and ELISA. At 20 days in vitro, protein (GFAP) was carried as described previously (Fer- both EGF and bFGF increased neurofilament expression rari et al., 1989). in a concentration-dependent manner, with saturation being achieved at 3-10 ng/ml for either EGF or bFGF (Fig. Quantitation of Neurofilament Expression 2B). Basic FGF appeared to be more active, producing about 2-2.5 times more reaction at all concentrations, in Neurofilament expression was quantified utilizing comparison to EGF. This greater activity of bFGF was an enzyme-linked immunosorbent assay (ELISA) with the monoclonal antibody RT97, which binds to the 150- more pronounced after 10 days in vitro, where EGF was at 30 ng/ml (Fig. 2A). Unlike kDa neurofilament subunit (Anderton et al., 1982). Mes- without effect-even encephalic cells were plated on polyornithine-coated, 96- bFGF, EGF did not appear to have any morphological well culture plates at 40,000 cells per well. At different effect at 10 days (not shown), thus corroborating the times, control and bFGF or EGF-treated cells were neurofilament results. Since these mesencephalic cultures consist of diwashed with phosphate-buffered saline (PBS), fixed with paraformaldehyde (4% in PBS) for 30 min at 25°C verse neuronal populations, it was of interest to examine washed twice with PBS, methanol-permeabilized at further the trophic effects of EGF on transmitter-identi-20°C (10 rnin), and washed again in PBS. Plates were fied cell types. Such uptake parameters provide a good stored at this stage at 4°C until a complete series was indication of the number of surviving neurons (Leon et ready for further processing. The fixed and permeabi- al., 1988). The uptakes of radioactively labeled dopalized cells were washed with PBS and blocked for 60 rnin mine and y-aminobutyric acid (GABA) were therefore with 10% (v/v) FCS in PBS. The monoclonal antibody assessed. Mesencephalic cultures treated for 20 days
EGF Effects on Mesencephalic Neurons
0
2
6
4
8
495
10
ngim1
Fig. 2. Concentration-dependent increase in neurofilament protein expression elicited by EGF ( 0 ) and bFGF (0) in mesencephalic neurons after 10 days (A) and 20 days (B) in vitro. Neurofilament was quantitated using the monoclonal antibody RT97 and ELISA. Values are expressed as percent of control (no factor): mean 2 SD ( n = 12). In arbitrary absorbance units x (492 nm), the control values were 271 27 (10 days) and 77 I+_ 7 (20 days).
*
with EGF (3 ng/ml) displayed an approximate 4-fold increase in both [3H]dopamine and [ 14C]GABA uptakes (Fig. 3). Culturing these cells with bFGF (3 ng/ml) also stimulated [ 14C]GABAuptake 4-fold, but [3H]dopamine uptake almost 20-fold (Fig. 3), as described previously (Ferrari et al., 1989). Thus, while growth factor enhancement of uptake in mesencephalic cells is not limited to a single neurotransmitter type, action on dopamine uptake is preferentially addressed by bFGF.
DISCUSSION
Fig. 1. Morphology of mesencephalic neurons in the presence of no additions (A), epidermal growth factor (3 ng/ml) (B), or basic FGF (3 ng/ml) (C). Neuronal cultures were established as described in Methods. Cells were maintained for 20 days, and then stained for neurofilament protein.
Very little is known concerning factors that may serve a trophic function in the CNS. With the exception of nerve growth factor (Barde, 1989), it has been difficult to identify putative trophic moIecuIes from brain tissue, as well as target neuronal populations. Epidermal and basic growth factors are polypeptide mitogens (Coheny 1962; Bohlen et al-9 l984) that have recently been found to have neuronotrophic activity for CNS neurons in vitro (Ferrari et al., 1989; Hatten et al., 1988; Morrison et al., 1986, 1987, 1988; Unsicker et al.,
496
Ferrari et al.
ulated [3H]dopamine uptake at 20 days in vitro in mesencephalic cells some 20-fold (see also Ferrari et al., 1989). An important difference thus becomes evident between the trophic effects of EGF and bFGF. The greater sensitivity of dopaminergic, in comparison to 1600GABAergic neurons, to the trophic effects of bFGF was substantiated here and shown not to extend to EGF. Us1200ing mesencephalic cultures prepared from embryonic day 16/17 rat fetuses, Knusel et al. (1990) observed only a 800modest (less than 2-fold) stimulation of dopamine uptake by both bFGF and EGF (100-1,000 ng/ml) at 7 days in vitro. This lack of specificity between the two growth factors could result from differences in embryonic age of the tissue used or region dissected, plating density, culC EGF bFGF C EGF bFGF ture age, or specific activity of the bFGF. The trophic effect of EGF probably reflects a direct Fig. 3. Stimulation of [3H]dopamine and y-[14C]aminobutyric action on mesencephalic neurons, although indirect efuptakes in mesencephalic cells by EGF and bFGF. Cultures fects mediated by contaminating nonneuronal cells can were treated for 20 days with 3 ng/ml of either EGF or bFGF, never be totally excluded. The percentage of glial conand [3H]dopamineand [I4C]GABA uptakes measured as detamination, measured by GFAP immunoreactive cells scribed under Materials and Methods. Values are expressed as (
Epidermal Growth Factor Exerts Neuronotrophic Effects on Dopaminergic and GABAergic CNS Neurons: Comparison With Basic Fibroblast Growth Factor G. Ferrari, G. Toffano, and S.D. Skaper Fidia Research Laboratories, Abano Terme (PD), Italy
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, neurotransmitteridentified 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 RT97 and ELISA confirmed the differential response, bFGF being 2-2.5 times more effective at all concentrations tested (ED,,,: 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. Key words: epidermal growth factor, trophic action, neurons, dopamine, GABA, mesencephalon INTRODUCTION Neuronotrophic factors, a class of polypeptide growth factors that acts on neurons of the peripheral and central nervous systems, maintain cell vitality and stimulate neurite outgrowth (Varon et al., 1988). Nerve growth factor (NGF), a trophic protein with a well-defined role in the peripheral nervous system (Levi-Montalcini, 1987), has, more recently, been identified within the central nervous system (CNS) and has been proposed to act as a trophic factor for particular subsets of cholinergic neurons (Barde, 1989; Dreyfus, 1989). 0 1991 Wiley-Liss, Inc.
The primary neuronotrophic factor for the majority of CNS neurons remains unknown. Additional trophic activities for various types of CNS neurons have been described, but extensive characterization has been lacking. However, two homogeneous proteins, namely basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), have been reported to enhance survival and neurite extension of CNS neurons in vitro (Ferrari et al., 1989; Hatten et al., 1988; Morrison et al., 1986, 1987, 1988; Unsicker et a]., 1987; Walicke, 1988; Walicke et al., 1986). Epidermal growth factor, a potent polypeptide mitogen originally purified from the male submaxillary gland (Cohen, 1962), has been identified in the mammalian CNS (Fallon et al., 1984; however, see Probstmeier and Schachner, 1986), along with its mRNA (Lazar et al., 1988) and receptor (Adamson and Meek, 1984; Werner et al., 1988; Nieto-Sampedro et al., 1988; Chabot et al., 1988). A nonmitogenic function for EGF in this tissue is supported by the demonstration that EGF enhances survival and neurite outgrowth of postnatal rat striatal, cortical, and cerebellar neurons in primary culture (Knusel et al., 1990; Morrison et al., 1987, 1988). Because of the relative heterogeneity of neuronal cell types obtained from CNS tissue taken from even limited anatomical areas, it is important to define the responses of biochemically identifiable (e.g., in terms of neurotransmitter traits) CNS neurons to the trophic actions of EGF. In this report, we show that EGF promotes survival and stimulates fiber outgrowth from rat mesencephalicderived dopaminergic and GAB Aergic neurons in primary culture.
Received October 3, 1990; revised February 11, 1991; accepted February 11, 1991. Address reprint requests to G. Ferrari, Fidia Research Laboratories, Via Ponte della Fabbrica 3/A, 35031 Abano Terme (PD), Italy.
494
Ferrari et al.
MATERIALS AND METHODS Materials Eagle's basal medium and Ham's F12 medium were obtained from Flow (Milan, Italy). Bovine pancreatic insulin, human transferrin, putrescine, progesterone, soybean trypsin inhibitor, and DNase were from Sigma (St. Louis, MO). Basic fibroblast growth factor was obtained from the FGF Company (San Francisco, CA) and EGF from Bethesda Research Laboratories (Gaithersburg, MD). Fetal calf serum (FCS) was obtained from Seromed (Berlin, FRG), and affinity-purified horseradish peroxidase-conjugated goat antimouse immunoglobulins from Vector. Mesencephalic Cell Cultures Rostra1 mesencephalic tegmentum was dissected from 14-day-old Sprague-Dawley rat embryos (Charles River, Como, Italy), dissociated, and cells cultured as previously described (Ferrari et al., 1989). Polyornithine-coated wells were seeded with 3 X lo5 cells per 22-mm well (12-well cluster) or 4 x lo4 cells per 6-mm well (96-well cluster). Where indicated, EGF or bFGF was added 4 hr after cell plating. No further additions or changes of medium were made. Dopaminergic and GABAergic neurons each represented about 1% of the initially plated neurons.
RT97 (1:1,000 dilution of ascites fluid in 10% FCSPBS) was then applied for 60 min at 25"C, washed three times with PBS containing 10% FCS, and then incubated with affinity-purified horseradish peroxidase-conjugated goat antimouse immunoglobulin (HRP-RAM, 1:1000 dilution) for a further 60 min. After three washes each with PBS and then with distilled water, wells were incubated with 50 p1 of 0.2% (w/v) O-phenylenediamine (OPD) and 0.02% (v/v) H20, in citrate-phosphate buffer, pH 5.0. The conversion of OPD to its oxidized product was stopped after 30 rnin by addition of 50 pl of 4.5 M H,S04. Chromogen formation was determined by measuring optical density at 492 nm with a titertek Multiskan@,and blanking against the appropriate control incubation mixture.
RESULTS
The addition of EGF (3 ng/ml) to dissociated cultures of mesencephalic neurons markedly enhanced cell survival and neurite outgrowth (Fig. 1A,B). Cultures treated with EGF for 20 days exhibited numerous cells with long, branched processes that stained positively for neurofilament protein. Cells immunopositive for GFAP were virtually nonexistent (less than 0.1 % of total cells present), suggesting that EGF does not exert its effects indirectly via astroglial cell proliferation. The fetal age of tissue used would not be expected to favor the growth Neurotransmitter Uptake of glial cells. Note, however, the more extensive neurite High-affinity uptake of [3H]dopamine and y-[ 14C]- field when these same mesencephalic cells are cultured aminobutyric acid was performed as described (Ferrari et for 20 days with 3 ng/ml of bFGF (Fig. 1C). al., 1989). The apparent differential abilities of EGF and bFGF in inducing neurite outgrowth at the microscopic Indirect Immunohistochemical Staining level were quantified using the neurofilament monoStaining of neurofilament and glial fibrillary acidic clonal antibody RT97 and ELISA. At 20 days in vitro, protein (GFAP) was carried as described previously (Fer- both EGF and bFGF increased neurofilament expression rari et al., 1989). in a concentration-dependent manner, with saturation being achieved at 3-10 ng/ml for either EGF or bFGF (Fig. Quantitation of Neurofilament Expression 2B). Basic FGF appeared to be more active, producing about 2-2.5 times more reaction at all concentrations, in Neurofilament expression was quantified utilizing comparison to EGF. This greater activity of bFGF was an enzyme-linked immunosorbent assay (ELISA) with the monoclonal antibody RT97, which binds to the 150- more pronounced after 10 days in vitro, where EGF was at 30 ng/ml (Fig. 2A). Unlike kDa neurofilament subunit (Anderton et al., 1982). Mes- without effect-even encephalic cells were plated on polyornithine-coated, 96- bFGF, EGF did not appear to have any morphological well culture plates at 40,000 cells per well. At different effect at 10 days (not shown), thus corroborating the times, control and bFGF or EGF-treated cells were neurofilament results. Since these mesencephalic cultures consist of diwashed with phosphate-buffered saline (PBS), fixed with paraformaldehyde (4% in PBS) for 30 min at 25°C verse neuronal populations, it was of interest to examine washed twice with PBS, methanol-permeabilized at further the trophic effects of EGF on transmitter-identi-20°C (10 rnin), and washed again in PBS. Plates were fied cell types. Such uptake parameters provide a good stored at this stage at 4°C until a complete series was indication of the number of surviving neurons (Leon et ready for further processing. The fixed and permeabi- al., 1988). The uptakes of radioactively labeled dopalized cells were washed with PBS and blocked for 60 rnin mine and y-aminobutyric acid (GABA) were therefore with 10% (v/v) FCS in PBS. The monoclonal antibody assessed. Mesencephalic cultures treated for 20 days
EGF Effects on Mesencephalic Neurons
0
2
6
4
8
495
10
ngim1
Fig. 2. Concentration-dependent increase in neurofilament protein expression elicited by EGF ( 0 ) and bFGF (0) in mesencephalic neurons after 10 days (A) and 20 days (B) in vitro. Neurofilament was quantitated using the monoclonal antibody RT97 and ELISA. Values are expressed as percent of control (no factor): mean 2 SD ( n = 12). In arbitrary absorbance units x (492 nm), the control values were 271 27 (10 days) and 77 I+_ 7 (20 days).
*
with EGF (3 ng/ml) displayed an approximate 4-fold increase in both [3H]dopamine and [ 14C]GABA uptakes (Fig. 3). Culturing these cells with bFGF (3 ng/ml) also stimulated [ 14C]GABAuptake 4-fold, but [3H]dopamine uptake almost 20-fold (Fig. 3), as described previously (Ferrari et al., 1989). Thus, while growth factor enhancement of uptake in mesencephalic cells is not limited to a single neurotransmitter type, action on dopamine uptake is preferentially addressed by bFGF.
DISCUSSION
Fig. 1. Morphology of mesencephalic neurons in the presence of no additions (A), epidermal growth factor (3 ng/ml) (B), or basic FGF (3 ng/ml) (C). Neuronal cultures were established as described in Methods. Cells were maintained for 20 days, and then stained for neurofilament protein.
Very little is known concerning factors that may serve a trophic function in the CNS. With the exception of nerve growth factor (Barde, 1989), it has been difficult to identify putative trophic moIecuIes from brain tissue, as well as target neuronal populations. Epidermal and basic growth factors are polypeptide mitogens (Coheny 1962; Bohlen et al-9 l984) that have recently been found to have neuronotrophic activity for CNS neurons in vitro (Ferrari et al., 1989; Hatten et al., 1988; Morrison et al., 1986, 1987, 1988; Unsicker et al.,
496
Ferrari et al.
ulated [3H]dopamine uptake at 20 days in vitro in mesencephalic cells some 20-fold (see also Ferrari et al., 1989). An important difference thus becomes evident between the trophic effects of EGF and bFGF. The greater sensitivity of dopaminergic, in comparison to 1600GABAergic neurons, to the trophic effects of bFGF was substantiated here and shown not to extend to EGF. Us1200ing mesencephalic cultures prepared from embryonic day 16/17 rat fetuses, Knusel et al. (1990) observed only a 800modest (less than 2-fold) stimulation of dopamine uptake by both bFGF and EGF (100-1,000 ng/ml) at 7 days in vitro. This lack of specificity between the two growth factors could result from differences in embryonic age of the tissue used or region dissected, plating density, culC EGF bFGF C EGF bFGF ture age, or specific activity of the bFGF. The trophic effect of EGF probably reflects a direct Fig. 3. Stimulation of [3H]dopamine and y-[14C]aminobutyric action on mesencephalic neurons, although indirect efuptakes in mesencephalic cells by EGF and bFGF. Cultures fects mediated by contaminating nonneuronal cells can were treated for 20 days with 3 ng/ml of either EGF or bFGF, never be totally excluded. The percentage of glial conand [3H]dopamineand [I4C]GABA uptakes measured as detamination, measured by GFAP immunoreactive cells scribed under Materials and Methods. Values are expressed as (
