Nerve Growth Factor (NGF)

Journal of Neuroscience Research 33:82-90 (1992)

Receptors in a Central Nervous System Glial Cell Line: Upregulation by NGF and Brain-Derived Neurotrophic Factor P.E. Spoerri, S. Romanello, L. Petrelli, A. Negro, R. Dal TOSO, A. Leon, and S.D. Skaper Department of Cellular Biology, Fidia Research Laboratories (P.E.S., S.R. , L.P., R.D.T., A.L., S .D.S.), and Advanced Technology Division (A.N.), Fidia S.p.A., Abano Terme (PD), Italy

The neurotrophic proteins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are related in their primary amino acid structures. In this study we investigated the extent to which the low-affinity NGF receptor (LNGFR) in C6 glioma cells can discriminate between the neurotro- phins NGF and BDNF. LNGFR-immunoreactivity (IR) was studied in C6 cells treated for 16 hr with NGF (50 ng/ml) or BDNF (10 ng/ml), using immu- nogold labeling and electron microscopic mor- phometric analysis. The cells were exposed to the anti-NGFR antibody 192-IgG, followed by immuno- globulin conjugated with colloidal gold. Untreated C6 cells exhibited some surface gold label (positive LNGFR-IR). Cells treated with NGF or BDNF dis- played significantly increased LNGFR-IR on all sur- faces in terms of gold labeling, which was more pro- nounced in NGF-treated cells. LNGFR-IR was also localized in coated endocytotic vesicles, in smooth en- doplasmic reticulum, and in secondary multivesicular lysosomes in neurotrophin-treated and untreated cells. The increase in LNGFR protein was further substantiated by a correspondingly higher content of LNGFR mRNA detected after 15 hr of either NGF or BDNF treatment. These results suggest that the LNGFR in glial cells can be upregulated by the struc- turally related neurotrophins NGF and BDNF. 0 1992 Wiley-Liss, Inc.

Key words: neurotrophins, low affinity, colloidal gold, immunoelectron microscopy, mRNA

INTRODUCTION Nerve growth factor (NGF) (Levi-Montalcini,

1987) is a member of a gene family termed neurotro- phins that encodes functionally and structurally related proteins: brain-derived neurotrophic factor (BDNF) and

neurotrophin-3 (NT-3) (Bothwell, 1991). BDNF has been purified (Barde et al., 1982) and its complete se- quence obtained by cDNA cloning (Leibrock et al., 1989). The high degree of sequence identity between NGF and BDNF has led to the cDNA cloning of another related protein, NT-3, also known as hippocampus-de- rived neurotrophic factor (HDNF) (Hohn et al., 1990; Ernfors et al., 1990; Maisonpierre et al., 1990; Rosenthal et al., 1990) or NGF-2 (Kaisho et al., 1990).

Neurotrophin effects are mediated by interaction with their heterogeneous receptors (referred to as NGFRs) present on specific neuronal cell populations (Thoenen, 199 1). Binding analyses have identified two receptor classes for NGF and BDNF: the high-affinity receptor (HNGFR) and the low-affinity receptor (LNGFR) (Sutter et al., 1979; Rodriguez-Tebar and Barde, 1988). The biological effects of NGF require in- ternalization of NGF via binding to the HNGFR (Bernd and Greene, 1984; Green et al., 1986). The finding that NGF-responsive neurons do not express high-affinity BDNF receptors suggests that BDNF utilizes a different high-affinity receptor than does NGF (Rodriguez-Tebar and Barde, 1988). HNGFRs exist that bind either NGF or BDNF; in contrast, the LNGFRs bind all three neu- rotrophins with similar affinities (Rodriguez-Tebar et al., 1990; Squint0 et al., 1991).

NGF increases the expression of LNGFRs on PC12 cells, as measured by NGFR protein (using ['251-NGF] binding or anti-NGFR antibody) (Bernd and Greene, 1984; Doherty et al., 1988) or LNGFR mRNA (Doherty et al., 1988). NGF also upregulates the expression of NGFR and NGFR mRNA in adult sensory neurons in

Received March 16, 1992; accepted April 20, 1992.

Address reprint requests to Dr. P.E. Spoeni, Department of Cellular Biology, Via Ponte della Fabbrica, 3/A, 35031 Abano Terme (PD), Italy.

0 1992 Wiley-Liss, Inc.

Neurotrophin Upregulation of NGFRs in GIial Cells 83

1981). After 3 days, the conditioned medium was cen- trifuged and filtered to remove cells and debris, and stored at -80°C until used. The biological activity of BDNF was measured in 48 hr neuronal survival assays of chicken embryo day 10 dorsal root ganglia (DRG) neu- rons on a polyornithine/laminin substratum (Skaper et al., 1990). Half-maximal activity was obtained at a 1:16 dilution of COS cell medium (approximately 3 ng/ml BDNF), and was not blocked by anti-mouse NGF anti- bodies. Medium from COS cells transfected with control vector alone failed to support DRG neuronal survival. BDNF (10 ng/ml as conditioned medium) was added to 24 hr cultures of C6 cells for 16 hr.

vitro (Higgins et al., 1989) and in central nervous system (CNS) neurons in vivo (Cavicchioli et al., 1989; Gage et al., 1989).

C6 rat glioma cells express LNGFRs which are immunoprecipitable with the antibody 192-IgG (Chand- ler et al., 1984; Kumar et al., 1990). Since little is known about the functional role of LNGFRs on CNS glial cells and their interaction with neurotrophins, the immunolo- calization of LNGFR on C6 cells was examined using 192-IgG, after treatment of the cells with either NGF or BDNF. Here we show that both neurotrophins signifi- cantly increase the amount of LNGFR immunoreactivity on the surface membrane of C6 cells and that this is accompanied by a concomitant increase in the content of LNGFR mRNA.

MATERIALS AND METHODS Materials

Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), glutamine, penicillin, EDTA, and Hanks’ balanced salt solution (HBSS) were obtained from Gibco (Grand Island, NY); goat anti-mouse (GAM) affinity purified antibody conjugated with 15 nm gold particles from Amersham (Buckinghamshire, UK); os- mium tetroxide, glutaraldehyde, and Epon 812 from Fluka (Buchs, Switzerland); ,’P-dCTP from NEN-Du- pont (Boston, MA); and random priming kit and Hybond nylon membranes from Amersham.

Cell Cultures and Experimental Treatments The rat C6 glioma cell line (C6: CCL 107) was

obtained from the American Type Culture Collection (Rockville, MD) and used between passage numbers 35 and 45. Stock cultures were maintained in DMEM sup- plemented with 10% (vh) heat-inactivated FCS, 44.4 rnM NaHCO,, 100 U/ml penicillin, and 2 mM L-glu- tamine, in 75 cm’ flasks in a humidified atmosphere of 5% C02/95% air at 37°C. Cells were subcultured prior to reaching confluency every 3-4 days, using 0.5 mM EDTA in Ca2’, Mg2+-free phosphate buffered saline (pH 7.2) to dislodge the cells.

For treatment cells were harvested, diluted into the above medium, and seeded at lo5 cells/35 mm dish. The beta subunit of NGF was purified from mouse submax- illary glands as described (Mobley et al., 1976). Twenty- four hours later, NGF (50 ng/ml) was added to the cul- tures for an additional 16 hr.

Recombinant rat BDNF was produced in our lab- oratories as follows: a BDNF cDNA was isolated from a rat brain cDNA library (Negro et al., unpublished obser- vations). Expression of BDNF was achieved by tran- siently introducing an SV40 expression vector containing the BDNF coding sequence into COS cells (Glutzman,

Immunoelectron Microscopy C6 cells grown as above were washed once with

HBSS. Fresh medium containing a 1500 dilution of rat anti-mouse antibody to NGFR (anti-NGFR; 192-IgG) (Chandler et al., 1984), generously supplied by Dr. Eu- gene Johnson, was added to the cultures and incubated with gentle agitation for 30 min at 37°C in a humidified atmosphere of 5% CO, and 95% air. After removal of the medium, cells were washed twice with HBSS and fresh culture medium was added containing a 1 : 15 dilution of GAM antibody conjugated with 15 nm gold particles and gently agitated, as above. The cultures were washed 5 times with HBSS and processed for transmission electron microscopy (TEM).

TEM For TEM, cells grown on plastic dishes were

drained of medium, fixed at room temperature in 2.5% glutaraldehyde in 0.08 M phosphate buffer (pH 7.3) for 30 min, and postfixed in 1% osmium tetroxide in 0.08 M phosphate buffer containing 0.1 M sucrose for 60 min. After dehydration and embedding, selected areas were sectioned and stained with a saturated solution of uranyl acetate, examined, and photographed using a Philips CM 12 electron microscope.

Morphometric Analysis Using the TEM conditions described above, a min-

imum of 45 electron micrographs were obtained from randomly selected samples of triplicate cultures of the variously treated C6 cells. From these electron micro- graphs ( X 22,000), data were extracted using direct planimetry measurements (Bendayan, 1984). After eval- uation of the surface distance (Sa) occupied by the plas- malemma in a defined compartment using a VIDS V semiautomatic image analysis system (AMS, UK), the number of gold particles (Ni) per unit area per electron micrograph was counted and the density of labeling (Ns) calculated: Ns = Ni/Sa. Quantitative evaluation was

84 Spoerri et al.

performed on specifically labeled tissue culture sections of treated and control cultures.

Northern Blot Hybridization Cells were harvested in 5 M guanidine isothiocy-

anate and total RNA isolated by the cesium chloride method (Chirgwin et al., 1979). Poly-(A+) RNA was separated by oligo (dt)-cellulose chromatography (Aviv and Leder, 1972), and 10 pg of glyoxylated poly-(A +) RNA was loaded onto each lane of 1% agarose gels (Maniatis et al., 1982). After electrophoresis, RNA was transferred to Hybond nylon membranes according to the manufacturer’s instructions. A 700 bp Bam HI fragment of the cDNA clone for the rat LNGFR (Radeke et al., 1987), obtained from Dr. Eric M. Shooter, and a 900 bp PstI fragment of the rat cyclophilin (plB15) probe (Mil- ner and Sutcliffe, 1983), obtained from Dr. Robert J. Milner, were 32P-labeled using a commercially available random priming kit. Filters were hybridized and washed (Dickson et al., 1986) and exposed to Kodak X-OMAT R films with intensifying screens at -80°C. For semi- quantitative estimations of NGFR and cyclophilin mRNA levels, the autoradiographs were scanned with a computerized densitometer (Model 300A, Molecular Dynamics).

Statistical Analysis One-way analysis of variance (ANOVA) was used

to test the effects of NGF or BDNF treatment. Where significant effects were found, Tukey’s test was em- ployed to evaluate differences between NGF and BDNF treatment groups. Significance was defined as P < 0.05.

RESULTS NGF and BDNF Upregulate the LNGFR Protein in C6 Cells

Ultrastructural immunolocalization of the LNGFR revealed that native C6 cells exhibited occasional gold labeling (Fig. la). Incubation of cells with NGF (50 ng/ml; 16 hr) increased gold labeling on all surfaces (Fig. lb). Similarly, exposure of cells to BDNF (10 ng/ml; 16 hr) augmented gold labeling (Fig. lc). These effects of BDNF are a direct action of the recombinant protein, as the biological activity of BDNF was not blocked by anti- mouse NGF polyclonal IgGs (Callegaro et al., 1990) or the monoclonal antibody aDll (Cattaneo et al., 1988); furthermore, nontransfected COS cells do not express detectable levels of NGF (by enzyme-linked immunosor- bent assay or bioassay) or NGF mRNA (data not shown).

Morphometric analysis of electron micrographs showed a significant 3.2- and 2.5-fold increase in gold particles [positive LNGFR-immunoreactivity (IR)] in the NGF- or BDNF-treated C6 cells, respectively, after 16 hr

compared to control cells (Fig. 2). The differences in LNGFR-IR between NGF- and BDNF-treated cells were also significant. In addition, indirect immunofluores- cence using 192-IgG showed enhanced LNGFR-IR on the surfaces of both NGF- and BDNF-treated cells after 16 hr (data not shown).

LNGFR-immunoreactive gold particles were found engulfed in endocytotic-coated vesicles of the plasma- lemma. Similarly, gold-containing coated vesicles were observed within the cytoplasm. The immunoreactive gold particles were localized within the smooth endo- plasmic reticulum (SER) and within secondary lyso- somes (multivesicular appearance) found in the perinu- clear region in control and NGF- or BDNF-treated cells (Fig. 3a-d).

NGF and BDNF Increase LNGFR mRNA Content Previous reports have shown that mRNA levels of

the LNGFR are positively regulated by NGF in periph- eral and central nervous tissue neurons (Cavicchioli et al., 1989; Higgins et al., 1989; Gage et al., 1989) and in C6 glial cells (Kumar et al., 1990). To further support and extend these results, C6 cells were incubated with NGF (50 ng/ml) for 12 and 15 hr and the quantity of LNGFR mRNA assessed by Northern blotting. As shown in Figure 4, this treatment increased the expres- sion of LNGFR mRNA above control at 15 hr. An in- crease of the LNGFR mRNA above control was also observed in cultures treated for 12 and 15 hr with BDNF (10 nglml), the kinetics of which depicted some similar- ities to NGF, suggesting that both trophic factors may exert their effects through common or converging sig- naling pathways. In the same experiments the levels of mRNA for cyclophilin, a constitutively expressed pro- tein, were not modified by neurotrophin treatment, indi- cating that the observed increases of LNGFR mRNA were not due to an overall metabolic activation but to a transcriptional activation of specific target genes.

DISCUSSION The present study demonstrates that C6 glioma

cells exhibit positive LNGFR-IR. Immunolocalization of NGFR with colloidal gold revealed short, nonrandom ar- rangements of gold particles on the plasmalemma. Treat- ment with NGF, followed by immunolocalization with anti-NGFR antibody (192-IgG), led to a significant in- crease of LNGFR-IR in terms of gold labeling, compared to untreated C6 cells. Exposure of C6 cells to BDNF also significantly enhanced LNGFR-IR on all surfaces. In- creased LNGFR-IR in response to NGF or BDNF oc- curred in association with an increase in LNGFR mRNA, indicating the involvement of an amplifying mechanism related to gene activation caused by the structurally re-

Neurotrophin Upregulation of NGFRs in Glial Cells 85

Fig. 1. Electron micrographs of C6 cells treated with NGF or BDNF and exposed to anti-NGFR (192-IgG) followed by GAM-IgG conjugated with 15 nm gold particles. Control cells exhibited occasional NGFR-IR on their surfaces (a); NGF (50

ng/mI; 16 hr)-treated cells (b); BDNF (10 ng/ml; 16 hr)-treated cells (c). NGF and BDNF increased the level of gold label (positive NGFX-IR) on all exposed surfaces. X 55,000.

86 Spoerri et al.

: 41 7 **I

" CONTROL NCF BDNF

Fig. 2. Quantitation of the data in Figure 1. Results are ex- pressed as means ? SEM of the number of gold particles from a minimum of 45 electron micrographs per treatment. These were obtained from random samples of triplicate cultures from three independent experiments. One-way ANOVA was used to analyze the effect of treatment which was significant (F = 32.50; D.F. = 2,130; P < 0.001). Tukey's test was employed to evaluate differences between groups. The differences be- tween the NGF- and BDNF-treated groups were also signifi- cant (P < 0.05).

lated neurotrophins. These effects of NGF or BDNF do not likely reflect cell surface expansion; i .e., cell process outgrowth, as NGF and BDNF, at the concentrations and times used, did not modify cellular morphology.

The ability of neuronal cells to respond to NGF or BDNF is dependent on the presence of specific cell sur- face NGFRs (Yankner and Shooter, 1982). Exposure of neural tissue in vivo or in vitro to NGF increases expres- sion of LNGFRs and LNGFR mRNA (Cavicchioli et al., 1989; Doherty et al., 1988; Higgins et a]. , 1989; Gage et al., 1989), reflecting an unmasking of a plastic response. The LNGFR has been identified and a molecular clone isolated, encoding a 75-80 kd membrane protein (gp75) (Chao et al., 1986; Johnson et al., 1986; Radeke et al., 1987). The LNGFR does not appear to transduce a func- tional response; rather, biological responses to NGF ne- cessitate binding to the HNGFR (Zimmerman et al., 1978; Sutter et al., 1979; Bernd and Greene, 1984).

The role of LNGFRs in the formation of a func- tional HNGFR binding site remains an area of active investigation (Hempstead et al., 1989; Bothwell, 1991; Klein et al., 1991a,b). A 140 kd receptor protein which crosslinks to NGF does not include the LNGFR. Recent studies have identified the protooncogene trk gene prod- uct gp140trkA as a critical component of the HNGFR. This protein is phosphorylated on tyrosine residues in response to NGF and contains intrinsic tyrosine kinase activity (Meakin and Shooter, 1991). NGF-induced pro-

tein tyrosine phosphorylation requires expression of gp75, even though the latter does not possess tyrosine kinase activity (Berg et al., 1991). These data are con- sistent with a model in which gp75 may interact directly with gpl40trkA tyrosine kinase, an interaction presum- ably required for high-affinity NGF binding and induc- tion of protein tyrosine phosphorylation (Berg et al., 1991; Thoenen, 1991).

The LNGFR binds NGF, BDNF, and NT-3 with similar affinities (Rodriguez-Tebar et al., 1990; Squint0 et al., 1991); however, BDNF and NT-3, but not NGF, bind to gpl45trkB (Klein et al., 1989; Middlemas et al., 1991). In this context, the newest member of the NGF family, neurotrophin-5 (NT-5), can activate tyrosine phosphorylation of both trkA and trkB (Berkemeier et al., 1991). The trkB gene product gpl45trkB mediates functional responses to both BDNF and NT-3 in trans- fected NIH 3T3 fibroblasts lacking LNGFR, implying that the LNGFR is not essential for survival and prolif- eraton in these cells (Hempstead et al., 1991; Klein et al., 1991b; Glass et al., 1991). These results, however, do not exclude the possibility that gpl45trkB may form complexes with gp75 (Hempstead et al., 1991) or with other molecules to mediate the neurotrophic effects of BDNF and NT-3.

The function of LNGFR on glial cells is obscure (Johnson et al., 1988). [1251-NGF] immunocytochemis- try with anti-NGFR antibody (192-IgG) reveals that NGFRs are not confined to neurons, as Schwann cells also express LNGFRs (Zimmerman and Sutter, 1983; Yasuda et al., 1987; DiStefano and Johnson, 1988; Tani- uchi et al., 1988). Increases in LNGFR expression have been observed following transection or crush of the sci- atic nerve (Taniuchi et al., 1988; Heumann et al., 1987). NGF reportedly increases NG-CAM expression on Schwann cells (Seilheimer and Schachner, 1987), sug- gesting that LNGFRs may also activate cellular re- sponses to NGF. Binding of ['251-NGF] to isolated C6 cell membranes indicated the presence of both high- and low-affinity association kinetics (Kumar et al., 1990). In our experiments, LNGFR-IR was not confined to the plasma membrane of C6 cells. Internalization of immu- nogold particles was commonly seen, as in a similar study using PC12 cells (Spoerri and Roisen, 1992). NGFR-like immunoreactive particles were observed in endocytotic vesicles, SER, and multivesicular lyso- somes, suggesting the presence of HNGFRs, as only HNGFRs appear to undergo internalization (Hosang and Shooter, 1987; Pioro et al., 1990, 1991; Spoerri and Roisen, 1992). Furthermore, C6 cells express trk immu- noreactive antigen (Zanellato et al., in preparation). The present results do not exclude the possibility that high- affinity receptors for NGF and BDNF react with the LNGFR and that the antibody 192-IgG, reacting with

Neurotrophin Upregulation of NGFRs in Glial Cells 87

Fig. 3. Profiles from NGFR-immunoreactive C6 cells reveal- ing the presence of reaction product (gold particles) beneath the plasmalemma or in the perinuclear region. NGFR-IR is seen

within (a,b) endocytotic-coated vesicles (CV); (c) the smooth endoplasmic reticulum (SER); and (d) multivesicular second- ary lysosomes (LY). X 55,000.

88 Spoerri et al.

I-12h-l +15h--1

LNGFR

18s

p1 B15

a m m 9 9 .

I I NGF-TREATED CELLS BDNF-TREATED CELLS

1.5 -: 12 I5 b TIME (hrs)

Fig. 4. Effect of NGF and BDNF on NGFR mRNA content in C6 cells. C6 cells were treated with NGF (50 ng/ml) or BDNF (10 ng/ml) for 12 and 15 hr. Poly-(A+) RNA was isolated as reported in Materials and Methods and transferred to nylon filters. a: Northern blot showing hybridization performed with LNGFR and cyclophilin cDNA probes. b: Results are ex- pressed as percent of control value at each time point, and represent the ratio between the densitometric intensities of LNGFR and cyclophilin hybridization bands. Cyclophilin lev- els remain unchanged by NGF and BDNF treatment at all times.

IgG-conjugated gold, may reveal also the high-affinity form of the receptor (presumably in the copresence of the low-affinity component).

To date, NGFRs have not been detected in CNS glia except for Muller retinal glia, and Bermann glia during development. Antibody 192-IgG reportedly failed to label astrocytes from other brain regions (DiStefano and Johnson, 1988; Carmignoto et al., 1991); however, in cultures of immature astrocytes from neonatal mice, NGF increased the expression of the neural adhesion molecule L1 (Saad et al., 1991). Moreover, neonatal rat cortical astrocytes do not express NGFR mRNA (Flani- gan and Skaper, unpublished data). Whether this absence of NGFR in primary cortical astrocytes in contrast to C6 glioma cells is a consequence of culture conditions, or reflects more profound cell lineage changes in vitro, re- mains to be determined.

C6 cells can synthesize NGF (Schwarz et al., 1977; Kumar et al., 1990). This induction of NGF is preceded by, and may be dependent on, c-fos expression (Moc- chetti et al., 1989). These cells also contain basal levels of NGFR mRNA which can be upregulated either by NGF (Kumar et al., 1990) or, as described here by BDNF, presumably via binding to the LNGFR. The present study provides an interesting example of one neurotrophic factor modulating the receptivity to another neurotrophic factor by the same target cell. Upregulation of LNGFR by its own ligand, or by a structurally related molecule, may be of relevance in the context of glial- mediated reparative processes of impaired functions of NGF or BDNF responsive neurons following CNS dam- age.

ACKNOWLEDGMENTS We thank Dr. Diego Guidolin for assistance with

the morphometric and statistical analyses and Ms. Lorenza Polito for typing of the manuscript.

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