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(The FASEB Journal. 1999;13:1611-1620.)
© 1999 FASEB

Neuronal survival activity of S100ßß is enhanced by calcineurin inhibitors and requires activation of NF-{kappa}B

ARSHAK R. ALEXANIAN and JAMES R. BAMBURG1

Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870, USA

1Correspondence: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA. E-mail: jbamburg{at}vines.colostate.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
S100ßß is a calcium binding, neurotrophic protein produced by nonneuronal cells in the nervous system. The pathway by which it enhances neuronal survival is unknown. Here we show that S100ßß enhances survival of embryonic chick forebrain neurons in a dose-dependent manner. In the presence of suboptimal amounts of S100ßß, neuronal survival is enhanced by the immunosuppressants FK506 and cyclosporin A at concentrations that inhibit calcineurin, which is present in these cells. Rapamycin, an immunosuppressant that does not inhibit calcineurin, did not enhance cell survival. Cypermethrin, a direct and highly specific calcineurin inhibitor, mimicked the immunophilin ligands in its neurotrophic effect. None of the drugs stimulated neuronal survival in the absence of S100ßß. In the presence of suboptimal amounts of S100ßß, FK506, cyclosporin A, and cypermethrin (but not rapamycin) also increased NF-{kappa}B activity, as measured by immunofluorescence of cells stained with antibody to the active subunit (p65) and by immunoblotting of nuclear extracts. Antioxidant and glucocorticoid inhibitors of NF-{kappa}B decreased both the amount of active NF-{kappa}B and the survival of neurons caused by S100ßß alone or in the presence of augmenting drugs. We conclude that S100ßß enhances the survival of chick embryo forebrain neurons through the activation of NF-{kappa}B.—Alexanian, A. R., Bamburg, J. R. Neuronal survival activity of S100ßß is enhanced by calcineurin inhibitors and requires activation of NF-{kappa}B.


Key Words: immunosuppressants • immunophilins • cyclosporin A • FK506 • rapamycin • cypermethrin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MANY NEUROTROPHIC PROTEINS are present in the central and peripheral nervous systems and play important roles in neuronal development, differentiation, and survival. Recent evidence implicates neurotrophic factors in normal functional activity and plasticity of nerve cells and in mediating protective responses to trauma and disease. There are three well-recognized families of neurotrophic factors: neurotrophins (1 , 2) ; the fibroblast growth factors (3) ; and the neuropoietic cytokines (4) . Cytokines, traditionally thought to be messengers within the immune system, are now also known to be neurotrophic factors that can influence neuronal survival and differentiation, protect neurons when injured or stressed, and alter neuronal phenotype in response to environmental signals. They are also important factors in the progression of neuropathologic changes (4 , 5) . Along with the cytokines, proteins of another class that were thought to be immune specific—immunophilins—have been identified in the nervous system. The amounts of some of these proteins are enriched 10- to 50-fold in the central nervous system compared with tissues of the immune system (6) , suggesting that cytokines and immunophilins contribute to the development and function of the nervous systems.

Central nervous system injury provokes a limited acute-phase cellular and molecular response, including enhanced expression of cytokines (7) and immunophilins (8) , as well as neurotrophic factors important in healing and repair. One of these neurotrophic factors is S100ßß, a calcium binding protein that is produced and secreted by glial cells in the central nervous system and by Schwann cells in the peripheral nervous systems (9 , 10) . Its levels are elevated in Down's syndrome and Alzheimer's disease, where its production is detrimental to neurons due to its ability to elevate cytoplasmic calcium (9) and activate astrocyte nitric oxide synthase (NOS)2 (11) . S100ßß is trophic for many neuronal populations (12 , 13) , so its increased production in times of stress may be a compensatory response (14) . However, nothing is currently known about how S100ßß enhances cell survival.

Within the immune system, some immunosuppressant drugs form complexes with immunophilins that inhibit the activity of protein phosphatase 2B, calcineurin (15 , 16) . This calcium-activated phosphatase regulates the nuclear translocation of a transcription factor (NF-ATc) required for T cell activation (15) . Immunophilins might also regulate neuronal function via the regulation of calcineurin. Calcineurin dephosphorylates several important targets in the brain, activating growth-associated protein GAP-43, NOS, and the inhibitor (I{kappa}B) of the transcription factor, NF-{kappa}B (17 18 19) . The nuclear uptake of NF-{kappa}B is regulated by the binding of the cytoplasmic inhibitor protein, I{kappa}B (20) , which is colocalized with NF-{kappa}B to dendrites and postsynaptic densities in the hippocampus and cerebral cortex (21) . Activators of NF-{kappa}B in brain include glutamate and neurotrophins, suggesting synaptic activity is important to its function and that it may have a role in synaptic plasticity of adult neurons (22 , 23) . The pathways that lead to the activation of NF-{kappa}B require the release of the inhibitory subunit I{kappa}B (24) , which unmasks the nuclear localization sequence on the NF-{kappa}B p50 and p65 (RelA) subunits. This DNA binding dimer is then translocated to the nucleus. This process can be triggered by alterations in the activity of certain protein kinases (24 , 25) or by a reduction/oxidation (redox) cascade (26) . The activation of NF-{kappa}B by a redox-sensitive step (26 , 27) is genetically separable from a second redox-sensitive step at the level of DNA binding (28) , but might work via regulation of protein kinases as well. Distribution of NF-{kappa}B between the nucleus and cytoplasm is thus controlled by I{kappa}B; nuclear uptake directly correlates with the increased phosphorylation of I{kappa}B, which targets it for degradation. Dephosphorylation of I{kappa}B by calcineurin inhibits the activation of NF-{kappa}B and prevents its entry into the nucleus.

NF-{kappa}B is present in the cytoplasm in many cells (29) , where it can be activated in response to signal transduction pathways. Expression of NF-{kappa}B has been reported in adult neurons of the substantia nigra, hippocampus, striatum, and cerebral cortex of the rat (30) . Its role in neuronal development is also suggested from studies that demonstrate NF-{kappa}B activation in neuronal postsynaptic densities of the hippocampus and cerebral cortex during neurogenesis (31) . Involvement of NF-{kappa}B in brain function, particularly after injury and in progressive neurodegenerative conditions, has recently been reported (32 , 33) . Significantly, NF-{kappa}B activation by secreted amyloid precursor protein can counteract the apoptotic effects of mutant presenilin-1 (34) , suggesting that NF-{kappa}B activation might be important to neuronal cell survival in Alzheimer's disease.

In the present study, we examined the ability of S100ßß to promote neuronal survival and neurite outgrowth of embryonic chick forebrain neurons and the signaling mechanism by which survival is enhanced. At suboptimal concentrations of S100ßß, we demonstrate that immunosuppressants that form calcineurin-inhibitory complexes with immunophilins enhance survival, an effect that is mimicked by a specific and direct calcineurin inhibitor. We then demonstrate that this survival is mediated, at least in part, by the activation of NF-{kappa}B.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
All chemicals were reagent grade and, unless otherwise stated, were obtained from Sigma Chemical Co. (St. Louis, Mo).

Purification of S100ß and preparation of S100ßß dimer
S100ß was either obtained from Sigma or purified from bovine brain by the method of Isobe et al. (35) , modified to include zinc-dependent affinity chromatography on phenyl-Sepharose CL-4B (36) . Briefly, bovine brain from which the meninges were removed was chopped and homogenized at 4°C in buffer A (30 mM Tris-Cl, pH 7.5, 1 mM EGTA) and centrifuged at 100,000 x g for 60 min. To the supernatant was added solid (NH4)2SO4 to 85% saturation and the pH was adjusted to 4.7 with HCl. The precipitate was collected by centrifugation, dissolved in buffer A, and dialyzed extensively against the same buffer. The extract was chromatographed on DEAE-Sepharose and S100ß was eluted with a gradient of 0–0.5 M NaCl. Fractions containing 9–10 kDa proteins were collected and pooled. ZnSO4 was added to a final concentration of 2 mM. The sample was applied to a column of phenyl-Sepharose CL-4B equilibrated with buffer B (30 mM Tris-Cl, pH 7.5, 300 mM NaCl and 250 µM ZnSO4). The column was washed with 2.5 bed vol of buffer B, followed by 2.5 bed vol of buffer A. S100ß was eluted with buffer A containing 2 mM EGTA and ran as a single band with an apparent size of 10 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

The dimer of S100ßß was prepared as described by Barger et al. (37) . Briefly, S100ß was incubated for 30 days at -20°C in 20 mM Tris-Cl, pH 7.5, 0.5 M NaCl, 3 mM CaCl2. The protein solution was then thawed and a sample electrophoresed on SDS-PAGE in the absence of reducing agents, as described below. The band corresponding to the dimer was excised from the gel, and the protein was extracted into 10 volumes (based on volume of gel slice) of 10 mM Tris-Cl, pH 7.5, during a 24 h incubation with agitation at 4°C. The extracted protein was concentrated on Millipore concentrators (5 k MWCO; Millipore Corporation, Bedford, Mass.), diluted 10x with 10 mM Tris-HCl, pH 7.5, and concentrated again to reduce SDS concentration.

Cell culture
Seven-day chick embryo forebrain was carefully stripped of meninges, chopped with a scalpel, and the pieces were incubated in 0.25% trypsin in Ca/Mg-free phosphate-buffered saline (PBS) (140 mM NaCl, 8 mM NaH2PO4 and 2.7 mM KCl, pH 7.2) at 37°C for 15 min. The tissue was gently triturated with a Pasteur pipette after addition of Ham's F-12 medium (Life Technologies, Grand Island, N.Y.) containing 10% fetal calf serum to inactivate the trypsin. Cell viability was >95% as determined by trypan blue exclusion. Cells were plated at density 104 cells/cm2 on glass coverslips coated with 5 µg/ml poly-D-lysine. Culture medium was Ham's F12 (no serum) with the following supplements: insulin (5 µg/ml), transferrin (5 µg/ml), putrescine (100 µM), sodium selenite (3 x10-8 M), and progesterone (2 x10-8 M) at 37°C. Cells were grown in a 5% CO2 atmosphere. The concentration of fetal calf serum in the final culture (from the dilution of the triturated cells) was <0.02%. In experiments in which rapamycin (Alexis Corporation, San Diego, Calif.), FK506 (kindly supplied by Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan), or cyclosporin A (CsA) were added, the drugs were dissolved in DMSO to make a 1000x stock. An equal volume of DMSO was added to the controls.

Fixation and immunostaining
Cells were fixed for 30 min in PBS containing 4% formaldehyde and 0.1% glutaraldehyde, followed by 5 min in methanol added at -20°C. The fixed cells were washed with TBS (10 mM Tris-Cl, pH 8.0, 150 mM NaCl) and incubated with 5% goat serum for 30 min. After washing in TBS, a 1:20 dilution of a monoclonal antibody (250 µg/ml) against the NF-{kappa}B p65 subunit (Transduction Laboratories, Lexington, Ky.) was applied for 30 min. The specificity of antibody raised against the active p65 subunit of NF-{kappa}B was established by Western blots of human brain homogenates (32) . After washing in TBS, bound antibody was detected with a 1:200 dilution of a secondary antibody (biotinylated anti-mouse IgG; Amersham International PLC), followed by staining with 1:40 dilution of streptavidin-fluorescein. Nuclei were counter stained with 4,6-diamidino-2-phenylindole (DAPI).

Microscopy and image analysis
Digitized immunofluorescence images were captured with a chilled CCD camera (PXL; Photometrics, Inc., Tucson, Ariz.) on a Nikon Diaphot microscope equipped with a 40x oil (1.3 na) immersion objective and analyzed with Metamorph software (Universal Imaging, Corp., West Chester, Pa.). Images of nuclear NF-{kappa}B staining were corrected for local background intensities before ratioing to controls, but were not corrected for the slight `nonspecific' immunofluorescence staining due to secondary antibody alone.

Preparation of nuclear and cell extracts
Chick forebrain cells, cultured on 10 cm dishes at low density (104 cells/cm2), were treated for 2 h with S100ßß, S100ßß+FK506, or left untreated. The cells were washed with PBS and harvested from the plate by scraping into 400 µl of cold buffer (10 mM HEPES, pH 7.5, 10 mM KCl, 0.1 mM EDTA. 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF). After incubating on ice for 15 min, 25 µl of 10% IGEPAL was added and the tubes were vigorously vortexed for 10 s. The lysed cells were centrifuged for 30 s in a microfuge (10,000 xg). The nuclear pellets were resuspended in 200 µl SDS buffer (2% SDS, 10 mM Tris-Cl pH 7.5, 10 mM NaF, 5 mM DTT, 2 mM EGTA). After boiling and sonicating the nuclei, extracted protein was precipitated with chloroform/methanol (38) . The precipitate was dissolved in SDS sample buffer (39) and protein concentrations were determined (40) . Whole cell extracts were prepared from washed forebrain cultures by adding the SDS buffer directly to the plate, scraping the cell extract to the edge, transferring it to a microfuge tube, and heating to boiling for 3 min. Proteins were precipitated, dissolved in SDS-sample buffer, and protein concentration was determined as above.

SDS-polyacrylamide gel electrophoresis and Western blotting
SDS-PAGE was performed on polyacrylamide mini-slab gels, as described (39) . For analysis of S100ßß, 2-mercaptoethanol was eliminated from the sample preparation buffer and the proteins were separated on 15% isocratic gels (15% T, 2.7% C). Equal amounts of protein extracted from cell nuclei (15 µg for NF-{kappa}B determination) or whole cells (10 µg for calcineurin determination) were resolved on 10% isocratic gels (10% T, 2.7% C). For Western blotting, proteins from whole cell or nuclear extracts were transferred to polyvinylidene difluoride membrane using the transfer buffer of Towbin et al. (41) . After blocking with 5% reconstituted dry milk in Tris-buffered saline and washing thoroughly between each step, blots were incubated with primary antibody (mouse monoclonal) against calcineurin (clone CN-A1 against {alpha}-subunit; Sigma) or the NF-{kappa}B subunit p65 (Transduction Laboratories), followed by alkaline phosphatase-conjugated goat anti-mouse IgG. The bands were detected using the chemiluminescent substrate CDP-star (Tropix, Bedford, Mass.) and by capturing and digitizing the images with a Photometrics AT200 chilled CCD camera. Quantification of bands was performed using Phoretix software (Newcastle, U.K.).

Statistical analysis of results
Error bars on all plots show the standard error of the mean for the replicate experiments described. Analysis of variance with Scheffe's post hoc test were used to compare treated samples with their appropriate controls to provide the levels of significance reported in the figure legends.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunophilin ligands act by augmenting the neurotrophic activity of S-100ßß
The 7-day embryonic chick forebrain is an excellent source of a relatively pure population of primary neurons (42) . Two hours after plating forebrain cells at a density of 11,000 cells/cm2, cells attached to the dish surface and viability was ~90% as measured by trypan blue exclusion (Fig. 1 ). The cultures were examined at 24 h by phase-contrast microscopy and three parameters were measured: the survival as determined by counting cells with rounded, phase-bright bodies and cells with neurites; the percentage of the cells with neurites (requires a process equal or greater in length than the diameter of the cell body); and the length of the longest neurite per cell. Ten fields were chosen at random from each duplicate culture/experiment and the complete experiment was repeated twice.



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  		Figure 1. Immunosuppressive immunophilin ligands potentiate the neurotrophic effects of S100ßß. Various concentrations of S100ßß (0.2, 2, and 20 µg/ml) were added to embryonic forebrain cells alone or in the presence of 100 nm FK506, 100 nM cyclosporin A, or 100 nM rapamycin. The same concentration of each immunosuppressant was also studied alone. After 24 h, two parameters were measured: survival and percentage of the cells with neurites. Results are expressed as the mean ± standard error of the mean (SE). #, P < 0.01 compared with untreated controls after 24 h. ##, P < 0.01 compared to cultures treated with 0.2 µg/ml S100ßß. ###, P < 0.01 compared to cultures treated with 2.0 µg/ml S100ßß. ####, P < 0.05 compared to cultures treated with 20 µg/ml S100ßß.

After 24 h, only 8% of the cells survived; of these, only 50% contained neurites (Fig. 1) . In contrast, cells cultured in the presence of S100ßß at concentrations from 0.2 µg/ml to 20 µg/ml for 24 h showed a dose-dependent survival response and enhanced neurite extension. None of the immunosuppressant drugs—rapamycin, FK506, or CsA—when added alone at a concentration of 100 nM had any significant effect on cell survival and neurite outgrowth (Fig. 1) . In separate experiments (not shown), we tested concentrations of each drug from 1 nM to 1 µM with similar results. However, when the calcineurin-inhibiting immunosuppressants CsA and FK506 were added in the presence of suboptimal amounts of S100ßß, a significant enhancement of both survival and neurite outgrowth occurred. FK506 at 100 nM increased the percentage of cells surviving in the presence of 0.2 µg/ml S100ßß from 26% to 73% (Fig. 1) . In the presence of 100 nM FK506, only a small increase in survival (to 81%) was achieved by increasing the amount of S100ßß by 100-fold (Fig. 1) . CsA at 100 nM also augmented the potency of 0.2 µg/ml S100ßß to a similar extent and increased survival to 95% when used with 2 µg/ml S100ßß (Fig. 1) . Most important from a mechanistic understanding of this phenomenon, 100 nM rapamycin, which does not form a calcineurin-inhibitory complex with immunophilins, gave no enhanced survival over the effect of S100ßß alone (Fig. 1) .

Activation of neuronal NF-{kappa}B in chick forebrain neurons with S-100ßß and FK506 or cyclosporin A, but not rapamycin
Since a role for NF-{kappa}B has been implicated in cell survival in several different systems, the effects of S100ßß alone and combined with immunosuppressants on NF-{kappa}B activity were examined. After different treatments of the cells, the relative activity of NF-{kappa}B was measured with an immunofluorescence assay (Fig. 2 ), using a monoclonal antibody specific for the NF-{kappa}B active subunit (p65) that is not complexed to I{kappa}B (32) . For quantitative analysis of NF-{kappa}B activity, the average fluorescence intensity of 50 cells whose digitized images were captured under identical conditions were compared. Forebrain neurons from 7-day embryonic chicks contain measurable levels of active NF-{kappa}B (Figs. 2 and 3) . Immunostaining of NF-{kappa}B was observed not only in nuclei but also in the soma (Fig. 2 , insets), indicating the release of active NF-{kappa}B from I{kappa}B prior to nuclear translocation. Confirmation of NF-{kappa}B activation and translocation to the nucleus was obtained by quantitative Western blotting. The relative levels of nuclear NF-{kappa}B in nuclear extracts of control cells (untreated) and cells treated for 2 h with S100ßß alone or S100ßß + FK506 or S100ßß + cypermethrin are 1.0, 1.3, 2.9, and 2.0, respectively. These values compare favorably with those from the immunomicroscopy assay (Fig. 3 ) of 1.0, 1.3, 1.85, and 1.6 for the same treatments. These results confirm the validity of the immuno microscopy assay for evaluating NF-{kappa}B activation, but suggest that it is slightly more conservative in its estimates of activation, probably because we did not correct the fluorescence images for the faint immunofluorescence due to secondary antibody alone.



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  		Figure 2. Effects of S100ßß, S100ßß + FK506, and 3,4-DCIC on activity of NF-{kappa}B in chick embryo forebrain cells 2 h after plating. Top row: Fluorescence micrographs of chick forebrain cell cultures stained for p65 NF-{kappa}B immunoreactivity with the activity-specific p65 monoclonal antibody. Middle row: DNA staining of same cells with DAPI. Bottom row: Phase-contrast micrographs of same fields. Insets are magnified images of single cells to show cytoplasmic localization of some of the immunostaining. The nucleus within these cells is nearly the diameter of the cell; the only cytoplasm is the thin layer around the nucleus most prominent in the S100ßß + FK506-treated cells stained for NF-{kappa}B. Calibration bar = 10 µM.



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  		Figure 3. Effects of S100ßß, FK506, rapamycin, cyclosporin, cypermethrin, 3,4-DCIC, and PDTC, as well as various combinations of these compounds, on activity of NF-{kappa}B in forebrain cells. Identical conditions were used to fix cells, immunostain, and obtain digitized images of cells. Average fluorescence intensity of the stained cells was corrected for background from a region outside the cell. These average intensities were then normalized by dividing by the average fluorescent intensity of the control cells. Determinations were made in three separate cultures with 50 cells per dish. Error bars = SE. #, P < 0.05 compared with untreated control. ##, P < 0.01 compared with untreated control.

The antioxidants pyrrolidine dithiocarbamate (PDTC; 10 µM) or 3,4-dichloroisocoumarin (3,4-DCIC; 5 µM) (Figs. 2 and 3) decrease the amount of active NF-{kappa}B, as previously reported (22) . Addition of the glucocorticoid dexamethasone also decreased the immunofluorescence staining of active p65 (data not shown). In contrast, treatment with S100 ßß (2 µg/ml) for 3 h increased the activity of NF-{kappa}B by ~32%, whereas combined treatment with S100ßß and FK506 or CsA increased active NF-{kappa}B staining by 82% and 54%, respectively (Figs. 2 and 3) . FK506 alone and rapamycin, either alone or with S100ßß, did not affect NF-{kappa}B activity (Fig. 3) . Antioxidants are also known inhibitors of NF-{kappa}B activation (22) . As expected, preincubation with antioxidants suppressed the NF-{kappa}B activating effects of S-100ßß and of S-100ßß + FK506 (Fig. 3) .

A calcineurin-specific inhibitor can substitute for immunophilin ligands in augmenting activity of S100ßß and enhancing activation of neuronal NF-{kappa}B.
To determine whether the effects of immunosuppressants FK506 and CsA are mediated by calcineurin, the highly specific calcineurin inhibitor cypermethrin (IC50=40 pM) was used (43) . We first demonstrated that the presence of calcineurin in the chick forebrain neuronal cultures by immunoblot analysis of whole cell extracts with a commercial calcineurin antibody (data not shown). S100ßß (2 µg/ml) for 3 h increased the activity of NF-{kappa}B by ~32%, whereas the combined treatment with S100ßß and cypermethrin (500 pM) increased active NF-{kappa}B staining by 63%, similar to that of immunophilin ligands (Fig. 3) . Cypermethrin also mimicked the immunophilin ligands in its neurotrophic effect on survival of chick forebrain neurons (Fig. 4 ). As with the immunophilin ligands, cypermethrin alone did not enhance either NF-{kappa}B activity (Fig. 3) or cell survival (Fig. 4) , suggesting that calcineurin inhibition alone is not sufficient for activating NF-{kappa}B.



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  		Figure 4. Cypermethrin, at concentrations specific for inhibition of calcineurin, potentiates the neurotrophic effect of S100ßß on chick embryo forebrain neurons. Cell survival in the presence of cypermethrin was measured 24 h after plating. Error bars = SE. #, P < 0.001 compared with control cultures after 24 h.

Inhibitors of NF-{kappa}B activation block enhanced survival of chick forebrain neurons.
Results presented so far are consistent with a model in which S100ßß, either alone at high concentration or at lower concentration in combination with calcineurin inhibitors, functions in cell survival by activating NF-{kappa}B. Here we show that the effect of S100ßß and immunosuppressants on survival is blocked by inhibiting activation of NF-{kappa}B. Both antioxidant and glucocorticoid inhibitors of NF-{kappa}B activation were used. The survival of forebrain neurons induced by S100ßß with and without FK506 was reduced to control values (3–12%) in the presence of 3,4 DCIC (5 µM), PDTC (10 µM), and dexamethasone (5 µM) (Fig. 5 ).



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  		Figure 5. The antioxidants 3,4-dichloroisocoumarin (3,4-DCIC) and pyrrolidine dithiocarbamate (PDTC), as well as the glucocorticoid dexamethasone (DEX), inhibit the survival of forebrain cells induced with S100ßß alone and with FK506 when analyzed 24 h after plating. Error bars = SE. #, P < 0.01 compared to control after 24 h. ##, P < 0.01 compared independently with cultures treated with S100ßß and those treated with S100ßß + FK506.

The effect of inhibitors of NF-{kappa}B on survival of forebrain neurons in high-density cultures.
Improved survival of neurons in high-density culture is common for different types of neurons and probably occurs through the release of neurotrophic factors from the cells. As NF-{kappa}B inhibitors inhibit the survival of cells in low-density cultures treated with S100ßß, we decided to test them on high-density (50,000/cm2) cultures, where the viability of cells after 24 h was ~85% without added neurotrophic factors (Fig. 6 ). NF-{kappa}B inhibitors significantly decreased the survival of cells (Fig. 6) , as we had observed in the case of forebrain cultures at low density (10,000/cm2) that had been treated with S100ßß or S100ßß + FK506, (Fig. 5) . These results suggest that the survival of chick forebrain neurons at either density depends on activation of NF-{kappa}B.



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  		Figure 6. The effects of inhibitors of NF-{kappa}B activation on survival of chick forebrain neurons in high-density (50,000/cm2) cell cultures. Both antioxidant (5 µM 3,4-dichloroisocoumarin and 10 µM pyrrolidine dithiocarbamate) and glucocorticoid (5 µM dexamethasone) inhibitors of NF-{kappa}B activation were added to the cultures 30 min after plating, and the surviving cells were quantified 24 h later. Error bars = SE. #, P < 0.001 compared with control.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have examined the influence of a number of ligands of immunophilins on S100ßß-induced survival in chick embryo forebrain cells. Even though S100ßß alone can enhance cell survival in a dose-dependent manner, the potency of lower concentrations of S100ßß is augmented by certain immunosuppressant drugs. Only those immunosuppressants that form calcineurin-inhibitory complexes with immunophilins enhanced survival and outgrowth from the forebrain cells. The augmenting potency of immunosuppressants was detected at concentrations from 10 to 100 nM, the concentration range in which they function in inhibiting the activity of calcineurin. In the absence of added S100ßß, none of the drugs enhanced cell survival. One of the known calcineurin substrates is the phosphorylated (inactive) form of I{kappa}B. Therefore, the next step of our investigation was to elucidate whether there is any correlation between NF-{kappa}B activity and neuronal survival induced by S100ßß in the presence and absence of immunosuppressants. S100ßß alone increased the activity of NF-{kappa}B, and the increase was augmented with FK506 and CsA, but not rapamycin. Since rapamycin does not form a calcineurin-inhibitory complex with immunophilins, these results suggested that calcineurin inhibition is important to the survival mechanism. This finding was confirmed by demonstrating immunophilin ligands could be replaced by cypermethrin, a more specific inhibitor of calcineurin.

It is becoming more and more obvious that the immune system and the nervous system have many components in common, especially with regard to signal transduction pathways. As signal transduction is so prominent and important in the brain, recent discoveries that immunophilin levels are 10- to 40-fold higher in the brain than in other tissues may not be surprising. However, the physiological role for the immunophilins FKBP12 and cyclophilin within a cell is still unknown. Nevertheless, evidence is accumulating that suggests they may play a role in normal regulation of calcineurin (44) . Both FKBP12 and cyclophilin colocalize with calcineurin in vivo (45) . FKBP12 binds to the ryanodine/IP3 receptors in the endoplasmic reticulum and may help regulate calcineurin activity by localizing this calcium/calmodulin-activated protein to these calcium release channels. A large cytoplasmic protein that binds and inhibits calcineurin, named cain, has recently been identified (46) and may serve as a scaffold for calcineurin. Thus, under physiological conditions in the absence of immunosuppressant drugs, the immunophilins may play a role in signaling pathways leading to calcineurin and NF-{kappa}B regulation. Additional studies are needed to elucidate this role. In addition, nonimmunosuppressive analogs of immunosuppressant drugs that bind to FKBP12 but have no calcineurin inhibitory activity have been shown to have potent effects on neurite outgrowth at very low concentration (8) . Thus, immunophilins are likely to have targets other than calcineurin through which they are able to effect neurotrophic effects.

In the absence of S100ßß, inhibition of calcineurin did not enhance cell survival. Since S100ßß is known to cause the elevation of cytoplasmic calcium in neurons (14) , it should activate calmodulin, leading to the activation of calcineurin. If S100ßß also stimulated the activation of NF-{kappa}B through activating kinases that phosphorylate I{kappa}B, high amounts of S100ßß might cause a net activation of NF-{kappa}B. Lower concentrations of S-100ßß might not fully stimulate the kinase, and the reversal of I{kappa}B phosphorylation by calcineurin could predominate and prevent the activation of NF-{kappa}B. Under these conditions, inhibitors of calcineurin would potentiate the effects of S100ßß. This mechanism for S100ßß action is supported by the finding that antioxidant and glucocorticoid inhibitors of NF-{kappa}B activity, both of which work by different mechanisms, strongly inhibit survival of forebrain cells by either high concentrations of S100ßß alone or lower concentrations in combination with a calcineurin inhibitor. These inhibitors of NF-{kappa}B have been used at much higher concentrations to treat other cultured cell types without apparent toxicity (22 , 47) , strongly suggesting that their ability to block survival in chick forebrain neurons is through their inhibition of NF-{kappa}B activation.

Multiple I{kappa}B kinases, IKKs, are responsible for the activation of NF-{kappa}B through the phosphorylation and removal of I{kappa}B. The serines at positions 32 and 36 on the alpha chain of I{kappa}B as well as those at positions 19 and 23 on the beta chain are the critical residues for phosphorylation (24 , 25) and target these molecules for degradation in the ubiquitin-26S-proteasome pathway (48) . The recent cloning of an essential component of the IKK complex, named NF-{kappa}B essential modulator (NEMO), suggests that the complex is of high molecular weight and possibly organized as a signaling scaffold (48) . Only one of the two major isoforms of I{kappa}B kinase associates with this complex. However, evidence is also accumulating for a role of other protein kinases and phosphatases in this emerging pathway (49 50 51 52 53) . Recently it has been shown that a Ca2+-dependent pathway involving the phosphatase calcineurin participates in the regulation of NF-{kappa}B in a cell-specific fashion. The calcineurin pathway works synergistically with both protein kinase C-dependent and independent pathways at the level of phosphorylation and degradation of the I{kappa}B alpha subunit (19) . Taken together, these results suggest that I{kappa}B may integrate the response from several signal transduction pathways through hierarchical phosphorylations at multiple serines.

In regions of brain from patients with Alzheimer's disease (33) and in dopaminergic neurons from patients with Parkinson's disease (32) , the proportion of neurons with nuclear NF-{kappa}B staining is significantly increased. In the latter case, a relationship was established between free radical formation, activation of NF-{kappa}B, and the apoptosis of dopaminergic neurons (32) . Other examples in the literature show cytokines mediating an acute and/or progressive neuronal injury cycle that is accompanied by an increase in NF-{kappa}B activity (54) . In this cycle, the immune cytokine interleukin 1, a key initiating and coordinating agent, activates astrocytes to synthesize inflammatory and neuroactive molecules, particularly S100ßß (55) . It also has been shown that NF-{kappa}B activation can be mediated through the low-affinity nerve growth factor receptor p75, which mediates cell death (23) . These studies suggest that NF-{kappa}B activation is a mediator of an apoptotic response. However, several recent reports have attributed an antiapoptotic function to NF-{kappa}B in both nonneuronal and neuronal cells (56 57 58 59 60) . Lin et al. (61) have shown that in the same cultured cell line, NF-{kappa}B expression could be either antiapoptotic or proapoptotic depending on the nature of the death stimulus. Since suppression of steady-state, but not stimulus-induced, NF-{kappa}B activity inhibited apoptosis, Lin et al. suggest that the cell death pathway is activated either by a product of an NF-{kappa}B-regulated gene or by a modification of this gene product brought about by alternative signaling pathways (61) . This latter model is attractive in that all cells would use NF-{kappa}B activation as a survival response, and only under conditions where an alternative pathway has been activated would the function of an NF-{kappa}B-regulated gene product be converted to have a proapoptotic affect. Such a model seems to function in triphenyltin-induced apoptosis in HL-60 cells in which a NF-kB-induced gene product actually is the apoptotic factor (47) . Perhaps cells showing enhanced NF-{kappa}B staining in their nuclei within the degenerating populations of cholinergic and dopaminergic neurons in Alzheimer's and Parkinson's disease, respectively, are trying to counteract the effects of other degenerative pathways.

We conclude that the NF-{kappa}B can play an important role in survival of chick embryo forebrain neurons and that at least part of the neurotrophic activity of S100ßß is mediated through its activation of NF-{kappa}B. In addition, the importance of calcineurin to the regulation of neuronal function suggests that its inhibition via immunophilin-immunosuppressant drug complexes within neurons can have profound affects on neuronal survival and outgrowth. Furthermore, the enhancement of neuronal survival by low concentrations of S100ßß suggests a strategy by which S100ßß might be useful as a neurotrophic agent at levels low enough to avoid the neurotoxicity resulting from excessive calcium uptake by neurons and activation of NOS in astrocytes.


   ACKNOWLEDGMENTS
 
The authors thank Ms. Laurie Minamide, Mr. Michael Brown, and Drs. Thomas Kuhn and Peter Meberg for technical assistance, helpful hints, and suggestions, and Drs. Barbara Bernstein, Howard Nornes, and Bruce Molitoris for critical reading of the manuscript. This work was supported in part by NIH grant GM35126 to J.R.B. and NIH Fogarty Fellowship TW05357 to A.R.A.


   FOOTNOTES
 
2 Abbreviations: CsA, cyclosporin A; DAPI, 4,6-diamidino-2-phenylindole; 3,4-DCIC, 3,4-dichloroisocoumarin; NOS, nitric oxide synthase; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PDTC, pyrrolidine dithiocarbamate.

Received for publication November 23, 1998. Revised for publication March 19, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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