Patricia M. Whitaker-Azmitia 1, Randall Murphy 2 and Efrain C. Azmitia 3. 1Department of ... an astroglial-specific protein first described by Moore in. 19659 ...
Brain Research, 528 (1990) 155-158
155
Elsevier
BRES 24290
Stimulation of astroglial 5-HT1A receptors releases the serotonergic growth factor, protein S-100, and alters astroglial morphology Patricia M. Whitaker-Azmitia 1, Randall Murphy 2 and Efrain C. Azmitia 3 1Department of Psychiatry, State University of New York, Stony Brook, NY 11794 (U.S.A.) and Departments of 2Chemistry and ~Biology, New York University, New York, NY 10003 (U.S.A.)
(Accepted 5 June 1990) Key words: Serotonin; Neuronal growth; S-100; Serotonin-lA receptor; Astroglial growth factor; Down's syndrome; Alzheimer's disease
Stimulation of astroglial 5-HTIAreceptors causes astroglial cells to acquire a more mature morphology and to release a factor (or factors) which promotes growth of serotonergic neurons. By using an antibody-blocking approach, we have shown that at least one of the growth-promoting factors thus released is the astroglial-specificprotein S-100. This may be a particularly important observation, in view of studies implicating S-100 in both Down's syndrome and Alzheimer's disease.
Serotonin neurons have been shown to autoregulate their own development 14. This is due in part to release of growth factors by stimulation of 5-HTtA receptors on astrocytes 16. We have recently shown that protein S-100, an astroglial-specific protein first described by Moore in 19659, added to primary cultures of serotonin neurons promotes their growth 3. S-100 had previously been reported to promote neurite extension in chick embryo cultures 7. Since S-100 production is increased in cultures of the astroglioma line C6 by dibutyryl cyclic AMP 8 and moreover this protein may be releasable H, we hypothesized that S-100 may be the factor released by 5-HTIA receptor stimulation. To test this hypothesis, we stimulated astroglial cells in primary culture with 100 nM ipsaperone (IPS), a 5-HTIA receptor agonist and collected the conditioned media (GCM-IPS). We then added the GCM-IPS to primary cultures of serotonin neurons, with and without the addition of an antibody to S-100, and assessed the effects on neuronal growth. Primary astroglial cultures were derived from newborn (1-3 day old) Sprague-Dawley rat pups as previously described ~6. After one week in culture, cultures were rinsed twice with Weymouth's media containing 5 /~g/ insulin and 0.5 mg/ml albumin. The cells were left to incubate at 37 °C for 12 h before replacing media with fresh serum-free media containing 100 nM ipsaperone, a selective 5-HTIA receptor agonist. After a further 24 h, the media (referred to as GCM-IPS) was collected and stored at -70 °C. until tested in neuronal cultures.
The growth-promoting properties of native bovine S-100 (10-1000 ng/ml; East Acres Biologicals, Southbridge, MA: guaranteed >99% homogenous by SDSPAGE) and of GCM-IPS (diluted 1/500) were compared. S-100 and GCM-IPS, or S-100 and GCM-IPS in the presence of a polyclonal antibody to S-100 (Accurate Chemical, Westbury, NY; final dilution 1/10,000; characterized in our laboratory for immunocytochemical staining of astrocytes in culture and brain) were added at the time of neuronal plating. Neuronal cultures were prepared from Sprague-Dawley rat embrYos at 13-14 days of gestation (Hilltop Breeding Laboratories) as previously described 2. After three days in culture, neuronal growth was assessed using the specific re-uptake capacity method. This indicator has been shown to reliably measure in vivo innervation density or the maturational state of specific neurons in culture 2"4"5. Briefly, cultures were incubated for 20 min with MEM containing 1% glucose and [3H]serotonin (26 Ci/mmol, New England Nuclear; final concentration 50 nM) with or without 50 nM fluoxetine. After removing the radiolabel, the cultures were allowed to dry and 200 ~1 of absolute ethanol was added for 1 h and then i50/A removed into 7 ml of Liquiscint for counting in a Beckman liquid scintillation counter (40% efficiency). Test cultures were pre-incubated with S-100 or GCM-IPS or S-100 antibody for 1 h before uptake was measured, to ensure that none of these had a direct effect on serotonin uptake. To visualize astroglial cultures after exposure to serum-
Correspondence: P.M. Whitaker-Azmitia, Department of Psychiatry, State University of New York, Stony Brook, NY 11794, U.S.A..
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Fig. 1. Effects of native bovine S-100 and of media from astroglial cells stimulated with the selective 5-HTIA agonist ipsaperone (GCM-IPS) on the growth of serotonergic neurons in culture as determined by selective uptake of [3H]serotonin. Hatched bars indicate the effects of S-100 and GCM-IPS in the presence of an antibody to S-100 (final dilution 1/10,000). Each bar represents the mean of four cultures, derived from different litters, and the error bars indicate standard errors of the mean. S-100, GCM-IPS and the antibody were all added at time of plating and the growth assessed 3 days later, df = 5; F = 1497, P < 0.0001. For individual values, P < 0.001.
free media with or without 100 nM ipsaperone, cultures were rinsed twice with Tris-buffered saline (TBS) at 4 °C before incubation with a polyclonal antibody to glial fibrillary acidic protein (GFA), (Accurate Chemicals; final dilution 1/800 in TBS with 0.2% Triton and 0.1% normal swine serum) a specific astroglial marker, for 2 h at 37 °. After rinsing with TBS, the cultures were stained using the avidin/biotin method prepared as Vectastain (Vector Labs) with final visualization using diaminobenzidine. Both S-100 (500 ng/ml) and GCM-IPS (diluted 1/500) produced an increase in the uptake capacity of the cultures for [3H]serotonin after 3 days exposure, but not when applied acutely. This stimulation was blocked by incubation, at the time o f plating, with a polyclonal antibody to S-100 (1/10,000; Fig. 1). Application of either the antibody alone or 0.5 nM ipsaperone (the maximum final concentration in the neuronal cultures after addition of GCM-IPS) was without significant effect. The GCMIPS had a greater effect in increasing the uptake of [3H]serotonin than did the pure S-100. Whether this was due to a greater amount of S-100 in the media or due to the presence of additional factors in the media is unclear. Fig. 2 gives representative fields of cultures treated
with serum-free media alone (A and C) or serum-free media containing 100 nM ipsaperone (B and D). The morphological alterations were characterized by an increase in process-bearing cells and an increased colonization of the cells. These changes were consistently observed in all eight primary cultures (i.e. from 8 different litters of animals) used to derive GCM. It has been previously demonstrated that serotonergic neurons can regulate their own growth through activation of a 5-HT 1 receptor TM. We have found that astroglial cells contain high levels of 5-HT 1 receptors in the immature state 15 and that activation of a subtype of these receptors, the 5-HTIA receptor, produces a GCM which can stimulate serotonergic maturation in dissociated tissue culture preparation 16. We now propose, based on our current studies, that 5-HTaA receptors localized to brain astroglial cells are involved in the release of S-100 and thus this protein is at least one of the means by which serotonin can autoregulate its own development. During fetal brain development, a transient intense immunoreactivity for S-100 is observable in the midline raphe region, where the serotonin cells are developing 13. Since in the process of producing and/or releasing S-100, the astroglial cells attain a mature morphology, our results suggest a functional interaction between astrocytes and neurons during development, whereby both cell types are matured through an action of the astroglial 5-HTIA receptor. The observed morphological change has also been seen after activation of other cAMP-producing receptors, such as the beta-adrenergic receptor 1° which is linked to the release of nerve growth factor (NGF) 12. It is interesting to note the parallels of these two systems, ie. receptor stimulation, cAMP production, astroglial morphological change and finally release of a growth factor. Finally, the gene for the beta subunit of S-100 has been mapped to the distal half of the long arm of chromosome 21, a candidate region for the pathology of Down's syndrome a and recent studies have shown an increase in S-100 immunoreactivity in postmortem Alzheimer's disease and Down's syndrome 6. A knowledge of the physiological effects of this protein and which factors regulate its release could therefore be of great importance to the understanding of both disorders. This work was supported by grants from the National Institute of Neurologial Disease and Stroke to PMW-A and from the National Science Foundation to ECA.
Fig. 2. Representative primary cultures of astroglial cells, immunocytochemically stained using an antibody to glial fibrillary acidic protein (GFA). A and C are cultures following 24 h exposure to serum-free Weymouth's media containing 5 #g/ml insulin and 0.5 mg/ml albumin (WM-IA). B and D are sister cultures exposed to WM-IA containing 100 nM ipsaperone for 24 h. A and B are 63 x and C and D are 400 × .
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158 1 Allore, R., O'Hanlon, D., Price, R., Neilson, K., Willard, H.E, Cox, D.R., Marks, A. and Dunn, R.J., Gene encoding the beta subunit of S-100 protein is on chromosome 21: implications for Down syndrome, Science, 239 (1988) 1311-1313. 2 Azmitia, E.C. and Whitaker-Azmitia, P.M., Target cell stimulation of dissociated serotonergic neurons in culture, Neuroscience, 20 (1987) 47-63. 3 Azmitia, E.C., Dolan, K. and Whitaker-Azmitia, P.M., S-10% but not NGF, EGF, insulin or calmodulin functions as a CNS serotonergic growth factor, Brain Research, in press. 4 Currie, D.N. and Dutton, G.R., 3H-GABA uptake as a marker for cell type in primary cultures of cerebellum and olfactory bulb, Brain Research, 199 (1980) 473-481. 5 Dreyfus, C.E, Sherman, D.L. and Gershon, M.D., Uptake of serotonin by intrinsic neurons of the myenteric plexus grown in organotypic tissue culture, Brain Research, 128 (1977) 124-139. 6 Griffin, W.S.T., Stanley, L.C., Ling, C., White, L., MacLeod, V., Perrot, L.J., White, C.L. and Araoz, C., Brain interleukin 1 and S-100 immunoreactivity are elevated in Down Syndrome and Alzheimer disease, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 7611-7615. 7 Kligman, D. and Marshak, D., Purification and characteriztion of a neurite extension factor from bovine brain, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 7136-7139. 8 Labourdette, G. and Mandel, P., Effect of norepinephrine and dibutyryl cyclic AMP on S-100 protein level in C6 glioma cells,
Biochem. Biophys. Res. Commun., 96 (1980) 1702-1709. 9 Moore, B., A soluble protein characteristic of the nervous system, Biochem. Biophys. Res. Commun., 19 (1965) 739-744. 10 Shain, W., Forman, D.S., Madelian, V. and Turner, J.N., Morphology of astroglial cells is controlled by beta-adrenergic receptors, J. Cell Biol., 105 (1987) 2307-2314. 11 Shashoua, V.E., Hesse, G.W. and Moore, B.W., Proteins of the brain extracellular fluid: evidence for release of S-100 protein, J. Neurochem., 42 (1984)1536-1541. 12 Schwartz, J.P. and Costa, E., Regulation of nerve growth factor content in C6 gloima cells by beta-adrenergic receptor stimulation, Naunyn. Schmied. Arch. Pharmacol., 300 (1977) 123-129. 13 Van Hartesveldt, C., Moore, B. and Hartman, B.K., Transient midline raphe glial structure in the developing rat, J. Comp. Neurol., 253 (1986) 175-184. 14 Whitaker-Azmitia, P.M. and Azmitia, E.C., Autoregulation of fetal serotonergic neuronal development: role of high affinity serotonin receptors, Neurosci. Lett., 67 (1986) 307-312. 15 Whitaker-Azmitia, P.M. and Azmitia, E.C., 3H-5-Hydroxytryptamine binding to brain astroglial cells: differences between intact and homogenized preparations and mature and immature cultures, J. Neurochem., 46(1986) 1186-1191. 16 Whitaker-Azmitia, P.M. and Azmitia, E.C., Stimulation of astroglial serotonin receptors produces media which regulates development of serotonergic neurons, Brain Research, 497 (1989) 80-85.
