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Jun 6, 2002 - Cerebrospinal Fluid, and Astrocyte Cultures of Rats. Francine Tramontina,1 Sabrina Conte,1 Daniela Gonçalves,1 Carmem Gottfried,1.
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C 2002) Cellular and Molecular Neurobiology, Vol. 22, No. 3, June 2002 (°

Short Communication

Developmental Changes in S100B Content in Brain Tissue, Cerebrospinal Fluid, and Astrocyte Cultures of Rats Francine Tramontina,1 Sabrina Conte,1 Daniela Gonc¸alves,1 Carmem Gottfried,1 Luis V. Portela,1 Lucia Vinade,1 Christianne Salbego,1 and Carlos-Alberto Gonc¸alves1,2 Received June 6, 2002; accepted July 22, 2002 SUMMARY 1. We investigated the content of S100B protein by ELISA in three brain regions (hippocampus, cerebral cortex, and cerebellum) and in cerebrospinal fluid of rats during postnatal development as well as the content and secretion of S100B in pre- and postconfluent primary astrocyte cultures. 2. An accumulation of S100B occurred in all brain regions with similar ontogenetic pattern between second and fourth postnatal weeks. However, we observed a decrease in the cerebrospinal fluid S100B after the critical period for synaptogenesis in rodents. 3. A similar profile of cell accumulation and decrease in basal secretion was also observed during aging of astrocyte cultures. 4. These data contribute to the proposal that S100B is an important glial-derived protein during brain development and that changes in extracellular levels of S100B may be related to glial proliferation and synaptogenesis. KEY WORDS: S100B; astrocyte; brain development.

INTRODUCTION S100B is a calcium-binding protein of 21 kDa expressed primarily in astrocytes and secreted by these cells (see Donato, 2001, for a review). Many studies have suggested an intracellular role for this protein particularly in the regulation of cytoskeleton and cell cycle. Extracellular S100B stimulates glial proliferation, survival of neurons, and extension of neurites in cell culture. Moreover, abnormal expression of S100B has supported its role in neurodegenerative disorders such as Alzheimer’s disease. 1 Departamento de Bioqu´ımica, ICBS, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil. 2 To

whom correspondence should be addressed at Departamento de Bioqu´ımica, UFRGS, Ramiro Barcelos, 2600-Anexo, 90.035-003 Porto Alegre, Brazil; e-mail: [email protected]. 373 C 2002 Plenum Publishing Corporation 0272-4340/02/0600-0373/0 °

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It has been shown that S100 (most probably S100B) increases in brain tissue during the first three postnatal weeks in rodents (Cicero et al., 1972; Haglid et al., 1977), a critical period for synaptogenesis. This increase occurs particularly in the synaptic membranes and in the astrocytic filaments. The S100B synaptic accumulation and its neurotrophic action in cultures suggest a role for this protein in synaptic plasticity. We have shown that postconfluent astrocytes from different brain regions have a basal secretion of S100B proportional to the cell immunocontent (Pinto et al., 2000). However, there are only few reports on S100B extracellular levels during brain development. In the present work we investigated the immunocontent of S100B in three brain regions (cerebral cortex, hippocampus, and cerebellum) and in cerebrospinal fluid (CSF) of rat brain during postnatal development as well as the basal secretion of S100B in pre- and postconfluent primary astrocyte cultures. METHODS Brain microslices (0.4 × 1.0 mm) from 2 to 60-day-old Wistar rats were homogenized in phosphate buffer saline (PBS) and stored at −20◦ C. Rats were anaesthetized by intraperitonial injection of pentobarbital sodium (50 mg/kg-body weight) and positioned in a stereotactic holder. Cerebrospinal fluid was obtained by cisterna magna punction, using a 0.33 mm of diameter insulin needle. The needle was inserted no more than 1.5 mm and a maximum volume of 30 µL was collected in a 3-min period to minimize risk of brainstem damage. CSF samples were frozen (−20◦ C) until further analysis. Primary cortical astrocyte cultures were prepared as previously described (Karl et al., 2000). At age 1, 3, or 8 weeks in vitro (WIV) the medium was replaced by DMEM with or without serum for 30 min. Then media were replaced by DMEM containing serum and S100B secretion was measured after 1 h. For intracellular content of S100B cells were scrapped in 0.25 mL of PBS. Fifty microliters of CSF (diluted 4× in PBS), homogenate of brain slices (diluted 100× in PBS), medium of culture or homogenate of astrocyte culture (diluted 100× in PBS) were used in an ELISA for S100B as described previously (Tramontina et al., 2000). Total protein was measured by Lowry’s method, using bovine serum albumin as a standard. RESULTS AND DISCUSSION All studied brain regions showed a similar ontogenetic pattern (Fig 1). S100B drops (∼20%, p < 0.05) after birth and increases from second postnatal week onwards. A linear regression showed a correlation between age and S100B content in all studied regions from 8 to 60 postnatal days ( p < 0.002). Similar values of S100B were found between 30 and 60 days, confirming that adult pattern is reached during the first four postnatal weeks. Absolute values of S100B (µg/mg of protein) in 60-day-old rats are approximately 1.8, 1.7, and 2.4 in hippocampus, cerebral cortex, and cerebellum, respectively. Considering that tissue content of S100B increases during synaptogenesis and remains elevated after this period, it would be interesting

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Fig. 1. Postnatal content of S100B in different brain regions. Immunocontent of S100B was measured in microslices from cerebral cortex (Cx), hippocampus (Hc), and cerebellum (Cb) of 2, 8, 15, 30, and 60-day-old rats. Each value is a mean (of five independent experiments) ±SE. a – Different from 2-day-old rats (assumed as 100%) for p < 0.05; b – different from 8-day-old rats for p < 0.05, but not from 2-dayold rats; c – different from 2 and 8-day-old rats for p < 0.01.

Fig. 2. S100B in cerebrospinal fluid of young and adult rats. Immunocontent of S100B in cerebrospinal fluid of 15, 30, 60, and 90-day-old rats. Each value is a mean (of six independent experiments) ±SE. ∗ Different from 15-day-old rats (assumed as 100%) for p < 0.01.

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Fig. 3. Cell content and secretion of S100B in cortical astrocyte cultures. Content (A) and secretion (B) of S100B in 1, 3, and 8-week-old primary astrocyte cultures were measured 1 h after a medium replacement containing or not serum (serum-deprivation) for 30 min. Each value is a mean of four independent experiments performed in triplicate. In A: a – Different from 1-week-old cultures ( p < 0.01); b – different from 3-week-old cultures ( p < 0.05). In B: a – different from 1-week-old cultures ( p < 0.01); b – different from 3-week-old cultures ( p < 0.01); c – different from cultures at same age without serum-deprivation ( p < 0.05).

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to investigate possible extracellular S100B changes, which could be related to its neurotrophic activity. S100B content in cerebrospinal fluid was studied from 15 to 90 postnatal days (Fig. 2). We were not able to obtain enough amount of CSF without brain damage in rats younger than 15-day-old. A negative correlation was observed between 15 and 90 days (r = −0.5131; p = 0.008). There was a mean 34% decrease in S100B from 30 days onwards. This pattern is opposite to the intracellular accumulation of S100B observed during this period (Fig. 1). Assuming its putative role in synaptogenesis it would be expected a decrease in extracellular levels of S100B during aging. Indeed, recently we showed that serum S100B in humans is dependent on age (Portela et al., 2002). We compare preconfluent astrocyte cultures of 1 WIV to postconfluent cultures of 3 and 8 WIV (Fig. 3). Immunocontent of S100B (expressed in ng/µg of total protein) increased from 1 to 8 weeks (r = 0.8864; p < 0.0001). After reaching confluence, the immunocontent increased from 3 to 8 weeks about 60% (Fig. 3(A)). Basal secretion (stimulated or not by serum-deprivation) did not accompany intracellular variations (Fig. 3(B)), showing a linear decrease with age of astrocytes in culture ( p < 0.01). Percentage of secretion (related to intracellular content) was about 1% in preconfluent cells independent of serum-deprivation. Postconfluent cells presented a lower percentage of secretion (between 0.04 and 0.8%) than preconfluent cells. These results confirm data from glioma cells (Van Eldik and Zimmer, 1987), whose secretion was more intense in preconfluent phase. S100B secretion was stimulated by serum-deprivation in postconfluent astrocytes. Therefore, cortical astrocytes in culture presented distinct intra- and extracellular changes of S100B levels dependent on age. Preconfluent astrocytes are more active in S100B secretion, which could be related to the role of this protein on glial proliferation. In summary we have shown that S100B presented an intense increase in hippocampus, cerebral cortex, and cerebellum from the second postnatal week onwards. A decrease in CSF S100B was observed after fourth postnatal week. A similar profile in S100B content was observed during aging of astrocytes in culture, i.e., cell accumulation and decrease in secretion. These data contribute to the proposal that S100B is an astrocyte-derived protein possibly involved in glial proliferation and synaptic plasticity during earlier development.

ACKNOWLEDGMENTS This work was supported by Brazilian funds from PRONEX, CNPq, CAPES, and FAPERGS.

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Haglid, K. G., Hansson, H. A., and Ronnback, L. (1977). S-100 in the central nervous system of rat, rabbit and guinea pig during post-natal development. Brain Res. 123:331–345. Karl, J., Gottfried, C., Tramontina, F., Dunkley, P. R., Rodnight, R., and Gon¸calves, C. A. (2000) GFAP phosphorylation studied in digitonin-permeabilized astrocytes: standardization of conditions. Brain. Res. 853:32–40. Pinto, S., Gottfried, C., Mendez, A., Gon¸calves, D., Karl, J., Gon¸calves, C. A., Wofchuk, S., and Rodnight, R. (2000). Immunocontent and secretion of S100B in astrocyte cultures from different brain regions in relation to morphology. FEBS Lett. 486:203–207. Portela, L. V. C., Tort, A. B. L., Schaf, D. V., Ribeiro, L., Walz, R., Rotta, L. N., Silva, C. T., Busnello, J. V., Kapczinski, F., Gon¸calves, C. A., and Souza, D. O. (2002). Serum S100B concentration is agedependent. Clin. Chem. 48:950–952. Tramontina, F., Karl, J., Gottfried, C., Mendez, A., Gon¸calves, D., Portela, L. V., and Gon¸calves, C. A. (2000). Digitonin-permeabilization of astrocytes in culture monitored by trypan blue exclusion and loss of S100B by ELISA. Brain Res. Protoc. 6:86–90. Van Eldik, L. J., and Zimmer, D. B. (1987). Secretion of S-100 from rat C6 glioma cells. Brain Res. 436:367– 370.