- Email: [email protected]
S100β promotes the extension of microtubule associated protein2 (MAP2)-immunoreactive neurites retracted after colchicine treatment in rat spinal cord culture
Neuroscience Letters 229 (1997) 212–214
S100b promotes the extension of microtubule associated protein2 (MAP2)immunoreactive neurites retracted after colchicine treatment in rat spinal cord culture Mayumi Nishi a , b ,*, Mitsuhiro Kawata a, Efrain C. Azmitia b a
Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kamigyou-ku, Kyoto 602, Japan b Department of Biology, New York University, 1009 Main Building, 100 Washington Square East, New York, NY 10003, USA Received 9 June 1997; accepted 17 June 1997
Abstract S100b, a glial derived calcium-binding protein with neurotrophic activity in the central nervous system, stimulates neurite extension of fetal raphe, cortex, spinal cord, and dorsal root ganglion neurons. The effects of S100b on neurite length and microtubule associated protein2 (MAP2) immunoreactivity (IR) after microtubule disruption with colchicine were investigated in primary rat spinal cord culture. The incubation with S100b (20 ng/ml) for 3 h after exposure to colchicine (10−6 M) for 30 min altered the distribution of MAP2-IR. The length of MAP2-IR neurites increased by 65% compared to that in colchicine treatment alone. MAP2-IR intensity in the cell body was reduced by 26% compared to that in colchicine treatment alone. These results indicate that neurites shrink when the microtubular cytoskeletal system is disrupted and S100b rapidly promotes re-assembly and/or stabilization. 1997 Elsevier Science Ireland Ltd. Keywords: Depolymerization; Polymerization; Spinal cord injury; Phosphorylation; Microtubular cytoskeletal system; Primary culture
S100b is a cytosolic calcium binding protein which is synthesized and stored in glial cells. Previous studies have shown that S100b acts mainly on neurite extension [2,9,16]. It is considered that this effect of S100b is caused by stabilization of microtubules by inhibiting phosphorylation of brain cytoskeletal proteins such as microtubules associated protein2 (MAP2), tau, growth associated protein43 (GAP43) [5,6,14]. Does S100b directly affect injured neurites and stimulate their extension? The present study investigated the effects of S100b on MAP2 immunoreactive (IR) neurite length and MAP2-IR intensity in the cell body region after microtubule disruption with colchicine in primary rat spinal cord cultures. Colchicine is a microtubule-disrupting agent which is known to activate mitogen activated protein kinase (MAP kinase; it is also called microtubule associated protein kinase) [15]. Microtubules are structural filaments in the cytoplasm which are spatially organized and extremely
* Corresponding author: Tel.: +81 75 2515301; fax: +81 75 2515306; e-mail: [email protected]
dynamic [11,12]. By using MAP2 immunoreactivity as a marker specific for the microtubule system, we show here that S100b stimulates MAP2-IR neurite extension after microtubule disruption with colchicine. Dissociated spinal cord primary cultures were prepared from 18-day-old Sprague–Dawley rat fetuses, according to the method of Azmitia and Hou [3]. Briefly, spinal cords were isolated and mechanically dissociated by triturating through a fire-polished glass pipette. The cells were plated at an initial plating density of 1 × 105 cells/cm2 on each well (area of 0.28 cm2) precoated with polyethyleneimine. The cultures were maintained in complete neuronal medium (CNM) containing 5% (v/v) fetal bovine serum (Sigma) for 48 h in a CO2 incubator at 37°C with 5% CO2/95% air. After 48 h the CNM was removed and the cultures were rinsed with serum-free media (MEM with 0.25% glucose, 1% non-essential amino acids, 20 mM putrescine, 15 nM sodium selenite, 5 mg/ml insulin, and 100 mg/ml transferrin). After incubation in the serum free medium for 48 h, the cultures were treated with 10−6 M colchicine (Sigma) for 30 min. Then colchicine was removed and the cultured cells were washed with serum-free media to wash out colchicine.
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0443-6
M. Nishi et al. / Neuroscience Letters 229 (1997) 212–214
213
The cultured cells were incubated with S100b (20 ng/ml) for 3 h in a CO2 incubator at 37°C. Control cultures were treated in the same way except that no drug was added. After drug treatment, cultured cells were fixed for 30 min at room temperature (RT) in 4% paraformaldehyde in phosphate buffer (PB, pH 7.4) and then blocked with 5% bovine serum in PB for 1 h at room temperature. Cultured cells were incubated with primary mouse anti-MAP2 monoclonal
Fig. 2. Effects of S100b on the length of MAP2-IR neurite in a spinal cord culture after exposure to colchicine. Values represent the average length of a MAP2-IR neurite (mean ± SEM obtained in a total of 300 neurites in 3 separate cultures; 100 neurites/culture). The results were analyzed using ANOVA followed by Tukey’s honest test. *P , 0.05 compared to colchicine treatment alone.
Fig. 1. Effects of S100b on MAP2-IR neurite and MAP2 immunoreactivity in the cell body of a colchicine-treated spinal cord culture. (A) Control culture was grown for 48 h in the presence of fetal bovine serum, then for 48 h in the absence of serum before fixation. (B) Culture treated with colchicine (10−6 M) for 30 min before fixation. Colchicine induced a retraction of MAP2-IR neurites (arrow head) and a concentration of MAP2 immunoreactivity in the cell body region. (C) Culture treated with S100b (20 ng/ml) for 3 h after exposure to colchicine. Treatment with S100b stimulated the extension of MAP2-IR neurite (arrow). MAP2IR intensity in the cell body region was reduced as compared to that observed in (B) (asterisk). Scale bar: 50 mm.
antibody (1:500 dilution; Sigma) for 48 h at 4°C. After rinsing with PB, the cultures were reacted with biotinylated goat anti-mouse antibody (1:250 dilution; Boehringer Mannheim) for 1 h at room temperature. The cultures were rinsed with PB and incubated in streptavidin–peroxidase conjugate (1:8000 dilution; Boehringer Mannheim) for 1 h at room temperature. The cells were visualized with 0.02% 3,3′-diaminobenzidine (Sigma)/0.2% nickel sulfate/0.006% H2O2 in 0.1 M Tris–HCl buffered (pH 7.6) saline. Slides were subjected to morphometric analysis as follows. The Optimas (ver. 4.0) image acquiring software program (Bioscan, Inc.) was used for all morphometric analyses according to previous papers [4,13]. For the analysis of process length, each process was captured and subjected to Optimas linelength measurement. For the analysis of MAP2-IR intensity in the cell body region, cell bodies were captured randomly and the mean area gray value of each cell body was measured. Statistical significance was determined using ANOVA followed by the post-hoc Tukey test. In the untreated control culture of 5 days in vitro, MAP2IR was localized to long neurites extending from neurons showing weak perikaryon MAP2-IR (Fig. 1A). Average length of MAP2-IR neurite is 80.6 ± 5.1 mm (Fig. 2). The length of MAP2-IR neurites in spinal cord neurons exposed to 10−6 M colchicine for 30 min was markedly reduced compared to that in control cultures, and MAP2-IR was concentrated in the cell body (Fig. 1B). The average length of MAP2-IR neurite was 31.7 ± 5.5 mm (Fig. 2). MAP2-IR intensity in the cell body region was increased compared to that of the control (Fig. 3). Incubation with S100b (20 ng/ ml) for 3 h after colchicine treatment altered the distribution of MAP2-IR (Fig. 1C). The length of MAP2-IR neurites increased by 65% compared to that in colchicine treatment alone (Fig. 2). MAP2-IR intensity in the cell body region was reduced by 26% compared to that in colchicine treatment alone (Fig. 3).
214
M. Nishi et al. / Neuroscience Letters 229 (1997) 212–214
undergo plastic changes [8]. The present findings suggest that S100b plays an important role in regulating dynamic changes of neuronal microtubules, which are essential in neural plasticity. The role of S100b in the spinal cord injury is intriguing. Although it remains to be determined to what degree our experimentally induced neuronal injuries in vitro are relevant to injuries in the adult brain, the results of the present study suggest a novel application of S100b for treating spinal cord injuries.
Fig. 3. Effects of S100b on MAP2-IR intensity in cell body region in a spinal cord culture after exposure to colchicine. The area gray values (ArGV) obtained from the computer-aided densitometric analysis have been subtracted from 100 (background setting). Values represent the average value of 100-Ar-GV (mean ± SEM obtained in a total of 300 neurons in 3 separate cultures; 100 neurons/culture). The results were analyzed using ANOVA followed by Tukey’s honest test. *P , 0.05 compared to colchicine
The present study demonstrated that S100b promoted the extension of MAP2-IR neurite retracted after exposure to colchicine in rat spinal cord cultures. It is known that colchicine causes neurite retraction in vitro [7]. The previous studies also indicated that colchicine activates MAP kinase by disrupting microtubules [15], which causes further phosphorylation of cytoskeletal proteins, resulting in depolymerization of microtubules [10]. It is known that S100b inhibits the phosphorylation of brain cytoskeletal proteins such as MAP2, tau, GAP43 [5,6,14] and promotes microtubule polymerization and stability, which are essential for neurite extension [1,11,16]. The results of the present study also showed that colchicine treatment altered the distribution of MAP2 immunoreactivity. Although the present study did not determine the alteration of total MAP2 content, it was observed that colchicine caused the concentration of MAP2 immunoreactivity in neuronal cell bodies and increased the MAP2-IR intensity in the cell body region compared to that in control culture. These observation indicate that colchicine causes retraction of MAP2-IR neurites by disrupting the microtubular cytoskeletal system of neurites. In contrast, S100b increased the length of MAP2-IR neurites and reduced MAP2-IR intensity in the cell body region after colchicine treatment. Taken together with the above observations, these results are consistent with the previous findings that S100b stimulates neurite extension by promoting microtubule polymerization and stabilization through interactions with MAP2. A recent study indicated that stabilization of microtubules by MAP2 has important consequences for the capacity of established neurites to
[1] Ainsztein, A.M. and Purich, D.L., Stimulation of tubulin polymerization by MAP-2, J. Biol. Chem., 269 (1994) 28465–28471. [2] Azmitia, E.C., Dolan, K. and Whitaker-Azmitia, P.M., S100b but not NGF, EGF, insulin or calmodulin is a CNS serotonergic growth factor, Brain Res., 516 (1990) 534–536. [3] Azmitia, E.C. and Hou, X.P., Steroid regulation of neurotrophic activity: primary microcultures of mid brain raphe and hippocampus. In E.R. DeKloet and W. Sutanto (Eds.), Neurobiology of Steroids, Methods in Neuroscience, Vol. 22, Academic Press, San Diego, CA, 1994, pp. 359–371. [4] Azmitia, E.C., Rubinstein, V.J., Strafaci, J.A., Riaos, J.C. and Whitaker-Azmitia, P.M., 5-HT1A agonist and dexamethasone reversal of parachloroamphetamine induced loss of MAP-2 and synaptophysin immunoreactivity in adult rat brain, Brain Res., 677 (1995) 181– 192. [5] Baudier, J. and Cole, R.D., Interactions between the microtubuleassociated tau proteins and S100b regulate tau phosphorylation by the Ca2+/Calmodulin-dependent protein kinase II, J. Biol. Chem., 263 (1988) 5876–5882. [6] Furmanski, P., Neurite extension by mouse neuroblastoma: evidence for two models of induction, Differentiation, 1 (1973) 319–322. [7] Hesketh, J.H. and Baudier, J., Evidence that S100b proteins regulate microtubule assembly and stability in rat brain extracts, Int. J. Biochem., 18 (1986) 691–695. [8] Kaech, S., Ludin, B. and Matus, A., Cytoskeletal plasticity in cells expressing neuronal microtubule-associated proteins, Neuron, 17 (1996) 1189–1199. [9] Kligman, D. and Marshk, D.R., Purification and characterization of neurite extension factor from bovine brain, Proc. Natl. Acad. Sci. USA, 82 (1985) 7136–7139. [10] Lindwall, G. and Cole, R.D., Phosphorylation affects the ability of tau protein to promote microtubule assembly, J. Biol. Chem., 259 (1984) 5301–5305. [11] Mattson, M.P., Effects of microtubule stabilization and destabilization on tau immunoreactivity in cultured hippocampal neurons, Brain Res., 582 (1992) 107–118. [12] Mitchison, T. and Kirschner, M., Dynamic instability of microtubule growth, Nature, 312 (1984) 237–242. [13] Nishi, M., Whitaker-Azmitia, P.M. and Azmitia, E., C, Enhanced synaptophysin immunoreactivity in rat hippocampal culture by 5HT1A agonist, S100b and corticosteroid receptor agonists, SYNAPSE, 23 (1996) 1–9. [14] Sheu, F.-S., Azmitia, E.C., Marshak, D.R. and Parker, P.J., Glialderived S100b protein selectively inhibits recombinant protein kinase C (PKC) phosphorylation of neuron-specific protein F1/ GAP43, Mol. Brain Res., 21 (1994) 62–66. [15] Shinohara-Goto, Y., Nishida, E., Hoshi, M. and Sakai, H., Activation of microtubule-associated protein kinase by microtubule disruption in quiescent rat 3Y1 cells, Exp. Cell Res., 193 (1991) 161–166. [16] VanEldik, L.J., Christie-Pope, B., Bolin, L.M., Shooter, E.M. and Whetsell, W.O. Jr., Neurotrophic activity of S100b in cultures of dorsal root ganglia from embryonic chick and fetal rat, Brain Res., 542 (1991) 280–285.
S100b promotes the extension of microtubule associated protein2 (MAP2)immunoreactive neurites retracted after colchicine treatment in rat spinal cord culture Mayumi Nishi a , b ,*, Mitsuhiro Kawata a, Efrain C. Azmitia b a
Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kamigyou-ku, Kyoto 602, Japan b Department of Biology, New York University, 1009 Main Building, 100 Washington Square East, New York, NY 10003, USA Received 9 June 1997; accepted 17 June 1997
Abstract S100b, a glial derived calcium-binding protein with neurotrophic activity in the central nervous system, stimulates neurite extension of fetal raphe, cortex, spinal cord, and dorsal root ganglion neurons. The effects of S100b on neurite length and microtubule associated protein2 (MAP2) immunoreactivity (IR) after microtubule disruption with colchicine were investigated in primary rat spinal cord culture. The incubation with S100b (20 ng/ml) for 3 h after exposure to colchicine (10−6 M) for 30 min altered the distribution of MAP2-IR. The length of MAP2-IR neurites increased by 65% compared to that in colchicine treatment alone. MAP2-IR intensity in the cell body was reduced by 26% compared to that in colchicine treatment alone. These results indicate that neurites shrink when the microtubular cytoskeletal system is disrupted and S100b rapidly promotes re-assembly and/or stabilization. 1997 Elsevier Science Ireland Ltd. Keywords: Depolymerization; Polymerization; Spinal cord injury; Phosphorylation; Microtubular cytoskeletal system; Primary culture
S100b is a cytosolic calcium binding protein which is synthesized and stored in glial cells. Previous studies have shown that S100b acts mainly on neurite extension [2,9,16]. It is considered that this effect of S100b is caused by stabilization of microtubules by inhibiting phosphorylation of brain cytoskeletal proteins such as microtubules associated protein2 (MAP2), tau, growth associated protein43 (GAP43) [5,6,14]. Does S100b directly affect injured neurites and stimulate their extension? The present study investigated the effects of S100b on MAP2 immunoreactive (IR) neurite length and MAP2-IR intensity in the cell body region after microtubule disruption with colchicine in primary rat spinal cord cultures. Colchicine is a microtubule-disrupting agent which is known to activate mitogen activated protein kinase (MAP kinase; it is also called microtubule associated protein kinase) [15]. Microtubules are structural filaments in the cytoplasm which are spatially organized and extremely
* Corresponding author: Tel.: +81 75 2515301; fax: +81 75 2515306; e-mail: [email protected]
dynamic [11,12]. By using MAP2 immunoreactivity as a marker specific for the microtubule system, we show here that S100b stimulates MAP2-IR neurite extension after microtubule disruption with colchicine. Dissociated spinal cord primary cultures were prepared from 18-day-old Sprague–Dawley rat fetuses, according to the method of Azmitia and Hou [3]. Briefly, spinal cords were isolated and mechanically dissociated by triturating through a fire-polished glass pipette. The cells were plated at an initial plating density of 1 × 105 cells/cm2 on each well (area of 0.28 cm2) precoated with polyethyleneimine. The cultures were maintained in complete neuronal medium (CNM) containing 5% (v/v) fetal bovine serum (Sigma) for 48 h in a CO2 incubator at 37°C with 5% CO2/95% air. After 48 h the CNM was removed and the cultures were rinsed with serum-free media (MEM with 0.25% glucose, 1% non-essential amino acids, 20 mM putrescine, 15 nM sodium selenite, 5 mg/ml insulin, and 100 mg/ml transferrin). After incubation in the serum free medium for 48 h, the cultures were treated with 10−6 M colchicine (Sigma) for 30 min. Then colchicine was removed and the cultured cells were washed with serum-free media to wash out colchicine.
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0443-6
M. Nishi et al. / Neuroscience Letters 229 (1997) 212–214
213
The cultured cells were incubated with S100b (20 ng/ml) for 3 h in a CO2 incubator at 37°C. Control cultures were treated in the same way except that no drug was added. After drug treatment, cultured cells were fixed for 30 min at room temperature (RT) in 4% paraformaldehyde in phosphate buffer (PB, pH 7.4) and then blocked with 5% bovine serum in PB for 1 h at room temperature. Cultured cells were incubated with primary mouse anti-MAP2 monoclonal
Fig. 2. Effects of S100b on the length of MAP2-IR neurite in a spinal cord culture after exposure to colchicine. Values represent the average length of a MAP2-IR neurite (mean ± SEM obtained in a total of 300 neurites in 3 separate cultures; 100 neurites/culture). The results were analyzed using ANOVA followed by Tukey’s honest test. *P , 0.05 compared to colchicine treatment alone.
Fig. 1. Effects of S100b on MAP2-IR neurite and MAP2 immunoreactivity in the cell body of a colchicine-treated spinal cord culture. (A) Control culture was grown for 48 h in the presence of fetal bovine serum, then for 48 h in the absence of serum before fixation. (B) Culture treated with colchicine (10−6 M) for 30 min before fixation. Colchicine induced a retraction of MAP2-IR neurites (arrow head) and a concentration of MAP2 immunoreactivity in the cell body region. (C) Culture treated with S100b (20 ng/ml) for 3 h after exposure to colchicine. Treatment with S100b stimulated the extension of MAP2-IR neurite (arrow). MAP2IR intensity in the cell body region was reduced as compared to that observed in (B) (asterisk). Scale bar: 50 mm.
antibody (1:500 dilution; Sigma) for 48 h at 4°C. After rinsing with PB, the cultures were reacted with biotinylated goat anti-mouse antibody (1:250 dilution; Boehringer Mannheim) for 1 h at room temperature. The cultures were rinsed with PB and incubated in streptavidin–peroxidase conjugate (1:8000 dilution; Boehringer Mannheim) for 1 h at room temperature. The cells were visualized with 0.02% 3,3′-diaminobenzidine (Sigma)/0.2% nickel sulfate/0.006% H2O2 in 0.1 M Tris–HCl buffered (pH 7.6) saline. Slides were subjected to morphometric analysis as follows. The Optimas (ver. 4.0) image acquiring software program (Bioscan, Inc.) was used for all morphometric analyses according to previous papers [4,13]. For the analysis of process length, each process was captured and subjected to Optimas linelength measurement. For the analysis of MAP2-IR intensity in the cell body region, cell bodies were captured randomly and the mean area gray value of each cell body was measured. Statistical significance was determined using ANOVA followed by the post-hoc Tukey test. In the untreated control culture of 5 days in vitro, MAP2IR was localized to long neurites extending from neurons showing weak perikaryon MAP2-IR (Fig. 1A). Average length of MAP2-IR neurite is 80.6 ± 5.1 mm (Fig. 2). The length of MAP2-IR neurites in spinal cord neurons exposed to 10−6 M colchicine for 30 min was markedly reduced compared to that in control cultures, and MAP2-IR was concentrated in the cell body (Fig. 1B). The average length of MAP2-IR neurite was 31.7 ± 5.5 mm (Fig. 2). MAP2-IR intensity in the cell body region was increased compared to that of the control (Fig. 3). Incubation with S100b (20 ng/ ml) for 3 h after colchicine treatment altered the distribution of MAP2-IR (Fig. 1C). The length of MAP2-IR neurites increased by 65% compared to that in colchicine treatment alone (Fig. 2). MAP2-IR intensity in the cell body region was reduced by 26% compared to that in colchicine treatment alone (Fig. 3).
214
M. Nishi et al. / Neuroscience Letters 229 (1997) 212–214
undergo plastic changes [8]. The present findings suggest that S100b plays an important role in regulating dynamic changes of neuronal microtubules, which are essential in neural plasticity. The role of S100b in the spinal cord injury is intriguing. Although it remains to be determined to what degree our experimentally induced neuronal injuries in vitro are relevant to injuries in the adult brain, the results of the present study suggest a novel application of S100b for treating spinal cord injuries.
Fig. 3. Effects of S100b on MAP2-IR intensity in cell body region in a spinal cord culture after exposure to colchicine. The area gray values (ArGV) obtained from the computer-aided densitometric analysis have been subtracted from 100 (background setting). Values represent the average value of 100-Ar-GV (mean ± SEM obtained in a total of 300 neurons in 3 separate cultures; 100 neurons/culture). The results were analyzed using ANOVA followed by Tukey’s honest test. *P , 0.05 compared to colchicine
The present study demonstrated that S100b promoted the extension of MAP2-IR neurite retracted after exposure to colchicine in rat spinal cord cultures. It is known that colchicine causes neurite retraction in vitro [7]. The previous studies also indicated that colchicine activates MAP kinase by disrupting microtubules [15], which causes further phosphorylation of cytoskeletal proteins, resulting in depolymerization of microtubules [10]. It is known that S100b inhibits the phosphorylation of brain cytoskeletal proteins such as MAP2, tau, GAP43 [5,6,14] and promotes microtubule polymerization and stability, which are essential for neurite extension [1,11,16]. The results of the present study also showed that colchicine treatment altered the distribution of MAP2 immunoreactivity. Although the present study did not determine the alteration of total MAP2 content, it was observed that colchicine caused the concentration of MAP2 immunoreactivity in neuronal cell bodies and increased the MAP2-IR intensity in the cell body region compared to that in control culture. These observation indicate that colchicine causes retraction of MAP2-IR neurites by disrupting the microtubular cytoskeletal system of neurites. In contrast, S100b increased the length of MAP2-IR neurites and reduced MAP2-IR intensity in the cell body region after colchicine treatment. Taken together with the above observations, these results are consistent with the previous findings that S100b stimulates neurite extension by promoting microtubule polymerization and stabilization through interactions with MAP2. A recent study indicated that stabilization of microtubules by MAP2 has important consequences for the capacity of established neurites to
[1] Ainsztein, A.M. and Purich, D.L., Stimulation of tubulin polymerization by MAP-2, J. Biol. Chem., 269 (1994) 28465–28471. [2] Azmitia, E.C., Dolan, K. and Whitaker-Azmitia, P.M., S100b but not NGF, EGF, insulin or calmodulin is a CNS serotonergic growth factor, Brain Res., 516 (1990) 534–536. [3] Azmitia, E.C. and Hou, X.P., Steroid regulation of neurotrophic activity: primary microcultures of mid brain raphe and hippocampus. In E.R. DeKloet and W. Sutanto (Eds.), Neurobiology of Steroids, Methods in Neuroscience, Vol. 22, Academic Press, San Diego, CA, 1994, pp. 359–371. [4] Azmitia, E.C., Rubinstein, V.J., Strafaci, J.A., Riaos, J.C. and Whitaker-Azmitia, P.M., 5-HT1A agonist and dexamethasone reversal of parachloroamphetamine induced loss of MAP-2 and synaptophysin immunoreactivity in adult rat brain, Brain Res., 677 (1995) 181– 192. [5] Baudier, J. and Cole, R.D., Interactions between the microtubuleassociated tau proteins and S100b regulate tau phosphorylation by the Ca2+/Calmodulin-dependent protein kinase II, J. Biol. Chem., 263 (1988) 5876–5882. [6] Furmanski, P., Neurite extension by mouse neuroblastoma: evidence for two models of induction, Differentiation, 1 (1973) 319–322. [7] Hesketh, J.H. and Baudier, J., Evidence that S100b proteins regulate microtubule assembly and stability in rat brain extracts, Int. J. Biochem., 18 (1986) 691–695. [8] Kaech, S., Ludin, B. and Matus, A., Cytoskeletal plasticity in cells expressing neuronal microtubule-associated proteins, Neuron, 17 (1996) 1189–1199. [9] Kligman, D. and Marshk, D.R., Purification and characterization of neurite extension factor from bovine brain, Proc. Natl. Acad. Sci. USA, 82 (1985) 7136–7139. [10] Lindwall, G. and Cole, R.D., Phosphorylation affects the ability of tau protein to promote microtubule assembly, J. Biol. Chem., 259 (1984) 5301–5305. [11] Mattson, M.P., Effects of microtubule stabilization and destabilization on tau immunoreactivity in cultured hippocampal neurons, Brain Res., 582 (1992) 107–118. [12] Mitchison, T. and Kirschner, M., Dynamic instability of microtubule growth, Nature, 312 (1984) 237–242. [13] Nishi, M., Whitaker-Azmitia, P.M. and Azmitia, E., C, Enhanced synaptophysin immunoreactivity in rat hippocampal culture by 5HT1A agonist, S100b and corticosteroid receptor agonists, SYNAPSE, 23 (1996) 1–9. [14] Sheu, F.-S., Azmitia, E.C., Marshak, D.R. and Parker, P.J., Glialderived S100b protein selectively inhibits recombinant protein kinase C (PKC) phosphorylation of neuron-specific protein F1/ GAP43, Mol. Brain Res., 21 (1994) 62–66. [15] Shinohara-Goto, Y., Nishida, E., Hoshi, M. and Sakai, H., Activation of microtubule-associated protein kinase by microtubule disruption in quiescent rat 3Y1 cells, Exp. Cell Res., 193 (1991) 161–166. [16] VanEldik, L.J., Christie-Pope, B., Bolin, L.M., Shooter, E.M. and Whetsell, W.O. Jr., Neurotrophic activity of S100b in cultures of dorsal root ganglia from embryonic chick and fetal rat, Brain Res., 542 (1991) 280–285.