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Inhibition of neuronal surface proteases decreases the requirement for GAP-43 in neurite outgrowth
DEVELOPMENTAL BRAIN RESEARCH
ELSEVIER
Developmental Brain Research 87 (1995) 87-90
Short communication
Inhibition of neuronal surface proteases decreases the requirement for GAP-43 in neurite outgrowth T h o m a s B. S h e a * Center for Cellular Neurobiology and Neurodegeneration Research, Department of Biological Sciences, University of Massachusetts at Lowell, Lowell, MA 01854, USA
Accepted 28 February 1995
Abstract
Intracellular delivery of anti-GAP IgG inhibits the elaboration of neurites by NB2a/dl cells. However, recent studies indicate that the extent of inhibition is minimized when cells were cultured on poly-L-lysine-coated or laminin-coated versus uncoated plates, suggesting that the role of GAP-43 in neuritogenesis may be specifically related to membrane adhesiveness. We therefore examined the influence of inhibition of thrombin, the neuronal surface protease that restricts neurite outgrowth, on GAP-43-dependent neurite outgrowth. Treatment of cells with the specific thrombin inhibitor hirudin in the presence of serum induced a similar percentage of neurite outgrowth as was observed following serum withdrawal. However, while neurite outgrowth induced by serum deprivation of cells was reduced following intracellular delivery of anti-GAP IgG, neurite outgrowth induced by hirudin treatment of cells was not. That inhibition of neuronal surface protease activity overcomes the inhibition of neurite outgrowth following intracellular delivery of anti-GAP IgG provides further evidence that the role that GAP-43 plays in neuritogenesis is related to membrane adhesiveness. Keywords: GAP-43; Neurite outgrowth; Neuronal maturation; Growth cone; Adhesion; Neuronal surface protease; Thrombin; Himdin
A number of in vivo and in vitro studies suggest that the neuronal growth-associated protein, GAP43 (also referred to as B50, F1, pp46, GAP48, p57 and neuromodulin) [4,23] plays an essential role in neuritogenesis. The highest GAP43 levels are observed during the embryonic and postnatal periods of active axonal outgrowth and synaptogenesis, after which GAP43 levels in most regions decline [3,9]. A resurgence in developmental levels of GAP43 expression accompanies axonal regeneration and reactive synaptogenesis [6,8,12,14]. Transfection with GAP-43 enhances neuritogenesis in PC12 and NG108-15 cells [10,25]. Similarly, expression of GAP43 enhances filopodial extensions in non-neuronal cells [24,26]. Neuritogenesis is inhibited following intracellular delivery of anti-GAP43 IgG in N B 2 a / d l cells [17]. However, conflicting results have appeared regarding the putative role for GAP-43 in neuritogenesis, including the observation of neurite outgrowth in a mutant PC12 cell line lacking GAP-43 [2] and apparently normal neurite outgrowth despite the diminution of GAP43 translocation
* Corresponding author. 0165-3806//95//$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0165-3806(95)00048-8
along developing neurites as a consequence of anti sense oligonucleotide-mediated inhibition of kinesin expression [7]. To address this controversy, we examined the effects of intracellular delivery of anti-GAP IgG on neurite outgrowth induced by different treatments of N B 2 a / d l neuroblastoma cells. We have previously shown that neurite elaboration by some, but not all, treatments is inhibited following intracellular delivery of anti-GAP IgG, and have suggested that the requirement for GAP-43 in neuritogenesis is related to membrane adhesiveness [17,20,21]. To probe further this potential relationship, we examined the effects on neuritogenesis of inhibition of neuronal surface protease activity following the intracellular delivery of anti-GAP IgG. We have previously shown that the neuronal surface protease thrombin (also present in serum), is normally responsible for restricting neuritogenesis, and that treatment of cells with the specific thrombin inhibitor, hirudin, will induce neurite outgrowth in the presence of serum [16,17]. We therefore examined whether or not the intracellular delivery of anti-GAP IgG prevented neuritogenesis following treatment of cells with hirudin. N B 2 a / d l mouse neuroblastoma cells, a subclone of
88
T.B. Shea / Deuelopmental Brain Research 87 (1995) 87-90
NB2a cells (originally derived from C1300 neuroblastoma cells) [16,17], were cultured in Dulbecco's Minimal Eagle's Medium containing 10% horse serum as described previously [16,17] and were induced to elaborate neurites exhibiting characteristics of axons following 4 h of serum deprivation or treatment with 5 - 5 0 U / m l hirudin in DMEM containing 0.8-10% serum [16,17]. Cells were transiently permeabilized cells as described [17]. Briefly, cells (50-75% confluent) in uncoated LabTek 8-well chamber slides were rinsed with serum-free DMEM and treated with 100 /.tl of pre-warmed (37 ° C) 1.2 M glycerol in phosphate-buffered saline (PBS; pH 7.4) and incubated at 0 ° C for 10 min (by placing the culture plates directly on ice). L-c~-lysophosphatidylcholine (LPC; final concentration 40 /zg/ml) was added and incubation was continued for an additional 8 min. Cultures then received 100 ~zl of prewarmed (37 ° C) serum-free DMEM containing either no further additions or 1:40 dilutions (relative to sheep serum IgG concentration) of anti-GAP IgG (raised in sheep) or pre-immune IgG. Anti-GAPA3 IgG recognizes only a single polypeptide migrating at 43kDa in immunoblot analysis of N B 2 a / d l cells; the generation of this antibody, and its specificity for GAP-43 in N B 2 a / d l cells has been described in our previous studies [17]. Following a 10 min. incubation at 37°C, all cultures received an additional pre-warmed 100 /xl aliquot of media with and without serum or hirudin, and were incubated for an additional 2 h. Cultures were then fixed (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4) and examined by phase-contrast microscopy for the presence or absence of neurites. Antibody uptake during permeabilization was confirmed by incubation of cultures permeabilized in the presence and absence of anti-GAP IgG, preimmune IgG, and non-permeabilized controls with antisheep IgG conjugated to horseradish peroxidase and visualization with diaminobenzidine in the presence of hydrogen peroxide as described [17]. Since the typical length of N B 2 a / d l neurites induced by a 2-h treatment with a variety of differentiating compounds is approximately one somal diameter [16], cells were scored for the presence or absence of one or more neurites of > 1 somal diameter. Between 200 and 300 Trypan blue-negative cells in multiple randomly selected microscopic fields in duplicate or triplicate plates from two separate experiments were scored, and the mean percentages of cells with neurites were statistically compared by Student's t-test [16,17]. All reagents were obtained from Sigma Chemical Co., St. Louis, MO. Only a small percentage ( 8 . 6 _ 2.5%) of N B 2 a / d l cells cultured in the presence of 10% serum elaborated neurites of length > their respective somal diameter. While reduction of the serum concentration to as little as 0.8% typically induced only a modest increase in the percentage of cells elaborating neurites (17.6_+ 4.7%), within 2 h of complete withdrawal of serum the majority of cells elaborated neurites (63.0 + 4.2%) (see also [16,17]).
Cells elaborated neurites in the presence of serum following the addition of hirudin, a specific inhibitor of the protease thrombin, at concentrations inversely proportional to the percentage of serum in the culture medium. For example, 5 U / m l induces neuritogenesis in 63.2 + 2.9% of cells cultured in the presence of 0.8% serum but did not induce neuritogenesis in the presence of 10% serum (not shown), However, 5 0 U / m l induced the majority of cells (74.2 ___15.3) to elaborate neurites in the presence of 10% serum. By contrast, none of these conditions induced significant alterations in perikaryal size or overall morphology. For example, in the absence of hirudin, perikaryal area of cells receiving pre-immune-IgG was 74.6 + 12.2, while the perikaryal area of cells receiving anti-GAP IgG was 6 9 . 8 _ 8.8 (measurements are in arbitrary relative units obtained via NIH Image analysis software); in the presence of hirudin, these values were 85.7__+ 8.8 and 78.8 + 12.3, respectively. Intracellular delivery of 1:40 dilution of anti-GAP IgG inhibited the elaboration of neurites followed by serum-deprivation of cells, while pre-immune serum did not perturb neuritogenesis (Fig. 1; see also [20,21]). By contrast, the intracellular delivery of the identical concentration of antiGAP IgG did not inhibit neurite outgrowth induced by hirudin treatment in the presence of serum (Fig. 1). The results of this, and our previous studies [20,21] indicate that GAP-43 plays an important role in neuritogenesis, and that this role is apparently related to adhesiveness. This line of reasoning is consistent with the association of GAP-43 with the membrane skeleton following fractionation [12], the preferential localization of GAP-43 within the most adhesive areas of the growth cone [12], and the elaboration of filopodia following transfection with GAP-43 [15,24,25]. While other studies have reported no inhibition of neuritogenesis following treatments that reduce GAP-43 expression [2] or translocation [7], it should be noted that cells in these studies were cultured on coated with adhesive substrates rather than uncoated plastic; we have observed that plating cells on poly-L-lysine or laminin drastically reduces any apparent requirement for GAP-43 in N B 2 a / d l neuritogenesis [20,21]. The reduction in growth cone size and the elaboration of neurites thinner caliber and turn less frequently following inhibition that accompanies inhibition of GAP-43 expression in sensory neurons [1] and N B 2 a / d l cells [22] has led to the suggestion that GAP-43 modulates interactions between developing neurites and the substratum. In this regard, certain aspects of the initial, non-target mediated stage(s) of neurite outgrowth are largely mediated by filopodia-substrate adhesiveness [13,16,18,19], and additional factors, including the presence of an adhesive substrate [17,18] are necessary to stabilize the putative neurite. Accordingly, it may be expected that any requirement for GAP-43-mediated membrane-substrate interactions may be reduced or supplanted by treatments that otherwise
T.B. Shea / Developmental Brain Research 87 (1995) 87-90
|
Acknowledgements This research was supported by the National Science Foundation.
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T Fig. 1. Quantitation of neurites elaborated following 2 h of serum-deprivation or hirudin treatment in the presence of 0.8% serum either without permeabilization, or after permeabilization in the presence of various dilutions of anti-GAP or pre-immune IgG. Values obtained for cultures receiving anti-GAP IgG that differ significantly ( P < 0.005 or less, Student's t-test) from those obtained in cultures receiving the respective concentration of pre-immune IgG are indicated by an asterisk. Note that the intracellular delivery of pre-immune lgG did not affect neurite outgrowth. Note further that the elaboration of neurites induced by serum-deprivation is inhibited by the intracellular delivery of anti-GAP IgG, while neurites induced by hirudin treatment are not inhibited.
increase membrane-substrate adhesiveness, such as decreased neuronal surface proteolysis following hirudin treatment or serum withdrawal (present study) a n d / o r culturing cells on more adhesive substrates [20,21]. The limited duration of intense GAP-43 staining within N B 2 a / d l neurites, coupled with the limited period of time ( < 24 h) during which intracellular delivery of anti-GAP IgG inhibits neurite elaboration [17] suggests that relatively high GAP-43 levels are required for only a relatively short period during initial neuritogenesis. One interpretation of the results of these and other studies is that GAP-43 may regulate early events of neuritogenesis in situ. It should be noted, however, that the expression of GAP-43 is in itself insufficient to induce neuritogenesis[5,11,17,19]. Accordingly, these findings are likely to underestimate the nature and extent of any requirement for GAP-43 in neuritogenesis in situ. The precise role of GAP-43, and the physiological relevance of these and other findings in cultured neuronal cells, remain to be determined.
[1] Aigner, L. and Caroni, P., Depletion of 43-kD growth-associated protein in primary sensory neurons leads to diminished formation and spreading of growth cones, J. Cell Biol., 123 (1993) 417-429. [2] Baetge, E.E. and Hammang, J.P., Neurite outgrowth in PC12 cells deficient in GAP-43, Neuron, 6 (1991) 21-30. [3] Basi, G.S., Jacobson, R.D., Virag, I., Schilling, J. and Skene, J.H.P., Primary structure and transcriptional regulation of GAP-43, a protein associated with nerve growth, Cell, 49 (1987) 785-791. [4] Benowitz, L.I. and Routtenberg, A., A membrane phosphoprotein associated with neural development, axonal regeneration, phospholipid metabolism, and synaptic plasticity, Trends Neurosci., 10 (1987) 527-532. [5] Burry, R.W. and Perrone-Bizzozero, N.I., Nerve growth factor stimulates GAP-43 expression in PC12 cell clones independently of neurite outgrowth, J. Neurosci. Res., 36 (1993) 241-251. [6] Dani, J.W., Armstrong, D.M. and Benowitz, L.I., Mapping the development of the rat brain by GAP-43 immunocytochemistry, Neuroscience, 40 (1991) 277-288. [7] Ferreira A., Niclas J., Vale R.D., Banker G. and Kosik K.S., Suppression of kinesin expression in cultured hippocampal neurons using antisense oligonucleotides, J. Cell Biol., 117 (1992) 595-606. [8] Goslin, K., Schreyer, D.J., Skene, J.H. and Banker, G., Development of neuronal polarity: GAP-43 distinguishes axonal from dendritic growth cones, Nature, 336 (1988) 672-674. [9] .lacobson, R.D., Virag, I. and Skene, J.H.P., A protein associated with axon growth, GAP-43, is widely distributed and developmentally regulated in rat CNS, J. Neurosci., 6 (1986) 1843-1855. [10] Kumagi, C., Tohda, M., Isobe, M. and Nomura, Y., Involvement of growth-associated protein-43 with irreversible neurite outgrowth by dibutyryl cycli AMP and phorhol ester in NG108-15 cells, J. Neurochem., 59 (1992) 41-47. [11] Megerian, J.T., Sullivan, J.P. and Klein, W.L., GAP-43 expression without neurite extension, Soc. Neurosci. Abstr., 15 (1989) 574. [12] Meiri, K.F. and Gordon-Weeks, P.R., GAP-43 in growth cones is associated with areas of membrane that are tightly bound to sbustrate and is a component a membrane skeleton subcellular fraction, J. Neurosci., 10 (1990) 256-266. [13] Monard, D., Implications of proteases and protease inhibitors in neurite outgrowth. In Neurobiochemistry, Selected Topics, Vol. 36, Springer-Verlag, Berlin, 1985, pp. 7-12. [14] Moya, K.L., Jhaveri, S., Schneider, G.E., Benowitz, L.I., Immunohistochemical localization of GAP-43 in the developing hamster retinofugal pathway, J. Comp. Neurol., 288 (1989) 51-58. [15] Nielander, H.B., French, P., Oestreicher, A.B., Gispen, W.H. and Scotman P., Spontaneous morphological changes by overexpression of the growth-associated protein B-50/GAP-43 in a PC12 cell line, Neurosci. Lett., 162 (1993) 46-50. [16] Shea, T.B., Beermann, M.L., Nixon, R.A., Multiple proteases regulate neurite outgrowth, J. Neurochem., 56 (1991) 842-851. [17] Shea, T.B., Perrone-Bizzozero, N.I., Beermann, M.L. and Benowitz, L.I., Phospholipid-mediated delivery of anti-GAP-43 antibodies into neuroblastoma cells prevents neuritogenesis, J. Neurosci., 11 (1991) 1685-1690. [18] Shea, T.B., Beermann, M.L. and Nixon, R.A., Sequential requirement for cytoskeletal constituents during axonal neurite initiation, elongation and stabilization, J. Cell Biol., 115 (1991) 163. [19] Shea, T.B., Beermann, M.L. and Nixon, R.A., Complementary
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effects of laminin and protease inhibitors on neurite outgrowth and maintenance, Neurosci. Res. Commun., 10 (1992) 53-61. [20] Shea, T.B. and Beermann, Intracellular delivery of anti-GAP-43 antibodies into neuroblastoma inhibits neurite outgrowth induced by serum deprivation more than outgrowth elicited by dbcAMP treatment, Mol. Cell Biol., 4 (1993) 171a. [21] Shea, T,B. and Beermann, Adhesive substrates minimize the requirement for GAP-43 in N B 2 a / d l neuritogenesis, Trans. Am. Soc. Neurochem., 25 (1993) 123. [22] Shea, T,B., Intracellular delivery of anti-GAP-43 antibodies reduces growth cone size, Biochem. Biophys. Res. Commun., 203 (1994) 459-464.
[23] Skene, J.H.P., Axonal growth-associated proteins, Ann. Re~:. Neurosci., 12 (1989) 127-156. [24] Widmer, F. and Caroni, P., Phosphorylation-site mutagenesis of the growth-associated protein GAP-43 modulates its effects on cell spreading and morphology, J. Cell Biol., 120 (1993) 503-512. [25] Yankner, B.A., Benowitz, L.I., Villa-Komaroff, L. and Neve, R.L., Transfection of PC12 cells with the human GAP-43 gene: effects on neurite outgrowth and regeneration, Mol. Brain Res., 7 (1990) 39-44. [26] Zuber, M.X., Goodman, D.W., Karns, L.R. and Fishman, M.C., The neuronal growth-associated protein GAP-43 induces filopodia in non-neuronal cells, Science, 244 (1989) 1193-1195.
ELSEVIER
Developmental Brain Research 87 (1995) 87-90
Short communication
Inhibition of neuronal surface proteases decreases the requirement for GAP-43 in neurite outgrowth T h o m a s B. S h e a * Center for Cellular Neurobiology and Neurodegeneration Research, Department of Biological Sciences, University of Massachusetts at Lowell, Lowell, MA 01854, USA
Accepted 28 February 1995
Abstract
Intracellular delivery of anti-GAP IgG inhibits the elaboration of neurites by NB2a/dl cells. However, recent studies indicate that the extent of inhibition is minimized when cells were cultured on poly-L-lysine-coated or laminin-coated versus uncoated plates, suggesting that the role of GAP-43 in neuritogenesis may be specifically related to membrane adhesiveness. We therefore examined the influence of inhibition of thrombin, the neuronal surface protease that restricts neurite outgrowth, on GAP-43-dependent neurite outgrowth. Treatment of cells with the specific thrombin inhibitor hirudin in the presence of serum induced a similar percentage of neurite outgrowth as was observed following serum withdrawal. However, while neurite outgrowth induced by serum deprivation of cells was reduced following intracellular delivery of anti-GAP IgG, neurite outgrowth induced by hirudin treatment of cells was not. That inhibition of neuronal surface protease activity overcomes the inhibition of neurite outgrowth following intracellular delivery of anti-GAP IgG provides further evidence that the role that GAP-43 plays in neuritogenesis is related to membrane adhesiveness. Keywords: GAP-43; Neurite outgrowth; Neuronal maturation; Growth cone; Adhesion; Neuronal surface protease; Thrombin; Himdin
A number of in vivo and in vitro studies suggest that the neuronal growth-associated protein, GAP43 (also referred to as B50, F1, pp46, GAP48, p57 and neuromodulin) [4,23] plays an essential role in neuritogenesis. The highest GAP43 levels are observed during the embryonic and postnatal periods of active axonal outgrowth and synaptogenesis, after which GAP43 levels in most regions decline [3,9]. A resurgence in developmental levels of GAP43 expression accompanies axonal regeneration and reactive synaptogenesis [6,8,12,14]. Transfection with GAP-43 enhances neuritogenesis in PC12 and NG108-15 cells [10,25]. Similarly, expression of GAP43 enhances filopodial extensions in non-neuronal cells [24,26]. Neuritogenesis is inhibited following intracellular delivery of anti-GAP43 IgG in N B 2 a / d l cells [17]. However, conflicting results have appeared regarding the putative role for GAP-43 in neuritogenesis, including the observation of neurite outgrowth in a mutant PC12 cell line lacking GAP-43 [2] and apparently normal neurite outgrowth despite the diminution of GAP43 translocation
* Corresponding author. 0165-3806//95//$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0165-3806(95)00048-8
along developing neurites as a consequence of anti sense oligonucleotide-mediated inhibition of kinesin expression [7]. To address this controversy, we examined the effects of intracellular delivery of anti-GAP IgG on neurite outgrowth induced by different treatments of N B 2 a / d l neuroblastoma cells. We have previously shown that neurite elaboration by some, but not all, treatments is inhibited following intracellular delivery of anti-GAP IgG, and have suggested that the requirement for GAP-43 in neuritogenesis is related to membrane adhesiveness [17,20,21]. To probe further this potential relationship, we examined the effects on neuritogenesis of inhibition of neuronal surface protease activity following the intracellular delivery of anti-GAP IgG. We have previously shown that the neuronal surface protease thrombin (also present in serum), is normally responsible for restricting neuritogenesis, and that treatment of cells with the specific thrombin inhibitor, hirudin, will induce neurite outgrowth in the presence of serum [16,17]. We therefore examined whether or not the intracellular delivery of anti-GAP IgG prevented neuritogenesis following treatment of cells with hirudin. N B 2 a / d l mouse neuroblastoma cells, a subclone of
88
T.B. Shea / Deuelopmental Brain Research 87 (1995) 87-90
NB2a cells (originally derived from C1300 neuroblastoma cells) [16,17], were cultured in Dulbecco's Minimal Eagle's Medium containing 10% horse serum as described previously [16,17] and were induced to elaborate neurites exhibiting characteristics of axons following 4 h of serum deprivation or treatment with 5 - 5 0 U / m l hirudin in DMEM containing 0.8-10% serum [16,17]. Cells were transiently permeabilized cells as described [17]. Briefly, cells (50-75% confluent) in uncoated LabTek 8-well chamber slides were rinsed with serum-free DMEM and treated with 100 /.tl of pre-warmed (37 ° C) 1.2 M glycerol in phosphate-buffered saline (PBS; pH 7.4) and incubated at 0 ° C for 10 min (by placing the culture plates directly on ice). L-c~-lysophosphatidylcholine (LPC; final concentration 40 /zg/ml) was added and incubation was continued for an additional 8 min. Cultures then received 100 ~zl of prewarmed (37 ° C) serum-free DMEM containing either no further additions or 1:40 dilutions (relative to sheep serum IgG concentration) of anti-GAP IgG (raised in sheep) or pre-immune IgG. Anti-GAPA3 IgG recognizes only a single polypeptide migrating at 43kDa in immunoblot analysis of N B 2 a / d l cells; the generation of this antibody, and its specificity for GAP-43 in N B 2 a / d l cells has been described in our previous studies [17]. Following a 10 min. incubation at 37°C, all cultures received an additional pre-warmed 100 /xl aliquot of media with and without serum or hirudin, and were incubated for an additional 2 h. Cultures were then fixed (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4) and examined by phase-contrast microscopy for the presence or absence of neurites. Antibody uptake during permeabilization was confirmed by incubation of cultures permeabilized in the presence and absence of anti-GAP IgG, preimmune IgG, and non-permeabilized controls with antisheep IgG conjugated to horseradish peroxidase and visualization with diaminobenzidine in the presence of hydrogen peroxide as described [17]. Since the typical length of N B 2 a / d l neurites induced by a 2-h treatment with a variety of differentiating compounds is approximately one somal diameter [16], cells were scored for the presence or absence of one or more neurites of > 1 somal diameter. Between 200 and 300 Trypan blue-negative cells in multiple randomly selected microscopic fields in duplicate or triplicate plates from two separate experiments were scored, and the mean percentages of cells with neurites were statistically compared by Student's t-test [16,17]. All reagents were obtained from Sigma Chemical Co., St. Louis, MO. Only a small percentage ( 8 . 6 _ 2.5%) of N B 2 a / d l cells cultured in the presence of 10% serum elaborated neurites of length > their respective somal diameter. While reduction of the serum concentration to as little as 0.8% typically induced only a modest increase in the percentage of cells elaborating neurites (17.6_+ 4.7%), within 2 h of complete withdrawal of serum the majority of cells elaborated neurites (63.0 + 4.2%) (see also [16,17]).
Cells elaborated neurites in the presence of serum following the addition of hirudin, a specific inhibitor of the protease thrombin, at concentrations inversely proportional to the percentage of serum in the culture medium. For example, 5 U / m l induces neuritogenesis in 63.2 + 2.9% of cells cultured in the presence of 0.8% serum but did not induce neuritogenesis in the presence of 10% serum (not shown), However, 5 0 U / m l induced the majority of cells (74.2 ___15.3) to elaborate neurites in the presence of 10% serum. By contrast, none of these conditions induced significant alterations in perikaryal size or overall morphology. For example, in the absence of hirudin, perikaryal area of cells receiving pre-immune-IgG was 74.6 + 12.2, while the perikaryal area of cells receiving anti-GAP IgG was 6 9 . 8 _ 8.8 (measurements are in arbitrary relative units obtained via NIH Image analysis software); in the presence of hirudin, these values were 85.7__+ 8.8 and 78.8 + 12.3, respectively. Intracellular delivery of 1:40 dilution of anti-GAP IgG inhibited the elaboration of neurites followed by serum-deprivation of cells, while pre-immune serum did not perturb neuritogenesis (Fig. 1; see also [20,21]). By contrast, the intracellular delivery of the identical concentration of antiGAP IgG did not inhibit neurite outgrowth induced by hirudin treatment in the presence of serum (Fig. 1). The results of this, and our previous studies [20,21] indicate that GAP-43 plays an important role in neuritogenesis, and that this role is apparently related to adhesiveness. This line of reasoning is consistent with the association of GAP-43 with the membrane skeleton following fractionation [12], the preferential localization of GAP-43 within the most adhesive areas of the growth cone [12], and the elaboration of filopodia following transfection with GAP-43 [15,24,25]. While other studies have reported no inhibition of neuritogenesis following treatments that reduce GAP-43 expression [2] or translocation [7], it should be noted that cells in these studies were cultured on coated with adhesive substrates rather than uncoated plastic; we have observed that plating cells on poly-L-lysine or laminin drastically reduces any apparent requirement for GAP-43 in N B 2 a / d l neuritogenesis [20,21]. The reduction in growth cone size and the elaboration of neurites thinner caliber and turn less frequently following inhibition that accompanies inhibition of GAP-43 expression in sensory neurons [1] and N B 2 a / d l cells [22] has led to the suggestion that GAP-43 modulates interactions between developing neurites and the substratum. In this regard, certain aspects of the initial, non-target mediated stage(s) of neurite outgrowth are largely mediated by filopodia-substrate adhesiveness [13,16,18,19], and additional factors, including the presence of an adhesive substrate [17,18] are necessary to stabilize the putative neurite. Accordingly, it may be expected that any requirement for GAP-43-mediated membrane-substrate interactions may be reduced or supplanted by treatments that otherwise
T.B. Shea / Developmental Brain Research 87 (1995) 87-90
|
Acknowledgements This research was supported by the National Science Foundation.
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T Fig. 1. Quantitation of neurites elaborated following 2 h of serum-deprivation or hirudin treatment in the presence of 0.8% serum either without permeabilization, or after permeabilization in the presence of various dilutions of anti-GAP or pre-immune IgG. Values obtained for cultures receiving anti-GAP IgG that differ significantly ( P < 0.005 or less, Student's t-test) from those obtained in cultures receiving the respective concentration of pre-immune IgG are indicated by an asterisk. Note that the intracellular delivery of pre-immune lgG did not affect neurite outgrowth. Note further that the elaboration of neurites induced by serum-deprivation is inhibited by the intracellular delivery of anti-GAP IgG, while neurites induced by hirudin treatment are not inhibited.
increase membrane-substrate adhesiveness, such as decreased neuronal surface proteolysis following hirudin treatment or serum withdrawal (present study) a n d / o r culturing cells on more adhesive substrates [20,21]. The limited duration of intense GAP-43 staining within N B 2 a / d l neurites, coupled with the limited period of time ( < 24 h) during which intracellular delivery of anti-GAP IgG inhibits neurite elaboration [17] suggests that relatively high GAP-43 levels are required for only a relatively short period during initial neuritogenesis. One interpretation of the results of these and other studies is that GAP-43 may regulate early events of neuritogenesis in situ. It should be noted, however, that the expression of GAP-43 is in itself insufficient to induce neuritogenesis[5,11,17,19]. Accordingly, these findings are likely to underestimate the nature and extent of any requirement for GAP-43 in neuritogenesis in situ. The precise role of GAP-43, and the physiological relevance of these and other findings in cultured neuronal cells, remain to be determined.
[1] Aigner, L. and Caroni, P., Depletion of 43-kD growth-associated protein in primary sensory neurons leads to diminished formation and spreading of growth cones, J. Cell Biol., 123 (1993) 417-429. [2] Baetge, E.E. and Hammang, J.P., Neurite outgrowth in PC12 cells deficient in GAP-43, Neuron, 6 (1991) 21-30. [3] Basi, G.S., Jacobson, R.D., Virag, I., Schilling, J. and Skene, J.H.P., Primary structure and transcriptional regulation of GAP-43, a protein associated with nerve growth, Cell, 49 (1987) 785-791. [4] Benowitz, L.I. and Routtenberg, A., A membrane phosphoprotein associated with neural development, axonal regeneration, phospholipid metabolism, and synaptic plasticity, Trends Neurosci., 10 (1987) 527-532. [5] Burry, R.W. and Perrone-Bizzozero, N.I., Nerve growth factor stimulates GAP-43 expression in PC12 cell clones independently of neurite outgrowth, J. Neurosci. Res., 36 (1993) 241-251. [6] Dani, J.W., Armstrong, D.M. and Benowitz, L.I., Mapping the development of the rat brain by GAP-43 immunocytochemistry, Neuroscience, 40 (1991) 277-288. [7] Ferreira A., Niclas J., Vale R.D., Banker G. and Kosik K.S., Suppression of kinesin expression in cultured hippocampal neurons using antisense oligonucleotides, J. Cell Biol., 117 (1992) 595-606. [8] Goslin, K., Schreyer, D.J., Skene, J.H. and Banker, G., Development of neuronal polarity: GAP-43 distinguishes axonal from dendritic growth cones, Nature, 336 (1988) 672-674. [9] .lacobson, R.D., Virag, I. and Skene, J.H.P., A protein associated with axon growth, GAP-43, is widely distributed and developmentally regulated in rat CNS, J. Neurosci., 6 (1986) 1843-1855. [10] Kumagi, C., Tohda, M., Isobe, M. and Nomura, Y., Involvement of growth-associated protein-43 with irreversible neurite outgrowth by dibutyryl cycli AMP and phorhol ester in NG108-15 cells, J. Neurochem., 59 (1992) 41-47. [11] Megerian, J.T., Sullivan, J.P. and Klein, W.L., GAP-43 expression without neurite extension, Soc. Neurosci. Abstr., 15 (1989) 574. [12] Meiri, K.F. and Gordon-Weeks, P.R., GAP-43 in growth cones is associated with areas of membrane that are tightly bound to sbustrate and is a component a membrane skeleton subcellular fraction, J. Neurosci., 10 (1990) 256-266. [13] Monard, D., Implications of proteases and protease inhibitors in neurite outgrowth. In Neurobiochemistry, Selected Topics, Vol. 36, Springer-Verlag, Berlin, 1985, pp. 7-12. [14] Moya, K.L., Jhaveri, S., Schneider, G.E., Benowitz, L.I., Immunohistochemical localization of GAP-43 in the developing hamster retinofugal pathway, J. Comp. Neurol., 288 (1989) 51-58. [15] Nielander, H.B., French, P., Oestreicher, A.B., Gispen, W.H. and Scotman P., Spontaneous morphological changes by overexpression of the growth-associated protein B-50/GAP-43 in a PC12 cell line, Neurosci. Lett., 162 (1993) 46-50. [16] Shea, T.B., Beermann, M.L., Nixon, R.A., Multiple proteases regulate neurite outgrowth, J. Neurochem., 56 (1991) 842-851. [17] Shea, T.B., Perrone-Bizzozero, N.I., Beermann, M.L. and Benowitz, L.I., Phospholipid-mediated delivery of anti-GAP-43 antibodies into neuroblastoma cells prevents neuritogenesis, J. Neurosci., 11 (1991) 1685-1690. [18] Shea, T.B., Beermann, M.L. and Nixon, R.A., Sequential requirement for cytoskeletal constituents during axonal neurite initiation, elongation and stabilization, J. Cell Biol., 115 (1991) 163. [19] Shea, T.B., Beermann, M.L. and Nixon, R.A., Complementary
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effects of laminin and protease inhibitors on neurite outgrowth and maintenance, Neurosci. Res. Commun., 10 (1992) 53-61. [20] Shea, T.B. and Beermann, Intracellular delivery of anti-GAP-43 antibodies into neuroblastoma inhibits neurite outgrowth induced by serum deprivation more than outgrowth elicited by dbcAMP treatment, Mol. Cell Biol., 4 (1993) 171a. [21] Shea, T,B. and Beermann, Adhesive substrates minimize the requirement for GAP-43 in N B 2 a / d l neuritogenesis, Trans. Am. Soc. Neurochem., 25 (1993) 123. [22] Shea, T,B., Intracellular delivery of anti-GAP-43 antibodies reduces growth cone size, Biochem. Biophys. Res. Commun., 203 (1994) 459-464.
[23] Skene, J.H.P., Axonal growth-associated proteins, Ann. Re~:. Neurosci., 12 (1989) 127-156. [24] Widmer, F. and Caroni, P., Phosphorylation-site mutagenesis of the growth-associated protein GAP-43 modulates its effects on cell spreading and morphology, J. Cell Biol., 120 (1993) 503-512. [25] Yankner, B.A., Benowitz, L.I., Villa-Komaroff, L. and Neve, R.L., Transfection of PC12 cells with the human GAP-43 gene: effects on neurite outgrowth and regeneration, Mol. Brain Res., 7 (1990) 39-44. [26] Zuber, M.X., Goodman, D.W., Karns, L.R. and Fishman, M.C., The neuronal growth-associated protein GAP-43 induces filopodia in non-neuronal cells, Science, 244 (1989) 1193-1195.