Re-examination of the local control by nerve growth factor of the outgrowth of neurites in PC12D cells

BRAIN RESEARCH ELSEVIER

Brain Research 730 (I 996) 212-222

Research report

Re-examination of the local control by nerve growth factor of the outgrowth of neurites in PC 12D cells Mamoru Sano *, Misao Iwanaga lnstitute fi)r Decelopmental Research, Aichi Human Sere'ice Center. Kamiya cho, Kasugai. Aichi 480-03. Japan

Accepted 9 April 1996

Abstract We have examined the local control by nerve growth factor (NGF) of the outgrowth of neurites from clonal cells, PC I 2D, a suhline whose phenotype resembles that of the parent PCI2 cell line in the NGF-primed state. We show here that (i) the outgrowth of neurites and their survival can be induced by NGF in enucleated P C I 2 D cells, (ii) individual neurites of a single 'giant cell', produced by cell fusion of P C I 2 D cells, can respond independently to the NGF in the local environment, (iii) dissected neurites from giant cells survive for longer in medium that contains NGF than in medium that does not, (iv) in P C I 2 D cells, the rapid formation of ruffles in response lo NGF, which appears to be based on increased cell-substratum adhesion, leads to the subsequent formation of neurites, and (v) upon addition of NGF, the movement of short processes displaces polylysine-coated beads in the vicinity of neurites. These observations suggest that the NGF-dependent maintenance or extension of neurites might be controlled within the neurites themselves and might not require the direct involvement of the cell body, even in PC12 cells. It seems possible that any NGF-induced changes that promote an increase in cell-substratum adhesion might be responsible for the initiation and elongation of neurites. It also seems possible that the growth of neurites towards a source of NGF might he based on repeated rounds of extension and retraction of filopodia and neurites in a manner that depends on the concentration of NGF. Keywords: Nerve growth factor: PC12D cell: Neurite; Growth cone: Ruffle

1. Introduction

Nerve growth factor (NGF) has been shown to play a crucial role in the development of sensory and sympathetic neurons, with numerous results confirming the hypothesis that NGF acts as a target-derived signal that regulates the survival and growth of afferent neurons [19]. With the increase in identified members of the neurotrophin family and the identification of the Trk receptor for each neurotrophin and a common signal-transduction pathway [3,7,9,15], a great deal of attention has been focused on the roles of neurotrophins in differentiation, survival and migration of neurons and in the pathfinding of axons [3,29]. In these studies, cultured neurons were in a mixture that included various types of cell and they required NGF or some neurotrophic factor for their basic survival. Accordingly, the PC12 clone of rat pheochromocytoma ceils,

* Corresponding author. Fax: (81) (568) 880829.

which responds to NGF by extending neurites, has been extensively used as a model system for studies of the NGF-mediated differentiation into cells that resemble sympathetic neurons. Results of some recent studies imply a requirement for stimulation of the MAP kinase cascade and other signals for the neuronal differentiation that are usually characterized by the outgrowth of neurites [8,23,37]. The NGF-dependent outgrowth of neurites from PC I2 cells requires long-term, transcription-dependent processes [4]. By contrast, in cultured embryonic sympathetic neurons, the elongation of neurites in response to NGF occurs quickly and in the absence of RNA synthesis. Furthermore, the concentration of NGF in the local environment of neurites exerts control on their local growth, which is independent of the growth of other regions of each neuron [5,6]. PCI2D cells, a subclone of PCI2 cells, extend neurites within a few hours in response to NGF, basic-fibroblast growth factor (bFGF), epidermal growth factor (EGF), drugs that raise levels of cAMP and to staurosporine in the absence of new gene expression [16,24,27]. In response lo

0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PH S 0 0 0 6 - 8 9 9 3 ( 9 6 ) 0 0 4 4 9 - 0

M. Sano, M. lu'at a ga / Brail Research 730 (1996) 212 222

2I~

N G F , ruffles fl~rm rapidly a r o u n d the p e r i p h e r y o f cells

cells that are e x p o s e d to an e l e v a t e d c o n c e n t r a t i o n o f N G F

and s u b s e q u e n t l y b e c o m e the g r o w t h c o n e s o f neurites

[26]. Thus, the P C 1 2 D cell line is very useful as a m o d e l

[25]. M o r e recently, w e o b s e r v e d that rapid s p r o u t i n g o f

s y s t e m for studies o f the N G F - i n d u c e d o u t g r o w t h o f neu-

neurites o c c u r s within m i n u t e s in local r e g i o n s o f P C I 2 D

rites, separately f r o m the initiation step, during the N G F -

Z~ 2 2 g

Fig. 1. Eflects of NGF on enucleated PC I2D cells, visualized by phase-contrast and fluorescence microscopy. Alter centrifugation in the absence (a d) or presence (e-h) of cytochalasin B, as described in Section 2, cells on discs were cultured in the absence la, b. e. f) or presence (c, d, g, h) of 50 ng/ml NGF for 20 h. The cells were fixed and nuclei were fluorescence stained with bisbenzimide as described in Section 2. Arrowheads (c) show enuclealed cells that have dcgeneraled. Arrows (g) show enucleated cells with neurites. Bar 50 l,xm.

214

M. Sano, M. lwanaga/Brain Research 730 (1996) 212 222

induced differentiation of PC12 cells into a cell type that resembles sympathetic neurons. In this study, we examined the rapid and local actions of PC12D cells in response to N G F to obtain clues to the mechanisms of the NGF-induced sprouting and elongation of neurites. In addition, we tried to reexamine the validity of the hypothesis that concentration gradients of N G F guide axons to their targets using this clonal cellular system.

2. Materials and methods

10 > g / m l of each solution in Ca 2+- and Mg 2+_free Hanks' solution was spread over a polylysine-coated coverslips and was incubated at 37°C overnight. Processes with lengths equivalent to two or more diameters of a cell body were counted as neurites. For the cell-attachment assay, a suspension of cells ( 1 - 2 X 105/200 >1) was spread on a coverslip (18 × 18 mm 2) that had been coated with one of various types of adhesion molecule. After incubation at 37°C for 30 s, 100 btl of the suspension of cells was collected and the number of cells was counted. Fluorescence staining of F-actin was performed as described previously [25].

2.1. M a t e r i a l s 2.3. E n u c l e a t i o n

D u l b e c c o ' s modified E a g l e ' s medium ( D M E M ) (high glucose) was from Gibco, Grand Island, NY. Fetal calf serum and horse serum were from M.A. Bioproducts, Walkersville, MD. Mouse N G F (7S form) was purified from salivary glands of male mice [36]. Rhodamine-labeled phalloidin was from Molecular Probes, Eugene, OR. PolyL-lysine was from Sigma, St Louis, MO. Laminin from mouse EHS sarcoma was from Biomed. Tech., Stoughton, MA. Collagen I was from Nitta Gel., Osaka, Japan. Collagen IV from EHS sarcoma was from Collaborative Research, Bedford, MA. Bovine fibronectin was from Chemicon, Temecula, CA. 2.2. Cell culture

Cultures of PC 12D cells were maintained on 90-mm-diameter culture dishes (Falcon, Lincoln Park, N J) at 37°C in a water-saturated atmosphere of 95% air and 5% CO 2. The medium used was D u l b e c c o ' s modified Eagle's medium supplemented 5% fetal calf serum and 10% horse serum. For coating coverslips with laminin, collagen, or fibronectin, 100 pA of a solution of the respective proteins at

Enucleated PC12D cells were prepared by the method of Nichols et al. [20]. Polystyrene discs (5 X 10 ram) were cut from the bottom of 90-mm culture dishes. Cells were cultured for about 18 h on these discs after the discs had been precoated with poly-IAysine, then they were further cultured in the presence of 10 p~g/ml cytochalasin B. Alter 2 days, the discs were centrifuged at 8000 × g for 15 min in a swinging-bucket rotor (model RPRS15 in a CRB-2 centrifuge; Hitachi, Tokyo) that had been prewarmed to 37°C, with the cell side of each disc facing down wards at an angle of 45 ° in the medium with or without 10 b t g / m l of cytochalasin B. The discs were then washed four times with medium and then were incubated in the presence or absence of N G F for 20 h. For staining of nuclei, cells were fixed in 1% glutaraldehyde in PBS fi)r 15 min at room temperature. After washing with PBS, cells were incubated with 0.1% Triton X-100 for 3 rain and then rinsed three times with PBS. Nuclei were stained with 0.2 mM bisbenzimide (Hoechst dye 33258; Sigma) for 10 rain. After rinsing, samples were mounted with glycerol jelly by observation of nuclear fluorescence.

Fig. 2. Effects of a concentration gradient of NGF on giant neurites from a multinucleate cell. Large neurites and a concentration gradient of NGF were prepared as described in Section 2. Phase-contrast photomicrographs are shown of large neurites fi'om a multinucleate cell before (a) and 4 h after (b) the onset of the steady release of a solution of NGF from a micropipette. Arrows show extending neurites with obvious growth cones. Arrowheads show shriveling neurites. Bar = 50 ~m.

M. Sam), M. lwanaga / Brabl Research 730 (1996) 212-222

215

Hg. 3. Effects of NGF on giant neurites dissected from multinucleate PC12 cells. Large neurites from multinucleate cells were prepared as described in Section 2. Neuriles were dissected from the cell bodies with a glass capillary tube controlled by a micromanipulator, A representative time-lapse sequential demonstration is shown of changes in morphology of separate neurites in the presence (a. c, e, g) and in the absence (b, d, f, h) c)f NGF (50 n g / m l ) . After ,lisseclion of neurile~ lYom cell bodies (a, b), cultures were maintained in serum-free condition for 0 h (c. d), 2 h (e, 1), and 3 h (g. h). Bar : 50 ~tm.

216

M. Sam), M. lwana?a/ Brain Research 730 ¢1996)212-222

2.4. Incubation with beads Carboxylate polystyrene beads (mean diameter, 0.5 I-tm; Polysciences, Warrington, PA) were coated with poly-klysine by the incubation of beads with a solution of poly-L-lysine (0.1 m g / m l ) for 1 h at room temperature and washed three times with sterile water. After overnight culture of cells in culture dishes of 60-mm diameter that had been precoated with poly-k-lysine, a drop of the suspension of beads was added. Then cultures were centrifuged at 270 × g for 5 min in a swing-bucket rotor (KS-8000; Kubota Centrifuge, Tokyo) at room temperature in order to sediment beads. Interactions between cells and beads were studied by time-lapse video recording with CCD camera (KY-F55; Victor, Tokyo) on the stage of a phase-contrast microscope (Diaphot-TMD; Nikon, Tokyo) under conditions of controlled temperature (37°C) and the ambient concentration of CO 2 (5%).

2.5. Giant neurites from multinucleate cells Multinucleate PC12D cells were produced by treatment of cells that had been cultured on 60-mm-diameter dishes (precoated with poly-k-lysine) with 50% polyethylene glycol for about 30 s (no. 1000; Nakarai, Japan) by the method of O ' L a g u e and Huttner [22]. For time-lapse video recording of cellular responses to a local gradient of NGF, fused cells were incubated in the presence of N G F until they extended neurites. After 1 or 2 days, cells were washed to remove N G F and were cultured on the stage of a phase-contrast microscope with controlled temperature (37°C) and the ambient concentration of CO 2 (5%). A concentration gradient of N G F was produced as described previously [26]: a solution of N G F (1 I x g / m l in phosphate-buffered saline with 1% bovine serum albumin) was steadily ejected from a micropipette controlled by a micromanipulator (NT88; Narishige, Tokyo) against the perfused flow of medium over the entire culture.

3. Results

3.1. Outgrowth of neurites.from enucleated P C I 2 D cells Cells on discs were cultured in the presence of cytochalasin B for 2 days. When discs were centrifuged at 8000 × g for 15 min in the presence of cytochalasin B, most cells (about 80%) were detached and remaining cells were enucleated. After centrifugation in the absence of cytochalasin B, no apparent changes were observed in the cells on discs. When the enucleated cells were cultured in the presence of N G F for 20 h, formation of extended processes was observed (Fig. 1). The enucleated cells degenerated during incubation for 20 h in the absence of NGF. These results suggest that the outgrowth of neurites and their survival can be induced by N G F in enucleated PC 12D cells.

3.2. Eff'ects of a concentration gradient of N G F on giant neurites A concentration gradient of N G F was applied to giant neurites that had extended from multinucleate cells, produced by cell fusion and incubation with N G F (Fig. 2). In around the tip of the micropipette, where the concentration of N G F was relatively high, neurites tended to extend and their growth cones developed within a few hours. Other neurites tended to shrivel up and their growth cones were missing. These observations indicate that each individual neurite from a single cell body can respond independently to the N G F in its local environment. 120

100

o~

8o

t.-

t-, Q)

60 -I

2.6. Dissection off large neurites

Z

40 Fused PC12D cells with large neurites were prepared as describe above. Cultures were washed three times with D M E M to remove serum and N G F just before the experiment. Neurites were quickly dissected from cell bodies with glass capillary tubes controlled by a micromanipulator on the stage of a phase-contrast microscope as described. The operation and subsequent incubation was carried out in the presence or absence of NGF. Time-lapse video-recording was carried out as described. Each photograph was made by a video printer (CVP-G7: Sony, Tokyo), and neurite lengths were quantitated from photographs using a ruler.

20

2 Incubation time (h)

Fig. 4. Cnmparison of the time course of degeneration of separate neuriles in the presence or absence of NGF. Experiments are described in the legend to Fig. 3. Relative lengths of neurites in video-micrographs after incubation for different hours in the absence of NGF ( n = 28: closed columns) or in the presence of NGF (11= 36" open columns) are shown. Bar SD.

M. Sam). M. lwana,~a/ Brain Research 730 ! 1996) 2 I2 222 3.3. El[eels ~/ NGF on giant neurites dissected from cell bodie.~

Giants neurites were dissected from multinucleate cell bodies with glass capillary tubes and sequential changes in their morphology in the absence and in the presence of N G F were observed by time-lapse video micrography (Fig. 3). The dissected neurites still extended and retracted filopodia in the presence of N G F (not shown), although most neurites gradually disintegrated. The disintegration of neurites appears very rapid in the absence of N G F (Fig. 3). When the changes in neurite length in cultures with N G F were compared with those in control cultures, the neurite length was lound to be maintained by the presence of N G F (Fig. 4). This result indicates that the growth and survival of even isolated neurites might be supported by NGF. 3.4. Cells cultured with coated beads

When P C l 2 D cells were cultured in the presence of polylysine-coated beads, beads around cells were absent in

217

NGF-treated cultures in which cells had extended neurites (Fig. 5). Such displacement of beads was particularly apparent around long neurites. Video-microscopic observation revealed that beads were displaced by retracting processes (Fig. 6A). Occasionally, beads were seen to be transported on the processes (Fig. 6B). In spite of only limited numbers of neurites, most beads around cells were displaced. These observations suggest that extension and retraction of processes or neurites might be extensively repeated all around each cell. Neurites or their processes might not extend continuously but might move over the surface of culture dish in the presence of an adequate concentration of NGF. 3.5. NGF-induced formation o f rl{ffles and the outgrowth of" neurite (m t'arious substrata

A few minutes after the addition of NGF, the rapid formation of ruffles around the periphery of PC I2D cells can be clearly revealed by actin-specific staining. The ruffles condense and become growth cones of neurites

Fig. 5. Phase-contrast micrographs of PC I2D cells cultured with poly-tAysine-coated polystyrene beads. Polystyrene beads were sedimented over tile cells as described in Seclion 2. Cells ~ere mcubated for 6 h (a, c) or 24 h (b, dt in the absence (a, c) or presence (b, d) of 50 ng/ml of NGF, Bar= 50 gem.

218

M. Sano, M. lwanaga / BraiH Research 730 (1996)212 222

within a few hours [25]. The formation of ruffles and subsequent outgrowth of neurites from PC12D cells in response to NGF were examined on various substrata (Figs. 7 and 8A). PC I2D cells attached more tightly to laminin, collagen IV and polylysine than to fibronectin or collagen I (Fig. 8B). The formation of ruffles and the outgrowth of neurites were more marked in the case of cells on the former substrata than on the latter (Figs. 7 and 8A). These observations indicate that the NGF-dependent initiation and elongation of neurites requires effective ad-

hesion of cells to the substratum. The formation of ruffles seems to be triggered by increased adhesiveness at the margin of cells. It is possible that local increases in cell-substratum adhesion caused by NGF might initiate the sprouting and elongation of neurites. 4. Discussion It has been demonstrated that the NGF-stimulated outgrowth neurites from PCI2 cells requires both transcrip-

Fig. 6. Time-lapse sequence demonstrating interactions between short processes extended from PC I2D cell and polystyrene beads. A: extension and retraction of a filopodium-like process draw a bead (arrow) towards cell body. (a) 0 time. (b) 3(1 rain, (c) 60 min. Bar = 15 p.m. B: a bead that is transported to the cell body on a process. (a) 0 time, (b) 1 rain 49 s, (c) 2 rain 24 s, (d) 3 mm 9 s. B a t ' = 20 p~m.

M. Sano, M. lwana?a / Brain Research 730 (1996) 212 222

tion-dependent and -independent pathways [4,13]. The transcription-independent pathway might be triggered in the plasma membrane and cytoplasm, while the transcription-dependent pathway might require interactions with the nucleus. PC12D cells extend neurites in response to NGF, even in the presence of an inhibitor of R N A synthesis [24] and, in the present study, the outgrowth of neurites from enucleated PC I2D cells was induced by NGF. An identical experiment was performed with native PC I2 cells cultured on an extracellular matrix from bovine endothelial cells [20]. Our P C I 2 D cells and the PC12 cells in the latter study might have been in a ' p r i m e d ' state. It was suggested that the promotion of the growth and survival of neurites from the sympathetic ganglia of newborn rats by N G F was confined to the distal neurite regions that were exposed to extracellular N G F [5,6]. Our recent work indicated that the rapid sprouting of neurites occurs locally on regions of PC I2D cells exposed to an elevated concentration of N G F [26]. We have also suggested that the activation and nuclear translocation of mitogen-activated protein kinase might be required for the transcription-dependent differentiation of native PC I2 cells but might not be necessary for the elongation of neurites [28]. The rapid outgrowth of neurites from PC 12D cells was unaffected by

21t~

the presence of cycloheximide (10 b t g / m l ) , which inhibited protein synthesis by 97%, as determined by monitoring the incorporation of [35S]methionine/cysteine into trichloroacetic acid-precipitable protein (unpublished data). These observations suggest the possibility that transcription- and translation-independent local actions of N G F might promote the outgrowth of neurites, even in PC I2 cells. Our present results with P C I 2 D cells show that (i) individual ueurites from a single cell can respond independently to N G F in the local environment, (ii) even survival of neurites isolated from cell bodies can be supported by NGF, (iii) neuronal processes move over surrounding areas in the presence of NGF, and (iv) cell-substratum adhesion seems to play a permissive role in the NGF-induced rapid changes in morphology of cells and the subsequent formation of neurites. These results indicate that the effect of N G F on the elongation of neurites might be local in clonal PC I2 cells as has been shown to be the case in cultured embryonic neurons [5,6]. The extension of growing and regenerating sensory and sympathetic axons along a concentration gradient of N G F has been definitively established by various experiments both in vitro and in vivo [19]. The observation by Gundersen and Barrett that thc

Fig. 7. Effects of various substrates on the NGF-induced rapid redistribution of F-actin in PCI2D cells. Cells were seeded on coverslips that had been coated with poly-tAysine (a, b), laminin (c), fibronectin (d), collagen I (e), or collagen IV (f) as described in Section 2. After about 20 tl. tile medium wa~, changed. After incubation in the absence (a) or presence (b-f) of NGF (50 ng/ml) for 4 rain, cells werc fixed and actm wa~, stained with rhodamine-phalhfidin. Bar - 2(I ~xm.

220

M. Satto, M. Iwanaga / Brain R e s e a r c h 730 ( 1996 J 2 1 2 - 2 2 2

0

20

0

10

40 60 Neurite-bearing cells (%)

80

i

20

30

40

cell attachment (%) Fig. 8. Effects of various substrates on the outgrowth of neurites and the attachment of cells. A: cells were seeded on coverslips that had been coated with various cell-adhesion proteins, as described in Section 2. After about 20 h, the medium was changed and cells were cultured in the presence of NGF (50 ng/ml) for 24 h. Neurite-bearing cells were counted. Data are averages_+S.D. (bars) of values from three determinations. B: the attachment of cells was compared on various substrates, as described in Section 2. Data are average of values from three determinations.

axons of cultured chick dorsal root ganglion neurons turned and grew toward a source of N G F (a micropipette) provides strong evidence for the chemotactic behavior of neurites [14]. However, it is difficult to imagine how a growth cone could recognize a concentration gradient of NGF. As shown in Fig. 5, the locomotive movements of filopodia and short processes could occur during the extension of neurites. Moreover, each neurite might respond individually to N G F in its local environment (Fig. 2). The retrograde movement of beads that had stuck to the surface of processes is shown in Fig. 6B. An increase in the clustering of beads on cell bodies was also observed (not shown). Although the mechanism of the movement was not analyzed in the present study, identical retrograde movements of particles on the surfaces of filopodia and lamelipodia have been reported by others [31,32]. The growth of neurites towards the source of N G F might be based on repeated rounds of extension and retraction of neurites in a manner that depends on the local concentration of NGF. Several studies have shown that the formation of neurites requires microtubules [1,2, 11 ]. It is possible that the

accumulation of materials might be required for the formation of neurites on PC I2 cells [4,35]. But others have found that neurites can extend from P C I 2 cells in the absence of microtubules [17], and that the initial steps in the formation of neurites do not depend on the assembly of microtubule [33]. Our previous study also showed that neither cytochalasin B nor colchicine prevented the NGFresponsive formation of protrusions in PC 12D cells. However, treatment of cells with both cytochalasin and colchicine completely prevented the change in morphology that normally occur in response to N G F [25]. Zheng et al. demonstrated that pulling on the margin of embryonic chick sensory neurons induces formation of neurites that contain normal arrays of microtubules [38]. It has also been demonstrated that actin polymerizes at the leading edges of the growth cones of nerves and then extends inwards [12,21]. These studies suggest a contribution by microtubules and microfilaments to the extension and stabilization of neurites after they have sprouted. There is general agreement that adhesion to a substrate is a prerequisite for growth of neurites, although a negative correlation between the growth of neurites and the adhesiveness of naturally occurring substrates has been indicated [10+18]. lntegrins have been shown to mediate the outgrowth of neuronal processes on components of the extracellular matrix. It has been shown that P C I 2 cells express two f31 integrins which function in attachment and the outgrowth of neurites on laminin and collagen [34]. Early studies of cultured sensory neurons and PC I2 cells showed that N G F enhanced c e l l - c e l l and cell-substratum adhesion+ leading to speculation that the outgrowth of neurites might be causally linked to increased cell adhesiveness [30]. In native PC I2 cells, however, the formation of neurites requires a long-term, transcription-dependent pathway, while an increase in cell adhesiveness is very rapidly induced by NGF. Furthermore, PC I2 cells, as well as PC12D cells, do not form neurites in the absence of N G F on highly adhesive substrata such as laminin or collagen. The present study indicated that the NGF-dependent outgrowth of neurites requires good adhesion of cells to the substratum. In PC12D cells, the rapid formation of ruffles in response to NGF, which appears to be based on increased cell-substratum adhesion, leads to the subsequent formation of neurites [25]. The NGF-dependent support of dissected neurites demonstrated in Fig. 3 might also be based on the NGF-induced enhancement of their adhesion to the substratum. We can speculate that any NGF-induced changes that promote an increase in cell-substratum adhesion could be responsible for the initiation and elongation of neurites by triggering the assembly or disassembly of the cytoskeleton. The hypothesis that N G F has local effects on axons and that growth of neurites requires adhesion to the substratum, are supported by various lines of evidences from cultures of embryonic neurons. The present results with clonal PC I2D cells also support these hypothesis. These cells provide a unique experimental

M. Sane, M. lwanaga / Braii Research 730 (1996) 212 222

system for further studies of the cellular mechanism of the NGF-induced sprouting and elongation of neurites, processes that can now be separated from the transcription-dependent differentiation of these cells.

[17]

[18]

Acknowledgements This work was supported by a grant from the Ministry of Education. Science and Culture of Japan (no. 06680781 ).

[19] [20]

[21]

References [22]

[1] Black, M.M., Aletta. J.M. and Greene, L.A., Regulation of microtubule composition and stability during nerve growth factor-promoted neuritc outgrowth, J. Cell Biol,, 103 (1986) 545 557. [2] Brugg, B. and Malus. A.. PC I2 cells express juvenile microtubuleassociated proteins during nerve growth factor-induced neurite outgrowth, J. ('ell Biol.. 107 (1988) 643-650. [3] Buchman. V.I,. and Davies, A.M., Diffi:rent neurotrophins are expressed and act in a developmental sequence to promote the survival of embryonic sensory neurons, l)ecelopment, 118 (1993) 989-1001. [4] Burstein, I).E. and Greene, L.A., Evidence for RNA synthesis-dependent and independent pathways in stimulation of neurite outgrowth by nerve growth factor, Prec. Natl. Acad. Sci. USA, 75 (1978) 6059-6063. [5] Campenot. R.B., Development of sympathetic neurons in compartmentized cultures. 1. Local control of neurite growth by nerve growth factor. 1)el. Biol., 93 (1982) 1-12. [6] Campenol. R.B., Development of sympathetic neurons in compartmentized cultures. II. Local control of neurite survival by nerve growth factor, lJel. Biol., 93 (1982) 13-21. [7] Chao, MV., Neurotmphin receptors: a window into neuronal differentiation. Neuron, 9 (1992) 583 593. [8] Cowley, S.. Paterson, H., Kemp, P. and Marshall, J., Activation of MAP kinasc kinase is necessary and sufficient for PCI2 differentialion and transformation of N1H 3T3 cells, Cell, 77 (1994) 841-852. [9] Davis, R..I.. The mitogen-activated protein kinase signal transduction pathway, ,I, Biol. ('hem.. 268 (1993) 14553-14556. [10] Do&t, .I. and .lessell, T.M.. Axon guidance and the patterning of neuronal projections in vertebrates. Science, 242 (1988) 692-699. [I I] Drubin, I)G.. Feinstein, S.C., Shooter, E.M, and Kirschner. M.W., Nerve growth factor-induced neurite outgrowth in PC I2 cells in,,elves the coordinate induction of microtubule assembly and assembly-promoting factors, J. ('ell Biol., 101 (1985) 1799-1807. [12] Forschcr. P.. 15n. C.H. and Thompson, C., Novel form of growth cone motility inw~lvmg site directed actin filament assembly. Nalure, 357119~21515 518. [13] Greene. 1, A. and Shooter, K S . , The nerve growth factor: biochemistry, synthesis, and mechanism of action, Annu. Rel'. Neurosei., 3 (1980) 353 402. [14] Gundersen. R. W. and Barrett, J.N., Characterization of the turning response ~,1 d~wsal root neuriles toward nerve growth factor, J. ( e l l Biol., 87 (1980) 546-554. [15] Ip, NY.. Still. T.N., "Fapley, P., Klein, R., Glass, D.I., Fandl, J., Greene, I, A.. Barbacid. M. and Yacopoulos, G.D., Similarities and difli_,rences m ihe way neurotrophins interact with the Trk receptors in neuronal and nonneuronalcells, Neuron, 10(1993) 137 149. [16] Katoh-Senlba.R., Kitajima,S. Yamazaki.Y. and Sano,M.. Neuritic

[23]

[24]

[25]

[26]

[27]

[281

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

22 1

growth from a new subline of PC 12 pheochromocytonla tells: cyclic AMP mimics the action of nerve gro,Ath factor..I. Neurosei. Res.. 17 (1987) 36 44. kamoureux, P., Steel, V.L.. Regal, C.. Adgate. L., Buxbaum. R.E, and Heidemaml. S.R., Extracellular matrix allows PCI2 neuritc ehmgation in the absence ,,/1"microtnbules, ,/. Cell Biol., 1 I0 (1990) 71 79. Lemmon, V., Burden. S.M., Paync, H.R., Ehnslie, G.J. and Hlavin, M.L., Neurite growth on different subs[rates: permissive "
222

M. &too, M. lwanaga / Brain Research 730 (1996) 212 222

nerve growth/'actor protein in a high molecular lk~rm, BiochemistJy, 6 (1967) 2202-2209. [37] Vaillancourt, R.R., Heasley, L.E., Zamarripa, J., Storey, B., Valius, M., Kazlauskas, A. and Johnson, G.L., Mitogen-activated protein kinase activation is insufficient for growth factor receptor-mediated PC12 cell differentiation, Mol. Cell Biol., 15 (1995) 3644-3653.

[38] Zheng, J., Buxbaum, R.E. and Heidemann, S.R., Investigation of microtubule assembly and organization accompanying tension-induced neurite initiation, J. Cell Sci,, 101 (1993) 1239-1250.