DEVELOPMENTAL

BIOLOGY

150,256-265 (19%)

Thrombospondin Promotes Process Outgrowth in Neurons from the Peripheral and Central Nervous Systems DONNA J. OSTERHOUT,* WILLIAM A. FRAZIER,? AND DENNIS HIGGINS* *Department of Phamrz~~ology and Therapeutics, School of Medicine, State University of New York, B@alo, New York l&V.& and +Department of Biodemistry and Molecular Biophysics, Washing&m University School of Medicine, St. Louis, Missouri 65110 Accepted December l7,1991 Thrombospondin (TSP) is a prominent constituent of the extracellular matrix of the developing nervous system. We have examined the effects of TSP on the morphological differentiation of neurons. In short-term cultures (~24 hr) of embryonic rat sympathetic neurons, TSP stimulated neurite outgrowth, causing significant increases in the number of processes and their length. Similar effects were observed in cultures of rat dorsal root ganglion, hippocampal, and cerebral cortical neurons. Moreover, in cultures of central neurons, TSP was more effective than laminin in enhancing process extension. Analysis of long-term (5-7 days) cultures of sympathetic neurons indicated that processes formed in the presence of TSP had the cytochemical characteristics of axons. Thus, TSP can influence neuronal development by selectively enhancing axonal growth. The neurite-promoting region of the molecule was identified using a panel of monoclonal antibodies targeted to different regions of the protein. Process outgrowth could be totally inhibited with antibody A4.1, which recognizes the stalk region of TSP. These data suggest that the neurite-promoting activity is localized to a single region of the TSP molecule. o 1992Academic PWSS. IIIC.

et ah, 1987). In smooth muscle, TSP acts as a positive regulatory growth factor and potentiates the mitogenic effects of epidermal growth factor (Majack et al, 1986). For endothelial cells, TSP strongly inhibits migration and proliferation in response to angiogenic mitogens like basic fibroblast growth factor (Good et al, 1990). The fact that TSP has acute regulatory effects on a variety of cell types has led to the suggestion that it may play a role in tissue formation and remodeling (Mosher, 1990). This hypothesis is supported by studies which have demonstrated that the concentration of TSP in the ECM is elevated in certain embryonic tissues during morphogenesis, and in areas of tissue injury and healing in adults, both times of significant cellular rearrangement and growth (O’Shea and Dixit, 1988; Wight et al., 1985; Raugi et uZ., 1987; Watkins et cd., 1990). In the embryo, TSP is present in the ECM of the developing central and peripheral nervous systems. Immunocytochemical analysis has revealed that TSP is particularly enriched in areas of cell migration (O’Shea and Dixit, 1988). In vitro studies using explants of cerebellar granule cells suggest a functional role for TSP, as antibodies to TSP inhibit granule cell migration in this system (O’Shea et UC, 1990). Many adhesive molecules of the ECM which promote cell migration also have the capacity to stimulate neurite outgrowth. Of the individual matrix components studied to date, laminin is the most potent. It induces rapid process outgrowth from most types of embryonic neurons, and increases the number of neurites extended in vitro (reviewed by Sanes, 1989;

INTRODUCTION

Thrombospondin (TSP)l is a large adhesive glycoprotein (M, 420,000) that was originally identified as a major secretory product of platelets (reviewed by Frazier, 1987; Mosher, 1990). Upon platelet activation, TSP facilitates the formation of a hemostatic plug at the site of injury by promoting platelet aggregation. Recent evidence indicates that TSP is not restricted to platelets; rather, it is secreted by a variety of cultured cells, including endothelial cells (McPherson et ok, 1981), smooth muscle icells (Raugi et al, 1982), fibroblasts (Jaffe et al, 1983), astrocytes (Asch et al, 1986), keratinocytes (Wikner et al., 1987), and melanoma cells (Roberts et al, 1987). Immunolocalization studies have demonstrated that secreted TSP is incorporated into the extracellular matrix (ECM), where it is found in association with both the cell surface and basement membrane (Jaffe et aH, 1983; Kramer et cd, 1985). As a component of the ECM, TSP has been implicated in the regulation of cell motility and growth. In vitro assays have revealed that TSP enhances adhesion of various nonneuronal cells (Murphy-Ullrich and Hook, 1989; Roberts et ah, 1987; Varani et uL, 1988); in this respect it resembles other matrix components, including laminin and fibronectin. Melanoma cell chemotaxis and haptotaxis are also facilitated by TSP (Taraboletti 1 Abbreviations matrix.

used: TSP, thrombospondin;

0012-1606/92 $3.00 Copyright All rights

0 1992 by Academic Press, Inc. of reproduction in any form reserved.

ECM, extracellular 256

OSTERHOUT, FRAZIER, AND HIGGINS

Reichardt and Tomaselli, 1991). Further studies have demonstrated a role for laminin in nerve regeneration in situ (Sandrock and Matthew, 1987). While laminin has strong neurite promoting activity, other evidence indicates that additional matrix components participate in modulating the elongation of neurites in situ (Reichardt et cd, 1989; Sanes, 1989). Since TSP has also been found to be concentrated in regions of process formation in situ (O’Shea and Dixit, 1988), it has the potential to influence the extension of neurites by embryonic neurons. Moreover, recent work has shown that TSP can promote process outgrowth from retinal ganglion cells in vitro (Neugebauer et al, 1991). The present studies demonstrate that TSP enhances the outgrowth of axons from several types of neurons in the central and peripheral nervous systems. These observations are consistent with the hypothesis that TSP is involved in the regulation of nervous system development and/or regeneration in situ. MATERIALS

Pur&ation

AND METHODS

of TSP

TSP was isolated from human platelets according to the procedure of Dixit et al. (1984). Platelets were activated with thrombin (1 U/ml) and the calcium ionophore A23187 [Z PLIM], and pelleted. The supernatant containing secreted platelet proteins was loaded onto agelatin-Sepharose (BioRad Laboratories, Cambridge MA) column directly linked to a heparin-Sepharose (BioRad) column. Both columns had been equilibrated with column buffer (5.0 mMTris, pH 7.6,150 mMNaC1, 1 mlM CaCl,) at 4°C. After the supernatant had been applied, the columns were disconnected, and the heparin-sepharose column was eluted stepwise with column buffer containing 0.25, 0.6, and 2.0 M NaCl. TSP eluted in the 0.6 M NaCl fraction, and was further purified on a BioGel A 0.5 m column. TSP eluted in the void volume, and was well separated from low molecular weight contaminants. The purity of the TSP preparation was evaluated using SDS polyacrylamide gel electrophoresis in the presence and absence of reducing agents. Protein bands were visualized with Coomassie blue stain. Under nonreducing conditions, TSP migrates as a high molecular weight band (Fig. 1); under reducing conditions, the subunits dissociate and migrate as a single band with an apparent molecular weight of 170 kDa. Protein concentrations were determined using the absorption coefficient of TSP as determined by Margossian et al. (1981) or by the method of Lowry et al. (1951). Preparation

of Polyclmml

Antisera

The 170 kDa band was excised from the gel and the protein was eluted using a Bio-Rad electroelution appa-

TSP Promotes Newite

257

Outgmwth

MroC10-31

200 11697 -

is*.si 12 FIG. 1. Electrophoretic analysis of TSP. The purity of TSP preparations was assessed using SDS polyacrylamide gel electrophoresis. Purified TSP (10 pg) was run on a 7.5% gel in the absence (lane 2) and presence (lane 1) of mercaptoethanol (5% v/v).

ratus according to manufacturer’s instructions. The eluted protein (100 pg) was suspended in complete Freund’s adjuvant and injected intradermally into female New Zealand rabbits. One booster injection was given 6 weeks after the initial immunization. The titer and specificity of antiserum was determined by dot blotting using a Vectastain ABC kit (Vector Laboratories, Burlinggame, CA). The antiserum reacted with purified TSP, but not with other matrix components such as laminin or fibronectin. Mono&ma1

Antibodies

A panel of monoclonal antibodies directed toward specific domains of the TSP molecule was used to identify the neurite promoting region. These antibodies (A4.1, A6.1, C6.7, and A2.5) have been characterized in previous work (Galvin et ab, 1985). Cell Culture Dissociated superior cervical ganglion cells (20-21 days) or lumbar dorsal root ganglion cells (15-16 days) were obtained from Holtzmann rat fetuses (Madison, WI) as previously described (Higgins et al, 1991; Lein et al., 1991). Dissociated cerebral cortical cells (16 days) were prepared according to the procedure of Ahmed et al (1983). Cells were plated onto poly-D-lysine (100 pg/ ml)-coated coverslips in the absence or presence of adsorbed matrix proteins (see below) and maintained in a defined serum-free medium. The medium used for sympathetic and sensory neurons contains a maximally effective concentration (100 rig/ml) of nerve growth factor which allows their long-term survival. Cortical neurons were plated into defined medium without nerve growth factor. Dissociated hippocampal neurons (19 days) were obtained using the method of Banker and Cowan (1979). Hippocampal cells were maintained in either medium containing 1% fetal calf serum (Mattson and Kater,

258

DEVELOPMENTALBIOLOGY vOLUME150.1992

1988) or in serum-free media (Manthorpe et aZ., 1983). To obtain optimal survival, it was necessary to plate these neurons onto matrix-coated plastic (Corning 12-well plates) rather than glass coverslips (Mattson and Kater, 1988). Cytosine D-arabinofuranoside (1 PM) was added to all long-term cultures on Days 2 and 3 to eliminate nonneuronal cells. Matrix proteins were adsorbed to poly-D-lysinecoated surfaces prior to plating the neurons. TSP, laminin, fibronectin, or fibrinogen was diluted to the appropriate concentration in sterile column buffer, and incubated with the coverslips for a minimum of 4 hr at 37°C. The coverslips were then rinsed a minimum of two times with sterile column buffer, and once with plating medium prior to adding cells. In antibody-blocking experiments, antibodies were diluted into the plating medium and incubated with the TSP-coated coverslips at 37°C for 1 hr prior to adding the cells. Neurons were then plated directly into the antibody-containing medium. Polyclonal antiserum was heat inactivated by warming to 60°C for 20 min prior to dilution. Monoclonal antibodies A4.1, A6.1, C6.7, and A2.5 were used at a concentration of 10 pg/ml. Mm-phornetry

In short-term (~1 day) cultures, cell morphology was assessed by phase contrast microscopy. To assess total neuritic length, camera lucida drawings of neurons were quantitated using Sigmascan software (Jandel Scientific, Corte Madera, CA). In all morphometric studies, only isolated neurons were analyzed (i.e., neurons whose cell bodies were at least 150 pm from the soma of their nearest neighbor), because earlier work has shown that density-dependent changes in morphology occur when cell bodies are separated by lesser distances (Bruckenstein and Higgins, 1988a). In long-term (a7 days) cultures, the number of processes was determined by injecting cells with Lucifer yellow. Axons were distinguished from dendrites using standard light microscopic criteria (Bruckenstein and Higgins, 1988b). Earlier work has shown that the processes identified as axons or dendrites by these criteria also express appropriate cytochemical and ultrastructural characteristics (Peng et ab, 1986; Bruckenstein and Higgins, 1988b; Tropea et al, 1989; Bruckenstein et a& 1990). To further identify the processes as axons or dendrites, cultures were immunostained with antibodies which selectively react with dendritic or axonal markers (Lein and Higgins, 1989). Axonal probes included monoclonal antibodies to synaptophysin (SY38; Boehringer-Mannheim Biochemicals, Indianapolis, IN); Weidemann and Franke, 1985) and phosphorylated forms of the H neurofilament subunit (NE14; Boehringer-Mannheim; Shaw et aZ.,

1986). A monoclonal antibody to nonphosphorylated forms of the M and H neurofilament subunits was used as a dendritic marker (SM132; Sternberger-Meyer Immunochemicals; Sternberger and Sternberger, 1983). Each experiment was duplicated in cultures obtained from at least two separate dissections. Unless otherwise indicated, all data are expressed as means & SEM and are from a single representative culture series. Analysis of variance using Scheffe’s test and x2 were used for statistical evaluation of the data. RESULTS

Effects of TSP on Sympathetic

Neurons

Sympathetic neurons grown in the presence of TSP exhibited a rapid increase in process formation. Only 23% of the neurons plated onto polylysine extended processes within the first 24 hr; such processes were usually broad, unbranched, and very short (Fig. 2). In contrast, 73% of the neurons grown on TSP had extended long, thin branched processes after 24 hr. TSP induced a threefold increase in the number of cells with neurites, a fourfold increase in the number of neurites per neuron, and a fourfold increase in the total neurite length (Table 1). Thus, TSP acts to increase process formation and branching, as well as increasing the rate at which sympathetic neurons initially extend neurites. The effects of TSP were concentration dependent (Fig. 3). Maximal neurite-promoting effects were observed at a precoating concentration of 100 pg/ml; halfmaximal effects were observed at 30 pg/ml. At concentrations above 100 Fg/ml, TSP had a slight toxic effect, in that the neurons did not appear to be healthy compared with cells exposed to lower concentrations of the protein. Consequently, the number of neurons extending processes was reduced at higher levels of TSP. Previous studies (Lein and Higgins, 1989) have shown that of the ECM proteins tested for neurite-promoting activity, laminin is the most effective in cultures of sympathetic neurons. Direct comparison of maximally effective concentrations of laminin and TSP revealed that TSP induced process outgrowth in a significantly smaller fraction of neurons than did laminin (Table 1). Laminin also caused larger increases in the mean number of processes/neuron and in total neuritic length. Embryonic rat sympathetic neurons readily attach to polylysine-coated coverslips, with approximately 80% of the cells adhering within the first hour (Lein et al, 1991). Adsorption of TSP to the polylysine substrate had no effect on cell attachment (Tables 2, 3). Thus, increases in the number of neurons bearing processes were independent of any alteration in cell attachment to the substratum.

OSTERHOUT, FRAZIER, AND HIGGINS

259

TSP Promotes Neurite Outgrowth

TABLE 1 EFFECTS OF THROMBOSPONDINON NEURITIC OUTGROWTH FROM SYMPATHETIC NEURONS AFTER 24hr IN VITRO % Neurons with neurites

Substrate Polylysine TSP (100 ,ug/ml) Laminin (10 h&ml) Fibrinogen (500 rg/ml)

23% 73%*

Number of neurites per neuron

Total length of neuritic plexus (rm)

0.27 + 0.05 1.02 * o.os*

106 + 10 494 f 62*

loo%*

2.85 AZ0.13*

1050 f 93*

21%

n.d.

n.d.

Note. Data are expressed as means + SEM. Unless otherwise specified, N = 100 neurons for each culture condition. ‘N = 30 neurons per culture condition; only neurons with neurites were included in the calculation of mean and SEM for this morphological parameter. * Denotes a difference at P < 0.01 from polylysine. Laminin and thrombospondin also differ from each other at P < 0.01 for all parameters.

FIG. 2. The effect of TSP on sympathetic neurons. Phase contrast microscopy revealed that sympathetic neurons had attached to polylysine but typically failed to extend neurites after 24 hr (A). In contrast, neurons plated onto polylysine in the presence of adsorbed TSP had extended long thin processes with prominent growth cones (B). Bar, 25 pm.

(Galvin et al, 1985), and target specific fragments of the protein. A4.1 binds to the stalk region between the two globular domains of the molecule, while A2.5 targets the heparin-binding domain. A6.1 is directed to a calciumbinding domain, and C6.7 binds the carboxy-terminal region. When neurons were plated in the presence of these monoclonal antibodies, the neurite-promoting activity of TSP was totally inhibited by A4.1, the antibody directed to the stalk region (Table 3). The other three

The action of TSP appeared to be specific because similar concentrations of fibrinogen, another protein found in platelet granules, failed to elicit process extension (Table 1). Further evidence for specificity was observed in experiments using a polyclonal antiserum to TSP. Addition of a 1:300 dilution of antiserum to the medium did not interfere with cell attachment to either polylysine or adsorbed TSP (Table 2). It did, however, inhibit the neurite-promoting effects of TSP by -80%. This inhibition was not due to a nonspecific retardation of growth, because the response to laminin was unaffected by TSP antiserum (Table 2). Localization

of the Neurite-Promoting

Region of TSP

To identify domains of TSP that might be involved in modulating neurite outgrowth, we employed a panel of monoclonal antibodies directed against individual regions of the TSP molecule. The monoclonal antibodies A4.1, A6.1, A2.5, and C6.7 have been well characterized

0

IO PRECOATINQ

I 30 CONCENTRATION

1 100

I 300

fJJQ/ML)

FIG. 3. The effects. of varying concentrations of TSP on neuritic outgrowth of sympathetic neurons. The percentage of cells bearing neurites was assessed after 24 hr in culture (N = 100 for each concentration).

260

DEVELOPMENTAL BIOLOGY TABLE 2 EFFECT OF POLYCLONAL ANTISERUM TO THROMJWSPONDINON NEURITIC OUTGROWTH FROM SYMPATHETIC NEURONS % Cells with neurites

Substrate Polylysine + Preimmune + Antiserum Laminin + Preimmune + Antiserum Thrombospondin + Preimmune + Antiserum

serum serum Serum

17% 25% 15% 98% 100% 98% 58% 57% 24%*

Neurites/cell 0.17 0.36 0.14 2.17 2.51 2.22 0.73 0.79 0.34

+ + + f + k + f f

0.04 0.07* 0.04 0.10 0.10 0.10 0.08 0.10 0.07*

Cells per mm2 16 21 23 15 18 19 14 16 14

+ 1.2 + 2.3 -c 3.1 + 1.4 + 1.7 + 2.5 + 1.1 + 1.7 * 1.4

Note. The antiserum and preimmune serum were used at a 1:300 dilution. The percentage inhibition in the presence of antibody was calculated as: 100 X (1 - (Growth on TSP in the presence of antiserum - Growth on polylysine in the presence of antiserum)/(Growth on TSP in the absence of antiserum - Growth on polylysine in the absence of antiserum)). Data are expressed as means f SEM, N = 90 neurons for each culture condition. * Denotes a difference at P < 0.05 from control cultures on the same substrate.

antibodies had no significant effects on the percentage of neurons forming processes, and C6.7 caused only a slight inhibition in the number of neurites/cell. The presence of the antibodies had a small effect on cell attachment; however, the slight decrease in cell number observed with antibody A4.1 was not sufficient to explain the profound block of process outgrowth. Similar results were seen in cultures of hippocampal neurons (see below); A4.1 abolished the neurite-promoting activity of TSP without affecting cell attachment (data not shown). Thus, the inhibition of neurite outgrowth was due to a specific blocking of the stalk domain of the TSP molecule.

VOLUME 150.1992

TSP contains the RGD sequence recognized by many integrins. However, it is located in a region of the calcium-binding domain which is distant from the site recognized by antibody A4.1. Consistent with the results of the antibody inhibition studies was the finding that the GRGDS peptide (1 mM) had no effect on neurite outgrowth on a TSP substrate (data not shown). Identity

of the Processes Formed

in the Prewnce of TSP

Previous studies have shown that ECM proteins can exert long-term effects on the morphological differentiation of sympathetic neurons. Laminin causes the stable expression of supernumerary axons, while other ECM components selectively promote the extension of dendrites (Lein and Higgins, 1989). To further characterize the effects of TSP, we examined neuronal morphology after long-term exposure to TSP. The morphology of neurons grown for 1 week in the presence of TSP is illustrated in Fig. 4A. The only apparent change observed in these cultures was a time-dependent increase in the density of the plexus formed by distal processes. Intracellular dye injections (Fig. 4B) revealed that most of these neurons remained unipolar (1.2 f 0.1 processes/cell after 1 week). The processes of unipolar neurons appeared axonal because they were long, thin, and of a constant diameter. Processes with the morphological characteristics of dendrites were observed in ~5% of the neurons. To confirm the light microscopic observations, long-term cultures were immunostained with antibodies which react with known axonal or dendritic markers. Nonphosphorylated forms of the M and H neurofilament subunits are localized primarily to the dendrites and somata in situ and in vitro (Peng et al, 1986; Sternberger and Sternberger, 1983). Figure 5B illustrates the staining pattern observed when cultures were stained with antibodies spe-

TABLE 3 IDENTIFICATION OF THE NEUFUTE PROMOTING REGION OF THROMBOSPONDIN Substrate

% Neurons with neurites

Polylysine TSP TSP and A2.5 TSP and C6.7 TSP and A6.1 TSP and A4.1

11% 66%* 62%* 59% * 60%* 11at

Number of neurites per neuron 0.15 1.04 0.97 0.75 0.93 0.12

+ + + + f *

0.03 0.07* 0.07* 0.05*+ 0.93* o.oq

Total length of neuritic plexus (pm) 133* 768 + 842 f 583 f 651 f 76 +

12 115* 123* 82* 73* 6*t

Cells per mm’ 2.0 f 2.3 f 1.7 f 1.6 f 1.7 f 1.3 +

0.23 0.18 0.30 0.06 0.19 0.01

Note. Data are expressed as means f SEM, data in columns 1 and 2 are pooled from two dissections (N = 180). However, neuritic length was measured in only one culture series. a N = 30 for neurons per culture condition. Only neurons with neurites were included in the calculation of the means and SEM for this morphological parameter. * Denotes a difference at P < 0.01 from polylysine. t Denotes a difference at P < 0.05 from TSP.

OSTERHOUT, FRAZIER, AND HIGGINS

FIG. 4. The morphology of sympathetic neurons in long-term culture on TSP. Phase contrast (A) and fluorescence (B) micrographs of a neuron injected with Lucifer yellow. After 1 week, neurons grown on TSP typically had one long axon and no dendrites. Bar, 50 pm.

TSPPromotesNewite Outgrowth

261

ments with hippocampal neurons grown in 1% serum with cell densities ranging from 132 to 571 cells/mm2. However, we have also observed comparable neuritepromoting effects in culture series maintained in serum-free media. The effects of TSP were most prominent during the first day in vitro; by the second day, the percentage of neurons with neurites in polylysine cultures (88%) approximated that of TSP-treated cultures (100%). Neurite outgrowth was enhanced in cultures of dorsal root ganglion neurons, with 74% of the cells responding to TSP after 24 hr in vitro. TSP induced a threefold increase in the number of neurites per cell (Table 5). TSP was also effective in stimulating process elongation from cerebral cortical neurons (Table 4), and their morphology resembled that of hippocampal neurons. Thus, it appears that this ECM component can exert similar

cific for the nonphosphorylated forms of neurofilaments. Typically, the cell soma stained intensely, but there was no staining of the processes. In contrast, virtually all of the processes were immunoreactive for axonal markers, namely the phosphorylated forms of the neurofilaments (Fig. 5C) and synaptophysin (Fig. 5D). These data suggest that TSP promotes the growth of one to two neurites which eventually assume the characteristics of axons.

The E$“ectsof TSP on Other Classesof Neurons TSP accelerated process formation in a variety of embryonic neurons. Hippocampal neurons generally fail to extend neurites on a polylysine substratum during the first 24 hr in culture. In the presence of TSP, neurite outgrowth was observed in 67% of hippocampal neurons and the mean number of neurites per cell increased fourfold (Table 4). Although some of the hippocampal neurons grown on TSP were unipolar, many neurons had multiple processes (Fig. 6). The multipolar cells typically extended several small neurites and one long process with a prominent growth cone. Since the scoring criterion used in analyzing these cultures stipulates that a process must be greater than one cell diameter in length, many of the minor neurites were not tabulated in compiling the data in Table 4. Thus, the neurite-promoting effects of TSP on hippocampal were somewhat underestimated. The effects of TSP on process outgrowth from hippocampal neurons were independent of cell adhesion, because the cell counts indicate that TSP had no effect on cell attachment to the substrate. The data shown in Table 4 are representative of four experi-

FIG. 5. Cytochemical analysis of processes formed in the presence of TSP. Phase contrast (A) and fluorescence (B) micrographs of a culture immunostained with antibody directed against the nonphosphorylated forms of the M and H neurofilament subunits, which are localized predominantly in the somata and dendrites of neurons in situ. There is intense staining of the somata, but little or no staining of the neuronal processes. In contrast, the processes of sister cultures are intensely stained by antibodies that recognize axon-selective antigens, quch as phosphorylated forms of the H subunit of neurofilaments (C) or synaptophysin (D). Bar, 25 pm.

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DEVELOPMENTAL BIOLOGY

TABLE 4 EFFECT OF THROMB~SPONDIN ON NEURITIC OUTGROWTH FROM EMBRYONIC CENTRAL NEURONS AFTER 24 hr IN VITRO Substrate

% Neurons with neurites

Number of neurites per neuron

Cells per mm2

Hippocampus Polylysine BSA Laminin TSP

23%

19% 46%* 67%*

* 0.07 I!I 0.08 0.92 -t 0.12* 1.38 Ii 0.13* 0.33 0.32

r 39 + 26 519 k 39 416 +24 571 532

VOLUME 150.1992 TABLE 5 EFFECTS OF THROMBOSPONDIN ON NEUFUTIC OUTGROWTH FROM DORSAL BOOT GANGLION NEURONS AFTER 24 hr IN VITRO

Substrate

% Neurons with neurites

Polylysine TSP

35% 74%*

Number of neurites per neuron 0.39 + 0.06 + 0.09*

1.15

Note. Data are expressed as means + SEM; N = 90 neurons for each culture condition. * Denotes a difference at P < 0.01.

Cerebral cortex Polylysine Laminin TSP

18% 36%* 48%*

0.19 f 0.04 0.46 do0.08* 0.91 * 0.13*

103 f 13 104 f 21 132 + 12

Note Data are expressed as means + SEM; N = 90 neurons for each culture condition. * Denotes a difference at P < 0.01 from polylysine. Laminin and thrombospondin are different (P < 0.05) in the percentage of neurons with neurites and the number of neurites per cell.

effects on many classes of central and peripheral neurons. However, one quantitative difference was evident. In the peripheral nervous system, laminin consistently evoked a larger response than TSP in cultures of sympathetic neurons (Table 1) and sensory neurons (data not shown). In contrast, in both of the central neurons examined, TSP stimulated neurite outgrowth more effectively than laminin (Table 4). DISCUSSION

During development, the expression of TSP is temporally and spatially regulated in both the peripheral and

FIG. 6. TSP promotes neurite outgrowth from hippocampal neurons in culture. Embryonic hippocampal neurons attach to polylysine but usually do not extend processes after 24 hr (A). In contrast, hippocampal neurons plated onto adsorbed TSP extend several neurites, often with one process much longer than the others (B). Bar, 25 Nrn.

central nervous systems (O’Shea and Dixit, 1988). Previous studies have demonstrated that TSP is involved in the migration of cerebellar granule cells (O’Shea et al, 1990), and that it can promote neurite outgrowth from retinal ganglion cells in culture (Neugebauer et ah, 1991). The present study demonstrates that TSP also enhances process outgrowth from a variety of neurons derived from both the central and peripheral nervous systems. These observations suggest that TSP may play an important role in neuronal growth and regeneration.

TSP Promotes

New-de Outgrowth

In short-term cultures of sympathetic neurons, TSP reduced the time to initial process extension, as well as increasing the number of primary processes and total neuritic length. In all assays, we utilized a polylysine substrate which caused maximal attachment of sympathetic neurons (Lein et ah, 1991). Under these conditions, TSP or other matrix proteins did not significantly alter cell number or viability. Thus, stimulation of neurite outgrowth by TSP was not the result of selective survival of subpopulations of sympathetic neurons. The fact that TSP also had no effect on the number of adherent central neurons suggests that the neurite-promoting activity of TSP was independent of cell attachment in cultures with more heterogeneous neural populations. TSP can interact with other proteins, including ECM components and plasma proteins (Frazier, 1987). Because most of our experiments were conducted with serum-free media, any effects of serum-derived proteins such as fibronectin and vitronectin were eliminated. The cultures of sympathetic neurons were maintained at low density and nonneuronal cells comprised a very small fraction (tlO%) of this population (Higgins, unpublished observations). Thus, the vast majority of neurons with processes were not in contact with glia and it is unlikely that glial cells could exert significant conditioning effects at such densities. It appears, then, that

OSTERHOUT, FRAZIER, AND HIGGINS

TSP acts directly upon sympathetic neurons to stimulate process extension without the involvement of other intermediates. In cultures of sensory and central neurons, glia were present in greater numbers, and it is not clear whether the effects of TSP are due solely to direct interaction with the neuron or involve additional gliaderived components.

Identity

of the Neurite-Promoting

Region of TSP

TSP, like many matrix proteins, has multiple discrete domains. The individual domains can mediate different effects, depending on the nature of the cell in contact with the protein. For example, endothelial cells bind and internalize TSP through the heparin-binding region at the amino terminus (Murphy-Ullrich and Mosher, 1987), while the carboxy-terminal cell-binding domain mediates the aggregation of platelets (Dixit et ak, 1985) and melanoma cell attachment (Roberts et ab, 1987). It is also possible for cells to interact with more than one domain of TSP. The platelet-binding domain promotes haptotaxis of tumor cells, while the heparin-binding domain of the molecule stimulates chemotaxis of the same tumor line (Taraboletti et aL, 1987). Because the effects of TSP could be totally blocked using an antibody to the stalk region (A4.1), it appears that neurite-promoting activity is primarily localized to a single domain of the molecule and that other domains make at most minor contributions. There are several peptide sequences contained in the stalk region which may mediate process outgrowth. One candidate is a properidin-like sequence which promotes adhesion of melanoma cells (Prater et al., 1991). There are also regions of the stalk which show homology to the N-terminal propeptide of type I(a1) collagen, the epidermal growth factor precursor, and a potential second heparin-binding site. Thus, it is possible that neurite outgrowth may involve binding to cell surface proteoglycans; alternatively, it may involve the properidin adhesive sites, or a combination of the two. Further studies using synthetic peptides will be necessary to precisely define the neurite-promoting sequence in this region of TSP.

TSP Selectively Promotes

the Growth of Axons

In situ, sympathetic neurons normally extend a single axon and multiple dendrites. However, the morphology of these neurons is subject to environmental regulation, and culture conditions can be manipulated such that the neurons assume different shapes. ECM molecules have been identified as factors which can regulate the morphogenesis of sympathetic neurons by selectively pro-

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moting the growth of a specific type of process. When these neurons are exposed to laminin or collagen IV, they only extend axons. In contrast, when these neurons are exposed to a basement membrane extract, they form multiple dendrites (Lein and Higgins, 1989; Lein et al, 1991). In this study, the identity of the processes formed in the presence of TSP was examined using both dye injection and immunocytochemical techniques. The results indicate that TSP exerts a selective morphogenic effect similar to that observed with laminin and collagen IV; namely, it promotes axonal but not dendritic growth in long-term cultures of sympathetic neurons. Laminin is capable of stimulating process elongation in most types of neurons (Lander, 1987). TSP likewise induces neurite outgrowth from several types of neurons, including those isolated from both the peripheral and central nervous systems. However, while there are similarities in the range of target cells for these two proteins, we observed quantitative differences in their effects. With peripheral neurons, maximally effective concentrations of TSP consistently evoked less outgrowth than laminin; with central neurons, TSP caused the greater response. The reasons for these quantitative differences are not yet evident; one possibility is a differential distribution of TSP receptors between the central and peripheral nervous systems. Regardless, the observations that the response of TSP is prominent in central neurons and that TSP levels are elevated in developing brain suggest a significant role for TSP in the morphogenesis of the central nervous system. Elevated levels of TSP are also observed in areas of tissue damage in the adult in nonneural tissues; it has, therefore, been suggested that TSP may have a role in wound healing (Raugi et ak, 1987; Watkins et al, 1990). The possibility also exists that TSP could participate in nerve regeneration after damage to the central nervous system. Although TSP is expressed at low levels in the normal adult brain, mature human astrocytes retain the ability to synthesize TSP (Asch et al., 1986; O’Shea and Dixit, 1988). Moreover, secretion of TSP in vitro is greatly enhanced by substances such as platelet derived growth factor that could be released in response to tissue damage (Asch et aZ., 1986; Grierson et al, 1991). Given our observations that TSP stimulates process outgrowth from embryonic central neurons, it may be that synthesis of TSP is involved in the normal sprouting response seen in neurons after injury. Further exploration of this possibility will require an examination of TSP synthesis in response to nerve damage. This work was supported by a grant from the National Science Foundation (BNS 8909373). We thank Johnna Metzler-Northrup for her expert technical assistance.

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