Protease inhibitors influence the direction of neurite outgrowth

Developmental Brain Research, 45 (1989) 203-209

203

Elsevier BRD 50859

Protease inhibitors influence the direction of neurite outgrowth Richard L. Hawkins and Nicholas W. Seeds Department of Biochemistry, Biophysics and Genetics, Universityof Colorado Health Sciences Center Medical School, Denver, CO 80262 (U.S.A.) (Accepted 13 September 1988)

Key words: Protease; Protease inhibitor; Neurite outgrowth; Dorsal root ganglion; Covalent derivatization

Addition of protease inhibitors to the culture medium has been shown to enhance neurite outgrowth by cultured mouse dorsal root ganglia (DRG). Those results are now extended to show that a diffusible source of soybean trypsin inhibitor (STI) or zones of immobilized STI can orient the direction of outgrowth towards the region of STI. However, a high concentration of diffusible STI promotes outgrowth in the opposite direction from the STI source. Immobilized leupeptin, L-lysine, or D-Phe-Pro-Arg-chloromethyl ketone can also direct outgrowth towards their immobilized areas, as do zones of laminin or fibronectin. However, derivatized zones containing urokinase or thrombin preferentially direct outgrowth away from those zones. These data support the hypothesis that a balance between extracellular protease and inhibitor is important in mediating interactions between neurite growth cone and extracellular matrix.

INTRODUCTION Proteases and protease inhibitors have been shown to influence the extent of neurite outgrowth from several neural cell types 3'8at'16. W e have p r o p o s e d that this effect of proteases m a y be due to adhesive protein contacts between cell and substratum and the r e q u i r e m e n t that these contacts be temporarily broken for neurite elongation to occur n,12a3. Thus, we have o b s e r v e d that addition of soybean trypsin inhibitor (STI) to the culture m e d i u m enhances the rate and extent of neurite outgrowth from normal mouse dorsal root ganglion ( D R G ) explants and that this effect can be reversed by addition of exogenous proteases 11. As a refinement of these previous studies, we have e x a m i n e d w h e t h e r a focal source of protease inhibitor could preferentially orient the direction of neurite outgrowth, in addition to simply enhancing the extent of outgrowth. To this end we have employed two systems. In one system, D R G are cultured on top of p o l y a c r y l a m i d e gels through which inhibitor can slowly diffuse from a localized source.

The o t h e r system utilizes zones of immobilized inhibitor or protease near which D R G are placed to see if outgrowth is directed toward or away from the derivatized zone. MATERIALS AND METHODS

Acrylamide gel well experiments Polyacrylamide gels (10% total, 2.6% cross-linker) were cast in 48-well (11.3 m m well diameter) tissue culture dishes (750/~l/well) with Duibecco's Modified Eagle M e d i u m ( D M E ) used to dilute a stock solution of acrylamide (1:1, v/v). Polymerization was initiated with a m m o n i u m persulfate and T E M E D . A f t e r the gels had set, they were rinsed with 1 mg/ml bovine serum albumin ( B S A ) and then a well (4 x 4 × 6 mm) was cut in the middle of the gel with a scalpel blade. The gels were soaked in 70% ethanol overnight and then transferred, using sterile technique, to 24-well (16 m m well diameter) tissue culture dishes. This leaves a large outer well surrounding the gel, in addition to the inner well cut into the gel. Gels were

Correspondence: N.W. Seeds, Department of Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center Medical School, 4200 E. 9th Avenue, B-121, Denver, CO 80262, U.S.A. 0165-3806/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

204 soaked twice (3 h each) in phosphate-buffered saline (PBS), followed by an additional soaking in DME, and finally they were soaked for 1-2 days at 37 °C in N2 9aedium 4, containing nerve growth factor (20 ng/ml) and penicillin/streptomycin (5 U/ml and 0.5 ~tg/ml). Laminin (2 #g/ml) or fibronectin (2 #g/ml) were also included in the N2 stock in some experiments, but were found not to affect outgrowth. However, NGF was found to be stimulatory and hence routinely included. Dorsal root ganglia (DRG) were removed from 1-2 day postnatal mice (strain C57BL/6J), the nerve roots excised, and the D R G placed on top of the acrylamide gel in between the inner and outer wells. The wells were then filled with either N2 alone or N2 with soybean trypsin inhibitor (STI). Medium in the inner well was replenished daily. After 3-8 days in vitro, the D R G were observed using phase-contrast microscopy, to assess whether the neurites had grown out predominantly oriented towards or away from the test well, or were randomly oriented. Directionality was scored if greater than 75% of the neurites were in a particular direction or if there was a greater than one ganglion diameter difference in neurite length between the two directions.

Immobilized inhibitor experiments Proteins or peptides were covalently coupled to glass coverslips by a modification of the methods of Alvin and Hughes 2, and Edelman et alJ'. Parallel lines (alternate 3 mm and 2 mm spacing) were scratched onto glass coverslips (25 mm) with a diamond pencil, washed with Cytoclean (Isolab, Akron, OH) and then immersed in 20% sulfuric acid overnight. Slips were washed twice in distilled water, followed by 0.1 M sodium hydroxide and blotted dry. Keeping the scratched side down, the slips were then placed in 3-aminopropyl triethoxy silane for 4 min at room temperature. The slips were then washed extensively in distilled water followed by a wash in PBS. Slips were then reacted with 2.5% glutaraldehyde in PBS for 30 min at room temperature, rinsed 3 times in PBS, and then placed in 0.1 M sodium phosphate, pH 7.2, containing 10/~g/ml BSA, for 1 h at room temperature. After 3 rinses in PBS, 3 mm wide filter paper strips were placed on the coverslips and aligned with the scratches on the opposite side. Immediately prior to placement on the coverslips, the

paper strips were soaked in the test inhibitor protein or peptide solution freshly mixed with 1-cyclohexyl3-(2-morpholino ethyl)-carbodiimide metho-p-toluene sulphonate (water-soluble carbodiimide (WSC) in 0.5 M NaC1, pH 6.0. Paper strips were left in place for 1 h at room temperature and then removed, and the coverslips rinsed 3 times in PBS. Slips were stored overnight in cold, sterile PBS containing antibiotics and then placed in 35 mm tissue culture dishes. Approximately 6 D R G were placed on each slip in 100/A N2 medium and cultured for 4-6 days in vitro. Neurite outgrowth was observed using phase-contrast microscopy and the location of the D R G noted with respect to protein treated or untreated zones, along with any preferential directionality of outgrowth. Mouse fibroblast extracellular matrix was prepared by the method of Fairbairn et al.7. RESULTS The use of high-percentage (10%) acrylamide gels allows D R G to be cultured on a substratum through which substances can diffuse. Preliminary experiments with low molecular weight dyes (Methyl orange and Bromphenol blue) indicated that when placed in separate (inner or outer) wells it took several hours for the dye to enter the gel and about 24 h before appreciable mixing of the two dyes occurred. Thus, if a protein were placed in N2 medium in one well and only N2 medium in the other, there should be a directional gradient of protein within the substratum for at least the first 24 h in vitro, during the period of initial neurite outgrowth from DRG. Fig. 1A shows that when STI (10#g/ml) is placed in the inner (+) well, a greater number of ganglia (twice as many at 3 days in vitro and 5 times as many after 8 days in vitro) show preferential neurite outgrowth towards the STl-containing inner well than toward the N2 medium alone outer (-) well. In either treatment condition, one-third of the ganglia displayed non-directed outgrowth. The average length of neurites, judged as previously described u, was 1.7, 1.8 and 1.5 in the + well, - well or non-directed orientation when exposed to N2 only and neurite length was 2.1, 2.0 and 2.0 in the + well, - well, or non-directed orientation when STI was in the + well. Thus, fiber length tended to be the same whether growing towards or

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Fig. 1. Neurite outgrowth toward a PA inhibitor. Preferential axonal growth toward a diffusible source of STI (10#g/ml) at 3 days and then at 8 days of the same culture. Analysis of the data by Fisher's exact test gave a non-significant difference between conditions at 3 days; however by 8 days of culture, P = 0.057 for a difference in outgrowth between N2 in both wells vs STI in + well conditions. In the absence of the inhibitor there is a preferential growth toward the outer (-) well. Panel B shows the combined results of several studies. A Chi-square analysis gave P < 0.001 for a difference in outgrowth between treatments. away from the STI source, but with a greater n u m b e r of ganglia whose fibers grew toward the STI source. It is noteworthy that when both wells contained either STI or N2 medium alone, there was a tendency for the neurites to orient towards the outer well. This was observed in several separate experiments (pooled data in Fig. 1B) and may be due to the greater volume of medium in the outer well which maintained a steady source of medium to counteract the tendency of the gel to dry at its exposed top surface; whereas the inner well was observed to deplete rapidly and required daily addition of medium. Fig. 2 shows the d o s e - r e s p o n s e relationship for STI in orienting outgrowth on acrylamide. STI at 10-100/~g/ml directed neurite outgrowth toward the STI-containing well, whereas at concentrations of 250 or 1000/~g/ml outgrowth was directed away from the STI well. This is similar to our previous 11 observations that high doses of STI are inhibitory towards

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25 50 I00 250 I000 STI in (~) well (#g/ml) Fig. 2. Dose-response relationship for protease inhibitor and neurite outgrowth. Increasing concentrations of STI in N2 (NGF) medium were added to the center (+) well and directional (i.e. >75% of the neurites were in a particular direction, or if neurite length differed in one direction by more than one ganglion diameter) neurite outgrowth scored on day 7 of culture. Although low doses of STI were stimulatory, high concentration of STI directed neurite growth away from the STI source.

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206 neurite outgrowth while lower doses are stimulatory. However, the cut-off dose for the switch from stimulation to inhibition is greater than 100/~g/ml for the current data while, our previous study showed the cut-off at 10/~g/ml. This difference may be due to the time it takes for STI to diffuse through the gel and hence reach full concentration in the current case. Since one cannot be sure of the concentration and location of the diffusional gradient in the above acrylamide gel experiments, we wanted to see if STI covalently attached to the substratum could also direct neurite outgrowth from DRG. As a first step, it was necessary to determine whether the bound STI retained inhibitory activity. This was determined two ways: (1) by derivatizing glass test tubes first with BSA and then attaching STI with a water-soluble carbodiimide (WSC). An amidolytic assay I1, which is known to be inhibited by soluble STI, composed of urokinase, plasminogen, and a chromogenic substrate (Kabi S-2251) was then carried out in the tubes. (2) The second activity check involved derivatization of STI to glass coverslips which were then placed on an acrylamide gel-containing plasminogen, casein, and urokinase, incubated overnight at 37 °C, and stained with Coomassie brilliant blue. Active protease produces non-staining caseinolytic zones, whereas active STI would inhibit the proteolysis and the gel would be stained. The test tube assay showed (Table I) that coupled STI retained inhibitory activity in a dose-dependent manner and that this inhibitory activity was greatest when attached to a surface previously coated with BSA at 10/~g/ml. The medium TABLE 1 Protease inhibitor coupling to glass

STI (10->1000 /~g/ml) was covalently coupled to glass test tubes (see Materials and Methods) that had been previously coated with different concentrations (10-1000/~g/ml) of BSA. The tubes were rinsed and used for an amidolytic assay of urokinase activity; the percent of non-inhibited urokinase activity compared to untreated glass tubes is indicated. Percent of urokinase activity S TI coating

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from the overnight incubation with PBS of derivatized tubes showed no inhibition of the amidolytic assay, indicating that the STI was firmly bound to the test tube. It should be noted that, in solution, concentrations of STI above 1 pg/ml show complete inhibition in the amidolytic assay, indicating that there is loss of activity or low coupling efficiency during the derivatization procedure, since ST1 derivatized test tubes were completely inhibitory at 1 mg/ml. The caseinolytic assay also showed that STI coupled to coverslips retained its protease inhibitory activity. Since covalently coupled STI was found to retain protease inhibitory activity, we next prepared coverslips with alternating bands of either STI or BSA alone, placed D R G near the border between bands, and observed whether neurite outgrowth would preferentially occur towards one band or whether it would be randomly oriented. Figs. 3 and 4 show that neurites displayed a significant preference to grow towards STI derivatized zones, whereas D R G on BSA (only) derivatized zones mostly displayed random outgrowth. When STI is inactivated by heat denaturation prior to its coupling, D R G showed preferential growth toward native STI-coated or BSAcoated sides of the coverslip and away from the denatured STI; thus suggesting that the preferential growth effect by STI is related to its protease inhibitory activity. The specificity of the outgrowth orientation by protease inhibitors was examined next. Fig. 5 shows that protease inhibitors which inhibit plasminogen activator, such as D-Phe-Pro-Arg-chloromethyl ketone, leupeptin, or L-lysine also can direct outgrowth, although STI gave the best response. Although trasylol retained its plasmin inhibitor activity after covalent coupling to the coverslip, the immobilized inhibitor was without effect on neurite outgrowth, similar to our previous report that trasylol did not affect basal neurite outgrowth 1~. Similarly, soybean lectin, included as a control for possible contaminating lectin activity in the STI preparations, had no effect. Ganglia cultured on BSA alone did not show preferential outgrowth from one zone to the next. Naturally occurring protease inhibitors of the protease nexin-type 1'9'19'21 are also known to promote neurite outgrowth by neuroblastoma 9 and sensory neurons (N.W. Seeds, D, Monard and R W . Scott, unpublished observations); thus far, attempts

207

Fig. 3. Preferential neurite outgrowth on immobilized protease inhibitor. Dorsal root ganglion placed on the BSA-coated side (A) of a coverslip shows preferential neurite growth towards the STI-treated side (B). The arrows indicate the line that demarcates the border between BSA and STI. tO couple active protease nexin to the substratum have been unsuccessful. The effect of proteases or extracellular matrix ( E C M ) coupled to coverslips is shown in Fig. 6. Neurite outgrowth tended to be away from zones coated with either urokinase or thrombin. Trypsin had a small positive outgrowth effect but this reflects slight outgrowth from one ganglion and is p r o b a b l y not significant. By the amidolytic assay, covalently coupled urokinase was found to retain activity; however, trypsin activity was completely abolished by the coupling procedure. E C M p r e p a r e d from cultured mouse fibroblasts had both positive and negative

orientation effects with the negative effect being definitely stronger. The meaning of this is unclear but could represent denaturation by sodium dodecyl sulfate of E C M proteins during the extraction process. However, the purified E C M components laminin or fibronectin were able to direct neurite outgrowth

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Fig. 4. Comparison of preferential or random neurite outgrowth on BSA- or STI-treated substrates. Dorsal root ganglia were placed on either BSA- or STI-treated zones of glass coverslips near the border between the two zones and neurite growth assessed after 4 days. A Chi-square analysis gave P < 0.005 for a difference between outgrowth on the treated zones.

Fig. 5. Influence of several immobilized protease inhibitors on neurite outgrowth. Solutions of the following substances were coupled to the coverslips: D-Phe-Pro-Arg-CH2CI ketone (1 mM), leupeptin (1 mg/ml), L-lysine (10 rng/ml), STI (1 mg/ml), trasylol (1000 U/ml), soy lectin (1 mg/ml). 8-10 ganglia were observed in each group. High concentrations of test substances were used since it was difficult to determine their coupling efficiency. The plasmin inhibitor, trasylol, did not influence growth, nor did soybean lectin. Preferential outgrowth was outgrowth towards the coupled zone from ganglia located near the borderline between inhibitor-coupled and BSA only zones.

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Fig. 6. Influence of proteases and matrix molecules on neurite outgrowth. Ganglion cultured on adjacent zones of BSA alone did not show preferential outgrowth from one zone to the next. Proteases: urokinase (100,ug/ml), thrombin (10/xg/ml), trypsin (1 mg/ml), and ECM produced by mouse embryo fibroblasts, laminin (1 mg/ml), flbronectin (1 mg/ml) or BSA (1 mg/ml) solutions were coupled to glass coverslips. The proteases urokinase (UK) and thrombin inhibited neurite outgrowth. Analysis by Fisher's exact test gave P = 0.016 for UK, P = 0.02 for ECM, P = ll.04 for laminin, and P = 0.033 for fibronectin for a difference in outgrowth compared to BSA treatment. Between 7-9 ganglia were observed in each group.

onto zones which had been derivafized with these substances. The length of neurite outgrowth 11 was similar in the preferential outgrowth vs non-directed outgrowth conditions (urokinase, 1.0/1.0; thrombin, 1.0/1.3; trypsin, 1.0/1.5; E C M , 1.7/1.6', laminin, 1.(I/1.0; Fbn, 1.5/1.8; BSA, 1.0/1.1, for preferential vs non-directed outgrowth, respectively). DISCUSSION The present data extend our previous results that protease inhibitors, especially soybean trypsin inhibitor, when in solution not only can enhance the extent of neurite outgrowth from cultured D R G but can serve also to orient the direction of outgrowth when immobilized on the substratum. This supports the hypothesis that neurite growth is, at least in part, regulated or directed by interactions between the growth cone, proteases and extracellular matrix. A d h e s i o n REFERENCES 1 Andreasen, P.A., Nielsen, L., Kristensen, P., GrondahlHansen, J., Skiver, L. and Dano, K., Plasminogen activator inhibitor from human fibrosarcoma cells binds uroki-

between growth cones and E C M is necessary for neurite growth to occur 15, purified E C M c o m p o n e n t s such as laminin p r o m o t e neurite outgrowth l°'~a, and substratum-attached extracellular material can direct outgrowth 5. But it is also necessary that for elongation to proceed, there not be obstructions in the path such as membranes or other cells and that the growth cone be able to temporarily break adhesive contact between cells and ECM. The growth cone has been shown to secrete plasmogen activator (pA)13; however, it appears that there is a balance 9" 10A6-19 between extracellular protease activity capable of clearing a path and/or detaching adhesive contacts, and endogenous secreted protease inhibitors 1' 9,19,21 which prevent wholesale destruction of the neurite or ECM. O u r results suggest that, in serum-free culture at least, there is excess protease activity (which can be observed as free P A in the conditioned medium) which tends to d a m p e n neurite outgrowth. The presence of protease inhibitor in the medium or on the substratum can then enhance or direct the outgrowth similar to the effect of adding laminin or fibronectin to the substratum. However, if the concentration of inhibitor is too great, then the p r o t e a s e - i n hibitor balance is again shifted and outgrowth is reduced. Although we have not observed regression or selective survival of fibers on one side of the coverslip, the rate of outgrowth on either side was similar, and, once established, neurites reached a steady state length by 5 - 8 days of culture and so it seems that the outgrowth pattern results from differences in adhesivity encountered during the initial outgrowth of the neurites. Thus, in addition to E C M or extracellular protease activity, it appears that protease inhibitors, whether soluble or substratum-bound, can affect neurite outgrowth. ACKNOWLEDGEMENTS These studies were s u p p o r t e d in part by grants from the National Science F o u n d a t i o n (BNS8607719) and the U . S . P . H . S . , NIH-NS09818, and the Muscular D y s t r o p h y Association. nase-type PA, but not its proenzyme, J. Biol. Chem., 261 (1986) 7644-7651. 2 Aplin, J.D. and Hughes, R.C., Protein-derivatized glass coverslips for the study of cell-to-substratum adhesion, Anal. Biochem.. 113 (1987) 144-148.

209 3 Becherer, P.R. and Wachsman, J.T., Increased neurite development and plasminogen activator expression by exposure of human neuroblastoma cells to a plasminogen-deficient growth medium, J. Cell Physiol., 104 (1980) 47-52. 4 Bottenstein, J.E. and Sato, G.H., Growth of a rat neuroblastoma cell line in serum-free supplemented medium, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 514-517. 5 Collins, F. and Lee, M.R., The spatial control of ganglionic neurite growth by the substrate-associated material from conditioned medium: an experimental model of haptotaxis, J. Neurosci., 4 (1984) 2823-2829. 6 Edelman, G.M., Rutishauser, U. and Millette, C.F., Cell fractionation and arrangement on fibers, beads, and surfaces, Proc. Natl. Acad. Sci. U.S.A., 68 (1971) 2153-2157. 7 Fairbairn, S., Gilbert, R., Ojakian, G., Schwimmer, R. and Quigley, J.P., The extracellular matrix of normal chick embryo fibroblasts: its effect on transformed chick fibro° blasts and its proteolytic degradation by the transformants, J. CellBiol., 101 (1985) 1790-1798. 8 Gibson, W.H., Burack, S.L. and Picciano, A., The effects of serine protease inhibitors on morphological differentiation of murine neuroblastoma cells (NB15), J. Cell. Physiol., 119 (1984) 119-126. 9 Guenther, J., Nick, H. and Monard, D., A glial-derived neurite promoting factor with protease inhibitory activity, EMBO J., 4 (1985) 1963-1966. 10 Hammarback, J.A., Palm, S.L., Furcht, L.T. and Letourneau, P.C., Guidance of neurite outgrowth by pathways of substratum-absorbed laminin, J. Neurosci. Res., 13 (1985) 213-220. 11 Hawkins, R.L. and Seeds, N.W., Effects of proteases and their inhibitors on neurite outgrowth from neonatal mouse sensory ganglia in culture, Brain Res., 398 (1986) 63-70.

12 Krystosek, A. and Seeds, N.W., Plasminogen activator secretion by granule neurons in cultures of developing cerebellum, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 7810-7814. 13 Krystosek, A. and Seeds, N.W., Plasminogen activator release at the neuronal growth cone, Science, 213 (1981) 1532-1533. 14 Lander, A.D., Fujii, D.K. and Reichardt, L.F., Purification of a factor that promotes neurite outgrowth: isolation of laminin and associated molecules, J. Cell Biol., 101 (1985) 898-913. 15 Letourneau, P.C., Cell-to-substratum adhesion and guidance of axonal elongation, Dev. Biol., 44 (1975) 92-101. 16 Monard, D., Niday, E., Limat, A. and Solomon, F., Inhibition of protease activity can lead to neurite extension in neuroblastoma cells, Prog. Brain Res., 58 (1983) 359-363. 17 Moonen, G., Grau-Wagemans, M.P. and Selak, I., Plasminogen activator-plasmin system and neuronal migration, Nature (Lond.), 298 (1982) 753-755. 18 Pittman, R.N., Release of plasminogen activator and a calcium-dependent metallo-protease from cultured sympathetic and sensory neurons, Dev. Biol., 110 (1985) 91-101. 19 Scott, R.W., Eaton, D., Duran, N. and Baker, J., Regulation of extracellular plasminogen activators by human fibroblasts - - the role of protease nexins, J. Biol. Chem., 258 (1983) 4397-4403. 20 Soreq, H. and Miskin R., Plasminogen activator in the developing rat cerebellum: biosynthesis and localization in granular neurons, Brain Res., 11 (1983) 149-158. 21 VonMourik, J.A., Lawrence, D. and Loskutoff, D., Purification of an inhibitor of plasminogen activator synthesized by endothelial cells, J. Biol. Chem., 259 (1984) 4914-492l.