Involvement of axonal phospholipase A2 activity in the outgrowth of adult mouse sensory axons in vitro

Pergamon PII:

Neuroscience Vol. 91, No. 4, pp. 1539–1547, 1999 Copyright 䉷 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00 S0306-4522(98)00684-8

INVOLVEMENT OF AXONAL PHOSPHOLIPASE A2 ACTIVITY IN THE OUTGROWTH OF ADULT MOUSE SENSORY AXONS IN VITRO ¨ M and A. EDSTRO ¨M M. HORNFELT,* P. A. R. EKSTRO Department of Animal Physiology, Lund University, S-223 62 Lund, Sweden

Abstract—The effect on axonal outgrowth of inhibition of phospholipase A2 activity was studied in a recently developed in vitro model, where dorsal root ganglia with attached spinal roots and nerve stumps from young adult mice were cultured in an extracellular matrix material (Matrigel). The phospholipase A2 inhibitors 4-bromophenacyl bromide and oleyloxyethyl phosphorylcholine dose-dependently reduced axonal outgrowth from the sciatic nerve stump. A similar inhibitory effect was seen when only the cut nerve end was exposed to the inhibitors in a compartmental culture system. The local effect of phospholipase A2 inhibition was further investigated on axons established in culture, using time-lapse recording. Exposure to phospholipase A2 inhibitors caused the retraction of filopodia extensions and a reduction in growth cone motility within a few minutes. After removal of inhibition, normal growth cone motility and axonal growth were regained. Nerve cell bodies and axons, in contrast to Schwann cells, showed immunoreactivity after staining with an antiserum against secretory phospholipase A2, and elevated levels of the enzyme could be detected after culture for 24 h. The immunoreactive protein was of approximately 170,000 molecular weight (phospholipase A2-170) as determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and immunoblotting. The localization of phospholipase A2-170 in axons growing into the Matrigel was also demonstrated by use of a whole-mount technique. The results of this study show the importance of continuous phospholipase A2 activity for growth cone motility and axonal outgrowth in the mammalian peripheral nerve, and suggest the involvement of an axonally localized enzyme. 䉷 1999 IBRO. Published by Elsevier Science Ltd. Key words: neurite outgrowth, peripheral nerve, growth cone, phospholipase A2-170.

Phospholipases A2 (PLA2s) are a heterogeneous group of enzymes catalysing the hydrolysis of the fatty acyl bond in the sn-2 position of phospholipids, liberating free fatty acids. Besides their participation in processes involving general phospholipid digestion, PLA2s are involved in membrane remodelling 2 and repair, 1 as well as protection of membranes from lipid peroxidation damage. 22 The PLA2s are also important in cellular signal transduction by their release of arachidonic acid, the rate-limiting precursor for the synthesis of inflammatory mediators like prostaglandins, thromboxanes and leukotrienes. Among enzymes with PLA2 activity are the wellcharacterized extracellular Group I, II and III secretory PLA2s (sPLA2s) of low molecular weight and the more recently described Group IV intracellular cytosolic PLA2s of high molecular weight. Several *To whom correspondence should be addressed. Abbreviations: BPB, 4-bromophenacyl bromide; BSA, bovine serum albumin; DRG, dorsal root ganglion; ECL, enhanced chemoluminescence; EDTA, ethylenediaminetetra-acetate; EGTA, ethyleneglycolbis(aminoethyl ether)tetra-acetate; HEPES, N-2-hydroxyethylpiperazine-N 0 -2-ethanesulphonic acid; OOPC, oleyloxyethyl phosphorylcholine; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PLA2, phospholipase A2; PLA2-170, phospholipase A2 of 170,000 mol. wt; sPLA2, secretory phospholipase A2.

forms of PLA2 enzymes have been characterized in the mammalian CNS, 6,10,13,14,17,18,25,27 and PLA2 activity or immunoreactivity has been identified in various peripheral nerves, 3,15,16 where it could play a role in nerve membrane maintenance and repair, 28 as well as in nerve degeneration events. 15 Some studies also support a role of these enzymes in nerve regeneration. Nakamura 11,12 showed that inhibitors of PLA2 activity can attenuate the antidepressantinduced regeneration of brain noradrenergic axons, while we have previously demonstrated a PLA2 sensitivity in the outgrowth of axons of the integrated frog sciatic nerve. 3 In addition, inhibition of PLA2 activity has been reported to impair neurite outgrowth from NG 108-15 cells. 19 The mechanisms behind these effects and the type(s) of PLA2 involved are still unknown. In the present report, we have used a preparation of adult mouse dorsal root ganglia (DRGs) with short segments of the sciatic nerve, cultured in an extracellular matrix material (Matrigel) which provides support for the axons during outgrowth. 20 This model system has the benefits of a maintained interaction between neurons and the various surrounding cell types in the ganglion and proximal sciatic nerve, at the same time that the outgrowth of solitary axons can be readily studied. The purpose of the study was to investigate the involvement of

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PLA2 activity in the regenerative processes of the mouse sciatic nerve with attention to its role at the level of individual axons. EXPERIMENTAL PROCEDURES

Materials Oleyloxyethyl phosphorylcholine (OOPC) was purchased from Biomol (U.K.) and 4-bromophenacyl bromide (BPB) from Sigma (St Louis, MO, U.S.A.). Matrigel was purchased from Collaborative Research (U.S.A.). l[ 4,5-3H]Leucine, [methyl- 3H]thymidine, Hybond-C extra nitrocellulose membranes, enhanced chemoluminescence (ECL) western blotting detection reagents and Hyperfilm ECL were purchased from Amersham (U.K.). Protein AAgarose was from Santa Cruz and porcine pancreatic PLA2 from ICN (both U.S.A.). Antiserum against Group I porcine pancreatic sPLA2 (rabbit, whole serum) was obtained from Upstate Biotechnology (U.S.A.). Peroxidase-conjugated antibodies (swine anti-rabbit) were from Dako (Denmark). Phosphatidylcholine (PC;, 1-stearoyl-2[1- 14C]arachidonyl) and phosphatidylethanolamine (PE; 1acyl-2-[1- 14C]arachidonyl) were purchased from Amersham (U.K.). Tissue-Tek 䉸 OCT Compound was from Miles (U.S.A.) and glass slides from Menzel-Gla¨ser (Germany). Mountex was purchased from Histolab (Sweden) and Glycergel 䉸 from Dako (Denmark). Axonal outgrowth in Matrigel Long-term effects of phospholipase A2 inhibitors. Young adult male NMRI mice (B&K, Sweden), aged four to five weeks (25–30 g), were intraperitoneally anaesthetized with mebumal (70 mg/kg body weight) and killed by exsanguination. Short lengths of the sciatic nerves, with their attached ganglia and roots, were removed by dissection and mounted in a small volume (⬃3 ml) of Matrigel, an extracellular matrix material. Preparations were cultured in RPMI 1640, supplemented with antibiotics (100 U/ml penicillin, 100 U/ml streptomycin and 250 ng/ml amphoretoricin B), and kept in a humidified atmosphere, saturated with 93.5% O2/6.5% CO2, at 36⬚C. When the experimental substance was dissolved in dimethylsulphoxide, an equal concentration (⬍0.2%) of the solvent was added to controls. A concentration of up to 2% of dimethylsulphoxide has no outgrowth effects per se (unpublished results from this laboratory). The outgrowth of axons from the sciatic nerve stump was evaluated by use of an eye-piece scale of an inverse microscope with phase contrast and dark-field optics. Experiments and controls were always performed on paired DRG preparations. The number of animals used was 113, and all possible efforts to reduce animal suffering were made. A compartmental method was used to investigate the site of the effect exerted by the PLA2 inhibitors. The ganglia, with dorsal and ventral roots, were separated from the sciatic nerve stump by a silicon grease barrier and BPB (10 mM) or OOPC (15 mM) subsequently added to the nerve compartment in the experimental situation. Short-term effects of phospholipase A2 inhibitors: timelapse recording of axonal growth. The local effect of PLA2 inhibitors on outgrowing axons was further investigated using time-lapse recording. DRGs were cultured, as above but without drugs, for 72 h, after which they were moved to a gas-tight incubation chamber in an inverse microscope with phase contrast and dark-field optics, equipped with a resistor-heated aluminium plate. Axonal and growth cone movements were monitored with a video camera (Sony, Digital Hyper Had) and recorded at a speed of 1/56 of normal on a video recorder (Sony, Time-Lapse 168). After a stabilizing period of 45 min, during which

basal axonal growth and behaviour were monitored, gentle additions of pre-warmed culture medium with or without the inclusion of BPB (at a final concentration of 5 and 10 mM) or OOPC (10 and 15 mM) were made and axonal outgrowth continuously recorded. At the end of experiments, the video tape was played back and analysed at normal speed. When reversibility of PLA2 inhibition was studied, the medium was removed after 1.5 h exposure, cultures washed, and axonal outgrowth and growth cone motility were evaluated after another 24-h incubation. Protein synthesis Ganglionic protein synthesis was evaluated by measuring the amount of [ 3H]leucine (1.85 MBq/ml) incorporated during the last 4 h of the culture period. Unincorporated amino acid was removed using 10% trichloroacetic acid for 24 h and the ganglia were dissolved in a tissue solubilizer (Soluen, Packard, U.S.A.) after a thorough rinse in trichloroacetic acid. The amount of radioactivity incorporated was then assayed by liquid scintillation counting. Cell proliferation In order to assess the effect of different PLA2 inhibitors on the proliferation of supportive cells, 4-mm pieces of sciatic nerve, cultured for 48 h with either BPB or OOPC, were incubated with [methyl- 3H]thymidine (1.85 MBq/ml) for an additional period of 2 h. The nerve segments were then prepared for scintillation counting as above (“Protein synthesis”). Assay for phospholipase A2 activity Radioactively labelled PC (2.00 GBq/mmol) or PE (1.96 GBq/mmol) were dried under N2 gas, resuspended in assay buffer (80 mM KCl, 10 mM HEPES, 1 mM EDTA, pH 8.5), with addition of 10% glycerol, 20 mg/ml aprotinin and 20 mg/ml leupeptin, and sonicated with ultrasound. The 100,000 × g supernatants of homogenized DRG preparations, preincubated for 1 h with different concentrations of BPB (5, 10, 20 mM) with equal amounts of dimethylsulphoxide present in the controls, or samples from immunoprecipitation (see below), were incubated in the presence of a PC or PE substrate (200 pmol), 1.6 mmol CaCl2 and 0.19 mg/ml bovine serum albumin (BSA; fatty acid free) in assay buffer to a total volume of 525 ml, at 37⬚C for 30 min. The reaction was terminated by the addition of 1.5 ml of chloroform/methanol/HCl (2:1:0.01, by volume) including 0.05% dithioerythritol and unlabelled phospholipids as carrier (0.1 mg/ml). After centrifugation, the lipid phase was loaded onto a silic acid column equilibrated with chloroform, and the free fatty acid fraction (A) eluted with 1 ml chloroform and the phospholipid fraction (B) with 2.1 ml methanol. The amount of radioactivity in the different fractions was assayed by liquid scintillation counting, and the PLA2 activity was calculated as the ratio between counts per minute values for A and A ⫹ B [A/(A ⫹ B)], as a mean of duplicate samples. The arachidonic acid release was linear with respect to time over the period of 0– 30 min and with respect to protein concentration in the range of 0–30 mg. In inhibition assays, 20 mg cytosolic fraction protein was used. The hydrolytic activity in the control situation, when using PC as substrate, was very low (⬍1%) compared to PE (⬃6.5%), and PE was therefore used regularly in inhibition studies and when assaying the PLA2 activity in anti-sPLA2 precipitated samples (see below). Immunoprecipitation Immunoprecipitation was performed according to the method described by Volonte´ et al., 23 with minor modifications. DRGs with attached nerve stumps were homogenized

Phospholipase A2 in axonal outgrowth

in a lysis buffer (50 mM Tris, 10% glycerol, 1% Triton X-100, 150 mM NaCl, 0.1 mM NaF, 1 mM Na3VO4, 10 mM EGTA, 2 mM phenylmethylsulphonyl fluoride, 1 mg/ml leupeptin, 200 KIU/ml aprotinin, 5 mM ZnCl2). Homogenized samples were centrifuged (30,000 × g) for 15 min and the supernatants were then incubated with Protein A-Agarose for 2 h and centrifuged (12,000 × g) for 20 min. The resulting supernatants were then incubated with either primary antiserum against sPLA2, non-immune rabbit whole serum or lysis buffer as a control, for 2 h. Protein A-Agarose, pre-blocked in 5% BSA/phosphate-buffered saline (PBS) and washed five times in lysis buffer, was added to the samples, which were then incubated for an additional 24 h and the immune complex precipitated by centrifugation as before. The precipitate was resuspended and thereafter centrifuged with six successive changes of washing buffer (20 mM Tris, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol). All incubations, centrifugations and washing steps were performed at 4⬚C. After a final centrifugation (12,000 × g), the precipitates were suspended in PLA2 assay buffer.

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were also performed. Following a wash in PBS for 5 h with successive exchanges of buffer, the cultures were incubated overnight with a peroxidase-conjugated secondary antibody (1:25 in 2% BSA/PBS). All incubation steps were carried out at 4⬚C. After a 5-h wash in PBS as above, preparations were developed in diaminobenzidine/(NH 4)2Ni(SO4)2 (2.5 mg/ml) and mounted in Glycergel 䉸. Statistics Values of axonal outgrowth were calculated and displayed as a ratio between the mean outgrowth length of the paired experiment and control preparations. Statistical significance of values of axonal outgrowth, protein synthesis and cell proliferation was tested with Student’s paired t-test. Values of PLA2 activity were calculated and displayed as a ratio between the mean PLA2 activity of experiments and controls. These data were subjected to ANOVA followed by Dunnett’s test for evaluation of statistical significance. All values are given as mean ^ S.E.M. Differences were considered significant if P ⬍ 0.05.

Western blotting and immunodetection Samples of DRGs, brain or pancreas were homogenized in sodium dodecyl sulphate-containing sample buffer. Purified pancreatic PLA2 was likewise dissolved and the samples boiled and separated by discontinuous sodium dodecyl sulphate–polyacrylamide gel electrophoresis (5– 17%) according to Laemmli, 8 under reducing conditions. Separated proteins were electroblotted to a Hybond-C extra nitrocellulose membrane. Membranes were blocked in 5% dry milk in PBS (pH 7.2) for 1 h at room temperature and incubated with a primary antiserum against sPLA2 (1:500 in 2% BSA/PBS) overnight at 4⬚C. After a rinse in PBS, the membranes were incubated with peroxidaseconjugated secondary antibody (1:10 000 in 2% BSA/ PBS ⫹ 0.1% Tween-20) for 1 h at room temperature. Controls with non-immune whole serum or exclusion of the primary antiserum were also performed. After extensive washing, the immunoreactive protein bands were detected with ECL and visualized on Hyperfilm ECL. Immunohistochemistry Tissue sections. Explants of DRGs, either freshly dissected or after 24 h regeneration in vitro, as well as pancreas tissue, were fixed in a solution of 4% paraformaldehyde in PBS for 4 h and cryoprotected with 20% sucrose/ PBS for 12 h, both at 4⬚C. The tissue was then embedded in Tissue-Tek 䉸, cut into 8-mm sections with a cryotome and dried onto poly-l-lysine-pretreated glass slides. Endogenous peroxidase activity was quenched by treatment with 1% H2O2 in purified water (Millipore, Milli-Q) for 30 min. Tissue sections were subsequently blocked in 2% BSA/PBS for 1 h at room temperature and incubated with primary anti-sPLA2 antiserum (1:500 in 2% BSA/PBS) overnight at 4⬚C. Controls with non-immune whole serum or exclusion of the primary antiserum were also performed. Immunoreactive sites were then identified with a peroxidase-conjugated secondary antibody (1:25 in 2% BSA/ PBS) for 1 h at 37⬚C, treated with diaminobenzidine/ (NH4)2Ni(SO4)2 (2.5 mg/ml), and subsequently dehydrated in a series of alcohol and mounted in Mountex. Whole mounts. Following normal culturing procedures, DRG preparations were fixed as above (“Tissue sections”). After a rinse in PBS, the preparations were blocked and permeabilized by incubation overnight in 5% dried milk/ 1% Triton X-100 in PBS. Preparations were then washed in PBS and incubated overnight with primary anti-sPLA2 antiserum (1:250 in 2% BSA/PBS). Controls with nonimmune whole serum or exclusion of the primary antiserum

RESULTS

Axonal outgrowth and the effects of phospholipase A2 inhibitors Long-term effects of phospholipase A2 inhibitors. The outgrowth length was determined as an average of three measurements, one at each of the outer edges of the outgrowth bulk and one in its centre. Since there was no difference in the effects of the PLA2 inhibitors on the axonal outgrowth from the dorsal root as compared to the sciatic nerve stump, nor from the lumbar fourth DRG preparation as compared to the fifth, only data from the sciatic nerve stump of the fourth DRG preparation are presented. In the control situation, numerous axons extended into the Matrigel from the cut ends of both the sciatic nerve stump and the dorsal root within 24 h. Although the axons varied in both length and direction of growth, a coherent bulk of axons was evident after 48 h culture, the time-point chosen for evaluation. BPB and OOPC, commonly used inhibitors of PLA2, both affected the outgrowth of axons from the cut end of the sciatic nerve stump in a dosedependent manner. An approximately 50% reduction of outgrowth distance was found with 10 mM BPB and 15 mM OOPC, respectively, after 48 h culture (Figs 1, upper panel; 2). The density, i.e. the number of outgrowing axons, was difficult to estimate due to both the three-dimensional growth pattern of axons within the Matrigel and the extensive axonal branching after entry into the gel. However, a general impression was that the density of axons was reduced by both inhibitors used (Fig. 2). In order to differentiate between central (ganglionic) as opposed to local (axonal) inhibitory effects, a compartmental culturing method was used. When applied only in the nerve-containing compartment, both BPB (10 mM) and OOPC (15 mM) caused a reduction in outgrowth from the sciatic nerve

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Fig. 1. (Upper panel) The effect of PLA2 inhibitors (BPB at 5, 10 or 20 mM; OOPC at 10, 15 or 20 mM) on three-dimensional axonal outgrowth over 48 h in vitro from the sciatic nerve stump of the lumbar fourth DRG preparation. Values are expressed as the ratio between experiment and untreated control, and represent mean ^ S.E.M. (n ˆ 6). Asterisk denotes a significant difference between experiment and control (Student’s paired ttest). (Lower panel) The effect of BPB (5, 10, 20 mM) on the PLA2 activity in cell-free tissue homogenates of DRG preparations. Aliquots of the cytosolic fraction (20 mg protein) were assayed for PLA2 activity with PE (1-acyl-2-[1- 14C]arachidonyl). Values are expressed as the ratio between experiment and control, and represent mean ^ S.E.M. (n ˆ 6). Asterisk denotes a significant difference between the mean values of the experiment and the control (Dunnett’s test). Absolute values of PLA2 activity ranged from 3% to 6.5% hydrolysis.

stump [BPB: 0.38 ^ 0.04 (ratio: experiment/ control ^ S.E.M.), P ⬍ 0.001, n ˆ 6; OOPC: 0.44 ^ 0.06, P ⬍ 0.01, n ˆ 6] similar to that when the whole preparation was exposed to the inhibitors

Fig. 2. Three-dimensional outgrowth of naked axons in Matrigel over 48 h in vitro from the sciatic nerve stump of the fourth lumbar DRG preparation, in the presence of either OOPC (15 mM; a), BPB (10 mM; b) or under control conditions (c). The outgrowth distance in the experimental situation was reduced to about half of that of controls. Pictures are representative of six separate experiments. Scale bars ˆ 250 mm.

(Fig. 1, upper panel). In the compartmental preparation, the outgrowth from the dorsal root was unaffected (BPB: 0.84 ^ 0.11, P ⬎ 0.05, n ˆ 6; OOPC:

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Table 1. Effect of 4-bromophenacyl bromide and oleyloxyethyl phosphorylcholine on ganglionic protein synthesis and proliferation of non-neuronal cells of the sciatic nerve Compound

Exp. ^ S.E.M.

Control ^ S.E.M.

Exp./control

n

Ganglionic protein synthesis 10 mM BPB 15 mM OOPC

49,778 ^ 1696 48,179 ^ 5043

55,273 ^ 4612 52,033 ^ 4651

0.90 0.93

5 6

Non-neuronal cell proliferation 10 mm BPB 35,867 ^ 3729 15 mm OOPC 32,259 ^ 3347

35,528 ^ 5974 28,580 ^ 3092

1.01 1.13

6 6

1.06 ^ 0.13, P ⬎ 0.05, n ˆ 5), which shows that the barrier effectively prevented leakage of the drugs.

BPB-inhibitable PLA2 was already blocked at 10 mM of the drug (Fig. 1, lower panel).

Short-term effects of phospholipase A2 inhibitors. To further elucidate the local effect of PLA2 inhibition, the axonal growth was studied at the level of the newly formed axons and their growth cones, using time-lapse recording. Axonal growth in Matrigel shows a three-dimensional pattern which makes it difficult to focus and monitor growth cone movement. However, after 48 h culture, the axons start to leave the Matrigel and, after 72 h, a network of axons growing twodimensionally on the plastic has been formed. At this stage, axon formation and growth cone movement, with the continuous extension and retraction of filopodia, can readily be studied with phase contrast optics. Both BPB (5 and 10 mM) and OOPC (10 and 15 mM) caused the retraction of filopodia extensions and a reduction in growth cone motility within a few minutes. Axonal advancement ceased completely within 30– 60 min exposure to the inhibitor, and the growth cone structures were condensed in appearance (Fig. 3a, b). PLA2 inhibition also caused the retraction of some axons (approximately 30 mm/30 min; Fig. 3b, c). However, the effect of PLA2 inhibition was reversible in the sense that, after a change to fresh medium, general growth cone motility and axonal growth were restored.

Specificity of the secretory phospholipase A2 antiserum

Test for toxicity and specificity of the phospholipase A2 inhibitors Using 10 mM BPB or 15 mM OOPC for 48 h, i.e. experimental situations in which axonal outgrowth distances were reduced to approximately 50% of control, ganglionic protein synthesis over the last 4 h was unaffected. Likewise, this treatment did not affect the proliferation of non-neuronal support cells (P ⬎ 0.05, Table 1; mainly Schwann cells 26). The effect of BPB on the PE-directed PLA2 activity in cell-free tissue homogenates of DRGs was tested. In these experiments, BPB at 5 and 10 mM reduced the enzymatic activity to about 75% and 60% of control, respectively (Fig. 1, lower panel). A further doubling of the BPB concentration did not lead to any further reduction of enzymatic activity, suggesting that most

An sPLA2 antiserum was used to study, by immunohistochemistry, the localization and expression of this enzyme in regenerating DRG preparations. As a positive control, sPLA2 was demonstrated immunohistochemically in the secretory cells of pancreatic acini (not shown). The specificity of the antiserum was further tested on immunoblots, where it detected a protein with a molecular weight of ⬃170,000 (PLA2-170), seen as a double band in homogenates of DRGs and as a single band in the brain (Fig. 4, lanes 1 and 2). In contrast, the same antiserum specifically recognized the 14,000 mol. wt purified pancreatic sPLA2 (Fig. 4, lane 3), as well as a protein in the same molecular weight range in pancreas homogenates (not shown). Controls, with non-immune rabbit whole serum or exclusion of the primary antiserum, were all negative (Fig. 4, lanes 4 and 5). The presence of a high- rather than low-molecularweight immunoreactive protein on immunoblots of nervous tissue was unexpected and may raise doubts as to its PLA2 nature. However, the immunoprecipitation experiments with DRG proteins showed that the antigen possesses PLA2 activity. Unprecipitated material usually has a hydrolysation of around 5% (e.g., 600 c.p.m. in the fatty acid phase and 10,800 c.p.m. in the phospholipid phase). In precipitated material, there is a risk that antiserum binding to the precipitated PLA2 will block or interfere with the hydrolytic activity of the latter. Furthermore, one should not expect a complete recovery of an antigen by a precipitation procedure that includes several washing steps. In spite of this, when the sPLA2 antiserum was used to immunoprecipitate DRG material, the hydrolysation values in two independent experiments were 0.24% and 1.20%, i.e. ⬃5–25% of the total activity, respectively. The two control situations, non-immune serum and buffer, gave identical hydrolysation values, which amounted to 0.16% and 0.63% in the two independent experiments, respectively. Thus, the use of an sPLA2 antiserum precipitated

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Fig. 4. Immunoreactivity for the sPLA2 antiserum after electrophoresis and immunoblotting of a homogenate of pooled DRGs (20 mg protein, lane 1). Homogenates of brain (30 mg protein, lane 2) and purified pancreatic PLA2 (5 mg protein, lane 3) were also separated and immunoblotted using the same antiserum. A protein with an approximate mol. wt of 170,000 appeared as a double band in DRGs (1) and as a single band in the brain (2). A protein of ⬃ 15,000 mol. wt was visualized in lane 3. Controls, with rabbit non-immune whole serum or the exclusion of the primary antiserum, were both negative (lanes 4 and 5). Bars indicate the migration distance of standard proteins, whose weights are given to the right. The immunoblot is representative of five separate experiments. The band broadening in lane 3 is a result of the high (NH4)2SO4 concentration in the sample.

immunohistochemically in nerve cell bodies of DRGs, where it was up-regulated after 24 h culture (Fig. 5a, b). Immunoreactivity was unequivocally and strictly located to neurons, as seen in crosssections, where axons but not Schwann cells were immunoreactive (Fig. 5c), and in whole-mount staining of growing axons in Matrigel (Fig. 6). Controls, as above, were all negative.

DISCUSSION

Fig. 3. Time-lapse recorded two-dimensional axonal outgrowth after 72 h in vitro, from the lumbar DRG immediately before (a), 10 min (b) or 30 min (c) after the addition of BPB to a final concentration of 10 mM. The treatment strongly affected growth cones (arrows), and in many cases the most distal part of the axons also seemed to retract (approximately 30 mm/30 min, arrowheads). Pictures are representative of five separate experiments. Scale bars ˆ 50 mm.

50–90% more PLA2 activity than control precipitations, strongly suggesting that the antiserum recognized an enzyme with PLA2 activity. The fact that non-immune serum did not yield more activity than buffer alone further argues against a non-specific precipitation by serum components in general. Localization and up-regulation of phospholipase A2 of 170,000 molecular weight The

PLA2-170

protein

was

demonstrated

Effects of phospholipase A2 inhibition on axonal outgrowth In the present study, a recently developed in vitro model was used to study the role of PLA2 in regeneration of adult mammalian peripheral nerves. Axotomized mouse sciatic sensory axons were allowed to grow three-dimensionally into an extracellular matrix-based gel, Matrigel, where they can be observed directly. 4 Under these conditions, BPB, a PLA2 inhibitor known to selectively bind to a histidine residue in the active site of PLA2, 24 dosedependently reduced the axonal outgrowth in longterm experiments. Similar effects were seen when the site-specific PLA2 substrate analogue OOPC 9 was used. These findings are in line with our previous observations made on frog nerves. 3 However, in the integrated in vitro preparation used in the latter study, axons regenerate inside the sciatic nerve in contact with neighbouring Schwann cells. This results in a

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Fig. 6. Immunoreactivity for the sPLA2 antiserum of whole preparations of DRGs with nerve stumps, cultured in Matrigel. Axons growing three-dimensionally into the Matrigel were highly immunoreactive. Controls, with rabbit non-immune whole serum or the exclusion of the primary antiserum, were both negative. The direction of axon growth is down and the edge of the nerve stump can be seen at the top of the figure. The picture is representative of five separate experiments. Scale bar ˆ 50 mm.

Fig. 5. Localization of PLA2-170 in lumbar DRGs and sciatic nerve. Some nerve cell bodies (large arrows) and axons (small arrows) showed PLA2-170 immunoreactivity. Increased levels of the enzyme were detected after 24 h in vitro (b), compared to freshly dissected controls (a). Cross-sections of the sciatic nerve showed the localization of PLA2-170 in axons, but not in Schwann cells (c). Pictures are representative of five separate experiments. Scale bars ˆ 50 mm (a, b), 100 mm (c).

complex experimental situation, where a distinction between outgrowth effects of PLA2 inhibition indirectly via Schwann cells and effects on the axons themselves cannot easily be made. In the present preparation, where axons grow without immediate contact with non-neuronal cells, it is possible to study the role of axonal PLA2 in more detail. PLA2 inhibitors blocked the growth of such axons, even when only the growing part of the preparation was exposed, while Schwann cell proliferation was unaffected. These facts, together with the rapid and dramatic effect of PLA2 inhibition on growth cones and newly formed axons in the

short-term experiments, strongly suggest that axonal PLA2 activity has an important role in outgrowth mechanisms. It could be argued that the reduction in outgrowth distances was the result of toxic and non-specific effects of the PLA2 inhibitors. The possibility that general toxic effects were responsible seems less likely, since neither BPB nor OOPC caused any significant reductions in ganglionic protein synthesis and Schwann cell proliferation at concentrations that reduced axonal outgrowth by 50%. The reversible effects in the short-term studies on axonal outgrowth and growth cone activity seen when 10 mM BPB or 15 mM OOPC were used are further arguments for non-toxic effects. Moreover, since the preparation contains a PLA2 activity that was strongly inhibited at a non-toxic concentration of BPB (10 mM), the specificity of the inhibitor is supported. In addition to its effects on PLA2 activity, high concentrations (⬎35 mM) of BPB have been shown to inhibit assembly of microtubules. 5 While this is likely to interrupt axonal outgrowth, it has probably not occurred here, since an impaired microtubule assembly should reduce cell proliferation, which was not the case at the concentrations of BPB used. The results from the time-lapse experiments suggested that continuous PLA2 activity is essential for the axonal growth processes. This also gains support from our demonstration of PLA2-170 in the newly formed axons (see below) and is in line with the high levels of PLA2 activity in neuronal growth cones during development. 13,14 Interestingly, in the short-term experiments, axons appeared more sensitive to the lower concentrations of the drugs than would be expected from the long-term studies, where the preparations were exposed continuously to the drugs. It is possible that some kind of compensatory mechanism is triggered during the constant

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PLA2 inhibition, perhaps involving the activation of other enzyme systems or simply the synthesis of more PLA2 enzyme(s). A PLA2 involvement in the axonal turnover of membranes, which is supposed to be necessary for axonal outgrowth, 21 could explain the outgrowth effects of PLA2 inhibition. On the other hand, the hydrolytic products of the substrates, such as fusogenic lyso-phospholipids, which may facilitate the resealing of the ruptured membrane, 28 or arachidonic acid and/or its metabolites, should also be expected to play important roles in this respect. The loss of these products may therefore have contributed to the observed inhibition. However, in the present experimental system, the application of exogenous arachidonic acid did not relieve the outgrowth block by OOPC (Hornfelt M., unpublished observations). The clarification of the exact property of the PLA2 activity that is of importance for the regeneration must await further experimentation. A high-molecular-weight form of phospholipase A2 sPLA2 is known as a low-molecular-weight protein. It was therefore most unexpected that immunoblotting with an antiserum against sPLA2 visualized two protein bands of approximately 170,000 mol. wt (PLA2-170) in homogenates of DRGs and brain, with no detection of the lowmolecular-weight form. This could be due to nonspecific binding of the antiserum, but the lack of reaction with control non-immune serum and the detection by the sPLA2 type I antiserum of lowmolecular-weight pancreatic sPLA2 in purified form, as well as a low-molecular-weight protein in a pancreatic tissue homogenate, make this assumption most unlikely. Furthermore, the results from PLA2 activity assays on immunoprecipitates show that the sPLA2 antiserum specifically recognizes proteins with PLA2 activity. It should be kept in mind, though, that the co-existence of multiple forms of PLA2 enzymes in the preparation is likely, since only up to 25% of the total PLA2 activity was precipitable with the antiserum and only about 40% of the total enzyme activity in cell-free homogenates was BPB sensitive. Whether or not a high-molecularweight variant of PLA2 plays a role in axonal growth therefore remains an open question. However, in this context it is interesting that Negre-Aminou et al. 13 recently detected a protein doublet of 160,000 and 180,000 mol. wts, with an antiserum against 85,000 mol. wt cytosolic PLA2 in a growth cone particle fraction of fetal rat brain. Furthermore, a PLA2

activity corresponding to a protein of ⬃180,000 mol. wt, with characteristics of both the secretory and cytosolic PLA2 forms, was recently found in the temporal cortex of the human brain, 18 and in a recent study by Kim et al. 7 a PLA2 activity compatible with a molecular mass of 170,000 was found in both membrane and cytosolic fractions of the cerebral cortex. These studies thus provide a precedent for the existence in nervous tissue of a PLA2 in the 160,000–180,000 mol. wt range. Axonal localization of phospholipase A2 of 170,000 molecular weight By the use of immunohistochemistry, PLA2-170 was demonstrated in nerve cell bodies, where it was up-regulated as a response to the injury. This may reflect the need for PLA2-170 protein in the growing axons, which were also shown to contain the protein. In contrast, no apparent immunoreactivity was observed in Schwann cells of the nerve trunk. Paul and Gregson 15 showed immunoreactivity in Schwann cells, but not in axons, during Wallerian degeneration, using an antiserum against Group II sPLA2. This suggests the existence of several PLA2 enzyme forms in the sciatic nerve, with strict but different localizations and perhaps functions. In the present study, PLA2 inhibition reduced the outgrowth of naked axons. This is noteworthy since these axons lack structural association with Schwann cells, and therefore the Group II sPLA2. 15 However, the strict axonal localization, the upregulation of PLA2-170 levels in nerve cell bodies during neurite outgrowth and the fact that a majority of the axons growing in the Matrigel were immunoreactive are good reasons to investigate whether PLA2-170 plays a role in axonal growth. CONCLUSION

This study shows the importance of continuous PLA2 activity for growth cone motility and axonal outgrowth in the mammalian peripheral nerve, and suggests the involvement of an axonally localized enzyme. The results further indicate the existence in nervous tissue of a high-molecular-weight PLA2 (PLA2-170). Its possible involvement in axonal growth warrants attention in future experiments.

Acknowledgements—The present study was supported by grants from the Swedish Natural Science Research Councils and the Royal Swedish Academy of Sciences. We thank Ms Inger Antonsson and Ms Judith Lindskog for excellent technical assistance.

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