Brain Research, 216 (1981) 361-374 © Elsevier/North-Holland Biomedical Press
P L A S M I N O G E N ACTIVATOR IN T H E R O D E N T BRAIN
HERMONA SOREQ and RUTH MISKIN Departments of Neurobiology and Biochemistry, The Weizmann Institute of Science, Rehovot (Israel)
(Accepted December 4th, 1980) Key words: plasminogen activator (PA) - - proteolysis - - brain development - - autoradiography - -
neuronal PA - - epithelial PA -- X-irradiation.
SUMMARY The cellular origin(s), the biochemical properties and the developmental pattern of the protease plasminogen activator (PA) were investigated in the rodent brain. PA activity was localized in frozen brain sections by a novel autoradiographic technique. PA levels and electrophoretic mobility were determined in homogenates plepared from major regions of the developing and the mature brain, and both the localization and the specific activity of the enzyme were examined in X-irradiated brain regions. PA activity was shown to be correlated with cell bodies in neuronal-enriched regions and also with endothelial, meningeal and ependymal layers. PA levels increased in a transient manner and at different rates and time periods in the various brain regions that were analyzed. PA in neuronal, but not in epithelial cell layers was affected by X-irradiation and one of the brain PA species had a similar molecular weight to that of neuroblastoma cells. Our findings suggest that in the brain PA is produced by neurons and by epithelial cells, and that it may have additional functions to that of thrombolysis both in the developing and the mature brain.
INTRODUCTION Plasminogen activators are highly specific serine proteases which convert the circulating zymogen plasminogen into plasmin, an active fibrinolytic protease s. The plasminogen activation system has for a long time been considered part of the mechanism controlling thrombolysis and the maintenance of blood fluidity. High levels of enzyme have also been associated with cell transformation and neoplasia. Recently, high rates of PA synthesis have been correlated with a variety of normal physiological processes, the common elements in which are cell migration and
362 rearrangement of tissues. It has therefore been suggested that the activation of plasminogen may provide a general physiological mechanism for extracellular proteolysis under normal and malignant conditions (for review see ref. 21). In the human brain, PA activity has been reported to be present in endothelial cells of small blood vessels, in the meninges, in the choroid plexus and in the cerebrospinal fluid 26. The PA level was also found to increase with development of the brain in human infants ~6. In the rabbit, PA levels in homogenates of various brain regions were found to be high compared with other organs 11. High levels of PA have also been reported in human 28 and mouse 12 neuroblastoma cell lines and in human glioblastomas, but PA was reported to be absent from primary cultures of human glial cells 27. The high levels of PA in the brain were attributed to the regulation of thrombolysis, whereas in brain tumors and cell lines of neuronal origin it was explained by the neoplastic nature of these sources. In order to reveal the possible involvement of PA in processes such as migration and differentiation of neurons, we initiated a characterization of this enzyme in the rodent brain. METHODS
Preparation of brain homogenate samples C57B6J mice and Wistar rats (from the Weizmann Institute Breeding Center) were killed by cervical dislocation, the brain was immediately removed and the various brain regions were dissected on ice. Tissue was rinsed twice in ice-cold PBS, weighed and homogenized (1 ml per 100 mg wet weight) in 0.5 ~ Triton X-100 in a glass-Teflon homogenizer driven by a Heidolph motor at 1500 rpm. Homogenates were stored at --20 °C, and PA analysis performed within one week of sample preparation. Protein concentration was determined according to Lowry et al. a6.
Analysis of PA activity In all PA assays used we measured the fibrinolytic activity of plasmin which was converted from plasminogen by PA. Plasmin activity was determined in homogenate samples by its ability to release -labeled fibrin degradation products from [125I]labeled fibrin-coated microwells. Iodination offibrin. Sixty/~l of 0.5 M phosphate buffer at pH 7.5 and 2.5 mCi of Na125I (IMS-30, The Radioehemical Centre, Amersham) were added to 0.5 ml cold solution of 10 mg/ml fibrinogen, pmified as described 23. lodination was started by the addition of 60 #1 of 10 mg/ml solution of chloramine T. Incubation was at 0 °C for 15 min. The reaction was stopped by the addition of 120 #1 of 10 mg/ml sodium bisulfite. Potassium iodide was then added (120 /zl of 17/o solution) and the mixture was extensively dialyzed against PBS at 4 °C. All solulions used were prepared immediately prior to addition in 50 mM phosphate buffer, pH 7.5. The extent of labeling was in the range of 2-3 × 105 cpm//~g fibrinogen in different preparations. Preparation offibrin plates. 250/d of a fibrinogen solution, containing 27 ,ug of non-radioactive fibrinogen and 1.5-3 × 105 cpm of [125I]fibrinogen were added to each well of a multi-wells plate (Linbro or Costar), and the plates were dried at 37 °C for
363 several days. Conversion of fibrinogen to fibrin was done prior to the assay by incubation at 37 °C for 2 h in a humidified atmosphere containing 5 9/oCO2 in air, with 0.5 ml of 0.5 % fetal calf serum in minimal essential medium for each well. The wells were then washed twice with 1 ml 0.1 M Tris.HC1 buffer, pH 8.1 (reaction buffer). Fibrin plate assay. Each reaction well contained 250 #1 of reaction buffer, 62/,g bovine serum albumin (necessary for obtaining linear kinetics), 20 ~g of human plasminogen (purified from human plasma 9 and treated with di-isopropylfluorophosphate 17) and 2-10 #1 of a tested sample. Each assay also included samples of trypsin, to determine the maximal releasable counts (usually about 70 % of the initial input into freshly coated wells) and urokinase (UK) (1-5 Ploug milliunits, Leo Pharmaceutical Products, Denmark). The reaction was initiated by incubation at 37 °C as described above. At appropriate times (usually 1.5 and 3 h) 50/zl aliquots were removed from each well and counted for soluble [125I]fibrin degradation products in a Packard gamma-spectrophotometer. Each sample was assayed at 3 dilutions containing protein in the range of 0.5-15/,g, and only results which showed linearity as a function of protein concentration were considered. Results were expressed as units of UK per mg of sample protein. Control assays from which plasminogen was omitted did not show any fibrinolytic reaction, indicating an absolute plasminogen dependence of the activity tested. Overlay assay of PA in frozen sections. A mouse or rat brain was mounted in a cryostat chuck and sectioned (15 #m) at --15 °C. Frontal sections throughout the brain were collected and mounted on glass slides. The sections could be kept at --20 °C for about one month with no detectable loss of activity. The overlay assay was done as described17. A reaction mixture contained 200 #1 of triple-strength Eagle's medium, 200 #1 of 2.5 % purified Agar (Difco, further purified according to Dulbecco and Vogtl°), 1 unit thrombin (Parke-Davis) 200/,1 of 10 mg/ml solution of fibrin, and 20 big human plasminogen. This volume was used for covering 3 sections. Incubation was at 37 °C, as described above, for 4-7 h. Autoradiography of PA activity. In order to localize PA activity in specific cell layers of brain sections, an autoradiographic modification of the fibrin overlay technique was developed 19. For autoradiography we included iodinated fibrin in the plasminogen-containing Agar overlays that were mounted on the analyzed sections. The adsorption of [125I]fibrin degradation products to cells was visualized as silver grains, either by direct film autoradiography or by emulsion autoradiography followed by light microscopy. This was carried out after the removal of the Coomassie brilliant blue-stained Agar overlay. It was shown that adsorption of [125I]labeled fibrin degradation products appeared only when plasminogen was present in the overlay. Molecular weight determination of active PA was performed according to Granelli-Piperno and ReichlL Briefly, crude samples were electrophoretically analyzed on SDS-polyacrylamide gels. Gels were then washed in 2.5 9/oTriton X-100 and mounted on Agar underlays containing fibrin and plasminogen. After 16 h incubation at 37 °C underlays were stained with Amido black. X-ray irradiation of rat brain. Rats were raised 8 per litter and irradiated as described by Altman and Anderson3. The left-hand side of the brain was shielded with
364 a 5 mm thick lead cover, which completely blocks irradiation (J. Ben-Barak, personal communication). The irradiated animals received 3 doses of 200 rad delivered on postnatal days 2, 3 and 5 followed by 4 doses of 150 rad on days 7, 9, 11 and 13. During sample preparation, the irradiated cerebellum and cerebral cortex were dissected on ice and separated from the control non-irradiated regions. The decrease in weight was 5- to 6-fold in irradiated as compared to control cerebellum and 20 °/o to 30 o~ in the cerebral cortex. RES U LTS Subregional localization of PA activity in rodent brain sections In order to localize PA activities in specific areas within the analyzed brain regions, we applied the fibrin overlay analysis to frozen sections prepared from PBSperfused mouse and rat brain. Prominent PA activity, detected as clear unstained lesions in the stained opaque fibrin overlays, was observed in certain regions of such sections, while the fibrin overlay covering other areas remained stained. The histochemical analysis of frozen sections from the brain of a 17-day-old mouse revealed the secretion offibrinolytic activity around the cerebral meninges and from regions in which the density of cell bodies is high. In lateral sagittal sections that cross the various layers of the hippocampus clear activity was observed around the dentate gyrus, close to the layer in which cell bodies of granular neurons are concentrated. Considerable fibrinolysis was also detected in such sections around the hippocampus and the lateral ventricle. The fibrinolytic activity of these sections was absolutely dependent on the presence of plasminogen (Fig. 1). PA localization was further resolved by autoradiography in sections of rat brain. The autoradiography revealed patterns of PA localization that were similar to those obtained by the non-radioactive overlays, but with increased resolution. Intense labeling was observed in the ependymal layer that surrounds the fourth ventricle, in the cells of the choroid plexus and in the granular layer of the cerebellar lobule I. Labeling was also detected in the meningeal cell layer and around blood vessels (Fig. 2). Significant labeling was also detected in the lateral ventricle, both in the choroid plexus and in the ependymal layer. Cytochemical staining of an adjacent section enabled to discriminate between myelin-containing regions and non-myelinated neuropil in the cerebral cell layers. Within the area surrounding the ventricle, cells were radioactively labeled and the labeling could be attributed to cell bodies, rather than to myelinated structures (Fig. 3). In the cerebellum, prominent labeling was clearly demonstrated in the granular layer, but not in the molecular layer. Intense labeling was also detected in the superficial layer of the white matter. This layer also displayed prominent myelin staining in histological staining of an adjacent section (Fig. 4). Variations in PA levels in regions o] developing mouse brain PA levels were determined in homogenates of whole developing mouse brain. The enzyme level increased about 2-3-fold from 14 days of gestation until birth and gradually declined after birth. Since it is well established that the various regions of the
Fig. 1. Overlay analysis of PA activity in mouse brain sections. Sagittal sections from a perfused brain of a 17-day-old mouse were either stained with cresyl violet (left-hand side) or incubated under fibrin overlay (right-hand side) with or without human plasminogen. Incubation was for 18 h at 37 °C. Overlaid sections were stained with Coomassie brilliant blue. Negative photographs are presented, in which dark zones represent PA fibrinolytic activity. Plasminogen-dependent diffused activity is seen around the lateral ventricle, the meningeal cell layer, the corpus callosum and the dentate gyrus. Magnification × 3.
mammalian brain differ in their rate and timing of development, as well as in cell-type composition 14, PA was characterized in homogenates of 4 major regions of the mouse brain. Different patterns of PA-specific activity were observed in various regions of the brain. In the cerebral cortex PA activity remained at a consistently low level. In the cerebellum and brain stem, PA activity was relatively high at birth and declined during the third postnatal week. In the developing thalamus PA level remained low for the first two weeks after birth, increased during the third week, decreased and reached a plateau level at 20 days (Fig. 5). In the brain of adult mice (120 days) the PA level was 2-3-fold higher in homogenates from the thalamus than in homogenates from the cerebral and cerebellar cortices or in homogenates from the braio stem. The low activity regions comprise the main part (over 75 ~ ) of the mature brain and these therefore most heavily influence the PA level in whole brain homogenates. This level (around 1 U K U / m g protein) is close to that of the highly active mouse organs, lung and kidney, and is considerably higher than that of the spleen, liver and smooth or skeletal muscle 25.
The effect of X-ray irradiation on PA activity Repeated X-ray irradiation during the first two postnatal weeks has been
Fig. 2. PA autoradiography in sections from rat cerebellum and brain stem. A: note prominent label, marked by arrows, in vermian lobule 1, in choroid plexus cells and in the ependymal layer that surrounds the fourth ventricle. Label is relatively absent from medial vestibular nucleus. B: note label in meningeal cells and in blood vessel. Some label can be observed in the corticospinal tract. Magnification ×50. Background gray stain originates from Coomassie staining of overlayed section.
Fig. 3. PA autoradiography in the lateral ventricle of rat brain. A: autoradiogram of lateral ventricle region. Note prominent labeling, marked by arrows, in choroid plexus cells, in ependymal layer and in surrounding cell bodies. Background gray stain represents total proteins. B: Luxol fast blue staining with red counterstain of an adjacent section 4. Note dark myelinated regions that surround the ventricle. Magnification x 50.
Fig. 4. PA autoradiography in the rat cerebellum. A: autoradiogram. Note prominent labeling marked by arrows in granular layer of parafloccular fissure and in a thin layer in the superficial layer of the white matter. Some activity can be observed in ependymal cells and deep in the white matter. Background gray stain originates from Coomassie brilliant blue staining of overlayed section. B: Luxol fast blue staining of an adjacent section. Note concentrated (dark) cell bodies in granular layer and dense myelin stain in the superficial layer of the white matter, in parallel to PA autoradiogram. Magnification x 50.
PA Levels in Developing Mouse Brain Regions i
-6 T e e ~
oo ~ ; . ~ - 2
Age, days Postnatal Fig. 5. PA levels in developing mouse brain regions. Preparation of extracts and PA assay were carried out under standard conditions (see Methods section). Data are presented as urokinase Ploug units per mg protein ( O - - O ) and as total protein content per region (- - -). Each point represents one experiment, in which activity was determined in an homogenate prepared from 5-10 pooled regions.
Effect of X-ray Irradiation on PA Activity in Rat Cortex and Cerebellum
\ \ (B' i
I_ _ •
12 35 0 I0 20 30 Age, days Postnatal
Fig. 6. Effect of X-ray irradiation on PA activity in rat cortex and cerebellum. Data are presented as urokinase Ploug units per mg protein (A, B) and as per cent of activity in the counterpart control region (C). Each point represents one experiment, in which activity was determined in homogenates prepared from 1-3 pooled cerebellum or cortex samples from irradiated i ~ , right-hand side) and control (E3, left-hand side) regions.
369 reported to selectively abolish most of the proliferating basket, steUate and granular neurons in the developing rat cerebellum, while its effect on the cerebral cortex has been found to be minimal2,3. We employed irradiation treatment to examine whether cerebral and cerebeUar cell-types that produce PA are affected by the irradiation. The irradiation effect was examined both in homogenates and in frozen sections. Generally, the overall effect of irradiation on PA levels in homogenates was stimulatory rather than inhibitory in both regions (Fig. 6). About a 2-fold increase in PA activity was observed in cerebellar homogenates of 4-day postnatal animals, after 2 doses of irradiation. The elevation was maximal at 12 days in cerebellar homogenates, following 7 irradiation doses. The stimulation then declined and reached a plateau level of about 125 ~ of control level at 35 days, two weeks after irradiation was terminated. In homogenates of the irradiated cerebral cortex, a small stimulatory effect was observed in homogenates after 4 days. PA levels then declined and reached a plateau showing a slight inhibition, compared to the non-irradiated control activity, at 25 days after birth. It thus appears that the effect of irradiation on PA levels in the rat cerebellum was more pronounced, lasted longer and was irreversible to a higher extent than in the cerebral cortex. Direct film autoradiograms of whole cerebellar sections revealed intense labeling in the vermian region of the rat cerebellum, including the medial lemniscus and dorsal vermian lobule (Fig. 7). Light microscopy autoradiograms showed PA activity in both the control and the X-ray-irradiated regions to be localized in specific cell layers. The cerebellar meninges displayed intense labeling in both the control and the X-ray irradiated region, indicating that the irradiation treatment had no effect on the activity of meningeal cells (Fig. 7). Within the floccular and the parafloccular fissures, labeling was observed in the granular layer but not in the molecular layer. Intense labeling was also observed within the X-irradiated region in the remnants of the granular layer, which was greatly reduced in size by the irradiation. In the superficial layer of the white matter of the non-irradiated side in the cerebellum, a thin layer of dense silver grains could be detected. The labeling as well as the intense myelin staining of this layer was completely eliminated in the X-ray-irradiated region. Molecular weight distribution of PA activity in mouse brain homogenates Molecular weight heterogeneity of plasminogen activators has been observed in samples of biological fluids and conditioned medium from cultured cells12,~9. In order to determine the molecular species of PA in mature and developing brain regions, we analyzed homogenate samples and compared them with extracts from cell lines of neuronal and glial origin and with the PA activity in rodent urine. In homogenates prepared from perfused mature whole mouse brain a major band of about 80,000 and a minor band of 50,000 molecular weight were observed. The major band co-migrated with the apparent PA from differentiated mouse neurobtastoma cells12. The minor band co-migrated with rat urine PA and was also observed in extracts from mouse lung, kidney and liver but was hard to detect in the neuroblastoma cell extracts which were testedzS. In extracts from rat glioma cells PA activity was below detection level under the assay conditions (Fig. 8). Prolonged incubation (up to 12 h at 37 °C) of a
Fig. 7. PA autoradiography in X-irradiated rat cerebellar section. A : left-hand side (control). B: right hand side (X-irradriated). Magnification >:30. Insert: direct film autoradiogram of whole section. Magnification ~ 3. Note reduction in size of X-irradiated side. Granular layer and meninges are prominently stained (see arrows). Note intense thin line of labeling in the superficial layer of the white matter in A: but not in B-side.
MW Species of PA O
Fig. 8. Detection of active molecular species of PA following electrophoretic separation. Homogenate samples were analyzed. Clear zones represent PA activity. (1) human urokinase (20 U K mU); (2) rat urine (10 mU); (3) whole mouse brain homogenate (20 mU); (4) rat glioma C6BU-1 cell extract; (5) hybrid (neuroblastoma-glioma)NG-108-15 cell extract (10 mU). Confluent cells were kindly donated by A. Zutra.
371 mixture of the liver extract, containing the single band of 50,000, with the neuroblastoma cell extract, containing only the 80,000 band, did not affect the migration pattern, suggesting that the different mobilities of PA species did not result from proteolytic digestion during the preparation and storage of the homogenates. Analysis of homogenates from various brain regions of adult and newborn mice also revealed the 80,000 and 50,000 bands to be of similar relative intensities to those observed to whole brain homogenates with no apparent variations with age. Similarly migrating PA species were also observed in homogenates from X-irradiated rat brain regions. DISCUSSION We have employed several experimental approaches in order to reveal the cellular origin(s) of PA in the mature and developing rodent brain and its possible correlation with developmental processes in the brain. These include a novel autoradiographic method designed to localize PA in frozen sections, homogenate assays and electrophoretic analysis of PA, all performed in normal and X-irradiated brain. All of our findings are consistent with the existence of at least two cellular sources for PA, of which one is likely to be nerve cells and the other, cells of epithelial origin. We localized PA activity by autoradiography in the developing and the adult brain in epithelial regions and in layers that are rich in neuronal cell bodies, such as the cerebellar granular layer and the dentate gyrus. Since primary glial cells do not produce PA 27, our data strongly suggest that developing and mature cell bodies of neurons synthesize large quantities of PA. This suggest;on is further substantiated by the analysis of rat brain following X-ray irradiation, which selectively eliminates dividing neurons but does not affect other cells in an irreversible manner a. The autoradiographic localization of PA in the irradiated cerebellum clearly revealed a marked reduction in silver grains in the granular layer, whereas the meningeal labeling was completely unaffected by the irradiation. The residual radioactive staining in the granular layer of the irradiated cerebellum can probably be attributed to the fraction of granular neurons that divide in prenatal days and are not affected by the irradiation 3. The irradiated cerebellum, which is much smaller in size, displays a higher PAspecific activity in homogenates than its non-irradiated counterpart cerebellum. A parallel, although smaller and briefer, increase was observed in homogenates from the irradiated cortex. The increase in PA levels may be due to induction of PA synthesis by the irradiation. DNA-damaging agents were in fact shown to induce the synthesis of PA in vitro in fibroblasts from several vertebrates 18. The intense labeling in the superficial layer of the cerebellar white matter is of particular interest, since myelinated regions in general did not show significant PA labeling. The heavy label in this layer suggests that in addition to cell bodies, certain brain regions rich in myelinated axons may contain high levels of PA. It should be noted that the labeled myelinated layer, the properties of which are not known, completely disappears from both histologically stained and autoradiographically analyzed sections following X-ray irradiation. Other techniques will be required to
372 determine whether myelinated structures within the granular layer itself also contain PA activity. PA activity has recently been shown to be required for the development of ganglia in vitro 15. In view of our findings, and since primary glial cells do not produce this enzyme27, it seems likely that PA produced by the differentiating neurons of developing ganglia may promote their wrapping by Schwann cells. The Purkinje cell layer in the normal and the X-irradiated cerebellum did not express high PA activity, suggesting that these cells are not the major source for the enzyme activity in the cerebellum. Cajal 7 proposed that the growth of Purkinje cell dendrites is stimulated by the migration of the granule cells. It was later postulated that an interaction between Purkinje cells and the migrating granule cells results in the formation of specific connections between these two cell types 22. It was demonstrated that the prevention of granule cells' migration by X-ray irradiation results in undeveloped dendrites of Purkinje cells5,24. Our findings suggest that the initiation of local proteolysis by PA secreted from granular neurons may participate in the stimulation of dendrite extention from Purkinje cells. Electrophoresis of rodent brain homogenates revealed two species of PA activity with the apparent molecular weights of 80,000 and 50,000. The co-migration of the 80,000 brain PA with the major enzyme species in mouse neuroblastoma cells and the co-migration of the 50,000 brain PA with rat urine enzyme may suggest that these two species are produced by different cell types. The involvement of PA in developmental processes in the postnatal cerebellum is suggested from the ontogenetic variability in the specific activity of the enzyme in cerebellar homogenates. The high PA levels in the early postnatal cerebellum appear during the period of extensive cell proliferation and migration 1,7,2°, while the decrease in PA-specific aclivity is concomitant with the stabilization of the total cerebellar protein content. This suggestion is reinforced by the consistently low level of PA in the developing cerebral cortex, where neuronal proliferation and migration occurs mostly in prenatal days 14. It is not clear yet why is there a peak in PA activity in the thalamus at day 20 or what is the meaning of the decline of PA activity with postnatal age in the brain stem. Our observation that neuronal-enriched layers in the mature brain produce high levels of PA suggests that this enzyme is required for the functioning of neurons. PA production by meningeal and ependymal cell layers, by choroid plexus cells and by blood vessels endothelium may supply the enzyme to the cerebrospinal fluid and the plasma, where it is required for the thrombolysis. The findings we present herein suggest that in addition to the involvement in thrombolysis, that has already been attributed to the brain plasminogen activation system, it may also have other functions both in developing and in the mature brain. ACKNOWLEDGEMENTS We thank C. Sotelo and U. Z. Littauer for critical reading of the manuscript. This research was supported by grants from United States-Israel Binational Science
373 F o u n d a t i o n (BSF) Jerusalem, Israel (to R.M.) a n d from the Israeli C o m m i s s i o n for Basic Research (to H. S.). H e r m o n a Soreq is a recipient of a Charles Revson Career D e v e l o p m e n t Chair.
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