American Journal ofPathology, VoL 13 7, No. 4, October 1990 Copyright C American Association ofPathologists
Rapid Communication Protease Nexin-1 Localization in the Human Brain Suggests a Protective Role Against Extravasated Serine Proteases
Ben H. Choi,* Michiyasu Suzuki,* Taiseung Kim,* Steven L. Wagner,t and Dennis D.
Cunninghamt From the Division ofNeuropathology, Departments of Pathology* and Microbiology and Molecular Genetics,t University of California, Irvine, California
Protease nexin-1 (PN-1) is a potent thrombin inhibitor that is identical to the glia-derived neuritepromotingfactor orglia-derived nexin. Here we report immunocytochemical studies of adult human cerebral cortex that revealed the presence of strong immunoreactivityfor PN- I in capillaries and in the smooth muscle cells of arteries and arterioles. Expression of PN-1 was also abundant in astroglial processes in the parenchyma and in perivascular astroglial endfeet of human cerebral cortex. In situ hybridization with an 35S-labeled RNA antisense probeforPN- I resulted in significant labeling ofastrocytes and blood vessels. Because thrombin is known to cause retraction of neurites and modification of astrocytic morphology at low concentrations, PN- 1 around blood vessels mayplay a major protective role against extravasation of thrombin and possibly other serineproteases into the human brain. (Am JPathol 1990, 13 7: 741- 74 7)
Protease nexin-1 (PN-1) is a 43-kd serine protease inhibitor that was identified in the conditioned medium of cultured human fibroblasts' and subsequently shown to be secreted by a variety of cultured extravascular cells,2 including astrocytes.3 PN-1 rapidly inhibits thrombin, urokinase, and plasmin by forming a complex with their catalytic site serine residues.1.4 The PN-1 -protease complexes then bind back to the cells that secrete PN-1 and are rap-
idly internalized and degraded.5 PN-1 binds to the cell surface and extracellular matrix (ECM)6; this accelerates its inactivation of thrombin7 and blocks its inactivation of urokinase and plasmin.e Thus PN-1 in interstitial fluid would be an effective thrombin, urokinase, and plasmin inhibitor, whereas it is a specific thrombin inhibitor when bound to the cell surface and ECM. Recent studies showed that PN-1 is identical to the glia-derived neurite-promoting factor or glia-derived nexin910 that stimulates neurite outgrowth in cultured neuroblastoma cells'1 and chick sympathetic ganglia.'2 The neurite outgrowth activity of this factor on cultured neuroblastoma cells depends on thrombin inhibition because it is blocked by thrombin and because other thrombin inhibitors produce neurite outgrowth."1 Subsequent studies showed that thrombin produces neurite retraction and that PN-1 has no detectable neurite outgrowth activity on neuroblastoma cells in the absence of thrombin.13'14 PN1 and thrombin also reciprocally regulate the stellation of cultured astrocytes; low concentrations of thrombin convert stellate astrocytes to a nonstellate flattened form and PN-1 reverses this action.15 Although PN-1 activity has been well characterized in vitro, its role in vivo has remained speculative. Recent studies on autopsy samples of human brain demonstrated that PN-1 activity and free, uncomplexed PN-1 was reduced approximately seven-fold in the brains of patients with Alzheimer's disease (AD) compared to agematched controls with similar postmortem intervals.16 This was not due to decreased PN-1 mRNA in the AD samples. Instead increased thrombin or thrombinlike protease activity seemed at least partly responsible because the AD Supported by NIH grants ES 02928 to Dr. Choi and GM 31609 to Dr. Cunningham. Dr. Wagner was supported by a postdoctoral fellowship from the George E. Hewitt Foundation for Medical Research. Accepted for publication July 12, 1990. Address reprint requests to B. H. Choi, MD, Division of Neuropathology, Department of Pathology, University of California, Irvine, CA 92717.
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brain samples contained increased levels of PN-1 -containing complexes that comigrated with PN-1 -thrombin complexes on Western blots. These results led to the hypothesis that reduced levels of PN-1 and increased thrombin or a thrombinlike protease in AD brain might lead to neuritic and astroglial alterations that could contribute to the pathogenesis of the disease.16 In view of this, it was of particular interest to determine the brain cell types and structures with which PN-1 is associated. Accordingly we performed immunocytochemical studies with well-characterized anti-PN-1 monoclonal and polyclonal antibodies and in situ hybridization experiments with an 35S-labeled antisense RNA probe for PN-1.
Immunoblot Analysis Human brain tissue was homogenized in 10 volumes of 10 mmol/I (millimolar) HEPES, 0.32 mol/I (molar) sucrose (pH 7.2), and supernatants were prepared as described previously.16 Aliquots of the supernatants containing 290 ,ug of protein per lane and various amounts of purified PN-1 were electrophoresed on 7.5% polyacrylamide gels according to the methods of Laemmli.19 The proteins subsequently were transferred to a nitrocellulose membrane, incubated overnight in 0.25% gelatin, and probed with anti-PN-1 polyclonal antisera (1/300) diluted in 1.0% ovalbumin (for 2 hours at 370 C). Biotinylated donkey antirabbit and streptavidin-biotinylated horseradish peroxidase preformed complex were used at 1:200 and 1:400
dilutions, respectively.
Materials and Methods The anti-PN-1 monoclonal antibodies used in these studies were previously described.17 Polyclonal antisera to PN-1 were generated by immunizing rabbits with highly purified PN-1 (200 ,g/dose) subcutaneously using Freund's adjuvant as a vehicle. PN-1 was purified from human fibroblast-conditioned medium or from postmortem human brain using immunoaffinity chromatography according to Van Nostrand et al.18
Enzyme-linked Immunosorbent Assay (ELISA) Ninety-six-well microtiter plates were coated with various concentrations of human brain PN-1 for 2 hours at 370 C. After washing three times with phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBS-Tween, Sigma Chemical Co., St. Louis, MO) the plates were incubated with anti-PN-1 MAbp 3017 ascites fluid diluted (1/5000) in PBS containing 1.0% ovalbumin for 60 minutes at 370 C. Unbound antibody was removed by washing four times with PBS-Tween. Biotinylated sheep anti-mouse IgG (Amersham, Arlington Heights, IL) was diluted in PBS-ovalbumin (1/400) and added to the wells, and the incubation was carried out as described above. The plates were washed four times with PBS-Tween and incubated with streptavidin-biotin-peroxidase complex (Amersham) diluted (1/800) in PBS-ovalbumin (for 30 minutes, at 300 C). After washing six times with PBS-Tween, the bound peroxidase activity was localized using o-phenylene diamine, quenched with an equal volume of 4N sulfuric acid, and the absorbance as read at 492 nm with a Tiertek Multiskan ELISA reader (Flow Laboratories, McLean, VA).
Immunocytochemical Studies Indirect immunofluorescence for PN-1 was performed on
8-,um cryostat sections of normal adult cerebral cortices freshly obtained from three temporal lobectomy specimens according to the method of Coons and Kaplan.20 The dilution of PN-1 MAb was 1:1. Normal mouse and rabbit sera were used as controls. Following a brief wash in PBS, the slides were covered with primary antibody or control serum for 30 minutes at 370 C. All sections then were washed in fresh PBS for 4 minutes and reincubated with fluorescein-labeled goat anti-mouse or anti-rabbit y globulin (diluted 1: 10) for 30 minutes at 37GC. They were washed again in fresh PBS for 4 minutes and mounted with glycerol-PBS (9:1). For double labeling of PN-1 and glial fibrillary acidic protein (GFAP), either fluorescein-labeled anti-mouse (for PN-1) and rhodamine-labeled antirabbit (for GFAP), or rhodamine-labeled anti-mouse (for PN-1) and fluoresein-labeled anti-rabbit (for GFAP) -y globulins were used interchangeably as secondary antisera. The slides were examined under a Nikon optiphot microscope (Nikon Inc., Garden City, NY) with episcopic fluorescence attachment using an HB-10101AF super highpressure mercury lamp light source, 450 to 490-nm exciter filter, and 520-nm barrier filter for fluorescein and 546nm exciter filter and 590-nm barrier filter for rhodamine. Immunoperoxidase staining, as described by Sternberger21 was carried out on 8-,um paraffin and 30-,um vibratome sections of samples fixed in 2% paraformaldehyde. The dilution of primary antiserum was 1:500. The incubation period in primary antisera varied from 24 to 48 hours, at 40 C.
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Figure 1. A: ELISA demontstratitng the biniding of anti-PN-1 MAbp3O to purified human brain PN-1. Human brain PN-J from postmortem cerebral cortex u'as purified by heparin-sepharose chromatography followed by anti-PN-1 MAbp9-immunoaffinitj' chromatography. The purified protein was then coated onito 96-well microtiter plates at the i'arious concenitrations showtn and the ELISA was carried out as described. B: Westerni blot of human cerebral cortex depictinig the specificity of anti-PN1 rabbit polyclonal an tisera. Lan e 1, temporal cortex; lane 2, visual cortex; latne 3, superior parietal cortex; lane 4, purified PN-1 (5,4g); lane 5, purified PN- 1 (IMg). A lower-molecular-weight protein is also fainztljy visible in the Western blot anid is probably afragment ofPN- 1.
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In Situ Hybridization A 603-base pair of cDNA insert from polymerase chain reaction amplification of human glioblastoma cDNA (containing the region of PN-1-encoding amino acids 149 to 350) was blunt end ligated into the Hindl site of pBS (Stratagene, La Jolla, CA). The plasmid vector was placed into the Escherichia coli strain XL1 blue and transformants were selected. The plasmid then was cut with Bam Hi or EcoRl. The Bam Hi linearized plasmid was used to synthesize sense riboprobe with T7 RNA polymerase. This 35S-UTP-labeled probe was used as a negative control. The plasmid cut with EcoRl was used to synthesize the 35S-UTP-labeled RNA probe. All subsequent steps were done under RNAse free conditions by treating all solutions with diethyl pyrocarbonate and by autoclaving. All glassware was autoclaved. In situ hybridization was performed on 10 to 20-,um frozen sections. The tissue was digested in 0.05 N HCI for 8 minutes at room temperature, washed in PBS, and digested in Proteinase K at a concentration of 5 ,ug/ml in 0.1 mol/l TRIS buffer 0.05 mol/l EDTA (pH 8.0) for 30 minutes at 370 C. The slides were acetylated by first rinsing in distilled water for 1 minute and transferred to freshly prepared 0.1 mol/l triethanolamine (pH 8.0) for 1 minute. The slides were quickly blotted dry and placed into 0.1 mol/l triethanolamine containing 2.5 mmol/l acetic anhydride for 10 minutes. The slides then were transferred to 2X saline sodium citrate (SSC) for a 1-minute wash and dehydrated. Prehybridization solution (50% formamide, 4x SSC, 5x Denhardts, 1% SDS, 25 ,g/ml tRNA, 0.1 mol/l DTT) then was applied for 1 hour at 550 C, and hybridization was carried out with approximately 50 Ml of hybridization solution containing 2 X 108 cpm of probe for 3 to 4 hours at 550 C. The tissue then was treated with 20 ,ig/ml RNAse A in a solution containing 0.5 mol/l NaCI, 0.01 mol/l TRIS buffer, and 1 mmol/l EDTA (pH 8.0) at 370 C for 30 minutes. Following SSC washes, the slides were
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dehydrated. The slides were dipped in Ilford (Ilford Polysciences, Warrington, PA) or NTB-2 (Kodak, Rochester, NY) photographic emulsions and exposed in the dark for 2 weeks before developing in D-19.
Results Biochemical Characterization of Anti-PN-1 Immunoglobulins The anti-PN-1 monoclonal antibody used in this study (MAbp 30) was prepared using PN-1 purified from cultured human fibroblast-conditioned medium.17 Therefore it was crucial to examine the reactivity of this MAb toward PN-1 present in human brain PN-1 was isolated from human brain tissue homogenates using essentially the same procedures used to immunopurify human fibroblast PN-1 1 Figure 1A shows that MAbp 30 bound to human brain PN-1 in a dose-dependent manner. Therefore the epitope to which MAbp 30 binds is present in both human brain and human fibroblast PN-1. Highly purified PN-1 isolated from human fibroblast-conditioned medium also was used to generate rabbit polyclonal antisera. Figure 1B is a Western blot showing that the rabbit anti-PN-1 polyclonal antibody recognized PN-1 in an homogenate of brain tissue. A lower molecular weight protein also is faintly visible in the Western blot. This is probably a fragment of PN-1. The polyclonal antibody used in this study has been shown to react with PN-1-protease complexes.16 The quantities of these complexes are very low in the human brain and is probably related to the fact that PN-1 -protease complexes are rapidly internalized and degraded by the same cells that secrete PN-1.5
Immunocytochemical Findings Both monoclonal and polyclonal antibodies for PN-1 gave strong PN-1 immunoreactivity in cryostat sections of hu-
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V-X
Figure 2. Photomicrographs showing indirect immuno]luorescence of PN-1 and GFAP in the cerebral cortex of adult human temporal lobe. a: Note strong PN- 1 im-
muno]luorescence of linear profiles in the
cerebral cortex that appear to be associated with astrocytic processes (arrows)
(X220). b: Photomicrograph of negative control (X220). cd: Double labeling of PN1 (Qluorescein) and GFAP (rhodamine) showing expression of PN- 1 in GFAP-positive astroglial processes (arrows). The processes extend into the capillarjy wall (V) (X440). ef: Double labeling of PN-1 and GFAP showing an astroglial cell (white arrow) extending two processes (arrows) into a medium-sized arteriole (V) showing
PN-1 immunofluorescence (X440). g,h: Double labeling of PN-1 (rhodamine) and GFAP (fluorescein) demonstrates that GFAP-positiz'e astrocyte also express PN-1 in the cytoplasm and its processes (arrows). White arrou'points to the nucleus of an astrocyte (X 440).
cerebral cortex with the use of the indirect immunofluorescent technique. The pattern of PN-1 immunofluorescence using monoclonal antibody (MAbp 30) was identical in all samples obtained from three different cases. Within the cerebral cortex, immunoreactivity was present within fibrillar processes that appeared to be associated with astroglial cells (Figures 2a, g). These cells extended their processes toward small capillaries and arterioles and appeared to form perivascular endfeet (Figures 2c-f). Double labeling for PN-1 and GFAP demonstrated coexpression of PN-1 and GFAP in the same astroglial cells (Figures 2g, f) and processes (Figures 2c-h). It should be emphasized that in all of the immunocytochemical studies there was no significant staining in control sections incubated in the absence of the primary antibody (Figure 2b). Immunoperoxidase staining for PN-1 in man
paraffin and vibratome sections fixed in 2% paraformaldehyde was only possible using polyclonal antisera. The presence of PN-1 immunoreactivity in the walls of capillaries and arterioles was well demonstrated by this method. Immunoreactive astroglial cells extending processes toward capillaries that showed immunoreactivity for PN-1 also were observed (Figure 3a). At higher magnification, the presence of specific PN-1 immunoreactivity was noted in the smooth muscle cells of arteries and arterioles both in the parenchyma and in the subarachnoid space (Figure 3b). Formalin-fixed and paraffin-embedded sections consistently gave negative results however.
In situ Hybridization As shown in Figure 4, intense labeling following the application of an antisense 35S-RNA probe for PN-1 was pres-
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Figure 3. Immunoperoxidase staining using a polyclonal antiserum for PN-1 in tissuesfixed in 2% paraformaldehyde. a: Note immunoperoxidase-positivle PN-1 in the walls ofsmall blood vessels (large arrows). Note also immunoreactive PN-1 in the cytoplasm ofastrocytes (arrow heads). An astrocytic process (small arrow) extends toward a small capillary that is also immunoperoxidase positivle for PN- 1 (X220). b: Photomicrograph showing intense PN-1 immunoreactivity in the smooth muscle cells (arrows) of the blood vessel in the subarachnoid space (X 440). Figure 4. In situ
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hybridization using 35S-labeled riboprobes for PN-1. a-c: Intense labeling of an antise'nse 35S-RNA PN-1 probe is noted in the vascular walls (empty arrows). Closed arrow in a points to a labeled astrocyte. (X440). d,e: Astrocytes (arrows) show strong labeling ofan antisense 35S-RNA PN1 probe over their nuclei. These cells were identified on the basis of general topographj, round to ovoid nuclei measuring 10 to 20 Am in diameter containing finely granular and evenly distributed chromatin and absence of distinct nucleoli, all of which are consistent with astrocytes rather than neurons, oligodendrocytes, or microglial cells (d: X220, e: X440). f: An adjacent conttrol section labeled with sentse 35S-RNA PN- 1probe shows only sparse backgrounid grains. Arrows poinlt to astrocytic niuiclei devoid of labeling. Ani empty arrouw points to a blood vesselshowing tno labelintg (X220).
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ent in the walls of blood vessels (Figures 4a-c) and in astrocytes (Figures 4d, e), while adjacent sections processed as controls using sense 35S-RNA PN-1 probe showed only sparse background grains (Figure 4f). These studies showed that the same brain cells and structures that contained PN-1 also contained PN-1 mRNA.
Discussion The present results show that much of the PN-1 and its mRNA in adult human brain are localized in and around blood vessels. This, along with results of several recent studies, suggest that it may play a protective role against extravasated prothrombin that would be converted to active thrombin. Thus it seems likely that PN-1 in the cerebral blood vessels functions as a thrombin inhibitor. Thrombin, in addition to playing a central role in the blood clotting system, is a serine protease with diverse bioregulatory activity. For example, it brings about retraction of neurites in cultured neuroblastoma cells,13-14 produces changes in astrocytic morphology,"5 and reverses morphologic differentiation of heterogeneous cells cultured from various regions in the human fetal brain.22 In each of these cell systems, thrombin, but not other proteases tested, brings about modification of cellular processes. More importantly, this occurs at thrombin concentrations that are
about five or six orders of magnitude lower than prothrombin concentrations in plasma. Thus minute amounts of extravasated thrombin have the potential to cause central nervous system (CNS) injury by altering neuronal and glial processes in a manner that may result in disruption of their function. Indeed such a mechanism may be operative in the pathogenesis of certain neurodegenerative diseases. In the brains of subjects with AD, for example, it has been reported 1) that neuritic plaques frequently cluster along the walls of blood vessels, 2) that blood-brain barrier function is compromised,23 25 and 3) that there is a significant reduction in active PN-1 levels and an increase in the levels of PN-1-thrombin complex.16 Thrombin also is a potent mitogen for astrocytes.1526 All of these findings underscore the importance of a protective mechanism to inactivate even minute amounts of thrombin that may enter the brain when the blood-brain barrier function is compromised. Although the manner in which PN-1 is localized within the brain suggests a role in blood-brain barrier function, it should be emphasized that PN-1 localization around blood vessels in other organs has not been investigated. The present studies also demonstrated the presence of significant amounts of PN-1 and its mRNA on astrocytes and their processes. The localization of PN-1 on perivascular astroglial endfeet tightly abutting the basal lamina of blood vessels is particularly significant. It sug-
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gests that PN-1 on the surfaces of astroglial processes in close association with the basal lamina of the vascular wall would be well suited to provide additional protection against extravasated thrombin. The PN-1 in astrocytes and their processes also may play significant roles in cellular events during neurogenesis, such as neuronal migration and final positioning of postmigratory neurons. It is known that migrating cells have more cell-surface proteolytic activity than stationary, differentiated cells. For example, it has been reported that dissociated cerebellar cultures possess plasminogen activator during active granule cell migration24 but not when granule cell migration is complete.27 It also has been reported that highly specific inhibition of external granule cell migration can be brought about by glia-derived nexin (PN-1) in explant cultures of cerebellum.28 The presence of PN-1 on astroglial processes is especially significant in this regard because radial glia are known to provide guidance for neuronal migration2934 and because radial glia give rise to astrocytes and express GFAP in humans and subhuman primates.3035 Although it is recognized that PN-1 bound to astrocytic cell surfaces is a specific thrombin inhibitor,8 it is possible that PN-1 secreted by astrocytes may be present in the extracellular spaces within the neuropil and interact with other serine proteases, such as urokinase, or that thrombin and/or thrombinlike proteases may be active within the neuropil during certain stages of normal or abnormal brain development. Thus it is possible that interplay between proteases and PN-1 may not only influence intricate neuronal-glial interactions and cellular events during neurogenesis but also may play crucial roles in the constant restructuring of neurites during development or in repair of injury in the nervous system. In this context it is significant that glia-derived nexin and its mRNA were detected in the olfactory system of the rat where continuous degeneration and regeneration of the CNS is taking place throughout life.36 Most of the glia-derived nexin in the rat olfactory system is thought to be synthesized and secreted by nonneuronal cells.6 Glia-derived nexin also may have a role in axonal regeneration of the peripheral nervous system. This was suggested by a study in which induction of glia-derived nexin was observed following an injury to a peripheral nerve.37 In summary, localization of PN-1 in brain blood vessels and astroglial cells suggests that it may play a major protective role against the extravasation of thrombin and possibly other serine proteases.
References 1. Baker JB, Low DA, Simmer RL, Cunningham DD: Proteasenexin: A cellular component that links thrombin and plasmin-
ogen activator and mediates their binding to cells. Cell 1980, 21:37-45 2. Eaton DL, Baker JB: Evidence that a variety of cultured cells secrete protease nexin and produce a distinct cytoplasmic serine protease-binding factor. J Cell Physiol 1983, 117: 175-182 3. Rosenblatt DE, Cotman CW, Nieto-Sampedro M, Rowe JW, Knauer DJ: Identification of a protease inhibitor produced by astrocytes that is structurally and functionally homologous to human protease nexin-1. Brain Res 1987, 415:40-48 4. Scott RW, Bergmann BL, Baipai A, Hersh RT, Rodrigues H, Barreda BN, Watts S, Baker JB: Protease nexin. Properties and a modified purification procedure. J Biol Chem 1985, 260:7029-7034 5. Low DA, Baker JB, Koonce WC, Cunningham DD: Released protease-nexin regulates cellular binding, internalization, and degradation of serine proteases. Proc Natl Acad Sci USA 1981, 78:2340-2344 6. Farrell DH, Wagner SL, Yuan RH, Cunningham DD: Localization of protease nexin-1 on the fibroblast extracellular matrix. J Cell Physiol 1988,134:179-188 7. Farrell DH, Cunningham DD: Human fibroblasts accelerate the inhibition of thrombin by protease nexin. Proc Natl Acad Sci USA 1986, 83:6858-6862 8. Wagner SL, Lau AL, Cunningham DD: Binding of protease nexin-1 to the fibroblast surface alters its target proteinase specificity. J Biol Chem 1989, 264:611-615 9. Gloor S, Odink K, Guenther J, Nick H, Monard D: A gliaderived neurite promoting factor with protease inhibitory activity belongs to the protease nexins. Cell 1986, 47:687-693 10. McGrogan M, Kennedy J, Li MP, Hsu C, Scott RW, Simonsen CC, Baker JB: Molecular cloning and expression of two forms of human protease nexin 1. Bio/technol 1988,6:172-177 11. Monard D, Niday E, Limat A, Solomon F: Inhibition of protease activity can lead to neurite extension in neuroblastoma cells. Prog Brain Res 1983, 58:359-364 12. Zurn AD, Nick H, Monard D: A glia-derived nexin promotes neurite outgrowth in cultured chick sympathetic neurons. Devl Neurosci 1988,10:17-24 13. Gurwitz D, Cunningham DD: Thrombin modulates and reverses neuroblastoma neurite outgrowth. Proc Natl Acad Sci USA 1988,85:3440-3444 14. Gurwitz D, Cunningham DD: Neurite outgrowth activity of protease nexin-1 on neuroblastoma cells require thrombin inhibition. J Cell Physiol 1990,142:155-162 15. Cavanaugh K, Gurwitz D, Cunningham D, Bradshaw R: Reciprocal modulation of astrocyte stellation by thrombin and protease nexin-1. J Neurochem 1990, 54:1735-1743 16. Wagner SL, Geddes JW, Cotman CW, Lau AL, Gurwitz D, Isackson PL, Cunningham DD: Protease nexin-1, an antithrombin with neurite outgrowth activity, is reduced in Alzheimer's disease. Proc Natl Acad Sci USA 1989,86:8284-8288 17. Wagner SL, Van Nostrand WE, Lau AL, Cunningham DD: Monoclonal antibodies to protease nexin 1 that differentially block its inhibition of target proteases. Biochem 1988, 27: 2173-2176
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18. Van Nostrand WE, Wagner SL, Cunningham DD: Purification of a form of protease nexin 1 that binds heparin with a low affinity. Biochem 1988, 27:2176-2181 19. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227: 680-685 20. Coons AH, Kaplan MH: Localization of antigen in tissue cells. ll. Improvements in a method for the detection of antigen by means of fluorescent antibody. J Exper Med 1950, 91:1-13 21. Sternberger LA: Immunocytochemistry. New York, Wiley, 1979, pp 104-169 22. Grand RJA, Grabham PW, Gallimore MJ, Gallimore PH: Modulation of morphological differentiation of human neuroepithelial cells by serine proteases: Independence from blood coagulation. EMBO J 1989, 8:2209-2215 23. Hardy JA, Mann DMA, Wester P, Winblad B: An integrative hypothesis concerning the pathogenesis and progression of Alzheimer's disease. Neurobiol Aging 1986, 7:489-502 24. Wisniewski HM, Kozlowski PB: Evidence for blood-brain barrier changes in senile dementia of the Alzheimer type (SDAT). Ann NY Acad Sci 1982, 396:119-129 25. Delacourte A, Defossez A, Persuy P, Peers MC: Observation of morphological relationships between angiopathic blood vessels and degenerative neurites in Alzheimer's disease. Virchows Arch A 1988, 411:199-204 26. Perraud F, Besnard F, Sensenbrenner M, Labourdette G: Thrombin is a potent mitogen for rat astroblasts but not for oligodendroblasts and neuroblasts in primary culture. Int J Devi Neurosci 1987, 5:181-188 27. Krystosek A, Seeds NW: Plasminogen activator secretion by granule neurons in cultures of developing cerebellum. Proc Natl Acad Sci USA 1981, 78:7810-7814
28. Moonen G, Gran-Wagemans M, Selak I: Plasminogen activator-plasmin system and neuronal migration. Nature 1982, 298:753-755 29. Linder J, Guenther J, Nick H, Zinser G, Antonicek H, Schachner M, Monard D: Modulation of granule cell migration by a glia-derived protein. Proc Natl Acad Sci USA 1986, 83:4568-4571 30. Choi BH: Glial fibrillary acidic protein in radial glia of early human fetal cerebrum. A light and electron microscopic immunoperoxidase study. J Neuropathol Exper Neurol 1986, 45:408-418 31. Choi BH: Developmental events during the early stages of cerebral cortical neurogenesis in man. A correlative light, electron microscopic, immunohistochemical and Golgi study. Acta Neuropathol (Berl) 1988, 75:441-447 32. Choi BH, Lapham LW: Radial glia in the human fetal cerebrum: A combined Golgi, immunofluorescent and electron microscopic study. Brain Res 1978,148:295-311 33. Choi BH, Matthias SC: Cortical dysplasia associated with massive ectopia of neurons and glial cells within the subarachnoid space. Acta Neuropathol (Berl) 1987, 73:105-109 34. Rakic P: Mode of cell migration to the superficial layers of fetal monkey neocortex. J Comp Neurol 1972,145:61-84 35. Levitt P, Rakic P: Immunoperoxidase localization of glial fibrillary acidic protein in radial glial cells and astrocytes of the developing rhesus monkey brain. J Comp Neurol 1980,193:
815-840 36. Reinhard E, Meier R, Halfter W, Rovelli G, Monard D: Detection of glia-derived nexin in the olfactory system of the rat. Neuron 1988,1:387-394 37. Meier R, Spreyer P, Ortmann R, Harel A, Monard D: Induction of glia-derived nexin after lesion of a peripheral nerve. Nature 1989, 342:548-550
Rapid Communication Protease Nexin-1 Localization in the Human Brain Suggests a Protective Role Against Extravasated Serine Proteases
Ben H. Choi,* Michiyasu Suzuki,* Taiseung Kim,* Steven L. Wagner,t and Dennis D.
Cunninghamt From the Division ofNeuropathology, Departments of Pathology* and Microbiology and Molecular Genetics,t University of California, Irvine, California
Protease nexin-1 (PN-1) is a potent thrombin inhibitor that is identical to the glia-derived neuritepromotingfactor orglia-derived nexin. Here we report immunocytochemical studies of adult human cerebral cortex that revealed the presence of strong immunoreactivityfor PN- I in capillaries and in the smooth muscle cells of arteries and arterioles. Expression of PN-1 was also abundant in astroglial processes in the parenchyma and in perivascular astroglial endfeet of human cerebral cortex. In situ hybridization with an 35S-labeled RNA antisense probeforPN- I resulted in significant labeling ofastrocytes and blood vessels. Because thrombin is known to cause retraction of neurites and modification of astrocytic morphology at low concentrations, PN- 1 around blood vessels mayplay a major protective role against extravasation of thrombin and possibly other serineproteases into the human brain. (Am JPathol 1990, 13 7: 741- 74 7)
Protease nexin-1 (PN-1) is a 43-kd serine protease inhibitor that was identified in the conditioned medium of cultured human fibroblasts' and subsequently shown to be secreted by a variety of cultured extravascular cells,2 including astrocytes.3 PN-1 rapidly inhibits thrombin, urokinase, and plasmin by forming a complex with their catalytic site serine residues.1.4 The PN-1 -protease complexes then bind back to the cells that secrete PN-1 and are rap-
idly internalized and degraded.5 PN-1 binds to the cell surface and extracellular matrix (ECM)6; this accelerates its inactivation of thrombin7 and blocks its inactivation of urokinase and plasmin.e Thus PN-1 in interstitial fluid would be an effective thrombin, urokinase, and plasmin inhibitor, whereas it is a specific thrombin inhibitor when bound to the cell surface and ECM. Recent studies showed that PN-1 is identical to the glia-derived neurite-promoting factor or glia-derived nexin910 that stimulates neurite outgrowth in cultured neuroblastoma cells'1 and chick sympathetic ganglia.'2 The neurite outgrowth activity of this factor on cultured neuroblastoma cells depends on thrombin inhibition because it is blocked by thrombin and because other thrombin inhibitors produce neurite outgrowth."1 Subsequent studies showed that thrombin produces neurite retraction and that PN-1 has no detectable neurite outgrowth activity on neuroblastoma cells in the absence of thrombin.13'14 PN1 and thrombin also reciprocally regulate the stellation of cultured astrocytes; low concentrations of thrombin convert stellate astrocytes to a nonstellate flattened form and PN-1 reverses this action.15 Although PN-1 activity has been well characterized in vitro, its role in vivo has remained speculative. Recent studies on autopsy samples of human brain demonstrated that PN-1 activity and free, uncomplexed PN-1 was reduced approximately seven-fold in the brains of patients with Alzheimer's disease (AD) compared to agematched controls with similar postmortem intervals.16 This was not due to decreased PN-1 mRNA in the AD samples. Instead increased thrombin or thrombinlike protease activity seemed at least partly responsible because the AD Supported by NIH grants ES 02928 to Dr. Choi and GM 31609 to Dr. Cunningham. Dr. Wagner was supported by a postdoctoral fellowship from the George E. Hewitt Foundation for Medical Research. Accepted for publication July 12, 1990. Address reprint requests to B. H. Choi, MD, Division of Neuropathology, Department of Pathology, University of California, Irvine, CA 92717.
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brain samples contained increased levels of PN-1 -containing complexes that comigrated with PN-1 -thrombin complexes on Western blots. These results led to the hypothesis that reduced levels of PN-1 and increased thrombin or a thrombinlike protease in AD brain might lead to neuritic and astroglial alterations that could contribute to the pathogenesis of the disease.16 In view of this, it was of particular interest to determine the brain cell types and structures with which PN-1 is associated. Accordingly we performed immunocytochemical studies with well-characterized anti-PN-1 monoclonal and polyclonal antibodies and in situ hybridization experiments with an 35S-labeled antisense RNA probe for PN-1.
Immunoblot Analysis Human brain tissue was homogenized in 10 volumes of 10 mmol/I (millimolar) HEPES, 0.32 mol/I (molar) sucrose (pH 7.2), and supernatants were prepared as described previously.16 Aliquots of the supernatants containing 290 ,ug of protein per lane and various amounts of purified PN-1 were electrophoresed on 7.5% polyacrylamide gels according to the methods of Laemmli.19 The proteins subsequently were transferred to a nitrocellulose membrane, incubated overnight in 0.25% gelatin, and probed with anti-PN-1 polyclonal antisera (1/300) diluted in 1.0% ovalbumin (for 2 hours at 370 C). Biotinylated donkey antirabbit and streptavidin-biotinylated horseradish peroxidase preformed complex were used at 1:200 and 1:400
dilutions, respectively.
Materials and Methods The anti-PN-1 monoclonal antibodies used in these studies were previously described.17 Polyclonal antisera to PN-1 were generated by immunizing rabbits with highly purified PN-1 (200 ,g/dose) subcutaneously using Freund's adjuvant as a vehicle. PN-1 was purified from human fibroblast-conditioned medium or from postmortem human brain using immunoaffinity chromatography according to Van Nostrand et al.18
Enzyme-linked Immunosorbent Assay (ELISA) Ninety-six-well microtiter plates were coated with various concentrations of human brain PN-1 for 2 hours at 370 C. After washing three times with phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBS-Tween, Sigma Chemical Co., St. Louis, MO) the plates were incubated with anti-PN-1 MAbp 3017 ascites fluid diluted (1/5000) in PBS containing 1.0% ovalbumin for 60 minutes at 370 C. Unbound antibody was removed by washing four times with PBS-Tween. Biotinylated sheep anti-mouse IgG (Amersham, Arlington Heights, IL) was diluted in PBS-ovalbumin (1/400) and added to the wells, and the incubation was carried out as described above. The plates were washed four times with PBS-Tween and incubated with streptavidin-biotin-peroxidase complex (Amersham) diluted (1/800) in PBS-ovalbumin (for 30 minutes, at 300 C). After washing six times with PBS-Tween, the bound peroxidase activity was localized using o-phenylene diamine, quenched with an equal volume of 4N sulfuric acid, and the absorbance as read at 492 nm with a Tiertek Multiskan ELISA reader (Flow Laboratories, McLean, VA).
Immunocytochemical Studies Indirect immunofluorescence for PN-1 was performed on
8-,um cryostat sections of normal adult cerebral cortices freshly obtained from three temporal lobectomy specimens according to the method of Coons and Kaplan.20 The dilution of PN-1 MAb was 1:1. Normal mouse and rabbit sera were used as controls. Following a brief wash in PBS, the slides were covered with primary antibody or control serum for 30 minutes at 370 C. All sections then were washed in fresh PBS for 4 minutes and reincubated with fluorescein-labeled goat anti-mouse or anti-rabbit y globulin (diluted 1: 10) for 30 minutes at 37GC. They were washed again in fresh PBS for 4 minutes and mounted with glycerol-PBS (9:1). For double labeling of PN-1 and glial fibrillary acidic protein (GFAP), either fluorescein-labeled anti-mouse (for PN-1) and rhodamine-labeled antirabbit (for GFAP), or rhodamine-labeled anti-mouse (for PN-1) and fluoresein-labeled anti-rabbit (for GFAP) -y globulins were used interchangeably as secondary antisera. The slides were examined under a Nikon optiphot microscope (Nikon Inc., Garden City, NY) with episcopic fluorescence attachment using an HB-10101AF super highpressure mercury lamp light source, 450 to 490-nm exciter filter, and 520-nm barrier filter for fluorescein and 546nm exciter filter and 590-nm barrier filter for rhodamine. Immunoperoxidase staining, as described by Sternberger21 was carried out on 8-,um paraffin and 30-,um vibratome sections of samples fixed in 2% paraformaldehyde. The dilution of primary antiserum was 1:500. The incubation period in primary antisera varied from 24 to 48 hours, at 40 C.
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Figure 1. A: ELISA demontstratitng the biniding of anti-PN-1 MAbp3O to purified human brain PN-1. Human brain PN-J from postmortem cerebral cortex u'as purified by heparin-sepharose chromatography followed by anti-PN-1 MAbp9-immunoaffinitj' chromatography. The purified protein was then coated onito 96-well microtiter plates at the i'arious concenitrations showtn and the ELISA was carried out as described. B: Westerni blot of human cerebral cortex depictinig the specificity of anti-PN1 rabbit polyclonal an tisera. Lan e 1, temporal cortex; lane 2, visual cortex; latne 3, superior parietal cortex; lane 4, purified PN-1 (5,4g); lane 5, purified PN- 1 (IMg). A lower-molecular-weight protein is also fainztljy visible in the Western blot anid is probably afragment ofPN- 1.
A
B
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In Situ Hybridization A 603-base pair of cDNA insert from polymerase chain reaction amplification of human glioblastoma cDNA (containing the region of PN-1-encoding amino acids 149 to 350) was blunt end ligated into the Hindl site of pBS (Stratagene, La Jolla, CA). The plasmid vector was placed into the Escherichia coli strain XL1 blue and transformants were selected. The plasmid then was cut with Bam Hi or EcoRl. The Bam Hi linearized plasmid was used to synthesize sense riboprobe with T7 RNA polymerase. This 35S-UTP-labeled probe was used as a negative control. The plasmid cut with EcoRl was used to synthesize the 35S-UTP-labeled RNA probe. All subsequent steps were done under RNAse free conditions by treating all solutions with diethyl pyrocarbonate and by autoclaving. All glassware was autoclaved. In situ hybridization was performed on 10 to 20-,um frozen sections. The tissue was digested in 0.05 N HCI for 8 minutes at room temperature, washed in PBS, and digested in Proteinase K at a concentration of 5 ,ug/ml in 0.1 mol/l TRIS buffer 0.05 mol/l EDTA (pH 8.0) for 30 minutes at 370 C. The slides were acetylated by first rinsing in distilled water for 1 minute and transferred to freshly prepared 0.1 mol/l triethanolamine (pH 8.0) for 1 minute. The slides were quickly blotted dry and placed into 0.1 mol/l triethanolamine containing 2.5 mmol/l acetic anhydride for 10 minutes. The slides then were transferred to 2X saline sodium citrate (SSC) for a 1-minute wash and dehydrated. Prehybridization solution (50% formamide, 4x SSC, 5x Denhardts, 1% SDS, 25 ,g/ml tRNA, 0.1 mol/l DTT) then was applied for 1 hour at 550 C, and hybridization was carried out with approximately 50 Ml of hybridization solution containing 2 X 108 cpm of probe for 3 to 4 hours at 550 C. The tissue then was treated with 20 ,ig/ml RNAse A in a solution containing 0.5 mol/l NaCI, 0.01 mol/l TRIS buffer, and 1 mmol/l EDTA (pH 8.0) at 370 C for 30 minutes. Following SSC washes, the slides were
a
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100 PN-I (ng)
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dehydrated. The slides were dipped in Ilford (Ilford Polysciences, Warrington, PA) or NTB-2 (Kodak, Rochester, NY) photographic emulsions and exposed in the dark for 2 weeks before developing in D-19.
Results Biochemical Characterization of Anti-PN-1 Immunoglobulins The anti-PN-1 monoclonal antibody used in this study (MAbp 30) was prepared using PN-1 purified from cultured human fibroblast-conditioned medium.17 Therefore it was crucial to examine the reactivity of this MAb toward PN-1 present in human brain PN-1 was isolated from human brain tissue homogenates using essentially the same procedures used to immunopurify human fibroblast PN-1 1 Figure 1A shows that MAbp 30 bound to human brain PN-1 in a dose-dependent manner. Therefore the epitope to which MAbp 30 binds is present in both human brain and human fibroblast PN-1. Highly purified PN-1 isolated from human fibroblast-conditioned medium also was used to generate rabbit polyclonal antisera. Figure 1B is a Western blot showing that the rabbit anti-PN-1 polyclonal antibody recognized PN-1 in an homogenate of brain tissue. A lower molecular weight protein also is faintly visible in the Western blot. This is probably a fragment of PN-1. The polyclonal antibody used in this study has been shown to react with PN-1-protease complexes.16 The quantities of these complexes are very low in the human brain and is probably related to the fact that PN-1 -protease complexes are rapidly internalized and degraded by the same cells that secrete PN-1.5
Immunocytochemical Findings Both monoclonal and polyclonal antibodies for PN-1 gave strong PN-1 immunoreactivity in cryostat sections of hu-
744 Choi et al AjP October 1990, Vol. 13 7, No. 4
V-X
Figure 2. Photomicrographs showing indirect immuno]luorescence of PN-1 and GFAP in the cerebral cortex of adult human temporal lobe. a: Note strong PN- 1 im-
muno]luorescence of linear profiles in the
cerebral cortex that appear to be associated with astrocytic processes (arrows)
(X220). b: Photomicrograph of negative control (X220). cd: Double labeling of PN1 (Qluorescein) and GFAP (rhodamine) showing expression of PN- 1 in GFAP-positive astroglial processes (arrows). The processes extend into the capillarjy wall (V) (X440). ef: Double labeling of PN-1 and GFAP showing an astroglial cell (white arrow) extending two processes (arrows) into a medium-sized arteriole (V) showing
PN-1 immunofluorescence (X440). g,h: Double labeling of PN-1 (rhodamine) and GFAP (fluorescein) demonstrates that GFAP-positiz'e astrocyte also express PN-1 in the cytoplasm and its processes (arrows). White arrou'points to the nucleus of an astrocyte (X 440).
cerebral cortex with the use of the indirect immunofluorescent technique. The pattern of PN-1 immunofluorescence using monoclonal antibody (MAbp 30) was identical in all samples obtained from three different cases. Within the cerebral cortex, immunoreactivity was present within fibrillar processes that appeared to be associated with astroglial cells (Figures 2a, g). These cells extended their processes toward small capillaries and arterioles and appeared to form perivascular endfeet (Figures 2c-f). Double labeling for PN-1 and GFAP demonstrated coexpression of PN-1 and GFAP in the same astroglial cells (Figures 2g, f) and processes (Figures 2c-h). It should be emphasized that in all of the immunocytochemical studies there was no significant staining in control sections incubated in the absence of the primary antibody (Figure 2b). Immunoperoxidase staining for PN-1 in man
paraffin and vibratome sections fixed in 2% paraformaldehyde was only possible using polyclonal antisera. The presence of PN-1 immunoreactivity in the walls of capillaries and arterioles was well demonstrated by this method. Immunoreactive astroglial cells extending processes toward capillaries that showed immunoreactivity for PN-1 also were observed (Figure 3a). At higher magnification, the presence of specific PN-1 immunoreactivity was noted in the smooth muscle cells of arteries and arterioles both in the parenchyma and in the subarachnoid space (Figure 3b). Formalin-fixed and paraffin-embedded sections consistently gave negative results however.
In situ Hybridization As shown in Figure 4, intense labeling following the application of an antisense 35S-RNA probe for PN-1 was pres-
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Figure 3. Immunoperoxidase staining using a polyclonal antiserum for PN-1 in tissuesfixed in 2% paraformaldehyde. a: Note immunoperoxidase-positivle PN-1 in the walls ofsmall blood vessels (large arrows). Note also immunoreactive PN-1 in the cytoplasm ofastrocytes (arrow heads). An astrocytic process (small arrow) extends toward a small capillary that is also immunoperoxidase positivle for PN- 1 (X220). b: Photomicrograph showing intense PN-1 immunoreactivity in the smooth muscle cells (arrows) of the blood vessel in the subarachnoid space (X 440). Figure 4. In situ
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hybridization using 35S-labeled riboprobes for PN-1. a-c: Intense labeling of an antise'nse 35S-RNA PN-1 probe is noted in the vascular walls (empty arrows). Closed arrow in a points to a labeled astrocyte. (X440). d,e: Astrocytes (arrows) show strong labeling ofan antisense 35S-RNA PN1 probe over their nuclei. These cells were identified on the basis of general topographj, round to ovoid nuclei measuring 10 to 20 Am in diameter containing finely granular and evenly distributed chromatin and absence of distinct nucleoli, all of which are consistent with astrocytes rather than neurons, oligodendrocytes, or microglial cells (d: X220, e: X440). f: An adjacent conttrol section labeled with sentse 35S-RNA PN- 1probe shows only sparse backgrounid grains. Arrows poinlt to astrocytic niuiclei devoid of labeling. Ani empty arrouw points to a blood vesselshowing tno labelintg (X220).
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ent in the walls of blood vessels (Figures 4a-c) and in astrocytes (Figures 4d, e), while adjacent sections processed as controls using sense 35S-RNA PN-1 probe showed only sparse background grains (Figure 4f). These studies showed that the same brain cells and structures that contained PN-1 also contained PN-1 mRNA.
Discussion The present results show that much of the PN-1 and its mRNA in adult human brain are localized in and around blood vessels. This, along with results of several recent studies, suggest that it may play a protective role against extravasated prothrombin that would be converted to active thrombin. Thus it seems likely that PN-1 in the cerebral blood vessels functions as a thrombin inhibitor. Thrombin, in addition to playing a central role in the blood clotting system, is a serine protease with diverse bioregulatory activity. For example, it brings about retraction of neurites in cultured neuroblastoma cells,13-14 produces changes in astrocytic morphology,"5 and reverses morphologic differentiation of heterogeneous cells cultured from various regions in the human fetal brain.22 In each of these cell systems, thrombin, but not other proteases tested, brings about modification of cellular processes. More importantly, this occurs at thrombin concentrations that are
about five or six orders of magnitude lower than prothrombin concentrations in plasma. Thus minute amounts of extravasated thrombin have the potential to cause central nervous system (CNS) injury by altering neuronal and glial processes in a manner that may result in disruption of their function. Indeed such a mechanism may be operative in the pathogenesis of certain neurodegenerative diseases. In the brains of subjects with AD, for example, it has been reported 1) that neuritic plaques frequently cluster along the walls of blood vessels, 2) that blood-brain barrier function is compromised,23 25 and 3) that there is a significant reduction in active PN-1 levels and an increase in the levels of PN-1-thrombin complex.16 Thrombin also is a potent mitogen for astrocytes.1526 All of these findings underscore the importance of a protective mechanism to inactivate even minute amounts of thrombin that may enter the brain when the blood-brain barrier function is compromised. Although the manner in which PN-1 is localized within the brain suggests a role in blood-brain barrier function, it should be emphasized that PN-1 localization around blood vessels in other organs has not been investigated. The present studies also demonstrated the presence of significant amounts of PN-1 and its mRNA on astrocytes and their processes. The localization of PN-1 on perivascular astroglial endfeet tightly abutting the basal lamina of blood vessels is particularly significant. It sug-
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gests that PN-1 on the surfaces of astroglial processes in close association with the basal lamina of the vascular wall would be well suited to provide additional protection against extravasated thrombin. The PN-1 in astrocytes and their processes also may play significant roles in cellular events during neurogenesis, such as neuronal migration and final positioning of postmigratory neurons. It is known that migrating cells have more cell-surface proteolytic activity than stationary, differentiated cells. For example, it has been reported that dissociated cerebellar cultures possess plasminogen activator during active granule cell migration24 but not when granule cell migration is complete.27 It also has been reported that highly specific inhibition of external granule cell migration can be brought about by glia-derived nexin (PN-1) in explant cultures of cerebellum.28 The presence of PN-1 on astroglial processes is especially significant in this regard because radial glia are known to provide guidance for neuronal migration2934 and because radial glia give rise to astrocytes and express GFAP in humans and subhuman primates.3035 Although it is recognized that PN-1 bound to astrocytic cell surfaces is a specific thrombin inhibitor,8 it is possible that PN-1 secreted by astrocytes may be present in the extracellular spaces within the neuropil and interact with other serine proteases, such as urokinase, or that thrombin and/or thrombinlike proteases may be active within the neuropil during certain stages of normal or abnormal brain development. Thus it is possible that interplay between proteases and PN-1 may not only influence intricate neuronal-glial interactions and cellular events during neurogenesis but also may play crucial roles in the constant restructuring of neurites during development or in repair of injury in the nervous system. In this context it is significant that glia-derived nexin and its mRNA were detected in the olfactory system of the rat where continuous degeneration and regeneration of the CNS is taking place throughout life.36 Most of the glia-derived nexin in the rat olfactory system is thought to be synthesized and secreted by nonneuronal cells.6 Glia-derived nexin also may have a role in axonal regeneration of the peripheral nervous system. This was suggested by a study in which induction of glia-derived nexin was observed following an injury to a peripheral nerve.37 In summary, localization of PN-1 in brain blood vessels and astroglial cells suggests that it may play a major protective role against the extravasation of thrombin and possibly other serine proteases.
References 1. Baker JB, Low DA, Simmer RL, Cunningham DD: Proteasenexin: A cellular component that links thrombin and plasmin-
ogen activator and mediates their binding to cells. Cell 1980, 21:37-45 2. Eaton DL, Baker JB: Evidence that a variety of cultured cells secrete protease nexin and produce a distinct cytoplasmic serine protease-binding factor. J Cell Physiol 1983, 117: 175-182 3. Rosenblatt DE, Cotman CW, Nieto-Sampedro M, Rowe JW, Knauer DJ: Identification of a protease inhibitor produced by astrocytes that is structurally and functionally homologous to human protease nexin-1. Brain Res 1987, 415:40-48 4. Scott RW, Bergmann BL, Baipai A, Hersh RT, Rodrigues H, Barreda BN, Watts S, Baker JB: Protease nexin. Properties and a modified purification procedure. J Biol Chem 1985, 260:7029-7034 5. Low DA, Baker JB, Koonce WC, Cunningham DD: Released protease-nexin regulates cellular binding, internalization, and degradation of serine proteases. Proc Natl Acad Sci USA 1981, 78:2340-2344 6. Farrell DH, Wagner SL, Yuan RH, Cunningham DD: Localization of protease nexin-1 on the fibroblast extracellular matrix. J Cell Physiol 1988,134:179-188 7. Farrell DH, Cunningham DD: Human fibroblasts accelerate the inhibition of thrombin by protease nexin. Proc Natl Acad Sci USA 1986, 83:6858-6862 8. Wagner SL, Lau AL, Cunningham DD: Binding of protease nexin-1 to the fibroblast surface alters its target proteinase specificity. J Biol Chem 1989, 264:611-615 9. Gloor S, Odink K, Guenther J, Nick H, Monard D: A gliaderived neurite promoting factor with protease inhibitory activity belongs to the protease nexins. Cell 1986, 47:687-693 10. McGrogan M, Kennedy J, Li MP, Hsu C, Scott RW, Simonsen CC, Baker JB: Molecular cloning and expression of two forms of human protease nexin 1. Bio/technol 1988,6:172-177 11. Monard D, Niday E, Limat A, Solomon F: Inhibition of protease activity can lead to neurite extension in neuroblastoma cells. Prog Brain Res 1983, 58:359-364 12. Zurn AD, Nick H, Monard D: A glia-derived nexin promotes neurite outgrowth in cultured chick sympathetic neurons. Devl Neurosci 1988,10:17-24 13. Gurwitz D, Cunningham DD: Thrombin modulates and reverses neuroblastoma neurite outgrowth. Proc Natl Acad Sci USA 1988,85:3440-3444 14. Gurwitz D, Cunningham DD: Neurite outgrowth activity of protease nexin-1 on neuroblastoma cells require thrombin inhibition. J Cell Physiol 1990,142:155-162 15. Cavanaugh K, Gurwitz D, Cunningham D, Bradshaw R: Reciprocal modulation of astrocyte stellation by thrombin and protease nexin-1. J Neurochem 1990, 54:1735-1743 16. Wagner SL, Geddes JW, Cotman CW, Lau AL, Gurwitz D, Isackson PL, Cunningham DD: Protease nexin-1, an antithrombin with neurite outgrowth activity, is reduced in Alzheimer's disease. Proc Natl Acad Sci USA 1989,86:8284-8288 17. Wagner SL, Van Nostrand WE, Lau AL, Cunningham DD: Monoclonal antibodies to protease nexin 1 that differentially block its inhibition of target proteases. Biochem 1988, 27: 2173-2176
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18. Van Nostrand WE, Wagner SL, Cunningham DD: Purification of a form of protease nexin 1 that binds heparin with a low affinity. Biochem 1988, 27:2176-2181 19. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227: 680-685 20. Coons AH, Kaplan MH: Localization of antigen in tissue cells. ll. Improvements in a method for the detection of antigen by means of fluorescent antibody. J Exper Med 1950, 91:1-13 21. Sternberger LA: Immunocytochemistry. New York, Wiley, 1979, pp 104-169 22. Grand RJA, Grabham PW, Gallimore MJ, Gallimore PH: Modulation of morphological differentiation of human neuroepithelial cells by serine proteases: Independence from blood coagulation. EMBO J 1989, 8:2209-2215 23. Hardy JA, Mann DMA, Wester P, Winblad B: An integrative hypothesis concerning the pathogenesis and progression of Alzheimer's disease. Neurobiol Aging 1986, 7:489-502 24. Wisniewski HM, Kozlowski PB: Evidence for blood-brain barrier changes in senile dementia of the Alzheimer type (SDAT). Ann NY Acad Sci 1982, 396:119-129 25. Delacourte A, Defossez A, Persuy P, Peers MC: Observation of morphological relationships between angiopathic blood vessels and degenerative neurites in Alzheimer's disease. Virchows Arch A 1988, 411:199-204 26. Perraud F, Besnard F, Sensenbrenner M, Labourdette G: Thrombin is a potent mitogen for rat astroblasts but not for oligodendroblasts and neuroblasts in primary culture. Int J Devi Neurosci 1987, 5:181-188 27. Krystosek A, Seeds NW: Plasminogen activator secretion by granule neurons in cultures of developing cerebellum. Proc Natl Acad Sci USA 1981, 78:7810-7814
28. Moonen G, Gran-Wagemans M, Selak I: Plasminogen activator-plasmin system and neuronal migration. Nature 1982, 298:753-755 29. Linder J, Guenther J, Nick H, Zinser G, Antonicek H, Schachner M, Monard D: Modulation of granule cell migration by a glia-derived protein. Proc Natl Acad Sci USA 1986, 83:4568-4571 30. Choi BH: Glial fibrillary acidic protein in radial glia of early human fetal cerebrum. A light and electron microscopic immunoperoxidase study. J Neuropathol Exper Neurol 1986, 45:408-418 31. Choi BH: Developmental events during the early stages of cerebral cortical neurogenesis in man. A correlative light, electron microscopic, immunohistochemical and Golgi study. Acta Neuropathol (Berl) 1988, 75:441-447 32. Choi BH, Lapham LW: Radial glia in the human fetal cerebrum: A combined Golgi, immunofluorescent and electron microscopic study. Brain Res 1978,148:295-311 33. Choi BH, Matthias SC: Cortical dysplasia associated with massive ectopia of neurons and glial cells within the subarachnoid space. Acta Neuropathol (Berl) 1987, 73:105-109 34. Rakic P: Mode of cell migration to the superficial layers of fetal monkey neocortex. J Comp Neurol 1972,145:61-84 35. Levitt P, Rakic P: Immunoperoxidase localization of glial fibrillary acidic protein in radial glial cells and astrocytes of the developing rhesus monkey brain. J Comp Neurol 1980,193:
815-840 36. Reinhard E, Meier R, Halfter W, Rovelli G, Monard D: Detection of glia-derived nexin in the olfactory system of the rat. Neuron 1988,1:387-394 37. Meier R, Spreyer P, Ortmann R, Harel A, Monard D: Induction of glia-derived nexin after lesion of a peripheral nerve. Nature 1989, 342:548-550
