tkuron,

Vol. 6, 575-581,

April,

1991, Copyright

0 1991 by Cell Press

rothrombin mRNA Is Expressed y Cells of the Nervous System Melitta Dihanich,* Matthew Kaser,+ Eva Dennis Cunningham,+ and Denis Monard* ’ Friedrich Miescher Institute CH-4002 Base1 ‘iwitzerland ‘Department of Microbiology and Molecular Genetics IJniversity of California Irvine, California 92717

Reinhard,*

Summary Thrombin, a serine protease of the blood coagulation system, has additional effects on cells in vitro. It is mitogenie for fibroblasts and astrocytes and contributes to the regulation of neurite outgrowth and astrocyte stellalion. Until now the expression of thrombin or its precursor prothrombin in tissues other than liver has not been demonstrated conclusively because of difficulty in avoiding serum contamination. Using sensitive mRNA detection methods, we show here that prothrombin is expressed not only in the liver, but also in the brain throughout development. Polymerase chain reaction, Northern, and in situ hybridization studies demonstrate the presence of prothrombin transcripts in the olfactory bulb, the cortex, the cerebellum, and other regions of the rat and human nervous system, as well as in neural cell lines. These results support an involvement of (pro)thrombin in the regulation of cellular events in the nervous system. Introduction Thrombin is a serine protease of the blood coagulation system. The synthesis of its precursor, prothrombin, in the liver is followed byglycosylation, secretion into the bloodstream, and subsequent posttranslational modification (Fenton, 1987). The cleavage of prothrombin to mature thrombin is part of a cascade of proteolytic events leading to fibrinogen activation (by thrombin itself) and blood clotting (Davie et al., 1979). In addition to its role in blood coagulation, thrombin has pronounced effects on cellular activation in vitro: it is mitogenic for fibroblasts and astrocytes (Chen and Buchanan, 1975; Cavanaugh et al., 1990), inhibits neurite outgrowth of neuronal cells (Monard et al., 1983; Gurwitz and Cunningham, 1988; Srand et al., 1989), and reverses the stellation of astrocytes (Cavanaugh et al., 1990). These data, together with the presence of serine protease inhibitors in the brain (Gloor et al., 1986; Abraham et al., 1988; Kitaguchi et al., 1988; Ponte et al., 1988; Reinhard et al., 1988; Tanzi et al., 1988), lead to the question of whether there is expression of any thrombin-like serine proteases in the brain. The present studies have addressed this point and have led to the finding of

prothrombin mental stages.

mRNA

in

rat

brain

at various

develop-

Results To obtain evidence for thrombin or a thrombin-like protease, we analyzed rat brain for prothrombin mRNA.Thisapproachwasemployed sincedirectmeasurements of (pro)thrombin could not exclude blood as a source of the protein. We prepared a pair of degenerate oligonucleotide primers corresponding to sequences conserved among serine proteases (Furie et al., 1982). The use of these in the polymerase chain reaction (PCR) with rat liver cDNA as a template yielded one major DNA fragment of 450 bp whose sequence was highly homologous to human thrombin. The same approach produced an identical thrombin fragmentwith cDNAfrom the brain of a 20-day-old (E20) rat embryo. No other sequences for thrombinlike proteases were found. With this 450 bp probe, we isolated and sequenced afull-length cDNAclone from a rat liver library in hZapll (Stratagene). The sequence encodes an open reading frame of 1851 bp (Dihanich and Monard, 1990) and has 80.9% identitywith human preprothrombin (Friezner Degen and Davie, 1987). To find out whether the prothrombin mRNA expressed in brain was the same as the one expressed in liver, DNA sequences obtained from brain and liver were compared; no significant differences were found. PCR was performed with cDNAs from various sources, including human brain and human neural cell lines. The expression of prothrombin mRNA in rat brain was detectable already early in development (E13), decreased to a minimum at birth, and increased after birth until adulthood (Figures la and lb). Prothrombin mRNA was also expressed in human brain and by both neuronal and glial cell lines of rat and human origin (Figure lb; Figure 2). Quantitative PCR was performed bycoamplification of the test template with a mutagenized control template to determine the relative amounts of prothrombin transcripts expressed by the different cell types (Table 1). This showed that the expression of prothrombin mRNA in brain and by neural cell lines was about 1% or less than the amount produced in liver. Similar results were obtained with Northern blots of poly(A)+-selected RNA from rat brain and rat neuroblastoma cells (Figure 3). Prothrombin transcripts of 2.3 kb were detected in liver from adult rats, as well as brains from El3 embryos and postnatal day 5 (P5) rats (Figures 3a and 3b). In addition, another prothrombin-related transcript of about 10 kb was found in brain. This transcript was identified as a partially spliced precursor of prothrombin mRNA, since it hybridized to both 5’and 3’end probes of the prothrombin cDNA (data not shown) and was enriched in nuclei

Neuron 576

a bp

b

BRAIN LIVER 133 17E 19E 20E 1PN 21PN p(A+)

bp

EPABCL

Figure

1. Prothrombin

mRNA

Is Expressed

in Rat Brain

throughout

Development

as well

as by Rat Neuronal

and Glial

Cell

Lines

(a) A 5% polyacrylamide gel with PCR products (224 bp prothrombin fragment, 506 bp B-actin fragment) from prothrombin primers 5’-GCATCCGTGCACAGCCAGCATCTCTFCCTG-3’ (sense) and 5’-GGATCCTCACAACCTGTGTACTFGGCCCAGAA-3’ (antisense: these primers bind to the Send of the prothrombin cDNA and also measure partially spliced prothrombin transcripts, but not genomic DNA) and from 8-actin primers (5’~CATACCGATCCAAGGCCCCCTTCCCGCGC-3’and S-GATAGGTCGACACGTCCCCCCCAGCCAGCCAGGTC-3’) after 50 and 25 cycles, respectively. Sources of cDNA templates are as follows: lanes labeled 13E, 17E, 19E, and 20E represent brains of 13-, 17-, 19-, 20day-old embryos; lanes labeled IPN and 21 P represent brains of PI and P21 rats (1 ng each); liver (0.001 ng). A densitometric scan of the gel yielded the following ratios of prothrombin@-actin products: 0.62 (13E), 0.75 (17E), 0.30 (19E), 0.08 (20E), 0.15 (IPN), 0.24 (21PN), and 0.60 (liver x 10-j). (b) Southern blot of PCR products obtained with rat prothrombin primers 5’-TACCTCAGGGCCCTGCT-3’ (nucleotides 492-508, sense) and S-CGTCCTCCCATCGACTCGTC-3’ (nucleotides 929-910, antisense) hybridized with 32P-labeled human prothrombin probe. These primers discriminate between the prothrombin transcripts and give a 437 bp product only with the mature mRNA. Sources of cDNA templates: E, E20 brain; P, P5 rat brain; A, adult rat brain; B, 8104 rat neuroblastoma cells; C, C6 rat glioma cells; L, adult rat liver.

isolated from neuroblastomacells (Figure 3c, PTP2), in which a full-length precursor of about 20 kb was also found (Figure 3c, PTP& Rat brain sections of various developmental stages were used for in situ hybridization with equal amounts (counts per minute) of prothrombin cDNA or control

CEHI3SOL-A

Figure2. Expression of Prothrombin by Human Cell Lines

mRNA

in Human

Brain and

Southern blot of PCR products with human cDNA templates. Hybridizationwas with a450 nucleotide 12P-labeled rat prothrombin probe (nucleotides 1252-1702). C, cortex; E, cerebellum; H, hippocampus; I, 118lNI human glioma cells; 3, LN-340 human glioma cells; S, SK-N-SH human neuroblastoma cells; 0, PCR without template (control); L, HepC2 human hepatoma cells; A, adult rat brain. All PCR products shown are derived from cDNA and hence from mRNA, since amplification of genomic DNA would yield larger products with all pairs of primers.

DNA probe (plasmid CEM3; Figure 4; Figure 6), as well as with antisense and sense RNA probes (Figure 5). In contrast to later developmental stages, El6 embryos were not perfused and gave higher background with control probes than perfused brains of later stages. As expected, liver was strongly positive (Figures 4a and 4b). Most regions of the brain and dorsal root ganglia were stained above background (Figures 4a4d; Figures6e and 6f). After birth, prothrombin mRNA was present in the olfactory bulb, the cortex, the coiliculus superior and inferior, the corpus striatum, and the thalamus (Figures 4e-4m). The substantia nigra and the nucleus subthalamicus were also stained, as well as a few cholinergic nuclei (Figures 4e and 4g). Figure 6 demonstrates the presence of prothrombin transcripts in cell bodies of the hippocampal formation and of the deep cerebellar nuclei in the brains of a P21 and a P4 rat, respectively. In the adult brain, in which neurite outgrowth ceased, prothrombin transcripts were still expressed (Figure 41; Figure 5). The expression pattern of prothrombin transcripts was similar to the staining pattern obtained with cresyl violet on adjacent sections (data not shown) and to that found for the neuronal and glial cell marker protein MAP1 and the neuronal cell marker protein MAP2 (Tucker et al., 1989); it differed significantly from the

llrain Prothrombin 1.77

mRNA

lable 1. Amounts of Mature Prothrombin i.1 Rat Organs and Rat Neural Cell Lines (Iells

Absolute (pmol/pg

Amount’ of Poly(A)’

Adult liver E20 brain P5 brain 8104 celIsc C 6 cell@

2.5-5 x IO-* 1.8-3.5 x 1O-4 3.5 x 10-a 1.7-3.5 x 10-a 1.4-2.8 x IO-’

mRNA

opmental stages. Most regions of the brain express prothrombin transcripts in situ; however, the white matter does not. The in situ hybridization pattern alone points toward mainly neuronal expression. However, considering the in vitro data, an expression by both neurons and glial cells cannot be ruled out. Possibly,

Expressed

Relative Amountb (% of Liver)

RNA)

100 0.8

1

the actual amount

0.8 0.5

iological state of the various cells, and same type might therefore be prothrombin and negative within the same tissue.

The primers used here (same as in Figure lb) amplify a 437 bp product with mature 2.3 kb mRNA only (mRNA precursors or genomic DNA give larger products). a Mean values of three experiments. b Calculated from the average of absolute values. j Rat neuroblastoma cell line. d Rat glioma cell line.

expression (data not prothrombin

pattern of glial fibrillary shown), which excludes by glial cells alone.

an

acidic protein expression of

Discussion Although the biochemistry of thrombin and its precursor prothrombin has been studied for decades, very little is known about their expression and regulation at the mRNA level. Until now, the synthesis of

prothrombin

was believed

liver (Fenton, mRNA is also

1987). Here we show that prothrombin expressed in the brain at various devel-

a

El3

L

to occur exclusively

b

P5 L

in the

of expression

depends

on the physcells

of the positive

Our finding of prothrombin mRNA expression in the brain has important implications. Prothrombin itself might have biological activity (Grand et al., 1989); it is also conceivable that it is activated locally by either factor X (via the intrinsic or extrinsic pathway; Davie et al., 1979) or another, unknown mechanism. Functionally, thrombin along with its inhibitor glia-derived nexinlprotease nexin 1 (GDNIPN-1; rat/human homolog) could regulate several activities of neural cells. The balance of thrombin and GDNIPN-1 can control neurite outgrowth (Monard et al., 1983; Guenther et al., 1985; Gurwitz and Cunningham, 1988) and astrocyte proliferation and stellation (Cavanaugh et al., 1990). Theeffectsof thrombin could result from directaction on cells or the extracellular matrix (McGuire and Seeds, 1990). Coexpression of thrombin and CDN could contribute to the adhesion and de-adhesion of filopodia and microspikes to extracellular matrix, thus supporting growth cone movement (Monard, 1988). Thus, it is important to understand possible sources of thrombin

in the

brain.

One

C

P5L

source

1234

is injury,

resulting

in

567

PTPPTP28S-

28SPT PT

-

-

-A

PT -

Figure 3. Brain-Derived Prothrombin Transcripts Are Also Detected on Northern Blots (a) Comparison of poly(A)+ RNA from El3 embryos (E13; 5 pg) and from liver (L; 0.1 pg). (P5; 9 pg) and from liver (L; 0.1 pg). (c) Prothrombin RNA precursors in isolated nuclei: neuroblastoma nuclear poly(A)’ RNA; lanes 4 and 7,0.1 pg of liver poly(A)+ RNA; lane 5,5 Hybridization probes were (a) full-length prothrombin riboprobe, (b) 450 nucleotide 450 nucleotide riboprobe. PTPI, ~20 kb prothrombin precursor; PTP*, 8-10 kb prothrombin A, actin mRNA (2 kb); 28S, large ribosomal RNA.

(b) Comparison of poly(A)‘- RNA from P5 rats lane 1, 5 wg; lane 2, 3 Hg; lane 3, 1 pg of 8104 pg; lane 6,2 fig of poly(A)’ RNA from P4 brain. cDNA probe (nucleotides 1252-1702), and (c) precursor; PT, prothrombin mRNA (2.3 kb);

Neuron 570

h h

Figure

4. In Situ Hybridization

of 35S-Labeled

Rat Prothrombin

cDNA

Probes

to Sections

of Rat Brain at Various

Developmental

Stages

(a and b) Sagittal section through an El6 embryo. (c and d) Coronal section through the head of an El6 embryo. (e and f) Coronal section through a P4 rat brain. (g) Coronal section of a P21 rat brain. (h) Scheme of coronal section. (i-m) Sagittal sections of (i and j) P11 rat brain, (k) P21 rat brain, and (I) adult brain. (m) Scheme of rat brain in sagittal section. (a, c, e, g, j, k, and I) Hybridization with a rat prothrombin probe (same as in Figure 3b, but at a fragment length of 1utJ nucleotides). (b, d, f, and i) Control (vector GEM3). Abbreviations: bo, bulbus olfactorius; co, cortex; cc, corpus callosum; ce, cerebellum; ci, colliculus inferior; cs, colliculus superior; gd, gyrus dentatus; h, hippocampus; ns, nucleus subthalamicus; s, corpus striatum; sn, substantia nigra; t, thalamus.

the conversion of plasma prothrombin to thrombin (Wagner et al., 1989). Our present results indicate that synthesis of prothrombin by brain cells might be a physiological source of thrombin, which could regulate their growth and development. Indeed, the expression of prothrombin mRNA correlates with that of GDN mRNA in the olfactory bulb (Reinhard et al., 1988), cortex, cerebellum, substantia nigra, nucleus

subthalamicus, and afewcholinergic nuclei (Reinhard et al., unpublished data). Both thrombin and GDN are regulated (Fenton, 1981; Meier et al., 1989): CDN, at the transcriptional level, and thrombin at possibly both the transcriptional and the posttranslational level. Our finding of a partially spliced prothrombin mRNA precursor suggests splicing as an additional regulatory mechanism

Brain Prothrombin 579

mRNA

r’sense

Figure

5. In Situ

The probes were (a and b) Coronal sections through

Hybridization

with

%-Labeled

Antisense

and

Sense

RNA

Probes

generated from the same prothrombin cDNA fragment as used in Figure 1. sections of adult rat brain (A car). (c and d) Sagittal sections of adult rat brain (A sag). The arrows an isolated olfactory bulb.

in (a) and (b) mark

Figure 6. In Situ Hybridization thrombin cDNA Probe to Sagittal of Rat Brain

of ProSections

Hybridization was with the same probes as used in Figure4. (a and b) Cerebellum of a P4 rat. (c and d) Hippocampal formation of a P21 rat. (e and f) Dorsal root ganglia of an El6 rat. (a, c, and e) Dark-field micrograph (stained cells appear white); (b, d, and 0 corresponding nuclear staining with Hoechst. Abbreviations: drg, dorsal root ganglia; h, hippocampal pyramidal cells; gd, gyrus dentatus; cn, cerebellar nuclei; ct. cartilage; wm, cerebellar white matter.

Neuron 580

of prothrombin biosynthesis in the brain. A breakdown of this regulation and resulting changes in the levels of this protease might contribute to the pathology of neurodegenerative diseases such as Alzheimer’s (Wagner et al., 1989; Sisodia et al., 1990). We are currently studying prothrombin regulation to test this hypothesis. Experimental

Procedures

Ceils LN-340 human glioma cells (Piguet et al., 1985) were grown in Dulbecco’s modified Eagle’s medium with 5% fetal calf serum; all other cell lines were grown in the same medium containing 10% fetal calf serum. Rat brain samples for preparation of mRNA were from Sprague-Dawley rats. Human samples from a 79-yearold man (2 hr postmortem) were obtained from the Institute of Pathology, University of Basel. Isolation of mRNA and PCR Poly(A)+ RNA (1 ug; Chomczynski and Sacchi, 1987; Maniatis et al., 1982) was reverse-transcribed as described by Frohman et al. (1988) usinga(dT),rprimer and SuperRT(Stehelin). Rat prothrombin transcripts were selectively amplified by PCR (35 cycles: 1 min at 94OC, 2 min at 55’C or .51°C, 3 min at 72OC) using rat prothrombin primers with cDNA templates (1 ng) transcribed from poly(A)’ RNA of different organs or cell lines and AmpliTaq polymerase (Perkin Elmer Cetus, Stratagene). Southern blots with these PCR products were hybridized with a 32P-labeled human prothrombin probe (5 x 106 cpm). For quantification (Gilliland et al., 1990), varying amounts of mutagenized control template (generated by introduction of an EcoRl site at nucleotide 751) were included, and PCR products were digested with EcoRl prior to gel electrophoresis. For amplification of human prothrombin transcripts, primers 5’-GGTGCCCATTGCCAACCAC-3’ (nucleotides 1269-1287) and 5’.AGGGTCCCCCACTGTCAC-3’ (nucleotides 1714-1697, antisense) were used with human cDNA templates (2 ng). Hybridization was with a 450 nucleotide32P-labeled rat prothrombin cDNA probe (nucleotides 1252-1702). Northern Hybridization Poly(A)” RNA prepared as described above was size-fractionated on denaturing I%-1.2% agarose, 6% formaldehyde gels and transferred to nitrocellulose. The filters were either hybridized with 3ZP-labeled riboprobes (specific activity IO9 cpmlug) at high stringency (50% formamide, 65OC) and washed in 0.2x SSC, 0.1% SDS at 65OC, or hybridized with 3*P-labeled cDNA probes (specific activity 5 x 108 cpmlug, 5 x IO5 cpmlml) in a hybridization buffer containing 0.5 M sodium phosphate (pH 7.2), 5% SDS, 1% bovine serum albumin, and 1 mM EDTA (pH 8) at 66°C (Mahmoudi and Lin, 1989). After washing in 40 mM sodium phosphate (pH 7.2), 1 mM EDTA (pH 8), 1% SDS, the filters were exposed on XAR-5 films (Kodak) for l-6 days (RNA probes) or for 7-10 days (DNA probes) between two intensifying screens. In Situ Hybridizations In Situ Hybridization with DNA Probes Sprague-Dawley rats (all stages except E16, which were fixed by immersion in the fixative) were perfused intracardially with 2% paraformaldehyde in PBS. Isolated brainswere immersed in 20% sucrose, PBS overnight at 4OC. Cryostat sections (IO-14 urn) were collected on polylysine-treated slides. Slides were washed in 4x SSC, 1 x Denhardt’s solution for 1 hr and prehybridized in 50% formamide, 4x SSC, 0.12 M phosphate buffer (pH 7), 0.65% Sarcosyl, 1 x Denhardt’s solution, 7.5% dextrane sulfate, 56 ug/ml calf thymus DNA, 1.125% B-mercaptoethanol for 2 hr at room temperature. Hybridization was in the same solution containing 1 ng of ‘SS-labeled probe (nucleotides 1252-1702 of rat prothrombin) per 100 ul at a specific activity of 5 x 108 cpmlug (fragment length -100 nucleotides) or the same amount of control probe (GEM3, the host vector of our prothrombin cDNA). Following

hybridization, slides were washed in 4x SSC 0.15% 8-mercaptoethanol (for 10 min) and 1 x SSC 0.15% B-mercaptoethanol (for 1 hr at room temperature and for 1 hr at 43OC). Dried sections were exposed on Amersham B Max films. For single-cell analysis, sections were coated with llford K5 emulsion. In Situ Hybridization with RNA Probes Cryostat sections (14 urn) were collected on slides treated with 3aminopropyltriethoxysilane, postfixed in 2% paraformaldehyde, PBS for IO min, washed in PBS for 5 min, immersed in 0.2 N HCI for 20 min at room temperature and in 2x SSC at 65°C for 30 min, and dehydrated in 65%, 75%, 85%, and 99% ethanol. Slides were prehybridized for 2 hr in 50% formamide, 10% dextrane sulfate, 10 mM Tris-HCI (pH 6.8), 10 mM sodium phosphate (pH 6.8), 0.3 M NaCl, 1 x Denhardt’s solution, 1 mglml E. coli tRNA, 20 mM dithiothreitol. Slides were hybridized overnight in the same solution containing 5 x IO6 cpmlml 35S-labeled riboprobe (transcribed from the same 450 bp prothrombin template described above and treated with sodium carbonate to generate 100 nucleotide fragments) at 54’C. Slides were then washed twice in 50% formamide, 0.3 M NaCI, 20 mM Tris (pH 6.8), 10 mM sodium phosphate (pH 6.8), 5 mM EDTA, 20 mM dithiothreitol for 30 min at 55OC. Washing was continued in STE (0.5 M NaCI, IO mM Tris [pH 7.51, 1 mM EDTA, 20 mM dithiothreitol) for 15 min at 37”C, in STE with 5 ug/ml RNAase A for 30 min at 37OC, in STE for 15 min and twice for 30 min in 2x SSC and 0.2x SSC at 37OC before dehydration in an increasing ethanol series containing 300 mM ammonium acetate. Acknowledgments We would like to thank Sara Kozma for the rat liver cDNA library (Stratagene); Werner Zuercher and Franz Fischer for oligonucleotide synthesis; Juerg Ullrich (Institute for Pathology, University of Basel) for the human brain samples; Christoph Nager and Martin Spiess for help with the computer work; Alicia Bleuel and Johnny Suidan for help with the animal work; Magda Rentsch, and Elisabeth Fries for technical help; and Pica Caroni, Matthias Chiquet, Jan Hofsteenge, Paul Isackson, Hans-Peter Saluz, Martin Schwab, Andrew Wallace, Steven L. Wagner, Bernhard Wehrle, and our colleagues for helpful discussions and reading of the manuscript. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact Received

July 18, 1990; revised

December

26, 1990.

References Abraham C. R., Selkoe, D. J., and Potter, H. (1988). Immunochemical identification of the serine protease inhibitor ar-antichymotrypsin in the brain amyloid deposits of Alzheimer’s disease. Cell 52, 487-501. Cavanaugh, K. P., Gurwitz, D., Cunningham, D., and Bradshaw, R. A. (1990). Reciprocal modulation of astrocyte stellation by thrombin and protease nexin-I. J. Neurochem. 54, 1735-1743. Chen, blood Acad.

L. B., and Buchanan, J. M. (1975). Mitogenic components. I. Thrombin and prothrombin. Sci. USA 72, 131-135.

activity of Proc. Natl.

Chomczynski, P., and Sacchi, N. (1987). Single-step method RNA-isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 762, 156-159. Davie, E. W., Fujikawa, K., Kurachi, K., and Kisiel, role of serine proteases in the blood coagulation Enzymol. Relat. Areas Mol. Biol. 48, 277-318. Dihanich, thrombin. Fenton, 495.

M., and Monard, D. (1990). cDNA Nucl. Acids Res. 78, 4251. J. W., II (1981). Thrombin.

Ann.

W. (1979). The cascade. Adv.

sequence

NY Acad.

of

of rat proSci. 370, 46&

Fenton, J. W., II (1987). Structural regions and bioregulatoryfunctions of thrombin. In Control of Animal Cell Proliferation,

Vol.

Brain Prothrombin 581

mRNA

II, A. L. Boynton and H. L. Leffert, Press, Inc.), pp. 133-151.

eds. (New

York,

NY: Academic

Friezner Degen, S. J., and Davie, E. W. (1987). Nucleotide quence of the gene for human prothrombin. Biochemistry j165-6177.

encoded by an amyloid protein precursor mRNA Alzheimer’s disease. Nature 337, 528-530. se26,

associated

with

Tucker, R. P., Garner, C. C., and Matus, A. (1989). In situ localization of microtubule-associated protein mRNA in the developing and adult rat brain. Neuron 2, 1245-1256.

:‘rohman, M. A., Dush, M. K., and Martin, C. R. (1988). Rapid production of full-length cDNAs from rare transcripts: amplificafion using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 89989002.

Wagner, S. L., Ceddes, J. W., Cotman, C. W., Lau, A. L., Gurwitz, D., Isackson, P. J., and Cunningham, D. D. (1989). Protease nexin1, an antithrombin with neurite outgrowth activity, is reduced in Alzheimer’s disease. Proc. Natl. Acad. Sci. USA 86, 8284-8288.

Furie, B., Bing, D. H., Feldmann, R. J., Robinson, D. J., Buirnier, J. P., and Furie, B. C. (1982). Computer generated models of blood coagulation factor Xa, factor IXa, and thrombin based upon structural homology with other serine proteases. J. Biol. Chem. :‘57, 38753882.

Wallace, A., Rovelli, C., Hofsteenge, I., and Stone, S. (1989). Effect of heparin on the glia-derived nexin-thrombin interaction. Biothem. J. 257, 191-196.

Gilliland, C., Perrin, S., and Bunn, H. F. (1990). Competitive PCR for quantitation of mRNA. In PCR Protocols, M. A. Innis, D. H. (;elfand, J. J. Sninsky, and T. J. White, eds. (New York, NY: Academic Press, Inc.), pp. 60-69. Floor, S., Odink, K., Guenther, J., Nick, H., and Monard, D. (1986). A glia-derived neurite promoting factor with protease inhibitory activity belongs to the protease nexins. Cell 47,687-693. (irand, R. J. A., Grabham, P. W., Callimore, M. J., and Gallimore, F. H. (1989). Modulation of morphological differentiation of human neuroepithelial cells by serine proteases: independence ff.om blood coagulation. EMBO J. 8, 2209-2215. Guenther, J., Nick, H., and neurite-promotingfactorwith J 4, 1963-1966.

Monard, D. (1985). A glia-derived proteaseinhibitoryactivity. EMBO

Curwitz, D., and Cunningham, and reverses neuroblastoma Acad. Sci. USA 85, 3440-3444.

D. D. (1988).Thrombin neurite outgrowth.

Kitaguchi, N., Takahashi, Y., Tokushima, P. (1988). Novel precursor of Alzheimer’s tein shows protease inhibitory activity.

modulates Proc. Natl.

Y., Shiojiri, S., and Ito, disease amyloid proNature 337, 530-532.

Mahmoudi, M., and Lin, V. K. (1989). Comparison of two different hybridization systems in Northern transfer analysis. BioTechn:ques 7, 332-333. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982). Cloning: A Laboratory Manual (Cold Spring Harbor, Soring Harbor Laboratory). McCuire, P. G., and Seeds, N. W. (1990). Degradation ing extracellular matrix by sensory neurons during growth. Neuron 4, 633-642. Meier, R., Spreyer, P., Ortmann, (1989). Induction of glia-derived nerve. Nature 342, 548-550. Monard, D. (1988). Cell-derived tors as regulators of neurite 541-544.

Molecular NY: Cold of underlyneurite out-

R., Harel, A., and Monard, D. nexin after lesion of a peripheral proteases outgrowth.

and protease inhibiTrends Neurosci. 77,

Monard, D., Niday, E., Limat, A., and Solomon, F. (1983). Inhibition of protease activity can lead to neurite extension in neuroblastoma cells. Prog. Brain Res. 58, 359-364. Piguet, V., Diserens, A.-C., Carrel, S., Mach, J.-P., and deTribolet, N. (1985). The immunology of human gliomas. Springer Sem. Immunopathol. 8, 111-127. Ponte, P., Gonzalez-DeWhitt, P., Schilling, J., Miller, J., Hsu, D., Greenberg, B., Davis, K., Wallace, W., Lieberburg, I., Fuller, F., and Cordell, B. (1988). A new A4 amyloid mRNA contains a domain homologous to serine proteinase inhibitors. Nature 337, 525-527. Reinhard, E., Meier, R., Halfter, (1988). Detection of glia-derived thfa rat. Neuron 7, 387-394.

W., Rovelli, G., and Monard, nexin in the olfactory system

D. of

Sisodia, S. S., Koo, E. H., Beyreuther, K., Unterbeck,A., and Price, D. L. (1990). Evidence that B-amyloid protein in Alzheimer’s disease is not derived by normal processing. Science 248,492-495. Tanzi, R. E., McClatchey, Gusella, J. F., and Neve,

A. I., Lamperti, E. D., Villa-Komaroff, L., R. L. (1988). Protease inhibitor domain