Detection of glia-derived nexin in the olfactory system of the rat.

Glia-derived nexin (GDN) is a 43 kd cell-secreted protease inhibitor with neurite promoting activity. We have raised specific polyclonal antisera to rat GDN. These antibodies stain a single band at 43 kd on immunoblots of concentrated C6 glioma-conditioned medium and have been used to demonstrate that GDN is present in the olfactory system of the rat. One band at 43 kd is recognized by the GDN antibodies on immunoblots of olfactory bulb homogenate. Immunohistochemistry shows that GDN occurs predominantly in the olfactory nerve layer of the olfactory bulb and in the olfactory submucosa. Comparative studies with antibodies against vimentin, GFAP, and fibronectin suggest that anti-GDN recognizes cells associated with the olfactory system, but not exclusively the olfactory neurons themselves. Data from the immunohistochemical studies were confirmed by RNA blots and GDN mRNA expression throughout development of the olfactory bulb. The high levels of GDN in the rat olfactory system may be related to the continuous degeneration and regeneration phenomena taking place in these structures.

Prothrombin mRNA is expressed by cells of the nervous system.

Thrombin, a serine protease of the blood coagulation system, has additional effects on cells in vitro. It is mitogenic for fibroblasts and astrocytes and contributes to the regulation of neurite outgrowth and astrocyte stellation. 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.

Thrombin causes neurite retraction in neuronal cells through activation of cell surface receptors.

The mechanism by which thrombin induces neurite retraction was studied in NB2a mouse neuroblastoma cells. The rapid effect of thrombin (completed within minutes) appears to involve an interaction between its anion-binding exosite and the thrombin receptor. Structural alterations of this site increase the EC50 for thrombin-mediated retraction, and a hirudin C-terminal peptide that blocks this site inhibits the response. The thrombin effect was mimicked by a 14 amino acid peptide starting with Ser-42, at the proposed cleavage site of the human thrombin receptor. The protein kinase inhibitors staurosporine and H-7 blocked thrombin-induced retraction. It is therefore proposed that thrombin-mediated neurite retraction is caused by cleavage-induced activation of the thrombin receptor and involves stimulation of a protein kinase(s).

Progressive neuronal and motor dysfunction in mice overexpressing the serine protease inhibitor protease nexin-1 in postmitotic neurons.

Perturbation of the homeostasis between proteases and their inhibitors has been associated with lesion-induced or degenerative neuronal changes. Protease nexin-1 (PN-1), a secreted serine protease inhibitor, is constitutively expressed in distinct neuronal cell populations of the adult CNS. In an earlier study we showed that transgenic mice with ectopic or increased expression of PN-1 in postnatal neurons have altered synaptic transmission. Here these mice are used to examine the impact of an extracellular proteolytic imbalance on long-term neuronal function. These mice develop disturbances in motor behavior from 12 weeks on, with some of the histopathological changes described in early stages of human motor neuron disease, and neurogenic muscle atrophy in old age. In addition, sensorimotor integration, measured by epicranial multichannel recording of sensory evoked potentials, is impaired. Our results suggest that axonal dysfunction rather than cell death underlies these phenotypes. In particular, long projecting neurons, namely cortical layer V pyramidal and spinal motor neurons, show an age-dependent vulnerability to PN-1 overexpression. These mice can serve to study early stages of in vivo neuronal dysfunction not yet associated with cell loss.

An octamer-binding site is crucial for the activity of an enhancer active at the embryonic met-/mesencephalic junction.

An enhancer sequence found in the Protease Nexin-1 (PN-1) gene was shown to drive lacZ expression specifically at the met-/mesencephalic junction in transgenic mouse embryos. A functional study of this enhancer has been performed to better understand the mechanisms regulating isthmic gene expression. An octamer-binding site for POU domain factors was found to be crucial for the activity of the enhancer in vivo. Comparative expression studies of POU domain factors, electrophoretic mobility shift assays and transient transfection experiments, strongly suggest that Brn-1/-2 regulate the enhancer activity in vivo. In addition, in vitro experiments indicated that FGF-8 was required for the maintenance of the enhancer activity, but not for the synthesis of Bn-1/-2. The data represents the first functional evidence for a role of POU factors in the regulation of met-/mesencephalic gene expression. It also implies that at least two regulatory pathways, namely the FGF-8 signaling and the octamer-binding site pathway, synergistically interact to control the PN-1 enhancer activity in vivo.

A Krox binding site regulates protease nexin-1 promoter activity in embryonic heart, cartilage and parts of the nervous system.

The rat protease nexin-1 (PN-1) promoter contains a GCGGGGGCG binding site for the transcription factors Krox-24, Krox-20 and NGFI-C. Mutations of this site abolished binding of Krox-24 in vitro. The wildtype protease nexin-1 promoter expressed beta-galactosidase similarity to the expression of protease nexin-1 mRNA. When the function of this Krox site was tested in vivo using transgenic F0 embryos, mutation had two opposite effects. beta-Galactosidase expression increased in cartilage and heart at both stages E11.5 and E13.5, but was abolished in nerves of the central and peripheral nervous system at stage E13.5. These results suggest that Krox factors are among the important transcription factors regulating protease nexin-1 expression and thereby intracellular proteolytic activity in embryonic heart, cartilage and parts of the nervous system.

Synthesis of glia-derived nexin in yeast.

Glia-derived nexin (GDN) is a 43-kDa glycoprotein isolated from rat glioma cell cultures. It promotes neurite extension in cultures of neuroblastoma cells and chick sympathetic neurons. Moreover, GDN is a potent serine protease inhibitor (serpin), belonging to the family of protease nexins. We report here the expression of rat GDN in the Saccharomyces cerevisiae strain GRF18 under the control of the PHO5 promoter. We describe the purification of more than 6 mg total GDN from the cellular extract of 1 liter of yeast culture. The amino acid composition and the sequence of CNBr-fragments of the recombinant protein correlate with the values deduced from the rat GDN cDNA. We provide evidence that the recombinant GDN has exactly the same properties as the glioma-derived protein with respect to its protease-inhibitory activity and its ability to promote the extension of neurites from neuroblastoma cells. The large amounts of recombinant protein obtained from this expression system will allow further biochemical and physiological analysis of GDN and of the serpins in general.

Immunocytochemical Localization of Glia-Derived Nexin, Laminin and Fibronectin on the Surface or Extracellular Matrix of C6 Rat Glioma Cells, Astrocytes and Fibroblasts.

The expression and cellular distribution of glia-derived nexin (GDN), laminin and fibronectin on C6 rat glioma cells, rat brain astrocytes and rat fibroblasts were investigated by immunoblotting and immunocytochemistry. Western blot analysis of C6 cell homogenates confirmed the specificities of the antibodies. Immunocytochemical staining of C6 cells, astrocytes and fibroblasts showed that laminin, fibronectin and GDN were abundant on the surface of glioma cells and astrocytes whereas on fibroblasts fibronectin was abundant though only traces of GDN and laminin could be detected. The light microscopy data were confirmed by ultrastructural studies showing that each antigen was present on the surface of the C6 rat glioma cells as numerous spots with slightly different distribution patterns for each of the antigens. In fibroblast cultures, the antigens were also localized in the extracellular matrix in the vicinity of the cells. Migrating fibroblasts but not migrating glioma cells or astrocytes deposit the matrix-proteins onto the substratum leaving behind a track of GDN, laminin and fibronectin. When the cells were treated with heparin prior to antibody incubation, the GDN immunoreactivity completely disappeared, whereas the distribution and abundance of laminin and fibronectin was not affected. Our data show that GDN binds, possibly by a heparin-like molecule, to the outer surface of cells or to the extracellular matrix and may protect cells and matrix proteins against proteolytic degradation.

Modulation of Human Neurite Outgrowth by Serine Proteases: A Comparison of the Interaction of Thrombin and Prothrombin with Glia-Derived Nexin.

Neurite outgrowth from cells of neuroepithelial origin is under the reciprocal control of thrombin and the thrombin inhibitor-glia-derived nexin (GDN). The neurite retraction activity of thrombin is blocked when GDN complexes with the enzyme and inhibits its proteolytic activity. However, we have previously shown that enzymically inactive proenzyme is also capable of inducing neurite retraction. We present evidence here to show that GDN does not bind to prothrombin in solution. When a mixture of prothrombin and GDN is subjected to either polyacrylamide gel electrophoresis or immunoprecipitation, a stable complex cannot be detected. This is in direct contrast to thrombin, which exhibits stable complexes with GDN under both conditions. At the cell surface, however, GDN is able to inhibit the biological activity of prothrombin. When a mixture of proenzyme and inhibitor is applied to previously differentiated transformed retinoblasts (Ad12 HER10), the ability of prothrombin to induce neurite retraction is blocked. Furthermore, following 1 h exposure to Ad12 HER10 cells, a solution of prothrombin was found to contain half the potential enzyme activity as detected by chromogenic assay. These results have been interpreted as evidence for the ability of neuronal cells to cleave prothrombin and subsequently release activated enzyme.

The prolonged presence of glia-derived nexin, an endogenous protease inhibitor, in the hippocampus after ischemia-induced delayed neuronal death.

The presence of glia-derived nexin and glia fibrillary acidic protein (GFAP) was investigated in the hippocampus of Mongolian gerbils (Meriones unguiculatus) after transient forebrain ischemia. Bilateral clamping of the common carotid arteries for 7 min resulted in selective degeneration of CA1 pyramidal cells after a delay of three to four days, the so-called delayed neuronal death. Immunoreactivity for glia-derived nexin was found in astrocytes of all CA1 layers and was detectable until day 90 (the longest survival time studied). Astroglial reactivity was demonstrated in parallel by staining for GFAP. The co-localization of glia-derived nexin and GFAP was confirmed by double immunocytochemistry. Ultrastructural studies showed the exclusive presence of glia-derived nexin in astrocytes, in the vicinity of degenerating and preserved neuronal structures. Perivascular glia was intensely stained, but endothelial cells were devoid of immunoreactivity. Glia-derived nexin is a potent protease inhibitor with in vitro neurite-promoting activity. During adulthood, it is mainly present in the olfactory system, where receptor neurons are constantly being replaced. The ability of astrocytes to renew the expression of glia-derived nexin after selective delayed neuronal death and the prolonged presence of the protease inhibitor in a zone where degeneration occurs in the immediate neighborhood of preserved neuronal elements indicate that glia-derived nexin may play a role in structural rearrangements of the central nervous system.

Inactivation of protease nexin-1 by xanthine oxidase-derived free radicals.

Neuronal viability is affected by reactive oxygen species. Lipid peroxidation is often defined as a major reason for cellular breakdown. Additionally, certain indispensable proteins are possible targets for excessively formed reactive oxygen species. Evidence is given here that protease nexin-1 (PN-1), an endogenous thrombin inhibitor and neurite outgrowth promoter, is inactivated by xanthine oxidase-derived free radicals. Varying protection by superoxide dismutase and catalase was observed, depending on the reaction conditions. The water-soluble alpha-tocopherol analogues MDL 74,406 (R(+)-3,4-dihydro-6-hydroxy-N,N,N-2,5,7,8-heptamethyl-2H-1-benzopy ran-2- ethanaminium 4-methylbenzenesulfonate), MDL 74,180DA (2,3-dihydro-2,2,4,6,7-pentamethyl-3-(4-methyl-piperazino)-1-benzo furan-5-ol dihydro-chloride) and trolox also protected PN-1. Neurodegeneration may be triggered by oxidative inactivation of protease inhibitors such as PN-1. Protection of PN-1 in Alzheimer's or Parkinson's diseases, could be a possible target for a therapeutic function of antioxidants in these diseases.

Molecular organization of the rat glia-derived nexin/protease nexin-1 promoter.

The first three exons and the promoter of rat glia-derived nexin, also called protease nexin-1 (GDN/PN-1), have been identified through analysis of rat genomic clones. A 1.6 kilobase (kb) fragment containing 105 base pairs of the first exon and 5'-flanking sequences was sequenced. The 5'-flanking sequence and the first exon were found to be GC-rich, indicating that the 5' region of the rat GDN/PN-1 gene resides within a CpG island. A TATA box-like sequence, but no CAAT box, was found. The rat GDN/PN-1 promoter contains five SP1 consensus sites, four consensus sites for the MyoD1 transcription factor, and one binding site for the transcription factors NGFI-A, NGFI-C, Krox-20, and Wilms tumor factor. The presence of these consensus sequences is consistent with the known expression pattern of GDN/PN-1. Primer extension and RNase protection assays identified one transcriptional start site. The 1.6 kb promoter fragment cloned in a reporter plasmid was found to induce firefly luciferase expression in a cell-specific manner. A positive regulatory element is localized in the region -1545 to -389. In vitro CpG methylation blocked transcription from the GDN/PN-1 promoter in rat hepatoma cells but not in C6 rat glioma cells.