Induction of the plasminogen activator system accompanies peripheral nerve regeneration after sciatic nerve crush.

Peripheral nerve regeneration is dependent on the ability of regenerating neurites to migrate through cellular debris and altered extracellular matrix at the injury site, grow along the residual distal nerve sheath conduit, and reinnervate synaptic targets. In cell culture, growth cones of regenerating axons secrete proteases, specifically plasminogen activators (PAs), which are believed to facilitate growth cone movement by digesting extracellular matrices and cell adhesions. In this study, the PA system was shown to be specifically activated in sensory neurons after sciatic nerve crush in adult mice. The number of sensory neurons expressing urokinase PA receptor (uPAR) mRNA levels increased above sham levels by 8 hr after crush, whereas the number of sensory neurons expressing uPA and tissue PA (tPA) mRNAs was significantly increased by 3 d after crush. PA mRNA levels were also increased at the crush site, with uPA mRNA elevated by 8 hr after crush and tPA and uPAR mRNA levels markedly increased by 7 d. PA-dependent enzymatic activity was significantly increased from 1 to 7 d after crush in nerves that had been crushed compared with uncrushed nerves. Immunohistochemistry showed that tPA was localized within regenerating axons of the sciatic nerve. There were no significant changes in plasminogen activator inhibitor 1 activity between crush and sham after the injury. These results clearly demonstrated that after injury the PA system was rapidly induced in sensory neurons, where it may play an important role in nerve regeneration in vivo.

Mice lacking tPA, uPA, or plasminogen genes showed delayed functional recovery after sciatic nerve crush.

Axonal outgrowth during peripheral nerve regeneration relies on the ability of growth cones to traverse through an environment that has been altered structurally and along a basal lamina sheath to reinnervate synaptic targets. To promote migration, growth cones secrete proteases that are thought to dissolve cell-cell and cell-matrix adhesions. These proteases include the plasminogen activators (PAs), tissue PA (tPA) and urokinase PA (uPA), and their substrate, plasminogen. PA expression and secretion are upregulated in regenerating mammalian sensory neurons in culture. After sciatic nerve crush in mice, there was an induction of PA mRNAs in the sensory neurons contributing to the crushed nerve and an upregulation of PA-dependent activity in crushed nerve compared with sham counterparts during nerve regeneration. To further assess the role of the PA system during peripheral nerve regeneration, PA-dependent activity as well as recovery of sensory and motor function in the injured hindlimb were assessed in wild-type, tPA, uPA, and plasminogen knock-out mice. Protease activity visualized by gel zymography showed that after nerve crush, the upregulation of PA activity in the tPA and uPA knock-out mice was delayed compared with wild-type mice. Recovery of sensory function was assessed by toe pinch, footpad prick, and the toe-spreading reflex. All knock-out mice demonstrated a significant delay in hindlimb response to these sensory stimuli compared with wild-type mice. For each modality tested, the uPA knock-out mice were the most dramatically affected, showing the longest delay to initiate a response. These studies clearly showed that PAs were necessary for timely functional recovery by regenerating peripheral nerves.

Plasminogen expression in the neonatal and adult mouse brain.

Tissue-type plasminogen activator (tPA) has been implicated in a variety of types of neural plasticity, including cell migration, occlusion-induced visual system plasticity, and learning. In the periphery, plasminogen serves as tPA's primary substrate; however, studies attempting to identify plasminogen in the central nervous system have produced mixed results. We have performed a comprehensive, multitechnique study examining plasminogen expression in the neonatal and adult mouse brain. Reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization reveal plasminogen mRNA in the cortex, hippocampus and cerebellum of both neonatal and adult C57BL/6 mice. Immunocytochemistry reveals plasminogen protein expression in these same brain regions. Notably, plasminogen expression in the cerebellum occurs in the granule cell and the Purkinje cell layers. tPA activity in these same regions is involved in granule cell migration during development and motor learning in adulthood. Therefore, these findings demonstrate that plasminogen is present in the central nervous system and localized to areas where it could serve as a substrate for plasticity-related increases in tPA activity.

Induction of tissue plasminogen activator in differentiated NG108-15 cells.

Plasminogen activators may facilitate neurite outgrowth and neuronal migration in the developing nervous system. The expression of tissue plasminogen activator by NG108-15 neuroblastoma grown under a variety of conditions has been explored. High tissue plasminogen mRNA expression correlates with growth conditions which induce morphological differentiation and neurite outgrowth; however, NG108-15 cells grown in suspension with dibutyryl-cAMP also show a high level of tissue plasminogen activator expression.

Tissue plasminogen activator (tPA) deficiency exacerbates cerebrovascular fibrin deposition and brain injury in a murine stroke model: studies in tPA-deficient mice and wild-type mice on a matched genetic background.

Although the serine protease, tissue plasminogen activator (tPA), is approved by the US Food and Drug Administration for therapy to combat focal cerebral infarction, the basic concept of thrombolytic tPA therapy for stroke was challenged by recent studies that used genetically manipulated tPA-deficient (tPA-/-) mice, which suggested that tPA mediates ischemic neuronal damage. However, those studies were potentially flawed because the genotypes of tPA-/- and wild-type control mice were not entirely clear, and ischemic neuronal injury was evaluated in isolation of tPA effects on brain thrombosis. Using mice with appropriate genetic backgrounds and a middle cerebral artery occlusion stroke model with nonsiliconized thread, which does lead to microvascular thrombus formation, in the present study we determined the risk for cerebrovascular thrombosis and neuronal injury in tPA-/- and genetically matched tPA+/+ mice subjected to transient focal ischemia. Cerebrovascular fibrin deposition and the infarction volume were increased by 8.2- and 6. 7-fold in tPA-/- versus tPA+/+ mice, respectively, and these variables were correlated with reduced cerebral blood flow up to 58% (P<0.05) and impaired motor neurological score by 70% (P<0.05). Our findings indicate that tPA deficiency exacerbates ischemia-induced cerebrovascular thrombosis and that endogenous tPA protects the brain from an ischemic insult, presumably through its thrombolytic action. In addition, our study emphasizes the importance of appropriate genetic controls in murine stroke research.

Neuronal migration is retarded in mice lacking the tissue plasminogen activator gene.

Neuronal migration is a critical phase of brain development, where defects can lead to severe ataxia, mental retardation, and seizures. In the developing cerebellum, granule neurons turn on the gene for tissue plasminogen activator (tPA) as they begin their migration into the cerebellar molecular layer. Granule neurons both secrete tPA, an extracellular serine protease that converts the proenzyme plasminogen into the active protease plasmin, and bind tPA to their cell surface. In the nervous system, tPA activity is correlated with neurite outgrowth, neuronal migration, learning, and excitotoxic death. Here we show that compared with their normal counterparts, mice lacking the tPA gene (tPA(-/-)) have greater than 2-fold more migrating granule neurons in the cerebellar molecular layer during the most active phase of granule cell migration. A real-time analysis of granule cell migration in cerebellar slices of tPA(-/-) mice shows that granule neurons are migrating 51% as fast as granule neurons in slices from wild-type mice. These findings establish a direct role for tPA in facilitating neuronal migration, and they raise the possibility that late arriving neurons may have altered synaptic interactions.

The expression of mRNAs for hepatocyte growth factor/scatter factor, its receptor c-met, and one of its activators tissue-type plasminogen activator show a systematic relationship in the developing and adult cerebral cortex and hippocampus.

The temporal and spatial expression in brain of the mRNAs for the pleiotropic cytokine hepatocyte growth factor/scatter factor (HGF/SF) and its receptor c-met were compared to those of a known HGF/SF activator, tissue-type plasminogen activator (tPA). In addition to the previously described expression in the developing and adult olfactory system [D.P. Thewke, N.W. Seeds, Expression of hepatocyte growth factor/scatter factor, its receptor, c-met, and tissue-type plasminogen activator during development of the murine olfactory system, J. Neurosci. 16 (1996) 6933-6944] two other regions of the mouse brain were found where the expression of tPA mRNA appeared to co-localized with HGF/SF and/or c-met mRNA. In the developing hippocampus, tPA mRNA was expressed coincident with HGF/SF and c-met mRNAs in the CA1 field. tPA mRNA was expressed in all areas of the adult hippocampus, while HGF/SF expression was restricted to the CA2 and CA3 fields, and c-met mRNA was seen primarily in the CA1 field. In the developing cerebral cortex, the expression of tPA mRNA was observed in the subplate and inner cortical plate between two layers of c-met expression, whereas HGF/SF mRNA was localized to the proliferative zone lining the lateral ventricle. Layer specific expression of both HGF/SF and c-met mRNA were observed in the adult cortex, where HGF/SF was expressed in layers IV and V and c-met in layers II-III, IV and V. The expression of tPA mRNA in the adult cortex was low and not layer specific, although homogenates of adult cortex did have detectable levels of tPA activity when subjected to zymography. Immunohistochemical analysis using HGF/SF and c-met antibodies on adult brain sections showed a distribution similar to the in situ hybridization results. C-met antibodies appeared to stain large neurons in the cortex and hippocampus. These results are consistent with the hypothesis that HGF/SF plays a role in the development and maintenance of both the cerebral cortex and hippocampus, and that tPA may act as a regulator of HGF/SF activity in these structures.

Neuronal extracellular proteases facilitate cell migration, axonal growth, and pathfinding.

The release of extracellular proteases by the axonal growth cone has been proposed to facilitate its movement by digesting cell-cell and cell-matrix contacts in the path of the advancing growth cone. The serine protease plasminogen activator (PA) has been shown to be secreted and focally concentrated at axonal growth cones of cultured mammalian neurons. Thus, PAs are well-placed to play an active role in growth cone movement and axonal pathfinding in development and regeneration. We discuss recent findings that suggest that the biological action of these proteases is more complex than originally thought.

Expression of hepatocyte growth factor/scatter factor, its receptor, c-met, and tissue-type plasminogen activator during development of the murine olfactory system.

The expression of hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, the c-met proto-oncogene product, was examined by in situ hybridization in the developing and adult murine olfactory system and compared with the expression of a known activator of HGF/SF, tissue-type plasminogen activator (tPA). In the developing olfactory canal, expression of both c-met and tPA was observed in the olfactory neuroepithelium, whereas HGF/SF expression appeared to be confined to the mucosa adjacent to the neuroepithelium. During development of the olfactory bulb, HGF/SF and tPA were expressed within the rostral migratory pathway leading to the olfactory bulb, whereas c-met expression was observed in the mitral cell layer (MCL) of the olfactory bulb and in the anterior olfactory nucleus. In the adult olfactory bulb, expression of HGF/SF was restricted to the periglomerular region of the glomerular layer, whereas c-met was expressed in the MCL and olfactory nerve fiber layers (ONL). tPA expression in the adult olfactory bulb was observed in the ONL, MCL, and granule cell layers. Therefore, tPA expression was relatively coincident with the expression of HGF/SF and/or c-met in the appropriate projection patterns of the developing and adult olfactory system. In addition, antibodies against tPA inhibited the olfactory bulb extract-mediated cleavage of single-chain HGF/SF. These results suggest that tPA may play a regulatory role in the development and maintenance of the olfactory system by activating HGF/SF in the immediate vicinity of its receptor.

Brain capillary tissue plasminogen activator in a diabetes stroke model.

BACKGROUND AND PURPOSE: Tissue plasminogen activator (TPA) is normally expressed in rat brain capillaries. This study examines the expression of TPA in brain capillaries of diabetic rats in relation to focal ischemic brain injury. METHODS: Diabetes type 1 was induced by streptozotocin for 7 days. Acute hyperglycemia was induced by 50% dextrose. Expression of TPA in brain capillaries was determined by Western blot and reverse transcription-polymerase chain reaction analyses. Focal stroke was produced by 1 hour of reversible middle cerebral artery occlusion. Physiological variables and cerebral blood flow were monitored during occlusion and within 1 hour of reperfusion. Neurological and neuropathologic examinations were performed after 24 hours of reperfusion. RESULTS: All rats developed comparable hyperglycemia (approximately 15 mmol/L). A complete depletion of TPA protein and 6.5-fold decrease in TPA mRNA were found in brain capillaries of diabetic rats, in contrast to normal TPA capillary levels in hyperglycemic rats. The blood flow in the periphery of the ischemic core was significantly reduced during reperfusion by 52% to 62% (P<.001) in diabetic rats and by 23% to 25% (P<.05) in hyperglycemic rats. The neurological score was worsened by 3.2-fold (P<.0003) by diabetes and by 24% by hyperglycemia only. Significant 41% (P<.007) and 29% (P<.05) increases in infarct volume and 163% (P<.007) and 60% increases in edema volume were found in diabetic rats relative to control and hyperglycemic rats, respectively. CONCLUSIONS: Diabetes type 1, but not acute hyperglycemia, produces downregulation of TPA in rat brain capillaries. This TPA reduction is associated with impaired restoration of blood flow after an ischemic insult, poor neurological outcome, and enhanced ischemic brain injury.

Modulated expression of plasminogen activator system components in cultured cells from dissociated mouse dorsal root ganglia.

The development and regeneration of the peripheral nervous system (PNS) is highly dependent on the migration of Schwann cells and the extension of axons toward their distant targets. Plasminogen activators (PAs) are associated with the surface of several cell types of neural origin where they are believed to mediate localized degradation of extracellular matrix, thus facilitating cell motility. In this study, we characterize the expression of tissue-type (tPA) and urokinase (uPA) PAs, as well as the urokinase cell surface receptor (uPAR) during differentiation of cultured cells from mouse dorsal root ganglia. During the first day in culture, the mRNA levels of all three components increase from 75- to 163-fold, as shown using a quantitative PCR method. By 72 hr, the mRNA levels decrease and approach basal levels. This transient increase is in direct correlation with the differentiation of neurons and Schwann cells and the formation of a neuritic network in these regenerating cultures. Densitometric analysis of gel zymographs demonstrates that the elevation in mRNA levels is accompanied by similar increases in the activity levels of tPA and uPA. Interestingly, in situ hybridization studies of the cultures show that tPA mRNA is restricted to small sensory neurons, whereas uPA mRNA is localized predominantly in large sensory neurons. uPAR mRNA is expressed by both neuronal subpopulations and, to a lesser extent, by Schwann cells and fibroblasts. Taken together, these results further support a role for the PA system in facilitating axon extension and cell migration during development and regeneration of the PNS.

Tissue plasminogen activator induction in Purkinje neurons after cerebellar motor learning.

The cerebellar cortex is implicated in the learning of complex motor skills. This learning may require synaptic remodeling of Purkinje cell inputs. An extracellular serine protease, tissue plasminogen activator (tPA), is involved in remodeling various nonneural tissues and is associated with developing and regenerating neurons. In situ hybridization showed that expression of tPA messenger RNA was increased in the Purkinje neurons of rats within an hour of their being trained for a complex motor task. Antibody to tPA also showed the induction of tPA protein associated with cerebellar Purkinje cells. Thus, the induction of tPA during motor learning may play a role in activity-dependent synaptic plasticity.

Expression of tissue plasminogen activator in cerebral capillaries: possible fibrinolytic function of the blood-brain barrier.

Previous work has shown that tissue plasminogen activator (tPA) is a key enzyme in the control of fibrinolysis within the vascular system. The sources of brain tPA and the mechanisms by which tPA secretion and production occur within cerebral microcirculation are not well established. In this study, expression of tPA was investigated in cerebral capillaries and capillary-depleted brain isolated from cortices of 4- to 5-week-old rats and guinea pigs. In both species, a single tPA band of M(r) 67,000 was detected in cerebral capillaries by Western blot analysis. The tPA signal was absent from capillary-depleted brain. These results were corroborated at the messenger ribonucleic acid level. Reverse transcription-polymerase chain reaction analysis revealed the presence of tPA complementary deoxyribonucleic acid in samples derived from cerebral microvessels and demonstrated very low or undetectable tPA expression in capillary-depleted brain. Immunohistochemical analysis confirmed tPA localization in endothelial cells of brain capillaries. We conclude that microvascular endothelium, i.e., the blood-brain barrier, may have a role in promoting plasmin-dependent fibrinolysis in brain microcirculation. Delineation of the molecular mechanisms of blood-brain barrier-mediated fibrinolysis will likely contribute to future stroke prevention efforts.

Tissue plasminogen activator mRNA expression in granule neurons coincides with their migration in the developing cerebellum.

Tissue plasminogen activator activity in the developing cerebellum, as quantified by zymography of cerebellar homogenates from embryonic day (E) 17 to adult mice, shows a peak of activity at postnatal day (P) 7, followed by a steady 75% decrease into adulthood. Northern blot analysis reveals a similar pattern for tissue plasminogen activator mRNA levels, which are low at E17 but increase dramatically, reaching their highest levels of specific mRNA/micrograms RNA in P1-P7 mice and declining about threefold in the adult mouse. In situ hybridization of whole mouse brain sections with a tissue plasminogen activator antisense cRNA probe shows pronounce reactivity in the cerebellum. Although some binding is associated with the cerebellar meninges, the external granule layer is devoid of tissue plasminogen activator mRNA at all ages. However, highly labeled elongated cells, which also bind antibody to neuronal nuclear antigen and are adjacent to Bergmann glial fibers (i.e., migrating granule neurons), are readily visible throughout the molecular and Purkinje layers at P7 and P14. In the adult mouse cerebellum, tissue plasminogen activator mRNA labeling is restricted to cells in the Purkinje/internal granule layers. Thus, tissue plasminogen activator gene expression is induced as granule neurons leave the external granule layer and begin their inward migration.

Tissue plasminogen activator expression in the embryonic nervous system.

Tissue plasminogen activator (tPA) expression during embryogenesis was determined by in situ hybridization of whole mouse embryos from E10.5 through E17.5. The strongest expression occurs in the basal midbrain and hindbrain, and continues posteriorly into the neural canal. This expression coincides with extensive cell migration and proliferation, and tissue remodelling of this region. tPA mRNA is also associated with cells that appear to be invading the cerebellar anlage. Presumptive proliferating and migrating cells in the olfactory neuroepithelium also express tPA. These results indicate that tPA is expressed by a number of different cell types in the developing nervous system and suggest a role for tPA in cell migration and tissue remodelling of the developing CNS.

Degradation of underlying extracellular matrix by sensory neurons during neurite outgrowth.

The ability of differentiating sensory neurons to remodel a fibronectin substratum was examined. During the early stages of neurite outgrowth, fibronectin was cleared from areas beneath the neuronal soma and processes. The removal of fibronectin occurred in the presence and absence of plasminogen and was associated with the release of fibronectin fragments into the culture medium. The degradation of fibronectin was dependent upon neuronal contact with the substratum. Extraction of cells with the nonionic detergent Triton X-114 identified plasminogen activator and plasmin associated with the cell surface. These findings suggest that the plasminogen activator/plasmin system may play an important role in the interaction of differentiating sensory neurons with the extracellular matrix during axonal outgrowth.

Characterization of 125I-tissue plasminogen activator binding to cerebellar granule neurons.

Mouse cerebellar cells in culture secrete tissue plasminogen activator (tPA) into the culture medium. Fibrin overlays have shown tPA to be associated with granule neurons in these cultures. This cell associated tPA can be displaced by extensive washing of the cells or by a brief lowering of the pH (less than 4), which leads to a loss of fibrinolytic activity by the cells. Incubation of these fibrinolytically inactive cells with exogenously added murine tPA leads to the restoration of the fibrinolytic activity, indicating the presence of tPA binding sites on these granule neurons. Using 125I-tPA, the binding to cerebellar granule neurons is rapid, saturable, specific, high affinity (Kd = 50 pM) and reversible. Both murine and human tPA compete with 125I-tPA for binding, with both murine and human urokinase (uPA) as well as human thrombin and plasminogen fail to compete. Neither the catalytic site nor the carbohydrate moiety of tPA appear to be involved in the binding, since both diisopropyl-fluorophosphate-treated tPA and endoglycosidase-H-treated tPA compete with 12I-tPA for binding. Furthermore, epidermal growth factor does not compete well with tPA for binding even at a 10:1 molar excess, suggesting that the epidermal growth factor-like (EGF) domain of tPA may not be involved in the binding mechanism. Autoradiography and antibody immunofluorescence show the specific tPA binding is to granule neurons in these cultures. Thus, granule neurons possess tPA receptors on their surface, where this protease binds retaining is functional activity and may play a role in cell and axon migration.