Nicholas W. Seeds

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.

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.

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.

Absence of Tissue Plasminogen Activator Gene or Activity Impairs Mouse Cerebellar Motor Learning

Motor learning is thought to involve modulation of synaptic inputs in the cerebellar cortex, including granule neuron/Purkinje neuron contacts. During a complex motor task requiring mice to walk across irregularly spaced pegs, cerebellar granule neurons show a rapid and transient induction of mRNA for the extracellular protease tissue plasminogen activator (tPA). This induction of tPA mRNA is cerebellar specific, is not seen in the cerebella of exercised or stressed animals, and is distinct from simple performance phenomena. Knock-out mice lacking the tPA gene show a significant reduction in both rate and extent of learning. Furthermore, blocking tPA activity during training dramatically impaired motor learning. Thus, tPA plays an important role in motor learning, in which tPA may facilitate remodeling of the active synaptic zone.

Effect of proteases and their inhibitors on neurite outgrowth from neonatal mouse sensory ganglia in culture.

Developing neurons and Schwann cells have been shown to secrete proteases. The influence of these proteases on neurite outgrowth by cultured sensory ganglia was examined by adding specific protease inhibitors. Neonatal mouse dorsal root ganglia were cultured directly on tissue-culture plastic dishes in serum-free N2 medium with different protease inhibitors. Soybean trypsin inhibitor was found to double the extent of neurite outgrowth by 4 days in vitro. Ovomucoid trypsin inhibitor and leupeptin also increased neurite outgrowth, while alpha 1-antitrypsin, antipain and phenylmethylsulfonyl fluoride elicited a smaller effect. Furthermore, added trypsin or thrombin inhibited neurite outgrowth and the inhibition could be reversed by soybean trypsin inhibitor, while exogenous plasminogen or urokinase were inhibitory only at high concentrations. Thus neurite outgrowth probably requires a closely regulated system of protease secretion and protease inhibitor production.

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

Abstract.u2002The 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.

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/μg 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 pronounced 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.

Transglutaminase and neuronal differentiation

SummaryDuring mouse brain maturation cellular transglutaminase specific activity increases 2.5 fold from day 3 to adulthood. A more pronounced increase is seen during morphological differentiation of mouse neuroblastoma cells, where serum withdrawal induces neurite outgrowth concomitant with a 10 fold increase in transglutaminase specific activity. In contrast, non-dividing neuroblastoma cells lacking neurites show only a 1.5 fold increase in enzyme specific activity. Transglutaminase activity does not reach maximal levels until extensive neurite formation has occurred. More than 80% of the transglutaminase activity is found in the soluble component of brain and neuroblastoma homogenates. Using [3H]-putrescine as the acyl acceptor, endogenous acyl donor substrates in the neuroblastoma cells included proteins that comigrated on SDS-PAGE with tubulin and actin; however, very high molecular weight crosslinked material is the major reaction product in vitro. When purified brain tubulin, microtubule associated proteins and microtubules were compared as exogenous substrates, only the polymeric microtubules were a good acyl donor substrate. Furthermore, preincubation of purified tubulin with transglutaminase and putrescine stimulated both the rate and extent of microtubule assembly. These findings suggest that transglutaminase may mediate covalent cross-linking of microtubules to other cellular components, or the post-translational modification of tubulin by the formation of γ-glutamylamines.

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 tPAs 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.