Be’eri Niego

Tissue‐type plasminogen activator requires a co‐receptor to enhance NMDA receptor function

Glutamate is the main excitatory neurotransmitter of the CNS. Tissue‐type plasminogen activator (tPA) is recognized as a modulator of glutamatergic neurotransmission. This attribute is exemplified by its ability to potentiate calcium signaling following activation of the glutamate‐binding NMDA receptor (NMDAR). It has been hypothesized that tPA can directly cleave the NR1 subunit of the NMDAR and thereby potentiate NMDA‐induced calcium influx. In contrast, here we show that this increase in NMDAR signaling requires tPA to be proteolytically active, but does not involve cleavage of the NR1 subunit or plasminogen. Rather, we demonstrate that enhancement of NMDAR function by tPA is mediated by a member of the low‐density lipoprotein receptor (LDLR) family. Hence, this study proposes a novel functional relationship between tPA, the NMDAR, a LDLR and an unknown substrate which we suspect to be a serpin. Interestingly, whilst tPA alone failed to cleave NR1, cell‐surface NMDARs did serve as an efficient and discrete proteolytic target for plasmin. Hence, plasmin and tPA can affect the NMDAR via distinct avenues. Altogether, we find that plasmin directly proteolyses the NMDAR whilst tPA functions as an indirect modulator of NMDA‐induced events via LDLR engagement.

t-PA-specific modulation of a human blood-brain barrier model involves plasmin-mediated activation of the Rho kinase pathway in astrocytes

Tissue-type plasminogen activator (t-PA) can modulate permeability of the neurovascular unit and exacerbate injury in ischemic stroke. We examined the effects of t-PA using in vitro models of the blood-brain barrier. t-PA caused a concentration-dependent increase in permeability. This effect was dependent on plasmin formation and potentiated in the presence of plasminogen. An inactive t-PA variant inhibited the t-PA-mediated increase in permeability, whereas blockade of low-density lipoprotein receptors or exposed lysine residues resulted in similar inhibition, implying a role for both a t-PA receptor, most likely a low-density lipoprotein receptor, and a plasminogen receptor. This effect was selective to t-PA and its close derivative tenecteplase. The truncated t-PA variant reteplase had a minor effect on permeability, whereas urokinase and desmoteplase were ineffective. t-PA also induced marked shape changes in both brain endothelial cells and astrocytes. Changes in astrocyte morphology coincided with increased F-actin staining intensity, larger focal adhesion size, and elevated levels of phosphorylated myosin. Inhibition of Rho kinase blocked these changes and reduced t-PA/plasminogen-mediated increase in permeability. Hence plasmin, generated on the cell surface selectively by t-PA, modulates the astrocytic cytoskeleton, leading to an increase in blood-brain barrier permeability. Blockade of the Rho/Rho kinase pathway may have beneficial consequences during thrombolytic therapy.

Plasmin-Dependent Modulation of the Blood–Brain Barrier: A Major Consideration during tPA-Induced Thrombolysis?

Plasmin, the principal downstream product of tissue-type plasminogen activator (tPA), is known for its potent fibrin-degrading capacity but is also recognized for many non-fibrinolytic activities. Curiously, plasmin has not been conclusively linked to blood–brain barrier (BBB) disruption during recombinant tPA (rtPA)-induced thrombolysis in ischemic stroke. This is surprising given the substantial involvement of tPA in the modulation of BBB permeability and the co-existence of tPA and plasminogen in both blood and brain throughout the ischemic event. Here, we review the work that argues a role for plasmin together with endogenous tPA or rtPA in BBB alteration, presenting the overall controversy around the topic yet creating a rational case for an involvement of plasmin in this process.

Oncostatin M is a neuroprotective cytokine that inhibits excitotoxic injury in vitro and in vivo

Oncostatin M (OsM) is a member of the interleukin (IL)‐6 family of cytokines and is well known for its role in inflammation, cell proliferation, and hematopoiesis. OsM, together with its glycoprotein 130 containing receptor complex, is expressed and regulated in most cells of the central nervous system (CNS), yet the function of OsM within this compartment is poorly understood. Here we have investigated the effect of OsM using in vitro and in vivo models of excitotoxic injury. Using primary cultures of mouse cortical neurons, OsM was shown to reduce N‐methyl‐D‐aspartate (NMDA) ‐induced neuronal death by 50% when added simultaneously with NMDA while pretreatment of neurons with OsM fully prevented NMDA toxicity indicating a profound protective effect of this cytokine. OsM was also shown to inhibit NMDA‐mediated increase in levels of free intracellular calcium and to selectively reduce neuronal expression of the NR2C subunit of the NMDA receptor. Finally, using an in vivo model of excitotoxic injury, OsM significantly reduced the NMDA‐induced lesion volume when coinjected with NMDA into the mouse striatum. Taken together, these results identify OsM as a powerful neuroprotective cytokine and provide a rational foundation to explore the therapeutic potential for OsM in diseases of the CNS.—Weiss, T. W., Samson, A. L., Niego, B., Daniel, P. B., Medcalf, R. L. Oncostatin M is a neuroprotective cytokine that inhibits excitotoxic injury in vitro and in vivo. FASEB J. 20, E1636 –E1645 (2006)

A nonfibrin macromolecular cofactor for tPA-mediated plasmin generation following cellular injury

Tissue-type plasminogen activator (tPA) is an extracellular protease that converts plasminogen into plasmin. For tPA to generate plasmin under biologic conditions, a cofactor must first bring tPA and plasminogen into physical proximity. Fibrin provides this cofactor for tPA-mediated plasmin generation in blood. Despite being naturally devoid of fibrin(ogen), tPA-mediated plasmin formation also occurs in the brain. The fibrin-like cofactor(s) that facilitates plasmin formation in the injured brain has remained unknown. Here we show that protein aggregates formed during neuronal injury provide a macromolecular, nonfibrin cofactor that promotes tPA-mediated plasmin formation and subsequent cell breakdown. The binding of plasminogen and tPA to these protein aggregates occurs via distinct mechanisms. Importantly, nonneuronal cell types also exhibit this cofactor effect upon injury, indicating a general phenomenon. This novel cofactor identified in nonviable cells has ramifications for ischemic stroke where tPA is used clinically and where plasmin activity within the injured brain is unwanted. A means of selectively inhibiting the binding of tPA to nonviable cells while preserving its association with fibrin may be of benefit for the treatment of ischemic stroke.

t-PA, but not desmoteplase, induces plasmin-dependent opening of a blood-brain barrier model under normoxic and ischaemic conditions.

Tissue-type plasminogen activator (t-PA) is the only thrombolytic treatment available for patients with acute ischaemic stroke. However, t-PA can increase permeability of the blood-brain barrier (BBB). Desmoteplase is a plasminogen activator derived from the common vampire bat, currently under clinical development for ischaemic stroke. We compared how t-PA and desmoteplase influenced BBB permeability using a human in vitro model where primary brain endothelial cells (BEC) and astrocytes are co-cultured on the opposite sides of a porous membrane. Permeability changes were evaluated 6 or 24h post-stimulation by passage of fluorescent albumin across the membrane. Under normoxic conditions, t-PA, but not desmoteplase, increased BBB permeability. Surprisingly, the ability of t-PA to affect the barrier was lost under conditions of oxygen-glucose deprivation (OGD). Addition of plasminogen re-sensitised the BBB to the action of t-PA under both normoxia and OGD, but did not affect the inert behaviour of desmoteplase, even when digested fibrinogen was added to ensure optimal plasmin generation. These observations coincided with plasmin-dependent changes in astrocyte and BEC morphology and disruption of tight junction proteins in BECs, specifically initiated by t-PA but not by desmoteplase. Finally, inhibition of plasmin post-stimulation with t-PA and plasminogen, especially within 2h, protected the BBB against t-PA-mediated barrier opening. Hence t-PA, but not desmoteplase, increases BBB permeability under both normoxic and OGD conditions in a reversible, plasmin-dependent process. The inability of desmoteplase to increase permeability despite its capacity to generate plasmin provides further support for its use as thrombolytic in patients with ischaemic stroke.

Two conserved regions within the tissue-type plasminogen activator gene promoter mediate regulation by brain-derived neurotrophic factor

Tissue‐type plasminogen activator (t‐PA) has recently been identified as a modulator of neuronal plasticity and can initiate conversion of the pro‐form of brain‐derived neurotrophic factor (BDNF) into its mature form. BDNF also increases t‐PA gene expression implicating t‐PA as a downstream effector of BDNF function. Here we demonstrate that BDNF‐mediated induction of t‐PA mRNA requires an increase in t‐PA gene transcription. Reporter constructs harboring 9.5 kb of the human t‐PA promoter conferred BDNF‐responsiveness in transfected mouse primary cortical neurons. This regulation was recapitulated in HEK 293 cells coexpressing the TrkB neurotrophin receptor. t‐PA promoter‐deletion analysis revealed the presence of two BDNF‐responsive domains, one located between −3.07 and −2.5 kb and the other within the proximal promoter. The upstream region was shown to confer BDNF responsiveness in a TrkB‐dependent manner when attached to a heterologous promoter. We also identify homologous regions within the murine and bovine t‐PA gene promoters and demonstrate that the equivalent upstream murine sequence functions as a BDNF‐responsive enhancer when inserted 5′ of the human proximal t‐PA promoter. Hence, BDNF‐mediated induction of t‐PA transcription relies on conserved modular promoter elements including a novel upstream BDNF‐responsive domain and the proximal t‐PA gene promoter.

Thrombin-induced activation of astrocytes in mixed rat hippocampal cultures is inhibited by soluble thrombomodulin

Thrombin, a serine protease known for its role in coagulation, also induces a variety of protease activated receptor (PAR)-mediated responses in the central nervous system that contribute to many brain pathologies. Since the proteolytic specificity of thrombin is uniquely controlled by thrombomodulin (TM), we investigated the mechanisms by which thrombin and a recombinant soluble form of human TM (Solulin, INN: sothrombomodulin alpha; rhsTM) could influence rat hippocampal cultures. Treatment of hippocampal cultures with thrombin for up to 48h resulted in a significant morphological rearrangement with the formation of expansive cell-free areas (CFAs) and a reduction in cell viability; both effects were blocked by rhsTM. Treatment with the selective PAR-1 agonist, TRAP (SFLLRN) caused the formation of CFAs, suggesting that CFA formation involved PAR-1 signaling. Astrocytes prepared from PAR-1(-/-) mice also had an attenuated CFA response to thrombin. Thrombin-induced CFA formation was a consequence of cell movement and substantial changes in cell morphology, rather than due to cell detachment. Immunocytochemical and functional analyses revealed that the thrombin-sensitive cells within these hippocampal cultures were astrocytes. The effects of thrombin on CFA development were mediated by astrocyte-specific release of intracellular calcium and signalling through ERK1/2. rhsTM was able to attenuate thrombin-induced ERK1/2 phosphorylation. Finally, astrocytes were shown to maintain thrombin-sensitivity following neuronal depletion with NMDA, a result which was confirmed with pure astrocyte cultures. Hence thrombin mediates PAR-1-induced activation of hippocampal astrocytes, but not neurons, in a process that can be modulated by rhsTM.

Fabrication of complex PDMS microfluidic structures and embedded functional substrates by one-step injection moulding

We report a novel injection moulding technique for fabrication of complex multi-layer microfluidic structures, allowing one-step robust integration of functional components with microfluidic channels, and fabrication of elastomeric microfluidic valves. This technique simplifies multi-layer microfluidic device fabrication, while significantly increasing device functionality. We demonstrate functional component integration through robust encapsulation of porous polyester membranes, in the context of an in vitro research platform intended to facilitate Blood Brain Barrier (BBB) research. We also demonstrate the fabrication of normally-closed, pneumatically actuated elastomer valves, integrated using the same one-step process. These valves are demonstrated in the context of variable flow resistors used to modulate flow in a pressure driven system.

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.