Search

Clot retraction refers to the process whereby activated platelets transduce contractile forces onto the fibrin network of a thrombus, which over time increases clot density and decreases clot size. This process is considered important for promoting clot stability and maintaining blood vessel patency. Insights into the mechanisms regulating clot retraction at sites of vascular injury have been hampered by a paucity of in vivo experimental models. By pairing localized vascular injury with thrombin microinjection in the mesenteric circulation of mice, we have demonstrated that the fibrin network of thrombi progressively compacts over a 2-hour period. This was a genuine retraction process, as treating thrombi with blebbistatin to inhibit myosin IIa-mediated platelet contractility prevented shrinkage of the fibrin network. Real-time confocal analysis of fibrinolysis after recombinant tissue-type plasminogen activator (tPA) administration revealed that incomplete proteolysis of fibrin polymers markedly facilitated clot retraction. Similarly, inhibiting endogenous fibrinolysis with tranexamic acid reduced retraction of fibrin polymers in vivo. In vitro clot retraction experiments indicated that subthreshold doses of tPA facilitated clot retraction through a plasmin-dependent mechanism. These effects correlated with changes in the elastic modulus of fibrin clots. These findings define the endogenous fibrinolytic system as an important regulator of clot retraction, and show that promoting clot retraction is a novel and complementary means by which fibrinolytic enzymes can reduce thrombus size. [ABSTRACT FROM AUTHOR]

Removal of dead cells in the absence of concomitant immune stimulation is essential for tissue homeostasis. We recently identified an injury-induced protein misfolding event that orchestrates the plasmin-dependent proteolytic degradation of necrotic cells. As impaired clearance of dead cells by the innate immune system predisposes to autoimmunity, we determined whether plasmin could influence endocytosis and immune cell stimulation by dendritic cells – a critical cell that links the innate and adaptive immune systems. We find that plasmin generated on the surface of necrotic cells enhances their phagocytic removal by human monocyte-derived dendritic cells. Plasmin also promoted phagocytosis of protease-resistant microparticles by diverse mouse dendritic cell sub-types both in vitro and in vivo. Together with an increased phagocytic capacity, plasmin-treated dendritic cells maintain an immature phenotype, exhibit reduced migration to lymph nodes, increase their expression/release of the immunosuppressive cytokine TGF-β, and lose their capacity to mount an allogeneic response. Collectively, our findings support a novel role for plasmin formed on dead cells and other phagocytic targets in maintaining tissue homeostasis by increasing the phagocytic function of dendritic cells while simultaneously decreasing their immunostimulatory capacity consistent with producing an immunosuppressive state. [ABSTRACT FROM AUTHOR]

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous and abundant protein that participates in cellular energy production. GAPDH normally exists in a soluble form; however, following necrosis, GAPDH and numerous other intracellular proteins convert into an insoluble disulfide cross-linked state via the process of "nucleocytoplasmic coagulation." Here, free radical-induced aggregation of GAPDH was studied as an in vitro model of nucleocytoplasmic coagulation. Despite the fact that disulfide cross-linking is a prominent feature of GAPDH aggregation, our data show that it is not a primary rate-determining step. To identify the true instigating event of GAPDH misfolding, we mapped the post-translational modifications that arise during its aggregation. Solvent accessibility and energy calculations of the mapped modifications within the context of the high resolution native GAPDH structure suggested that oxidation of methionine 46 may instigate aggregation. We confirmed this by mutating methionine 46 to leucine, which rendered GAPDH highly resistant to free radical-induced aggregation. Molecular dynamics simulations suggest that oxidation of methionine 46 triggers a local increase in the conformational plasticity of GAPDH that likely promotes further oxidation and eventual aggregation. Hence, methionine 46 represents a "linchpin" whereby its oxidation is a primary event permissive for the subsequent misfolding, aggregation, and disulfide cross-linking of GAPDH. A critical role for linchpin residues in nucleocytoplasmic coagulation and other forms of free radical-induced protein misfolding should now be investigated. Furthermore, because disulfide-cross-linked aggregates of GAPDH arise in many disorders and because methionine 46 is irrelevant to native GAPDH function, mutation of methionine 46 in models of disease should allow the unequivocal assessment of whether GAPDH aggregation influences disease progression. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

For over a decade, tissue-type plasminogen activator (t-PA), a serine protease classically known for its profibrinolytic role in the vasculature, has been implicated in numerous aspects of the synaptic plasticity process. But despite being the most intensively studied protease of the CNS, the mechanisms and molecular mediators behind the action of t-PA on synaptic efficacy remain largely undefined. Rather than examine the role of t-PA in proteolytic remodeling of the synaptic extracellular matrix, this review will focus on the evidence that defines t-PA as a direct modulator of neurotransmission and synaptic plasticity by impacting on glutamatergic and dopaminergic pathways. [Copyright &y& Elsevier]

This article discusses a study which investigated the effect of oncostatin M (OsM) using in vitro and in vivo models of excitotoxic injury. The effect of OsM was determined on neurons treated with kainic acid. The study found that changes in the expression of individual subunits can affect NMDA receptor function. Moreover, it found that NMDA-induced cell death is preceded by an increase in the level of free intracellular calcium.

The Open Field (OF) test is one of the most commonly used assays for assessing exploratory behaviour and generalised locomotor activity in rodents. Nevertheless, the vast majority of researchers still rely upon costly commercial systems for recording and analysing OF test results. Consequently, our aim was to design a freely available program for analysing the OF test and to provide an accompanying protocol that was minimally invasive, rapid, unbiased, without the need for specialised equipment or training. Similar to commercial systems, we show that our software-called MouseMove-accurately quantifies numerous parameters of movement including travel distance, speed, turning and curvature. To assess its utility, we used MouseMove to quantify unilateral locomotor deficits in mice following the filament-induced middle cerebral artery occlusion model of acute ischemic stroke. MouseMove can also monitor movement within defined regions-of-interest and is therefore suitable for analysing the Novel Object Recognition test and other field-related cognitive tests. To the best of our knowledge, MouseMove is the first open source software capable of providing qualitative and quantitative information on mouse locomotion in a semi-automated and high-throughput fashion, and hence MouseMove represents a sound alternative to commercial movement analysis systems. [ABSTRACT FROM AUTHOR]

The article presents a study which explores that Tankyrase-mediated ADP-ribosylation, a regulator of Tumor necrosis factor (TNF)-induced death . It mentions that findings of the study suggests that disruption of ADP-ribosylation during an infection can prime a cell to retaliate with an inflammatory cell death.

The article presents a study which explores about the Tankyrase-mediated ADP-ribosylation as a regulator of Tumor necrosis factor (TNF)-induced death. It mentions that TNF, a key component of the innate immune response and suggests that disruption of ADP-ribosylation during an infection can prime a cell to retaliate with an inflammatory cell death.

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. [ABSTRACT FROM AUTHOR]

The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) "killer" domain and neighboring first brace helix of human MLKL with nanomolar affinity. When expressed as genetically encoded reagents in cells, these monobodies potently block necroptotic cell death. However, they did not prevent MLKL recruitment to the "necrosome" and phosphorylation by RIPK3, nor the assembly of MLKL into oligomers, but did block MLKL translocation tomembranes where activatedMLKL normally disrupts membranes to kill cells. An X-ray crystal structure revealed a monobodybinding site centered on the a4 helix of the MLKL 4HB domain, which mutational analyses showed was crucial for reconstitution of necroptosis signaling. These data implicate the a4 helix of its 4HB domain as a crucial site for recruitment of adaptor proteins that mediatemembrane translocation, distinct from known phospholipid binding sites. [ABSTRACT FROM AUTHOR]

Abstract: Purkinje neurons are a sensitive and specialised cell type important for fine motor movement and coordination. Purkinje cell damage manifests as motor incoordination and ataxia — a prominent feature of many human disorders including spinocerebellar ataxia and Huntington's disease. A correlation between Purkinje degeneration and excess cerebellar levels of tissue-type plasminogen activator (tPA) has been observed in multiple genetically-distinct models of ataxia. Here we show that Purkinje loss in a mouse model of Huntington's disease also correlates with a 200% increase in cerebellar tPA activity. That elevated tPA levels arise in a variety of ataxia models suggests that tPA is a common mediator of Purkinje damage. To address the specific contribution of tPA to cerebellar dysfunction we studied the T4 mice line that overexpresses murine tPA in postnatal neurons through the Thy1.2 gene promoter, which directs preferential expression to Purkinje cells within the cerebellum. Here we show that T4 mice develop signs of cerebellar damage within 10weeks of birth including atrophy of Purkinje cell soma and dendrites, astrogliosis, reduced molecular layer volume and altered gait. In contrast, T4 mice displayed no evidence of microgliosis, nor any changes in interneuron density, nor alteration in the cerebellar granular neuron layer. Thus, excess tPA levels may be sufficient to cause targeted Purkinje cell degeneration and ataxia. We propose that elevated cerebellar tPA levels exert a common pathway of Purkinje cell damage. Therapeutically lowering cerebellar tPA levels may represent a novel means of preserving Purkinje cell integrity and motor coordination across a wide range of neurodegenerative diseases. [Copyright &y& Elsevier]

The neurovascular unit provides a dynamic interface between the circulation and central nervous system. Disruption of neurovascular integrity occurs in numerous brain pathologies including neurotrauma and ischaemic stroke. Tissue plasminogen activator is a serine protease that converts plasminogen to plasmin, a protease that dissolves blood clots. Besides its role in fibrinolysis, tissue plasminogen activator is abundantly expressed in the brain where it mediates extracellular proteolysis. However, proteolytically active tissue plasminogen activator also promotes neurovascular disruption after ischaemic stroke; the molecular mechanisms of this process are still unclear. Tissue plasminogen activator is naturally inhibited by serine protease inhibitors (serpins): plasminogen activator inhibitor-1, neuroserpin or protease nexin-1 that results in the formation of serpin:protease complexes. Proteases and serpin:protease complexes are cleared through high-affinity binding to low-density lipoprotein receptors, but their binding to these receptors can also transmit extracellular signals across the plasma membrane. The matrix metalloproteinases are the second major proteolytic system in the mammalian brain, and like tissue plasminogen activators are pivotal to neurological function but can also degrade structures of the neurovascular unit after injury. Herein, we show that tissue plasminogen activator potentiates neurovascular damage in a dose-dependent manner in a mouse model of neurotrauma. Surprisingly, inhibition of activity following administration of plasminogen activator inhibitor-1 significantly increased cerebrovascular permeability. This led to our finding that formation of complexes between tissue plasminogen activator and plasminogen activator inhibitor-1 in the brain parenchyma facilitates post-traumatic cerebrovascular damage. We demonstrate that following trauma, the complex binds to low-density lipoprotein receptors, triggering the induction of matrix metalloproteinase-3. Accordingly, pharmacological inhibition of matrix metalloproteinase-3 attenuates neurovascular permeability and improves neurological function in injured mice. Our results are clinically relevant, because concentrations of tissue plasminogen activator: plasminogen activator inhibitor-1 complex and matrix metalloproteinase-3 are significantly elevated in cerebrospinal fluid of trauma patients and correlate with neurological outcome. In a separate study, we found that matrix metalloproteinase-3 and albumin, a marker of cerebrovascular damage, were significantly increased in brain tissue of patients with neurotrauma. Perturbation of neurovascular homeostasis causing oedema, inflammation and cell death is an important cause of acute and long-term neurological dysfunction after trauma. A role for the tissue plasminogen activator–matrix metalloproteinase axis in promoting neurovascular disruption after neurotrauma has not been described thus far. Targeting tissue plasminogen activator: plasminogen activator inhibitor-1 complex signalling or downstream matrix metalloproteinase-3 induction may provide viable therapeutic strategies to reduce cerebrovascular permeability after neurotrauma. [ABSTRACT FROM PUBLISHER]

Cytoplasmic accumulation of TDP-43 is a disease hallmark for many cases of amyotrophic lateral sclerosis (ALS), associated with a neuroinflammatory cytokine profile related to upregulation of nuclear factor κB (NF-κB) and type I interferon (IFN) pathways. Here we show that this inflammation is driven by the cytoplasmic DNA sensor cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) when TDP-43 invades mitochondria and releases DNA via the permeability transition pore. Pharmacologic inhibition or genetic deletion of cGAS and its downstream signaling partner STING prevents upregulation of NF-κB and type I IFN induced by TDP-43 in induced pluripotent stem cell (iPSC)-derived motor neurons and in TDP-43 mutant mice. Finally, we document elevated levels of the specific cGAS signaling metabolite cGAMP in spinal cord samples from patients, which may be a biomarker of mtDNA release and cGAS/STING activation in ALS. Our results identify mtDNA release and cGAS/STING activation as critical determinants of TDP-43-associated pathology and demonstrate the potential for targeting this pathway in ALS. • TDP-43 enters mitochondria, triggers mtDNA release via the mPTP • TDP-43-induced cytosolic mtDNA accumulation activates the cGAS/STING pathway • Evidence of cytoplasmic mtDNA was found in ALS patient cells and disease models • Blocking STING prevents inflammation and neurodegeneration in vitro and in vivo TDP-43 causes inflammation in ALS by stimulating mitochondrial DNA release, which is subsequently sensed by the cytosolic cGAS/STING pathway, suggesting that inhibition of cGAS/STING could help alleviate inflammation-related damage in ALS. [ABSTRACT FROM AUTHOR]