Thrombin orchestrates cellular events after injury to the vascular system and extravasation of blood into surrounding tissues. The pathophysiological response to thrombin is mediated by proteaseactivated receptor-1 (PAR-1), a seven-transmembrane G proteincoupled receptor expressed in the nervous system that is identical to the thrombin receptor in platelets, fibroblasts, and endothelial cells. Once activated by thrombin, PAR-1 induces rapid and dramatic changes in cell morphology, notably the retraction of growth cones, axons, and dendrites in neurons and processes in astrocytes. The signal is conveyed by a series of localized ATP-dependent reactions directed to the actin cytoskeleton. How cells meet the dynamic and localized energy demands during signal transmission is unknown. Using the yeast two-hybrid system, we identified an interaction between PAR-1 cytoplasmic tail and the brain isoform of creatine kinase, a key ATP-generating enzyme that regulates ATP within subcellular compartments. The interaction was confirmed in vitro and in vivo. Reducing creatine kinase levels or its ATP-generating potential inhibited PAR-1-mediated cellular shape changes as well as a PAR-1 signaling pathway involving the activation of RhoA, a small G protein that relays signals to the cytoskeleton. Thrombin-stimulated intracellular calcium release was not affected. Our results suggest that creatine kinase is bound to PAR-1 where it may be poised to provide bursts of site-specific high-energy phosphate necessary for efficient receptor signal transduction during cytoskeletal reorganization. P rotease activated receptor-1 (PAR-1) mediates the cellular responses to thrombin during blood coagulation, cell proliferation, vascular permeability changes, tumor metastasis, and nervous system injury (1-3). PAR-1 is a seven-transmembrane G protein-coupled receptor with a novel activation mechanism. Proteolysis at a thrombin cleavage site in the extracellular amino terminus exposes a new amino terminus containing the peptide ligand SFLLRN, which binds intramolecularly to initiate intracellular signals (4). Although originally detected in platelets, endothelial cells, and fibroblasts, PAR-1 is also expressed in the nervous system in a developmentally regulated manner and by specific subpopulations of neurons and astrocytes that are especially vulnerable to neurodegeneration and ischemic injury (1,5,6).In most cells expressing PAR-1, activation of the receptor transmits signals to the actin cytoskeleton that profoundly alter cell shape. Platelets, for example, convert from a spherical to disk shape and extend filopodia (7), endothelial cells contract (8), neurons retract axons, and astrocytes resorb processes and flatten their cell bodies (9-11). These signals also regulate changes in actin-related cell motility observed in neurons (10), fibroblasts (12), and tumor cells (3). The morphological response is mediated by a key signaling pathway that uses serine͞ threonine kinases, G␣12͞13, RhoA, and myosin light chain kinase; actomyosin contraction ...
A single amino acid mutation near the active site of the CAPN5 protease was linked to the inherited blinding disorder, autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV, OMIM #193235). In homology modeling with other calpains, this R243L CAPN5 mutation was situated in a mobile loop that gates substrate access to the calcium-regulated active site. In in vitro activity assays, the mutation increased calpain protease activity and made it far more active at low concentrations of calcium. To test whether the disease allele could yield an animal model of ADNIV, we created transgenic mice expressing human (h) CAPN5(R243L) only in the retina. The resulting hCAPN5(R243L) transgenic mice developed a phenotype consistent with human uveitis and ADNIV, at the clinical, histological and molecular levels. The fundus of hCAPN5(R243L) mice showed enhanced autofluorescence (AF) and pigment changes indicative of reactive retinal pigment epithelial cells and photoreceptor degeneration. Electroretinography showed mutant mouse eyes had a selective loss of the b-wave indicating an inner-retina signaling defect. Histological analysis of mutant mouse eyes showed protein extravasation from dilated vessels into the anterior chamber and vitreous, vitreous inflammation, vitreous and retinal fibrosis and retinal degeneration. Analysis of gene expression changes in the hCAPN5(R243L) mouse retina showed upregulation of several markers, including members of the Toll-like receptor pathway, chemokines and cytokines, indicative of both an innate and adaptive immune response. Since many forms of uveitis share phenotypic characteristics of ADNIV, this mouse offers a model with therapeutic testing utility for ADNIV and uveitis patients.
Hyperopia (farsightedness) is a common and significant cause of visual impairment, and extreme hyperopia (nanophthalmos) is a consequence of loss-of-function MFRP mutations. MFRP deficiency causes abnormal eye growth along the visual axis and significant visual comorbidities, such as angle closure glaucoma, cystic macular edema, and exudative retinal detachment. The Mfrp rd6 /Mfrp rd6 mouse is used as a pre-clinical animal model of retinal degeneration, and we found it was also hyperopic. To test the effect of restoring Mfrp expression, we delivered a wild-type Mfrp to the retinal pigmented epithelium (RPE) of Mfrp rd6 /Mfrp rd6 mice via adeno-associated viral (AAV) gene therapy. Phenotypic rescue was evaluated using non-invasive, human clinical testing, including fundus auto-fluorescence, optical coherence tomography, electroretinography, and ultrasound. These analyses showed gene therapy restored retinal function and normalized axial length. Proteomic analysis of RPE tissue revealed rescue of specific proteins associated with eye growth and normal retinal and RPE function. The favorable response to gene therapy in Mfrp rd6 /Mfrp rd6 mice suggests hyperopia and associated refractive errors may be amenable to AAV gene therapy.
Thrombin is a serine protease that evokes various cellular responses involved in injury and repair of the nervous system through the activation of protease-activated receptor-1 (PAR-1). Signals that modulate cell morphology precede most PAR-1 effects, but the initial signal transduction molecules are not known. Using the yeast two-hybrid system, we identified Hsp90, a chaperone with known signaling properties, as a binding partner of PAR-1. The interaction was confirmed by glutathione Stransferase pull-down, overlay, and co-immunoprecipitation assays. Morphological assays in mouse astrocytes were carried out to evaluate the importance of Hsp90 during cytoskeletal signaling. Reducing Hsp90 levels by antisense treatment or disruption of the Hsp90⅐PAR-1 complex by the Hsp90-specific drug geldanamycin attenuated thrombinmediated astrocyte shape changes. Furthermore, overexpression of the PAR-1 cytoplasmic tail abrogated thrombininduced cytoskeletal changes in neuronal cells. Treatment with geldanamycin specifically inhibited activation of RhoA without affecting thrombin-mediated intracellular calcium release, revealing the regulation of a distinct signaling pathway by Hsp90. Taken together, these studies demonstrate that Hsp90 may be essential for PAR-1-mediated signaling to the cytoskeleton.Thrombin is a serine protease involved in a number of pathophysiological processes that include blood clotting, inflammation, repair processes, and tumor metastasis (1-4). In brain, thrombin regulates the viability of neurons and astrocytes by increasing survival under conditions of hypoglycemia and oxidative stress and inducing apoptosis under other conditions (5-8). Thrombin is also chemotactic for macrophages and mitogenic for smooth muscle cells, fibroblasts, and astrocytes and induces secretion of growth factors and cytokines from fibroblasts and smooth muscle cells (9). Most of the thrombinmediated effects are preceded by morphological changes in cells that follow activation of a seven-transmembrane G proteincoupled receptor called protease-activated receptor-1 (PAR-1)
IntroductionAfter spending several billion dollars and a decade or more in development and clinical trial, only approximately 10% of new drugs show efficacy and reach the market for patient use. This apparent inefficiency, however, masks enormous opportunities to repurpose existing drugs to treat diseases for which they were not originally intended, especially diseases with limited therapeutic options (1, 2). Therapies for inflammatory diseases are an attractive choice for drug repositioning because of the complexity of the immune response, the hit-or-miss nature of treatment (which is often by trial and error), and the number of traditional drugs and new biologics for inflammatory diseases.Determining which drugs to reposition, however, is not trivial. Among a heterogeneous patient population that seems affected by the same autoimmune condition, one cannot be sure if the disease is in fact the same, or at what progressive stage a patient is presenting. Currently, most predictions of patients that would benefit from drug repositioning are drawn from retrospective computational methods, genomic analyses, BACKGROUND. In patients with limited response to conventional therapeutics, repositioning of already approved drugs can bring new, more effective options. Current drug repositioning methods, however, frequently rely on retrospective computational analyses and genetic testing -time consuming methods that delay application of repositioned drugs. Here, we show how proteomic analysis of liquid biopsies successfully guided treatment of neovascular inflammatory vitreoretinopathy (NIV), an inherited autoinflammatory disease with otherwise poor clinical outcomes.
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