Diversification of PAR signaling through receptor crosstalk.

Protease activated receptors (PARs) are among the first receptors shown to transactivate other receptors: noticeably, these interactions are not limited to members of the same family, but involve receptors as diverse as receptor kinases, prostanoid receptors, purinergic receptors and ionic channels among others. In this review, we will focus on the evidence for PAR interactions with members of their own family, as well as with other types of receptors. We will discuss recent evidence as well as what we consider as emerging areas to explore; from the signalling pathways triggered, to the physiological and pathological relevance of these interactions, since this additional level of molecular cross-talk between receptors and signaling pathways is only beginning to be explored and represents a novel mechanism providing diversity to receptor function and play important roles in physiology and disease.

The PKC-ζ pseudosubstrate peptide induces glutamate release from retinal pigment epithelium cells through kinase- independent activation of Best1.

AIMS: The retinal pigment epithelium (RPE) is a highly specialized cell monolayer, that plays a key role in the maintenance of photoreceptor function and the blood-retina barrier (BRB). In this study, we found that a myristoylated pseudosubstrate of PKC-ζ (PKCζ PS), considered as a PKC-ζ inhibitor, plays a distinct role in RPE. MAIN METHODS: We demonstrated that PKCζ PS stimulates the release of Glutamate (Glu) using in vitro3H-Glutamate release experiments. By western blot, kinase assays, and Fluoresence Ca+2 Concentration Measurements, we determined the cellular mechanisms involved in such release. KEY FINDINGS: Surprisingly, PKCζ PS has no effect on either phosphorylation of T560, essential for catalytic activity, nor it has an effect on kinase activity. It induces the dose-dependent release of Glu by increasing intracellular Ca+2 levels. Interestingly, this release was not observed upon stimulation by other non-competitive PKC-ζ inhibitors. We here demonstrated that the PKCζ PS stimulates the release of Glutamate from RPE by activating the Ca2+-dependent Cl channel Bestrophin 1 (Best1). SIGNIFICANCE: These results question PKCζ PS specificity as an inhibitor of this enzyme. Furthermore, the present results underline the relevance of clarifying the molecular mechanisms involved in glutamate release from the retina under conditions derived from excitotoxic stimuli.

Thrombin-activated PAR1 membrane expression is regulated by Rab11a-RCP complex dissociation.

PAR1 activation by thrombin promotes intracellular signaling leading to RPE cell transformation, proliferation, and migration, characteristic of fibroproliferative eye diseases. Due to the cleavage of PAR1 N-terminal domain, carried by thrombin, the arrest of PAR1 signaling is achieved by transport into lysosomes and degradation. Recent findings suggest that the GTPase Rab11a in conjunction with its effector RCP may direct PAR1 to lysosomes. Hereby we demonstrate that thrombin-induced PAR1 internalization and lysosomal targeting requires the disassembly of the Rab11a/RCP complex, and that this process depends on thrombin-induced intracellular calcium increase and calpain activation. These findings unveil a novel mechanism that regulates thrombin activated PAR1 internalization and degradation.

Thrombin induces Ca2+-dependent glutamate release from RPE cells mediated by PLC/PKC and reverse Na+/Ca2+ exchange.

Purpose: We analyzed the molecular mechanisms leading to glutamate release from rat primary cultures of RPE cells, under isosmotic conditions. Thrombin has been shown to stimulate glutamate release from astrocytes and retinal glia; however, the effect of thrombin on glutamate release from RPE cells has not been examined. Our previous work showed that upon the alteration of the blood-retina barrier, the serine protease thrombin could contribute to the transformation, proliferation, and migration of RPE cells. In this condition, elevated extracellular glutamate causes neuronal loss in many retinal disorders, including glaucoma, ischemia, diabetic retinopathy, and inherited photoreceptor degeneration. Methods: Primary cultures of rat RPE cells were preloaded with 1 µCi/ml 3H-glutamate in Krebs Ringer Bicarbonate (KRB) buffer for 30 min at 37 °C. Cells were rinsed and super-perfused with 1 ml/min KRB for 15 min. Stable release was reached at the 7th minute, and on the 8th minute, fresh KRB containing stimuli was added. Results: This study showed for the first time that thrombin promotes specific, dose-dependent glutamate release from RPE cells, induced by the activation of protease-activated receptor 1 (PAR-1). This effect was found to depend on the Ca2+ increase mediated by the phospholipase C-ß (PLC-ß) and protein kinase C (PKC) pathways, as well as by the reverse activity of the Na+/Ca2+ exchanger. Conclusions: Given the intimate contact of the RPE with the photoreceptor outer segments, diffusion of RPE-released glutamate could contribute to the excitotoxic death of retinal neurons, and the development of thrombin-induced eye pathologies.

Antisense oligonucleotides targeting mutant Ataxin-7 restore visual function in a mouse model of spinocerebellar ataxia type 7.

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disorder characterized by cerebellar and retinal degeneration, and is caused by a CAG-polyglutamine repeat expansion in the ATAXIN-7 gene. Patients with SCA7 develop progressive cone-rod dystrophy, typically resulting in blindness. Antisense oligonucleotides (ASOs) are single-stranded chemically modified nucleic acids designed to mediate the destruction, prevent the translation, or modify the processing of targeted RNAs. Here, we evaluated ASOs as treatments for SCA7 retinal degeneration in representative mouse models of the disease after injection into the vitreous humor of the eye. Using Ataxin-7 aggregation, visual function, retinal histopathology, gene expression, and epigenetic dysregulation as outcome measures, we found that ASO-mediated Ataxin-7 knockdown yielded improvements in treated SCA7 mice. In SCA7 mice with retinal disease, intravitreal injection of Ataxin-7 ASOs also improved visual function despite initiating treatment after symptom onset. Using color fundus photography and autofluorescence imaging, we also determined the nature of retinal degeneration in human SCA7 patients. We observed variable disease severity and cataloged rapidly progressive retinal degeneration. Given the accessibility of neural retina, availability of objective, quantitative readouts for monitoring therapeutic response, and the rapid disease progression in SCA7, ASOs targeting ATAXIN-7 might represent a viable treatment for SCA7 retinal degeneration.

OCT Angiography Helps Distinguish Between Proliferative Macular Telangiectasia Type 2 and Neovascular Age-Related Macular Degeneration.

BACKGROUND AND OBJECTIVE: To demonstrate the advantage of optical coherence tomography angiography (OCTA) for the diagnosis and management of proliferative macular telangiectasia type 2 (MacTel2) masquerading as neovascular age-related macular degeneration (AMD). PATIENTS AND METHODS: This is an observational cases series. Three patients referred with the diagnosis of neovascular AMD were identified in this retrospective study. In addition to color fundus, fluorescein angiography, and spectral-domain OCT (SD-OCT) imaging, SD-OCTA (AngioPlex; Carl Zeiss Meditec, Dublin, CA) was performed. RESULTS: SD-OCTA revealed bilateral parafoveal retinal microvascular changes in three patients and unambiguously confirmed the diagnosis of MacTel2. CONCLUSION: OCTA is an important tool for the correct diagnosis of MacTel2 in older patients with the concomitant or masquerading diagnosis of AMD. [Ophthalmic Surg Lasers Imaging Retina. 2018;49:303-312.].

Thrombin-Induced Calpain Activation Promotes Protease-Activated Receptor 1 Internalization.

The serine protease thrombin activates Protease-Activated Receptors (PARs), a family of G-protein-coupled receptors (GPCRs) activated by the proteolytic cleavage of their extracellular N-terminal domain. Four members of this family have been identified: PAR1-4. The activation of Protease-Activated Receptor 1(PAR1), the prototype of this receptor family, leads to an increase in intracellular Ca+2 concentration ([Ca+2]i) mediated by Gq11α coupling and phospholipase C (PLC) activation. We have previously shown that the stimulation of PAR1 by thrombin promotes intracellular signaling leading to RPE cell transformation, proliferation, and migration which characterize fibroproliferative eye diseases leading to blindness. Within this context, the elucidation of the mechanisms involved in PAR1 inactivation is of utmost importance. Due to the irreversible nature of PAR1 activation, its inactivation must be efficiently regulated in order to terminate signaling. Using ARPE-19 human RPE cell line, we characterized thrombin-induced [Ca+2]i increase and demonstrated the calcium-dependent activation of µ-calpain mediated by PAR1. Calpains are a family of calcium-activated cysteine proteases involved in multiple cellular processes including the internalization of membrane proteins through clathrin-coated vesicles. We demonstrated that PAR1-induced calpain activation results in the degradation of α-spectrin by calpain, essential for receptor endocytosis, and the consequent decrease in PAR1 membrane expression. Collectively, the present results identify a novel µ-calpain-dependent mechanism for PAR1 inactivation following exposure to thrombin.

Paxillin: a crossroad in pathological cell migration.

Paxilllin is a multifunctional and multidomain focal adhesion adapter protein which serves an important scaffolding role at focal adhesions by recruiting structural and signaling molecules involved in cell movement and migration, when phosphorylated on specific Tyr and Ser residues. Upon integrin engagement with extracellular matrix, paxillin is phosphorylated at Tyr31, Tyr118, Ser188, and Ser190, activating numerous signaling cascades which promote cell migration, indicating that the regulation of adhesion dynamics is under the control of a complex display of signaling mechanisms. Among them, paxillin disassembly from focal adhesions induced by extracellular regulated kinase (ERK)-mediated phosphorylation of serines 106, 231, and 290 as well as the binding of the phosphatase PEST to paxillin have been shown to play a key role in cell migration. Paxillin also coordinates the spatiotemporal activation of signaling molecules, including Cdc42, Rac1, and RhoA GTPases, by recruiting GEFs, GAPs, and GITs to focal adhesions. As a major participant in the regulation of cell movement, paxillin plays distinct roles in specific tissues and developmental stages and is involved in immune response, epithelial morphogenesis, and embryonic development. Importantly, paxillin is also an essential player in pathological conditions including oxidative stress, inflammation, endothelial cell barrier dysfunction, and cancer development and metastasis.

PKC-ζ Regulates Thrombin-Induced Proliferation of Human Müller Glial Cells.

PURPOSE: To investigate the effect of thrombin on the proliferation of human Müller glial cells (MCs) and define the possible signaling mechanisms involved in this process. METHODS: Protease-activated receptor (PARs 1-4) expression was analyzed using RT-PCR and Western blot in the MIO-M1 Müller cell line (MC). Müller cell proliferation was assessed by the MTS reduction method. Wound healing and immunoreactivity to Ki67 antigen were used to dissociate proliferation and migration. Cell migration was examined using transwell migration assays. The involvement of extracellular signal-regulated kinase (ERK1/2) phosphorylation/activation in thrombin-induced human MC proliferation was determined by Western blot. Intracellular pathways involved in ERK1/2 activation were analyzed by pharmacologic inhibition. RESULTS: We first demonstrated that human MCs express PARs 1 to 4. Our results show that thrombin dose-dependently stimulates MC proliferation by 44%, with a calculated Ec50 of 0.86 nM. Müller cell maximal proliferation required sustained thrombin treatment for 72 hours, in contrast to our previous findings in RPE cells showing maximal thrombin-induced proliferation at 24-hour stimulation. We demonstrate that thrombin induces MC cell proliferation through the Ras-independent activation of the Raf/MEK/ERK cascade, under the control of protein kinase C (PKC)-ζ. CONCLUSIONS: The breakdown of blood-retina barrier (BRB) exposes MCs to thrombin contained in serum. Our findings further strengthen the critical involvement of thrombin in the development of proliferative retinopathies and may provide pharmacologic targets for the prevention or treatment of these diseases.

Glutamate Receptor Stimulation Up-Regulates Glutamate Uptake in Human Müller Glia Cells.

Glutamate, the main excitatory amino acid in the vertebrate retina, is a well know activator of numerous signal transduction pathways, and has been critically involved in long-term synaptic changes acting through ionotropic and metabotropic glutamate receptors. However, recent findings underlining the importance of intensity and duration of glutamate stimuli for specific neuronal responses, including excitotoxicity, suggest a crucial role for Na(+)-dependent glutamate transporters, responsible for the removal of this neurotransmitter from the synaptic cleft, in the regulation of glutamate-induced signaling. Transporter proteins are expressed in neurons and glia cells, albeit most of glutamate uptake occurs in the glial compartment. Within the retina, Müller glia cells are in close proximity to glutamatergic synapses and participate in the recycling of glutamate through the glutamate/glutamine shuttle. In this context, we decided to investigate a plausible role of glutamate as a regulatory signal for its own transport in human retinal glia cells. To this end, we determined [(3)H]-D-aspartate uptake in cultures of spontaneously immortalized human Müller cells (MIO-M1) exposed to distinct glutamatergic ligands. A time and dose-dependent increase in the transporter activity was detected. This effect was dependent on the activation of the N-methyl D-aspartate subtype of glutamate receptors, due to a dual effect: an increase in affinity and an augmented expression of the transporter at the plasma membrane, as established via biotinylation experiments. Furthermore, a NMDA-dependent association of glutamate transporters with the cystoskeletal proteins ezrin and glial fibrillary acidic protein was also found. These results add a novel mediator of the glutamate transporter modulation and further strengthen the notion of the critical involvement of glia cells in synaptic function.