PAR1 activation induces the release by Schwann cells of factors promoting cell survival and neuritogenesis

Protease-activated receptor 1 (PAR1) is a member of a family of four G-protein-coupled receptors which are activated by proteolytic cleavage of their N-terminal extracellular domain. The expression and the role of PAR1 in peripheral nervous system (PNS) is still poorly investigated, although high PAR1 mRNA expression was found in the dorsal root ganglia and in the non-compacted Schwann cell myelin microvilli at the nodes of Ranvier. Schwann cells (SCs) are the principal population of glial cells of the PNS which myelinate axons and play a key role in axonal regeneration and remyelination. Aim of the present study was to determine if the activation of PAR1 affects the neurotrophic properties of SCs. By double immunofluorescence we observed a specific staining for PAR1 in S100ȕ-positive cells of rat sciatic nerve and sciatic teased fibers. Moreover, PAR1 was highly expressed in SC cultures obtained from both neonatal and adult rat sciatic nerves. When PAR1 specific agonists were added to these cultures an increased proliferation rate was observed. Moreover, the conditioned medium obtained from primary SCs treated with PAR1 agonists increased cell survival and neurite outgrowth on PC12 cells respect to controls. By proteomics, western blot and RT-PCR analyses we identified five proteins which are released by SCs following PAR1 stimulation: Macrophage migration inhibitory factor (Mif), Aldose reductase (Akr1b1), Matrix metalloproteinase-2 (Mmp2), Syndecan-4 (Sdc) and Decorin (Dcn). Conversely, a significant decrease in the level of three proteins was observed: Complement C1r subcomponent (C1r) and Complement component 1 Q subcomponent-bindingprotein (C1qbp). When PAR1 expression was silenced by siRNA the observed pro-survival and neurotrophic properties of SCs appear to be reduced respect to controls. References PAR1 activation affects the neurotrophic properties of Schwann cells. Pompili E1, Fabrizi C2, Somma F2, Correani V3, Maras B3, Schininà ME3, Ciraci V2, Artico M4, Fornai F5, Fumagalli L2. 2017 Jan 4;79:23-33. doi: 10.1016/j.mcn.2017.01.001.Schwann cells (SCs) regulate a wide variety of axonal functions in the peripheral nervous system, providing a supportive growth environment following nerve injury (1). Here we show that rat SCs express the protease-activated receptor-1 (PAR1) both in vivo and in vitro. PAR1 is a G-protein coupled receptor eliciting cellular responses to thrombin and other proteases (2). To investigate if PAR1 activation affects the neurotrophic properties of SCs, this receptor was activated by a specific agonist peptide (TFLLR) and the conditioned medium was transferred to PC12 pheocromocytoma cells for assessing cell survival and neurite outgrowth. Culture medium from SCs treated with 10 µM TFLLR reduced significantly the release of LDH and increased the viability of PC12 cells with respect to the medium of the untreated SCs. Furthermore, conditioned medium from TFLLR-treated SCs increased neurite outgrowth on PC12 cells respect to control medium from untreated cells. To identify putative neurotrophic candidates we performed proteomic analysis on SC secretoma and real time PCR experiments after PAR1 activation. Stimulation of SCs with TFLLR increased specifically the release of a subset of five proteins: Macrophage migration inhibitory factor (Mif), Aldose reductase (Akr1b1), Matrix metalloproteinase-2 (Mmp2), Syndecan-4 (Sdc) and Decorin (Dcn). At the same time there was a significant decrease in the level of three proteins: Complement C1r subcomponent (C1r), Complement component 1 Q subcomponent-binding protein (C1qbp) and Angiogenic factor with G patch and FHA domains 1 (Aggf1). These data indicate that PAR1 stimulation does induce the release by SCs of factors promoting cell survival and neuritogenesis. Among these proteins, Mif, Sdc, Dcn and Mmp2 are of particular interest

Degeneration and regeneration of peripheral nerves: role of thrombin and its receptor PAR-1

The peripheral nervous system has a striking regeneration potential and after damage extensive changes in the differentiation state both of the injured neurons and of the Schwann cells are observed. Schwann cells, in particular, undergo a large scale change in gene expression becoming able to support axonal regeneration. Nerve injury is generally associated to inflammation and activation of the coagulation cascade. Thrombin acts as a polyfunctional signalling molecule exerting its physiological function through soluble target proteins and G-protein-coupled receptors, the protease-activated receptors (PARs) [1]. Recently, we have demonstrated that the activation of the main thrombin receptor, PAR-1, in Schwann cells favours their regenerative potential determining the release of factors which promote axonal regrowth [2]. The pro-regenerative potential of thrombin seems to be exerted in a narrow range of concentrations (pM-nM range). In fact, our preliminary data indicate that high levels of thrombin in the micromolar range slow down Schwann cell proliferation and induce cell death. On the contrary, PAR-1 activating peptides mimic the pro-survival but not the pro-apoptotic effects of thrombin. Controlling thrombin concentration may preserve neuronal health during nerve injury and represent a novel target for pharmacologic therapies

PAR1 activation induces the release by Schwann cells of factors promoting cell survival and neuritogenesis

Protease-activated receptor 1 (PAR1) is a member of a family of four G-protein-coupled receptors which are activated by proteolytic cleavage of their N-terminal extracellular domain. The expression and the role of PAR1 in peripheral nervous system (PNS) is still poorly investigated, although high PAR1 mRNA expression was found in the dorsal root ganglia and in the non-compacted Schwann cell myelin microvilli at the nodes of Ranvier. Schwann cells (SCs) are the principal population of glial cells of the PNS which myelinate axons and play a key role in axonal regeneration and remyelination. Aim of the present study was to determine if the activation of PAR1 affects the neurotrophic properties of SCs. By double immunofluorescence we observed a specific staining for PAR1 in S100ȕ-positive cells of rat sciatic nerve and sciatic teased fibers. Moreover, PAR1 was highly expressed in SC cultures obtained from both neonatal and adult rat sciatic nerves. When PAR1 specific agonists were added to these cultures an increased proliferation rate was observed. Moreover, the conditioned medium obtained from primary SCs treated with PAR1 agonists increased cell survival and neurite outgrowth on PC12 cells respect to controls. By proteomics, western blot and RT-PCR analyses we identified five proteins which are released by SCs following PAR1 stimulation: Macrophage migration inhibitory factor (Mif), Aldose reductase (Akr1b1), Matrix metalloproteinase-2 (Mmp2), Syndecan-4 (Sdc) and Decorin (Dcn). Conversely, a significant decrease in the level of three proteins was observed: Complement C1r subcomponent (C1r) and Complement component 1 Q subcomponent-bindingprotein (C1qbp). When PAR1 expression was silenced by siRNA the observed pro-survival and neurotrophic properties of SCs appear to be reduced respect to controls. References PAR1 activation affects the neurotrophic properties of Schwann cells. Pompili E1, Fabrizi C2, Somma F2, Correani V3, Maras B3, Schininà ME3, Ciraci V2, Artico M4, Fornai F5, Fumagalli L2. 2017 Jan 4;79:23-33. doi: 10.1016/j.mcn.2017.01.001.Schwann cells (SCs) regulate a wide variety of axonal functions in the peripheral nervous system, providing a supportive growth environment following nerve injury (1). Here we show that rat SCs express the protease-activated receptor-1 (PAR1) both in vivo and in vitro. PAR1 is a G-protein coupled receptor eliciting cellular responses to thrombin and other proteases (2). To investigate if PAR1 activation affects the neurotrophic properties of SCs, this receptor was activated by a specific agonist peptide (TFLLR) and the conditioned medium was transferred to PC12 pheocromocytoma cells for assessing cell survival and neurite outgrowth. Culture medium from SCs treated with 10 µM TFLLR reduced significantly the release of LDH and increased the viability of PC12 cells with respect to the medium of the untreated SCs. Furthermore, conditioned medium from TFLLR-treated SCs increased neurite outgrowth on PC12 cells respect to control medium from untreated cells. To identify putative neurotrophic candidates we performed proteomic analysis on SC secretoma and real time PCR experiments after PAR1 activation. Stimulation of SCs with TFLLR increased specifically the release of a subset of five proteins: Macrophage migration inhibitory factor (Mif), Aldose reductase (Akr1b1), Matrix metalloproteinase-2 (Mmp2), Syndecan-4 (Sdc) and Decorin (Dcn). At the same time there was a significant decrease in the level of three proteins: Complement C1r subcomponent (C1r), Complement component 1 Q subcomponent-binding protein (C1qbp) and Angiogenic factor with G patch and FHA domains 1 (Aggf1). These data indicate that PAR1 stimulation does induce the release by SCs of factors promoting cell survival and neuritogenesis. Among these proteins, Mif, Sdc, Dcn and Mmp2 are of particular interest

Anti-proliferative effect of Rosmarinus officinalis L. extract on human melanoma A375 cells

Rosemary (Rosmarinus officinalis L.) has been used since ancient times in traditional medicine, while nowadays various rosemary formulations are increasingly exploited by alternative medicine to cure or prevent a wide range of health disorders. Rosemary's bioproperties have prompted scientific investigation, which allowed us to ascertain antioxidant, anti-inflammatory, cytostatic, and cytotoxic activities of crude extracts or of pure components. Although there is a growing body of experimental work, information about rosemary's anticancer properties, such as chemoprotective or anti-proliferative effects on cancer cells, is very poor, especially concerning the mechanism of action. Melanoma is a skin tumor whose diffusion is rapidly increasing in the world and whose malignancy is reinforced by its high resistance to cytotoxic agents; hence the availability of new cytotoxic drugs would be very helpful to improve melanoma prognosis. Here we report on the effect of a rosemary hydroalcoholic extract on the viability of the human melanoma A375 cell line. Main components of rosemary extract were identified by liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS) and the effect of the crude extract or of pure components on the proliferation of cancer cells was tested by MTT and Trypan blue assays. The effect on cell cycle was investigated by using flow cytometry, and the alteration of the cellular redox state was evaluated by intracellular ROS levels and protein carbonylation analysis. Furthermore, in order to get information about the molecular mechanisms of cytotoxicity, a comparative proteomic investigation was performed

Redox proteomics analysis of HNE-modified proteins in Down syndrome brain: clues for understanding the development of Alzheimer disease.

Down syndrome (DS) is the most common genetic cause of intellectual disability, due to partial or complete triplication of chromosome 21. DS subjects are characterized by a number of abnormalities including premature aging and development of Alzheimer disease (AD) neuropathology after approximately 40 years of age. Several studies show that oxidative stress plays a crucial role in the development of neurodegeneration in the DS population. Increased lipid peroxidation is one of the main events causing redox imbalance within cells through the formation of toxic aldehydes that easily react with DNA, lipids, and proteins. In this study we used a redox proteomics approach to identify specific targets of 4-hydroxynonenal modifications in the frontal cortex from DS cases with and without AD pathology. We suggest that a group of identified proteins followed a specific pattern of oxidation in DS vs young controls, probably indicating characteristic features of the DS phenotype; a second group of identified proteins showed increased oxidation in DS/AD vs DS, thus possibly playing a role in the development of AD. The third group of comparison, DS/AD vs old controls, identified proteins that may be considered specific markers of AD pathology. All the identified proteins are involved in important biological functions including intracellular quality control systems, cytoskeleton network, energy metabolism, and antioxidant response. Our results demonstrate that oxidative damage is an early event in DS, as well as dysfunctions of protein-degradation systems and cellular protective pathways, suggesting that DS subjects are more vulnerable to oxidative damage accumulation that might contribute to AD development. Further, considering that the majority of proteins have been already demonstrated to be oxidized in AD brain, our results strongly support similarities with AD in DS

COVID-19: a review of the literature and the experience of INMI Lazzaro Spallanzani two months after the epidemic outbreak

COVID-19 disease often presents as an acute respiratory syndrome with a very varied and still nonspecific radio-logical signs. The characteristic radiological signs begin to be known and codified and include the ground glass opacities in the initial phase of the disease, which are complicated by the thickening of the inter lobar and intra lobular septa with a “crazy paving” paintings, the appearance of areas of parenchymal consolidation up to full-blown acute respiratory distress syndrome states. The purpose of this paper is to review the international literature and the analysis of the first 70 patients studied at the National Institute for Infectious Diseases Lazzaro Spallanzani in Rome

Calcium binding promotes Prion Protein fragment 90-231 conformational change toward a membrane destabilizing and cytotoxic structure

The pathological form of prion protein (PrPSc), as other amyloidogenic proteins, causes a marked increase of membrane permeability. PrPSc extracted from infected Syrian hamster brains induces a considerable change in membrane ionic conductance, although the contribution of this interaction to the molecular mechanism of neurodegeneration process is still controversial. We previously showed that the human PrP fragment 90–231 (hPrP90–231) increases ionic conductance across artificial lipid bilayer, in a calcium-dependent manner, producing an alteration similar to that observed for PrPSc. In the present study we demonstrate that hPrP90–231, pre-incubated with 10 mM Ca++ and then re-suspended in physiological external solution increases not only membrane conductance but neurotoxicity as well. Furthermore we show the existence of a direct link between these two effects as demonstrated by a highly statistically significant correlation in several experimental conditions. A similar correlation between increased membrane conductance and cell degeneration has been observed assaying hPrP90–231 bearing pathogenic mutations (D202N and E200K). We also report that Ca++ binding to hPrP90–231 induces a conformational change based on an alteration of secondary structure characterized by loss of alpha-helix content causing hydrophobic amino acid exposure and proteinase K resistance. These features, either acquired after controlled thermal denaturation or induced by D202N and E200K mutations were previously identified as responsible for hPrP90–231 cytotoxicity. Finally, by in silico structural analysis, we propose that Ca++ binding to hPrP90–231 modifies amino acid orientation, in the same way induced by E200K mutation, thus suggesting a pathway for the structural alterations responsible of PrP neurotoxicity