Novel interactions of transglutaminase-2 with heparan sulphate proteoglycans: reflection on physiological implications

This mini-review brings together information from publications and recent conference proceedings that have shed light on the biological interaction between transglutaminase-2 and heparan sulphate proteoglycans. We subsequently draw hypothesis of possible implications in the wound healing process. There is a substantial overlap in the action of transglutaminase-2 and the heparan sulphate proteoglycan syndecan-4 in normal and abnormal wound repair. Our latest findings have identified syndecan-4 as a possible binding and signalling partner of fibronectinbound TG2 and support the idea that transglutaminase-2 and syndecan-4 acts in synergy

Interplay between transglutaminases and heparan sulphate in progressive renal scarring

Transglutaminase-2 (TG2) is a new anti-fibrotic target for chronic kidney disease, for its role in altering the extracellular homeostatic balance leading to excessive build-up of matrix in kidney. However, there is no confirmation that TG2 is the only transglutaminase involved, neither there are strategies to control its action specifically over that of the conserved family-members. In this study, we have profiled transglutaminase isozymes in the rat subtotal nephrectomy (SNx) model of progressive renal scarring. All transglutaminases increased post-SNx peaking at loss of renal function but TG2 was the predominant enzyme. Upon SNx, extracellular TG2 deposited in the tubulointerstitium and peri-glomerulus via binding to heparan sulphate (HS) chains of proteoglycans and co-associated with syndecan-4. Extracellular TG2 was sufficient to activate transforming growth factor-β1 in tubular epithelial cells, and this process occurred in a HS-dependent way, in keeping with TG2-affinity for HS. Analysis of heparin binding of the main transglutaminases revealed that although the interaction between TG1 and HS is strong, the conformational heparin binding site of TG2 is not conserved, suggesting that TG2 has a unique interaction with HS within the family. Our data provides a rationale for a novel anti-fibrotic strategy specifically targeting the conformation-dependent TG2-epitope interacting with HS

Neurochemical Changes in the Mouse Hippocampus Underlying the Antidepressant Effect of Genetic Deletion of P2X7 Receptors.

Recent investigations have revealed that the genetic deletion of P2X7 receptors (P2rx7) results in an antidepressant phenotype in mice. However, the link between the deficiency of P2rx7 and changes in behavior has not yet been explored. In the present study, we studied the effect of genetic deletion of P2rx7 on neurochemical changes in the hippocampus that might underlie the antidepressant phenotype. P2X7 receptor deficient mice (P2rx7-/-) displayed decreased immobility in the tail suspension test (TST) and an attenuated anhedonia response in the sucrose preference test (SPT) following bacterial endotoxin (LPS) challenge. The attenuated anhedonia was reproduced through systemic treatments with P2rx7 antagonists. The activation of P2rx7 resulted in the concentration-dependent release of [3H]glutamate in P2rx7+/+ but not P2rx7-/- mice, and the NR2B subunit mRNA and protein was upregulated in the hippocampus of P2rx7-/- mice. The brain-derived neurotrophic factor (BDNF) expression was higher in saline but not LPS-treated P2rx7-/- mice; the P2rx7 antagonist Brilliant blue G elevated and the P2rx7 agonist benzoylbenzoyl ATP (BzATP) reduced BDNF level. This effect was dependent on the activation of NMDA and non-NMDA receptors but not on Group I metabotropic glutamate receptors (mGluR1,5). An increased 5-bromo-2-deoxyuridine (BrdU) incorporation was also observed in the dentate gyrus derived from P2rx7-/- mice. Basal level of 5-HT was increased, whereas the 5HIAA/5-HT ratio was lower in the hippocampus of P2rx7-/- mice, which accompanied the increased uptake of [3H]5-HT and an elevated number of [3H]citalopram binding sites. The LPS-induced elevation of 5-HT level was absent in P2rx7-/- mice. In conclusion there are several potential mechanisms for the antidepressant phenotype of P2rx7-/- mice, such as the absence of P2rx7-mediated glutamate release, elevated basal BDNF production, enhanced neurogenesis and increased 5-HT bioavailability in the hippocampus

Role of sphingolipids in the biogenesis and biological activity of extracellular vesicles

Extracellular vesicles (EVs) are membrane vesicles released by both eukaryotic and prokaryotic cells; they not only serve physiological functions, such as disposal of cellular components, but also play pathophysiologic roles in inflammatory and degenerative diseases. Common molecular mechanisms for EV biogenesis are evident in different cell biological contexts across eukaryotic phyla, and inhibition of this biogenesis may provide an avenue for therapeutic research. The involvement of sphingolipids (SLs) and their enzymes on EV biogenesis and release has not received much attention in current research. Here, we review how SLs participate in EV biogenesis by shaping membrane curvature and how they contribute to EV action in target cells. First, we describe how acid and neutral SMases, by generating the constitutive SL, ceramide, facilitate biogenesis of EVs at the plasma membrane and inside the endocytic compartment. We then discuss the involvement of other SLs, such as sphingosine-1-phosphate and galactosyl-sphingosine, in EV formation and cargo sorting. Last, we look ahead at some biological effects of EVs mediated by changes in SL levels in recipient cells

Modelling the prion protein-mediated transport of extracellular vesicles on the neuron surface

Neurodegenerative diseases (NDs) are among the leading causes of global mortality, characterized by the progressive deterioration of specific neuron populations, ultimately leading to cognitive decline and dementia. Extracellular vesicles (EVs) are believed to play a role in the early stages of these diseases, acting as carriers of pathogens and contributing to neuroinflammation and disease propagation. This study presents a mathematical model aimed at characterizing the movement of EVs bearing prion protein (PrP) on their surface along neuronal surfaces. The model, informed by experimental data, investigates the influence of PrP and actin polymerization on EV transport dynamics and explores the possible interplay between passive and active mechanisms. EVs isolated from non-human astrocytes were analysed under three conditions: untreated control (Ctrl), neurons treated with Cytochalasin D (CytoD-HN) and EVs treated with Cytochalasin D (CytoD-EV). The mathematical model is data-driven, testing different hypotheses regarding the underlying transport mechanisms. In the CytoD-EV dataset, EV movement was modelled using a flashing Brownian ratchet to represent directed motion. For active transport in the CytoD-HN set, a symmetric periodic potential was used to describe EV rolling along the neuron surface. The Ctrl scenario incorporates both mechanisms, reflecting a more complex transport behaviour. A sensitivity analysis and comparison between numerical predictions and experimental data suggest that the model effectively captures key features of EV motion, providing a quantitative framework to interpret different transport regimes. While some variability remains, the approach offers a promising basis for future investigations into the role of cytoskeletal dynamics in EV-mediated disease propagation

Cross-talk between microglia and oligodendroglial progenitors in cerebral ischemia: implications for new purinergic strategies to brain repair

Oligodendrocytes, the myelin-forming cells in the brain, are severely affected by ischemia [1], contributing to stroke- associated deficits. The possibility to implement spontaneous post-injury repair mechanisms by targeting myelin still represents an unexplored field. In this respect, GPR17, a P2Y-like receptor transiently expressed on Oligodendrocyte Progenitor Cells (OPCs) has emerged as a target to implement stroke repair through stimulation of OPC maturation [2]. Results obtained by fate-mapping analysis using the conditional GPR17-iCreERT2xCAG-eGFP transgenic mice showed that the subpopulation of adult OPCs expressing GPR17 (GFP+-cells) represents “a reserve pool” that is maintained for repair purposes after brain damage [3]. In particular, we recently demonstrated that, after brain ischemia, GFP+-cells actively respond to injury increasing their proliferation rate and migratory capacity. However, at later stages, only a low percentage of these cells undergoes maturation [4]. This limited post-stroke repair is likely due to local unfavourable inflammatory milieu mediated by macrophages and resident microglia, which participate to post-ischemic inflammation assuming both detrimental and beneficial phenotypes. Here, we aimed at: (i) characterizing the spatio-temporal distribution of GFP+-cells in relation to microglia/macrophage polarization in transgenic mice after middle cerebral artery occlusion (MCAo); (ii) exploring the cross-talk between microglia and OPCs, by assessing how vesicles released extracellularly (EVs) by microglia, polarized toward a pro- or anti- inflammatory state, influence OPC behaviour. In vivo studies showed that GFP+-cells accumulate at the border of the ischemic lesion starting from 72h after ischemia, when microglia and macrophages show both pro- and anti-inflammatory features. One week after stroke, the absolute number of pro-inflammatory cells increases, while myeloid cells with pro-regenerative phenotype do not significantly change. In vitro studies pointed out that EVs produced by pro-inflammatory microglia only slightly limit OPC proliferation, whereas EVs produced by pro-reparative microglia tend to increase it. Preliminary data showed that all types of EVs (from unstimulated, pro-inflammatory or pro-regenerative microglia) are able to induce OPC migration, indicating that EVs provide attractive guidance cues independently of the activation state of donor microglia. Interestingly, EVs from pro-regenerative microglia have a higher chemotactic effect on the subpopulation of cells expressing GPR17, suggesting that EVs may also contain purinergic signals able to influence OPC migration via GPR17. Finally, exposure to EVs from either pro- or anti-inflammatory microglia (but not resting cells) promote OPC maturation. However, only EVs released by pro-regenerative cells significantly foster myelin deposition in an in vitro system of OPCs co-cultured with DRG neurons. Shedding light on these signals is important for developing combined therapeutic interventions where a purinergic approach, aimed at implementing recovery after stroke, is potentiated by agents promoting a better microglia phenotype with pro-regenerative effects on OPCs

Tissue carcinoembryonic antigen and oestrogen receptor status in breast carcinoma: an immunohistochemical study of clinical outcome in a series of 252 patients with long-term follow-up.

Carcinoembryonic antigen (CEA) is a well-known tumour marker whose immunohistochemical expression could be prognostically relevant in breast carcinomas. We evaluated CEA immunohistochemical expression, using the specific T84.66 monoclonal antibody, in a series of 252 consecutive cases of infiltrating breast carcinomas (104 N0, 148 N1/2) with median follow-up of 84 months. Oestrogen receptor (ER) status has been evaluated with the immunohistochemical method (ER1D5 antibody, 10% cut-off value): 121 cases were ER negative, 128 cases were ER positive and in three cases ER status was unknown. CEA staining was cytoplasmic; staining intensity and percentage of reacting cells were combined to obtain a final score (CEA score). The difference between the distribution of CEA score within the modalities of the other variables was not statistically significant. Univariate survival analysis has been performed on the series of node-negative and node-positive patients. In the latter subgroup, this has been performed separately for patients treated with systemic adjuvant hormonal therapy or chemotherapy. A multivariate analysis was only performed for node-positive patients treated with adjuvant therapy. CEA immunoreactivity was not prognostically relevant in any subset of analysed patients. The most important prognostic markers were nodal status and tumour size

Multiparametric analysis of cell-free DNA in melanoma patients.

Cell-free DNA in blood (cfDNA) represents a promising biomarker for cancer diagnosis. Total cfDNA concentration showed a scarce discriminatory power between patients and controls. A higher specificity in cancer diagnosis can be achieved by detecting tumor specific alterations in cfDNA, such as DNA integrity, genetic and epigenetic modifications.The aim of the present study was to identify a sequential multi-marker panel in cfDNA able to increase the predictive capability in the diagnosis of cutaneous melanoma in comparison with each single marker alone. To this purpose, we tested total cfDNA concentration, cfDNA integrity, BRAF(V600E) mutation and RASSF1A promoter methylation associated to cfDNA in a series of 76 melanoma patients and 63 healthy controls. The chosen biomarkers were assayed in cfDNA samples by qPCR. Comparison of biomarkers distribution in cases and controls was performed by a logistic regression model in both univariate and multivariate analysis. The predictive capability of each logistic model was investigated by means of the area under the ROC curve (AUC). To aid the reader to interpret the value of the AUC, values between 0.6 and 0.7, between 0.71 and 0.8 and greater than 0.8 were considered as indicating a weak predictive, satisfactory and good predictive capacity, respectively. The AUC value for each biomarker (univariate logistic model) was weak/satisfactory ranging between 0.64 (BRAF(V600E)) to 0.85 (total cfDNA). A good overall predictive capability for the final logistic model was found with an AUC of 0.95. The highest predictive capability was given by total cfDNA (AUC:0.86) followed by integrity index 180/67 (AUC:0.90) and methylated RASSF1A (AUC:0.89).An approach based on the simultaneous determination of three biomarkers (total cfDNA, integrity index 180/67 and methylated RASSF1A) could improve the diagnostic performance in melanoma