In the Rat Hippocampus, Pilocarpine-Induced Status Epilepticus Is Associated with Reactive Glia and Concomitant Increased Expression of CD31, PDGFRß, and Collagen IV in Endothelial Cells and Pericytes of the Blood-Brain Barrier.

In humans and animal models, temporal lobe epilepsy (TLE) is associated with reorganization of hippocampal neuronal networks, gliosis, neuroinflammation, and loss of integrity of the blood-brain barrier (BBB). More than 30% of epilepsies remain intractable, and characterization of the molecular mechanisms involved in BBB dysfunction is essential to the identification of new therapeutic strategies. In this work, we induced status epilepticus in rats through injection of the proconvulsant drug pilocarpine, which leads to TLE. Using RT-qPCR, double immunohistochemistry, and confocal imaging, we studied the regulation of reactive glia and vascular markers at different time points of epileptogenesis (latent phase-3, 7, and 14 days; chronic phase-1 and 3 months). In the hippocampus, increased expression of mRNA encoding the glial proteins GFAP and Iba1 confirmed neuroinflammatory status. We report for the first time the concomitant induction of the specific proteins CD31, PDGFRß, and ColIV-which peak at the same time points as inflammation-in the endothelial cells, pericytes, and basement membrane of the BBB. The altered expression of these proteins occurs early in TLE, during the latent phase, suggesting that they could be associated with the early rupture and pathogenicity of the BBB that will contribute to the chronic phase of epilepsy.

Neurotensin receptor 2 is induced in astrocytes and brain endothelial cells in relation to neuroinflammation following pilocarpine-induced seizures in rats.

Neurotensin (NT) acts as a primary neurotransmitter and neuromodulator in the CNS and has been involved in a number of CNS pathologies including epilepsy. NT mediates its central and peripheral effects by interacting with the NTSR1, NTSR2, and Sort1/NTSR3 receptor subtypes. To date, little is known about the precise expression of the NT receptors in brain neural cells and their regulation in pathology. In the present work, we studied the cellular distribution of the NTSR2 protein in the rat hippocampus and questioned whether its expression was modulated in conditions of neuroinflammation using a model of temporal lobe epilepsy induced by pilocarpine. This model is characterized by a rapid and intense inflammatory reaction with reactive gliosis in the hippocampus. We show that NTSR2 protein is expressed in hippocampal astrocytes and its expression increases together with astrocyte reactivity following induction of status epilepticus. NTSR2 immunoreactivity is also increased in astrocytes proximal to blood vessels and their end-feet, and in endothelial cells. Proinflammatory factors such as IL1ß and LPS induced NTSR2 mRNA and protein in cultured astroglial cells. Antagonizing NTSR2 with SR142948A decreased NTSR2 expression as well as astroglial reactivity. Together, our results suggest that NTSR2 is implicated in astroglial and gliovascular inflammation and that targeting the NTSR2 receptor may open new avenues in the regulation of neuroinflammation in CNS diseases.

An Evolution-Guided Analysis Reveals a Multi-Signaling Regulation of Fas by Tyrosine Phosphorylation and its Implication in Human Cancers.

Demonstrations of both pro-apoptotic and pro-survival abilities of Fas (TNFRSF6/CD95/APO-1) have led to a shift from the exclusive "Fas apoptosis" to "Fas multisignals" paradigm and the acceptance that Fas-related therapies face a major challenge, as it remains unclear what determines the mode of Fas signaling. Through protein evolution analysis, which reveals unconventional substitutions of Fas tyrosine during divergent evolution, evolution-guided tyrosine-phosphorylated Fas proxy, and site-specific phosphorylation detection, we show that the Fas signaling outcome is determined by the tyrosine phosphorylation status of its death domain. The phosphorylation dominantly turns off the Fas-mediated apoptotic signal, while turning on the pro-survival signal. We show that while phosphorylations at Y232 and Y291 share some common functions, their contributions to Fas signaling differ at several levels. The findings that Fas tyrosine phosphorylation is regulated by Src family kinases (SFKs) and the phosphatase SHP-1 and that Y291 phosphorylation primes clathrin-dependent Fas endocytosis, which contributes to Fas pro-survival signaling, reveals for the first time the mechanistic link between SFK/SHP-1-dependent Fas tyrosine phosphorylation, internalization route, and signaling choice. We also demonstrate that levels of phosphorylated Y232 and Y291 differ among human cancer types and differentially respond to anticancer therapy, suggesting context-dependent involvement of Fas phosphorylation in cancer. This report provides a new insight into the control of TNF receptor multisignaling by receptor phosphorylation and its implication in cancer biology, which brings us a step closer to overcoming the challenge in handling Fas signaling in treatments of cancer as well as other pathologies such as autoimmune and degenerative diseases.

OFIP/KIAA0753 forms a complex with OFD1 and FOR20 at pericentriolar satellites and centrosomes and is mutated in one individual with oral-facial-digital syndrome.

Oral-facial-digital (OFD) syndromes are rare heterogeneous disorders characterized by the association of abnormalities of the face, the oral cavity and the extremities, some due to mutations in proteins of the transition zone of the primary cilia or the closely associated distal end of centrioles. These two structures are essential for the formation of functional cilia, and for signaling events during development. We report here causal compound heterozygous mutations of KIAA0753/OFIP in a patient with an OFD VI syndrome. We show that the KIAA0753/OFIP protein, whose sequence is conserved in ciliated species, associates with centrosome/centriole and pericentriolar satellites in human cells and forms a complex with FOR20 and OFD1. The decreased expression of any component of this ternary complex in RPE1 cells causes a defective recruitment onto centrosomes and satellites. The OFD KIAA0753/OFIP mutant loses its capacity to interact with FOR20 and OFD1, which may be the molecular basis of the defect. We also show that KIAA0753/OFIP has microtubule-stabilizing activity. OFD1 and FOR20 are known to regulate the integrity of the centriole distal end, confirming that this structural element is a target of importance for pathogenic mutations in ciliopathies.

A monoclonal antibody directed against a conformational epitope of the HIV-1 trans-activator (Tat) protein neutralizes cross-clade.

The identification of a neutralizing mAb against extracellular HIV-1 transactivator of transcription (Tat) is important for the development of an efficient HIV-1 treatment. Tat plays an essential role in HIV-1 pathogenesis, not only for HIV-1 replication but also as an extracellular toxin able to disrupt the immune system. We showed previously that immunization of rabbits with Tat Oyi, a variant cloned from an African woman who did not develop AIDS following HIV-1 infection, raised antibodies able to recognize different Tat variants. We carried out mice immunization with Tat Oyi and selected a mAb named 7G12, which had the capacity to cross-recognize heterologous Tat variants by a common three-dimensional epitope. These results highlighted that Tat variants were able to acquire a structure, in contrast to a number of studies showing Tat as an unfolded protein. mAb 7G12 also had the capacity to neutralize the biological activities of these Tat variants by blocking the cellular uptake of extracellular Tat. This is the first study using Tat Oyi to produce a mAb able to neutralize effectively activities of extracellular Tats from different HIV-1 subtypes. This mAb has an important potential in therapeutic passive immunization and could help HIV-1 infected patients to restore their immunity.

The actin binding protein α-actinin-2 expression is associated with dendritic spine plasticity and migrating granule cells in the rat dentate gyrus following pilocarpine-induced seizures.

α-actinin-2 (α-actn-2) is an F-actin-crosslinking protein, localized in dendritic spines. In vitro studies suggested that it is involved in spinogenesis, morphogenesis, actin organization, cell migration and anchoring of the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptors in dendritic spines. However, little is known regarding its function in vivo. We examined the levels of α-actn-2 expression within the dentate gyrus (DG) during the development of chronic limbic seizures (epileptogenesis) induced by pilocarpine in rats. In this model, plasticity of the DG glutamatergic granule cells including spine loss, spinogenesis, morphogenesis, neo-synaptogenesis, aberrant migration, and alterations of NMDA receptors have been well characterized. We showed that α-actn-2 immunolabeling was reduced in the inner molecular layer at 1-2 weeks post-status epilepticus (SE), when granule cell spinogenesis and morphogenesis occur. This low level persisted at the chronic stage when new functional synapses are established. This decreased of α-actn-2 protein is concomitant with the recovery of drebrin A (DA), another actin-binding protein, at the chronic stage. Indeed, we demonstrated in cultured cells that in contrast to DA, α-actn-2 did not protect F-actin destabilization and DA inhibited α-actn-2 binding to F-actin. Such alteration could affect the anchoring of NR1 in dendritic spines. Furthermore, we showed that the expression of α-actn-2 and NR1 are co-down-regulated in membrane fractions of pilocarpine animals at chronic stage. Last, we showed that α-actn-2 is expressed in migrating newly born granule cells observed within the hilus of pilocarpine-treated rats. Altogether, our results suggest that α-actn-2 is not critical for the structural integrity and stabilization of granule cell dendritic spines. Instead, its expression is regulated when spinogenesis and morphogenesis occur and within migrating granule cells. Our data also suggest that the balance between α-actn-2 and DA expression levels may modulate NR1 anchoring within dendritic spines.

Expanding the tools for identifying mononuclear phagocyte subsets in swine: Reagents to porcine CD11c and XCR1.

Pig is a domestic species of major importance in the agro-economy and in biomedical research. Mononuclear phagocytes (MNP) are organized in subsets with specialized roles in the orchestration of the immune response and new tools are awaited to improve MNP subset identification in the pig. We cloned pig CD11c cDNA and generated a monoclonal antibody to pig CD11c which showed a pattern of expression by blood and skin MNP subsets similar to humans. We also developed a porcine XCL1-mCherry dimer which specifically reacted with the XCR1-expressing dendritic cell subset of the type 1 lineage in blood and skin. These original reagents will allow the efficient identification of pig MNP subsets to study their role in physiological and pathological processes and also to target these cells in novel intervention and vaccine strategies for veterinary applications and preclinical evaluations.