Etude de l'activation et de l'inactivation pH-dépendantes des canaux ASICs (Acide-Sensing Ion Channels)

RESUME: Etude de l'activation et de l'inactivation pH-dépendantes des canaux ASICs (Acid-Sensing Ion Channels) Benoîte BARGETON, Département de Pharmacologie et de Toxicologie, Université de Lausanne, rue du Bugnon 27, CH-1005 Lausanne, Suisse Les canaux sodiques ASICs (Acid-Sensing Ion Channels) participent à la signalisation neuronale dans les systèmes nerveux périphérique et central. Ces canaux non voltage dépendants sont impliqués dans l'apprentissage, l'expression de la peur, la neurodégénération consécutive à une attaque cérébrale et la douleur. Les bases moléculaires sous-tendant leur activité ne sont pas encore totalement comprises. Ces canaux sont activés par une acidification du milieu extracellulaire et régulés, entre autres, par des ions tels que le Ca2+, le Zn2+ et le CI". La cristallisation de ASIC inactivé a été publiée. Le canal est un trimére de sous-unités identiques ou homologues. Chaque sous-unité a été décrite en analogie à un avant bras, un poignet et une main constituée d'un pouce, d'un doigt, d'une articulation, une boule β et une paume. Nous avons appliqué une approche bioinformatique systématique pour identifier les pH senseurs putatifs de ASICIa. Le rôle des pH senseurs putatifs a été testé par mutagénèse dirigée et des modifications chimiques combinées à une analyse fonctionnelle afin de comprendre comment les variations de ρ H ouvrent ces canaux. Les pH senseurs sont des acides aspartiques et glutamiques éparpillés sur la boucle extracellulaire suggérant que les changements de pH contrôlent l'activation et l'inactivation de ASIC en (dé)protonant ces résidus en divers endroits de la protéine. Par exemple lors de l'activation, la protonation des résidus à l'interface entre le pouce, la boule β et le doigt d'une même sous-unité induit un mouvement du pouce vers la bouie β et le doigt. De même lors de l'inactivation du canal les paumes des trois sous-unités formant une cavité se rapprochent. D'après notre approche bioinformatique, aucune histidine n'est impliquée dans la détection des variations de pH extracellulaire c'est-à-dire qu'aucune histidine ne serait un pH-senseur. Deux histidines de ASIC2a lient le Zn2+ et modifient l'affinité apparente du canal pour les protons. Une seule des deux est conservée parmi tous les ASICs, hASICIa H163. Elle forme un réseau de liaison hydrogène avec ses voisins conservés. L'étude détaillée de ce domaine, Pinterzone, montre son importance dans l'expression fonctionnelle des canaux. La perturbation de ce réseau par l'introduction d'un résidu hydrophobe (cystéine) par mutagénèse dirigée diminue l'expression du canal à la membrane plasmique. La modification des cystéines introduites par des réactifs spécifiques aux groupements sulfhydryle inhibe les canaux mutés en diminuant leur probabilité d'ouverture. Ces travaux décrivent les effets de l'acidification du milieu extracellulaire sur les canaux ASICs. ABSTRACT: Study of pH-dependent activation and inactivation of ASIC channels Benoîte BARGETON, Department of Pharmacology and Toxicology, University of Lausanne, Rue du Bugnon 27, CH-1G05 Lausanne, Switzerland The ASIC (Acid-Sensing Ion Channels) sodium channels are involved in neuronal signaling in the central and peripheral nervous system. These non-voltage-gated channels are involved in learning, the expression of fear, neurodegeneration after ischemia and pain sensation. The molecular bases underlying their activity are not yet fully understood. ASICs are activated by extracellular acidification and regulated, eg by ions such as Ca2+, the Zn2+ and CI". The crystallization of inactivated ASIC has been published. The channel is a trimer of identical or homologous subunits. Each subunit has been described in analogy to a forearm, wrist and hand consisting of a thumb, a finger, a knuckle, a β-ball and a palm. We applied a systematic computational approach to identify putative pH sensor(s) of ASICIa. The role of putative pH sensors has been tested by site-directed mutagenesis and chemical modification combined with functional analysis in order to understand how changes in pH open these channels. The pH sensors are aspartic and glutamic acids distributed throughout the extracellular loop, suggesting that changes in pH control activation and inactivation of ASIC by protonation / deprotonation of many residues in different parts of the protein. During activation the protonation of various residues at the interface between the finger, the thumb and the β-ball induces the movement of the thumb toward the finger and the β-ball. During inactivation of the channel the palms of the three subunits forming a cavity approach each other. No histidine has been shown to be involved in extracellular pH changes detection, i.e. no histidine is a pH- sensor. Two histidines of ASIC2 bind Zn2+ and alter the apparent affinity of channel for protons. Only one of the two His is conserved among all ASICs, hASICIa H163. This residue is part of a network of hydrogen bonding with its conserved neighbors. The detailed study of this area, the interzone, shows its importance in the functional expression of ASICs. Disturbance of this network by the introduction of hydrophobic residues decreases the cell surface channel expression. Chemical modification of the introduced cysteines by thiol reactive compounds inhibits the mutated channels by a reduction of their open probability. These studies describe the effects of extracellular acidification on ASICs. RESUME GRAND PUBLIC: Etude de l'activation et de l'inactivation pH-dépendantes des canaux ASICs (Acid-Sensing Ion Channels) Benoîte BARGETON, Département de Pharmacologie et de Toxicologie, Université de Lausanne, rue du Bugnon 27, CH-1005 Lausanne, Suisse La transmission synaptique est un processus chimique entre deux neurones impliquant des neurotransmetteurs et leurs récepteurs. Un dysfonctionnement de certains types de synapses est à l'origine de beaucoup de troubles nerveux, tels que certaine forme d'épilepsie et de l'attention. Les récepteurs des neurotransmetteurs sont de très bonnes cibles thérapeutiques dans de nombreuses neuropathologies. Les canaux ASICs sont impliqués dans la neurodégénération consécutive à une attaque cérébrale et les bloquer pourraient permettre aux patients d'avoir moins de séquelles. Les canaux ASICs sont des détecteurs de l'acidité qui apparaît lors de situations pathologiques comme l'ischémie et l'inflammation. Ces canaux sont également impliqués dans des douleurs. Cibler spécifiquement ces canaux permettrait d'avoir de nouveaux outils thérapeutiques car à l'heure actuelle l'inhibiteur de choix, l'amiloride, bloque beaucoup d'autres canaux empêchant son utilisation pour bloquer les ASICs. C'est pourquoi il faut connaître et comprendre les bases moléculaires du fonctionnement de ces récepteurs. Les ASICs formés de trois sous-unités détectent les variations de l'acidité puis s'ouvrent transitoirement pour laisser entrer des ions chargés positivement dans la cellule ce qui active la signalisation neuronale. Afin de comprendre les bases moléculaires de l'activité des ASICs nous avons déterminé les sites de liaison des protons (pH-senseurs), ligands naturels des ASICs et décrit une zone importante pour l'expression fonctionnelle de ces canaux. Grâce à une validation systématique de résultats obtenus en collaboration avec l'Institut Suisse de Bioinformatique, nous avons décrit les pH-senseurs de ASICIa. Ces résultats, combinés à ceux d'autres groupes de recherche, nous ont permis de mieux comprendre comment les ASICs sont ouverts par une acidification du milieu extracellulaire. Une seconde étude souligne le rôle structural crucial d'une région conservée parmi tous les canaux ASICs : y toucher c'est diminuer l'activité de la protéine. Ce domaine permet l'harmonisation des changements dus à l'acidification du milieu extracellulaire au sein d'une même sous-unité c'est-à-dire qu'elle participe à l'induction de l'inactivation due à l'activation du canal Cette étude décrit donc quelle région de la protéine atteindre pour la bloquer efficacement en faisant une cible thérapeutique de choix.

Activation of c-Jun in the nuclei of neurons of the CA-1 in thrombin preconditioning occurs via PAR-1.

Recently it has been shown that the c-Jun N-terminal kinase (JNK) plays a role in thrombin preconditioning (TPC) in vivo and in vitro. To investigate further the pathways involved in TPC, we performed an immunohistochemical study in hippocampal slice cultures. Here we show that the major target of JNK, the AP-1 transcription factor c-Jun, is activated by phosphorylation in the nuclei of neurons of the CA1 region by using phospho-specific antibodies against the two JNK phosphorylation sites. The activation is early and transient, peaking at 90 min and not present by 3 hr after low-dose thrombin administration. Treatment of cultures with a synthetic thrombin receptor agonist results in the same c-Jun activation profile and protection against subsequent OGD, both of which are prevented by specific JNK inhibitors, showing that thrombin signals through PAR-1 to JNK. By using an antibody against the Ser 73 phosphorylation site of c-Jun, we identify possible additional TPC substrates.

Activation of PPAR delta Regulates Ywhae (Encoding 14-3-3 epsilon Protein), a Gene Required for Ventricular Morphogenesis

Cardiac ventricular morphogenesis is a key developmental stage during which the ventricles grow considerably in size, but the transcriptional pathways controlling this process remains poorly understood. 14-3-3_ is a member of a conserved protein family that regulates a wide range of processes such as transcription, apoptosis and proliferation by binding to the phospho-serine/threonine residues of its target proteins. We found that deletion of the Ywhae gene (encoding 14-3-3_) in mice leads to abnormal ventricular morphogenesis and an embryonic cardiomyopathy (Cieslik KA et al, Circ. Res. 2008, abstract). Interestingly, we recently showed in cultured cells that the Ywhae gene is regulated directly by peroxisome proliferator-activated receptor _ (PPAR_) (Brunelli L et al, Circ. Res. 2007), a ligand-inducible nuclear receptor that controls energy metabolism and development. Postnatal cardiac-specific deletion of the Ppard gene in mice causes a lethal dilated cardiomyopathy, but it is still unknown whether PPAR_ regulates genes involved in heart development. We hypothesized that the expression of the Ywhae gene is responsive to PPAR_ during heart development. We confirmed that PPAR_ is expressed in the heart during development, and found higher expression at E10.5 compared to later gestational ages. We showed by immunofluorescence that a PPAR_ agonist (50 _M L-165,041 for 24 hr) upregulates 14-3-3_ in primary cardiomyocytes. We showed that when P19CL6 cells are driven towards cardiomyocyte lineage by dimethyl sulfoxide (DMSO), 14-3-3_ levels increase 4-fold, while L-165,041 treatment increases levels by an additional 50%. Based on previous work in mice (Leibowitz MD et al, FEBS Lett. 2000; Letavernier E et al, J. Am. Soc. Nephrol. 2005), we tested the response of Ywhae to PPAR_ in vivo . We fed 30 mg/kg/day L-165,041 to 14-3-3__/_ adult pregnant mice for 3 days starting at E9.5 and assessed Ywhae mRNA levels in embryonic hearts at E12.5. Baseline mRNA levels in Ywhae_/_ hearts were double that of Ywhae_/ hearts, while L-165,041 upregulated Ywhae mRNA levels in both Ywhae_/_ and Ywhae_/ hearts by 65%. These results indicate that Ywhae responds to PPAR_ in vivo, and suggest that PPAR_ regulates Ywhae during ventricular morphogenesis.

Molecular mechanisms implicated in NF-κB activation and antiviral defense

SUMMARY Nuclear factor kappa B (NF-κB) transcription factors control many aspects of cell fate through induction of inflammatory, immune or survival molecules. We have identified two novel proteins, named receptor interacting protein (RIP)-4 and caspase recruitment domain (CARD) adaptor inducing interferon-β (Cardif), which activate NF-κB. Further, we have found that Cardif plays a prominent antiviral function. Antiviral innate immunity is mounted upon recognition by the host of virally associated structures like double-stranded (ds) RNA, which constitutes a viral replication product of many viruses within infected cells. dsRNA, depending on its subcellular localization, can be sensed by two separate arms of host defense. Firstly, Toll-like receptor (TLR)-3, a member of the type I transmembrane TLR family, recognizes endosomally-located dsRNA. Secondly, cytoplasmic dsRNA is detected by the recently identified RNA helicase retinoic acid inducible gene I (RIG-I). Triggering of TLR3- and RIG-I-dependent pathways results in the activation of the transcription factors NF-κB and Interferon regulatory factor (IRF)-3, which cooperatively transduce antiviral immune responses. We have demonstrated that RIP1, a kinase previously shown to be required for TNF signaling, transmits TLR3-dependent NF-κB activation. Further we have identified and characterized Cardif as an essential adaptor transmitting RIG-I-mediated antiviral responses, including activation of NF-κB and IRF3. In addition, we showed that Cardif is cleaved and inactivated by a serine protease of hepatitis C virus, and therefore may represent an attractive target for this virus to escape innate immune responses. RESUME Les facteurs de transcription "nuclear factor kappa B" (NF-κB) contrôlent divers aspects du devenir cellulaire à travers l'induction de molécules inflammatoires, immunitaires ou de survie. Nous avons identifié deux nouvelles protéines, nommées "receptor interacting protein" (RIP)-4 et "caspase recruitment domain (CARD) adaptor inducing interferon-β" (Cardif), qui activent NF-κB. En outre, nous avons trouvé que Cardif joue un rôle antiviral crucial. L'immunité innée antivirale s'établit au moment de la reconnaissance par l'hôte de structures virales, comme l'ARN double brin, qui constitue un produit de réplication de beaucoup de virus à l'intérieur de cellules infectées. L'ARN double brin, dépendant de sa localisation subcellulaire, peut être détecté par deux branches de défense distinctes. Premièrement, le récepteur transmembranaire "Toll-like" (TLR), TLR3, reconnaît l'ARN double brin lorsque localisé dans les endosomes. Deuxièmement, l'ARN double brin cytoplasmique est reconnu par l'ARN hélicase récemment décrite "retinoic acid inducible gene I" (RIG-I). Le déclenchement de voies dépendantes de TLR3 et RIG-I active les facteurs de transcription NF-κB et IRF3, qui coopèrent afin de transduire des réponses immunitaires antivirales. Nous avons démontré que RIP1, une kinase décrite précédemment dans le signalement du TNF, transmet l'activation de NF-κB dépendante de TLR3. De plus, nous avons identifié et caractérisé Cardif comme un adapteur essentiel transmettant les réponses antivirales médiées par RIG-I, qui incluent l'activation de NF-κB et IRF3. De surcroît, Cardif est clivé et inactivé par une sérine protéase du virus de l'hépatite C, et ainsi pourrait représenter une cible attractive pour ce virus afin d'échapper aux réponses immunitaires innées.

Lymphocyte specificity to protein antigens. V. Conformational dependence of activation of cytochrome c-specific T cells.

Variations in the immunogenic and antigenic properties of native and denatured forms of cytochrome c were observed depending on the strain of mouse tested. In C57BL/6 and (C57BL/6 X DBA/2)F1 (BDF1) mice, priming with either native or denatured cytochrome c (apocytochrome c) gave rise to T cell blasts responding in a similar fashion to the two forms of the antigen and to different peptides derived from CNBr cleavage of the protein when tested for proliferation in the presence of C57BL/6 or BDF1 accessory cells. A different pattern of proliferation was observed when apocytochrome c-specific DBA/2 or BDF1 T cell blasts were tested with DBA/2 accessory cells. In this case, no response was obtained to heme peptide 1-65. This was not due to an inability of DBA/2 macrophages to process and present heme peptide 1-65, as they were able to present this antigen to native cytochrome c-specific BDF1 T cell blasts. Thus, it seems that different sets of clones are generated upon priming BDF1 mice with denatured cytochrome c which are able to recognize different sets of peptides depending on the nature of the accessory cells. The results obtained are consistent with the hypothesis that degradation and presentation of native and denatured cytochrome c by macrophages is dependent on the three-dimensional conformation of the protein.

Event-related potentials and changes of brain rhythm oscillations during working memory activation in patients with first-episode psychosis.

Background: Earlier contributions have documented significant changes in sensory, attention-related endogenous event-related potential (ERP) components and θ band oscillatory responses during working memory activation in patients with schizophrenia. In patients with first-episode psychosis, such studies are still scarce and mostly focused on auditory sensory processing. The present study aimed to explore whether subtle deficits of cortical activation are present in these patients before the decline of working memory performance. Methods: We assessed exogenous and endogenous ERPs and frontal θ event-related synchronization (ERS) in patients with first-episode psychosis and healthy controls who successfully performed an adapted 2-back working memory task, including 2 visual n-backworking memory tasks as well as oddball detection and passive fixation tasks. Results: We included 15 patients with first-episode psychosis and 18 controls in this study. Compared with controls, patients with first-episode psychosis displayed increased latencies of early visual ERPs and phasic θ ERS culmination peak in all conditions. However, they also showed a rapid recruitment of working memory-related neural generators, even in pure attention tasks, as indicated by the decreased N200 latency and increased amplitude of sustained θ ERS in detection compared with controls. Limitations: Owing to the limited sample size, no distinction was made between patients with first-episode psychosis with positive and negative symptoms. Although we controlled for the global load of neuroleptics, medication effect cannot be totally ruled out. Conclusion: The present findings support the concept of a blunted electroencephalographic response in patients with first-episode psychosis who recruit the maximum neural generators in simple attention conditions without being able to modulate their brain activation with increased complexity of working memory tasks.

Phosphorylation of neurofilament subunit NF-M is regulated by activation of NMDA receptors and modulates cytoskeleton stability and neuronal shape.

The cytoskeleton is essential for the structural organization of neurons and is influenced during development by excitatory stimuli such as activation of glutamate receptors. In particular, NMDA receptors are known to modulate the function of several cytoskeletal proteins and to influence cell morphology, but the underlying molecular and cellular mechanisms remain unclear. Here, we characterized the neurofilament subunit NF-M in cultures of developing mouse cortical neurons chronically exposed to NMDA receptor antagonists. Western blots analysis showed that treatment of cortical neurons with MK801 or AP5 shifted the size of NF-M towards higher molecular weights. Dephosphorylation assay revealed that this increased size of NF-M observed after chronic exposure to NMDA receptor antagonists was due to phosphorylation. Neurons treated with cyclosporin, an inhibitor of the Ca(2+)-dependent phosphatase calcineurin, also showed increased levels of phosphorylated NF-M. Moreover, analysis of neurofilament stability revealed that the phosphorylation of NF-M, resulting from NMDA receptor inhibition, enhanced the solubility of NF-M. Finally, cortical neurons cultured in the presence of the NMDA receptor antagonists MK801 and AP5 grew longer neurites. Together, these data indicate that a blockade of NMDA receptors during development of cortical neurons increases the phosphorylation state and the solubility of NF-M, thereby favoring neurite outgrowth. This also underlines that dynamics of the neurofilament and microtubule cytoskeleton is fundamental for growth processes.

Promoter recognition and activation by the global response regulator CbrB in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the CbrA/CbrB two-component system is instrumental in the maintenance of the carbon-nitrogen balance and for growth on carbon sources that are energetically less favorable than the preferred dicarboxylate substrates. The CbrA/CbrB system drives the expression of the small RNA CrcZ, which antagonizes the repressing effects of the catabolite repression control protein Crc, an RNA-binding protein. Dicarboxylates appear to cause carbon catabolite repression by inhibiting the activity of the CbrA/CbrB system, resulting in reduced crcZ expression. Here we have identified a conserved palindromic nucleotide sequence that is present in upstream activating sequences (UASs) of promoters under positive control by CbrB and σ(54) RNA polymerase, especially in the UAS of the crcZ promoter. Evidence for recognition of this palindromic sequence by CbrB was obtained in vivo from mutational analysis of the crcZ promoter and in vitro from electrophoretic mobility shift assays using crcZ promoter fragments and purified CbrB protein truncated at the N terminus. Integration host factor (IHF) was required for crcZ expression. CbrB also activated the lipA (lipase) promoter, albeit less effectively, apparently by interacting with a similar but less conserved palindromic sequence in the UAS of lipA. As expected, succinate caused CbrB-dependent catabolite repression of the lipA promoter. Based on these results and previously published data, a consensus CbrB recognition sequence is proposed. This sequence has similarity to the consensus NtrC recognition sequence, which is relevant for nitrogen control.

Aggregation of integrins and RhoA activation are required for Thy-1-induced morphological changes in astrocytes.

Thy-1, a cell adhesion molecule abundantly expressed in mammalian neurons, binds to a beta(3)-containing integrin on astrocytes and thereby stimulates the assembly of focal adhesions and stress fibers. Such events lead to morphological changes in astrocytes that resemble those occurring upon injury in the brain. Extracellular matrix proteins, typical integrin ligands, bind to integrins and promote receptor clustering as well as signal transduction events that involve small G proteins and cytoskeletal changes. Here we investigated the possibility that the cell surface protein Thy-1, when interacting with a beta(3)-containing integrin on astrocytes, could trigger signaling events similar to those generated by extracellular matrix proteins. DI-TNC(1) astrocytes were stimulated with Thy-1-Fc immobilized on beads, and increased RhoA activity was confirmed using an affinity precipitation assay. The effect of various inhibitors on the cellular response was also studied. The presence of Y-27632, an inhibitor of Rho kinase (p160ROCK), a key downstream effector of RhoA, significantly reduced focal adhesion and stress fiber formation induced by Thy-1. Similar effects were obtained when astrocytes were treated with C3 transferase, an inhibitor of RhoA. Alternatively, astrocytes were transfected with an expression vector encoding fusion proteins of enhanced green fluorescent protein with either the Rho-binding domain of Rhotekin, which blocks RhoA function, or the dominant-negative N19RhoA mutant. In both cases, Thy-1-induced focal adhesion formation was inhibited. Furthermore, we observed that RhoA activity after stimulation with soluble Thy-1-Fc molecule was augmented upon further cross-linking using protein A-Sepharose beads. The same was shown by cross-linking beta(3)-containing integrin with anti-beta(3) antibodies. Together, these results indicate that Thy-1-mediated astrocyte stimulation depended on beta(3) integrin clustering and the resulting increase in RhoA activity.

Peroxisome proliferator-activated receptor gamma activation is required for maintenance of innate antimicrobial immunity in the colon.

Crohn's disease (CD), a major form of human inflammatory bowel disease, is characterized by primary immunodeficiencies. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is essential for intestinal homeostasis in response to both dietary- and microbiota-derived signals. Its role in host defense remains unknown, however. We show that PPARgamma functions as an antimicrobial factor by maintaining constitutive epithelial expression of a subset of beta-defensin in the colon, which includes mDefB10 in mice and DEFB1 in humans. Colonic mucosa of Ppargamma mutant animals shows defective killing of several major components of the intestinal microbiota, including Candida albicans, Bacteroides fragilis, Enterococcus faecalis, and Escherichia coli. Neutralization of the colicidal activity using an anti-mDefB10 blocking antibody was effective in a PPARgamma-dependent manner. A functional promoter variant that is required for DEFB1 expression confers strong protection against Crohn's colitis and ileocolitis (odds ratio, 0.559; P = 0.018). Consistently, colonic involvement in CD is specifically linked to reduced expression of DEFB1 independent of inflammation. These findings support the development of PPARgamma-targeting therapeutic and/or nutritional approaches to prevent colonic inflammation by restoring antimicrobial immunity in CD.

Immune activation alters cellular and humoral responses to yellow fever 17D vaccine.

Background. Defining the parameters that modulate vaccine responses in African populations will be imperative to design effective vaccines for protection against HIV, malaria, tuberculosis, and dengue virus infections. This study aimed to evaluate the contribution of the patient-specific immune microenvironment to the response to the licensed yellow fever vaccine 17D (YF-17D) in an African cohort. Methods. We compared responses to YF-17D in 50 volunteers in Entebbe, Uganda, and 50 volunteers in Lausanne, Switzerland. We measured the CD8+ T cell and B cell responses induced by YF-17D and correlated them with immune parameters analyzed by flow cytometry prior to vaccination. Results. We showed that YF-17D-induced CD8+ T cell and B cell responses were substantially lower in immunized individuals from Entebbe compared with immunized individuals from Lausanne. The impaired vaccine response in the Entebbe cohort associated with reduced YF-17D replication. Prior to vaccination, we observed higher frequencies of exhausted and activated NK cells, differentiated T and B cell subsets and proinflammatory monocytes, suggesting an activated immune microenvironment in the Entebbe volunteers. Interestingly, activation of CD8+ T cells and B cells as well as proinflammatory monocytes at baseline negatively correlated with YF-17D-neutralizing antibody titers after vaccination. Additionally, memory T and B cell responses in preimmunized volunteers exhibited reduced persistence in the Entebbe cohort but were boosted by a second vaccination. Conclusion. Together, these results demonstrate that an activated immune microenvironment prior to vaccination impedes efficacy of the YF-17D vaccine in an African cohort and suggest that vaccine regimens may need to be boosted in African populations to achieve efficient immunity. Trial registration. Registration is not required for observational studies. Funding. This study was funded by Canada's Global Health Research Initiative, Defense Threat Reduction Agency, National Institute of Allergy and Infectious Diseases, Bill & Melinda Gates Foundation, and United States Agency for International Development.

Characterization of sustained BOLD activation in the rat barrel cortex and neurochemical consequences.

To date, only a couple of functional MR spectroscopy (fMRS) studies were conducted in rats. Due to the low temporal resolution of (1)H MRS techniques, prolonged stimulation paradigms are necessary for investigating the metabolic outcome in the rat brain during functional challenge. However, sustained activation of cortical areas is usually difficult to obtain due to neural adaptation. Anesthesia, habituation, high variability of the basal state metabolite concentrations as well as low concentrations of the metabolites of interest such as lactate (Lac), glucose (Glc) or γ-aminobutyric acid (GABA) and small expected changes of metabolite concentrations need to be addressed. In the present study, the rat barrel cortex was reliably and reproducibly activated through sustained trigeminal nerve (TGN) stimulation. In addition, TGN stimulation induced significant positive changes in lactate (+1.01μmol/g, p<0.008) and glutamate (+0.92μmol/g, p<0.02) and significant negative aspartate changes (-0.63μmol/g, p<0.004) using functional (1)H MRS at 9.4T in agreement with previous changes observed in human fMRS studies. Finally, for the first time, the dynamics of lactate, glucose, aspartate and glutamate concentrations during sustained somatosensory activation in rats using fMRS were assessed. These results allow demonstrating the feasibility of fMRS measurements during prolonged barrel cortex activation in rats.

Salmonella virulence factor SipB induces activation and release of IL-18 in human dendritic cells.

Interleukin-18 (IL-18) plays an important role in innate and acquired immunity, in particular against intracellular pathogens. However, little is known about the microbial factors that trigger IL-18 secretion by dendritic cells (DCs). To determine the influence of bacterial virulence factors on the activation and release of IL-18, we infected human monocyte-derived DCs with virulence mutants of the facultative intracellular pathogen Salmonella typhimurium. Our results show that infection by S. typhimurium causes caspase-1-dependent activation of IL-18 and triggers the release of IL-18 in human DCs. The secretion of IL-18 by the DCs was closely correlated with the ability of the S. typhimurium strains to induce apoptosis. We demonstrate that activation and release of IL-18 are blocked by mutations in the Salmonella sipB gene, which encodes a virulence factor that activates caspase-1 to induce apoptosis. These findings indicate that the activation and release of IL-18 induced by bacterial virulence factors may represent one component of innate immunity against the intracellular bacteria.

Intracellular ubiquitylation of the epithelial Na+ channel controls extracellular proteolytic channel activation via conformational change.

The epithelial Na(+) channel ENaC is a key player in the maintenance of whole body Na(+) balance, and consequently of blood pressure. It is tightly regulated by numerous signaling pathways including ubiquitylation via the ubiquitin-protein ligase Nedd4-2. This mechanism is itself under the control of several kinases, which phosphorylate Nedd4-2, thereby interfering with ENaC/Nedd4-2 interaction, or by Usp2-45, which binds to and deubiquitylates ENaC. Another, different regulatory mechanism concerns the proteolytic activation of ENaC, during which the channel is cleaved on its luminal side by intracellular convertases such as furin, and further activated by extracellular proteases such as CAP-1. This process is regulated as well but the underlying mechanisms are not understood. Previously, evidence was provided that the ubiquitylation status of ENaC may affect the cleavage of the channel. When ubiquitylation of ENaC was reduced, either by co-expressing Usp2-45, or mutating either the ENaC PY-motifs (i.e. the binding sites for Nedd4-2) or intracellular lysines (i.e. ubiquitylation sites), the level of channel cleavage was increased. Here we demonstrate that lysine-mutated ENaC channels are not ubiquitylated at the cell surface, are preferentially cleaved, and Usp2-45 does not affect their cleavage efficiency. We further show by limited proteolysis that the intracellular ubiquitylation status of ENaC affects the extracellular conformation of αENaC, by demonstrating that non-ubiquitylated channels are more efficiently cleaved when treated with extracellularly added trypsin or chymotrypsin. These results present a new paradigm in which an intracellular, post-translational modification (e.g. ubiquitylation) of a transmembrane protein can affect its extracellular conformation.

In vivo activation of PPAR target genes by RXR homodimers.

The ability of a retinoid X receptor (RXR) to heterodimerize with many nuclear receptors, including LXR, PPAR, NGF1B and RAR, underscores its pivotal role within the nuclear receptor superfamily. Among these heterodimers, PPAR:RXR is considered an important signalling mediator of both PPAR ligands, such as fatty acids, and 9-cis retinoic acid (9-cis RA), an RXR ligand. In contrast, the existence of an RXR/9-cis RA signalling pathway independent of PPAR or any other dimerization partner remains disputed. Using in vivo chromatin immunoprecipitation, we now show that RXR homodimers can selectively bind to functional PPREs and induce transactivation. At the molecular level, this pathway requires stabilization of the homodimer-DNA complexes through ligand-dependent interaction with the coactivator SRC1 or TIF2. This pathway operates both in the absence and in the presence of PPAR, as assessed in cells carrying inactivating mutations in PPAR genes and in wild-type cells. In addition, this signalling pathway via PPREs is fully functional and can rescue the severe hypothermia phenotype observed in fasted PPARalpha-/- mice. These observations have important pharmacological implications for the development of new rexinoid-based treatments.