Cytosolic sensing of extracellular self-DNA transported into monocytes by the antimicrobial peptide LL37.

The intracellular location of nucleic acid sensors prevents recognition of extracellular self-DNA released by dying cells. However, on forming a complex with the endogenous antimicrobial peptide LL37, extracellular DNA is transported into endosomal compartments of plasmacytoid dendritic cells, leading to activation of Toll-like receptor-9 and induction of type I IFNs. Whether LL37 also transports self-DNA into nonplasmacytoid dendritic cells, leading to type I IFN production via other intracellular DNA receptors is unknown. Here we found that LL37 very efficiently transports self-DNA into monocytes, leading the production of type I IFNs in a Toll-like receptor-independent manner. This type I IFN induction was mediated by double-stranded B form DNA, regardless of its sequence, CpG content, or methylation status, and required signaling through the adaptor protein STING and TBK1 kinase, indicating the involvement of cytosolic DNA sensors. Thus, our study identifies a novel link between the antimicrobial peptides and type I IFN responses involving DNA-dependent activation of cytosolic sensors in monocytes.

The activity of the anti-apoptotic fragment generated by the caspase-3/p120 RasGAP stress-sensing module displays strict Akt isoform specificity.

The caspase-3/p120 RasGAP module acts as a stress sensor that promotes pro-survival or pro-death signaling depending on the intensity and the duration of the stressful stimuli. Partial cleavage of p120 RasGAP generates a fragment, called fragment N, which protects stressed cells by activating Akt signaling. Akt family members regulate many cellular processes including proliferation, inhibition of apoptosis and metabolism. These cellular processes are regulated by three distinct Akt isoforms: Akt1, Akt2 and Akt3. However, which of these isoforms are required for fragment N mediated protection have not been defined. In this study, we investigated the individual contribution of each isoform in fragment N-mediated cell protection against Fas ligand induced cell death. To this end, DLD1 and HCT116 isogenic cell lines lacking specific Akt isoforms were used. It was found that fragment N could activate Akt1 and Akt2 but that only the former could mediate the protective activity of the RasGAP-derived fragment. Even overexpression of Akt2 or Akt3 could not rescue the inability of fragment N to protect cells lacking Akt1. These results demonstrate a strict Akt isoform requirement for the anti-apoptotic activity of fragment N.

Enhanced compressed sensing recovery with level set normals.

We propose a compressive sensing algorithm that exploits geometric properties of images to recover images of high quality from few measurements. The image reconstruction is done by iterating the two following steps: 1) estimation of normal vectors of the image level curves, and 2) reconstruction of an image fitting the normal vectors, the compressed sensing measurements, and the sparsity constraint. The proposed technique can naturally extend to nonlocal operators and graphs to exploit the repetitive nature of textured images to recover fine detail structures. In both cases, the problem is reduced to a series of convex minimization problems that can be efficiently solved with a combination of variable splitting and augmented Lagrangian methods, leading to fast and easy-to-code algorithms. Extended experiments show a clear improvement over related state-of-the-art algorithms in the quality of the reconstructed images and the robustness of the proposed method to noise, different kind of images, and reduced measurements.

A novel approach to reducing number of sensing units for wearable gait analysis systems.

Gait analysis methods to estimate spatiotemporal measures, based on two, three or four gyroscopes attached on lower limbs have been discussed in the literature. The most common approach to reduce the number of sensing units is to simplify the underlying biomechanical gait model. In this study, we propose a novel method based on prediction of movements of thighs from movements of shanks. Datasets from three previous studies were used. Data from the first study (ten healthy subjects and ten with Parkinson's disease) were used to develop and calibrate a system with only two gyroscopes attached on shanks. Data from two other studies (36 subjects with hip replacement, seven subjects with coxarthrosis, and eight control subjects) were used for comparison with the other methods and for assessment of error compared to a motion capture system. Results show that the error of estimation of stride length compared to motion capture with the system with four gyroscopes and our new method based on two gyroscopes was close ( -0.8 ±6.6 versus 3.8 ±6.6 cm). An alternative with three sensing units did not show better results (error: -0.2 ±8.4 cm). Finally, a fourth that also used two units but with a simpler gait model had the highest bias compared to the reference (error: -25.6 ±7.6 cm). We concluded that it is feasible to estimate movements of thighs from movements of shanks to reduce number of needed sensing units from 4 to 2 in context of ambulatory gait analysis.

Central glucose sensing and the control of energy homeostasis

Glucose is an important signal that regulates glucose and energy homeostasis but its precise physiological role and signaling mechanism in the brain are still uncompletely understood. Over the recent years we have investigated the possibility that central glucose sensing may share functional similarities with glucose sensing by pancreatic beta-cells, in particular a requirement for the expression of the glucose transporter Glut2. Using mice with genetic inactivation of Glut2, but rescued pancreatic beta-cell function by transgenic expression of a glucose transporter, we have established that extrapancreatic glucose sensors are involved: i) in the control of glucagon secretion in response to hypoglycemia, ii) in the control of feeding and iii) of energy expenditure. We have more recently shown that central Glut2-dependent glucose sensors are involved in the regulation of NPY and POMC expression by arcuate nucleus neurons and that the sensitivity to leptin of these neurons is enhanced by Glut2-dependent glucose sensors. Using mice with genetic tagging of Glut2-expressing cells, we determined that the NPY and POMC neurons did not express Glut2 but were connected to Glut2 expressing neurons located most probably outside of the arcuate nucleus. We are now defining the electrophysiological behavior of these Glut2 expressing neurons. Our data provide an initial map of glucose sensing neurons expressing Glut2 and link these neurons with the control of specific physiological function.

Modulatory effects of acid-sensing ion channels on action potential generation in hippocampal neurons.

Extracellular acidification has been shown to generate action potentials (APs) in several types of neurons. In this study, we investigated the role of acid-sensing ion channels (ASICs) in acid-induced AP generation in brain neurons. ASICs are neuronal Na(+) channels that belong to the epithelial Na(+) channel/degenerin family and are transiently activated by a rapid drop in extracellular pH. We compared the pharmacological and biophysical properties of acid-induced AP generation with those of ASIC currents in cultured hippocampal neurons. Our results show that acid-induced AP generation in these neurons is essentially due to ASIC activation. We demonstrate for the first time that the probability of inducing APs correlates with current entry through ASICs. We also show that ASIC activation in combination with other excitatory stimuli can either facilitate AP generation or inhibit AP bursts, depending on the conditions. ASIC-mediated generation and modulation of APs can be induced by extracellular pH changes from 7.4 to slightly <7. Such local extracellular pH values may be reached by pH fluctuations due to normal neuronal activity. Furthermore, in the plasma membrane, ASICs are localized in close proximity to voltage-gated Na(+) and K(+) channels, providing the conditions necessary for the transduction of local pH changes into electrical signals.

A quorum sensing regulated small volatile molecule reduces acute virulence and promotes chronic infection phenotypes.

A significant number of environmental microorganisms can cause serious, even fatal, acute and chronic infections in humans. The severity and outcome of each type of infection depends on the expression of specific bacterial phenotypes controlled by complex regulatory networks that sense and respond to the host environment. Although bacterial signals that contribute to a successful acute infection have been identified in a number of pathogens, the signals that mediate the onset and establishment of chronic infections have yet to be discovered. We identified a volatile, low molecular weight molecule, 2-amino acetophenone (2-AA), produced by the opportunistic human pathogen Pseudomonas aeruginosa that reduces bacterial virulence in vivo in flies and in an acute mouse infection model. 2-AA modulates the activity of the virulence regulator MvfR (multiple virulence factor regulator) via a negative feedback loop and it promotes the emergence of P. aeruginosa phenotypes that likely promote chronic lung infections, including accumulation of lasR mutants, long-term survival at stationary phase, and persistence in a Drosophila infection model. We report for the first time the existence of a quorum sensing (QS) regulated volatile molecule that induces bistability phenotype by stochastically silencing acute virulence functions in P. aeruginosa. We propose that 2-AA mediates changes in a subpopulation of cells that facilitate the exploitation of dynamic host environments and promote gene expression changes that favor chronic infections.

Pattern-based sensing of nucleotides in aqueous solution with a multicomponent indicator displacement assay

A multicomponent indicator displacement assay ( MIDA) based on an organometallic receptor and three dyes can be used for the identification and quantification of nucleotides in aqueous solution at neutral pH.

Sensing the environment : a role for the mouse olfactory subsystems

Summary : Four distinct olfactory subsystems compose the mouse olfactory system, the main olfactory epithelium (MOE), the septal organ of Masera (SO), the vomeronasal organ (VNO) and the Grueneberg ganglion (GG). They are implicated in the sensory modalities of the animal and they evolved to analyse and discriminate molecules carrying chemical messages, such as odorants and pheromones. In this thesis, the VNO, principally implicated in pheromonal communications as well as the GG, which had no function attributed until this work, were investigated from their morphology to their physiological functions, using an array of biochemical and physiological methods. First, the roles of a particular protein, the CNGA4 ion channel, were investigated in the VNO. In the MOE, CNGA4 is expressed as a modulatory channel subunit implicated in odour discrimination and adaptation. Interestingly, this calcium channel is the unique member of the cyclic nucleotide-gated (CNG) family to be expressed in the VNO and up to this work its functions remained unknown. Using a combination of transgenic and knockout mice, as well as histological and physiological approaches, we have characterized CNGA4 expression in the VNO. A strong expression in immature neurons was found as well as in the microvilli of mature neurons (putative site of chemodetection). Interestingly and confirming its dual localisation, the genetic invalidation of the CNGA4 channel has, as consequences, a strong impairment in vomeronasal maturation as well as deficit in pheromone sensing. Thus the CNGA4 channel appears to be a multifunctional protein in the mouse VNO playing essential role(s) in this organ. During the second part of the work, the morphology of the most recently described olfactory subsystem, the Grueneberg ganglion, was investigated in detail. Interestingly we found that glial cells and ciliated neurons compose this olfactory ganglion. This particular morphological aspect was similar to the olfactory AWC neurons from C. elegans which was used for further comparisons. Thus as for AWC neurons, we found that GG neurons are sensitive to temperature changes and are able to detect highly volatile molecules. Indeed, the presence of alarm pheromones (APs) secreted by stressed mice, elicit strong cellular responses, as well as a GG dependent behavioural changes. Investigations on the signaling elements present in GG neurons revealed that, as for AWC neurons, or pGC-D expressing neurons from the MOE, proteins participating in a cGMP pathway were found in GG neurons such as pGC-G and CNGA3 channels. These two proteins might be implicated in chemosensing as well as in thermosensing, two apparent properties of this organ. In this thesis, the multisensory modalities of two mouse olfactory subsystems were described and are related to a high degree of complexity required for the animal to sense its environment

Quorum-sensing systems in staphylococci as therapeutic targets.

The staphylococci are an ever-present threat in our world, capable of causing a wide range of infections, and are a persistent presence in the clinical environment. As the number of antimicrobial compounds effective against staphylococci decreases, because of the acquisition and spread of antibiotic resistance, there is a growing need for novel therapeutic molecules. Intra and inter-species communication (quorum sensing) is a biologically significant phenomenon that has been associated with virulence, intracellular survival, and biofilm formation. Quorum sensing molecules of staphylococci and other species (e.g. Pseudomonas aeruginosa) can inhibit virulence factor production and/or growth of staphylococci, leading to the possibility that interference with staphylococcal quorum-sensing systems could be a way of controlling the diverse infections caused by the staphylococci. In this article, we discuss the potential of quorum-sensing systems of staphylococci as therapeutic targets.

Sensing pheromones : a role for the CNGA4 and TRPC2 proteins

Chez les mammifères, les phéromones sont des molécules clés dans la régulation des comportements sociaux au sein d'une espèce. Chez la souris, la détection de ces molécules se fait dans l'organe voméronasal (VNO] et implique le canal TRPC2 afin de dépolariser les neurones. Des différences de comportement entre des souris Trpc2-/- et des souris sans VNO suggèrent l'implication d'une autre protéine effectrice dans la voie de signalisation des phéromones. L'hypothèse étant que cette protéine formerait un canal hétéromérique avec TRPC2. CNGA4 est une protéine sans fonction connue dans le VNO des rongeurs. Elle appartient à la famille des protéines CNG qui joue un rôle important dans différentes voies de signalisation comme la vision ou l'olfaction. Etant donné sa présence dans le VNO, son rôle inconnu dans cet organe et son rôle important dans de nombreuses voies de signalisation, nous avons décidé d'étudier CNGA4 afin de connaître sa localisation, ses propriétés ou encore sa structure. Nous avons découvert que CNGA4 est exprimée dans les axons, les neurones immatures ainsi que sur les microvillosités des neurones de VNO. A l'aide de souris portant une version non fonctionnelle de CNGA4, nous avons pu montrer que cette protéine joue un rôle majeur dans la voie de signalisation des phéromones. Ainsi, les neurones du VNO portant une version non fonctionnelle de CNGA4 répondent moins fréquemment aux phéromones et par conséquent les phéromones activent également moins de neurones dans le bulbe olfactif accessoire, premier relais du VNO avec le cortex. Cette détection défaillante se traduit par une absence d'agressivité des souris mutantes ainsi que par une incapacité de ces souris à discriminer le sexe de leur conspécifique. Etant donné les propriétés similaires de CNGA4 et de TRPC2, nous avons supposé que les deux protéines pourraient interagir. Cette hypothèse a été confortée par l'observation que CNGA4 n'est plus exprimée dans les microvillosités du VNO des souris Trpc2-/-. A l'aide d'expériences d'expression hétérologue, nous avons pu observer que les deux protéines interagissent et forment un canal activé par un analogue du diacylglycérol suggérant que ce canal est fonctionnel. Ces résultats indiquent que CNGA4 formerait un canal hétéromérique avec TRPC2 et aurait dans ce canal une fonction modulatrice. Des expériences complémentaires sont nécessaires afin de connaître le rôle de chacune de ces protéines dans la voie de signalisation des phéromones. Sensing pheromones: a role for the CNGA4 and TRPC2 proteins Mammalian pheromones are key chemical signals in the regulation of intraspecies social behaviors. Detection of these pheromones, which takes place in sensory neurons of the vomeronasal organ (VNO), implies the activation of the transient receptor potential canonical channel 2 (TRPC2) as the final effector. Interestingly, discrepancies between Trpc2 /- mice and mice lacking a VNO suggest the implication of another protein in the pheromone signaling pathway. This protein could either form a heteromeric channel with TRPC2 or a separate homomeric ion channel. The cyclic nucleotide-gated channel subunit CNGA4 is also expressed in the rodent VNO but its role and properties in this organ remain unknown. CNGA4 belongs to the CNG channel family which is playing an important role in different sensory pathways such as in light and odorant detection. We thus decided to study the role of the CNGA4 protein in the mouse VNO. We found CNGA4 to be expressed in axons, dendrites and in the sensory microvilli. Using mice bearing a non-functional form of CNGA4 we further demonstrated the importance of the CNGA4 protein for the pheromone signaling pathway as neurons from mutant mice were responding less frequently to chemosensory cues. As a result, mutant mice displayed a non-aggressive behavior and an impaired sexual discrimination ability. Based on the CNGA4 localization and its role in the pheromone signaling pathway we hypothesized a possible interaction between CNGA4 and TRPC2 forming a heteromeric channel. First evidences for this interaction came from the absence of CNGA4 expression in the sensory microvilli of Trpc2-/- mice. Second, using transfected HEK cells as an expression system we could observe that CNGA4 and TRPC2 interact and translocate to the plasma membrane. Perfusion of a DAG analogue on co-transfected HEK cells resulted in a strong calcium entry suggesting that the two proteins form a functional channel. These results might suggest a modulatory role for CNGA4 in a heteromeric TRPC2+CNGA4 ion channel. Further experiments will give more insights on the combined role of these transduction ion channels in pheromone detection.

Membrane lipid composition affects plant heat sensing and modulates Ca ( 2+) -dependent heat shock response.

Understanding how plants sense and respond to heat stress is central to improve crop tolerance and productivity. Recent findings in Physcomitrella patensdemonstrated that the controlled passage of calcium ions across the plasma membrane regulates the heat shock response (HSR). To investigate the effect of membrane lipid composition on the plant HSR, we acclimated P. patens to a slightly elevated yet physiological growth temperature and analysed the signature of calcium influx under a mild heat shock. Compared to tissues grown at 22°C, tissues grown at 32°C had significantly higher overall membrane lipid saturation level and, when submitted to a short heat shock at 35°C, displayed a noticeably reduced calcium influx and a consequent reduced heat shock gene expression. These results show that temperature differences, rather than the absolute temperature, determine the extent of the plant HSR and indicate that membrane lipid composition regulates the calcium-dependent heat-signaling pathway.

ACID-SENSING ION CHANNELS: functional and molecular characterization in putative nociceptors, and modulation by nerve injury and serine protease

Résumé Les canaux ioniques ASICs (acid-sensing ion channels) appartiennent à la famille des canaux ENaC/Degenerin. Pour l'instant, quatre gènes (1 à 4) ont été clonés dont certains présentent des variants d'épissage. Leur activation par une acidification rapide du milieu extracellulaire génère un courant entrant transitoire essentiellement sodique accompagné pour certains types d'ASICs d'une phase soutenue. Les ASICs sont exprimés dans le système nerveux, central (SNC) et périphérique (SNP). On leur attribue un rôle dans l'apprentissage, la mémoire et l'ischémie cérébrale au niveau central ainsi que dans la nociception (douleur aiguë et inflammatoire) et la méchanotransduction au niveau périphérique. Toutefois, les données sont parfois contradictoires. Certaines études suggèrent qu'ils sont des senseurs primordiaux impliqués dans la détection de l'acidification et la douleur. D'autres études suggèrent plutôt qu'ils ont un rôle modulateur inhibiteur dans la douleur. De plus, le fait que leur activation génère majoritairement un courant transitoire alors que les fibres nerveuses impliquées dans la douleur répondent à un stimulus nocif avec une adaptation lente suggère que leurs propriétés doivent être modulés par des molécules endogènes. Dans une première partie de ma thèse, nous avons abordé la question de l'expression fonctionnelle des ASICs dans les neurones sensoriels primaires afférents du rat adulte pour clarifier le rôle des ASICs dans les neurones sensoriels. Nous avons caractérisé leurs propriétés biophysiques et pharmacologiques par la technique du patch-clamp en configuration « whole-cell ». Nous avons pu démontrer que près de 60% des neurones sensoriels de petit diamètre expriment des courants ASICs. Nous avons mis en évidence trois types de courant ASIC dans ces neurones. Les types 1 et 3 ont des propriétés compatibles avec un rôle de senseur du pH alors que le type 2 est majoritairement activé par des pH inférieurs à pH6. Le type 1 est médié par des homomers de la sous-unité ASIC1 a qui sont perméables aux Ca2+. Nous avons étudié leur co-expression avec des marqueurs des nocicepteurs ainsi que la possibilité d'induire une activité neuronale suite à une acidification qui soit dépendante des ASICs. Le but était d'associer un type de courant ASIC avec une fonction potentielle dans les neurones sensoriels. Une majorité des neurones exprimant les courants ASIC co-expriment des marqueurs des nocicepteurs. Toutefois, une plus grande proportion des neurones exprimant le type 1 n'est pas associée à la nociception par rapport aux types 2 et 3. Nous avons montré qu'il est possible d'induire des potentiels d'actions suite à une acidification. La probabilité d'induction est proportionnelle à la densité des courants ASIC et à l'acidité de la stimulation. Puis, nous avons utilisé cette classification comme un outil pour appréhender les potentielles modulations fonctionnelles des ASICs dans un model de neuropathie (spared nerve injury). Cette approche fut complétée par des expériences de «quantitative RT-PCR ». En situation de neuropathie, les courants ASIC sont dramatiquement changés au niveau de leur expression fonctionnelle et transcriptionnelle dans les neurones lésés ainsi que non-lésés. Dans une deuxième partie de ma thèse, suite au test de différentes substances sécrétées lors de l'inflammation et l'ischémie sur les propriétés des ASICs, nous avons caractérisé en détail la modulation des propriétés des courants ASICs notamment ASIC1 par les sérines protéases dans des systèmes d'expression recombinants ainsi que dans des neurones d'hippocampe. Nous avons montré que l'exposition aux sérine-protéases décale la dépendance au pH de l'activation ainsi que la « steady-state inactivation »des ASICs -1a et -1b vers des valeurs plus acidiques. Ainsi, l'exposition aux serine protéases conduit à une diminution du courant quand l'acidification a lieu à partir d'un pH7.4 et conduit à une augmentation du courant quand l'acidification alleu à partir d'un pH7. Nous avons aussi montré que cette régulation a lieu des les neurones d'hippocampe. Nos résultats dans les neurones sensoriels suggèrent que certains courants ASICs sont impliqués dans la transduction de l'acidification et de la douleur ainsi que dans une des phases du processus conduisant à la neuropathie. Une partie des courants de type 1 perméables au Ca 2+ peuvent être impliqués dans la neurosécrétion. La modulation par les sérines protéases pourrait expliquer qu'en situation d'acidose les canaux ASICs soient toujours activables. Résumé grand publique Les neurones sont les principales cellules du système nerveux. Le système nerveux est formé par le système nerveux central - principalement le cerveau, le cervelet et la moelle épinière - et le système nerveux périphérique -principalement les nerfs et les neurones sensoriels. Grâce à leur nombreux "bras" (les neurites), les neurones sont connectés entre eux, formant un véritable réseau de communication qui s'étend dans tout le corps. L'information se propage sous forme d'un phénomène électrique, l'influx nerveux (ou potentiels d'actions). A la base des phénomènes électriques dans les neurones il y a ce que l'on appelle les canaux ioniques. Un canal ionique est une sorte de tunnel qui traverse l'enveloppe qui entoure les cellules (la membrane) et par lequel passent les ions. La plupart de ces canaux sont normalement fermés et nécessitent d'être activés pour s'ouvrire et générer un influx nerveux. Les canaux ASICs sont activés par l'acidification et sont exprimés dans tout le système nerveux. Cette acidification a lieu notamment lors d'une attaque cérébrale (ischémie cérébrale) ou lors de l'inflammation. Les expériences sur les animaux ont montré que les canaux ASICs avaient entre autre un rôle dans la mort des neurones lors d'une attaque cérébrale et dans la douleur inflammatoire. Lors de ma thèse je me suis intéressé au rôle des ASICs dans la douleur et à l'influence des substances produites pendant l'inflammation sur leur activation par l'acidification. J'ai ainsi pu montrer chez le rat que la majorité des neurones sensoriels impliqués dans la douleur ont des canaux ASICs et que l'activation de ces canaux induit des potentiels d'action. Nous avons opéré des rats pour qu'ils présentent les symptômes d'une maladie chronique appelée neuropathie. La neuropathie se caractérise par une plus grande sensibilité à la douleur. Les rats neuropathiques présentent des changements de leurs canaux ASICs suggérant que ces canaux ont une peut-être un rôle dans la genèse ou les symptômes de cette maladie. J'ai aussi montré in vitro qu'un type d'enryme produit lors de l'inflammation et l'ischémie change les propriétés des ASICs. Ces résultats confirment un rôle des ASICs dans la douleur suggérant notamment un rôle jusque là encore non étudié dans la douleur neuropathique. De plus, ces résultats mettent en évidence une régulation des ASICs qui pourrait être importante si elle se confirmait in vivo de part les différents rôles des ASICs. Abstract Acid-sensing ion channels (ASICs) are members of the ENaC/Degenerin superfamily of ion channels. Their activation by a rapid extracellular acidification generates a transient and for some ASIC types also a sustained current mainly mediated by Na+. ASICs are expressed in the central (CNS) and in the peripheral (PNS) nervous system. In the CNS, ASICs have a putative role in learning, memory and in neuronal death after cerebral ischemia. In the PNS, ASICs have a putative role in nociception (acute and inflammatory pain) and in mechanotransduction. However, studies on ASIC function are somewhat controversial. Some studies suggest a crucial role of ASICs in transduction of acidification and in pain whereas other studies suggest rather a modulatory inhibitory role of ASICs in pain. Moreover, the basic property of ASICs, that they are activated only transiently is irreconcilable with the well-known property of nociception that the firing of nociceptive fibers demonstrated very little adaptation. Endogenous molecules may exist that can modulate ASIC properties. In a first part of my thesis, we addressed the question of the functional expression of ASICs in adult rat dorsal root ganglion (DRG) neurons. Our goal was to elucidate ASIC roles in DRG neurons. We characterized biophysical and pharmacological properties of ASIC currents using the patch-clamp technique in the whole-cell configuration. We observed that around 60% of small-diameter sensory neurons express ASICs currents. We described in these neurons three ASIC current types. Types 1 and 3 have properties compatible with a role of pH-sensor whereas type 2 is mainly activated by pH lower than pH6. Type 1 is mediated by ASIC1a homomultimers which are permeable to Ca 2+. We studied ASIC co-expression with nociceptor markers. The goal was to associate an ASIC current type with a potential function in sensory neurons. Most neurons expressing ASIC currents co-expressed nociceptor markers. However, a higher proportion of the neurons expressing type 1 was not associated with nociception compared to type 2 and -3. We completed this approach with current-clamp measurements of acidification-induced action potentials (APs). We showed that activation of ASICs in small-diameter neurons can induce APs. The probability of AP induction is positively correlated with the ASIC current density and the acidity of stimulation. Then, we used this classification as a tool to characterize the potential functional modulation of ASICs in the spared nerve injury model of neuropathy. This approach was completed by quantitative RT-PCR experiments. ASICs current expression was dramatically changed at the functional and transcriptional level in injured and non-injured small-diameter DRG neurons. In a second part of my thesis, following an initial screening of the effect of various substances secreted during inflammation and ischemia on ASIC current properties, we characterized in detail the modulation of ASICs, in particular of ASIC1 by serine proteases in a recombinant expression system as well as in hippocampal neurons. We showed that protease exposure shifts the pH dependence of ASIC1 activation and steady-state inactivation to more acidic pH. As a consequence, protease exposure leads to a decrease in the current response if ASIC1 is activated by a pH drop from pH 7.4. If, however, acidification occurs from a basal pH of 7, protease-exposed ASIC1a shows higher activity than untreated ASIC1a. We provided evidence that this bi-directional regulation of ASIC1a function also occurs in hippocampal neurons. Our results in DRG neurons suggest that some ASIC currents are involved in the transduction of peripheral acidification and pain. Furthermore, ASICs may participate to the processes leading to neuropathy. Some Ca 2+-permeable type 1 currents may be involved in neurosecretion. ASIC modulation by serine proteases may be physiologically relevant, allowing ASIC activation under sustained slightly acidic conditions.

Glut2-dependent glucose-sensing controls thermoregulation by enhancing the leptin sensitivity of NPY and POMC neurons.

The physiological contribution of glucose in thermoregulation is not completely established nor whether this control may involve a regulation of the melanocortin pathway. Here, we assessed thermoregulation and leptin sensitivity of hypothalamic arcuate neurons in mice with inactivation of glucose transporter type 2 (Glut2)-dependent glucose sensing. Mice with inactivation of Glut2-dependent glucose sensors are cold intolerant and show increased susceptibility to food deprivation-induced torpor and abnormal hypothermic response to intracerebroventricular administration of 2-deoxy-d-glucose compared to control mice. This is associated with a defect in regulated expression of brown adipose tissue uncoupling protein I and iodothyronine deiodinase II and with a decreased leptin sensitivity of neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons, as observed during the unfed-to-refed transition or following i.p. leptin injection. Sites of central Glut-2 expression were identified by a genetic tagging approach and revealed that glucose-sensitive neurons were present in the lateral hypothalamus, the dorsal vagal complex, and the basal medulla but not in the arcuate nucleus. NPY and POMC neurons were, however, connected to nerve terminals from Glut2-expressing neurons. Thus, our data suggest that glucose controls thermoregulation and the leptin sensitivity of NPY and POMC neurons through activation of Glut2-dependent glucose-sensing neurons located outside of the arcuate nucleus.