The overall objective of our project is to understand the role of newly identified apical Planar Cell Polarity (PCP) signaling dependent on Gai-proteins during the maturation of the inner ear of mammals, in physiological and pathophysiological conditions. In the Western world, the proportion of the population that suffers from hearing loss is around 7 to 8%. Statistics collected from different countries show that out of 1,000 births, 1 to 1.5 will suffer profound hearing loss or deafness. Because the mechanoreceptive hair cells which mediate the sensory transduction in the inner ear can be injured or definitively lost after exposure to noise, otoxic drugs, or as part of normal aging, hearing losses are the fastest growing, and one of the most prevalent chronic conditions facing an aging population. Developing knowledge on the genetic and molecular bases of auditory cells differentiation that could guide strategies for regeneration and protection has the potential to lead to the establishment of new tools for prognosis and diagnosis of deafness, but also has the potential to open new avenue of research for inner ear pathologies in the hope to explore opportunities for preventive and therapeutic interventions. Recently, we have identified a new PCP signaling pathway, which we called G-protein-dependent PCP signaling (Ezan et al., 2013).During the course of this original study, we observed that in later stages of maturation, the hair bundles topping the hair cells appeared malformed, shorter and fragmented in two of the studied mice mutants, suggesting the involvement of certain genes of this PCP pathway in the late maturation of the hair cells and more generally in hearing function. As a first step, our proposal will explore this hypothesis, notably through the use of transgenic mouse models and in particular via Cre-lox technology that will allow us to study the postnatal development of the inner ear, its maturation and its function, or disruption of function. As a second step, we will explore the hypothesis that the apical complex Crumbs controls the dynamic of tubulin and actin, at least in part via the recruitment of certain of the apical PCP signaling pathway. For this, we will build on a multidisciplinary and multi-model approach that will bring us the benefits of three species: Xenopus, mouse and Drosophila. The results of our project will lead to the identification of a new family of candidate genes for deafness, to the elucidation of the molecular mechanism leading to these deafnesses and to decipher new protein networks at the crossroads between the apico-basal polarity, the Planar Cell Polarity and the cilium.

Pain is a physiological signal that contributes to the protection of individuals from potentially harmful stimuli. In pathological conditions, however, maladaptive plasticity of primary afferent neurons conveying sensory information, or of neuronal networks transmitting this information towards the brain, can induce long lasting intense pain sensations without any protective value. Sensory and noxious information is transmitted from the periphery of the organism by morphologically and functionally specialized neurons. Among these neurons, a specific class of mechanoreceptors, the C-Low Threshold Mechanoreceptors (C-LTMRs), has received recent emphasis through studies unraveling their role in the modulation of pain transmission. Hence, this particular population of neurons is likely to constitute an important yet unexplored target for the treatment of chronic pathological pain. Our project will combine molecular, cellular, electrophysiological and behavioral approaches to investigate how C-LTMRs control the integration of nociceptive information in spinal cord, and how their dual function in mediating both pleasant aspects of touch and unpleasant painful information can be achieved. Using optogenetics, we will decipher neuronal and synaptic pathways involved in the modulation of pain transmission by C-LTMRs. In parallel, we will first launch a wide genome screen combining FACS sorting and RNA Seq to identify novel genes specifically expressed in C-LTMRs, alter their expression using knock-down and overexpression experiments, and determine their roles in modulating cell excitability, spinal networks physiology, and pain sensation. The project comprises 2 main tasks addressed by three partner teams. In Task 1: We will combine genetic, morphological and functional approaches to resolve the organization of spinal networks processing C-LTMRs-triggered information, and how, within these networks, C-LTMRs-triggered information interferes with noxious information to attenuate its propagation to higher brain structures. i) Using genetically engineered mice expressing the trans-synaptic tracer WGA specifically in C-LTMRs, we will perform a first characterization of spinal neurons connected to C-LTMRs. ii) By combining spinal slice patch clamp recordings with cre-lox AAV based strategy to target the expression of channel rhodopsin to C-LTMRs, we will confirm the identification of spinal interneurons receiving inputs from C-LTMRs, characterize the pharmacology, short and long term plasticity of these inputs. iii) We will determine how these inputs propagate to the spinal projection neurons identified by retrograde labelling. iv) Finally, using highly challenging dual optical control, we will decipher the mechanisms by which noxious (controlled with TRPV1 permeant ion channel photoswitch) and non-noxious (controlled with genetically targeted channelrhodopsin expression in C-LTMRs) information gets integrated in dorsal horn networks. In Task 2: We will determine how the fine tuning of nociceptive transmission by C-LTMRs is altered in chronic pain conditions. i) We will use FACS sorting followed by RNA Seq to identify selective markers of C-LTMRs, characterize their expression in neuropathic animals using qRT-PCR and histological techniques, thus enabling the selection of few functionally relevant genes. ii) Using knock-down and overexpression approaches, we will characterize how these genes shape the activity of C-LTMRs and their response to mechanical or chemical stimuli, in control and neuropathic animals. iii) We will determine how these markers alter sensory-nociceptive transmission in spinal networks and pain behavior in acute and neuropathic pain models. This project will not only extend our knowledge on the physiology of C-LTMRs but also on the role of this unique population of primary sensory neurons in pain processing very likely leading to the identification of new potential therapeutic targets.