Life-long homeostasis is essential for multicellular organisms, especially within long-living cells such as neurons. In particular, maintaining the structural and functional complexity of neurons over the lifespan of an organism requires finely tuned regulatory mechanisms that are purely understood. The goal of our project is to uncover the function of polyglutamylation as a fine-tuning mechanism of the microtubule cytoskeleton by bridging different biological scales, from intracellular transport to cellular morphology and function to animal behaviour. Following our discovery that deregulation of the posttranslational modification polyglutamylation leads to neurodegeneration in mice and humans, we will now determine the physiological roles of polyglutamylation in neurons in the whole-organism context by combining three model systems – C. elegans, zebrafish and mice - within the interdisciplinary settings of our consortium.

Pain is the most common symptom for which patients seek medical attention. According to the International Society of Pain (IASP) the prevalence of chronic pain is 18% of general population. In Europe, the pain just does not weigh on individuals and their families. Indeed, the social burden of chronic pain costs about billions of euros in Europe: probably not less than 300 billion euros to the EU as overall, 1.5 - 3% of gross domestic product. Nowadays, chronic pain is seen in industrialized countries as a broad public health problem particularly due to its high social cost. Although a number of pain medications are available on the pharmaceutical market, it is still today unfortunately observed that some chronic pain syndromes have completely escape these medicines, thus affecting considerably the quality of life for these patients. Indeed, many clinical cases of neuropathic pain are relatively insensitive to classical medicine. In addition, chronic use of morphine or its derivatives, in cases of the most rebellious pain, causes states of tolerance and dependence. It is important to explore new therapeutic approaches to block the mechanisms underlying chronic pain and to relieve its syndromes. Recently, it has been established that ongoing pain associated with peripheral nerve lesions is not only linked to a heightened excitability of sensory neurons but would also be the result of a persistent inflammation affecting nociceptive pathways. Patients suffering from these chronic pain syndromes are subject to hyperalgesia and/or allodynia. Thus, our incomplete understanding of the mechanisms underlying chronic pain sensitivity accounts for the general ineffectiveness of current options for the treatment of chronic pain syndromes. Nowadays, neuroimmune interactions are increasingly recognized as important factor in alteration of the nociceptive processing during neuropathic pain. As part of this neuro-immune relation, recent evidences suggest that the up-regulated expression of inflammatory chemotactic cytokine (chemokine) in association with tissue damage or infection serves not only in the capacity of leukocytes chemotaxis but also in the generation of exacerbated hyperexcitability state of sensory neurons. This interdisciplinary project proposal brings together two research fields: neuroscience and immunopathology. Indeed, the overall objective of this research project is to define, the functional role of chemokines CCL2 and/or CX3CL1 in the interactions between immune cells (in particular monocytes/macrophages) and sensory neurons and their implications in the mechanisms of initiation and maintenance of painful syndromes. We hypothesize that these chemokines after peripheral nerve injury could induce, via theirs chemotactic properties, the recruitment of immune cells locally at the lesion and Dorsal root ganglia levels, and thus could participate in mechanisms leading to Wallerian degeneration and chronicity of pain. Thus, directly through its neuro-modulatory effects and indirectly, through its influence on immune-competent cells that broadly alter the neuronal function, these chemokines may fundamentally impact on nociceptive information processing. In this context, this project presents three aspects: i) - the fundamental one, devoted to the understanding of the relationship between effects exerted by chemokines (ie CCL2 and CX3CL1) on neurons, glial, schwann and immune cells, and importance of these relations in the initiation of pain, then the shift from acute to chronic pain syndromes; ii) –deciphering in an appropriate preclinical models of peripheral neuropathic pain, the cellular and molecular mechanisms leading to chronic inflammation to understand the pathogenesis of this disease III) bring in these appropriate preclinical models of pain the rational use of chemokine receptor antagonists as potential therapeutic agents for the treatment of pathological chronic pain.

At the back of the eye lies a monolayer of cells essential for vision: the retinal pigment epithelium (RPE). Apical microvilli from these polarized cells make close contact with the photosensitive photoreceptor outer segments (POS). POS are constantly renewed to fight the high levels of oxidative damage they are subjected to, and one of RPE cell main roles to maintain lifelong vision is the daily elimination of used POS tips by phagocytosis. Absence or failure to complete this task leads to the development of blinding diseases for which no treatment exists, such as early-onset retinal dystrophies or age-related macular degeneration (AMD). An important feature of RPE phagocytosis is its rhythmic activity. We showed previously that the alphavbeta5 integrin receptor controls the rhythmic activation of RPE phagocytosis. Subsequently, an intracellular signaling cascade activates the Mer tyrosine kinase (MerTK) internalization receptor. Our recent studies suggest that MerTK also controls the amounts of POS that can be tethered by RPE cells, including via the extracellular cleavage of MerTK. Interestingly, the known machinery for RPE phagocytosis is close to the clearance of apoptotic cells by macrophages. In macrophages, many molecules intervene in such processes, suggesting that similar intricate protein networks could operate in RPE cells. However, tissue specificity exists, including the permanent contact between photoreceptors and RPE cells, thus regulation of the machinery has to be controlled very tightly via several mechanisms to launch and stop phagocytosis at the proper time. Our recent data on the tissue-specific opposite role of MerTK ligands reinforce this idea. Moreover, several other receptors have been shown to be expressed by RPE cells, but their participation in POS phagocytosis has not been investigated yet. So far, studies on the phagocytic machinery have been performed on nocturnal rodent species using mostly rod photoreceptors sensitive to dim light. However, central vision in humans is mostly due to cones that give us details resolution and color vision. Rod and cone POS membrane structures are different and they are used for vision almost in exclusion of each other, either at night or during the day, respectively. Therefore, our hypothesis is that the elimination of used cone POS could take place at a different timing and with a different molecular machinery than for rod POS. For these reasons, with the REPHAGO project we plan on identifying the contribution of new membrane receptors in controlling the daily activation of rod (Aim 1) and cone (Aim 2) POS phagocytosis by RPE cells. Candidate receptors for rod POS phagocytosis will be validated in vitro and then in vivo for interesting candidates according to their implication during the phagocytic process. We will characterize cone-specific molecules for POS clearance using transcriptome studies associated with functional validation assays. For this project, we will use multiple state-of the art approaches (functional phagocytosis assays, in vivo phagocytosis assessment, RNAseq, visual animal phenotyping…) as well as new and innovative animal models (RPE-specific knockout mouse models for rod receptor candidates, cone-rich diurnal rodent model). Identified receptors will be then explored in other phagocytic cells. Understanding the complexity and specificity of protein networks and interactions to complete daily this crucial task will enlighten us both on normal retinal function and on the consequences of phagocytic defects. This will help us consider new avenues for therapies and therapeutic targets for these pathologies for which no treatment exists. As well, the sequential activation of the RPE machinery can help us decipher molecular pathways that are used in other phagocytic cells, and in particular macrophages. Thus, our results could contribute to the understanding of phagocytic processes occurring in other tissues or pathologies such as atherosclerosis.

Repairing nerve damage in the peripheral nervous system (PNS) is a significant challenge in regenerative medicine. Traumas, degenerative diseases, and surgical interventions often lead to debilitating consequences for patients. Despite significant advances in neurobiology and neurosurgery, current treatments often fall short in promoting effective nerve regeneration. Our project aims to address this gap by developing an innovative electroactive biomaterial specifically designed to stimulate axonal growth and facilitate the repair of damaged nerves. To achieve this, we adopt a multidisciplinary approach, bringing together expertise in cellular biology, materials engineering, and neurophysiology. The primary objective is to understand the underlying mechanisms of the beneficial effect of a local electric field (LEF) on nerve regeneration and apply this knowledge to the design of a novel therapeutic device. i) Phase 1 of the proposal : Characterization of Neuron-Substrate Interactions under the Influence of LEF In this first phase, our goal is to comprehensively understand the interactions between neurons and the electroactive substrate in the presence of LEF. We have developed an original experimental setup that allows precise control of the applied electric field intensity and real-time visualization of neurite growth. This device is highly adaptable, enabling the incorporation of bioactive molecules that promote axonal growth. Simultaneously, we employ advanced imaging techniques such as fluorescence microscopy and scanning electron microscopy to study cellular interactions at the neuron-substrate interface. We also conduct in-depth biological studies to characterize the effects of LEF on various cellular parameters, such as neurite elongation, neuronal adhesion, and membrane-cytoskeleton coupling. By modifying the adhesive, biochemical, and elastic properties of the substrate, we aim to determine the optimal conditions to promote nerve regeneration while minimizing inflammatory reactions and rejection phenomena. ii) Phase 2 of the proposal : Development of a Cylindrical Implant Prototype In this second phase, we plan to design a prototype cylindrical implant integrating electrodes into a biocompatible and bioresorbable tube. This device will allow targeted application of LEF to the injured area, thereby promoting the regeneration of nerve fibers. We intend to test this prototype in vivo using animal models of nerve injuries to validate its effectiveness and safety. iii) Expected Results : We anticipate that our innovative approach will lead to a better understanding of nerve regeneration mechanisms and the development of more effective therapeutic strategies for nerve injuries. We hope to demonstrate that the application of LEF can stimulate axonal growth and improve the functionality of regenerated neurons. Additionally, we expect our implantable device to be well-tolerated by the body and capable of promoting significant nerve regeneration in patients with severe nerve injuries. iv) Conclusion : In conclusion, our project represents a significant advancement in the field of nerve regeneration by combining multidisciplinary approaches to develop an innovative electroactive biomaterial. We are confident that our work will contribute to improving the quality of life for patients suffering from nerve injuries by offering more effective therapeutic solutions tailored to their specific needs.

Most animals are born with a minimal set of behaviors that is progressively enriched during development to accommodate new internal needs, physical and environmental constraints. The emergence of these new functional capacities reflects the concurrent maturation of the brain circuitry, as new neurons become functional and new connections are formed, but also results from changes in the synaptic strengths that modify the computational capacity of given networks. How innate developmental programs and on-going interactions with the environment concur to the self-organization of extended functional circuits remains an open question. In the present project, we will combine genetic developments, behavioral and functional monitoring, and data-driven graphical models to unveil fundamental mechanisms that orchestrate the maturation of brain-wide neural circuits leading to the progressive emergence of new computational capabilities and behaviors during early development. To address this question, we will leverage the assets of Danionella translucida (DT), a novel vertebrate model whose brain remains small and transparent up to the adult stage, and is thus amenable to large-scale monitoring of brain activity during a significant development period. The phylogenetic and anatomical proximity of DT with Zebrafish - a well-established model vertebrate - will facilitate the identification of functional neuronal circuits and allow for fruitful cross-species comparison. We will focus on the maturation of the neuronal circuits that organize spontaneous and light-driven exploration. We will first quantify how this behavior evolves from the larval to the juvenile stage using freely-swimming behavioral assays. We will characterize in particular the phenotypic transition, from continuous to bout-like navigation, that takes place during this developmental period. We will then use whole-brain calcium imaging and optogenetic activation to probe the collective dynamics of the neuronal circuits engaged in this process in partially tethered, fictively swimming animals across the same age range. We will use morphological registration between individuals of same age and across development to produce generic coarse-grained brain-scale dynamics, at successive stages of development. This unique dataset, that will include whole-brain neural activity, sensory and motor signals, will be used to train energy-based graphical models. Such models have generative powers: we will produce synthetic activity and fictive motor signals that will recapitulate in silico the brain-scale neural dynamics and associated sensory-motor computations. By training these models at various developmental stages, and comparing the inferred parameters, we aim to illuminate basic developmental rules that could account for the observed changes in behavior. The project will be carried out by a consortium of three teams, with highly complementary expertise. Filippo Del Bene is a biologist who has made key contributions to the understanding of molecular processes that control the development of neural circuits in the larval zebrafish brain. He has played a major role in establishing Danionella Translucida as an important emerging model system in neuroscience. Georges Debrégeas is an experimental physicist, who develops state-of-the art optical systems for whole-brain imaging in zebrafish larvae, high-throughput behavioral assays and who develops efficient data processing methods to analyze these high-dimensional datasets. Rémi Monasson is a theoretical physicist who applies statistical-physics and machine-learning based approaches to biological data analysis and modelling, particularly in neuroscience.