Neurodegenerative disorders are primarily characterized by neuron loss of function and are associated with neuroinflammation and aging. Although there are several medicines approved for managing neurodegenerative disorders, a large majority of them only help with associated symptoms. This lack of pathogenesis-targeting therapies is primarily due to the restrictive effects of the blood–brain barrier, which keeps close foreign substances out of the brain. Antimicrobial peptides (AMPs) are molecules of the innate immune system known for their anti-infectious, immunomodulatory, and physiological functions; they have been identified in the brain in an infectious context but their role on brain function in sterile condition remains poorly defined. Our project aims to define the cathelicidin related antimicrobial peptide (CRAMP) and the secretory leukocyte protease inhibitor (SLPI), two AMPs expressed in the brain, as key molecule for the control of neuroinflammation and cognitive function. Our preliminary data in mice show that CRAMP and SLPI are expressed in several brain-resident cell types and particularly in the hippocampal neurons and that CRAMP deficiency results in neuroinflammation and cognitive defects at steady state. Based on these observations, we aim to characterize the spatiotemporal expression of CRAMP and SLPI in the brain and the pathway(s) regulating their expression with a special focus on gut-microbiota-derived metabolites that are known to induce AMP expression in several tissues. We further want to determine the mode of action of these AMPs regulating the crosstalk between brain cells and to demonstrate the therapeutic potential of AMP modulation on neuroinflammation-associated cognitive decline. Our project will enhance our understanding of brain function and provide further insight into innovative therapy avenues for neurodegenerative diseases.

Autophagy is a lysosomal degradation and recycling pathway that controls the quality and quantity of cytoplasmic material. Autophagy requires the formation of a double-membrane vacuole called the autophagosome that sequesters proteins and other cytoplasmic components to be delivered into the lysosome. Autophagy plays crucial roles in cellular and tissue homeostasis, and dysfunctional autophagy has been linked to human pathologies including kidney diseases. Autophagy is now recognized to have non-cell autonomous functions via the secretion of cytosolic molecules sequestered in autophagosomes or autophagosome-like structures. By controlling the release of a large panel of molecules (e.g., cytokines, amino acids, nucleotides), non-cell-autonomous autophagy (NCAA) mediates communication between cells within the same organ and between organs. Recently, studies have paved the way for research into the function of NCAA in human physiology and pathophysiology; however, little is known about the role of NCAA in kidney physiology. In 2016, we demonstrated that shear stress generated by urinary fluid flow induces a primary cilia-dependent autophagy in kidney epithelial cells and that this induction of autophagy influences kidney epithelium homeostasis by regulating cell volume. We now propose to investigate the contribution of non-cell autonomous autophagy to kidney physiology. We will focus our attention on nucleotides (such as ATP and its metabolites) as these extracellular messengers are known to participate in the control of renal electrolyte tubular transport. This hypothesis is based on our preliminary results and on published data showing that inhibition of autophagy blocks ATP secretion under different stress conditions. This proposal is organized in three specific aims: (1) We will determine whether NCAA controls kidney epithelial cell size upon shear stress. We will first decipher the molecular machinery required for the release of ATP and identify the purinergic receptors responsible for cell size regulation upon shear stress. Next we will determine whether this autophagic-dependent secretion of ATP affects cell volume in autocrine or/and paracrine dependent manners. (2) We will study how NCAA regulates kidney cell size in vivo using mouse and zebrafish models incompetent for autophagy. For this purpose we will analyze the accumulation of ATP-positive vesicles in kidney epithelial cells of chloroquine-treated mice. We will also determine the sizes of distal tubular and collecting duct cells in mice in which autophagy is conditionally impaired in the proximal tubules (PEPCK-Cre Atg7fl/fl mice). Finally, in zebrafish we will track the induction of autophagy and the release of ATP in the tubular pronephros during physiological urinary fluid flow (at 48 hours post fertilization) using transgenic LC3 (CMV:GFPLC3) zebrafish to enable monitoring of autophagy; Atg5-deficient larvae will be used as a negative control. (3) Finally, we will determine whether NCAA controls another aspect of kidney physiology by regulating its endocrine role. We will focus our attention on the release of renin because adenosine (produced by extracellular breakdown of ATP, which is secreted by cells from the macula densa) inhibits the release of renin from the juxtaglomerular cells. In conclusion, this proposal will determine how NCAA affects kidney physiology. We expect to show that NCAA regulates kidney cell size in cultured cells and in vivo. We also anticipate that NCAA will prove to be a mediator of crosstalk within cells in the same organ and also between different organs, which will represent a conceptual breakthrough in the autophagy and the physiology fields.

Protein misfolding diseases (PMDs) are associated with either aggregation of misfolded proteins leading to toxic gain-of-function phenotypes or with protein degradation leading to loss-of-function phenotypes. Cystic Fibrosis (CF) is an example of a loss of function resulting from genetic mutations within CFTR. F508del, by far the most frequent mutation, is associated with protein misfolding, reduced channel function and cell surface stability. Mutant channel maturation and function can be partially rescued with small molecules referred to as correctors, while channel function is efficiently enhanced by potentiators. Nonetheless, this treatment combination is still suboptimal in clinics and there are still no means to increase channel cell surface stability. Our working hypothesis is to increase channel rescue by targeting specific protein-protein interactions, which retain abnormally folded proteins. This novel strategy is supported by our previous work, which revealed that an interaction with intermediary filament, Keratin 8, retains F508del-CFTR in the endoplasmic reticulum (ER) and that disruption of this interaction restores CFTR-F508del functional expression, leading to the identification of a new class of CFTR correctors. Preliminary data indicate that the two classes of correctors, targeting either CFTR directly or protein-protein interactions, present an additive effect. Our new results identified PRAF2 as a novel key regulator of CFTR exit from the ER, suggesting that PRAF2/F508del-CFTR interaction could also be a target for pharmacotherapy. We have previously shown that PRAF2 controls the ER exit of cargo transmembrane proteins (i.e. receptors, transporters…) in a stoichiometric manner and that this regulation involves the presence of a molecular code-bar (RXR motifs) located in the intracellular domains of PRAF2-regulated proteins. The first aim of this project is therefore to understand the role of PRAF2 in the ER exit of CFTR by identifying both the specific binding motifs and the additional molecular actors implicated. This will set the basis for a molecular screen aimed at identifying chemical compounds targeting the PRAF2/CFTR interaction. In parallel, we will identify new (i) differential (WT or mutant CFTR-specific) interatomics and (ii) compartment-specific protein-protein interactions for F508del-CFTR using the newest proteomic approaches: proximity labeling with APEX2 and APEX2-complementation coupled to mass spectrometry. The importance of these “new” molecular partners will be evaluated both functionally and biochemically. The best targets will be combined with CFTR modulators available today to enhance treatment efficacy and tested on primary epithelial cells. The end point to these studies would be the identification of new drugs capable of enhancing the efficacy of current treatments. We believe, indeed, that the release of partially functional misfolded proteins from specific protein-protein interactions will restore to some extent functional activity. For cystic fibrosis, this could be further enhanced by combination with available CFTR modulators. The data obtained so far support our strategy of targeting protein-protein interactions in PMDs and provide the flowchart to study other loss of function PMDs such as alpha-1-antitrypsin deficiency, diabetes, nephritic syndrome, and also to some extend other diseases such Chronic obstructive pulmonary disease (COPD)…. Consequently, the methodology developed in this project could in fine lead to the identification of key protein-protein interactions in other diseases which could be targeted and represent potentially treatments, which could similarly be improved be improved with a new class of drugs.

Liver is a vital organ, ensuring hundreds of metabolic functions and thus finely specialized for its metabolism, which consequently need from birth to adulthood to acquire its metabolic zonation and gradually enter into quiescence. Nevertheless, in response to liver damages, hepatocytes have the capacities to proliferate to maintain the hepatic mass and function, in cooperation with hepatic stem cells. This adaptative response driven by hepatocytes could favor the development of a chronic disease in case of persistent injuries. Chronic liver diseases emerge these recent years as a major health problem regarding to change in our eating habits, but also to abuse in alcohol consumption and infections by hepatitis viruses. Liver disease initiates with a single steatosis which could deteriorate into steatohepatitis (steatosis with inflammation, fibrosis and necrosis), and, finally, to cirrhosis and cancer. These last years, cumulative evidence showed that various microRNAs (miRNAs) extensively contribute to liver pathogenesis, from liver disease to cancer, and thus should constitute potent biomarkers and therapeutic targets for these pathologies. In particular, a cluster of 54 miRNAs originated from the imprinted DLK1/DIO3 locus appear as an attractive player for liver disease establishment since it is crucial both for stem cell and hepatocyte fate in mouse liver following activation of the WNT/ß-catenin signaling, a master regulator pathway for liver zonation and stem cell maintenance. Using deep-sequencing approaches in our mouse models, we importantly demonstrated that ß-catenin orchestrates a specific transcriptional program, resulting in a metabolic reprogramming and an exit from quiescence of hepatocytes, which could favor the establishment of a cancerous switch – an event observed in a third of hepatocellular carcinoma. In particular, we observed that ß-catenin promptly induced the expression of all the constituents of the locus, either expressed from the maternal or the paternal strand, dependently on methylation process and more importantly through direct binding of ß-catenin on its promoter region. In the next three years, using our mouse models, we will unveil how ß-catenin induces a loss of imprinting in the DLK1/DIO3 locus through epigenetic modifications and/or chromatin remodeling, and which factors are recruited in space and time by ß-catenin to modify the locus expression during liver pathogenesis. We will also address the functional consequences of the silencing of this locus by genome editing on hepatocyte metabolic and proliferative properties in different liver pathological contexts. This project deciphering the crosstalk between ß-catenin and epigenetic events (miRNAs, imprinting and chromatin remodeling) for gene expression should highlight new promising targets for regenerative medicine but also for different diseases associated to WNT/ß-catenin signaling such as metabolic syndromes or cancers.

Memory formation is critical for normal adaptive functioning and is at the center of many cognitive disorders. The discovery that memory becomes labile again when reactivated, and needs to go through a process of reconsolidation to become stable, has strengthened the idea that memory stabilization is a highly plastic process. The great interest in the reconsolidation process is that it offers a window of opportunity to disrupt memories a long time after the initial encoding and suggests that memory reactivation may play a role in modulating memory strength and in the updating of memory content. The understanding of the reconsolidation process is therefore of considerable importance to provide further insights into the development of therapeutic tools for treatment of pathological memories. To date, many studies have investigated the molecular and cellular bases of reconsolidation as well as the synaptic mechanisms underlying this process within the hippocampus in particular. Recent progress has been made towards finding the engram, in particular the populations of neurons that are active during memory encoding and retrieval (defined as “engram cells”). Surprisingly, the process of reconsolidation has not been considered in the context of ongoing adult neurogenesis, which is known to confer a new support to memory processes. Adult neurogenesis is the generation of new granular neurons in the dentate gyrus of the adult hippocampus. A decade of research has proven the role of adult neurogenesis in memory formation, specifically in processes such as spatial learning, Pattern separation, forgetting, etc…However, the role of the continuous addition of granular neurons in the reconsolidation process of established memory has not been investigated. Thus the role of hippocampal neurogenesis in the stabilization of memory remains to be defined. Here, I propose to demonstrate that adult-born neurons play a key role in memory reconsolidation. Preliminary data demonstrated that adult-born neurons are activated by spatial memory reconsolidation. Therefore, the first goal of this project will be to generalize the role of adult-born neurons in reconsolidation of hippocampal-dependent memories, using two different behavioral hippocampal-dependent paradigms—the water maze (WM) and the contextual fear conditioning (CFC). Then I will demonstrate a causal relationship between adult neurogenesis and memory reconsolidation. Towards this aim, we developed a new tool to specifically modulate adult-born neurons activation at the time of reconsolidation. It is based on the pharmacogenetic approach of the DREADDs (Designer Receptors Exclusively Activated by Designer Drugs). We developed a new retrovirus that expresses GFP and contains the DREADD coupled to either the Gi or Gs proteins. Considering retroviruses incorporate DNA into dividing cells only, this new tool allows the selective inactivation (DREADD-Gi) or stimulation (DREADD-Gs) of the GFP-tagged newly generated neurons at a specific time point. Recently, a great deal of experimental investment is directed towards the mechanisms of memory storage. Memory engram technology allows the labeling and subsequent manipulation of cells ensembles, demonstrating that memory is indeed held in specific populations of neurons. My preliminary results suggest that a specific population of neurons that are immature, and thus not activated, at the time of learning, becomes necessary when functional maturity is reached, to stabilize the memory trace after reactivation. Therefore, the third aim of the project will be to understand how this immature population of neurons that is not activated by learning can become part of the engram, and to uncover the specific connectivity between immature adult-born neurons and “engram cells” at the time of learning. Together, these experiments will lead to a better understanding of the reconsolidation process and the role of adult neurogenesis in the dynamics of memory.