Detection of salient stimuli or environmental changes is a key mechanism that facilitates associative learning by allocating attentional resources toward biologically significant. The detection of salience is highly dynamic and influenced by a wide range of factors, including past experiences, arousal, emotions, stressful events, and internal homeostatic needs or environmental changes. However, in some cases, irrelevant events acquire saliency, whereas biologically significant events do not. Such misallocation of the salience of external and internal signals is often associated with altered inhibition of automatic responses and impaired goal-directed behaviors which constitute core symptoms shared by several neurodevelopmental and psychiatric disorders. Widely distributed circuits in the animal and human brain are activated by salient stimuli and increasing evidence indicates that dorsal subicular circuits are essential for the detection and processing of salient contextual stimuli. These regions comprising the prosubiculum receive projections from the locus coeruleus and the ventral tegmental area, two monoaminergic nuclei also highly reactive to stimuli perceived as salient. Although recent findings indicate that hippocampal monoamine transmission might be critical for the detection of contextual salient stimuli, dorsal subicular circuits, and the underlying cellular mechanisms through which monoamine signaling modulates salience detection remain to be elucidated. By combining advanced circuit-mapping, intersectional genetics, ex and in vivo electrophysiological recording and behavior, SubDOPA aims to parse the role of the dorsal Prosubiculum (PSd) monoamine signaling not only in the detection of environmental changes but more generally in the detection and processing of salient stimuli and to understand how this may elicit attentional-behavioral switches allowing animals to select the most appropriate behavioral reactions toward biologically significant events. More specifically we will characterize the connectivity and function of PSd neurons responding to monoamines. We will monitor the dynamic of monoaminergic neuronal activity and release during environmental changes or salient stimuli presentation. We will investigate the impact of the detection of biologically salient events on the PSd neuronal network. The completion of SubDOPA will provide new insights into the neural circuits and cellular mechanisms controlled by monoamines involved in salience detection and will contribute to a better understanding of the emergence of deficits associated with aberrant salience which are core symptoms shared by several neurodevelopmental and psychiatric disorders.

Post-traumatic stress disorder (PTSD) is the fourth most prevalent psychiatric diagnosis encompassing three clusters of symptoms: re-experiencing the traumatic event, avoidance symptoms and hyper arousal. PTSD is characterized by a high-rate of treatment resistance and inter-individual variability, with heightened prevalence and severity amongst women. The most effective treatment for PTSD consists in cognitive behavioral therapy such as exposure therapy. However, excessive and persistent avoidance of trauma related cues (people, conversations, places, situations) contributes to the maintenance of PTSD by preventing patients from reappraising their perception of the threat. Alleviating avoidance symptoms is therefore a prerequisite to improve the outcome of exposure therapy. In strike contrast with the study of Pavlovian defensive reactions (such as freezing behavior), active avoidance and its neural correlate remain poorly understood. Recent work in rodents suggests that active avoidance involves deep brain regions such as the amygdala, ventral striatum and midbrain motor centers. In contrast, the dentate gyrus in the hippocampus -a brain region implicated in learning and memory, spatial navigation and emotionality- plays an important role in preventing the persistence of active place avoidance. This is especially important as neuroimaging studies in patients suffering from PTSD point to the hippocampus as a critical site of vulnerability to stress. The NEURAVOID project aims at investigating how the hippocampus and its downstream partner the lateral septum prevent persistent place avoidance by inhibiting the activity of the ventral striatum. We will use optical tools in freely moving male and female mice to characterize this neural circuit with high spatial and temporal resolution. These approaches will be implemented in an active place avoidance paradigm to (i) determine whether the activity of lateral septal neurons projecting to the ventral striatum predict persistent place avoidance; (ii) characterize the lateral septum microcircuit linking the hippocampus and ventral striatum which controls persistent active place avoidance; (iii) devise a closed loop system in this circuitry to prevent persistent place avoidance in real time. In the short term, we hope that these findings will guide functional brain imaging studies in order to optimize treatment response across male and female patients suffering from PTSD. In the long term, we hope that this body of work will help refining deep brain stimulation protocols as well as brain-machine interfaces to curb excessive avoidance behavior in PTSD.

The asymmetric distribution of lipids between the two leaflets of cell membranes is a fundamental feature of eukaryotic cells. For instance, while phosphatidylcholine and sphingomyelin are restricted to the outer leaflet of membranes of the late secretory/endocytic pathways in most cell types, phosphatidylserine (PS), phosphatidylethanolamine, and phosphatidylinositol-4,5-bisphosphate are only found in the cytosolic leaflet. Regulated exposure of PS in the outer leaflet of the plasma membrane is an early signal for clearance of apoptotic cells by macrophages or triggering of the blood coagulation cascade. Inside the cell, PS plays critical roles since the high negative surface charge conferred by PS on the cytosolic leaflet of membranes facilitates the recruitment of polybasic motif-containing proteins such as the small GTPase K-Ras and the membrane fission protein EHD1, providing a link between PS distribution and regulation of cell signalling and vesicular trafficking. For transbilayer lipid asymmetry to be maintained, cells have evolved the so-called lipid flippases, transmembrane proteins from the P4-ATPase family which are responsible for the active transport of lipid species from the exoplasmic to the cytosolic leaflet of membranes, at the expense of ATP. Most P4-ATPases require association with transmembrane proteins from the Cdc50 family for proper localization and lipid transport activity. The yeast lipid flippase complex Drs2-Cdc50 has been shown to specifically transport PS and this transport is crucial for bidirectional vesicle trafficking between the endosomal system and the trans-Golgi network (TGN). Mutations in human P4-ATPases have been linked to severe neurological disorders, reproductive dysfunction as well as metabolic and liver disease, underlining the essential role of transbilayer lipid asymmetry in cell physiology. We previously showed, using a combination of limited proteolysis, genetic truncation, and structural approaches, that the catalytic activity of purified Drs2-Cdc50 complex is autoinhibited by its two unstructured N- and C-terminal extensions and activated by phosphatidylinositol-4-phosphate (PI4P). Yet, the molecular mechanism underlying activation of Drs2-Cdc50-dependent lipid transport activity remains unknown. Recently, the small GTPase Arl1 and the Arf-GEF Gea2, a GDP/GTP exchange factor for Arf, were shown to physically interact with the N- and C-termini of Drs2, respectively, and to be required for Drs2-Cdc50-catalyzed lipid transport in isolated TGN vesicles. Arl1 also binds to Gea2, suggesting an intricate mechanism for the regulation of Drs2-mediated transbilayer lipid transport. Based on previous work and our preliminary results, our working hypothesis is that binding of Arl1 and Gea2 to the N- and C-termini of Drs2 relieves autoinhibition and thus activates lipid transport by Drs2-Cdc50. Hence, combining biochemical, in silico and medium/high-resolution structural approaches, FLIPPER aims to dissect this regulatory mechanism, using in vitro reconstitution of the lipid transport machinery. This will be achieved by combining our expertise in the structural and biochemical analysis of small GTPases and Arf-GEFs (J. Cherfils) with structural mass spectrometry techniques, including hydrogen-deuterium exchange mass spectrometry (C. Bechara), structure determination of the Drs2-Cdc50-Arl1-Gea2 complex by cryo-EM (J. Lyons/P. Nissen) and know-how into the biochemistry and functional investigation of lipid flippases (G. Lenoir). Altogether, our proposal aims to provide a mechanistic basis for Drs2 activation in vivo and reveal new functions for understudied small GTPases and large Arf-GEFs such as Arl1 and Gea2.

Heart Failure (HF) is an epidemic of the 21st century. This chronic disease is a major public health problem due to its high prevalence (26 million patients worldwide), frequent hospitalizations and economic impact of associated direct and indirect costs. With the aging and industrialization of the population, this value will continue to increase. The clinical syndrome of HF represents the final stage of the continuum of cardiovascular diseases such as arterial hypertension or myocardial infarction (MI), which is a leading cause of cardiovascular mortality. Infarct size is a major determinant of mortality after MI and its limitation is required to prevent post-ischemic HF and to improve survival. Prompt revascularization by reperfusion (using primary coronary angioplasty or thrombolysis) has improved functional myocardial recovery and increased patient survival dramatically. However despite beneficial effects on ischemic lesions, reperfusion leads to ischemia-reperfusion (IR) injury due to abrupt restoration of blood flow and oxygen. Unfortunately, there is no treatment to specifically abolish IR-induced lesions leading to apoptosis of cardiomyocytes previously weakened by ischemia. Due to improved revascularization techniques, MI is a major provider of HF patients because decreased mortality results in increased morbidity leading to HF. Indeed, although most patients are doing well for the long term after reopening of the occluded artery, some patients undergo an adverse remodeling of the left ventricle leading to the chronic pathology of HF despite the implementation of secondary prevention therapies. A new concept has recently emerged: a large infarct is neither necessary nor sufficient for progressive adverse LV remodeling and HF to occur. Beside beneficial effects during cardiac healing, inflammation is a driver of post-MI remodeling leading to HF when overactivated for a longer time than required. We have demonstrated the crucial role of the apoptotic pathway during myocardial IR mediated by the FAS receptor, which binds to the FADD protein to trigger the caspase-dependent apoptotic cascade. FADD is also described as a mediator of inflammation and its inactivation in transgenic animal models prevents the appearance of HF. Our working hypothesis is to specifically target the FAS:FADD interaction activated during MI in order to develop an innovative therapeutics to decrease IR lesions and the ventricular remodeling leading to HF. Based on the long collaboration between both partners of the project, we have designed a specific inhibitor peptide (Tat-FADDp) of the FAS:FADD complex, which shows anti-apoptotic effect in vitro and a reduction in infarct size and apoptosis in an in vivo murine model of acute myocardial IR (EP2982685 A1). The HFADD project will confirm the cardioprotective effects of the patented peptide and evaluate its vasculoprotective and anti-inflammatory properties involved in the pathology of HF. More specifically, we plan to: (1) Investigate the anti-remodeling effect of Tat-FADDp in a mouse model of HF, (2) Develop second-generation Tat-FADDp, (3) And study in vivo the therapeutic time window, pharmacokinetics and biodistribution of the peptide. Final products will be well-characterized cardio- and vasculoprotective peptides able to limit infarct size and the pro-inflammatory phase that is over-activated during HF. This innovative strategy specifically targeting the FAS:FADD interaction involved in deleterious mechanisms such as apoptosis, inflammation, hypertrophy and fibrosis will enable the early inhibition of IR lesions and subsequent adverse LV remodeling, in order to reduce morbi-mortality and associated economic costs. The HFADD project will have a real strong impact by improving human health care, mortality and societal costs. Our valorization plan includes the filling of new patents and the creation of our own start-up with the help of the SATT AxLR valorization structure of the CNRS.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common adverse side effect of anticancer agents that seriously compromises the patient’s quality of life, limits dosage, and leads to changes in treatment to non-neurotoxic agents with the risk of limiting the effective clinical outcome. Among these compounds, oxaliplatin (used in the treatment of several solid tumors such as colorectal cancer) induces a quasi-systematic acute neurotoxicity which persists in more than 30% of survivors. The pathophysiology of sensory and motor symptoms induced by anticancer chemotherapeutic drugs is still poorly understood and no effective preventive or curative treatment is available. Hyperpolarization-activated cyclic nucleotide–gated (HCN) channels family is composed of four members (HCN1-4) widely distributed throughout pain pathways and playing an important role in the development and maintenance of neuropathic pain. Non-selective HCN blockers ae known to alleviate pain symptoms in CIPN rodent models but their potential cardiac side effects limit the clinical translation of their use in CIPN patients. Interestingly, both the function and membrane expression of HCN are tightly regulated by its auxiliary subunit, tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), which is not expressed in the heart, hence limiting the risk of cardiac side effects. Therefore, the recent development of molecules disrupting the TRIP8b-HCN interaction should provide new understanding regarding the involvement of these proteins in neuropathic pain signaling and propose the modulation of HCN function as a new innovating and better tolerated therapeutic analgesic strategy. The aims of this project are (i) to gain insight into the role played by TRIP8b-HCN interaction in the pathophysiology of CIPN, (ii) to demonstrate their pharmacological interest for the treatment of neuropathic pain using compounds with peripheral and/or central distribution specificity. To achieve these aims, experiments will be performed using in vivo rodent models of CIPN that will be challenged using TRIP8b-HCN interactions disrupting reference molecules as well as new and specific peptidomimetic compounds, coupled to up to date electrophysiological techniques and behavior studies. In this project we will (i) perform an extensive cellular and molecular characterization of TRIP8b-HCN interactions in peripheral, spinal and supraspinal neurons in CIPN; (ii) assess the functional and neurophysiological consequences of disrupting TRIP8b-HCN interactions in naïve and CIPN animals; (iii) evaluate the well-tolerated effect of this strategy. Altogether, the completion of this proposal will foster our understanding of the role of HCN channels and their protein partner TRIP8b in the pain pathways and critically help the development of innovative therapeutic strategies in pain treatment that remains an unmet medical need today. Specifically, the project will provide:-a detailed characterization of HCN channels and of their partner protein TRIP8b involvement in the sensori-discriminative and emotional neuronal circuits sustaining neuropathic pain-a validation of the disruption of HCN-Trip8b interaction as an innovative and well tolerated strategy to treat or prevent CIPN The outcome of our project and its therapeutic potential would have a great impact on improving the quality of life of patients suffering from chemotherapy-induced neuropathic pain and possibly their survival. This will consequently have important industrial and economic outcomes. Moreover, since HCN channels have also been involved in other neuropathic pain and in inflammatory pain, this project will pave the way toward new classes of analgesic drugs, active on chronic pain. .