Organophosphorus compounds (OP), initially developed as plant protection products, are used for military purposes as nerve agents (NA) (Sarin, VX or Novitchok). They pose a threat to military and civilian populations. They cause irreversible inhibition of peripheral and central cholinesterases, blocking the breakdown of acetylcholine. The majority of neuroprotective research focuses on seizures induced by high doses of OP. The effects of lower, less symptomatic or even asymptomatic doses are little studied. However, very long-term persistent mood disorders are observed after a single exposure to an asymptomatic dose of OP in animals. Cognitive and neuropathic deficits have been observed in victims exposed to low doses. There is a crucial need for exposure biomarkers that allow i) early pre-symptomatic diagnosis and ii) evaluation and/or treatment of organophosphate and related compounds intoxications in the long term. To date, the neurotoxicity of OPs has been mainly addressed through the lens of neuronal and synaptic perturbations. The blood-brain barrier (BBB) represents the main exchange surface between the circulation and the brain tissue. Damage to the BBB and its role in neurotoxicity following OP intoxication remains poorly understood and constitutes an original diagnostic target and potential therapeutic strategy. The B-BOP project aims to study the integrity, transport and activation properties of the BBB following OP exposure and to evaluate them as a biomarker for the detection and prediction of brain damage. This will allow the evaluation of novel therapeutic strategies aimed at taking advantage of BBB damage, either by allowing the cerebral passage of antidote or by restoring the physiological properties of the BBB. We have chosen to focus on in vivo imaging methods in order to i) perform longitudinal studies and limit the number of animals needed for the project, ii) allow a 3-dimensional mapping of the effect of OPs in all brain regions, and iii) to inscribe this project in a translational approach with clinical diagnostic and/or theranostic perspectives in exposed patients. Moreover, the data obtained by in vivo imaging in animals will be confronted with histological data obtained post-mortem to allow a molecular interpretation. The comparison of the response intensity of the different biomarkers and their temporal course will allow to evaluate the hypothesis that there is a synchronicity between the regulation of the BBB and the neurological and behavioral consequences of intoxication. This will allow to evaluate the contribution of BBB perturbations to the neurotoxic mechanisms of action of OPs. The relative contribution of each biomarker will allow to establish their relevance for the detection, monitoring or prediction of the neurotoxic consequences of OPs in the long term. The longitudinal approach will also allow to define an optimal temporal window to estimate the degree of BBB damage from non-invasive imaging data in order to optimize therapeutic strategies. The work program is based on the synergy and expertise of 4 groups recognized in 1/ imaging of the integrity and transport properties of the BBB (CEA/SHFJ), 2/ imaging of the integrity and inflammatory activation of BBB endothelial cells (INSERM U1237-PhIND), 3/ the study of molecular interactions between BBB endothelial cells and glial cells (INSERM U1034) and 4/ the neurophysiological and behavioral study of OP intoxication and the validation of a mouse model of exposure to OP (IRBA).

Organophosphates (OPs) constitute a class of synthetic molecules still widely used as an insecticide but also as chemical weapons. This class has been the subject of numerous investigations by the chemical industry in search of pesticides that are more effective and potentially less harmful to the environment. In addition, the military authorities are interested in these molecules to counteract their effects. Indeed, a number of OPs pose serious environmental toxicity problems as well as the civilian or military populations targeted by attacks perpetrated with such compounds. Globally, there are around 250,000 fatal cases of OP poisoning per year. OPs interfere with the esterase activity essential for the degradation of certain active molecules in organisms. The most studied is acetylcholinesterase (AChE) which hydrolyzes acetylcholine. This neurotransmitter is essential in the cholinergic transmission of nerve impulses in the central (CNS) and peripheral (PNS) nervous systems, the failure of which leads to a major cholinergic syndrome which combines peripheral manifestations and epileptic seizures. In the event of accidental poisoning by an OP or during terrorist attacks, conventional emergency medical treatment consists of the injection of antidotes composed of pyridinium oxime for the reactivation of AChE and substances countering the effects of excess acetylcholine. However, pyridinium oxime remains ineffective in reactivating CNS AChE due to its poor passage through the blood brain barrier (BBB). The BHE-OP-Antidotes project aims to discover transient opening treatments for BBB or vector drug delivery systems to be used to facilitate the passage of BBB through the AChE reactivating molecules of the CNS. The project uses two complementary and validated in vivo models for this type of work, the zebrafish larva and the mouse. These two models are predictive for applications in humans given the very high evolutionary conservation of the biological processes studied. The zebrafish opens up the possibility of carrying out many combinations of experiments that will make it possible to amend the mouse model and whose results will be validated with the mammalian model. These models will be used to test the conditions for opening the BBB according to a genetic or pharmacological approach in association with a decrease in neurotoxicity induced by conventional pyridinium oximes following intoxication by OPs. The ability of vectorized biomimetic nanoparticles (NPs) loaded with conventional pyridinium oximes or new antidotes to prevent and treat the effects on the CNS of poisoning by OPs will be evaluated. For the two animal models used, the combination of biochemical, molecular and cellular studies, functional imaging, electroencephalographic studies and locomotor tests will be implemented to assess the restoration of the functional integrity of the CNS. The project mobilizes is carried out in collaboration between four teams which have internationally recognized expertise in chemistry, biochemistry, molecular biology, neurosciences, toxicology, pharmacology and the use of zebrafish and mouse animal models including the effects of OPs on the CNS and PNS, study of AChE reactivators, and study of BBB permeability: Pr. P. Babin, project coordinat or (Partner n ° 1, University of Bordeaux, INSERM U1211) (zebrafish model, OPs and antidotes, cholinergic and neuropathic effects), Dr. N. Soussi-Yanicostas (Partner n ° 2, NeuroDiderot INSERM U1141, Hôpital Robert Debré) (OPs and models of epilepsy in zebrafish), Dr. A.-G . Calas (Partner n ° 3, Toxicology & Chemical Risks Department, IRBA, Brétigny) (mouse model, OPs and antidotes, effects on the CNS and SNP) and Dr. C. Chapouly (Partner n ° 4, INSERM U1034, University de Bordeaux) (mouse model, in vitro and in vivo studies of BBB and its permeability).

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) which represents a major public health issue as current treatments do not prevent neurodegeneration. During MS, CD8+ T cells and T helper (Th1/Th17) CD4+ T cells actively participates to inflammatory CNS pathology. After crossing the blood brain barrier, T cells are trapped in the perivascular space where contact mediated signals with astrocytes regulate trafficking and immune-modulatory pathways critical to CNS neuro-inflammation. Literature shows that Notch signaling regulates T cell subset development, promoting autoimmunity in experimental autoimmune encephalomyelitis (EAE). Interestingly, we showed that the Notch ligand, Delta like 4 (Dll4), is upregulated in reactive astrocytes and sustains astrogliosis in EAE and MS. We now hypothesize that astrocytic Dll4 interacts with its receptors Notch1 and 2 on infiltrated T cells to control subset function during neuropathology. Aim1 and 2 test if reactive astrocytes use Dll4 to control perivascular and parenchymal immune cell function in acute and progressive phases of EAE and in MS. The project combines expertise of C. Chapouly in neurovascular biology with these of A. Astier and R. Liblau in immunology. The methodology relies on (1) mice knockout for Dll4 in astrocytes induced with EAE to study relapses and secondary progression, (2) co-cultures of human T cells (from constituted biobanks) with human astrocytes and (3) CNS tissues from person with MS versus non pathological CNS tissues. The project relies on cutting edge technologies such as FACS (17-color standardized panels), RNAseq and spatial transcriptomics combined with more traditional analysis. Overall, our project aims to understand how acute neuro-inflammatory changes prime the CNS for subsequent injury or repair in order to identify novel translational strategies for MS pathology.

Venous Thromboembolic Events (VTE), are serious, life-threatening conditions. VTE are estimated to be the cause of 12% of death per annum in European countries, and 10k-20k deaths in France. Clinical and sometimes biological risk factors of thrombosis can be sought, but in about 50% of patients no cause will be found, making it difficult to assess the risk of recurrence. Three major conditions referred to as Virchow's triad are well recognized to participate in the pathophysiology of VTE: (i) hypercoagulability, (ii) perturbation of blood flow, and (iii) endothelial dysfunction. To compensate the misunderstanding of the causes of thrombosis, different approaches have been developed to better assess hypercoagulability, the most important cause of recurrence. Thrombin generation tests (TGT) have been proposed to characterize the kinetics and amplitude of thrombin formation but there is currently no strong evidence that it can help in the diagnosis of patients with unexplained VTE. It is thus urgent to implement new strategies to better prevent and treat VTE. In this context, the pluridisciplinary consortium of Thrombosense will develop a new test that will enable probing hypercoagulability, in real time, using an endothelialized mimetic blood vessel, in flow conditions, thus satisfying the 3 parameters of the Virchow’s triad. The scientific objectives of the project are: (i) To develop 3-D endothelialized mimetic vessels, (ii) To develop a vein-on-a-chip platform for thrombosis. For this, optomechanical sensors will be co-integrated in the mimetic vessels and used as thromboelastography sensors, and (iii) To validate the platform with whole blood from normal controls and patients with prothrombotic conditions. The improved knowledge on thrombosis in Thrombosense will be used to propose a breakthrough, in-vitro diagnosis tool. Such a test would improve the assessment of hypercoagulability and, hence, prediction of recurrence after anticoagulant treatment is stopped.

Myeloproliferative disorders (MPD) are acquired haematological diseases often due to the acquisition of the activating JAK2V617F mutation in a hematopoietic stem cell, that leads to increased production of platelets, red cells and leukocytes. Thrombosis reveals MPD in about 30% of patients and is the main cause of morbidity and mortality. Understanding the mechanisms underlying the MPD thrombotic diathesis is therefore crucial to develop efficient anti thrombotic strategies. JAK2V617F red cells, platelets, neutrophils and endothelial cells (EC) were shown to participate in the pathogenesis of thrombosis in MPDs but, it is still unclear which of these actors is the most relevant and thus needs to be targeted. Our research hypothesis is that certain cell types are more important than others in the occurrence of thrombosis in JAK2V617F MPD. We will first quantify the JAK2V617F allele burden in each cell type in patients with and without thrombosis and compare this “clonality profile” with the occurrence of thrombosis and the type of thrombosis (arterial, venous, splanchnic thrombosis). Our second objective will be to generate mouse models with different “clonality profiles”, and analyze the occurrence of thrombosis using various mouse models of thrombosis (ie, stroke, inferior vena cava stenosis and cerebral venous thrombosis). Our third objective will be to test different therapies to find the most efficient ones to prevent thrombosis depending on the thrombus localization and the clonality profile of the mouse. The ACTOR project gathers one team specialized in MPD-associated thrombosis and one with an international expertise in platelet biology and mouse models of hemostasis and thrombosis. The project will use state-of-the art techniques and a bed to benchside strategy to identify the most relevant actors of thrombosis in JAK2V617F MPDs. It will allow the development of preclinical mouse models to precisely identify the best antithrombotic strategy