We propose to decipher the mechanisms of action of neuroendothelial N-methyl-D-aspartate receptors (NMDAR). In addition to neurons, where they drive glutamatergic neurotransmission, NMDAR are expressed in a variety of cell types. In particular, brain endothelial cells express NMDAR, which could be involved in blood-brain barrier maintenance and alteration. In a recent paper, we identified an unexpected population of NMDAR in endothelial cells, expressed at the luminal side of microvessels and located at the vicinity of blood/spinal cord barrier-forming tight junctions. We developed a monoclonal antibody (Glunomab®), directed against a specific site of NMDAR (aminoacids 176-180), which blocked the entry of leukocytes to the inflamed spinal cord, thus providing therapeutic effects in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS). Nevertheless, the downstream targets which link endothelial NMDAR function to leukocyte migration across the blood/brain and blood/spinal cord barriers are not fully understood yet. Interestingly, our current studies show that these receptors have an unconventional composition, including the presence of the rare GluN3 subunit, which provides response to glycine (in addition to glutamate) and metabotropic signaling (in addition to ionotropic function). Interestingly, naturally occurring circulating auto-antibodies against NMDAR are present in ~10% of human subjects and are overexpressed in neuropsychiatric and neurological diseases. Beyond these quantitative data, qualitative information are needed concerning the regions of NMDAR recognized by these antibodies. In fact, circulating autoantibodies against NMDAR could provide either beneficial or deleterious effects, depending on the region that they target. In line with this, we postulate that identifying the regions targeted by NMDAR auto-antibodies could have prognosis value in neurological diseases. Given our recent work concerning NMDAR in animal models of MS, we believe that investigating circulating antibodies would be particularly relevant for prognosis of MS. Thus the goals of this project are i) To characterize the signaling pathways and downstream targets triggered by NMDAR activation in endothelial cells, in relation to leukocyte penetration towards the spinal cord, ii) To identify the repertoire of NMDAR antibodies in MS patients (based on their target regions on NMDAR) and to determine whether different clusters of antibodies are associated with different outcomes, iii) To investigate the effects of these different clusters on the function of endothelial NMDAR and in animal models of MS, and iv) To bridge experimental data and clinical observations.

Ischemic stroke is a sudden neurological disorder which constitutes the third leading cause of death and the leading cause of acquired disability in adults in industrialized countries. The current treatment for the acute phase of ischemic stroke is to remove the blood clot obstructing the cerebral bloodflow by enzymatic degradation (thrombolysis) or by removing it mechanically through catheterization (thrombectomy). To guide clinical practitioners in their choice of treatment, magnetic resonance imaging (MRI) is essential. But MRI has its limits and does not allow the diagnosis of microthrombi, which are however partly responsible for post stroke sequelae. This project aims to (i) study the physiopathology of microthrombi in ischemic stroke, (ii) develop a contrast agent to reveal microthrombi on MRI and (iii) establish a treatment for micrthrombi. 1. Study of microvascular thrombosis in ischemic stroke The presence and impact of distal microthrombi will be studied in 2 models of ischemic stroke in mice. The first model we will use is thromboembolic, it is obtained by injection of thrombin into the middle cerebral artery (MCA). In the second model, cerebral ischemia is induced by occlusion of the MCA with a filament; removing the filament will allow us to reproduce the abrupt recanalization encountered in patients who benefited from thrombectomy. We will study precisely on histological sections the quantity, the stability over time and the composition of microthrombi in these 2 models. 2. Synthesis of a novel MRI contrast agent to reveal microthrombi Previous work in the PhIND laboratory has demonstrated the great potential of the molecular magnetic resonance imaging (MRI) strategy with microparticles of iron oxide (MPIO). Despite the promises of this strategy, the MPIOs used in these studies are composed of inert and toxic materials. The development of biodegradable and non-toxic MPIOs is therefore necessary to make this technology applicable to humans. In this project we propose to synthesize MPIOs from iron oxide nanoparticles assembled in a biodegradable matrix. We will use an emulsion coupled to a crosslinking process to cluster the iron oxide nanoparticles in the biodegradable matrix. We refer to these particles by the acronym PHysIOMIC. To optimize this synthesis and characterize the particles obtained, we will work with the organic chemistry department of the pharmaceutical research laboratory of Caen (CERMN). We will then use this novel MRI contrast agent to reveal the microthrombi present in ithe schemic stroke models using the molecular MRI technique. Preliminary results confirm that this method is effective in revealing the occlusive microthrombi present in the ischemic thromboembolic stroke model. In order to increase the specificity of our system, we will work on functionalizing the PHysIOMIC with antibodies specific to the platelets contained in the microthrombi. 3. Preclinical study of a thrombolytic therapy for the treatment of microthrombi We will test 3 different thrombolytic treatments that are known to be effective in degrading platelet and von Willebrand factor rich clots. We will test N-acetylcystein, the powerful thrombolytic effect of which has recently been demonstrated in the PhIND laboratory, and 2 treatments whose fibrinolytic activity is triggered by the presence of thrombin, which is generated in very large quantities by the activated platelets present in microthrombi.

The challenge of this project is the development of a multimodal treatment against thrombosis (stroke) using innovative theranostic inorganic nano-objects. Our approach is based on the design of biodegradable nanomaterials of core@shell type (iron oxide@porous silica with large pores) allowing a localized hyperthermia action, combined in a first strategy with the delivery of antithrombotic protein (tPA) and in a second strategy with a neuroprotective antioxidant effect. This project is structured on the judicious complementarity of a consortium bringing together : i) the nanoengineering of degradable mesoporous silica around activatable iron oxide cores (IPCMS, CNRS, Strasbourg) allowing magnetic hyperthermia and optimized protein loading/delivery from these core@shell nano-objects (size 50-100 nm); ii) the synthesis of small inorganic nanoparticles (size 2-10 nm) grafted or incorporated into the pores of the core@shell NPs, namely on the one hand quantum dots (QDs) making it possible to evaluate the local temperature of the core@shell (nanothermometry) and used as measurement tools and on the other hand platinum nanoparticles having an antioxidant effect ensuring the neutralization of radical species (ROS) deleterious to antithrombotic treatment (LPCNO, INSA, Toulouse) and iii) the biological evaluation of these systems on in vitro models of thrombosis obtained from human blood and ultimately of their thrombolytic action improved by magnetic hyperthermia on a mouse model of ischemic stroke (PhIND, INSERM, Caen).

Tertiary lymphoid structures (TLS) are ectopic lymphoid tissues that drive immune responses at sites of chronic inflammation. They are of widespread interest in biology and medicine since their presence dramatically influences disease course in autoimmune disorders, infection, and cancer. However, due to their small size and their development in chronically altered tissues, TLS cannot be selectively targeted or detected by imaging. The current project aims at developing new methods to map and eventually destroy the TLS in a non-invasive manner. To this aim, a new formulation of injectable superparamagnetic particles will be produced and targeted to the TLS. A set of innovations in contrast agent design and material chemistry will be developed to dramatically increase targeting efficiency compared to existing methods. By combining these innovations, intravenously injected superparamagnetic particles will accumulate in TLS in large numbers, thereby allowing their non-invasive detection by magnetic resonance imaging (MRI) and magnetic particle imaging (MPI). This will also open the possibility of thermal destruction of TLS using magnetic hyperthermia, providing a way to suppress a local immune response without affecting systemic immunity. Experimental models involving the formation of TLS will be performed in mice to demonstrate the applicability of the approach. The biomedical innovations of the ATILA project will have clinically relevant applications, such as diagnosis of chronic inflammation, prediction of the response to immune checkpoint inhibitors in cancer and spatially selective suppression of immunity in autoimmune diseases.

The goal of this project is to develop and characterize an innovative combined thrombolytic and anticoagulant approach for ischemic stroke. Ischemic stroke is a neurological deficit resulting from an insufficient blood supply to the brain due to the presence of a clot in a cerebral artery. In France, stroke is the 3rd leading cause of mortality (first in women) and the 1st cause of disability in adults, and it is estimated that there are 85,000 new cases of ischemic stroke per year, with a 20% increase in the upcoming years. The current standard of care of patients presenting an ischemic stroke is thrombectomy combined or not with thrombolysis. Thrombectomy is an endovascular procedure consisting in mechanically removing the clot with a stent retriever in a large occlusion of a cerebral artery. Thrombolysis, is the only pharmacological approach used in stroke patients to dissolve the clot. rtPA, which is the main clinically used pharmacological agent for thrombolysis, will activate plasminogen and convert it into plasmin that cleaves fibrin within the clot to destabilize it and restore the blood flow. Even though thrombolysis is the gold standard, it has many limitations. Therefore, there is an unmet medical need for more efficient therapeutic strategies to promote recanalization of cerebrovascular arteries with less harm to the patient. The main objective of this project is to develop and characterize new innovative thrombolytic strategies by specifically targeting an intravascular thrombus. The agents we developed will be characterized in in vitro and in vivo models of arterial thrombosis to evaluate their ability to bind a thrombus, to inhibit thrombin and to promote fibrin lysis. The thrombolytic potential will be then assessed in relevant mouse models of ischemic stroke and efficient doses will be determined. We will also determine whether these doses reduce the risk of bleeding as compared to currently used therapeutic agents in mouse models.