Inflammaging

The pro-inflammatory phenotype of neuroinflammation (NI) is a complex cellular and molecular process that contributes significantly to neurodegenerative and neuropsychiatric conditions. It is therefore essential to be able to measure it precisely to understand its mechanisms and develop new therapies targeting NI and monitor them in vivo. The only way to monitor NI in vivo is positron emission imaging (PET). To date, clinical PET imaging uses the translocator protein 18kDa (TSPO) as a biomarker of NI. However, TSPO is not specific to the pro-inflammatory phenotype of microglia because TSPO is also expressed in the anti-inflammatory phenotype and by activated astrocytes and endothelial cells. At the heart of pro-inflammatory processes is the inflammasome, induced in the microglia by many stimuli, such as neuronal death, or protein aggregates present in Alzheimer's disease (AD) or Parkinson's disease. The NLRP3 (NOD-like receptor family, pyrin domain containing 3) protein, an essential constituent of the inflammasome, and is therefore an ideal target for specific imaging of the pro-inflammatory phenotype of microglia. As noted by the referees and the evaluation committee, this area of research is extremely active and has produced since our Phase 1 project new and particularly interesting chemical structures targeting NLRP3, which we have integrated into this project. The iBraiN and ICOA laboratories have a long history of collaboration in the development of new tracers for PET imaging. Our pilot research has identified different chemical structures and possible structural modification pathways. Our pilot data show increased NLRP3 expression in the TgF344-AD model used in this project, confirming the published data. The objectives of the project are to: 1. Design and synthesize a library of NLRP3-targeting fluorinated ligands by in silico modelling based on the chemical structures of NLRP3 inhibitors 2. Identify and characterize fluorinated NLRP3 ligands in vitro by membrane binding assays 3. Characterize the 3 best candidates by in vivo PET imaging 4. Validate the best candidate in clinically relevant models of acute (stroke) and chronic (Alzheimer's) brain disease The project is structured in 6 Work Packages: • WP1: Project Management & Coordination • WP2: Design and synthesis of a library of fluorinated ligands targeting NLRP3. In silico prediction tools will be used to evaluate structural modulations of target affinity and BBB permeability for the design and synthesis of novel fluorinated compounds. • WP3: In vitro evaluation of new compounds In vitro screening to determine the affinity of new compounds for NLRP3. The best molecules will be tested for their ability to cross the BBB by mass spectrometry. • WP4: Synthesis of precursors and radiolabeling [18F] of the 3 best compounds identified in WP3. • WP5: In vivo evaluation of the top 3 candidates [18F] Characterization of the top 3 compounds by in vivo PET imaging in a model of LPS injection-induced neuroinflammation in the striatum of rats. • WP6: Validation of the lead compound [18F] in two clinically relevant animal models. Demonstration of the utility and robustness of NLRP3 PET imaging and our novel PET tracer in a model of acute (middle cerebral artery occlusion in rats) and chronic (Alzheimer's disease model TgF344-AD) neuroinflammation. The main expected result is a NLRP3 PET radiotracer fully characterized in vivo in animal models, patented, and rapidly transferable to clinical imaging locally but also via our many French and European collaborators.