PROJECT SUMMARY/ABSTRACT The glymphatic-lymphatic system plays an essential role in immune surveillance and clearance of metabolic waste. Dysfunction of the system is closely associated with neurodegenerative disorders. While the existence of brain lymphatic vessels (mLVs) was proposed more than 200 years ago, it was only reliably confirmed with solid experimental evidence in 2015. Fluorescent imaging and gadolinium-based contrast agents (GBCA) have since been used to probe the brain’s glymphatic-lymphatic system in rodents and nonhuman primates. However, current strategies for imaging the glymphatic-lymphatic system have several limitations that preclude their use for in vivo imaging and application in the clinical setting. The vascular endothelial growth factor receptor 3 (VEGFR3), a tyrosine kinase receptor, is expressed exclusively by the lymphatic endothelium. Deletion of VEGFR3 in mouse pups led to a nearly complete lack of mLVs and compromised waste clearance. In addition, VEGFR3 may also contribute to clearing parenchymal wastes in the glymphatic system through the bidirectional water channel aquaporin-4. Positron emission tomography (PET) is a sensitive and noninvasive imaging modality (nM to pM) used to clinically assess metabolism, enzymes, receptors, and transporters at the molecular level. In response to the Notice of Special Interest (NOSI): Availability of Administrative Supplements to INBRE Awards to Fund Research Collaborations (NOT-GM-22-001), we assembled a team with the Delaware State University (DSU). We hypothesize that fluorine-18 labeled VEGFR3-specific radiotracers can be used for real- time PET imaging of the whole brain’s glymphatic-lymphatic system. To test our hypothesis, we propose two aims. In Aim 1, the Co-Project Leader from DSU will use their knowledge of biomolecules and biochemical pathways to identify a systematic biomarker relevant to whole-brain waste clearance and protein aggregation in neurodegenerative disorders. In Aim 2, the Co-Project Leader from Nemours will design and synthesize new imaging agents targeting the biomarkers with high binding affinity and selectivity and optimize the imaging agents in terms of stability, BBB penetration, easy radiolabeling, and metabolic profile. The innovative proposal and interdisciplinary collaboration will lay the foundation for further real-time live animal imaging in neurodegenerative disorders and clinical translation for new diagnostics and therapeutics development. The pilot project will lead to the discovery of new biomarkers in the CNS, improved imaging techniques, and ultimately, better diagnosis and treatment of CNS diseases. Furthermore, preliminary data from this INBRE supplement will inform an R01 application by targeting PAR-23-165.

PROJECT NARRATIVE The glymphatic-lymphatic system in the brain was recently discovered to play an essential role in immune surveillance and clearance of metabolic waste. Our proposed preclinical studies will provide preliminary evidence of biomarker validation and corresponding imaging findings in neurodegenerative diseases.