ENGINEERING VASOACTIVE PROTEASE SENSORS FOR BRAIN-WIDE BIOMARKER IMAGING

Proteases such as fibroblast activation protein (FAP) and matrix metalloprotease-9 (MMP9) play crucial roles in neural plasticity, network remodeling, blood–brain barrier regulation, neurodegeneration and tumors. Yet their precise spatiotemporal molecular activity remains difficult to measure. To address this challenge, we are developing genetically encodable vasoactive sensors that will enable brain-wide mapping of protease activity in living mammals. The sensors consist of a blocking domain, a protease-specific cleavage site, and a vasoprobe. Proteolytic cleavage releases the vasoprobe, triggering receptor-mediated vasodilation causing hemodynamic signals detectable by fMRI (Fig1.A). We screened multiple FAP-sensitive sensor designs in vitro to assess activity and specificity, followed by in vivo testing in rodents. In vitro assays showed concentration-dependent activation of the FAP sensor with nanomolar EC₅₀ value (Fig1.C) and high specificity for FAP (Fig1.D). To facilitate widespread and sustained sensor expression, we further used adeno-associated viral (AAV) vectors to deliver the genetically encoded sensors into the caudate putamen. Four weeks post-injection, rats expressing the FAP sensor AAV (FAP AAV) showed robust fMRI signal changes upon stimulation with FAP compared to controls, with signals coinciding with AAV-mediated sensor expression confirmed by histology (Fig1.E, F). Based on this FAP sensor, we also developed a functional genetically encodable MMP9 sensor, demonstrating the versatility of this approach (Fig1.G). This work represents the first genetically encoded protease sensors compatible with hemodynamic imaging and fMRI. Prospectively, these sensors enable minimally invasive, brain-wide imaging of enzymatic activity relevant to neural plasticity, network remodeling, and disease.