(607c) Calamri: Calcium Activated MRI Reporters for Imaging Neuroactivity

Calcium is a pivotal signaling molecule with critical roles in regulating several neurological functions including learning, memory, stress, and addiction. For this reason, genetically encodable calcium-responsive fluorescent proteins have found extensive use for studying calcium dynamics in cultured cells using fluorescence microscopy. Unfortunately, visible light penetrates tissues poorly beyond ~1 mm – thus, fluorescent reporters are of limited utility for studying calcium activity deep inside the living brain. Unlike fluorescence imaging, magnetic resonance imaging (MRI) uses tissue penetrant radiofrequency photons to safely and noninvasively image intact organisms. However, lack of MRI-compatible genetic reporters for detecting cellular calcium has hindered the use of MRI for studying brain-wide calcium activity in live animals. To bridge this critical gap, we have characterized and engineered a naturally evolved calcium-sensing protein to develop the first genetically encodable Calcium activated MRI sensor – to be known as CalaMRI. This protein possesses a unique ability to bind paramagnetic Mn(II) ions exclusively in the presence of calcium and based on these observations, we hypothesized that this calcium-induced paramagnetic switching provides an operational mechanism for emitting MR signals in the presence of calcium. For this purpose, we firstly expressed this protein in E. coli and purified it. We measured MRI signal strength of wild type CalaMRI in vitro in the absence and presence of saturating amounts of calcium and observed ~5-fold change in contrast-to-noise ratio. We also tested dynamic range of the sensor and showed that CalaMRI can detect calcium in physiologically relevant concentrations. Our current efforts are focused on both optimization of sensor performance using directed evolution and testing the performance of CalaMRI in Chinese hamster ovary (CHO) cells before we move to proof-of-concept assessment in living animals. The most recent results, including in vitro, cellular, and in vivo performance of CalaMRI, will be presented. These promising results will provide a transformative capability to medical research for connecting molecular-scale cellular activity with behavior, neurological function, and disease in animal models.