Imaging astrocytic ATP:ADP in vitro and in vivo using PercevalHR (RPA Pilot / Seed Project)

Our short-term goal is to obtain preliminary data for a larger grant proposal (P01) focusing on the impact of astrocyte metabolism on the control of vascular tone in the brain, and particularly, in the somatosensory cortex (S1) of mice. Our long-term goal is to study the largely underappreciated impact of astroglial cells on neuronal network activity in vivo, and in the context of disease states such as Alzheimer’s disease (AD) and type 2 diabetes (T2D), as well as in aging. The preliminary data presented shows the use of astrocyte/ neuron co-cultures to characterize a new tool for measuring ATP-to-ADP ratios (fluoresescent nanosensor PercevalHR) in response to glucose changes; should an award be made, experiments will expand to in vivo studies of associated changes during neuronal activation. PercevalHR was developed at Harvard in the lab of Dr. Gary Yellen, to measures bioenergetics changes in the cytosol of multiple cell types [1]. We have obtained the plasmid for this sensor and have recently packaged the construct into a lentivirus equipped with a non-specific neuron/ astrocyte promoter (CMV). The data obtained will provide dynamic ATP and ADP measures in neurons and astrocytes first in culture, then in vivo and will develop a strong dataset for presentation of preliminary data in our planned P01 submission.