Most neurons in your brain are irreplaceable and must function in their circuits for life. To maintain the brain health, stressed or damaged neurons must receive support from surrounding cells to increase their chances of survival. To communicate with each other, both neurons and non-neuronal cells called glia release molecules that communicate their health status and elicit appropriate responses. Remarkably, one of the communication strategies that glia and neurons use involves the transfer of entire cellular organelles called mitochondria whose primary job is to produce energy. While the transfer of healthy, intact mitochondria improves neuronal function, the transfer of faulty mitochondria is toxic. How glia and neurons coordinate requests for and deliveries of mitochondria to ensure brain health is an unanswered question but one of vital importance. This project will elucidate the triggers for mitochondrial transfer, the cellular machinery used in their release, and the uptake mechanisms that integrate mitochondria into recipient cells. In doing so, the research will provide a window into a key process that could in the future be exploited to improve brain function throughout life. To perform aspects of this project, undergraduate students will participate in a unique credit-bearing, team-based research experience focused on neuron-glia communication and using the fruit fly as a model system. Undergraduate students will also participate in an outreach event to local elementary and middle school students. Taken together, the project will reveal new modes of cellular communication that underlie brain health and will provide training opportunities for the future scientific workforce. This project will establish the mechanisms and regulation of mitochondrial trans-cellular transfer (mitoTCT) in vivo by deploying an innovative combinatorial strategy of genetics, 3D microscopy, and imaging flow cytometry (iFACS) in the Drosophila nervous system. Using dual binary expression systems, neuronal and glial membranes and mitochondria will be labeled with four colors, facilitating unambiguous identification of the sources and directions of mitoTCT. In Aim 1, the project will elucidate key regulators of mitoTCT rate in vivo. This aim will test the hypothesis that nerve injury and/or excess neuronal activity can trigger mitoTCT from glia to neurons. In Aim 2, the project will determine the mechanism(s) by which mitochondria are transferred between glia and neurons. Three proposed modes of transfer (tunneling nanotubes, extracellular vesicles, and release of free mitochondria) will be evaluated by inhibition of each mechanism in a cell type specific manner. In both aims, the health status of mitochondria will be evaluated using genetically encoded redox sensors, which will determine whether the mode of action is leading to rescue or toxicity of recipient cells. This research will advance fundamental knowledge of neuron-glia communication via mitochondria and will illuminate how we might harness mitochondrial transfer for the benefit of neuronal health. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.