The goal of this project is to determine the contribution of mitochondria to synaptic transmission between neurons in the mammalian brain. Mitochondria provide energy in all cells, and are important to support brain function. However, the large size and extended shapes of neurons represents a major challenge for these cells to maintain energy at distant sites of activity, especially at synapses where brain cells communicate with each other. At synapses, mitochondria are assumed to produce energy to support the release of neurotransmitter, but this remains untested. Knowing the mechanisms used to maintain cellular energy in neurons is essential to comprehend brain function and understand how these mechanisms break down in various disease states. This project aims to provide new, critical, and fundamental knowledge on the routes of energy supply in healthy neuronal synapses, and illustrate how synapses compensate for loss of mitochondrial function. These results will inform experiments to correct systems where mitochondrial function is impaired, such as in neurodegenerative disease and aging, providing a significant benefit to society. This information will be essential to better comprehend the impact of neurological disease states in an increasingly aging population. Additionally, undergraduates and graduate students will be trained in state-of-the-art techniques for cell and molecular biology, with a focus on neuroscience techniques, providing training opportunities for the next generation of researchers. In addition, this project will generate a novel card game-based learning tool to help teach fundamental mechanisms of synaptic function to primary and college-level students.
Recent reports suggest that glycolysis and ATP buffering can compensate for loss of mitochondrial function at the presynaptic terminal, even during bouts of activity. Thus, the prevailing view of mitochondrial function at the presynaptic terminal is inaccurate, requiring a fundamental re-evaluation of the role of presynaptic mitochondria. The research team will utilize the exceptional experimental accessibility of the calyx of Held synapse as a model to evaluate the role of presynaptic mitochondria. In the first objective, mitochondrial ATP synthase will be acutely inhibited, and synaptic transmission and ATP maintenance will be measured using electrophysiology and fluorescence-based ATP-imaging methods. The second objective will determine how presynaptic mitochondrial Ca2+-buffering shapes short-term synaptic plasticity, by eliminating the mitochondrial Ca2+ uniporter (MCU) from the presynaptic terminal using in vivo viral-mediated genetic deletion at the mouse calyx of Held. The effect of MCU elimination on mitochondrial localization, presynaptic cytosolic Ca2+ buffering, and synaptic plasticity will be measured using confocal microscopy, electrophysiology, and fluorescent Ca2+ imaging. The third objective will determine how presynaptically localized mitochondria fine-tune synaptic transmission. Viral expression of a dominant-negative TRAK2 peptide sabotages the interaction between mitochondria and kinesin motor proteins, and will be used to specifically impair axonal trafficking of mitochondria, depleting them form the presynaptic terminal of selected neuronal populations. Synaptic mitochondria will be visualized using confocal microscopy, and short-term plasticity of synaptic transmission will be evaluated using electrophysiology. Fluorescent imaging of ATP and Ca2+ will be used to evaluate the effect of mislocalized presynaptic mitochondria on transmission and synaptic maturation.
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.
