Mechanism and function of retrograde mitochondrial transport in axons

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Project Summary Mitochondria are essential for cellular function and organism viability. These organelles are well known for their production of ATP, the primary energy currency of most eukaryotic cells. Less well known are the plethora of other functions these organelles have including production of signaling molecules, regulation of apoptotic signaling cascades, serving as a calcium sink, and also being the primary storage and utilization site of iron in the cell. To serve these diverse functions, mitochondria must be properly localized in all cells; however, this organelle is particularly critical in neurons. Neurons are highly metabolically active, electrically polarized, and can have an enormous volume making regulation of the mitochondrial population particularly challenging. Likely due to the high metabolic demands of this cell, precise control of mitochondrial localization and maintenance of mitochondrial health are essential for neuronal survival. Abnormal mitochondrial localization, health, and function have been linked to many neurodegenerative diseases including Alzheimer’s disease. In Alzheimer’s, defects in mitochondrial calcium load and contacts with the endoplasmic reticulum have both been noted. Additionally, advanced neuroimaging of early-stage patients revealed defects in mitochondrial function, making understanding how mitochondrial function is maintained in neurons paramount to understanding disease biology. While the last several decades have revealed fascinating insights into mitochondrial biology in neurons, we still do not have a thorough understanding of how the population of mitochondria is maintained over the long life of the neuron. Anterograde transport is critical for bringing healthy organelles from the cell body into the long axonal process which can extend a meter from the cell body in humans. Conversely, retrograde transport moves aged or damaged organelles towards to cell body. Once damaged organelles reach the cell body, some undergo targeted degradation. The fate of the bulk of these organelles and the source of healthy mitochondria has not been defined. We have developed an in vivo system to address these long-standing questions in the field. Using zebrafish neurons, we can image mitochondrial localization, health, and transport in vivo in a fully intact neural circuit. We have developed transgenic lines, genetic tools, and imaging approaches to individually label mitochondria to track them and follow their lifetime and biogenesis in neurons. This will allow us to determine the source of healthy mitochondria necessary for maintenance of the population in neurons (Aim 1). Independently, we designed a strategy to define the mechanism of motor-mitochondria attachment specifically necessary for retrograde transport of the organelle (Aim 2). Together, the proposed experiments will provide mechanistic insight into how and why mitochondria move in the retrograde direction while also defining the source of healthy organelles necessary for maintenance of the mitochondrial population in neurons. The knowledge gained will enhance our insight into the basic biology of the cell that can be repurposed for potential therapeutic interventions.