The goal of our research is to understand how mitochondria are transported, positioned, maintained and replicated in nerve cells. Although neuronal organelle transport has been intensively studied using cell-free systems, and numerous candidates for the motors that drive organelle movement have been identified, we still know very little about how transport is controlled and coordinated in intact neurons. Mitochondria, more than any other neuronal organelles, compel our interest because both their functions and their motility patterns are complex and tightly regulated. Mitochondria not only produce ATP via oxidative phosphorylation, but also sequester Ca2+, produce the majority of the neuron's reactive oxygen species, and play a critical role in apoptosis. Thus, any disruption of their distribution within the axon can be expected to compromise the physiological homeostasis of the neuron. Altogether, these features make the mitochondria life cycle a focal point for understanding neuronal development, injury and neurodegenerative disease. Our studies will employ cultured chick embryonic neurons, which produce bona fide axons with active outgrowth and abundant mitochondrial transport. The first two aims of this study are: (1) to determine how mitochondrial movement and docking are regulated in axons; (2) to dissect the specific intracellular signaling pathways that direct mitochondrial transport and docking. We will pursue these goals using a newly developed procedure in which focal nerve growth factor stimulation along the axon shaft induces a profound change in mitochondrial transport and positioning.
The final aim of the study is: (3) to elucidate the spatial pattern of mitochondrial transmembrane potential, protein import, and DNA synthesis in neurons. In these experiments we will apply vital dyes and molecular probes to a ganglion culture system in which axons remain viable for hours after separation from their cell bodies. In support of experiments in all three aims, we have recently built avian retroviral vectors that deliver transgenes to the genome of cultured neurons so that proteins and functions of interest can be monitored directly in live cells by fluorescence microscopy. Together, the proposed studies should provide detailed insight into the mitochondrial life cycle in neurons. The information provided by these experiments in chick neurons may aid in understanding the molecular and cellular basis of human nerve injury and neurodegenerative diseases.
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