Regulatory mechanisms of the Ca2+-dependent transcription factor NFAT in sensory neurons
Ca 2+ -mediated regulation of gene expression plays an important role in neuronal plasticity. NFAT (Nuclear Factor of Activated T-cells) is a Ca 2+ /calcineurin (CaN)dependent transcription factor that has been implicated in a number of neuronal functions including axon outgrowth, presynaptic remodeling and neural survival. NFAT is activated by Ca 2+ /CaN-dependent dephosphorylation, whereas re-phosphorylation by glycogen synthase kinase-3! (GSK3!) and several other protein kinases deactivates
... inases deactivates NFAT and triggers its export from the nucleus. In addition to electrically-mediated Ca 2+ signals, neurotrophins can potently regulate NFAT function in neurons as well. However the mechanisms of NFAT activation by electrical activity and neurotrophins are not completely understood. In aim 1, I found that electrical stimulation produced a mitochondrial Ca 2+ cycling-mediated prolonged [Ca 2+ ] i elevation (plateau), which profoundly affected NFAT activity. The elimination of the [Ca 2+ ] i plateau by blocking mitochondrial Ca 2+ uptake or release strongly reduced nuclear import of NFAT. Furthermore, preventing Ca 2+ mobilization from mitochondria diminished NFAT-mediated transcription. In aim 2, I found that NGF, a family of neurotrophins, potentiated NFAT-dependent transcription triggered by electrical activity through the TrkA-PI3K-Akt-GSK3! pathway and this effect was mediated primarily by NFATc3. Monitoring NFATc3 movement in DRG neurons in real time showed that NGF slowed the rate of NFATc3 nuclear export, which was mimicked by inhibiting GSK3!, whereas blockade of PI3K prevented this effect. Taken together, I proposed that mitochondrial Ca 2+ cycling functions as a novel regulatory mechanism for NFAT activation and NFATc3 serves as an integrator of electrical activity and neurotrophin signaling in the regulation of gene expression in DRG neurons. ___________________________________ Toshihiro Kitamoto ii To my family iii ACKNOWLEDGEMENTS Above all, I would like to thank my wonderful advisor Yuriy M. Usachev for his personal and professional counsel throughout my graduate school journey. Without his continuous guidance and patience with me, it would not be possible to overcome ups and downs during the dissertation process. Your tireless counsel will never be forgotten. I was also deeply indebted to my amazing thesis committee members, Donna Hammond, Steven Green, Stefan Strack and Toshihiro Kitamoto. Their feedbacks and comments on my projects provided me ideas during challenging periods of my research and ultimately helped improve the quality of the research.