| Nuclear transcription factors are critical regulators of gene expression, and their localization is primarily thought to be restricted to the nucleus and surrounding cell soma. With the advent of more sensitive detection assays, a small number of nuclear transcription factor proteins and mRNAs have been found in neuronal dendrites. This raises a number of interesting questions regarding the functional significance of such sub-cellular localization. This thesis investigates the localization and function of the nuclear transcription factor Elk-1 (E-26 like protein 1) in neuronal dendrites to specifically address whether (a) Elk-1 is a multifunctional protein with roles independent of direct transcriptional regulation in neurons, and/or whether (b) Elk-1 can move from dendrites into the nucleus to regulate nuclear gene transcription. Elk-1, originally classified as a proto-oncogene, is phosphorylated by multiple MAP kinase cascades and has been implicated in tumorigenesis, apoptosis, neuronal differentiation and synaptic plasticity.; Using immunocytochemistry and in situ hybridization, Elk-1 protein and mRNA were localized to proximal and distal dendrites of primary neurons. Using co-immunoprecipitation from mouse brain, we found a novel association of Elk-1 protein with proteins forming the mitochondrial permeability transition pore complex (PTP), a structure involved in both apoptotic and necrotic cell death. Electron microscopy in rat brain section confirmed an association of Elk-1 with mitochondria in both cell bodies and in dendrites, and DNA damage increased Elk-1's association with mitochondria in primary neurons. To more closely examine the function of Elk-1 specifically in dendrites, we developed the phototransfection technique to focally introduce mRNA into discrete sub-regions of live, intact neurons. Elk-1 mRNA introduced and translated in dendrites initiated cell death, whereas Elk-1 mRNA introduced and translated in cell bodies had no effect on cell viability. Consistent with the previously proposed dendritic imprinting hypothesis, additional data indicated that following introduction and translation in dendrites, Elk-1 traveled to the nucleus and induced a transcription-dependent cell death. These data highlight the multifunctional nature of the Elk-1 protein and show that Elk-1 synthesized in dendrites uniquely impacts neuron viability. |
