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Date of Award

Spring 5-15-2012

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Neurosciences)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

The growth of axons is an intricately regulated process involving intracellular signaling cascades and gene transcription. Studies thus far have elucidated the various extracellular cues and intracellular cellular signaling cascades that play critical roles in neurite initiation and axon outgrowth. However, very little is known about the identities of transcription factors that are important for neurite formation and axon growth. Serum response factor (SRF) is a stimulus-dependent transcription factor highly enriched in the brain. SRF deletion in adult neurons results in severe deficits in activity-dependent expression of several immediate early genes, synaptic plasticity and learning and memory. The role of SRF in neuronal development remains poorly studied. We found that deletion of SRF in the developing nervous system or developing neurons results in highly attenuated axonal growth both in vivo and in vitro. To understand the mechanisms by which SRF regulates axon growth, we initially undertook a bioinformatics approach to identify post-translational modifications that are critical for SRF transcriptional activity. From this in silico analysis, we identified a highly conserved serine residue (Ser224) as a major phosphorylation site for glycogen synthase kinase-3 (Figure 13) (GSK-3). GSK-3 is one of the major intracellular signal-integrating molecules for regulating cellular responses to extracellular cues. Recent studies have described a critical role for GSK-3 in axon growth. Currently, the widely-studied mechanism by which glycogen synthase kinase-3 (GSK-3) regulates axon growth is through regulation of local microtubule dynamics. We found that SRF and GSK-3 interact in the brain and that GSK-3 efficiently phosphorylates SRF both in vitro and in cells only on the predicted ser224 residue. We further found that SRF-Ser224 phosphorylation is necessary for the formation of a functional SRF-MKL transcriptional complex. Axonal growth deficits caused by GSK-3 inhibition can be rescued by SRF both in vitro and in ex vivo organotypic brain slices. Using high-density microarray analysis, we identified Vinculin (Vcl) and microtubule-associated protein 1B (MAP1B) as SRF target genes that were capable of promoting axon growth in SRF-deficient neurons. However, Vcl expression alone was able to rescue the enlarged growth cone and cell soma morphologies exhibited by SRF-deficient neurons. Furthermore, shRNA knockdown of Vcl attenuated axon growth in cultured hippocampal neurons while Vcl expression could partially rescue axon growth of GSK-3 inhibited neurons. Together, our findings demonstrate that GSK-3-SRF together constitute a novel signaling pathway critical for axon growth in developing central nervous system.

Language

English (en)

Chair and Committee

Narendrakumar Ramanan

Committee Members

Paul Bridgman, Valeria Cavalli, Tim Holy, Steve Mannerick, Karen O'Malley

Comments

Permanent URL: https://doi.org/10.7936/K75X26VJ

Available for download on Sunday, May 15, 2112

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