THE AXONAL TRANSCRIPTOME OF HUMAN EMBRYONIC STEM CELL DERIVED NEURONS AND THE CELL AUTONOMOUS TRANSCRIPTIONAL RESPONSE TO AXON INJURY Public Deposited

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  • March 22, 2019
Creator
  • Bigler, Rebecca
    • Affiliation: School of Medicine, Curriculum in Genetics and Molecular Biology
Abstract
  • During development and adulthood neurons rapidly react to isolated events sensed by and affecting the most distal portion of the neuron, the axon. Prompt local responses at axons to extracellular signaling molecules and mechanical contacts suggest limited immediate involvement of the neuron cell body. The delayed response of the cell bodies may include altered gene expression and mRNA trafficking, which may influence the availability of mRNA at distal axons. The primed axonal transcriptome is critical for the rapid and local responses influenced by local protein synthesis. To establish the presence of mRNA within human axons and quantify the axonal transcriptome I evaluated human embryonic stem cell derived neurons (hESC-neurons) grown in axon-isolating microfluidic chambers. The hESC-neuron axonal transcriptome was significantly different from the somatic transcriptome suggesting functions within the axons that depend on local translation. The enriched functional categories within the axonal transcriptome of hESC-neurons were similar to those of primary rodent neurons demonstrating conservation of translation-dependent axonal functions, while features unique to hESC-neurons are also present. This evidence supports the use of hESC-neurons as a model system to further investigate mechanisms establishing and modifying the human axonal transcriptome and the function of local translation within human axons. The axonal transcriptome encodes locally translated proteins required for axon injury signaling. Axon injury initiates a multi-phased injury response that begins locally at the injury site, is transmitted to the somata and eventually expands to other regions of the neural network. The structural and functional changes of the delayed injury response have been investigated in vivo but the molecular signaling of this response is poorly understood. I evaluated the effect of sparse direct axotomy to cultured primary rat neurons grown in two-compartment axon-isolating microfluidic chambers. Directly injured axons regenerated in vitro and axotomy did not affect culture viability but did induce a delayed, persistent synaptic function change in the in vitro neural network, consistent with previously reported in vivo findings. This functional change was dependent on a transcriptional response generated within the first hour after injury. Gene expression 24 hours after axotomy in this model system showed similarities to gene expression 24 hours after in vivo injury and suggested expression of proteins that function at the synapse might mediate the synaptic changes. Our in vitro axotomy model system demonstrates significant similarities to in vivo model systems but greatly enhances our ability to investigate the transcription-dependent mechanisms of injury signaling within neurons.
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  • In Copyright
Advisor
  • Miller, C. Ryan
  • Taylor, Anne
  • Crews, Stephen
  • Cohen, Todd
  • Sullivan, Patrick
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill
Graduation year
  • 2017
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