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Post-transcriptional gene regulation in mature neurons

  • Author(s): Ho, Victoria
  • Advisor(s): Martin, Kelsey C
  • et al.
Abstract

Neurons are highly-polarized cells with processes that span great distances and many independent subcompartments. Despite their challenging morphology, neurons are able to respond to external stimuli in a local or cell-wide manner. They are also able to form stable connections with other neurons, while remaining plastic and adaptable to change. To meet these demands, neurons use complex, post-transcriptional mechanisms to regulate gene expression. Two approaches are taken in this dissertation to study post-transcriptional gene regulation in neurons.

First, a cell biological approach was used to study a specific interaction between the microRNA, miR-124, and GluA2 mRNA in dissociated hippocampal neurons. The subcellular localization pattern of miR-124 and GluA2 mRNA was determined by extracting RNA from synaptosome fractions and by direct visualization with fluorescence in situ hybridization. The effect of miR-124 overexpression on endogenous GluA2 protein was determined by immunoblotting and again by direct visualization with quantitative immunocytochemistry. These experiments reveal that miR-124 is dendritically-localized while GluA2 mRNA is somatically-restricted. Both RNAs are present in the cell body, where they interact to downregulate GluA2 proteins in the soma by ~30 %. Synaptic GluA2 protein was not affected by this manipulation, suggesting that post-translational regulatory mechanisms (e.g. trafficking) are in place to maintain appropriate concentrations of GluA2 at the synapse.

Second, a high-throughput sequencing approach was taken to identify general mechanisms of gene regulation during chemical long-term potentiation (chemLTP) in acute hippocampal slices, as well as to screen for interesting candidates for further investigation. RNA was extracted from chemLTP-treated and control slices, and changes in steady-state RNA levels were determined. This study is ongoing but so far, preliminary results suggest that non-neuronal cells may play an important role in the strengthening of neuronal connections. Future analysis will focus on 3' untranslated regions, which determine the post-transcriptional fate of genes.

The two approaches provide different levels of information on the regulation of gene expression in neurons and yield unexpected findings. Together, they illustrate the complexity of regulatory mechanisms in neurons, and that there is much we have left to understand.

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