STUDYING mRNA TRANSPORT AND REGULATION OF RETROGRADE INJURY SIGNALING ASSOCIATED AXONAL TRANSCRIPT LEVELS IN THE CONTEXT OF AXONAL REGENERATION

Abstract

This dissertation focuses on a detailed mechanism of mRNA transport during development of hippocampal neurons, and regulation of retrograde injury signaling (RIS)-associated genes in the context of regeneration. Damaged neurons in the CNS are unable to regenerate leading to neuronal degeneration and cell death. Identifying mechanisms that promote axonal regeneration of the damaged fibers is beneficial. First, I set to explore the detailed quantification of mRNA transport during the development of hippocampal neurons. Rigorous quantitative assessment of mRNA transport concluded that mRNA transport is driven by the functional demands of the cell. I measured the velocity, directionality and the duration of mRNA particles. In the axons, net velocity was highest at day 7 in vitro, which coincides with the initial stage of synapse formation. Within dendrites, it continues to increase through day 12 in vitro coinciding with an increased duration of synaptic contact, suggesting role of protein synthesis in context of sustained synaptic connectivity. Next, I set to explore regulations of genes involved in RIS process, a process stimulated upon injury and required for axonal regeneration. Investigation of regulation of RIS associated axonal transcript levels led to development of a whole hippocampal explant culture system. The hippocampal explant culture system enabled examination of axonal gene and protein expression independent of neuronal cell bodies. The study of RIS process suggests a novel biphasic increase in axonal gene expression (1 & 24 hrs post-injury). These genes are tightly and differentially regulated contributing to early synthesis of corresponding axonal proteins in hippocampal neurons. Additionally, importin β-dependent activity at the nucleus then appears to modulate a second wave (24 hrs) of RIS-associated transcripts, which are likely to further support axonal outgrowth. These studies provide insight into a powerful set of axonal processes that may be exploited to enhance CNS regeneration and repair.