Comparative analysis of the molecular pathways involved in spinal cor injury in axolotls vs. mammaals

University of Minnesota Ph.D. dissertation. April 2015. Major: Molecular, Cellular, Developmental Biology and Genetics. Advisor: Karen Echeverri. 1 computer file (PDF); 196 pages.Spinal cord injuries (SCI) in mammals are major causes of physical disabilities. In contrast the Mexican salamander (axolotl) possesses amazing regenerative capabilities and can regenerate a fully functional spinal cord after injury. We have attempted to identify the molecular determinants underlying this evolutionary divergence by undertaking a detailed comparative analysis of a regenerative model, the axolotl spinal cord, and a corresponding non-regenerative system, rat spinal cord after injury. This approach identified a small number of highly conserved microRNAs that are differentially regulated in axolotl versus. Detailed in vivo studies of one of these microRNAs, miR-125b that is highly expressed in axolotl but low in rat has identified it as a key regulator of the regenerative response in axolotl. We also found that upregulation of miR-125b after injury improves SC repair in rats. In addition, we have identified SEMA4D as a target gene regulated by this microRNA after SCI in both axolotl and rat. We also studied how this miRNA is regulated in the axolotl to promote regeneration after SCI. We found that in the absence of injury, actin cytoskeleton de-polymerization induced by Cytochalasin D (Cyto D) induced a decrease in miR-125b expression similar to the decrease induced by injury in axolotl. Analyses of the regulatory region of the miR-125b axolotl gene revealed predicted binding sites for c-Fos. When we performed SCI in axolotls, we observed an increase in the expression of c-Fos 1 day after injury. As expected, we found that in the absence of injury, treatment with Cyto D induced similar changes in c-Fos expression similar to injury. Lastly, we proposed the development of a 3D in vitro co-culture model system to study at the cellular and molecular level how miR-125b SCI regulates the response to injury in rats and whether miR-125b expression is regulated by biomechanical activation of the Rho-c-Fos pathway. The proposed model will allow better imaging, better spatial and temporal control over modulating microRNA and gene expression in different cells at different time points. Our overall data suggest that dynamic changes in the actin cytoskeleton after injury induces changes in miR-125b expression, possibly through activation of cFos in the RhoA pathway, to create a permissive environment for regeneration in axolotls