Abstract Background: Following surgical resection for intractable epilepsy caused by malformations of cortical development (MCDs), over 30% of children continue to have life-changing seizures. Mutations in genes of the mechanistic target of rapamycin (mTOR) pathway lead to these disorders that include focal cortical dysplasia (FCD), tuberous sclerosis complex and hemimegalencephaly. Therefore, there is a critical need to discover the specific mechanisms by which increased mTOR signaling leads to the development of epilepsy to develop specific therapies for children with MCDs. The central hypothesis of this proposal is that mTOR pathway upregulation in pyramidal cells causes axon overgrowth, creating an epileptogenic network in both mouse and human MCDs Methods: The first step in testing the central hypothesis will be to determine the effect of mTOR pathway upregulation on axonal length in mouse FCD and human MCDs via quantifiable analysis of fluorescently labeled murine axons of and by 3D reconstruction of individually filled human neurons. A second set of experiments will be performed to determine whether some pyramidal neurons act as operational hub cells in human and mouse MCDs. Calcium imaging on ex vivo brain slices of mouse and human tissue will be used to define the local network connectivity and identify hub cells. Finally, axonal degeneration will be induced in vivo in dysplastic cells in mouse FCD to determine the effect of targeted reduction in axon length on seizure frequency. Implications: The proposed research will help to determine whether axonal overgrowth is a key mechanism by which mTOR upregulation leads to the generation of pharmacoresistant epilepsy in children with MCDs. Additionally, this project will demonstrate whether targeting axonal overgrowth offers the potential for treatment of seizures. Thus, the results of this research will provide further biological understanding of MCD and provide hope for rational development of future successful therapies for children with this set of disorders. Career Development Plan: During the five years of this award, Dr. Alexander will work with a team of mentors to achieve six specific career goals which allow her to accomplish this project and will also cement her transition to independent investigator. With hands-on mentorship and a focused set of coursework, she will work towards three training goals. These are as follows: 1) to obtain training and mentorship in the principles and techniques of molecular neuroscience; 2) to obtain mentorship and training in neuronal network analysis using calcium imaging; and 3) to obtain training and mentorship in laboratory management as a neurosurgeon- scientist. Dr. Alexander is a pediatric neurosurgeon at Children's Hospital Colorado. Her chairman and division chief continue to support 50% protected time through on-campus resources which include dedicated research space, a rodent EEG core, shared equipment and a startup package for laborato...