? ? Malformations of brain development are increasingly recognized as a common cause of epilepsy, mental retardation and cerebral palsy, major factors in neurological disease. The doublecortin (OCX) gene is critical for neuronal development in humans, as mutations result in lissencephaly in males and double cortex in females, producing severe neurocognitive deficits. The Gleeson Lab identified its role as a microtubule (MT)-associated protein and its involvement in several signaling pathways through phosphorylation dependent mechanisms. My work has established that Dcx is part of a gene family containing Dclk and Dck2, each encoding a brain-expressed protein with DCX and serine-threonine kinase domain. In this application, I hypothesize genetic functional redundancy and a requirement for kinase function in brain. This application is focused on helping me achieve research independence through a rigorous scientific training period aimed at completing a full repertoire of research skills needed for future success as an Principal Investigator at a major US research institute. The scientific goals are to elucidate the molecular and cellular mechanisms of the Dcx gene family in brain development and function through the study of loss of- function alleles and identification of kinase targets. The career development goals are to provide a rigorous scientific training period with which to complement already in-depth research skills by supplementing these with advanced microscopy and proteomics and further development of a network of research collaborations. ? Aim 1. Test the degree of functional redundancy of Dcx homologues Dclk and Dck2 in multiple stages of brain development (Mentored phase). The function of Dcx, Dclk and Dck2 are at least partially redundant in mouse in neuronal proliferation, migration and axonal growth. I will analyze the degree of functional redundancy through the analysis of phenotype of Dcx, Dclk and Dck2 single, double and triple knockout mice and compare these results with siRNA-mediated gene knockdown approaches.
Aim 2. Test for involvement of kinase activity and substrates in the Dcx family gene pathway (Independent Phase). My preliminary data indicates that DCLK and DCK2 are potent brain-expressed kinases, about which previous data has indicated strong negative-regulation of DCX function through phosphorylation. I will test the requirement of kinase function through analysis of genetically modified mutants I have already created and will create. I will apply novel chemical genetics techniques combined with proteomics and genomics to identify direct kinase substrates and transcriptional targets. I will create novel knock-in mice that will allow for temporal specific regulation of kinase activity to provide the first data on the function of the kinase domain of the DCX family. ? ? ?
| Koizumi, Hiroyuki; Fujioka, Hiromi; Togashi, Kazuya et al. (2017) DCLK1 phosphorylates the microtubule-associated protein MAP7D1 to promote axon elongation in cortical neurons. Dev Neurobiol 77:493-510 |
