Transcription Factor Complex Formation in CNS Regeneration

In fish, central nervous system (CNS) neurons are able to regenerate after injury. Previous work has identified a neuronal protein, growth associated protein-43 or GAP43, which is involved in developmental and regenerative growth. Using zebrafish as a model organism, our work has focused on the elucidation of molecular pathways involved in the re-expression of GAP43 in response to CNS injury. GAP43 is expressed in both CNS and peripheral nervous system (PNS) neurons until the animal reaches adulthood. Afterwards, the re-expression of GAP43 is induced by neuronal injury, which then contributes to the ability of the damaged tissue to grow through the injured site leading to a full functional recovery. Previously we identified three regions within the larger gene regulatory region of the GAP43 gene that contain possible binding sites for five specific transcription factors, P53, ATF3, MASH1, STAT3, and c-Jun. More recently we verified that three of these five (c-Jun, MASH1, and ATF3) function as major factors in the functional regeneration of CNS axons. c-Jun and ATF3 both contain leucine-zipper dimerization domains, and have been shown to form heterodimers with each other in other contexts. Based on previous results in our lab, we have observed that knock-down (KD) of c-Jun and atf3 expression in regenerating neurons prevents the re-expression of GAP43 in response to optic nerve injury. Our hypothesis is that these proteins, once activated after injury, form heterodimers within the regenerating retinal ganglion cells (RGC), to activate the transcription of GAP-43. In order to test this hypothesis we will use co-immuno-precipitation assays, to test for the physical interaction of these proteins in the regenerating RGCs of zebrafish. We anticipate finding the presence of c-Jun/ATF3 dimers in regenerating RGC within the re-generational time-frame, post injury. Currently we are optimizing protocols for using the antibodies against mammalian c-Jun and ATF3 in immunopreciptation and Western blot assays using zebrafish tissues