Characterization of the role of ATF3 in nerve regeneration

It has long been known that axons of the central nervous system of higher vertebrates have a limited capacity to regenerate themselves after trauma or re-innervate their target compared to the axons of the peripheral nervous system. Conversely, lower vertebrates (fish, amphibians) retain striking central and peripheral nervous systems regeneration capabilities. This may be because in adulthood of higher vertebrates, many genes are developmentally silenced, while in lower vertebrates, these genes remain transcriptionally active during their lifespan. Proteins are upregulated during axonal regeneration, which are called growth associated proteins. In zebrafish, regeneration induced CNPase homolog, is a protein that is highly upregulated during optic nerve regeneration. This protein shares significant homology with the mammalian 2', 3'-cyclic-nucleotide 3'-phosphodiesterases (CNPases). CNPases are well-established mammalian myelin markers, suggesting a role in the functioning of the nervous system. Activating transcription factor 3 (ATF3) has been shown to be an important mediator of nerve regeneration. It belongs to the ATF/c-AMP responsive element binding (CREB) family of transcription factors. Members of this family bind to ATF/CRE promoter regulatory elements and thereby control specific target genes whose products regulate functions in the immediate response to injury. In the case of neurons, the metabolic machinery is primarily turned to survival and regeneration by expression of these specific target genes. In intact neurons, ATF3 expression is very low or does not occur but its expression is rapidly upregulated in response to neuronal stress or injury. To improve upon the techniques previously used to detect protein expression and for morphometric analysis, a RFP-zRICH(H334A) fusion protein was used to detect protein expression in differentiated neurons under fluorescence microscopy. ImageJNeuronJ software was used to efficiently detect effects on neuritogenesis in neurons expressing this fusion protein. Another goal of this study was to assess whether ATF3 plays a role in nerve regeneration. With this purpose, stable transfectant pools of the PC12 cell line overexpressing ATF3 fused to the marker protein green fluorescent protein were established. The ability of ATF3 to affect neuritogenesis of PC12 cells in response to nerve growth factor was determined by conducting morphometric assays with these stable transfectant pools using ImageJ software