MDM2-P53 ANTAGONISTS IN AXONAL REGENERATION - DESCRIPTION (provided by applicant): Axonal and neuronal injuries are relevant to human pathogenesis of several disorders including brain, spinal cord, or peripheral nerve traumatic injuries and neurodegenerative diseases. Promotion of axonal regeneration is essential for functional recovery in these diseases. Mechanisms of regeneration following axonal injury and transection are largely regulated by gene expression changes, which are characterized by two pathogenic phases: a first wave is characterized by an inflammatory reaction, axonal degeneration and cell death, and a second long lasting one (starting several days after injury) is exemplified by pro-plasticity and pro-regenerative changes, along with reactive gliosis. In both phases, transcription factors play an important role as upstream regulators of the wide gene expression response. Strategies aimed to modulate the activity of key transcription factors could prove useful for nerve regeneration, as they would influence a complex biological response. We matched gene expression analysis with the search for specific pro- plasticity transcription factor targets combining microarrays temporal gene expression examination following Spinal Cord Injury (SCI) with bioinformatics analysis for transcription factors binding sites (TBS). We identified common TBS for the transcription factor p53 on select novel and established pro-plasticity and pro- regeneration genes, induced from one to four weeks after SCI, which include GAP-43, Coronin 1b, and Rab13. Subsequent in vitro and in vivo experiments have established that p53 a) drives expression of these genes; b) is required for neuronal differentiation; c) is required for physiological nerve regeneration. P53 protein is not only highly unstable, but also its transcriptional activity is highly regulated at the protein level. The major inhibitor of p53 transcriptional activity and protein stability is the ubiquitin ligase MDM2. In the present proposal, we hypothesize that stabilization and increase of p53 transcriptional activity using drug inhibitors of MDM2-p53 interaction (Nutlins) would amplify the p53 dependent pro-plasticity, and regenerative molecular response after injury, and therefore promote axonal regeneration and functional recovery, he specific aims are to demonstrate the therapeutic potential of the inhibition of MDM2-p53 interaction using Nutlin-3 in a mouse model of facial and sciatic nerve transection including the use of p53 null and WT mice. Specifically: 1) study nerve regeneration in WT and p53 null mice to address both the effect of the drug on regeneration in WT animals, and the specificity of the mechanism using p53 null mice, which should not respond to the administration of the drug; 2) evaluate the amount of cell loss and reactive gliosis in WT mice, as p53 transcriptional activity is pleiotropic and its enhancement can affect plasticity as well as cell death and proliferation. This experimental therapeutic approach could set the ground for a future clinical application to boost nerve regeneration in neurodegenerative disorders.
