Abstract:
It was a long held belief that adult mammalian central nervous is unable to regenerate
in any condition and reiterated in Ramon y Cajal’s seminal work (Santiago Ramón y Cajal, 1991). This idea was disproved when injured CNS axons were able to regenerate in PNS lesion environment and embryonic grafts shedding light on the reasons contributing to CNS regeneration failure (Richardson et al., 1980, Aguayo et al., 1981). Regeneration in the CNS is inhibited by myelin and astrocyte based inhibitors along with the presence of an inhibitory transcriptional environment, elicited and/or enhanced by the cascades induced by injury (Yiu and He, 2006). Extensive research has identified transcription factors and proteins which when modulated enhance regeneration of the injured adult CNS axons (Liu et al., 2011). Traditional approaches to promote a permissive molecular environment in neurons have provided crucial leads but not therapeutic options. Hence, novel approaches and targets need to be identified by studying molecules involved in developmental processes like neurogenesis, axon path-finding and neuronal morphogenesis. Ubiquitin ligases andubiquitin ligase like proteins have been identified to play a role in neuronal morphogenesis, connectivity and degeneration after injury (Yamada et al., 2013). MDM4, a ubiquitin ligase
like enzyme, has p53 as its prime substrate and interacts also with molecules like PTEN, Smads, p21, previously implicated in regeneration(Toledo and Wahl, 2006, Eva et al., 2012). MDM4 occludes the transcriptional activation domain of p53 limiting its transactivation while another E3 ubiquitin ligase MDM2 reduces the level and hence the activity of p53 (Marine, 2011, Marine and Jochemsen, 2004). In this study, we have investigated the effect of modulating novel factors MDM2 and MDM4 on CNS regeneration using optic nerve crush as an injury model. Genetic ablation of
MDM4 and pharmacological inhibition of MDM2 in retinal ganglion cells induced regeneration of optic axons, without substantially affecting neuronal survival. Genome wide gene expression analysis from FACS sorted pure RGCs revealed up-regulation of IGF1R gene and its role was confirmed by its specific pharmacological inhibition. Hence this study represents MDM2-MDM4-p53-IGF1R as a neuronal signaling pathway that might present novel therapeutic targets for neuro-trauma patients.
Along with identifying the role of p53 and its negative regulators MDM2 and MDM4 in
regeneration, we also studied the role of histone acetyl transferases P/CAF and p300 which are known to be epigenetic modulators in neurons (as collaboration between colleagues at the same lab). Expression of p300, which acetylates specific lysine residues of p53 and histone H3, was decreases in RGCs upon maturation and hence was a potential valid target. Viral overexpression of p300 in RGCs enhanced regeneration after optic nerve crush coupled with boosting the pre-conditioning effect of lens injury. The pre-conditioning lesion primes the neurons to enter a regenerative state and enables the axons to overcome the inhibitory extrinsic environment. Pre-conditioning lesion effect can be induced in the spinal system (i.e in the dorsal root ganglia) by lesioning the peripheral axons which permits the regeneration of their central branches in the CNS. Regenerative effect of the conditioning lesion is elicited due to the expression of regeneration associated genes (RAGs), but the mechanism controlling their expression remains unknown. Here, we were able to clarify a unique role of p300/ CBP associated Factor (PCAF) following conditioning lesion. PCAF dependent acetylation at histone H3 lysine 9 (H3K9) along with a reduction in methylation of
H3K9 (H3K9me2), was observed at the promoters of RAGs exclusively after PNS axonal
injury. PCAF dependent acetylation of theses promoters increased RAGs expression, which was mediated by extracellular signal regulated kinase (ERK) axonal retrograde signaling. Hence we have established a unifying role for PCAF as a broad regulator for regeneration, following a conditioning lesion. Viral PCAF overexpression also promoted axonal regeneration after CNS injury in spinal ascending sensory fibers, though such an effect was not observed in the ONC system, owing mainly due lower PCAF expression levels observed.
To conclude, in this study we were able to identify novel ubiquitin ligases, MDM4 and
MDM2 which when deleted promote regeneration in the adult CNS. Additionally
9 overexpression of epigenetic modulators p300 and P/CAF was found to induce regeneration in the CNS. Development and validation of drugs that can specifically modify the activity of these targets can present novel therapeutic options.
