Overexpression of ATF3 or the combination of ATF3, c-Jun, STAT3 and Smad1 promotes regeneration of the central axon branch of sensory neurons but without synergistic effects

Abstract

Peripheral nerve injury results in the activation of a number of transcription factors (TFs) in injured neurons, some of which may be key regulators of the regeneration-associated gene (RAG) programme. Among known RAG TFs, ATF3, Smad1, STAT3 and c-Jun have all been linked to successful axonal regeneration and have known functional and physical interactions. We hypothesised that TF expression would promote regeneration of the central axon branch of DRG neurons in the absence of a peripheral nerve lesion and that simultaneous overexpression of multiple RAG TFs would lead to greater effects than delivery of a single TF. Using adeno-associated viral vectors, we overexpressed either the combination of ATF3, Smad1, STAT3 and c-Jun with farnesylated GFP (fGFP), ATF3 only with fGFP, or fGFP only, in DRG neurons and assessed axonal regeneration after dorsal root transection or dorsal column injury and functional improvement after dorsal root injury. ATF3 alone and the combination of TFs promoted faster regeneration in the injured dorsal root. Surprisingly, however, the combination did not perform better than ATF3 alone. Neither treatment was able to induce functional improvement on sensory tests after dorsal root injury or promote regeneration in a dorsal column injury model. The lack of synergistic effects among these factors indicates that while they do increase the speed of axon growth, there may be functional redundancy between these TFs. Because axon growth is considerably less than that seen after a conditioning lesion, it appears these TFs do not induce the full regeneration programme.

Phenotypic screening with primary neurons to identify drug targets for regeneration and degeneration

“…A different phenotypic screening campaign tested cDNAs of genes more highly expressed in a regenerating neuron (sensory neurons) than in a poorly regenerating CNS neuron (cerebellar granule cells). High content analysis confirmed that STAT3 was a positive regulator of neurite growth (Smith et al, 2011); the idea that STAT3 may enhance axon regeneration is also consistent with a number of other studies (Schwaiger et al, 2000; Fagoe et al, 2015; Nagata et al, 2014). Building on the STAT3 results and the VP16-KLF7 results, we have recently shown that overexpression in retinal ganglion cells of constitutively active STAT3 (STAT3CA) modified with VP16 increases optic nerve regeneration in vivo compared to STAT3CA alone (Mehta et al, 2016).…”

Epigenetic profiling reveals a developmental decrease in promoter accessibility during cortical maturation in vivo

“…AP-1 factors are widely implicated in axon growth and known to work interactively to induce pro-regenerative transcriptional programs (9, 10, 22-32). Specifically, ATF3 and JUN show cooperative actions in regulating axon outgrowth in peripheral neurons (9, 33-35).…”

Intrinsic mechanisms of neuronal axon regeneration

“…Although interactions between the known pro-regenerative transcription factors have been extensively studied in other cell types, it is critical to understand their interactions in the context of axon injury. This need is further highlighted by recent studies in which combinatorial co-expression of a few of these transcription factors had limited to no ability to boost regeneration above single-factor overexpression in poorly regenerating neurons 110,148 .…”

“…Genetic manipulations that have focused on overexpression of regeneration-associated genes or transcription factors have yielded limited success 114,143,146,148 . This lack of success could be because more than one factor is necessary to fully stimulate the growth process; however, another explanation is that the cellular context dictates the competency of the neurons to respond to a given genetic manipulation.…”