Combinatorial expression of transcription factors to promote axon regeneration

Society for Neuroscience Chicago 2015 Central Nervous System Regeneration Transplantation and Regeneration
Support: International Spinal Research Trust Netherlands Organization for Scientific Research

Title: Combinatorial expression of transcription factors to promote axon regeneration

Abstract: Objective. Dorsal root ganglia (DRG) neurons are ideal for research into the intrinsic regeneration associated gene (RAG) program. Injury to the central projection of these neurons in the spinal cord does not result in regeneration, but after a lesion to the peripheral projection an increase in growth of the central projection is observed. We hypothesised that the RAG program is coordinated by combinations of transcription factors (TFs) that act synergistically or in a complementary fashion. This hypothesis was tested in two approaches: 1. In vivo AAV vector mediated simultaneous overexpression of ATF3, c-Jun, STAT3 and Smad1, four TFs in injured DRG neurons which have been functionally linked to axon regeneration and are known to physically and functionally interact. 2. Promoter analysis of the RAG program which resulted in the identification of nine key TF families, some of which were previously linked to regeneration (AP1, ATF, KLF) and some of which are not (e.g. the MEF family). We systematically screened combinations of these TFs in vitro. Methods: 1. ATF3 alone or the combination of ATF3, c-Jun, STAT3 and Smad1 were over expressed in the left L4/L5 DRG of rats using an AAV5-dual vector1,2 that also expresses GFP. The L4, L5 dorsal roots were transected and repaired. Axonal regeneration was assessed histologically and sensory recovery tested functionally. 2. Nine TFs identified by promoter analysis of the RAG program were screened in various combinations in the F11 neuronal cell line with Cellomics neurite length analysis. All single TFs, all possible pairs, and selected combinations of 3 and 4 TFs were tested. Results: 1. Over-expression of ATF3 or the combination of ATF3, c-Jun, STAT3 and Smad1 together resulted in faster axonal growth after dorsal root injury, but no additional benefits of the combination over ATF3 alone were observed. No sensory recovery was observed. 2. Of the nine selected TFs screened in F11 cells several combinations of transcription factors promoted synergistic increases in neurite growth, which included combinations of TFs of the ATF, KLF, MEF and STAT families. Conclusion: We successfully delivered multiple TFs to DRG neurons in vivo. Simultaneous overexpression of four known pro-regenerative TFs promoted faster axon regeneration in the dorsal root in vivo, but without apparent synergistic effects. Identification of key TFs by RAG promoter analysis followed by systematic combinatorial screening has yielded several more promising combinations of TFs, which we are currently testing in vivo for effects on regeneration and gene expression.

1.Fagoe et al Gene Ther. 2014 Mar;21(3):242-52 2. Mason et al Mol Ther. 2010 Apr;18(4):715-24
Disclosures: C.L. Attwell: None. N. Fagoe: None. R.E. van Kesteren: None. A.B. Smit: None. J. Verhaagen: None. M.R.J. Mason: None.

Authors: *C. L. ATTWELL1, N. FAGOE1, R. E. VAN KESTEREN2, A. B. SMIT2, J. VERHAAGEN1, M. R. J. MASON1; 1Neuroregeneration, Netherlands Inst. For Neurosci., Amsterdam Zuidoost, Netherlands; 2Dept of Mol. and Cell. Neurobio., Ctr. for Neurogenomics and Cognitive Res., Amsterdam, Netherlands

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