An integrated in silico pipeline identifies a novel TF combination that promotes enhanced CST growth following injury

Authors
*I. VENKATESH1, Z. WANG2, V. MEHRA1, E. EASTWOOD1, M. SIMPSON1, A. CHAKRABORTY1, D. GROSS1, Z.BEINE1, M. CABAHUG1, G. OLSON1, M. G. BLACKMORE3;2Dept. of Biomed. Sci., 3Biomed. Sci., 1Marquette Univ., Milwaukee, WI

Ishwariya Venkatesh PhD Marquette University

Lab Abstract
Embryonic and peripheral neurons respond to axonal injury with activation of transcriptional networks conducive to re-growth. In contrast, injured mature CNS neurons fail to re-induce appropriate transcriptional networks, resulting in failed regeneration and permanent damage. We have previously shown that forced re-expression of single transcription factors (TFs) such as KLF6 promotes axon outgrowth following injury and is a promising strategy for therapeutic neural repair. However, with single TF treatments the overall number and regenerative speed of axons remains sub-optimal, and likely insufficient for full functional recovery. Because TFs rarely function in isolation, we hypothesized that supplying combinations of TFs that synergize with KLF6 may boost growth phenotypes. To this end, we developed a bioinformatics pipeline to detect TFs that may synergize with KLF6 to drive the expression of pro-growth genes. To validate our bioinformatic predictions, we next systematically co-expressed candidate TFs in combination with KLF6 in assays of neurite outgrowth in post-natal CNS neurons. Remarkably, nearly 20% of the TF synergized with KLF6 to promote neurite outgrowth, validating the bioinformatics approach. To prioritize TFs for in vivo testing, we performed TF – target gene network analyses that identified 4 core TFs – EOMES, RARB, NKX32 and NR5A2 that are predicted to be critical to the regulation of pro-growth gene networks. Finally, we tested in vivo the ability of the 4 core TFs to promote CST axon growth individually or in combination with KLF6 following pyramidotomy injuries. Individually, RARB overexpression lead to increased CST sprouting following pyramidotomy. Importantly, we observed that although NR5A2 had no effect on growth by itself, forced co-expression with KLF6 promoted a robust increase in midline crossing by transduced CST axons, significantly above the level of KLF6 alone. Ongoing experiments are aimed at clarifying the molecular mechanisms underlying KLF6/NR5A2 synergy in driving axon growth. Overall, we describe a novel bioinformatics-based approach that has identified a completely novel TF combination that drives enhanced sprouting in the injured CST.

Grant Support
R21NS106309
R01NS107807
R01NS083983
Craig Nielsen Post doc fellowship (Venkatesh)

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