Characterization of Wnt Directed Axon Guidance in Human and Rat Peripheral Nerve Injury
Fred De Winter, PhD1; Arie C Van Vliet, Msc1; Martijn R Tannemaat, PhD, MD2; Martijn Malessy, MD, PhD3; Joost Verhaagen, PhD1
1Netherlands Institute for Neuroscience, Amsterdam, Netherlands, 2Leiden University Medical Center, Leiden, Netherlands, 3Department of Neurosurgery, Leiden University Medical Center, Leiden, Netherlands
Neonatal brachial plexus palsy (NBPP) results in ~10% of the patients in the formation of a neuroma-in-continuity (NIC) that is thought to disturb axonal outgrowth and prevent functional recovery. To increase our knowledge of NIC we characterized gene expression in NIC tissue that was resected during reconstructive surgery. In a microarray study we compared gene expression in the NIC to that in the proximal nerve. The expression of 722 genes was differentially regulated between both tissues. Gene ontology overrepresentation analysis and ingenuity pathway analysis showed that genes involved in processes like fibrosis, cell adhesion, cell movement but also axon guidance were overrepresented in the NIC.
To learn more about axon guidance genes in the NIC we focused on the role of members of Wnt gene family and more specifically on its most prominently upregulated (4 fold) member Wnt5a. We observed that Schwann cells in the NIC express Wnt5a and axons that traverse the NIC express Wnt5a receptor Ryk. In a parallel study we found that also in the rat sciatic nerve Wnt genes (including Wnt5a) are highly expressed after injury and in addition, their receptors (including Ryk) show regeneration related changes in expression in the DRG. With the help of in vitro assays we discovered that these changes in Wnt receptor expression can determine whether Wnt5a either stimulates or inhibits neurite outgrowth. Based on these results we propose that regenerating axons in vivo are initially attracted by Wnt5a and subsequently repelled distally towards their targets when regeneration is progressing. In vivo manipulation of the rodent regenerating nerve revealed that Schwann cells specific Wnt5a knockout or sequestering of all Wnt ligands by virally overexpressing WifI, reduces axonal outgrowth and delays the return of CMAPs respectively. This shows that Wnt(5a) expression observed after peripheral nerve injury is normally beneficial for regeneration.
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