Identification of proteins that bind to LDL Receptor-related Protein-1 (LRP1) in Schwann cells and activate c-Jun In vitro and In vivo

After injury, Schwann cells (SCs) serve as “first responders” by transdifferentiating into a repair phenotype that supports proliferation, cell migration, secretion of growth factors and extracellular matrix and clearance of myelin. These processes are essential for functional nerve regeneration and are referred to as activation of the SC Repair Program (Jessen et al. 2015 Dev Cell 34:613-20). LRP1 is up-regulated in SCs after injury and orchestrates many elements of the SC Repair Program (Campana et al., 2006 J Neurosci 26(43):1197-207). Cell signaling pathways activated by SC LRP1 include ERK1/2, PI3K, Rac1, and the unfolded protein response (UPR). The transcription factor c-Jun plays an essential of role in the activation the SC Repair Program (Arthur-Farraj et al., 2012 Neuron 75(4):663-47), however SC signaling receptors that regulate c-Jun have not been identified. To address this question, primary SCs were treated with known LRP1 ligands, tissue-type plasminogen activator (tPA) or the hemopexin domain of MMP-9 (PEX) for 5-30 min. Both LRP1 ligands robustly phosphorylated c-Jun that were blocked by RAP, a LRP1 antagonist. To confirm that LRP1 activates c-Jun in vivo, sciatic nerve injury was induced in Sprague Dawley rats. 24 hours later, when LRP1 is substantially up-regulated in SCs, tPA or PEX was injected directly into the nerves. After 15 min, both ligands significantly increased c-Jun activation as determined by immunoblots (p<0.05). To identify novel LRP1 ligands, responsible for activating the SC Repair Program, we applied a proteomics-based discovery approach. Extracts of injured sciatic nerves, prepared so that intact cells are not disrupted, were immunoprecipitated with Fc-fusion proteins containing the extracellular ligand-binding domains of LRP1 (CCR2 and CCR4). Free Fc protein served as a control. Tandem mass spectrometry (LC-MS/MS) revealed potential novel LRP1 ligands including Pacsin-1, the axonal protein NFM, and von Willebrand factor. The LRP1-interactome was computationally scored with MiST to exclude false positives and to narrow down candidates for in vitro binding validation. Novel LRP1 ligands involved in c-Jun activation may be used therapeutically to augment the SC Repair Program and thus, enhance functional nerve repair.