Primary afferent derived PACSIN1 binds the Schwann cell survival receptor, LRP1, and transactivates TrkC to promote gliatrophic activities

Introduction: Low-density lipoprotein receptor-related protein (LRP1) binds numerous ligands, some of which transactivate receptor tyrosine kinases (RTKs) via Src family kinases (SFKs) in neurons. Using affinity precipitation and LC-MS/MS, we identified PACSIN1 as an intracellular neuronal protein released from injured primary afferents, which binds to SC LRP1. Binding of ligands to LRP1 may support Schwann cell (SC) reprogramming into the Repair Phenotype. The goal of this study was to determine whether PACSIN1 activates cell-signaling via LRP1 and thus, trigger activities associated with SC reprogramming in PNS injury. Methods: Injured sciatic nerve extracts were isolated using an approach that does not cause de novo cell lysis and affinity-precipitated with Fc-fusion proteins containing the ligand-binding domains of LRP1 (CCR2 and CCR4). By this approach, PACSIN1 was identified as an LRP1 ligand. Immunohistochemistry of dorsal root ganglia (DRGs) confirmed that PACSIN1 is uniquely located in primary sensory neurons and not peripheral glia. The cell-signaling pathway activated by PACSIN1 was initially characterized in primary cultured SCs. The effects of Lrp1 silencing, the SFK inhibitor, PP2, and the Trk inhibitor K252a on PACSIN1-induced ERK1/2, C-Jun, and TrkC phosphorylation were examined. Transgenic mice with conditional deletion of LRP1 in SCs (scLRP1-/-) were used to determine the specificity of PACSIN1 cell signaling in injured nerves. We also tested the ability of PACSIN1 to promote SC gene expression using bulk RNASeq, survival (Tunnel, Cell Death ElisaÔ) and migration (TranswellÔ) assays were performed. Results: PACSIN1 promoted phosphorylation of c-Jun and ERK1/2 in a concentration- and time-dependent manner in primary cultured SCs. Silencing Lrp1 expression blocked PACSIN1 induced c-Jun and ERK1/2 phosphorylation without affecting the activity of neurotrophin-3, a direct TrkC ligand. PP2 and K252a blocked PACSIN1-induced TrkC, c- Jun, and ERK1/2 phosphorylation in SCs, indicating an essential role for SFKs and transactivation. In crush-injured nerves, PACSIN1 activated c-Jun in scLRP1+/+ mice, but not scLRP1-/- mice, demonstrating specificity for SC LRP1. PACSIN1 promoted significant changes in the SC transcriptome within 4 hours. These genes were associated with PACSIN1 induced SC survival and migration. Conclusion: PACSIN1 activates cell-signaling in SCs via LRP1. The effects of PACSIN1 on TrkC phosphorylation demonstrate that RTK transactivation is conserved in the LRP1 signaling pathway across different nervous system cells. The specific RTK targeted in SCs (TrkC) is novel. Activation of c-Jun by PACSIN1 in vitro and in vivo, suggests that PACSIN1 can activate the SC Repair program in PNS injury and specifically contribute to SC survival and mobility.