Alpha-1 catenin regulation of the mammalian blood-nerve barrier

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Our long-term objective is to discover how the blood-nerve barrier (BNB) recovers after nerve injury. We have recently observed a relationship between loss of α1-catenin (CTNNA1), BNB dysfunction and chronic neuropathic pain in peripheral neuropathy. This project is designed to address a fundamental question: What is the role of CTNNA1 in BNB recovery and reduced nociception following peripheral nerve injury? Based on our published and exciting Preliminary Data, including murine sciatic nerve BNB permeability assays and reflexive neurobehavioral nociception tests in conditional GDNF transgenic wildtype and knockout mice following sciatic nerve crush injury, we propose the following hypotheses: GDNF (via RET-Tyrosine kinase/ Ras-MAPK signaling pathways) phosphorylates transcription factor CREB1 with resultant increase in CTNNA1, cortactin (CTTN) and tight junction protein claudin-4 (CLDN4) gene transcription. GDNF-RET- Tyrosine kinase also activates SRC kinase which phosphorylates membrane-bound CTTN that binds to CTNNA1. This induces CTNNA1 binding to CLDN4, resulting in tight junction formation. CTNNA1 is the critical adapter molecule that connects the tight junctional complex to the cytoskeleton during BNB recovery. This process restores BNB function and reduces nociception after peripheral nerve injury. In order to address this hypothesis, we will determine CTTN, phosphorylated CTTN, CTNNA1 and CLDN4 expression dynamics by western blot of confluent primary human endoneurial endothelial cell membrane extracts following injury with and without exogenous GDNF in vitro. We will subsequently inhibit specific gene transcription using commercially available siRNAs targeting CTTN, SRC kinase and CTNNA1 and determine effect on the GDNF-mediated BNB recovery via continuous transendothelial electrical resistance and solute permeability assays using low and high molecular weight fluoresceinated molecules. To validate the in vitro work and demonstrate a direct relationship between BNB recovery and reduced nociception after injury, we will perform sciatic nerve crush injury in tamoxifen-inducible microvascular-specific conditional CTTN and CTNNA1 transgenic knockout mice and block SRC kinase with Bosutinib in wildtype mice, with appropriate controls. We will quantify % permeable endoneurial microvessels to horseradish peroxidase by electron microscopy and perform reflexive neurobehavioral tests of nociception in a blinded manner, with appropriate controls and technical replicates to demonstrate scientific rigor, and data reproducibility and inference validity. Structural changes at the BNB are associated with chronic peripheral neuropathies, and we have observed a direct relationship with chronic neuropathic pain. Understanding mechanisms of BNB recovery after nerve injury could translate to novel therapies for chronic neuropathic pain achieved by restoring endoneurial homeostasis. BNB recovery could also provide a more conductive microenvironment for axonal regeneration.