Schwann cells (SC) are the most important accessory cells of neurons in the vertebrate peripheral nervous system and are involved in every aspect of peripheral nerve structure and healthy function. SCs develop in intimate contact with axons and they depend on axons for signals that promote their genesis, survival and subsequent differentiation in myelinating and non-myelinating SCs. The close interaction between SCs and neurons results in the morphological and functional specializations in both cell types. In particular, SCs produce the highly specialized insulating myelin sheath that is essential for rapid propagation of action potentials. Additionally, SCs provide essential metabolic support to neurons, illustrating the reciprocal relationships between neurons and SCs (Salzer 2015 PMID 26054742). Ensheathment of axons by SCs and initiation of myelination is critically dependent on a number of coordinated molecular interactions. One of those consist of the protein LGI4, which is secreted from Schwann cells and binds the ADAM22 transmembrane receptor that spans the axonal membrane. Deletion of Lgi4 or Adam22 results in severe hypomyelination as part of a condition called Arthrogryposis Multiplex Congenita. However, how LGI4/ADAM22 drives SC development and myelination is largely unknown. We will explore the hypothesis that LGI4/ADAM22 binds to a receptor on the Schwann cell membrane to initiate cellular differentiation and myelination (Kegel et al, 2014 PMID 24715463). As ADAM22 is a receptor with an extensive cytoplasmic domain we will identify ADAM22 axoplasmic interactors and explore their contribution to myelination. To identify potential Schwann cell receptors (extra-cellular) and neuronal-axoplasmic (intra-cellular) interactors for LGI4/ADAM22 we have adopted a proximity Biotin labelling (BioID, Roux et al 2012 PMID 22412018) and mass spectrometry approach in transgenic mice carrying a ADAM22-BirA* transgene and identified a number of neuronal candidate proteins. In this project we will build on these results to pursue two major aims: 1) Confirm the interaction of candidate proteins with the ADAM22 cytoplasmic domain and establish the functional role these interactions in myelinating sensory neuron/SC cultures using CRISPR/Cas9 mediated deletion of candidate genes in neurons (Booth et al., 2019 PMID 31110056). 2) We will use a secreted form of the ADAM22 extracellular domain fused to BirA* (is biotin ligase) and complexed with LGI4 as a bait to purify potential LGI4/ADAM22 receptors expressed on cultured SCs and establish their identity through mass spectrometry. The functional role of these potential LGI4/ADAM22 receptors in Schwann cell development and myelination will be established through CRISPR/Cas9 mediated gene deletion in Schwann cells in co-culture with DRG neurons. In pursuing these questions, we hope to provide a comprehensive understanding of LGI4/ADAM22s role in myelination and anticipate that this will have wider resonance in the neurosciences as LGI/ADAM proteins play essential roles in other parts of the nervous system. The project provides a training in modern molecular genetic techniques (CRISPR/Cas9; provided by Dr MacKenzie), neuronal cell biology (myelinating DRG cultures), biochemistry (mass spectrometry) and bioinformatics.