The Role Of Schwann Cell Derived Lrp4 In Nerve Regeneration And Terminal Schwann Cell Marker Screening

Schwann cells（SCs）,originated from neural crest cells during development,are glial cells in the peripheral nervous system.Myelinated SCs（m SCs）and terminal SCs（t SCs）are the main two types of SCs.MSCs wrap motor axons to form myelin,while t SCs cover the motor nerve terminal.Both SCs are indispensable not only for normal innervation but also for regeneration after nerve injury.Peripheral nerve injury caused by traumatic or nontraumatic factors is a common disease,which leads to severe disability of patients.Promoting the recovery after injury is still a huge challenge.Unlike the central nerve,one of the characteristics of peripheral nerve axons is their ability to regenerate.During regeneration,m SCs provide necessary signals,spatial cues for axons to regrowth.MSCs are demyelinated and transformed into immature SCs with similar morphology.These immature SCs proliferate quickly and initiate a series of repair-related signals,such as up-regulating the expression of neurotrophic factors,activating myelin autophagy,and recruiting macrophages.Besides,these cells line up to form pathways for guiding axons to their targets.After repairing,these immature SCs can re-form myelin sheath to wrap axons in appropriate microenvironment.TSCs,a subset of non-myelinated synaptic glia,wrap the nerve terminal of the neuromuscular junction（NMJ）.TSCs have been reported to play critical roles in motor axon innervation,synaptogenesis,synaptic pruning and transmission.In general,although SCs have many morphological and transcriptional changes in nerve regeneration,the underlying mechanisms are still unclear.Low-density lipoprotein receptor-related protein 4（Lrp4）,a member of the low-density lipoprotein（LDL）receptor family,is critical for NMJ formation and maintenance.In the astrocytes of the central nervous system,Lrp4 regulates ATP release.Besides,Lrp4deficiency in astrocytes plays a protective role in ischemic brain injury.However,the role of Lrp4 in the peripheral nervous system glial cells is unknown.NMJs are chemical synapses formed between motor neurons and muscle fibers.NMJ usually is used as a regeneration model to study peripheral nerve regeneration.In the first part of this study,we established a regeneration model based on NMJ to study the nerve terminal guidance by t SCs after injury.The current researches on SCs are mainly focused on m SCs,while fewer attention is paid to t SCs.Lacking the specific marker for t SCs,the studies on t SCs are still at the morphological observation level.We hardly know anything about the molecular mechanisms underlying t SCs participating in NMJ regeneration.In the first part of this study,we investigated the role of SCs-derived Lrp4 in peripheral nerve regeneration.To knock out Lrp4 in SCs,we crossed floxed Lrp4 mice with Dhh::Cre,where Cre recombinase is expressed prominently in SCs precursors of the developing peripheral nerves,to obtain Dhh::Cre;Lrp4^f/f(Dhh-LRP4^-/-)mice.The ablation of Lrp4 in SCs promoted peripheral axon regeneration caused by the proliferation of demyelinated SCs.Further investigation found that SCs in the distal end of injured nerve segments demyelinated faster in Dhh-LRP4^-/-mice after nerve injury.Electron microscopy（EM）results demonstrated that mutant mice had fewer myelin debris and more regenerated axons.These results suggest that the deletion of Lrp4 in SCs promotes peripheral nerve regeneration by increasing SCs proliferation and myelin debris clearance.To further study the role of t SCs in NMJ regeneration,we intend to identify the marker for t SCs.In the second part of this study,we used single-cell sequencing technology to screen specific markers of t SCs.We built a platform for single-cell isolation and isolated m SCs or t SCs,respectively.Furtherly,we optimized our experimental methods for amplification and library construction of the transcripts.Analysis of the sequencing results revealed that the expression levels of myelin-related transcripts were different between m SCs and t SCs.Further analysis is still in progress.Taken together,our study provides novel insight into the roles of SCs,especially t SCs,in nerve regeneration.