American Society for Peripheral Nerve

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Proteome of the Distal Nerve: Its Implication in Delayed Repair and Poor Functional Recovery
Guo Song, MBBS; M. Cristine Charlesworth, PhD; Raymond M Moore, MS; Robert J Spinner, MD; Huan Wang, MD, PhD
Mayo Clinic, Rochester, MN

Introduction: Nerve regeneration and functional recovery is poorer when nerve repair is delayed. Among the many contributing factors, chronic denervation mainly affects Schwann cell function and axon outgrowth. After losing axonal contact, Schwann cells proliferate and secrete neurotrophic factors to provide a growth-supportive environment. With chronic denervation the permissive factors progressively decline. Those factors/proteins were individually studied. To better understand the global protein expression profiles, proteomics analysis of chronically denervated nerves was carried out in this study to delineate proteins that were contributory to this detrimental effect.

Methods: Rat sciatic nerve repair model was used. In 4 rats nerve repair was done immediately after transection, while in the other 4 rats repair was done after a 12 week delay. After 16 weeks, nerve samples distal to the repair site were harvested. Proteins were individually extracted using bead mill homogenizer containing SDS lysis buffer. 25 ug of protein from each sample was fractionated by SDS-PAGE gels. Gels were stained, excised, digested, and peptides extracted for analysis by nanoLC-MS/MS. Individual protein expression level of the surgery side was compared to that of the control side using label-free analysis (MaxQuant software). Any protein with a P value less than 0.05 and a fold change of ≥4 was considered as differentially expressed. Ingenuity Pathway Analysis (IPA) and Cytoscape softwares were used for pathway/network analysis.

Results: The distal nerve stump proteome contained 5754 detectable proteins. Differential expression analysis showed significant increase of immune and inflammatory response related proteins and decrease of proteins related to axon regeneration and lipid metabolism process in the delayed repair model. Proteins related to Schwann cell function and axonal outgrowth that were down-regulated included CBL, MPZ, PTGDS, MADD, IGDF1R, DHH, HSPB8, GJB1, SHH, MAPK11, ADGRG6, LGALS8, PAK3, CNTF, CAMKK1, TNPV1 and ARPP19. Proteins associated with inflammatory response and apoptosis that were up-regulated were S100A8, S100A9, PLA2G4A, CASP6, CASP3, IGFBP5 and C6. IPA revealed that protective pathways involved in LXR/RXR activation, RAC signaling, ERK/MAPK signaling, CNTF signaling, IL-6 signaling, and FGF signaling were inhibited in the delayed repair group, while 3 detrimental pathways including complement system, PTEN signaling and apoptosis signaling were activated.

Conclusions: The poorer regeneration in delayed repair may be attributed to down-regulation of beneficial proteins and up-regulation of detrimental proteins. The proteins and pathways identified in this study can be potential therapeutic targets.

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