Proteomic and functional annotation analysis of injured peripheral nerves reveals ApoE as a protein upregulated by injury that is modulated by metformin treatment

BACKGROUND Peripheral nerve injury (PNI) results in a fundamental reorganization of the translational machinery in the injured peripheral nerve such that protein synthesis is increased in a manner linked to enhanced mTOR and ERK activity. We have shown that metformin treatment, which activates adenosine monophosphate-activated protein kinase (AMPK), reverses tactile allodynia and enhanced translation following PNI. To gain a better understanding of how PNI changes the proteome of the sciatic nerve and ascertain how metformin treatment may cause further change, we conducted a range of unbiased proteomic studies followed by biochemical experiments to confirm key results. RESULTS We used multidimensional protein identification technology (MUDPIT) on sciatic nerve samples taken from rats with sham surgery, spinal nerve ligation (SNL) surgery or SNL + 200 mg/kg metformin treatment. MUDPIT analysis on these complex samples yielded a wide variety of proteins that were sorted according to their peptide counts in SNL and SNL + metformin compared to sham. These proteins were then submitted to functional annotation analysis to identify potential functional networks altered by SNL and SNL + metformin treatment. Additionally, we used click-chemistry-based labeling and purification of nascently synthesized proteins followed by MUDPIT to further identify peptides that were synthesized within the injured nerve. With these methods, we identified apolipoprotein E (ApoE) as a protein profoundly increased by PNI and further increased by PNI and metformin. This result was confirmed by Western Blot of samples from SNL rats and spared nerve injury (SNI) mice. Furthermore, we show that 7-day treatment with metformin in naïve mice leads to an increase in ApoE expression in the sciatic nerve. CONCLUSIONS These proteomic findings support the hypothesis that PNI leads to a fundamental reorganization of gene expression within the injured nerve. Our data identify a key association of ApoE with PNI that is regulated by metformin treatment. We conclude from the known functions of ApoE in the nervous system that ApoE may be an intrinsic factor linked to nerve regeneration after PNI, an effect that is further enhanced by metformin treatment.