The objective of this study was to obtain an unbiased overview of how PNI changes the proteome and functional annotation network of the sciatic nerve and how these changes are further modified by metformin treatment. Here, we report an overview based on MUDPIT and DAVID technology of how pathways are altered in the sciatic nerve distal to PNI and how these pathways are further modified by treatment with metformin. Using this technology, we identified ApoE as a protein profoundly increased and nascently synthesized in the distal sciatic nerve following PNI in rats and mice. Moreover, our results from DAVID analysis reveal that ApoE is a component of a number of functional annotations linked either positively or negatively to metformin treatment that may play a key role in peripheral nerve regeneration and repair or neuronal excitability, respectively. Our results clearly link metformin treatment to further increases in ApoE following PNI and increased expression of ApoE in sciatic nerves of naïve mice. Hence, we conclude that although ApoE has long been linked to PNI, this molecule is a potential regulator of neuropathic pain and/or regeneration following PNI.
Traditional proteomics methodologies separate complex protein samples by isoelectric point and molecular weight in 2-dimensional gels. Patterns are compared between samples by isolating individual protein spots, followed by proteolytic digestion, and analyzing the mass of each peptide by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. The measured peptide masses are searched against the predicted mass values for theoretical digestion of proteins in a sequence database, and the protein is identified by a statistically significant number of matches. MUDPIT, which we have utilized here, eliminates gel separations. Instead, biochemical fractions containing many proteins are directly proteolyzed and the enormous number of peptides generated, are separated by 2-dimensional liquid chromatography (repeated cycles of fractionation that extend for 22 hours) before entering the mass spectrometer. Instead of MALDI-TOF, the procedure employs tandem mass spectrometry (MS-MS) so that, after the mass of a peptide is measured, the peptide is fragmented using a collision-induced dissociation cell and the masses of the fragmentation products are determined. Using computational analysis, the data is transformed into an amino acid sequence. Although one peptide is often sufficient to identify a protein using this very sensitive technique, the specificity of this approach improves with increased number of assigned peptides to a particular protein. The advantage of this approach is the identification of minor proteins in a biological fraction that cannot be visualized on 2-dimensional gels. Recent studies have identified 1,000 to 2,000 proteins in a single fraction with MUDPIT .
ApoE is a 34 kDa glycoprotein and is a major determinant of lipid transport and metabolism . ApoE is strongly up-regulated after sciatic nerve crush injury where it increases several hundred fold  and then declines once regeneration is complete . In this model, it has been proposed that macrophages migrating into the injured site synthesize ApoE, which is then released concurrently with cholesterol and lipids derived from the degenerating myelin as ApoE lipoproteins. ApoE is then taken up by low-density lipoprotein (LDL) receptors on the surface of Schwann cells  for recycling and regeneration. However, further studies reveal an important role for ApoE in neuroregeneration and remyelination [24, 28, 29] suggesting a direct effect of ApoE on neuronal function and, possibly, regeneration. ApoE, for example, is well known as an important mediator of Alzheimer’s disease . ApoE has also emerged as an important regulator of nerve sprouting and nerve regeneration as well as neuroprotection. Several studies have indicated that the addition of ApoE to cultured neurons enhances nerve sprouting [28, 31], including enhanced sprouting of dorsal root ganglion (DRG) neurons . After traumatic injury, genetic depletion of ApoE leads to worse outcomes [32–34], whereas enhancing ApoE activity is beneficial [35–40]. More recently, an ApoE mimetic, COG112, was shown to significantly improve recovery of motor and sensory function following sciatic nerve crush in C57BL/6 mice . This indicates a positive modulatory role of ApoE in nerve regeneration. This finding also suggests that further enhancing ApoE expression or action in addition to increases that occur from injury alone are beneficial. This suggests that enhancements observed with metformin may be important in functional recovery after nerve injury. While this hypothesis requires further testing, it is very strongly supported by functional annotation analysis. Finally, ApoE has potent anti-inflammatory properties. Mice lacking ApoE have a substantially exaggerated response to LPS with regard to tumor necrosis factor α (TNFα), interleukin-6 (IL-6), (IL-12) and interferon gamma (IFNγ) . Reconstitution of plasma levels of ApoE in ApoE-knockout mice normalizes LPS-induced IL-12 and significantly reduces LPS-induced TNFα plasma levels. Sustained chronic inflammation is known to be detrimental for functional recovery following PNI . PNI and neuropathic pain are associated with changes in proinflammatory cytokine expression in the PNS, where these factors may play a direct role in sensitizing injured sensory afferents . Thus, stimulating endogenous expression of ApoE, as can be done with metformin administration, may provide benefits by repairing damaged nerves and modulating pain. This effect of metformin may also have important benefits in other neurological pathologies where ApoE might either be deficient or play a beneficial role therapeutically.
There is strong evidence that changes in translation regulation may underlie pathology leading to and maintaining neuropathic pain [5–8, 44]. PNI induces a complete reorganization of translational machinery in the PNS . This change is functionally linked to altered sensory processing, mainly allodynia  and pin-prick hyperalgesia [5, 6], as revealed by behavioral pharmacology studies. One possible drawback of utilizing pharmacological mechanisms to block translation regulation pathways for the treatment of neuropathic pain is a detrimental effect on nerve regeneration due to the key role that translation regulation pathways play in this event, at least in vitro. We argue that activating AMPK to achieve regulation of enhanced translation following nerve injury is unlikely to create these adverse consequences. Again, findings using the DAVID algorithm very strongly support this conclusion as they show that metformin-induced increases in ApoE are linked to functional annotations that are predicted to enhance peripheral nerve regeneration and repair. Here several key findings should be considered: 1) while metformin treatment blocks dysregulated translation after PNI, it does not reduce normal translation , 2) profiling of the effects of metformin on the translatome reveals that metformin targets only a subset of mRNAs to alter the proteome  (consistent with our findings here) and 3) metformin increases ApoE expression which is linked to enhanced functional recovery after PNI. In that regard, it is critical to note that while ApoE participates in a wide range of cellular functions, after metformin treatment, the shift in the proteome changes the context of overall cellular functions such that a set of functional annotations (Figure 2, red bars) containing ApoE and highly enriched in regeneration and repair is revealed. Moreover, metformin treatment reduced functional annotations linked to neuronal excitability induced by SNL (Figure 2, blue bars) consistent with its effect on reducing neuropathic allodynia in rats in this model of neuropathic pain . While we cannot rule out other possible mechanisms, with the exception of AMPK, for these effects of metformin, the safety, clinical availability and tolerability of this drug make it an attractive candidate for human trials for the treatment of neuropathic and possibly other forms of pain [7, 47].