Proteomics of the Injured Rat Sciatic Nerve Reveals Protein Expression Dynamics During Regeneration

Abstract

Using proteomics, we investigated the temporal expression profiles of proteins in rat sciatic nerve after experimental crush. Extracts of sciatic nerves collected at 5, 10, and 35 days after injury were analyzed by two-dimensional gel electrophoresis and quantitative image analysis. Of the ∼1,500 protein spots resolved on each gel, 121 showed significant regulation during at least one time point. Using cluster analysis, these proteins were grouped into two expression profiles of down-regulation and four of up-regulation. These profiles mainly reflected differences in cellular origins in addition to different functional roles. Mass spectrometric analysis identified 82 proteins pertaining to several functional classes, i.e. acute-phase proteins, antioxidant proteins, and proteins involved in protein synthesis/maturation/degradation, cytoskeletal (re)organization, and in lipid metabolism. Several proteins not previously implicated in nerve regeneration were identified, e.g. translationally controlled tumor protein, annexin A9/31, vitamin D-binding protein, α-crystallin B, α-synuclein, dimethylargininases, and reticulocalbin. Real-time PCR analysis of selected genes showed which were expressed in the nerve versus the dorsal root ganglion neurons. In conclusion, this study highlights the complexity and temporal aspect of the molecular process underlying nerve regeneration and points to the importance of glial and inflammatory determinants. Using proteomics, we investigated the temporal expression profiles of proteins in rat sciatic nerve after experimental crush. Extracts of sciatic nerves collected at 5, 10, and 35 days after injury were analyzed by two-dimensional gel electrophoresis and quantitative image analysis. Of the ∼1,500 protein spots resolved on each gel, 121 showed significant regulation during at least one time point. Using cluster analysis, these proteins were grouped into two expression profiles of down-regulation and four of up-regulation. These profiles mainly reflected differences in cellular origins in addition to different functional roles. Mass spectrometric analysis identified 82 proteins pertaining to several functional classes, i.e. acute-phase proteins, antioxidant proteins, and proteins involved in protein synthesis/maturation/degradation, cytoskeletal (re)organization, and in lipid metabolism. Several proteins not previously implicated in nerve regeneration were identified, e.g. translationally controlled tumor protein, annexin A9/31, vitamin D-binding protein, α-crystallin B, α-synuclein, dimethylargininases, and reticulocalbin. Real-time PCR analysis of selected genes showed which were expressed in the nerve versus the dorsal root ganglion neurons. In conclusion, this study highlights the complexity and temporal aspect of the molecular process underlying nerve regeneration and points to the importance of glial and inflammatory determinants. Injury to a peripheral nerve induces cellular and molecular changes in the injured neurons and in the microenvironment of their axon stumps, which together support axonal regeneration (for a review see Refs. 1Fawcett J.W. Keynes R.J. Peripheral nerve regeneration..Annu. Rev. Neurosci. 1990; 13: 43-60Google Scholar, 2Fu S.Y. Gordon T. The cellular and molecular basis of peripheral nerve regeneration..Mol. Neurobiol. 1997; 14: 67-116Google Scholar, 3Hokfelt T. Zhang X. Wiesenfeld-Hallin Z. Messenger plasticity in primary sensory neurons following axotomy and its functional implications..Trends Neurosci. 1994; 17: 22-30Google Scholar). During the first week after injury, Wallerian (anterograde) degeneration occurs whereby distal axons and their myelin sheaths are phagocytosed by invading macrophages (4Lazarov-Spiegler O. Solomon A.S. Schwartz M. Peripheral nerve-stimulated macrophages simulate a peripheral nerve-like regenerative response in rat transected optic nerve..Glia. 1998; 24: 329-337Google Scholar). Schwann cells, deprived of axonal contact, play a key role in regulating macrophage recruitment (5Tofaris G.K. Patterson P.H. Jessen K.R. Mirsky R. Denervated Schwann cells attract macrophages by secretion of leukemia inhibitory factor (LIF) and monocyte chemoattractant protein-1 in a process regulated by interleukin-6 and LIF..J. Neurosci. 2002; 22: 6696-6703Google Scholar, 6Shamash S. Reichert F. Rotshenker S. The cytokine network of Wallerian degeneration: Tumor necrosis factor-α, interleukin-1α, and interleukin-1β..J. Neurosci. 2002; 22: 3052-3060Google Scholar). Moreover, following injury, Schwann cells proliferate and migrate to provide a growth-permissive substrate onto which axons regenerate through the expression of cell adhesion molecules and growth factors (7Trapp B.D. Hauer P. Lemke G. Axonal regulation of myelin protein mRNA levels in actively myelinating Schwann cells..J. Neurosci. 1988; 8: 3515-3521Google Scholar, 8White F.V. Toews A.D. Goodrum J.F. Novicki D.L. Bouldin T.W. Morell P. Lipid metabolism during early stages of Wallerian degeneration in the rat sciatic nerve..J. Neurochem. 1989; 52: 1085-1092Google Scholar, 9Scherer S.S. The biology and pathobiology of Schwann cells..Curr. Opin. Neurol. 1997; 10: 386-397Google Scholar, 10Frostick S.P. Yin Q. Kemp G.J. Schwann cells, neurotrophic factors, and peripheral nerve regeneration..Microsurgery. 1998; 18: 397-405Google Scholar). Thus, the molecular and cellular changes that underly peripheral nerve regeneration are complex and identification of proteins involved will contribute significantly to our understanding of neuroregeneration. To study the molecular mechanisms that govern regeneration of the sciatic nerve, global gene expression profiling of dorsal root ganglia (DRG) 1The abbreviations used are: DRG, dorsal root ganglia; 2DGE, two-dimensional gel electrophoresis; O/N, overnight; qPCR, quantitative PCR; Ct, cycle threshold. 1The abbreviations used are: DRG, dorsal root ganglia; 2DGE, two-dimensional gel electrophoresis; O/N, overnight; qPCR, quantitative PCR; Ct, cycle threshold. and sciatic nerve has been employed recently. These studies revealed that the regeneration-associated genes can be categorized into several groups, including cytoskeletal proteins, neurotransmitter metabolizing enzymes, neuropeptides, growth factors, and signal transduction molecules in the DRG (11Bonilla I.E. Tanabe K. Strittmatter S.M. Small proline-rich repeat protein 1A is expressed by axotomized neurons and promotes axonal outgrowth..J. Neurosci. 2002; 22: 1303-1315Google Scholar, 12Costigan M. Befort K. Karchewski L. Griffin R.S. D’Urso D. Allchorne A. Sitarski J. Mannion J.W. Pratt R.E. Woolf C.J. Replicate high-density rat genome oligonucleotide microarrays reveal hundreds of regulated genes in the dorsal root ganglion after peripheral nerve injury..BMC Neurosci. 2002; 3: 16Google Scholar, 13Xiao H.S. Huang Q.H. Zhang F.X. Bao L. Lu Y.J. Guo C. Yang L. Huang W.J. Fu G. Xu S.H. Cheng X.P. Yan Q. Zhu Z.D. Zhang X. Chen Z. Han Z.G. Identification of gene expression profile of dorsal root ganglion in the rat peripheral axotomy model of neuropathic pain..Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8360-8365Google Scholar, 14Tanabe K. Bonilla I. Winkles J.A. Strittmatter S.M. Fibroblast growth factor-inducible-14 is induced in axotomized neurons and promotes neurite outgrowth..J. Neurosci. 2003; 23: 9675-9686Google Scholar), as well as genes encoding proteins involved in inflammation, proliferation, and myelination in the nerve (15Araki T. Nagarajan R. Milbrandt J. Identification of genes induced in peripheral nerve after injury. Expression profiling and novel gene discovery..J. Biol. Chem. 2001; 276: 34131-34141Google Scholar, 16Kubo T. Yamashita T. Yamaguchi A. Hosokawa K. Tohyama M. Analysis of genes induced in peripheral nerve after axotomy using cDNA microarrays..J. Neurochem. 2002; 82: 1129-1136Google Scholar, 17Nagarajan R. Le N. Mahoney H. Araki T. Milbrandt J. Deciphering peripheral nerve myelination by using Schwann cell expression profiling..Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8998-9003Google Scholar). Proteomic analysis of the protein expression pattern of the regenerating nerve is in many ways additive to gene expression profiling. It analyzes proteins and protein isoforms directly, and detects proteins derived from the nerve, neuronal cell bodies, and also from external sources such as the circulation. A quantitative and qualitative assessment of the proteome can be achieved by combining two-dimensional gel electrophoresis (2DGE) generating a high-resolution, quantitative protein expression map, and MS to identify regulated proteins (18Clauser K.R. Hall S.C. Smith D.M. Webb J.W. Andrews L.E. Tran H.M. Epstein L.B. Burlingame A.L. Rapid mass spectrometric peptide sequencing and mass matching for characterization of human melanoma proteins isolated by two-dimensional PAGE..Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5072-5076Google Scholar, 19Shevchenko A. Jensen O.N. Podtelejnikov A.V. Sagliocco F. Wilm M. Vorm O. Mortensen P. Boucherie H. Mann M. Linking genome and proteome by mass spectrometry: Large-scale identification of yeast proteins from two dimensional gels..Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14440-14445Google Scholar). Previous metabolic labeling experiments employing 2DGE (20Skene J.H. Shooter E.M. Denervated sheath cells secrete a new protein after nerve injury..Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4169-4173Google Scholar, 21Muller H.W. Gebicke-Harter P.J. Hangen D.H. Shooter E.M. A specific 37,000-dalton protein that accumulates in regenerating not in Scholar, H.W. Hangen D.H. Shooter E.M. Expression of specific sheath cell proteins during peripheral nerve growth and regeneration in Biol. Scholar, Gebicke-Harter P.J. J.H. J.W. Shooter E.M. Expression of during nerve degeneration and Natl. Acad. Sci. U. S. A. several proteins regulated by nerve injury, of which one protein, identified Gebicke-Harter P.J. J.H. J.W. Shooter E.M. Expression of during nerve degeneration and Natl. Acad. Sci. U. S. A. Scholar). In the using 2DGE, we analyzed the temporal protein expression profiles in sciatic nerve distal to a injury versus sequencing by to the identification of proteins involved in a of to nerve injury and the role of Schwann cells and macrophages as well as proteins in the response to injury as well as the of the regeneration and were to the of the experimental The were in on a and and were were using The sciatic nerve at and for by a the distal of the a through the to identify the of the were at 5, 10, and 35 days time after by were at the of to the that changes in protein levels gene expression can be to of the sciatic nerve, nerve of and distal of the and a of the nerve, were and at root ganglia at levels and were and at were from were on into and into on and in of and using a The on for by at The to a to which and were a of A used to the protein in the were of and after were in and at to were in for by in for The using the in at gel at after were in for and A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of proteins Chem. 1996; Scholar). of protein were in were for in and were for in by in The were in a at a of the were in The were using a and pattern analysis using image analysis using a were groups, four to of time after nerve days after days after 35 days after Analysis of protein expression profiles in the nerve at one time days after injury, of the protein by the of the of 5, 10, and 35 days and protein expression profiles were by of a P. C. M. H. of and by gene expression Scholar, P. D. J. Zhu Q. S. of gene expression and to Natl. Acad. Sci. U. S. A. Scholar). To this by the for The protein spots of were from the and at at as A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of proteins Chem. 1996; were from the gel in for by for were and using the were in of in for MS in of of in for the were in a from a onto a on the of a a at MS and were as previously K. R. S. analysis of rat of the protein functional for the of Biol. Chem. 2003; Scholar). MS and were the using from to identify the The at and were used for of the The for the and the mass The used for of the for of MS and the mass at and isolated from sciatic nerve distal to the from sciatic nerve of the and from DRG from the and of each time P. N. of by Scholar), in the after to a of by by gel electrophoresis and by each time of of were in two and which were for and cDNA for by gel Of each of and leukemia to the of the of A and cDNA were and at Real-time quantitative PCR on a PCR and were used in a of using on cDNA to genes were selected for the two analyzed by a analysis of the cycle of genes to and The of and used to the sciatic nerve DRG, and were used in in a new To study regeneration of peripheral nerve after injury, proteome analysis at 5, 10, and 35 days following sciatic nerve crush. In this in Refs. 2Fu S.Y. Gordon T. The cellular and molecular basis of peripheral nerve regeneration..Mol. Neurobiol. 1997; 14: 67-116Google and C. Peripheral nerve 1996; that are for The Wallerian in the first G. H.W. injury, axonal degeneration and Scholar), Schwann cell at after R. Le N. Mahoney H. Araki T. Milbrandt J. Deciphering peripheral nerve myelination by using Schwann cell expression profiling..Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8998-9003Google Scholar), macrophage at days and at days after R. Le N. Mahoney H. Araki T. Milbrandt J. Deciphering peripheral nerve myelination by using Schwann cell expression profiling..Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8998-9003Google Scholar, G. H.W. injury, axonal degeneration and Scholar), axonal regeneration first the distal of the and days after to functional at and after nerve R. Le N. Mahoney H. Araki T. Milbrandt J. Deciphering peripheral nerve myelination by using Schwann cell expression profiling..Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8998-9003Google Scholar). The sciatic nerve proteome protein spots of a gel of a nerve and nerve distal to the analysis of the protein of distal nerve at 5, 10, and 35 days after nerve and revealed a of spots that were significantly in and spots that were significantly for the and 35 days after and days after in at least one of the time significant differences in were the nerve days after injury, and the The expressed proteins were using into of temporal regulation profiles Expression profiles and proteins of and in the and of down-regulation and to Expression profiles and proteins that in the and of up-regulation. for each expression profile a selected in of the as of the different temporal regulation profiles and functional of regulated proteins in the sciatic nerve distal to the in a new of temporal changes in protein levels as by nerve crush. of the protein spots that in levels that are for each of the temporal regulation profiles as in The the in protein levels of each of the at days days and 35 days after nerve crush. of The regulated spots by the were identified as and of the protein identification and abbreviations see To identify proteins expression in injured sciatic the spots were from the and from that were and The a of the which the identification of The were analyzed using MS and of the peptide mass protein were by analysis using and for of the protein A of of the regulated proteins be identified, and are categorized to their in to the of different functional of proteins, the of metabolic proteins is in the of proteins, the of acute-phase proteins and proteins involved in protein and and lipid metabolism is in the of proteins of proteins regulated by sciatic nerve and identified by peptide mass and factor controlled tumor protein molecular to protein protein D-binding response protein protein protein response protein to in a new In cluster protein levels were at days after and protein levels at 35 days after injury two proteins expressed in myelinating Schwann cells, a protein in and that were by and after nerve crush. Expression profile proteins that were at and days after and expression at 35 days after nerve injury. Several neuronal proteins such as protein and in expression profile which is Wallerian degeneration and axon proteins of the and were well in expression profiles and 5, of and These proteins vitamin D-binding protein and the and and at a molecular mass of which is the molecular mass of Expression profile of that on after the at that is expressed in Schwann cells and macrophages injury F. A. Rotshenker S. Peripheral nerve injury induces Schwann cells to two macrophage and the Neurosci. 1994; 14: Scholar). protein is the that is expressed in macrophages and The role of in injury and J. 1994; Scholar, Q. induces in 1997; Scholar, S.S. D. in macrophages and A role in Scholar). The at days in expression profile the at days in expression profile Schwann cell proteins involved in cytoskeletal i.e. and also in expression profile expression profile as well as and 5, several proteins involved in protein and such as protein factor two protein and protein and the proteins and The of these proteins Schwann cell and levels of two isoforms of and four isoforms of after injury. the temporal protein regulation profiles were in expression profile of and in profile of and and isoforms and in profile of and up-regulation. as a of four in P. of and its on lipid and 1994; Scholar), the of spots in our to as of of the antioxidant proteins and A. K. A. of expression in response to Biol. 2001; Scholar, M. S. A. T. of during after can be during after Biol. Chem. 2003; Scholar). The of and were by the of by in of a in A. K. A. of expression in response to Biol. 2001; Scholar). expression analysis of selected proteins in to the of expression in to the neuronal in the versus peripheral cells and cells invading the PCR showed that of the selected genes regulation in the nerve and regulation in the DRG the of the and the and and translationally controlled tumor protein as well as the down-regulation of α-crystallin and Thus, the changes in gene expression of these proteins the changes in protein and their regulation is by peripheral cells, Schwann cells and invading The mRNA levels of response and protein not changes in the nerve, showed down-regulation at the protein is in the that these proteins are of neuronal and in the not mRNA for and in the sciatic nerve, which is in the these proteins are derived from the circulation. Using proteomics, we investigated the of peripheral nerve injury on the global protein expression in sciatic nerve at 5, 10, and 35 days after injury. cluster analysis of the temporal expression In the of down-regulation and mainly reflected changes in protein in the nerve in Schwann cells and invading and the of regulation and revealed mainly in levels of proteins from the DRG The identified regulated proteins in such as inflammation, protein lipid and cytoskeletal These proteins well be involved in the of Wallerian axon and of axonal nerve injury Schwann cells to and In two proteins of cluster to be expressed in myelinating Schwann cells, e.g. and were after and levels of and at 35 days after injury. down-regulation is in our and the of Schwann cell that occurs of axonal and myelin by macrophages of in and regenerating sciatic nerve..J. Biol. 52: Scholar, J.F. T. N. 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A protein to the and nerve Neurosci. 1988; 8: Scholar), were also in sciatic nerve after crush. that these proteins be by e.g. Schwann cells, the regenerating axons by axonal is in cluster of down-regulation together Schwann cell which the first is a of and is as a protein to the N. L. M. N. P. J. and functional Neurosci. Scholar). the gene has been implicated in and the protein accumulates in and bodies, the of and The A of proteins involved in and Neurosci. 1998; Scholar). The functional of down-regulation of in the sciatic nerve to be axon several proteins are distal to the of the induced proteins in cluster is which two different in nerve on Schwann cells and macrophages of myelin F. A. Rotshenker S. Peripheral nerve injury induces Schwann cells to two macrophage and the Neurosci. 1994; 14: adhesion and neurite P. S. 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C. expression in the sciatic nerve of experimental Scholar). a to be Several proteins i.e. and the proteins and were in the functional of protein and that days after crush. are implicated in protein in H. The a novel of proteins to the of and be for protein and of e.g. and growth factors in Schwann In and been implicated in cell adhesion H. M. Identification of the in protein 1997; and in H. C. Scholar). these of and are of importance for peripheral nerve regeneration to be Schwann cell and into the injury of the distal nerve of cell and The protein and mRNA expression of and at days after be for are proteins that by from the of of expression of isoforms been in human G. D.M. changes in cell and Opin. 2001; Scholar), and in neuronal P. R. P. and isoforms in 1997; Scholar). During nerve of are for axonal regeneration and levels of and after injury, including of and Expression profile and that are in macrophages and which is in our that a in the In and been to the injured nerve from the of in the regenerating and peripheral Identification of and Biol. Chem. 1990; Scholar), which is our that to their in the nerve as well as the in expression profiles that studies a role for in of from nerves for of axonal and myelin R.E. P.J. role for and in during regeneration and of the rat sciatic nerve..J. 1989; Scholar, P.J. R.E. Shooter E.M. in nerve injury and Scholar, Shooter E.M. R.E. by neuronal growth in Scholar, R.S. of for axonal growth of Biol. Chem. Scholar, P. H. G. T. A. K. H.W. expression of mRNA in of peripheral J. 1990; Scholar). The and of and their role in and during the regeneration Injury to a of the in of proteins into the of the acute-phase proteins and as well as the proteins and and are a specific macrophage S. a for Biol. 2001; Scholar), which the cells of the regenerating nerve the of from the can also be induced in Schwann cells and invading macrophages in injured peripheral nerves L. N. in expression and of and its in injured A of and peripheral 1998; 82: Scholar). is a protein that of macrophages R. monocyte to is by Biol. 1994; and is a for factor N. protein as a for macrophage factor in the macrophage in 1996; and in the injured sciatic nerve attract and are that and to a of These are and in the S. A. M. J. H. W.J. J. The gene expression of two in the Biol. Chem. 1998; Scholar). the levels of a of after nerve has been to nerve growth neurite and of cells P.H. nerve growth neurite and gene expression of cells..J. Neurochem. and a role in nerve our showed of and after nerve crush. our in the has been during sciatic nerve and a role as of Schwann cell and peripheral nerve myelination has been K. P. S. peripheral nerve by regulating Schwann cell 2002; Scholar). proteins antioxidant we of the antioxidant and A. K. A. of expression in response to Biol. 2001; Scholar, M. S. A. T. of during after can be during after Biol. Chem. 2003; and identified as a protein in macrophages T. M. H. M. S. K. S. and characterization of a macrophage Biol. Chem. and is expressed in the H. T. S. is and expressed in the rat Scholar). The levels of antioxidant after nerve a In this a of proteins resolved in studies employing 2DGE of sciatic nerve H.W. Hangen D.H. Shooter E.M. Expression of specific sheath cell proteins during peripheral nerve growth and regeneration in Biol. Scholar, D. A two-dimensional gel study of proteins and by sciatic Neurol. Scholar), to in and a of its we not of proteins such as and growth factors, which are to be induced in Schwann cells nerve injury. we identify many novel proteins regulated by sciatic nerve in identification is the first in their functional role in the nerve of the cellular as well as and are the to our to the process of nerve