Differential Proteomics Reveals Multiple Components in Retrogradely Transported Axoplasm After Nerve Injury

Information on axonal damage is conveyed to neuronal cell bodies by a number of signaling modalities, including the post-translational modification of axoplasmic proteins. Retrograde transport of a subset of such proteins is thought to induce or enhance a regenerative response in the cell body. Here we report the use of a differential 2D-PAGE approach to identify injury-correlated retrogradely transported proteins in nerves of the mollusk Lymnaea. A comprehensive series of gels at different pI ranges allowed resolution of ∼4000 spots by silver staining, and 172 of these were found to differ between lesioned versus control nerves. Mass spectrometric sequencing of 134 differential spots allowed their assignment to over 40 different proteins, some belonging to a vesicular ensemble blocked by the lesion and others comprising an up-regulated ensemble highly enriched in calpain cleavage products of an intermediate filament termed RGP51 (retrograde protein of 51 kDa). Inhibition of RGP51 expression by RNA interference inhibits regenerative outgrowth of adult Lymnaea neurons in culture. These results implicate regulated proteolysis in the formation of retrograde injury signaling complexes after nerve lesion and suggest that this signaling modality utilizes a wide range of protein components. Information on axonal damage is conveyed to neuronal cell bodies by a number of signaling modalities, including the post-translational modification of axoplasmic proteins. Retrograde transport of a subset of such proteins is thought to induce or enhance a regenerative response in the cell body. Here we report the use of a differential 2D-PAGE approach to identify injury-correlated retrogradely transported proteins in nerves of the mollusk Lymnaea. A comprehensive series of gels at different pI ranges allowed resolution of ∼4000 spots by silver staining, and 172 of these were found to differ between lesioned versus control nerves. Mass spectrometric sequencing of 134 differential spots allowed their assignment to over 40 different proteins, some belonging to a vesicular ensemble blocked by the lesion and others comprising an up-regulated ensemble highly enriched in calpain cleavage products of an intermediate filament termed RGP51 (retrograde protein of 51 kDa). Inhibition of RGP51 expression by RNA interference inhibits regenerative outgrowth of adult Lymnaea neurons in culture. These results implicate regulated proteolysis in the formation of retrograde injury signaling complexes after nerve lesion and suggest that this signaling modality utilizes a wide range of protein components. Injured neurons from mammalian peripheral or invertebrate central nerves retain a capacity for functional regeneration that is dependent on extrinsic cues as well as on factors intrinsic to the cell (1Caroni P. Neuro-regeneration: Plasticity for repair and adaptation..Essays Biochem. 1998; 33: 53-64Google Scholar, 2Goldberg J.L. How does an axon grow?.Genes Dev. 2003; 17: 941-958Google Scholar, 3Snider W.D. Zhou F.Q. Zhong J. Markus A. Signaling the pathway to regeneration..Neuron. 2002; 35: 13-16Google Scholar). The latter have been postulated to include injury signals emanating from the lesion site of the damaged axons. Such injury signals may include axoplasmic proteins activated by post-translational modifications at the injury site and conveyed by retrograde transport to the cell body (4Ambron R.T. Walters E.T. Priming events and retrograde injury signals—A new perspective on the cellular and molecular biology of nerve regeneration..Mol. Neurobiol. 1996; 13: 61-79Google Scholar, 5Perlson E. Hanz S. Medzihradszky K.F. Burlingame A.L. Fainzilber M. From snails to sciatic nerve: Retrograde injury signaling from axon to soma in lesioned neurons..J. Neurobiol. 2004; 58: 287-294Google Scholar). Placement of ligatures between the neuronal cell bodies and the lesion site in an injured nerve allows the collection of axoplasm enriched in retrogradely transported components, and studies by Ambron and colleagues in the mollusk Aplysia have sought to identify such components by a candidate protein approach (6Zhang X.P. Ambron R.T. Positive injury signals induce growth and prolong survival in Aplysia neurons..J. Neurobiol. 2000; 45: 84-94Google Scholar). The identity of most Aplysia retrograde injury signaling proteins has not yet been determined, although at least one appears to belong to the mitogen-activated protein kinase family (7Sung Y.J. Povelones M. Ambron R.T. RISK-1: A novel MAPK homologue in axoplasm that is activated and retrogradely transported after nerve injury..J. Neurobiol. 2001; 47: 67-79Google Scholar, 8Lin H. Bao J. Ying J.S. Walters E.T. Ambron R.T. Rapid electrical and delayed molecular signals regulate the serum response element after nerve injury: Convergence of injury and learning signals..J. Neurobiol. 2003; 57: 204-220Google Scholar). An importin-dynein complex was recently shown to transport retrograde injury signal proteins in mammalian nerve, but the identity of these signaling proteins is still unknown (9Hanz S. Perlson E. Willis D. Zheng J.Q. Massarwa R. Huerta J.J. Koltzenburg M. Kohler M. van-Minnen J. Twiss J.L. Fainzilber M. Axoplasmic importins enable retrograde injury signaling in lesioned nerve..Neuron. 2003; 40: 1095-1104Google Scholar). The candidate protein approach can occasionally be very successful, but the anatomical and molecular complexity of the nervous system suggest that most signaling systems are not likely to be critically dependent on single proteins (10Grant S.G. Synapse signalling complexes and networks: Machines underlying cognition..Bioessays. 2003; 25: 1229-1235Google Scholar). Thus for example the protein complexes involved in synaptic signal transduction are typically made up of ∼100 interacting proteins, and most recent estimates suggest that 700–1000 proteins are required to create the signaling network for a working synapse (10Grant S.G. Synapse signalling complexes and networks: Machines underlying cognition..Bioessays. 2003; 25: 1229-1235Google Scholar, 11Husi H. Ward M.A. Choudhary J.S. Blackstock W.P. Grant S.G. Proteomic analysis of NMDA receptor-adhesion protein signaling complexes..Nat. Neurosci. 2000; 3: 661-669Google Scholar, 12Jimenez C.R. Eyman M. Lavina Z.S. Gioio A. Li K.W. van der Schors R.C. Geraerts W.P. Giuditta A. Kaplan B.B. van Minnen J. Protein synthesis in synaptosomes: A proteomics analysis..J. Neurochem. 2002; 81: 735-744Google Scholar). It is very likely that long-range retrograde transduction of signals along axons will require molecular machines of similar complexity in composition. We therefore sought to characterize retrograde injury signal complexes by using a comprehensive differential proteomics approach in the freshwater mollusk Lymnaea stagnalis. This invertebrate model nervous system is advantageous for both in vitro and in vivo analyses of regeneration in single neurons and small networks (13Syed N.I. Ridgway R.L. Lukowiak K. Bulloch A.G. Transplantation and functional integration of an identified respiratory interneuron in Lymnaea stagnalis..Neuron. 1992; 8: 767-774Google Scholar, 14Koert C.E. Spencer G.E. van Minnen J. Li K.W. Geraerts W.P. Syed N.I. Smit A.B. van Kesteren R.E. Functional implications of neurotransmitter expression during axonal regeneration: Serotonin, but not peptides, auto-regulate axon growth of an identified central neuron..J. Neurosci. 2001; 21: 5597-5606Google Scholar, 15Lukowiak K. Haque Z. Spencer G. Varshay N. Sangha S. Syed N. Long-term memory survives nerve injury and the subsequent regeneration process..Learn Mem. 2003; 10: 44-54Google Scholar), and both Lymnaea and other mollusks have served as pioneer preparations for development of proteomic approaches in the nervous system (16El Filali Z. Hornshaw M. Smit A.B. Li K.W. Retrograde labeling of single neurons in conjunction with MALDI high-energy collision-induced dissociation MS/MS analysis for peptide profiling and structural characterization..Anal. Chem. 2003; 75: 2996-3000Google Scholar, 17Jimenez C.R. van Veelen P.A. Li K.W. Wildering W.C. Geraerts W.P. Tjaden U.R. van der Greef J. Neuropeptide expression and processing as revealed by direct matrix-assisted laser desorption ionization mass spectrometry of single neurons..J. Neurochem. 1994; 62: 404-407Google Scholar, 18Page J.S. Rubakhin S.S. Sweedler J.V. Single-neuron analysis using CE combined with MALDI MS and radionuclide detection..Anal. Chem. 2002; 74: 497-503Google Scholar). We used two-dimensional electrophoresis to compare retrogradely concentrated axoplasm from lesioned and control Lymnaea nerve and identified differential spots by high-performance tandem mass spectrometry (MS). 1The abbreviations used are: MS, mass spectrometry; 2D-PAGE, two-dimensional PAGE; CID, collision-induced dissociation; DTT, dithiothreitol; HRP, horseradish peroxidase; IF, intermediate filament; IPG, immobilized pH gradient; LPS, Lymnaea physiological saline; RACE, rapid amplification of cDNA ends; RGP, retrograde protein; dsRNA, double-stranded RNA. The results indicate that retrograde injury signaling may be mediated by soluble protein complexes arising from cleavage or modification of a wide variety of axonal proteins coupled with the loss of vesicular signal components normally trafficked in the nerve. Central ganglia of Lymnaea were dissected to expose the main nerve tracts radiating to peripheral targets. Nerves were first ligated close to the ganglia and then crush-lesioned with fine forceps ∼2 cm from the ligation site. The animals were incubated in a bath of Lymnaea physiological saline (LPS: NaCl 53 mm, KCl 1.7 mm, CaCl2 4.1 mm, MgCl2 1.5 mm, HEPES 5.0 mm, pH 7.9) at 20 °C, and at given time points axoplasm was collected from ligated nerve segments by gentle squeezing into clean sterilized LPS with 1 mm orthovanadate and protease inhibitors mixture (Merck, West Point, PA). The extract was clarified by a 1-min centrifugation at 20,000 × g to obtain pellet 1. The supernatant was then subjected to ultra-centrifugation for 1 h at 250,000 × g to obtain pellet 2 and soluble fractions. Both pellets were solubilized separately in lysis buffer with proteinase and phosphatase inhibitors. The three extracts were then processed separately for two-dimensional PAGE (2D-PAGE) analysis. Aliquots of 250–750 μg of protein were precipitated with 10% trichloroacetic acid in acetone and 20 mm dithiothreitol (DTT), and protein pellets were resuspended in rehydration solution (7 m urea, 2 m thiourea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 60 mm DTT, 2% Pharmalyte). First-dimension isoelectric focusing was carried out on an IPGphor system (Amersham Biosciences, Piscataway, NJ) essentially as described by the manufacturer. Precast immobilized pH gradient (IPG) strips (18 cm; 3.5–4.5, 4–5, 4.5–5.5, 5–6, 5.5–6.7, 4–7, 3–10, 6–11) were used in first-dimensional separation for a total of 35–70 kV-h. After the first-dimension separation, the strips were equilibrated with a solution containing 6 m urea, 50 mm Tris-HCl, pH 8.8, 30% glycerol, 2% SDS, 1% DTT, and bromophenol blue for 15 min. A second equilibration was then performed in DTT-free solution containing 4% iodoacetamide for 15 min. The strips were then directly applied to 10% Duracryl high-tensile strength acrylamide gels (Genomic Solutions, Ann Arbor, MI) for electrophoresis. Strips were overlaid with 0.5% low-melting-point agarose in running buffer containing bromophenol blue. Gels were run in a Hoefer DALT system (Amersham Pharmacia Biotech, Uppsala, Sweden), fixed and stained by a mass spectrometric-compatible silver stain, and then scanned and analyzed using ImageMaster 2D Elite software (Amersham Pharmacia Biotech) or Z3 software (Compugene, St. Louis, MO). Gels were always run in duplicate, and each experiment was repeated at least three times. Protein spots revealing over 3-fold change (after normalization) between lesion and control samples were excised and digested in-gel with trypsin (donatello.ucsf.edu/ingel.html). The digests were analyzed by liquid chromatography MS/MS using an Ultimate high-performance liquid chromatography system equipped with a FAMOS autosampler and a C18 PepMap 75-μm × 150-mm column (Dionex-LC-Packings, San Francisco, CA). Solvent A was 0.1% formic acid in water, and B was 0.1% formic acid in acetonitrile, at a flow rate of ∼350 nl/min. Approximately one-tenth of each digest (1 μl) was injected at 5% B, then the organic content of the mobile phase was to over min. The column was to a tandem mass equipped with a the MS were by collision-induced dissociation in were by the as and the most trypsin products were The was to the mass and of the The were mollusk has yet been protein assignment was carried out with the of M.A. Burlingame A.L. Functional assignment of the 20 from using mass spectrometry and new Chem. 2001; such as and comprising for the and were used for on cDNA from Lymnaea central nervous A single of was and on this and of were then used to obtain a cDNA by and and of RGP51 and were using was to obtain a RGP51 cDNA on both with the The was then used as a for of double-stranded RNA using RNA The was applied to a of in of neurons as described G. Z. van Minnen J. Syed N.I. Fainzilber M. of over of Neurobiol. 2004; Scholar). neurons were incubated in a for up to h and for an were to have at least 1 with growth and that were at least 2 soma in tracts radiating from the central ganglia of adult Lymnaea were ligated close to the and incubated with or nerve in a bath of physiological saline at 20 Retrograde transport in the system were to be in the range of by horseradish in the nerves were on 2 cm from the the to time was for collection of axoplasm at the ligation into three as in comprising soluble components from centrifugation and vesicular and components arising from and the soluble components not precipitated by centrifugation three were on × gels control to lesion samples to obtain the most comprehensive we used strips resolution and protein different of pI and molecular mass ranges were run to a total of ∼4000 axoplasmic protein spots by silver staining, 172 of were in lesioned versus control digests of the differential spots by peptide sequencing using in peptide from a total of 134 spots that be by to over 40 different proteins with cellular post-translational modifications in some of these proteins were revealed by MS, including and not and a novel of and K.F. Z. Perlson E. Fainzilber M. H. D. M. Burlingame A.L. of and spectrometric and of a new modification in proteins the 2004; 3: Scholar). to these directly of the spots cleavage of the identified proteins or with mass that may or PAGE of axoplasm samples from nerve from control and injured preparations were by centrifugation into three different and then over a first-dimension pH 4–7, by a 10% mass are at Gels were gels of Lymnaea nerve ligation axoplasm using strips to enhance resolution and of First-dimension strips from pH 3–10, 4–7, and mass are at The central column of the by the gels each is from the is from the injured indicate differential proteins identified in the peptide used for protein assignment are in the of kinase protein B dissociation The peptide used for protein assignment are in the in a new modifications in mass mass is to the Lymnaea protein or the in the mass on are from different in the protein are from different in the protein mass is to the Lymnaea protein or the in the The are from different in the protein in a new of the identified proteins to the axoplasm that the differential proteins in pellet 2 were in axoplasm from lesioned nerves. 2 was by an to from the and a number of the components in this are proteins, including a and These results indicate that vesicular components that normally retrogradely in the nerve are blocked by the a signal to the cell of differential proteins in the to the number of differential for each and may differ from the number of protein to different or modifications of a single protein in or to one protein identified in a single retrograde to mass on for example for retrograde protein of This is used for spots containing peptide with in the protein dissociation RNA protein protein The to the number of differential for each and may differ from the number of protein to different or modifications of a single protein in or to one protein identified in a single retrograde to mass on for example for retrograde protein of This is used for spots containing peptide with in the in a new The most proteins by on the peptide were from the intermediate filament and The that these a of after the is not other components to be highly in such as or were not found in of the spots from the spots were used to for and subsequent of the cDNA and spots were found to from a single RGP51 (retrograde protein 51 kDa). RGP51 the of the D. S. E. of intermediate filament Scholar), and the analyses indicate that the RGP51 components up-regulated by lesion are cleavage products of and 6 and the main spots up-regulated by lesion are cleavage in this of and 15 from the mass spectrometric analyses enable of the cleavage products on the protein and of one of the cleavage in RGP51 This cleavage site does not the protease calpain is to be activated nerve injury and has been to the of proteins P.A. calpain and loss of Scholar, to after injury in Neurosci. 17: Scholar, D. of for calpain growth formation after of Aplysia neurons..J. Neurobiol. 2002; Scholar). We therefore in vitro cleavage of by in vitro and incubated with axoplasm from injured Lymnaea nerve. shown in was in and the cleavage was blocked in the of the or the calpain The of the cleavage products in this experiment well with the in the 2D-PAGE and for segments indicate from mass spectrometric segments indicate used for of and indicate the and B, a using the of RGP51 to other of the peptide from digests of the and of as the for these B, of the peptide of of RGP51 and of cleavage products on on a of the The of is by nerve other from both proteins are up-regulated by the of the peptide from MS are each these served to the on the cleavage The of the and of RGP51 was by MS/MS the cleavage site is therefore by an in vitro cleavage of RGP51 in of or of inhibits the cleavage to the functional of RGP51 for neuronal regeneration in we carried out of RGP51 expression in of adult Lymnaea shown in neurons not in over a we of control The RGP51 not the of the neuronal cell bodies and not their survival over the time of the experiment not a axonal protein cleavage and retrograde transport nerve lesion and is for an regenerative response by lesioned The of this was to obtain a comprehensive of the retrogradely transported protein ensemble after nerve injury by using 2D-PAGE to compare control versus lesion axoplasm retrogradely concentrated at nerve are differential expression after nerve most such is at axonal regeneration in the central nervous system by the cell body response to Neurosci. 2002; Scholar, A new for regeneration: 2002; and does not the and post-translational thought to in lesioned axons E. Hanz S. Medzihradszky K.F. Burlingame A.L. Fainzilber M. From snails to sciatic nerve: Retrograde injury signaling from axon to soma in lesioned neurons..J. Neurobiol. 2004; 58: 287-294Google Scholar). to on retrograde injury signaling proteins in lesioned nerve have on candidate proteins to of their signaling or in other in the of in this (7Sung Y.J. Povelones M. Ambron R.T. RISK-1: A novel MAPK homologue in axoplasm that is activated and retrogradely transported after nerve injury..J. Neurobiol. 2001; 47: 67-79Google Scholar, 8Lin H. Bao J. Ying J.S. Walters E.T. Ambron R.T. Rapid electrical and delayed molecular signals regulate the serum response element after nerve injury: Convergence of injury and learning signals..J. Neurobiol. 2003; 57: 204-220Google Scholar). some of the novel proteins found in of signaling proteins, the results implicate a range of components in retrograde injury signaling and These include a injury vesicular ensemble comprising proteins normally transported in axons that are by the lesion to the of at the nerve to this vesicular other proteins were identified in soluble axoplasm and new candidate signaling proteins for the These proteins to from a number of post-translational the most of are in the mass of the proteins, including mass that may be to or and mass that are of proteolysis in the axoplasm of the most of the identified proteins in was the termed of were identified in of the differential of a Lymnaea cDNA this protein mass spectrometric and the of MS and allowed of a cleavage site that to the of of RGP51 in injury this cleavage site does not to protease we the of the protease is to be activated nerve injury P.A. calpain and loss of and to at a number of that are most likely proteolysis of Scholar). Axoplasmic cleavage of in RGP51 was blocked by with or by a calpain at least some of the products transported retrogradely nerve and may the in calpain has been in after nerve A. inhibitors as in nerve and N. Scholar, of and in neuronal of Neurobiol. 2000; 62: Scholar), of calpain is not calpain is required for events in neuronal and such as to after injury in Neurosci. 17: Scholar), the of S. A. Scholar, R. of in injured mammalian 2000; Scholar), and growth formation and D. of for calpain growth formation after of Aplysia neurons..J. Neurobiol. 2002; Scholar, E. A. regulate growth and of 2003; Scholar). These studies suggest that calpain may a at the lesion site that and growth formation R. A. D. the of an axonal into a growth after of Aplysia neurons..J. 2003; Scholar). that an for calpain at the lesion site be the cleavage of other to enable of the to the retrograde transport to the retrograde transport be the signaling of the identified intermediate filament candidate retrograde injury signaling have been thought to be H. Bao J. Ying J.S. Walters E.T. Ambron R.T. Rapid electrical and delayed molecular signals regulate the serum response element after nerve injury: Convergence of injury and learning signals..J. Neurobiol. 2003; 57: 204-220Google Scholar), the proteomic approach points out We used to that RGP51 is required for regeneration of adult Lymnaea neurons in and are the structural components of the axonal the a for RGP51 in signaling events underlying be that we out a structural for RGP51 in axon or The between structural E. A structural of intermediate in and 1998; versus signaling J.L. intermediate cell 2002; Scholar, P. and Chem. of is a in the and both may be in were shown to retrogradely after from an network to a in J.L. A. of intermediate filament revealed by protein 1998; Scholar). The on gels may such a structural in the the other or products may as for the transport of signaling proteins such as S. M. of a cellular protein that with the of Chem. 2000; or may signaling or by direct to or to R.L. R. P. of the acid of the intermediate filament protein by Scholar, R.L. P. of the intermediate filament protein in and in the of 2001; Scholar). will be to such for cleavage products in axonal retrograde signaling after nerve results implicate regulated proteolysis in the formation of retrograde injury signaling complexes after nerve lesion and suggest that this signaling modality utilizes a wide range of protein components. with

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