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Physiol. Genomics 29: 215-230, 2007. First published January 9, 2007; doi:10.1152/physiolgenomics.00255.2006 Free Article
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Received 6 November 2006; accepted in final form 6 January 2007.
Physiological Genomics 29:215-230 (2007)
1094-8341/07 $8.00 © 2007 American Physiological Society

Proteomics study of neuropathic and nonneuropathic dorsal root ganglia: altered protein regulation following segmental spinal nerve ligation injury

Naoka Komori1, Nobuaki Takemori1, Hee Kee Kim3, Anil Singh1, Seon-Hee Hwang3, Robert D. Foreman2, Kyungsoon Chung3, Jin Mo Chung3 and Hiroyuki Matsumoto1

1 Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
2 Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
3 Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
Peripheral nerve injury is often followed by the development of severe neuropathic pain. Nerve degeneration accompanied by inflammatory mediators is thought to play a role in generation of neuropathic pain. Neuronal cell death follows axonal degeneration, devastating a vast number of molecules in injured neurons and the neighboring cells. Because we have little understanding of the cellular and molecular mechanisms underlying neuronal cell death triggered by nerve injury, we conducted a proteomics study of rat 4th and 5th lumbar (L4 and L5) dorsal root ganglion (DRG) after L5 spinal nerve ligation. DRG proteins were displayed on two-dimensional gels and analyzed through quantitative densitometry, statistical validation of the quantitative data, and peptide mass fingerprinting for protein identification. Among {approx}1,300 protein spots detected on each gel, we discovered 67 proteins that were tightly regulated by nerve ligation. We find that the injury to primary sensory neurons turned on multiple cellular mechanisms critical for the structural and functional integrity of neurons and for the defense against oxidative damage. Our data indicate that the regulation of metabolic enzymes was carefully orchestrated to meet the altered energy requirement of the DRG cells. Our data also demonstrate that ligation of the L5 spinal nerve led to the upregulation in the L4 DRG of the proteins that are highly expressed in embryonic sensory neurons. To understand the molecular mechanisms underlying neuropathic pain, we need to comprehend such dynamic aspect of protein modulations that follow nerve injury.

circulatory proteins; dorsal root ganglion protein expression profiles; isozyme switching; myogenic proteins; peripheral neuropathy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
PERIPHERAL NERVE INJURY OFTEN develops into multiple symptoms of neuropathic pain. Shortly after injury, injured peripheral nerves undergo Wallerian degeneration, the degeneration of axons distal to the injury accompanying inflammatory processes. Degenerating nerves and myelin debris are phagocytosed by recruited macrophages and Schwann cells (49), i.e., the myelinating glia of the peripheral nervous system. To facilitate nerve degeneration and regeneration, injured nerves and macrophages secrete a variety of immune/inflammatory mediators. A large number of studies demonstrate a strong correlation between the severity of neuropathy and the extent of nerve degeneration accompanied by the elevated levels of cytokines and neurotrophic factors (29, 115). Abnormal levels of nerve growth factors available following injury to individual cells, either injured or noninjured, in the dorsal root ganglia (DRG) cause altered expression of neuropeptides, receptors, and channel proteins, leading to phenotypic changes of the primary sensory neurons. Neuronal cell death, due to the primary injury itself or neurochemical imbalance secondary to injury, proceeds over an extended period of time. Sites of abnormal sensory inputs extend from the peripheral location of injury to the higher order neurons via the primary sensory neurons in DRG.

Using animal models of neuropathic pain, many studies demonstrated altered regulation of individual proteins implicated in abnormal pain transmission in neuropathy. Moreover, recent DNA array and proteomics studies have reported a large number of genes and proteins that are regulated in vivo after peripheral nerve injury (5, 11, 22, 58, 89, 129, 137, 143). These studies have provided better pictures of cellular and molecular changes in the degenerating nerve and its microenvironment after sciatic nerve injury. The studies also demonstrated the regulation of different sets of genes and proteins, which might have resulted from the use of different species, the different types of injury, or the slight differences in their experimental designs, such as the time points of sampling and the analytical tools like microarray chips. In addition, mRNAs and proteins are subject to diverse regulations and modifications, and it is not always feasible to correlate the levels of certain mRNAs and the corresponding proteins (4, 43, 53) or the data between DNA array and proteomics studies. It is also true that, in proteomics, no single technique is currently capable of analyzing the levels of all proteins expressed in a cell (31) mainly because proteins with diverse physicochemical properties cannot be solubilized totally by a single solvent or detergent. For these reasons, more proteomics studies are needed to generate clearer pictures of global changes that follow nerve injury. Since there are apparent behavioral and morphological differences among the most utilized animal neuropathy models (38, 61, 68, 74), comparison of protein expression patterns from different types of injuries might help us understand elaborate molecular and cellular mechanisms underlying diverse peripheral neuropathy.

A segmental spinal nerve ligation injury model is one of the commonly used peripheral nerve injury models, and the time course and symptomatology of the neuropathy have been well defined. Rats develop symptoms of neuropathic pain within a few days after ligation of the 5th and 6th lumbar (L5/L6) spinal nerve (45, 62). The hypersensitivities are generally maintained for weeks following ligation. The number of DRG cells decreases in the first 6 wk to 70% of the original value, and the retrograde degeneration of dorsal root axons is observed for several months (70). Both injured and noninjured sensory nerve fibers comingle in the sciatic nerve distal to a ligation site, and nerve impulses generated from both injured L5 DRG and noninjured L4 DRG contribute for sensitization at the spinal cord level (20). Because we have limited knowledge as to how DRG, as a whole, would cope with a devastating injury that threatens the existence of the primary sensory neurons, the segmental spinal nerve ligation injury model will be an ideal system to study differential protein regulations in two different neuronal populations, one directly injured and another not injured, but in close contact with injured nerve fibers. Therefore, in this paper we analyzed, using a proteomic approach, the proteins that exhibited differential expression after ligation of L5 spinal nerve to obtain insight into what cellular mechanisms underlie this pathophysiological condition.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animals and surgical procedure.
Young adult male Sprague-Dawley rats ({approx}9–10 wk old, total of 55 rats) from Harlan Sprague-Dawley (Indianapolis, IN, or Prattville, AL) were used in the study conducted at University of Oklahoma Health Sciences Center and University of Texas Medical Branch. Animals were housed in groups of two in plastic cages with bedding and were provided with free access to food and water. Peripheral nerve injury was introduced by the ligation of L5 spinal nerve (62), and sham-operation was the same as the ligation procedures except for the actual ligation of the nerve. All animals were subjected to a behavior test (20) throughout the study period to observe the changes in their mechanical thresholds. The animal protocols used in this study have been approved by the Institutional Animal Care and Use Committee of The University of Oklahoma Health Sciences Center and that of The University of Texas Medical Branch. They also meet the requirements of the National Institute of Health Guide for the Care and Use of Laboratory Animals. For the analysis of protein regulation, 30 animals were divided into three experimental groups, and 10 animals per each experimental group were further divided into ligation (n = 5) and sham-operation (n = 5). One week after ligation, the animals from each experimental group were anesthetized with pentobarbital sodium (90 mg/kg ip) and perfused transcardially with prechilled saline. The ipsilateral (Ipsi) and contralateral (Cont) L4 and L5 DRGs from ligated (LG) and sham (SH) animals were carefully removed under a microscope, frozen immediately on dry ice, and stored at –80°C until they were analyzed. Eight groups of DRG samples thus prepared were designated as LG-L5-Ipsi, LG-L5-Cont, LG-L4-Ipsi, LG-L4-Cont, SH-L5-Ipsi, SH-L5-Cont, SH-L4-Ipsi, and SH-L4-Cont.

Two-dimensional gel electrophoresis.
We prepared three individual sets of DRG samples. Each set, consisting of eight groups of DRG samples prepared from ligated (n = 5) and sham-operated (n = 5) animals, was processed separately from other two sets during two-dimensional gel electrophoresis (2-DGE) procedures. Two-DGE was performed as previously described (82) with some modifications. In brief, eight DRG samples, each containing five DRGs, were homogenized in 150 µl of isoelectric focusing lysis buffer containing 8 M urea, 2 M thiourea, 4% CHAPS, 20 mM spermine base, and 10 mM Tris (2-carboxyethylphosphine) hydrochloride. After homogenization followed by centrifugation at 15,000 g for 10 min, the supernatants were loaded onto isoelectric focusing tube gels and subjected to the first dimension for {approx}6,500 Vh. The tube gels were transferred onto 11% SDS-PAGE gels for the second dimension. Molecular weight (MW) marker proteins used in SDS-PAGE were SeeBlue Plus2 prestained protein standard (Invitrogen Life Technologies, Carlsbad, CA). After SDS-PAGE, protein spots were visualized with Coomassie brilliant blue-G250 for protein expression analysis, and the gels were dried with cellophane sheets (Bio-Rad Lab., Hercules, CA). Supplemental gels were generated and stained with Coomassie brilliant blue-R250 for presentation and identification of low abundant protein spots.

Quantification of protein expression levels.
Images of gels were captured by the use of a 14-bit transmission scanner PowerLook III (UMAX Technologies, Dallas, TX) and the PowerScan program (Nonlinear Dynamics, Newcastle upon Tyne, UK). The scanner was precalibrated up to the transmission optical density of 3.1 using a Kodak Photographic Step Tablet No. 2 (catalog #152 3398; Eastman Kodak, Rochester, NY). The gel images from a total of 24 samples, i.e., three sets of eight DRG groups, were analyzed together with the Progenesis Workstation program (Nonlinear Dynamics), which automatically detects all protein spots on each gel and assigns the same protein identification number to the corresponding protein spots on all gels in an analysis. The protein spot boundaries and number assignments performed by the Progenesis program were visually inspected and manually corrected when necessary. When we detected isoelectric point (pI) variants of a same protein on a gel and if they were clearly separated from their pI variants, we considered each spot as an individual protein spot. When a protein appeared on a gel as a beaded string of spots with clear overlap between the adjacent spots and if spot separation would significantly skew the volume of one or all the spots involved, as happens when one spot is significantly larger than its neighboring spot(s), we considered the whole beaded structure as one protein spot provided that all spots were confirmed in mass analysis to be the same protein. After background subtraction and normalization, the expression level of each protein in SH-L4-Cont DRG was used as a standard to analyze differential protein expression of the protein in other DRGs. The mean ± SD (n = 3) of the expression level of a protein in each group was compared by one-way ANOVA followed by Tukey's multiple comparison test (GraphPad Prism; GraphPad Software, San Diego, CA). When the expression level of a certain protein was significantly different (P < 0.05) from the standard level, the protein was considered to be significantly regulated. When one or some, but not all, of the pI variants of a protein were significantly regulated, it was inspected if the significant density change in one spot was not due to a pI shift of another spot as often happens in the case of phosphorylation. Protein spots near the extreme basic or acidic ends of a gel were excluded from mass analysis even if they appeared to be significantly regulated.

Mass spectrometry.
Protein spots were excised from gels and subjected to mass spectrometry (MS) analysis. In-gel digestion, mass spectrometry, and database search were performed as previously described in detail (82, 128) with minor modifications. Each excised gel piece was digested with modified trypsin (catalog #V5111; Promega, Madison, WI) to generate tryptic peptides of the protein in the gel. After repeated extractions from the gel, the tryptic peptide sample was reconstituted with 0.2% (vol/vol) trifluoroacetic acid for both MS and MS/MS analyses. For MS analysis, individual samples were mixed with a matrix solution consisting of 10 mg/ml {alpha}-cyano-4-hydroxycinnamic acid in 50% (vol/vol) acetonitrile/0.1% (vol/vol) trifluoroacetic acid. Mass spectra were obtained by a matrix-assisted laser desorption/ionization time-of-flight MS (Voyager Elite; Applied Biosystems, Foster City, CA), and monoisotopic peaks were assigned by PerSeptive GRAMS/386 version 3.02. Resulting peptide mass fingerprinting data were submitted to the MASCOT peptide mass fingerprint program (Matrix Science, London, UK) to search for a protein candidate for each protein spot. Database searches were performed against the National Center for Biotechnology Information (NCBI) nonredundant database (version 20060325) using the following parameters; 1) the protein database under mammals (452,404 sequences), 2) unlimited protein MW/pI ranges, 3) presence of protein modifications including acrylamide modification of cysteine, methionine oxidation, protein NH2 terminus acetylation, and pyroglutamic acid, and 4) peptide mass tolerance of ± 0.25 Da. Experimental peptide mass values were screened against the predicted peptide masses of each entry in the database, and the significance of the peptide matching between an experimental protein and its foremost candidate protein was presented by a MASCOT, or MOWSE, score. In the current study, scores ≥69 correspond to P < 0.05. To confirm the identity of protein candidates acquired in MS analysis, peptide fragmentation, or MS/MS analysis, of at least two or three peptides per protein was performed using a matrix-assisted laser desorption/ionization quadrupole-ion-trap time-of-flight MS (AXIMA QIT; Shimadzu/Kratos, Manchester, UK) and Shimadzu/Kratos Launchpad software version 2.3.4. Prior to the analysis, individual tryptic peptide samples were desalted with C18 ZipTip (Millipore, Bedford, MA) and mixed with a matrix solution consisting of 2% (wt/vol) 2,5-dihydroxy benzoic acid in 50% (vol/vol) acetonitrile/0.1% (vol/vol) trifluoroacetic acid. The database search parameters were as described for MS analysis except that a mass tolerance of ± 0.5 Da was selected for precursor ions and ± 0.8–2.0 Da for fragment ions. We considered the confirmation to be positive when the peptides that we analyzed for confirmation obtained, either individually or together, a significant MASCOT score (≥40, P < 0.05).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
GRANTS
 REFERENCES
 
Regardless of the differences in the nature of injury or disease, neuronal cell death impacts not only injured neurons, but also neighboring intact neurons as well as nonneuronal cells surrounding them (25, 114). We now know that peripheral nerve injury could eventually lead to altered regulation of a large number of molecules. Although several gene array studies have been directed to investigate the genes regulated in the DRG following nerve injury (22, 129, 137, 143), proteomics studies are needed to corroborate the gene expression profiles in DRG. In this study, we performed a proteomics investigation of neuropathic DRG using the spinal nerve ligation model in which animals fully developed signs of neuropathy by 1 wk postligation of L5 spinal nerve. All spinal nerve-ligated animals demonstrated typical neuropathic behaviors as evidenced by a dramatic decrease in mechanical thresholds on the side of nerve ligation. Neither sham-operated rats nor the contralateral side of ligated rats showed behavioral signs of neuropathic pain. A schematic illustration of the primary sensory neurons in L4 and L5 DRG and of the site of nerve ligation is shown in Fig. 1. Nearly 15,000 sensory cell bodies are present in rat L4/L5 DRG (70), giving rise to the most prevalent cellular component of the DRGs. One week after the ligation of L5 spinal nerve, we collected DRG samples by transecting both the dorsal root and spinal nerve very close to the DRG enlargement (Fig. 1). We compared the protein expression in the ipsilateral and contralateral L4 and L5 DRGs from ligated and sham-operated animals. Figure 2 presents gel images of (A) a set of eight DRG samples and (B) enlargements of an area where two proteins were significantly regulated due to nerve ligation. On average we detected >1,300 protein spots from each DRG sample. Although the highest spot number [1,345 ± 53.11 (mean ± SD, n = 3)] was obtained from LG-L5-Ipsi DRG, the number was not significantly different from the other groups.


Figure 1
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  		Fig. 1. Schematic illustration of the primary sensory neurons in the 4th and 5th lumbar (L4 and L5) dorsal root ganglion (DRG) and of the site of nerve ligation. Following ligation of L5 spinal nerve (an arrow in gray), noninjured L4 sensory nerves (nerve fibers in black) in the sciatic nerve coexist with injured L5 sensory nerves undergoing degeneration (nerve fibers in gray). For the protein expression analysis, both ipsilateral and contralateral L4 and L5 DRGs were excised by transecting the dorsal root and spinal nerve very close to the DRGs (dotted lines) 1 wk postligation.

 

Figure 2
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  		Fig. 2. Two-dimensional gel profiles of DRG proteins. A set of 8 DRG samples (A) and enlarged images of an area (B, a rectangular box in A) where 2 proteins (arrows) exhibited significant regulation. One week postligation, both ipsilateral (Ipsi) and contralateral (Cont) sides of the L4 and L5 DRGs, i.e., L5-Ipsi, L5-Cont, L4-Ipsi, and L4-Cont DRG, were collected from ligated (LG, n = 5) and sham-operated (SH, n = 5) animals. Eight DRG samples in 1 set were processed simultaneously, but separately from other sets, in two-dimensional gel electrophoresis (2-DGE) analysis, and DRG gel images from 3 experimental sets were analyzed together by the Progenesis program. kDa, Apparent molecular weights of molecular weight marker proteins.

 
Approximately 100 protein spots exhibited significant changes in their expression levels 1 wk after ligation and sham-operation, of which 90 protein spots were positively identified in our MS analyses (Table 1). Although some proteins were not successfully analyzed in MS/MS analysis due to their minute quantity, they all had highly significant MS scores. The expression profiles and the gel positions of the 90 protein spots are presented in Table 2 and Fig. 3, respectively. In Table 2, we grouped the identified proteins into seven classes based on their established physiological roles. The 90 protein spots belong to 67 proteins because some of the proteins existed as pI variants. As evident with serum proteins (33), on a 2-DGE gel many proteins exhibit isoelectric heterogeneities caused mostly by posttranslational modification(s) such as glycosylation and phosphorylation. Consistent with this, we detected the pI variants of, e.g., fibrinogens that were significantly upregulated (Table 2). We also detected many high-MW cytoskeletal proteins (Fig. 3) despite an earlier concept that 2-DGE poorly resolves cytoskeletal proteins and high-MW proteins above {approx}100 kDa. In ligated animals, three major patterns of protein regulation were observed: 1) proteins regulated solely in the ipsilateral L5 DRG directly affected by nerve ligation, 2) some proteins regulated, whether significantly or not, in all four DRGs of ligated animals compared with sham animals, and 3) a small number of proteins regulated exclusively in L4-Ipsi DRG without significant change in their levels in L5-Ipsi DRG (Table 2). In sham animals, a number of protein spots exhibited significant regulation in the ipsilateral L5 DRG; however, almost all were regulated more significantly in ligated animals. No significant change in protein expression was detected among L5-Cont, L4-Ipsi, and L4-Cont of sham animals (Table 2). In our supplemental data, we present proteins that were regulated moderately to nearly significantly because their regulation might be important in a small subset of DRG neurons. (The online version of this article contains supplemental material.) We also present, as comparison, some proteins whose regulations have been reported in other studies.


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  		Table 1. List of proteins significantly regulated after nerve ligation and sham-operation

 

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  		Table 2. Expression profiles of DRG proteins 1 wk after nerve ligation and sham-operation. Proteins significantly regulated were grouped based on their established physiological roles

 

Figure 3
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  		Fig. 3. Gel images presenting protein spots identified in our mass analyses. Protein spots significantly upregulated (A) and downregulated (B) were designated by their abbreviated names listed in Table 1. kDa, Apparent molecular weights of molecular weight marker proteins.

 
Group 1: circulatory proteins participating in homeostasis and inflammatory response.
Group 1 consists mainly of circulatory proteins participating in homeostasis. Albumin, apolipoproteins, fibrinogens, {alpha}2-HS-glycoprotein/fetuin A, plasminogen, and transferrin are normally present at high levels in the serum as well as other biological fluids, such as cerebrospinal fluid and the vitreous humor of the eye. Because of relatively high vascular permeability to serum proteins in peripheral DRG and nerves compared with the central nervous system (80, 95), we could detect the circulatory proteins on all our gels. In agreement with our data, previous histological studies demonstrate the elevated levels of albumin (94), fibrinogen (2), and transferrin (103) in the vicinity of sciatic nerve lesion. The upregulation/recruitment of apolipoproteins was apparently to recycle lipids from degenerating myelin for use by regenerating axons. Apolipoproteins may also exert neuroprotective effects through their antioxidative properties (77, 104). In addition, APO E is essential for normal sensory functions including thermal nociception (41). The relatively small increase in the level of APO E compared with APO AI and APO AIV in our study may be because a massive upregulation of APO E is usually restricted to the distal side of nerve injury (118). On the other hand, APO AI and APO AIV might have been in large part recruited from the circulation as previously observed after sciatic nerve lesion (12). {alpha}1-Macroglobulin is present in plasma and inhibits a broad spectrum of proteinases. Interestingly, the protein was reported to enhance nerve growth factor-promoted neurite outgrowth (69). After cleavage of its bait region by a target proteinase, macroglobulin entraps the proteinase for clearance from the circulation through a cell surface receptor (144). Although rat {alpha}1-macroglobulin is a {approx}170-kDa protein, two spots of {alpha}1-macroglobulin observed on our gels were proteolytic fragments of the COOH terminus containing the receptor binding domain (144). The patterns of the pI variants exhibiting MW of {approx}36 kDa and pI of {approx}5 on our gels (Fig. 3) are similar to those found in rat serum (33).

Shortly after peripheral nerve injury, the nerve distal to the injury and a short segment of the proximal nerve undergo axonal degeneration (40). Accordingly, we observed a large number of proteins implicated in axonal degeneration and phagocytosis. Many of these proteins are immunomodulatory proteins found in group 1, like complement component 3 (C3) (14, 27, 28), galectin-3 (37, 111), hemopexin (78, 125), {alpha}2-HS-glycoprotein/fetuin A (57), plasminogen (2), and vitamin D-binding protein (50, 131). C3 is one of the most versatile and multifunctional complements (110) in host defense and local inflammatory mechanisms. It is also synthesized in the peripheral nervous system for normal regeneration and remyelination of axons (28). The activation of C3 is regulated through sequential proteolytic cleavages that produce a number of active peptide fragments. The protein spot detected in our study contains the ß-chain peptide of either C3b or C3c (56). Galectin-3, a member of the galectin family of ß-galactoside-binding lectins, participates in diverse biological events (65), and it is expressed in various cells including a subset of small DRG neurons (106). Recent studies reveal that galectin-3 plays a critical role in phagocytosis (37, 111). Hemopexin in heme transport and iron recovery is an acute-phase protein. It is not only present in the extracellular matrix of intact peripheral nerves and ganglia (17), but also upregulated in Schwann cells and invading macrophages during axonal degeneration (17, 78). Hemopexin is hardly detected either before or after injury in the central nervous system, indicating its special role in the peripheral nervous system (125). Vitamin D-binding protein, also known as group-specific component or Gc-globulin, is a multifunctional plasma protein that binds diverse lipophilic ligands, scavenges G-actin released into circulation after tissue injury (26), and plays a key role in macrophage activation (50). Because nerve degeneration involving phagocytosis is mostly restricted to the distal part of injury site, the upregulation of these proteins in the injured DRG could have been largely due to their elevated levels in the circulation. However, it is possible that some of the proteins are upregulated at both sides of nerve injury and involved in DRG cell death which is observed for weeks postligation.

As clearly demonstrated in Table 2, the majority of the proteins upregulated in sham animals belong to group 1. The increased levels of the proteins in both ligated and sham-operated animals were consistent with their enhanced infiltration through the blood-nerve barrier at the time of inflammation and nerve injury. Because of the tissue damage by sham operation just adjacent to the L5 spinal nerve, these proteins were apparently recruited to the DRG to prepare the sensory neurons for potential neuronal damage and repair. The upregulated proteins in sham-operated animals may help us distinguish proteins important for the general host-defense mechanisms from those specific to nerve injury.

Group 2: extracellular matrix proteins.
Several types of collagens, including type I (116) and VI collagens (98), are present in the peripheral nerves. In this study, a beaded filamentous type VI collagen was upregulated in the ipsilateral and contralateral L5 DRG, while a fibril-forming type I collagen was upregulated moderately to significantly in all the LG DRGs except for the ipsilateral L5 DRG (Table 2). In support of our data, previous studies have demonstrated that collagens are strongly upregulated at both sides of nerve injury (32, 116). Type VI collagen mediates the three-dimensional organization of the extracellular matrix, and therefore, its upregulation must have promoted extracellular matrix organization and Schwann cell migration and myelination. On the other hand, we do not know why type I collagen was upregulated in all the DRGs, except for the ipsilateral L5 DRG, of ligated animals (Table 2). Although we failed to observe transforming growth factor (TGF)-ß on our gels, TGF-ß is secreted after nerve injury by injured DRG cells and axons (122, 130), and it modulates the expression of extracellular proteins including type I collagen (135). In addition, nerve ligation in this study upregulated a number of proteins known to regulate the expression and/or functions of extracellular matrix-associated proteins (30, 60, 113, 139). Therefore, it is possible that type I collagen was under a complex chain of regulations leading to its downregulation in the ipsilateral L5 DRG. Further study is, however, needed to corroborate the regulation of type I collagen observed after nerve ligation. The last protein in group 2 is transforming growth factor-ß-induced protein (TGFBI), also known as Big-h3. TGFBI has been studied extensively in human cornea because mutations of this protein cause several phenotypically different corneal dystrophies (87). Recent studies demonstrate that TGFBI is upregulated in rat cerebral cortex by stab wounds (146) and in rheumatoid arthritis (90). It appears to enhance cell adhesion and migration through interaction with integrins (90).

Group 3: proteins important for the functional and structural integrity of peripheral nerves.
Group 3 consists of proteins that are strongly associated with cytoskeletal and axonal structures. The proteins in this group were further divided into those upregulated (up) and those downregulated (down) in Table 2. Actin and tropomyosin are the major components of neuronal microfilaments. In this study we detected the upregulation of actin and tropomyosin-2ß, -3{gamma}, and -4{delta}. The tropomyosin spot we identified as tropomyosin-3{gamma} is also known as nonmuscle tropomyosin-5 NM-1. Two different actin isoforms, ß and {gamma}, and at least 10 tropomyosin isoforms including 3{gamma} and 4{delta} are present in neurons, and different combinations of actin and tropomyosin isoforms are found in functionally distinct regions of the neurons (42). Tropomyosins stabilize actin cytoskeleton by competing with actin depolymerizing molecule(s), and therefore, their upregulation in the DRG was apparently to stabilize and reconstruct, if necessary, actin cytoskeleton and neuronal microfilaments. In cells, actin cytoskeleton is dynamic, constantly exchanging actin subunits via polymerization and depolymerization. Consistent with this, we observed a range of actin-associated proteins with distinct functional roles. Trp-Asp-repeat protein 1 (WDR1) is a member of the WD/WD40 repeat protein family, and it is thought to facilitate actin turnover in cooperation with an actin depolymerization factor, ADF/cofilin. Interestingly, the upregulation of WDR1 has been observed following the degeneration of the auditory sensory epithelium in birds (92). Many of the downregulated proteins in group 3b are associated with axonal outgrowth and synaptic rearrangement, like advillin/pervin that may regulate filopodia of neuronal growth cone (105) and the collapsin response mediator protein (CRMP) family members that mediate growth cone collapse during development. They are expressed in developing as well as adult DRG neurons (3, 105, 138). In the adult nervous systems, CRMP 3 and 4 may participate in calpain-mediated neuronal cell death in response to stress (51, 64). Another downregulated protein periaxin is expressed by myelinating Schwann cells and localized at the abaxonal membrane of mature myelin to support the linkage between the myelin and extracellular matrix. In agreement with our data, periaxin is strongly downregulated during axonal demyelination (58, 112).

We identified seven intermediate filament proteins on our gels, and our study demonstrates their differential regulations following L5 nerve ligation. One week after nerve ligation, we observed the upregulation of lamin A and vimentin and the downregulation of neurofilament medium chain. Neurofilament light chain was also downregulated, almost significantly, to the extent very similar to the downregulation of the medium chain (Supplemental Tables A, B). Their downregulation in this study is compatible with a previous study showing the decreased levels of the neurofilament mRNAs in the DRG neurons following nerve lesion (142). Lamin A appeared on our gel as a string of {approx}6 spots, of which three spots were significantly upregulated in the ipsilateral L5 DRG. The analysis of the entire string as one spot also attained statistical significance (P < 0.001). The upregulation of lamin A in the injured DRG cells might have been to maintain normal sensory mechanotransduction (66). Although vimentin, another upregulated intermediate filament protein, is generally considered specific for glial cells, the presence of vimentin has been reported in the developing neurons in the central nervous system and peripheral DRG (21, 76) as well as in the sciatic nerve axoplasm after injury (99, 141). Intermediate filament proteins are differentially regulated in diverse cell types like neurons, glia, and muscles during development (24, 59, 145) and after different types of injuries (71, 81, 99). To understand the roles of intermediate filament proteins in injury, it may be necessary to explore factors like the energy requirements or the states of differentiation, besides the types, of cells that each protein is strongly associated with. Further investigation of their regulation after different types of injuries may give insight into a precise role(s) of each intermediate filament protein during development and under pathological conditions.

Group 4: membrane-associated proteins in signal transduction.
Group 4 is composed of channel proteins and signaling proteins that can modulate the functional and structural integrity of membranes (Table 2). The annexin family of Ca2+/phospholipid binding proteins performs a wide range of cellular functions from the regulation of membrane organization/traffic to the regulation of Ca2+ flux/signaling. We observed the upregulation of two of the members, annexin A1 and A3, which are differently expressed in DRG sensory neurons (88). In addition to the diverse functions they perform, annexin A1 has been reported to act as an apoptotic signal to phagocytes (97), while annexin A3 may assist fusion of phagosomes in neutrophils (35). Recent studies have yet uncovered annexin A1 as an acute-phase protein indispensable for anti-inflammatory responses (97), and therefore, this protein may actually play multiple roles in injured DRG cells and their microenvironment. In this report we grouped both annexins under signal transduction because their functions generally involve cell surface receptors and membrane-associated proteins (86, 97). Spinal nerve ligation also upregulated two members of the chloride intracellular ion channels (CLICs) that are involved in a wide range of cellular functions (36). They have been placed under the glutathione S-transferase superfamily, and upon oxidation, CLIC 1 forms functional channels that are likely controlled by redox-dependent processes (46). CLIC 4 may interact with ß-actin, dynamin, {alpha}-tubulin, and creatine kinase in the neuronal plasma membrane (124) for cell motility inhibition (108) and/or release of neuroactive molecules (19). The last protein in group 4 is Rho GDP dissociation inhibitor-ß, which modulates the activities of Rho family of GTPase and thereby the signals that regulate cell morphology (93) and adhesion (30).

Group 5: proteins associated with transcription and translation.
Proteins in group 5 are ribonucleoproteins that regulate transcription and/or translation (Table 2). Heterogeneous nuclear riboprotein L (HNRP-L) controls mRNA stability and splicing (109, 113). Major vault protein constitutes >70% of the total mass of vault, i.e., a conserved 13-MDa ribonucleoprotein particle of undetermined function. They are highly expressed in neural tissues and enriched in the nerve terminals (48) and lamellipodia (119). Recent studies strongly indicate the association of major vault protein with signaling pathways that modulate gene/protein expression (60, 123). The upregulation of HNRP-L and major vault protein in our study may be supported by their upregulation under pathological conditions (6, 9, 113). Poly (rC) binding protein 3 is a part of a ribonucleoprotein complex necessary for unconventional cap-independent translation, and it has been reported to regulate virus RNAs (117). Unlike HNRP-L or major vault protein, this protein was downregulated in this study. Although the exact functional roles of the above proteins in the peripheral nervous system, especially following nerve injury, are not clear, HNRP-L and major vault protein may participate in extracellular matrix remodeling via the regulation of extracellular matrix-associated proteins (60, 113, 121, 123).

Group 6: proteins in defense against oxidation and neuronal cell death.
Group 6 consists of antioxidants and chaperones (Table 2), and their upregulation was apparently to defend the sensory neurons and the neighboring cells against oxidative attacks. In the nervous system, nerve injury as well as stressful conditions can easily cause oxidative damage to the cytoskeletal and membrane structures, and it is comprehensible that the upregulated proteins have strong associations with antioxidative processes and the structural integrity of neurons. The presence of several chaperons in the injury-conditioned DRG neurons has been reported (141), of which HSP27 and HSP70–2 exhibited significant upregulation in the ipsilateral L5 DRG of ligated animals in this study. HSP27 stabilizes actin microfilaments and protects actin cytoskeleton against oxidative stress (52). The upregulation of HSP27 in this study is compatible with the significant increase in its immunoreactivity in the DRG cells, especially small-size neurons, after sciatic nerve transection (23) and corroborates the importance of HSP27 in the survival of peripheral sensory neurons during pathological events (8).

Group 7: cellular metabolic enzymes.
Proteins in group 7 are enzymes in metabolic pathways, and they were further subgrouped into those upregulated (up) and those downregulated (down) in Table 2. Many key enzymes in energy metabolism exist in several isoforms to adapt different cellular and tissue requirements. An embryonic isoform generally remains ubiquitous in many adult tissues, while other isoforms become dominant in tissues with high and fluctuating energy requirements, such as brain and muscles. Different isoforms often coexist in cells and even form heteromers (34, 102, 132) for functional diversity. This may be similar to the presence of {approx}10 tropomyosin isoforms in functionally distinct regions of neurons (42). In fact, most of the muscle/nonneuronal isozymes observed in this study have been reported to be present in the neurons of the brain (44, 47, 79, 136) or in the embryonic DRG sensory neurons (3). In addition to cellular compartmentalization, cytosolic enzymes form functionally coupled enzyme complexes in the plasma, organellar, and synaptic membranes where the immediate availability of energy is required (15, 54, 63). For example, ubiquitous {alpha}- and neuronal {gamma}-enolases both exist in the synaptic plasma membrane (132) and may interact with creatine kinase and pyruvate kinase (73). In the ipsilateral L5 DRG of ligated animals, we observed the upregulation of {alpha}-enolase, but not {gamma}-enolase, presumably because {alpha}-enolase can bind other enzymes with high affinity (84), resulting in tight coupling of enzymes and improved glycolytic flux. Pyruvate kinase M1 and M2 were also upregulated in the ipsilateral L5 DRG of ligated animals (Table 2). M2 is the predominant isoform found in metabolically active cells, such as proliferating cells and regenerating tissues, because the isoform allows cells to survive under suboptimal conditions (83), supporting the substantial upregulation of M2 over M1 isoform in our study. Downregulation of aldolase at the same time as the upregulation of other key glycolytic enzymes and glucose 6-phosphate dehydrogenase clearly points toward synchronized activation of the glycolytic and pentose phosphate pathways. The pentose phosphate pathway has been proven crucial for cellular defense against oxidative damage through recycling of glutathione (96). The regulations of the cellular enzymes were, thus, coordinated in such a way that the DRG cells could efficiently and simultaneously generate ATP for ATP-dependent cellular processes and NADPH for protection against oxidative insults. On our gels two isozymes of lactate dehydrogenase were detected. Neuronal lactate dehydrogenase B in brain is known to produce pyruvate in neurons from lactate released from astrocytes by glial-type lactate dehydrogenase A (10). In this study, the neuronal isozyme was significantly downregulated (Table 2). It should be noted that the upregulation of {alpha}-enolase and pyruvate kinase (1, 83) and the downregulation of lactate dehydrogenase B (16) may reflect the presence of hypoxic or similarly stressful conditions in the injured DRG. Like those previously observed in the spinal cord after injury (133), the upregulated enzymes, transferrin, and hemopexin may represent a group of hypoxia- and heme-responsive proteins in the injured DRG.

In the ipsilateral L5 DRG of ligated animals, we observed the downregulation of a number of mitochondrial proteins, including hydroxymethylglutaryl-CoA synthase 1, a cholesterogenic enzyme that is upregulated during the myelination of Schwann cells (134), and ATP synthase ß-subunit, an enzyme in the ATP biosynthesis. In addition, we observed fairly obvious downregulation of monoglyceride lipase, essential for the storage lipid synthesis in the peripheral nerves (134), and acetyl-CoA acetyltransferase 2, in fatty acid metabolism (Supplemental Tables A, B). The downregulation of the enzymes in the lipid biosynthesis in this study is consistent with the inhibition of the de novo lipid synthesis due to the increased free fatty acid levels from degenerating axons (140).

Another intriguing aspect of enzyme regulation observed in the current study was that L5 spinal nerve ligation upregulated proteins that are considered muscle-type in L4 DRG. The significant upregulation of carbonic anhydrase, muscle-type creatine kinase, muscle-type ß-enolase, and muscle-type tropomyosin-2ß was observed exclusively in the ipsilateral L4 DRG. Their similar expression patterns are shown in Fig. 4. The two spots of muscle-type isozyme (Table 2) share a relatively wide border between them, and therefore, they were analyzed as one spot in Fig. 4B (P < 0.001). In the search of genes that determine a peripheral sensory neuron phenotype, Akopian and Wood (3) isolated mRNA transcripts enriched in neonatal rat DRG. One of the DRG neuron-specific genes is now known to be advillin/pervin (105), which was significantly downregulated in this study. Surprisingly, the neonatal DRG-enriched clones included a number of genes normally associated with muscles like carbonic anhydrase, muscle-type creatine kinase, myosin, and troponin-T/-I (3). Carbonic anhydrase is expressed in proprioceptive sensory neurons in DRG (67), and the presence of myosin and troponin in DRG neurons is corroborated by other studies (85, 107). It is currently unknown if carbonic anhydrase, muscle-type creatine kinase, ß-enolase, and tropomyosin-2ß engage in a special function in a cell, which might have been particularly important for the L4 DRG cells. We speculate that one reason for the upregulation of so-called muscle-type isozymes is their capability to strongly interact with each other and with other neighboring proteins (39, 84, 100) and therefore to uphold functional and structural stability of cells under stress. Selective localization of muscle-type creatine kinase in human hippocampal neurons (44) and chicken Purkinje neurons (47) has been postulated to reflect the specific energy requirements of the specialized cells (47). Besides maintaining pH homeostasis, carbonic anhydrase 3 is a potent antioxidant (72); both functions are unquestionably vital to metabolically active cells. Because carbonic anhydrase is a marker of proprioceptive DRG neurons (67), the upregulation of the myogenic proteins seen in this study might have been confined in the proprioceptive sensory cells in the L4 DRG. Since the majority of the sensory fibers in the sciatic nerve originate from the L4 and L5 DRG (126), after nerve ligation noninjured L4 sensory fibers in the sciatic nerve are physically in close contact with injured L5 sensory fibers undergoing axonal degeneration. The loss of sensory innervation of muscles by the proprioceptive L5 sensory fibers might have led to the altered regulations of the myogenic proteins in the L4 DRG. The exact cause of their altered expression is currently under investigation. The sensory neurons in DRG can be categorized into 1) unmyelinated C-fibers or myelinated A{delta}- or Aß-fibers based on their sizes, myelination, and electrophysiological properties, 2) the somatic or visceral fibers based on their target tissues/organs, and 3) the nociceptors, mechanoreceptors, or proprioceptors based on the types of sensation they monitor. They can be further grouped into several subsets based on their biochemical properties (67, 120). In addition, the sensory cell bodies are partly surrounded by small glial cells called satellite cells. Therefore, extensive immunohistochemical analyses will be necessary to elucidate the exact cell type(s) in which the proteins observed in this study were regulated.


Figure 4
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  		Fig. 4. Proteins significantly upregulated in the ipsilateral L4 DRG of ligated animals. Carbonic anhydrase 3 (A), creatine kinase muscle-type (B), ß-enolase (C), and tropomyosin-2ß (D) all show similar expression profiles. Their expression levels were overall higher in all the DRGs from ligated animals than those from sham-operated controls, and the levels were relatively higher at the L4 level than the L5 level. DRG gel images from 3 experimental sets were analyzed together with the Progenesis program, and the expression level, as expressed as a normalized density (arbitrary units) of each protein spot, was determined. The average density of a protein spot in each DRG sample was compared against that in SH-L4-Cont (n = 3; *P < 0.05; ***P < 0.001). The graphs of muscle-type creatine kinase in B were generated from the combined data of the 2 isoelectric point variants.

 
In conclusion, as expected at the time of injury, we observed the regulation of a large population of proteins in host/cellular defense mechanisms and antioxidation. Many of the proteins were apparently recruited from the circulation or secreted by invading macrophages, while others might have been upregulated in the injured nerves and Schwann cells. The upregulation of many proteins in the actin cytoskeletal organization strongly supports the concept that stabilization of actin cytoskeleton is one of the priorities in a fight against injury or stress. We also observed the upregulation of several proteins which are regarded as muscle-, embryonic-, or glial-type. In support of our data, a large number of studies demonstrate that the proteins regulated in this study are subject to differential regulation in diverse cell types like neurons, glia, and muscles during development and after injury. Nearly half of the proteins that we identified in this study have been shown to be similarly regulated either at the mRNA or protein levels after other types of peripheral nerve injury, validating our experimental procedures and also substantiating the functional significance of the proteins in peripheral nerve injury. These proteins include annexins (129, 143), apolipoproteins (12, 13, 58, 118), C3 (14, 27, 28, 89), hemopexin (17, 78, 125), HSP27 (8, 23, 143), tropomyosins (58, 143), and vimentin (58, 91, 99). Many of the studies that supported our data at protein levels were the histochemical studies of individual proteins after sciatic nerve injury. The regulation of C3, CRMP 3, and mitochondrial creatine kinase in this study is corroborated by their changes at the mRNA levels in a study using the same injury model (137). Our proteomics data of the injured DRG are overall very comparable with those of the injured sciatic nerve by Jiménez et al. (58) except that there are quite a few differences in the expression profiles of cellular enzymes. Although this might have been due to the fact that we used a different injury model, we believe the discrepancy largely reflects different regulations of cellular enzymes in the cell bodies proximal to the injury site from those in the distal axons.

The proteins whose regulation following peripheral nerve injury has not been previously reported and whose regulations were specific to ligated animals include chloride intracellular channels, contrapsin-like protease inhibitor 21, HSP70–2, major vault protein, Rho GDP dissociation inhibitor-ß, and TGFBI/Big-h3. Further studies would elucidate if their regulations are specific to the nerve ligation model used in this study or common to other types of nerve injury as well. In the nervous system, levels of neuronal activity correlate well with the levels of glucose uptake. To respond to stress or injury, metabolic pathways that are normally inactive under steady-state conditions are activated while some of the regular pathways may be halted or bypassed. In addition, a substantial amount of energy is needed to make changes in protein expression and to build up cellular defense mechanisms. For these reasons, it is conceivable that we observed the regulation of many cellular enzymes, especially those in the energy metabolism. Although isozyme switching of some metabolic enzymes has been characterized during early developmental stages or in rapidly growing tumor cells, this study may provide more insight into how widely cellular energy metabolism would be affected by nerve injury and what kinds of cellular changes might be orchestrated to fight against neuronal degeneration. As many cellular enzymes have been revealed to engage in roles unrelated to their conventional functions (1, 7, 18, 75, 101, 127), the isozyme switching observed in this study may have other functional meanings. Since certain metabolic enzymes are now known to be the fundamental parts of neuron-specific functions (15, 54, 55, 132), it will be of great importance to elucidate how they are regulated after nerve injury.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by Presbyterian Health Foundation Grant 1401 (N. Komori) and National Institutes of Health Grants EY-13877, RR-17703 (H. Matsumoto), NS-035471 (R. D. Foreman), NS-11255 (K. Chung), and NS-31680 (J. M. Chung).


   ACKNOWLEDGMENTS
 
We thank Drs. Albert Chandler, Margaret J. Chandler, Jay P. Farber, and Robert A. Steinberg for reading this manuscript and for very helpful comments. We are grateful for the participation of Dr. Sadamu Kurono and NSF EPSCoR Oklahoma Biotechnology Network Laser Mass Spectrometry Facility, University of Oklahoma Health Sciences Center, Oklahoma City, during the initial part of this study.


   FOOTNOTES
 
Address for reprint requests and other correspondence: N. Komori, Dept. of Biochemistry and Molecular Biology, Univ. of Oklahoma Health Sciences Center, Oklahoma City, OK 73190 (e-mail: naoka-komori{at}ouhsc.edu).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


   REFERENCES
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