update on the pathobiology of neuropathic pain

799

Review

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Patients suffering from neuropathic pain due to metabolic disorders (e.g., diabetes, herpes zoster and infections), traumatic injury (chronic or acute), inflammation and neurotoxicity of periph-eral or central nerves are often only insufficiently treated, although a number of analgesic drugs are available [1–3]. The current pharmacological treatment of neuropathic pain includes opioids at higher doses; and tricyclic antidepressants such as amitriptyline, which act by modulation of ion channels and monoamine regulators. Built on these mechanisms, serotonin–norepinephrine reuptake inhibitors such as duloxetine are used for treatment of chronic depression comorbidity and diabetic neuropathy. Furthermore, ion chan-nel blockers such as anticonvulsants (e.g., gaba-pentin or pregabalin) are approved for neuro-pathic pain treatment. However, all these drugs have limited efficacy combined with a number of side effects (e.g., sedation and weight gain in the case of anticonvulsants) and the mechanism of their analgesic properties is not completely understood. This unsatisfactory therapy can drastically decrease the quality of life. Thus, there is a need for the development of novel and more effective drugs with low side effects in the treatment of neuropathic pain.

Neuropathic pain is defined as pain initiated or caused by a primary lesion or dysfunction in the nervous system, and is characterized by spontaneous pain, hyperalgesia (increased pain

response to a noxious stimulus) and allodynia (pain elicited by a normally innocuous stimulus), which can persist long after the initial injury has healed. These symptoms are related to peripheral and central sensitization mechanisms that lead to increased excitability of nerves due to changes in the expression and activity of ion channels, such as voltage-gated sodium channels or ligand-gated transient receptor potential channels; peripheral and central receptors (e.g., cannabinoid 1 or NMDA, respectively). Furthermore, induction of apoptosis and activation of microglia has been observed, and endogenous inhibitory as well as immune mechanisms are modulated and also contribute to sensitization [4,5]. These known mechanisms are taken into account for research on the development of novel analgesic drugs. Several new drugs have already entered clinical studies; however, all of these new substances are also associated with partially severe side effects. Many details on the molecular mechanisms of the pathology of neuropathies are missing, which is a great challenge for the treatment strategies of neuropathies [6–8]. Therefore, further insights into the molecular pathophysiology of neuropa-thies are still urgently needed and might sup-port the development of novel therapeutics for an effective treatment of neuropathic pain.

The knowledge of protein regulation involved in various diseases is an essential prerequisite for the discovery, design and evaluation of new

Ellen Niederberger†, Hilmar Kühlein and Gerd Geisslinger†Author for correspondence Pharmazentrum Frankfurt/ZAFES, Institut für Klinische Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universität Frankfurt, Theodor Stern Kai 7, 60590 Frankfurt am Main, Germany Tel.: +49 696 301 7616 Fax: +49 696 301 7636 [email protected]

Nerve injury or dysfunction in the peripheral and central nervous systems are the leading causes for the development of neuropathies, which are frequently associated with allodynia and hyperalgesia. Treatment of these disorders is often unsatisfactory due to side effects or insufficient analgesia of the currently available drugs. Therefore, elucidating the molecular mechanisms of neuropathic pain is an important prerequisite for the rational development of novel analgesic drugs for the therapy of neuropathic pain. Several proteomic approaches have been performed to explore protein modifications in the nervous system associated with neuropathies in different animal models, which might contribute to the detection of new drug targets. Furthermore, there are proteomic studies investigating human cerebrospinal fluid from patients suffering from neuropathies. The results of these studies and the potential clinical value of the proteomic data are summarized and discussed in this review.

Keywords: allodynia • analgesics • drug development • hyperalgesia • neuropathy • sensitization

Update on the pathobiology of neuropathic painExpert Rev. Proteomics 5(6), 799–818 (2008)

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drugs, which is underlined by the fact that pharmacologically active drugs very often target proteins because of their pathophysi-ological relevance. On the other hand, it has to be noted that among over 3000 proteins that have been suggested as reasonable drug targets, only approximately 500 are indeed used for pharma-cological therapy thus far [9]. In the clinical context, proteomics is suggested as a promising tool to identify biomarkers of vari-ous diseases. Clinical proteomics can be divided into expression proteomics, which can be utilized for the detection of diagnostic, prognostic and predictive biomarkers of diseases, and functional proteomics which should help elucidating novel drug targets. These parts of proteomics have already been applied in medical research for the examination of neurological and cardiovascular diseases, infections and, in particular, cancer [10]. As an example, a number of cancer biomarkers in different cancer types have been identified by SELDI-TOF mass spectrometry (MS) [11]. In addi-tion to the newly detected drug targets, the proteomic technique provides the possibility to screen already known drugs for effects apart from their known actions [12].

Since it is well known that neuropathic pain leads to sensitiza-tion mechanisms in the peripheral and central nervous systems, which involve transcriptional and post-transcriptional modifica-tions in sensory nerves [1,5], the ana lysis of protein modifications in nervous tissue using animal models of neuropathy might support the identification of pain-related proteins that can then be used as diagnostic markers or drug targets. If this aim can be reached in the future, the treatment conditions for patients suffering from neuropathic pain might be improved.

This review delivers an update and extension of a recent review that summarized a number of proteomic approaches that have been performed after applying animal models of neu-ropathic pain [13]. Results of these studies provide several new hypotheses regarding proteins involved in nerve injury, nerve degeneration and regeneration, and will thus increase the basic knowledge on neuropathic pain and provide hints for further drug development strategies.

Neuropathy models in rodentsIn the last two decades, a number of animal models have been developed, which cover a variety of different causes for neuro-pathic pain. The models are most frequently performed in rats and mice, and can be regarded as analogs to specific human pain-ful conditions. In most cases, traumatic injuries to the spinal cord or peripheral nerves are produced which result in chronic pain [14]. Animal models that have been applied to proteomic studies are summarized in Table 1.

Neuropathy models in the peripheral nervous systemSeveral disturbances such as trauma, compression, infections, metabolic diseases, neurotoxins, tumors and others can be respon-sible for peripheral neuropathic pain in humans. The human conditions of neuropathy can be mimicked in established rodent models of peripheral neuropathic pain, which comprise spinal nerve ligation (SNL), partial nerve injury, chronic constriction injury (CCI) and spared nerve injury (SNI). Peripheral animal

models are more frequently used than central neuropathy models since, in patients, peripheral neuropathies are also more frequent than central neuropathic pain states. Most of these models are illustrated in Figure 1.

Chronic compression of the dorsal root ganglionIn this model the L4 and L5 dorsal root ganglia (DRGs) are com-pressed by a stainless steel rod. This chronic compression leads to an ipsilateral cutaneous allodynia associated with increased excitability of neurons in the compressed ganglion. In patients, a herniated disk or spinal stenosis can compress these DRGs, and thus contribute to low back pain and sciatica [15,16].

Spared nerve injuryThe SNI model is based on section and ligation of two of the three peripheral branches of the sciatic nerve: the tibial and common peroneal nerves are ligated and the sural nerve remains intact. The difference to several other neuropathy models is that the co-ming ling of distal intact axons with degenerating axons is restricted, thus making behavioral testing of the noninjured skin territories adjacent to the denervated areas possible. SNI leads to a quick sensory hypersensitivity (<24 h) that lasts for at least 6 months. The mechanical and thermal hyperalgesia is increased in the ipsilateral sural and to a lesser extent saphenous territories, without any change in heat thermal thresholds [17].

L4

L5

L6

Dorsal root ganglion

Spinal nerve ligation

Spared nerve injury

Partial nerve injury

Chronic constriction injury

Sci

atic

ner

ve

Tibialnerve

Suralnerve

Commonperonealnerve

Figure 1. Animal models of neuropathic pain.

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ReviewProtein regulation in neuropathic pain

Neuroma formationTo induce neuroma generation the saphenous nerve is tightly ligated at the mid-thigh level with silk sutures and then cut distal to the ligature. The distal end of the tube is left open. Approximately 5 mm of the distal nerve stump is excised to pre-vent regeneration. Nerve endings in neuromas have been shown as hyperexcitable and mechanically sensitized and, therefore, provide an attractive model of nerve injury [18,19].

Spinal nerve ligationIn this experimental model of mononeuropathy the L5 and L6 spinal nerves are unilaterally ligated [20,21], resulting in symptoms

of hyperalgesia and allodynia within 24 h that last for at least 4 months. The model is not associated with autotomy, and de-afferentation is moderate. In a variant of this model only the L5 spinal nerve is ligated [20]. This surgery is easier to perform in comparison with combined L5/L6 ligation but also leads to the same symptoms. SNLs in rodents can be compared with the clinical conditions of injury to the nerve plexus or the dorsal root in humans.

Partial sciatic nerve ligation Unilateral ligation of 33–50% of the sciatic nerve high in the thigh leads to the required partial nerve injury. Following

Table 1. Overview of animal models of neuropathy applied in proteomic studies.

Study (year) Animal model Method Effect Comparable human disease

Ref.

Central models

Kang (2006) Spinal cord injury (traumatic)

Partial cut into the dorsal horn of the spinal cord at Th9–10 level [39]

Paralysis that recovers in approximately 2 weeks

Traumatic or ischemic injury of the spinal cord

[61]

Ding (2006) Spinal cord injury (complete transection)

Complete transection of the spinal cord at the thoracic level (Th9–10)

Complete paralysis of the hind legs; no regeneration possible

Traumatic or ischemic injury of the spinal cord

[42]

Peripheral models

Lee (2003); Alzate (2004)

L5/L6 spinal nerve ligation

Unilateral ligation of L5 and L6 spinal nerves [20]

Hyperalgesia and allodynia Injury to the nerve plexus or the dorsal root

[50,51]

Komori (2007) L5 spinal nerve ligation

Unilateral ligation of the L5 spinal nerve [20]

Hyperalgesia and allodynia Injury to the nerve plexus or the dorsal root

[52]

Zhang (2008) Chronic compression of DRGs

L4 and L5 DRGs are compressed by a steel rod [15,16]

Ipsilateral cutaneous allodynia

Herniated disk or spinal stenosis

[49]

Jimenez (2005) Peripheral nerve crush

Crush of the sciatic nerve at the mid-thigh level by hemostatic forceps [29,30]

Tactile and thermal hyperalgesia and allodynia; Wallerian degeneration of nerves

Sciatic nerve contusion [29]

Katano (2006) Partial nerve injury Unilateral ligation of 33–50% of the sciatic nerve high in the thigh [22]

Spontaneous pain; mechanical allodynia, mechanical and thermal hyperalgesia

Complex regional pain syndrome

[57]

Kuhlein et al. Spared nerve injury

Tibial and common peroneal nerves of sciatic nerve are ligated; sural nerve remains intact (spared nerve) [17]

Sensory hypersensitivity, mechanical and thermal hyperalgesia

Various peripheral neuropathies

[Kuhlein et al.

Unpublished data]

Kunz (2005) Chronic constriction injury

Constriction of the sciatic nerve through loose ligatures at the mid-thigh level [24]

Nerve inflammation; substantial loss of myelated and unmyelated fibres; spontaneous pain; thermal and mechanical hyperalgesia; cold and tactile allodynia

Lumbar disk herniation or nerve entrapment, heavy metal poisoning, anoxia and metabolic disorders

[58]

Huang (2008) Neuroma generation

Ligation of the saphenous nerve at the mid-thigh level [18,19]

Hyperexcitability and mechanical sensitization of neurons

Traumatic injury of peripheral nerves

[19]

DRG: Dorsal root ganglion.



surgery, animals develop spontaneous pain characterized by guarding behavior of the ipsilateral hind paw and licking start-ing within a few hours and lasting for several months. In addi-tion, animals suffer from allodynia to mechanical stimulation and hyperalgesia to thermal and mechanical noxious stimuli. Autotomy is absent in most cases and de-afferentation is mod-erate. Behavioral changes and sensory disorders in the partial sciatic nerve ligation model correlate with symptoms of com-plex regional pain syndrome in humans after peripheral nerve injury [5,22,23].

Chronic constriction injury of the sciatic nerveIn this painful peripheral mononeuropathy the sciatic nerve is unilaterally constricted through loose ligatures at the mid-thigh level [24,25], which results in nerve inflammation associated with extensive de-afferentation distal to the placement of the liga-tures. The CCI-model is similar to clinical conditions of chronic nerve compression in humans that can occur after lumbar disk herniation or nerve entrapment, heavy metal poisoning, anoxia and metabolic disorders [26,27].

Animals develop behavioral signs of spontaneous pain, hyper-algesia in response to thermal and mechanical nociceptive stim-ulation, as well as cold and tactile allodynia within the first 24 h after surgery lasting over a period of at least 2 months. It is suggested that this model also comprises an inflammatory component in the development of neuropathic pain because anti-inflammatory treatment of CCI-rats results in decreased hyperalgesia [28].

Experimental nerve crushIn this model the sciatic nerve is exposed at the mid-thigh level and crushed by hemostatic forceps with grooved jaws. Within a period of 3 weeks tactile and thermal hyperalgesia and allodynia are manifested and last for at least 52 weeks. The nerve crush leads to Wallerian degeneration with subsequent regeneration processes [29,30].

Neuropathy models in the CNSSpinal cord injury (SCI) is the leading model in the central neuro-pathies and corresponds to human conditions resulting from trau-matic or ischemic injury of the spinal cord. This neuropathy is most frequently associated with dysesthesia as well as spontaneous and evoked pain.

A number of animal models are available to induce SCI including weight drop [31,32], spinal cord compression [33], crushing [34], photochemically induced injury [35,36], excitatory neurotoxin methods [37,38] and spinal hemisection [39–41], as well as complete transection [42]. These models lead to spontaneous and evoked pain as well as allodynia and hyperalgesia. Here, only models that have been applied in proteomic studies are described.

Spinal cord hemisection In this model the spinal cord is only partially dissected at the Th 13 level, resulting in immediate flaccid paralysis of the

ipsilateral hindpaw. This paralysis is recovered 15 days after injury, thus allowing the investigation of the nociceptive behavior, which shows signs of hyperalgesia and allodynia [39–41].

Complete spinal cord transection Complete transection of the spinal cord at the thoracic level Th 9–10 leads to complete paralysis of the hind legs, which cannot be regenerated. This model is suitable to investigate the mechanism of the adult inability to regenerate neurons in the CNS [42].

Human neuropathy Human neuropathies can arise from a number of diseases, such as diabetes, herpes zoster infections and cancer, which are associated with nerve damage. However, since most of these diseases involve multiple signal transduction mechanisms it is difficult to distinguish between those that are related to nerve injury and those related to the underlying disease. Furthermore, the major problem in the investiga-tion of human neuropathic conditions is the extraction of sample tissue, which is not possible in most cases. To gain a better insight in human neuropathies, several studies have been performed using cerebrospinal fluid (CSF) as material for proteomic approaches. The CSF is in direct contact with the CNS and contains proteins that are involved in brain metabolism. Therefore, it might have a great impact on diagnostics of human neuropathies and might provide new basic knowledge for the development of novel therapies. CSF was mostly collected from the lumbar subarachnoid space of healthy volunteers and patients, respectively.

ProteomicsMost of the proteomic studies summarized in this review have applied the classical method of 2D polyacrylamide gel electropho-resis (PAGE) combined with protein identification by MS. The 2D-PAGE tehnique is composed of an isoelectric focusing in the first dimension and sodium dodecyl sulphate gel electrophoresis in the second dimension, allowing the separation and visualization of complex protein mixtures (>1000 proteins/gel) according to their isoelectric point (pI), molecular weight, solubility and relative abundance [43]. Quantification of the protein levels is performed by quantitatively analyzing the intensity of the protein stain. Regulated protein spots are subsequently separated and identification is gener-ally performed by MS specifically MALDI-TOF MS on the basis of peptide mass matching [44–46].

This method is generally well accepted as a powerful tool to analyze a great amount of proteins. However, there are several limitations that have to be taken in account. It is notable that low-level regulatory proteins cannot be well analyzed with this technique. Therefore, a number of regulations will probably not be detected with proteomic approaches. Furthermore, it is quite difficult to display high- and low-molecular-weight proteins as well as hydrophobic membrane proteins on the 2D gels. Since it is well known that most of regulated proteins in neuropathic pain, such as receptors and ion channels are membrane proteins, this might constitute another restriction for proteomic approaches. Moreover, preparations of nervous system tissues consist of a



hetero geneous mix of cells (e.g., neurons, astrocytes and micro-glia) that cannot be distinguished on the gels, thus making it difficult to draw a conclusion on the distinct cellular function of the respective regulated proteins.

Nevertheless, proteomics can deliver a huge amount of infor-mation on protein regulations that might help to identify novel mechanisms and can be used as a starting point to investigate the protein functions in further studies in more detail.

Protein modifications in nerve tissues in different animal models of neuropathyThe aforementioned animal models of neuropathy have been applied to assess regulations of the protein expression pattern in several tissues of the nervous system. Consistent with the fact that most of the applied animal neuropathy models show clear behavioral and morphological differences [47,48] the protein modifications differed strongly among the respective models and different tissues. This is also in accordance with different pain syndromes and a number of different causes of neuropathic pain in humans, which indicates that a specific nerve injury might have specific underlying mechanisms. An overview of the regu-lated proteins in all proteomic studies of neuropathy to date is provided in Table 2.

Peripheral neuropathyChronic constriction of dorsal root ganglia In the model of chronic compression of the DRGs (CCD) in rats, the protein regulations in the L4 and L5 DRGs have been investigated 28 days after CCD. From approximately 400 spots on the 2D gels, the authors found 98 proteins regulated after CCD, of which 15 proteins have been successfully identified by applying MALDI-TOF MS [49]. In this study, the regulations of Annexin a2, protein p11, PKCε, glyceraldehyde phosphate dehydrogenase and heat-shock protein 70 are emphasized. The authors conclude that Annexin a2, protein p11 and PKCε are involved in the regulation of membrane trafficking, as well as the expression and sensitivity of ion channels and might, therefore, contribute to hyperexcitability of DRG neurons in neuropathic pain. Glyceraldehyde phosphate dehydrogenase and heat-shock protein 70 regulations are suggested to be secondary events due to processes of nerve injury and neuroprotection.

Spared nerve injuryIn a recent study by our group, proteomes of lumbar dorsal horn samples from SNI- and sham-operated rats were analyzed by 2D-DIGE. Among an average of 2300 protein spots/gel, we found 55 significantly regulated proteins (at least 40% regulation) from which one was up- and 54 were downregulated 7 days after nerve injury [Kuhlein et al.; Unpublished Data]. From the 55 different pro-teins that were analyzed by MALDI-TOF-MS, 38 were success-fully identified. Functionally, most of these proteins are involved in energy metabolism, cellular structure, signal transduction and DNA binding. Additional gene expression ana lysis of a number of selected proteins has been performed by applying RT-PCR to further assess the regulation on the transcriptional level. The

results showed that a number of proteins that have been found regulated in the 2D-DIGE ana lysis are similarly modified on the transcriptional level. However, several genes were not regulated in the RT-PCR, indicating that the regulation is instead due to translational or post-translational modifications or to subcellular localization [Kuhlein et al.; Unpublished Data].

NeuromaThe protein expression pattern of hyperexcitable neuromas has been investigated in rats and mice 2 days, and 1, 2 and 3 weeks, after neuroma induction, respectively, in comparison with untreated control animals. Using DIGE ana lysis for mouse neur-oma samples, the authors found approximately 200 regulated pro-teins (>1.75-fold difference) among approximately 1800 protein spots/gel at each time point. More than half of the proteins were upregulated in neuroma samples. In total, 55 spots were identi-fied by combined MALDI-TOF MS and quadrupole-TOF liquid chromatography-MS/MS and have been classified into functional groups. Different expression of several of the identified proteins was then investigated by immunoblotting neuromas from rats. Since results delivered similar regulations as compared with the mouse 2D-DIGE experiments, the authors concluded that the observed changes can be translated across species [19].

Spinal nerve ligationTwo alternative models of spinal nerve ligation have been applied by three groups. Protein modifications were monitored in the spinal cord, the brainstem or the DRGs [50–52]. Interestingly, none of the proteins that have been found regulated in the brainstem was also regulated in the spinal cord and the DRGs and vice versa. Comparing DRGs and the spinal cord, there was only one over-lapping regulation, namely a downregulation of creatine kinase B. These results indicate that the nociceptive transmission involves different proteins at different levels and tissues.

In detail, a model of L5/L6 nerve ligation revealed five pro-teins with different expression levels (no minimum regulation threshold indicated) in the spinal cord after nerve injury [50]. The downregulation of creatine kinase B has been suggested as particularly important for the development and maintenance of neuropathic pain, since creatine has been described to reduce glutamate levels and to exhibit neuroprotective properties [53–55]. Therefore, the authors concluded that creatine kinase might be a reasonable target in the therapy of neuropathic pain.

Investigation of the differential protein expression patterns in the brainstem by applying the same model led to the detection of 14 upregulated and seven downregulated proteins (≥30% regulation) 7 days after nerve ligation in comparison with sham-operated rats. The proteins serve diverse functional properties and the authors suggested that the protein modifications might reflect primary and secondary events that could contribute to the progression of neuropathic pain [51].

A study that applied the modified SNL model by ligation of the L5 DRG only analyzed protein changes in the L4 and L5 dorsal root ganglia. They found 67 regulations (no minimum regulation threshold indicated) among approximately 1300 separated proteins.



Those proteins were involved in mechanisms critical for structural and functional integrity of neurons and the defense against oxidative damage as well as the regulation of metabolic processes [52].

Partial nerve injuryThe model of partial nerve injury has been applied in rats to inves-tigate the polarity of primary afferent fibers. In total, 12 proteins have been uniquely found in the spinal nerves peripheral to the DRG and three in the central region. The authors concentrated on characterization of the collapsin-response mediator protein (CRMP-2), which was only peripherally regulated while total CRMP-2 levels remained unchanged after nerve injury. Owing to this result, and since it is already well known that CRPM-2 acts as a regulator of neuronal polarity, axonal growth and regeneration after nerve injury [22,56], it has been suggested that periCRMP-2 might be related to pathophysiological changes in the CNS (spinal nerves) and regeneration processes in the periphery [57].

Chronic constriction injuryThe CCI model in rats has been used by our group to analyze the protein expression pattern in the lumbar spinal cord after nerve injury. We found approximately 500 protein spots on the 2D gels from which five were significantly regulated (≥40% regulation) 14 days after induction of CCI. In this study, we compared regula-tions of proteins in the spinal cord associated with neuropathy with regulations in the context of inflammatory pain (zymosan-induced paw inflammation). Since only one overlapping regulation was observed, we concluded that inflammatory and neuropathic pain have distinct regulatory mechanisms in the spinal cord [58].

Sciatic nerve crushProteomics have been applied to assess the protein expression pro-file of the rat sciatic nerve in a model of experimental nerve crush. The main aim of this study was the detection of proteins involved in regeneration processes of the sciatic nerve. Tissue was prepared at 5, 10 and 35 days after injury to cover immediate responses to injury as well as regeneration processes. Approximately 1500 spots were resolved on each gel, and at least 121 protein modifications (no minimum regulation threshold indicated) have been observed during at least one time point. Proteins were divided in several functional classes such as protein synthesis/maturation/degrada-tion, cytoskeletal (re)organization or lipid metabolism, and it was concluded that the detected proteins might reflect the complexity and the temporal aspects of nerve regeneration [29].

Central neuropathySpinal cord injuryDing et al. performed a model of complete spinal cord transec-tion that leads to paralysis of the hind legs. Therefore, this study was not intended to investigate pain related to the nerve injury but mainly focused on proteins that might be responsible for the inability of the adult CNS to regenerate nerve damage. These proteins are of great importance since it has to be suggested that axonal regeneration might enhance the rewiring of the neuronal network and thus promote functional recovery [59,60]. The authors

found an upregulation 5 days after injury of more than 30 proteins (≥1.5-fold regulation) in the spinal cord, which might be involved in injury and regeneration processes. Correspondingly, the proteins have been subdivided in different functional groups concerning stress response and metabolic changes, lipid and protein degen-eration, and neural survival and regeneration. In particular, two upregulated proteins (11-zinc-finger protein and glypican) are highlighted as inhibitors for axonal growth and regeneration [42].

In another study, protein regulations in the injured spinal cord of rats have been assessed after traumatic injury [61]. Among 947 protein spots on the 2D gels, 39 upregulated and 29 down-regulated proteins (≥ twofold regulation) have been detected 24 h after spinal cord injury. Proteins have also been categorized in the categories transporters, signal transduction, protein synthesis and processing, metabolism, angiogenesis/circulatory system, apoptosis, cell adhesion and migration, cell cycle, neural func-tion, carcinogenic and DNA binding. A number of these protein regulations could be confirmed by additional immunohisto-chemical ana lysis, which led to the conclusion that secondary events after spinal cord injury include apoptotic cell death as well as regeneration processes.

Protein modifications in human cerebrospinal fluidConti et al. investigated CSF from healthy controls in comparison with patients with different peripheral neuropathies associated or not associated with pain (PN and NPN, respectively) [62]. It has been stated that the disease course was chronically progressive in all cases. They found ten differentially regulated proteins (at least 1.5-fold variations) from which six were identified. In pain patients, cystatin C was increased in comparison with controls and nonpain patients, whereas the IgM autoantibody IgM cold agglutinin was decreased when compared with controls. FAM3C, a soluble cytokine-like protein, was present in all PNs but only in approximately 50% of NPNs. Three isoforms of pigment epithelium-derived factor (PEDF) were decreased in nonpain patients as compared with controls. Interestingly, western blot experiments revealed that three other isoforms of PEDF are not regulated in these patients indicating a differential expression of PEDF isoforms in different pathologies. The authors considered regulation of the cysteine protease inhibitor cystatin C and the glycoprotein PEDF as the most interesting observations since cystatin has already been described as a marker of pain and PEDF as neurotrophic and neuroprotective factor [62].

Another approach compared CSF from healthy controls with CSF from patients with amyotrophic lateral sclerosis (ALS) and patients with purely motor peripheral neuropathy. The main aim of this study was the identification of biomarkers for ALS. As a biomarker for ALS, the authors focused on three proteins that are stably and reliably decreased in ALS (4.8; 6.7 and 13.4 kD; ‘three protein model’). The 4.8 and 14.4 kD proteins have been identified as cys-tatin C and a proteolytic fragment of the neuro endocrine-specific protein, VGF. In patients with peripheral neuropathies these pro-teins have not been regulated in the CSF [63]. This is in contrast to the increase of cystatin expression as shown earlier, indicating that different neuropathies show differential regulation of this protein.



Table 2. Summary of proteins regulated after nerve injury.

Animal model

Regulated protein Protein identification number

Isoelectric point

MW Change in expression

Ref.

Heat-shock proteins/chaperones/antioxidants

N Calreticulin precursor 100123639 4.09 47.9 2.67 [19]

SCI Chaperonin containing TCP1, subunit 3

40018616M 6.2 60.6 -4 [61]

SCI Similar to chaperonin containing TCP-1 ζ-subunit

34872057 6.6 58.02 -2.78 [61]

CCI/NC α-B-crystallin 117388 6.8 20.1 0.53/decreased

[58,29]

SNL γ-crystallin C P02529 7.6 20.9 Increased [51]

NC DnaK-type molecular chaperone hst 70

92355 5.4 69.5 Increased [29]

SCI Guanidinoacetate methyltransferaseAdenylyl cyclase-associated protein homo

6978873 5.7 26.4 -2.65 [61]

SCI/NC/SNL Heat-shock 27 kDa protein 1 14010865 6.1 22.8 -1.64/decreased/increased

[29,52,61]

SNL Heat-shock 70 kDa protein 2 11177910 5.4 69.8 Increased [52]

CCD 70 kD Heat-shock-like protein 204667 5.37 71.06 Increased [49]

SNI/CCD Prohibitin 13937353 5.57 29.9 -2.12/decreased [Kuhlein et al., Unpublished

Data]; [49]

N Protein DJ-I 100117264 6.32 20 9.91 [19]

SNL Superoxide dismutase 2 8394331 9.0 24.9 Increased [52]

N T-complex protein I subunit-β 100320217 6.37 57.3 2.22 [19]

Neuronal function and structural proteins

SNL/N Actin-β or -γ 13592133 5.3 42.1 -1.8/increased [19,52]

SNL Actin-related protein 2 62655196 7.7 40.0 Increased [52]

CCD Similar to actin, cytoplasmic (γ-actin)

62645364 5.31 42.1 Decreased [49]

SNL Advillin/pervin 13242318 n.i. n.i. Decreased [52]

SNL Capping protein gelsolin-like 61556900 6.1 39.1 Increased [52]

NC/PNL/SNL Collapsin response mediator protein 2

135126 5.9 62.8 Decreased [29,52,57]

SNL Collapsin response mediator protein 3

34861163 6.5 62.7 Decreased [52]

SNL Collapsin response mediator protein 4

14518293 6.0 62.4 Decreased [52]

SCI Dynamin 1 18093102 6.3 95.9 -2.98 [61]

NC F-actin capping protein Z-β 4826659 5.7 31.1 Decreased [29]

SNL Fascin 1 30023548 6.6 52.2 Increased [52]

The regulations are indicated as described in the respective publication and show the fold increase or decrease (indicated by ‘-‘ or values <1 ) of the protein level. CCD: Chronic constriction of dorsal root ganglion; CCI: Chronic constriction injury of the sciatic nerve; CG-NAP: Centrosome and Golgi-localized PKN-associated protein; GAPDH: Glyceraldehyde phosphate dehydrogenase; HS: Heparin sulphate; LHON: Leber´s hereditary optic neuropathy; MW: Molecular weight; N: Neuroma; NADH: Nicotinamide adenine dinucleotide, reduced form; NC: Nerve crush; n.i.: Not indicated; NSF: N-ethylmaleimide-sensitive factor; PN: Peripheral neuropathies; PNL: Partial nerve ligation; SCI: Spinal cord injury; SNI: Spared nerve injury; SNL: Spinal nerve ligation; TCR: T-cell receptor; UDP: Uridine diphosphate.




Animal model


Isoelectric point


Ref.

Neuronal function and structural proteins

SCI Glial fibrillary acidic protein δ 5030428 5.8 48.8 26.71 [61]

SNL GTP-binding protein Rab 3A 7689363 4.8 24.9 Increased [51]

SNL Lamin A 1072002 6.3 74.6 Increased [52]

N Macrophage capping protein 100136906 6.7 39.2 2.64 [19]

SNL Myelin protein zero 8393778 9.5 27.9 Increased [52]

SNI Myelin basic protein 4454311 11.75 14.2 -1.84 [Kuhlein et al., Unpublished

Data]

CCD Myosin light polypeptide 6 2842665 4.46 17.14 Decreased [49]

SCI/N Neurofilament, light polypeptide

13929098 4.6 61.3 3.13/-5.93 [19,61]

SCI/N Neurofilament triplet M protein P12839 4.8 95.8 4.83/-4.69 [19,61]

SCI/SNL Neurofilament 3, medium 8393823 4.8 95.7 5/decreased [52,61]

SCI Neuronal differentiation-related gene

21326455 6.2 55.2 -3.55 [61]

NC/SNL Periaxin 9506999 6.4 16.1 Decreased [29,52]

SCI Peripherin 6981416 5.4 53.6 Only SCI/decreased

[19,61]

SNI Phosphohippolin 7672728 5.08 10.5 -1.57 [Kuhlein et al., Unpublished

Data]

N Plastin-2 100118892 4.96 70 1.75/4.03 [19]

SNL Ras-related protein Rab-1B 131803 5.6 22.1 Increased [51]

SCI Septin 2 16924010 6.1 41.6 -2.19 [61]

NC α-synuclein 9507125 4.7 14.5 Decreased [29]

N γ-synuclein 100271440 4.68 13.16 -1.87 [19]

SCI/PNL/N Tubulin, α 1 38328248 4.9 50.2 Only SCI/peripheral fraction/-2.59

[19,57,61]

N Tubulin, α 2 100117348 4.94 50.1 -3.77 [19]

N Tubulin, α 6 100403810 4.96 49.9 -3.29 [19]

SCI Tubulin β-chain 15 92930 4.8 49.9 2.67 [61]

NC Tubulin β-1 21746161 4.8 50.7 Increased [29]

N Tubulin β-2C 100169463 4.52 49.8 2.47 [19]

PNL Tubulin β-3 10998840 4.8 50.4 n.i. [57]

CCD/N Tubulin β-5 56754676 4.78 50.09 -2.41/decreased [19,49]

SNL Transgelin 2 61557028 8.4 22.6 Increased [52]

SNL Tropomyosin 2-β 20178269 4.7 33.0 Increased [52]

SNL Tropomyosin 3 29336093 4.8 29.3 Increased [52]





Animal model


Isoelectric point


Ref.

Neuronal function and structural proteins (cont.)

NC/SNL Tropomyosin 4, α 6981672 4.7 28.7 Increased [29,52]

SNI Predicted: tropomyosin isoform 6

62643678 29.2 -2.4 [Kuhlein et al., Unpublished

Data]

SNL Trp-Asp-repeat protein 1 62078997 6.2 67.0 Increased [52]

NC/SNL/N Vimentin 860908 4.8 44.7 Increased [19,29 52]

Proteins related to cellular homeostasis and metabolism

SCI/NC Aconitase 2, mitochondrial 40538860 7.9 85.4 3/increased

[29,61]

SCI/NC/ SNL/LHON

Albumin 19705431 6.1 68.7 15.24/ increased/decreased

[29,52,61,64]

NC/SNL Adenine phosphoribosyltransferase

114075 6.3 19.7 Increased [29,51]

SNI Adenylate kinase 1 59808167 5.14 21.7 -2.04 [Kuhlein et al., Unpublished

Data]

NC Aldehyde dehydrogenase 25990263 5.7 53.9 Decreased [29]

NC Aldehyde reductase 1 6978491 6.3 36.3 Decreased [29]

NC Aldolase A 6978487 8.3 39.8 Decreased [29]

NC/SNL Aldose C 1334163 6.8 39.7 Decreased [29,52]

N Aspartyl-tRNA synthetase 100122743 6.06 57.1 1.84 [19]

CCD ATP synthase α-subunit precurser

203055 9.22 58.9 Decreased [49]

SCI/NC Mitochondrial H1-ATP synthase α subunit

40538742 9.2 59.8 Only SCI/decreased

[29,61]

SCI Similar to ATPase, H1 transporting, V1 subunit A, isoform 1

34869154 5.4 68.3 2.66 [61]

NC/SNL/N ATP synthase β-subunit 92350 4.9 50.9 Increased/decreased/2.08

[19,29,52]

SNL/N Carbonic anhydrase 3 31377484 6.9 29.8 Increased/decreased

[19,52]

PNL/SNL Collagen-α 1 27688933 5.7 139.1 n.i./increased [52,57]

SNL Collagen-α 2 62665835 5.7 99.6 Increased [52]

SNL/N Creatine kinase B 417208 5.4 42.9 0.1 /-1.89/4.71 [19,50]

SNL Creatine kinase, mitochondrial 60678254 5.2 93.5 Decreased [52]

CCI/SNL Creatin kinase MM (muscle form)

6978661 6.6 43.2 0.63/increased [52,58]

SNI Similar to Cytochrom C oxidase VIb

62644353 6.72 18.2 -2.37 [Kuhlein et al., Unpublished

Data]





Animal model


Isoelectric point


Ref.

Proteins related to cellular homeostasis and metabolism (cont.)

SNI Cyt P-450 554434 5.8 12.5 5.16 [Kuhlein et al., Unpublished

Data]

SCI Similar to dihydropyrimidinase-related protein-2

34874349 6.3 81.4 3.13 [61]

NC Dimethylargininase 1 6912328 5.5 31.1 Decreased [29]

NC Dimethylargininase 2 27704430 5.7 30.1 Decreased [29]

SCI/SNL Eno1 protein 50926833M 6.2 47.1 2.1/increased [52,61]

N α-enolase 100462072 6.37 47 Increased [19]

SNL β-enolase, muscle-type 54035288 7.1 47.4 Increased [52]

N γ-enolase 100331704 4.73 47.2 2.08 [19]

SNL Fatty acid-binding protein, brain P55051 5.4 15.0 Increased [51]

NC/SNL Fibrinogen α-chain 71824 6.6 60.6 Increased [29,52]

SNL Fibrinogen β-chain 56971493 7.9 55.0 Increased [52]

SNL Fibrinogen γ-chain 61098186 5.3 53 Incrased [52]

NC γ enolase 182118 5 47.6 Decreased [29]

NC GAPDH 27661099 8.4 36.2 Decreased [29]

NC/SNL Galectin 3 1346429 8.9 25.6 Increased [29,52]

SNL Glucose-6-phosphate dehydrogenase

8393381 6.0 59.9 Increased [52]

SNL Glutathione peroxidase 1 2654236 7.7 22.5 Increased [52]

CCD Glutathione peroxidase 5 62663617 9.64 21.47 Decreased [49]

SNL Glutathione synthetase 25742757 5.5 52.7 Increased [52]

SNI Glutathione S-transferase, mu 5 25282395 6.33 27.1 - 2.12 [Kuhlein et al., Unpublished

Data]

SNI Glutathione S-transferase, mu 3 13592152 6.84 25.8 - 2.07 [Kuhlein et al., Unpublished

Data]

CCD Glyceraldehyde-3-phosphate dehydrogenase

8393418 8.14 36.09 Increased [49]

SNL Guanine deaminase 7533042 5.5 51.6 Increased [52]

NC/SNL Hemopexin 16758014 7.6 51.3 Increased [29,52]

SNL Hydroxymethylglutaryl-CoA synthase 1

8393538 5.6 58.2 Decreased [52]

SNL l-lactate dehydrogenase 120975 5.3 36.8 1.9 [50]

NC/SNL/N Lactate dehydrogenase B 6981146 5.7 37 -2.79/decreased [19,29,52]

SNI Latexin 14269568 5.77 25.7 -1.97 [Kuhlein et al., Unpublished

Data]





Animal model


Isoelectric point


Ref.


SCI Similar to leucine aminopeptidase

34878080 8.0 64.5 1.81 [61]

NC α-1 macroglobulin 202857 6.5 167.1 Increased [29]

NC Malat dehydrogenase 15100179 6.2 36.8 Decreased [29]

SNI Nucleoside diphosphate kinase 2


Data]

N Dipeptidase, unspecific 100315879 5.38 52,8 -2.62 [19]

SNI Peptidylprolyl isomerase A 8394009 8.34 18.1 -2.24 [Kuhlein et al., Unpublished

Data]

N Peroxiredoxin 1 100121788 8.26 22.1 Increased [19]

SCI/N Peroxiredoxin 2 34849738 5.3 21.8 2.581/1.79 [19,61]

SNI Peroxiredoxin 5, precursor 51261175 7.66 22.5 -1.57 [Kuhlein et al., Unpublished

Data]

SNL/SNI Peroxiredoxin 6 16758348 5.6 24.9 -1.88/increased [52]; [Kuhlein et al.,

Unpublished Data]

SCI NADH dehydrogenase 1-α subcomplex 10-like protein

30171809M 7.1 40.54 -1.55 [61]

SNL/N 3-phosphoglycerate dehydrogenase

55562727 6.3 57.5 2.5/decreased [19,52]

SCI/SNI Phosphoglycerate mutase type B subunit

8248819 7.1 28.8 1.97/-2.33 [61]; [Kuhlein et al.,

Unpublished Data]

NC/SNL Phosphoglycerate kinase 16757986 7.5 45.1 Decreased/increased

[29,52]

N Prolyl 4-hydroxylase-β 100122815 4.77 57.4 2.24 [19]

CCI/NC/N Protein disulfide isomerase 1352384 5.9 57.0 3.08/increased [19,29,58]

SNI Protein-l-isoaspartate O-methyltransferase 1


Data]

SNL Purine-nucleoside phosphorylase

34869683 6.5 32.6 Increased [52]

SCI/SNI Pyruvate dehydrogenase-β 50925725M 6.2 38.98 -2.47/-1.46 [61]; [Kuhlein et al.,

Unpublished Data]

SNL Pyruvate kinase M1 56929 6.6 58.4 Increased [52]





Animal model


Isoelectric point


Ref.


SNL/N Pyruvate kinase M2 62665891 7.6 58.6 Increased/mostly decreased

[19,52]

SCI Pyruvate kinase 3 16757994M 6.6 57.8 2.86 [61]

NC Sialic acid synthetase 27714479 6.3 40 Decreased [29]

SNL Nervous system cytosolic sulfotransferase

14522868 5.6 19.1 Increased [51]

NC Thioredoxin peroxidase 1 2499469 5.2 21.8 Increased as well as decreased

[29]

NC Thioredoxin peroxidase 2 16923958 8.3 22.4 Increased as well as decreased

[29]

SNL Transferrin 6175089 6.9 79.1 Increased [52]

NC/SNI Triose phosphate isomerase 12621074 6.5 26.9 Decreased/ -1.89

[29]; [Kuhlein et al.,

Unpublished Data]

CCI Ubiquinol-cytochrome c reductase iron–sulfur subunit, mitochondrial precursor (Rieske iron–sulfur protein).

136708 8.9 28.0 0.48 [58]

NC/CCD/SNI/N Ubiquitin C-terminal hydrolase 92934 5.1 24.7 Decreased/ -1.54/-2

[19,29,49, Kuhlein et al.,

Unpublished Data]

NC/SNL UDP glucose dehydrogenase 13786146 7.5 54.9 Increased [29,52]

NC/SNL Vitamin D-binding protein 476569 5.6 55.3 Increased [29,52]

Immune system

PN Chain A, Fab fragment human monoclonal IgM cold agglutinin

10835792 7.24 24.7 -1.8 [62]

SNL Complement component 3 8393024 6.1 188.2 Increased [52]

SNL α-2-HS-glycoprotein 6978477 6.3 39.0 Increased [52]

SNL Immunoglobulin heavy chain 1685249 5.2 13.3 Increased [51]

SNL Immunoglobulin-γ heavy chain V region

5051137 5.7 13.7 Increased [51]

NC Interferon inducible protein 10 receptor

20984919 5.1 89.7 Decreased [29]

SNL IL-1 Q9NZH8 5.1 18.7 Increased [51]

SNL α-1-macroglobulin 202857 6.5 168.7 Increased [52]

SNL Major histocompatibility complex class 1

1906678M 5.2 19.1 Increased [51]

SNL TCR 23194212 5.7 10.9 Decreased [51]





Animal model


Isoelectric point


Ref.

Signaling proteins

Calmodulin SNL/NC

P02593 4.1 16.7 Decreased [29,51]

SNL Chloride intracellular channel 1 13929166 5.9 28.9 Increased [52]

SNL Chloride intracellular channel 4 50657380 5.1 27.4 Increased [52]

SNL Contrapsin-like protease inhibitor 21

220698 5.3 46.7 Increased [52]

SNL Delayed rectifier potassium channel subunit IsK

116416M 6.8 14.6 Increased [51]

PN FAM3C 3334194 6.08 20.7 ~50% absent [62]

SNL/N Guanine nucleotide-binding protein G(o), α-subunit

120975 5.3 40.6 2.2 /-2.3 [19,50]

SNI Integrin-linked kinase 19173772 8.3 51.8 -1.56 [Kuhlein et al., Unpublished

Data]

SNL Nitric oxide synthase 3 3747079 6.0 13.2 Decreased [51]

NC Phosphatidylethanolamine binding protein

8393910 5.5 20.9 Decreased [29]

CCD Protein kinase C epsilon 49359177 6.62 84.89 Increased [49]

N Rab GDP dissociation inhibitor-α

100323179 4.7 50.5 -2.01 [19]

SNL Rho GDP dissociation inhibitor-β

56789330 5.0 22.9 Increased [52]

SNL Serin proteinase inhibitor B-1a 71051053 5.9 42.9 Increased [52]

SCI Similar to RhoGDI-1 34875656 5.1 23.5 5.75 [61]

SCI/NC

Voltage-dependent anion channel 1

13786200M 8.6 30.8 3.45/increased [29,61]

SNI Voltage-dependent anion channel 2


Data]

Proteins related to cell cycle/apoptosis and nerve degeneration

SNL/N Annexin A1 6978501 7.0 39.2 1.78/increased [19,52]

SCI/CCD Annexin A2 9845234M 7.5 38.7 2.52/increased [49,61]

SNL Annexin A3 51980303 6.0 36.6 Increased [52]

SNI/N Annexin A4 55742832 5.42 36.2 -2.02/2.4 [Kuhlein et al., Unpublished

Data]

NC Annexin A9/31 4502103 5.5 37.6 Increased [29]

NC Annexin V 1421099 4.9 35.8 3.11/decreased

[29,61]





Animal model


Isoelectric point


Ref.

Proteins related to cell cycle/ apoptosis and nerve degeneration (cont.)

SCI/NC Apolipoprotein A-I precursor 113997 5.5 30.1 1.71/increased

[29,61]

SNL Apolipoprotein AI 6978515 5.5 30.1 Increased [52]

NC/SNL/LHON

Apolipoprotein A-IV 114008 5.1 44.4 Increased [29,52,64]

NC Apolipoprotein D 6978523 4.9 21.6 Increased [29]

NC/SNL

Apolipoprotein E 114041 5.2 35.8 Increased [29,52]

SCI Cyclic nucleotide phosphodiesterase 1

34873724M 9.0 47.3 Only SCI [61]

SCI Similar to programmed cell death 6 interacting protein

34866400 7.5 70.5 -2.25 [61]

SNL Transforming growth factor-β induced

62663187 8.6 100.2 Increased [52]

Protein synthesis and processing

NC Elongation factor 1β 461991 4.5 24.7 Increased [29]

CCD Elongation factor 2 21322619 6.04 34.31 Increased [49]

NC Endoplasmatic reticulum protein 29

16758848 6.2 28.6 Increased [29]

SCI Similar to translation initiation factor eIF-4A II

55716055M 5.3 46.4 -2.27 [61]

SCI Phgdh protein 55562727M 6.3 56.5 -3.06 [61]

SNL Poly (rC) binding protein 3 62665831 7.1 39.6 Decreased [52]

NC Heterogenous nuclear ribonucleoprotein H1

10946928 5.9 49.2 Decreased [29]

NC/ SNL Heterogenous nuclear ribonucleoprotein L

20824058 6.7 60.9 Increased [29,52]

SNL Major vault protein 7514005 5.7 100.1 Increased [52]

CCD Similar to 40S ribosomal protein S17

62659539 6.32 50.49 Increased [49]

NC 40S ribosomal protein SA 125970 4.8 32.7 Increased [29]

Miscellaneous

N Calpain small subunit 100130992 5.34 28.46 4.83 [19]

NC Calumenin 6680840 4.5 37.1 Increased [29]

NC Cathepsin B 1127276 5.1 27.7 Increased [29]

CCD Centrosomal protein CG-NAP 22761814 5.03 52.2 Increased [49]

PN Cystatin C 14278690 9 15.8 1.82 [62]

SCI FK506-binding protein 4 22324680 5.7 45.9 1.63 [61]





Animal model


Isoelectric point


Ref.

Miscellaneous (cont.)

SCI Fscn1 protein 30023548 6.6 51.4 -2 [61]

SNI Glycoprotein 5 6980974 8.98 63.8 -1.89 [Kuhlein et al., Unpublished

Data]

NC Haptoglobin 6981042 6.3 38.5 Increased [29]

N Mimecan precursor 100120848 5.52 34 -1.8 [19]

SNI Msx-interacting-zinc finger 50925461 8.37 64.3 -2.26 [Kuhlein et al., Unpublished

Data]

SNI Similar to Nit protein 2 62657780 8.94 31 -1.89 [Kuhlein et al., Unpublished

Data]

SCI Similar to β-soluble NSF attachment protein

34859344 5.8 38.97 -1.6 [61]

NPN Pigment epithelium-derived factor

20178323 5.3 50.4 Decreased [62]

SNL Proteasome subunit 3914438 5.3 28.6 2.6 [50]

SCI Reticulocalbin 2 8394171 4.3 31.2 -2.21 [61]

NC Reticulocalbindin 6677691 4.3 37.2 Increased [29]

SNI Slc27a4-predicted protein 60688260 5.03 26.5 -1.7 [Kuhlein et al., Unpublished

Data]

SNL TCR α-chain precursor V region 88674 7.7 14.5 Increased [51]

SNL TCR-β variable region P04435 6.9 14.9 Decreased [51]

NC Translationally controlled tumor protein

27686473 5 15.6 Increased [29]

LHON Transthyretin 55669576 5.2 12.8 Decreased [64]

SCI Tumor rejection antigen gp96 34862435 4.7 92.8 4.21 [61]


A recent study dealt with Leber’s hereditary optic neuropa-thy (LHON), which is sometimes associated with the devel-opment of multiple sclerosis-like illness caused by mechanisms that are yet to be elucidated. The authors compared CSF from healthy controls with samples from LHON and multiple scle-rosis patients, and found seven significantly regulated protein spots (> threefold changes). Of these proteins, human serum albumin, a dimer of transthyretin and apolipoprotein A-IV, was differentially regulated in LHON patients in comparison with healthy controls, indicating that it is involved in this neuropathic disease. Since LHON and multiple sclerosis patients revealed different protein expression patterns, the authors concluded that there are different molecular mechanisms on the basis of the two disorders [64].

Potential markers of neuropathyAs summarized previously, the proteomic studies of different neur-onal tissues in various neuropathy models in rodents and CSF sam-ples in humans delivered a large amount of proteins that might be associated with the pathogenesis of neuropathy. These proteins are mostly involved in neuronal degeneration, regeneration and inflam-matory processes, and can be roughly arranged into subgroups with respect to their physiological functions. Figure 2 shows clearly that, consistent with data from genomic studies [65], the bulk of regulated proteins are related to cellular homeostasis and metabolism, fol-lowed by proteins responsible for neuronal function and structure. Other groups of regulated proteins comprise heat-shock proteins, chaperones and antioxidants, proteins related to the cell cycle, apop-tosis and neurodegeneration, signaling proteins, proteins related to



the immune system and proteins related to protein synthesis and processing. Up- and downregulated proteins are equally distributed within the groups, with the exception of signaling proteins and pro-teins related to cell cycle/apoptosis and nerve degeneration. In these groups, the bulk of proteins are upregulated (12 out of 17 and 13 out of 16, respectively). One might have expected that at least after applying the same models there should be great overlaps in protein regulation, although interestingly, among over 210 regulated pro-teins found, there were only approximately 50 overlapping protein regulations. This result indicates that the different models induce differential protein regulation and that these regulations are again variably pronounced in different neuronal tissues. Another explana-tion might be that the different approaches to investigate pain are very heterogeneous. Nevertheless, there are also several regulations found in multiple studies that might emphasize the importance of these proteins [13]. A number of the regulated proteins have not been related to pain so far and may, therefore, present interesting new fields in this context.

The disturbance of the neuronal integrity after nerve injury con-stitutes a massive interference in the cellular metabolism, which is surely one explanation for the great amount of regulated protein associated with cellular metabolism and homeo stasis. Another reason could simply be the high abundance of these proteins in all cells. However, this fact reflects one of the biggest pharmacologi-cal problems, since high-abundance proteins often play essential roles in cell functions and are therefore not appropriate as drug targets in most cases. As an alternative, changes in their regulation in nerve cells might be used as a biomarker for certain diseases of the nervous system and provide important information regarding the metabolic status of the cell.

Most neuronal function proteins are cytoskeleton proteins. These proteins serve essential functions in axonal outgrowth and conductivity, and signaling from the axon terminals to the cell bodies. Owing to these functions, they are playing important roles in regeneration and repair after nerve injury. As an example, tubulin is upregulated in the PNS and CNS after nerve injury, which has been connected to regeneration processes of injured neurons [66,67]. On the other hand, GFAP and vimentin are described in astrocyte activation after nerve injury, thus contributing to inhibited regeneration and axonal plasticity [68]. This is further sup-ported by a study which showed vimentin synthesis in axonal preparations of injury conditioned DRG cultures, indicating that it might play a role in axonal growth [69]. In another report it has been shown that vimentin is involved in axonal transporta-tion of phosphorylated kinases after nerve injury, thus providing a possibility for retro-grade injury signaling mechanisms that are required for regeneration processes [70].

Antioxidant proteins and heat-shock proteins are stress proteins that stabilize other proteins and mediate cell protection under conditions of environmental stress. An upregulation of several heat-shock proteins has been observed in the nervous system after stress induction or injury [71,72]. In particular, the small heat-shock protein 27 protects neurons against neurotoxic stimuli and inhibits neurodegeneration as revealed from overexpression experiments [73–75]. Interestingly, several of the proteomic studies summarized here found decreased levels of heat-shock protein 27, indicating an irreversible damage of the nerve tissue. Antioxidants are involved in a variety of neurological disorders such as Alzheimer and Parkinson’s disease and might, therefore, also reflect interesting proteins in the context of chronic pain [76,77].

Several modified proteins are associated with apoptosis, which is a well-known response to nerve injury in neurons of the peripheral and central nervous systems. The progress of neuropathic pain might be related to neuronal sensitization and a disturbance of inhibitory systems that can be triggered by apoptotic processes. Furthermore, neurodegeneration is often accompanied by the formation of toxic protein aggregates that subsequently induce apoptosis and neuronal loss [78]. Therefore, inhibition of apoptosis by regulation of pro- or anti-apoptotic proteins might provide a future strategy against the development of neuropathic pain [26]. In most of the proteomic studies, proteins in this group were found upregulated after nerve injury, in particular different isoforms of annexins and apolipopro-teins, indicating that a number of compensatory mechanisms are activated to prevent cells from apoptosis or to induce nerve repair.

Signaling proteins are one of the most important and interest-ing categories, since they are playing fundamental roles in the development of sensitization processes that are switched on by

38%Proteins related to cellularhomeostasis and metabolism

21%Neuronal function andstructural proteins

10%Miscellaneous

8%Signaling proteins

7% Proteins related to cell cycle/apoptosis and nerve degeneration

7% Heat-shock proteins/chaperones/antioxidants

5% Protein synthesisand processing

4%Immune system

Figure 2. Functional groups of regulated proteins after nerve injury.



chronic pain states. It is well known that a number of kinases such as PKC, ERK and c-Jun kinase are upregulated or activated after nerve injury, respectively [26,79,80].

Expert commentaryTo date, the treatment of pathological pain coming along with neuropathy is insufficient, although a number of analgesic drugs are available. This deficiency might be explained by the fact that the molecular mechanisms associated with neuropathies are only incompletely understood so far. Therefore, clarifying these mecha-nisms will represent a great step forward in the development of new therapies of neuropathic pain patients. Particularly protein modifications induced by nerve injury are of outstanding interest in this context [81]. To elucidate these modifications, a number of proteomic studies have been performed using various well-estab-lished animal models of central and peripheral neuropathies, which reflect several forms of human neuropathies, as well as human CSF samples from patients suffering from neuropathies. The studies identified a large number of modified proteins following nerve injury. However, owing to the varying assay conditions concern-ing the choice of the models or tissues, the regulations differed largely among the respective studies. The variable protein patterns can also be ascribed to diverse time points for tissue dissection, sample buffers and lysis conditions, differing isoelectric focus-ing (e.g., pH-range and separation protocol) and sodium dodecyl sulphate PAGE.

Certainly, it can be stated that proteomics appears to be a useful technique to investigate proteins associated with pain. However, for an enhanced reproducibility and comparability, the assay con-ditions as well as validation of the results should be equalized among the laboratories. A network of scientists who work together with standardized protocols would be very helpful to put forward progress in proteomic pain research. A good example for such an organization is the Human Brain Proteome Project, which focuses on the characterization of proteins in the healthy and diseased human brain.

Nevertheless, the results that are summarized here can be regarded as a starting point for further ana lysis of the func-tional roles of these proteins in neuropathic pain, which could then provide new insights into the molecular mechanisms of neuropathy in the near future and hopefully to the develop-ment of novel analgesics that are suitable for the treatment of

neuropathic pain. Some of the protein regulations found, such as heat-shock or cytoskeleton proteins, are already known in the context of neuropathic pain, and may, therefore, serve as a proof of concept for the proteomic approaches. In several of the proteomic studies, an outlook on further investigations to analyze the functional relevance of the protein alteration has already been indicated. Lee et al. focused on the role of cre-atin kinase B [50], Ding et al. emphasized the role of 11-zinc finger protein and glypican [42] and Katano et al. concentrated on collapsin response mediator 2 [57]. Interestingly, this protein has been identified in another proteomic approach as a binding partner of the sodium channel blocker lacosamide, which has been described for antihyperalgesic effects [82].

However, it is clear that most of the changes have not been investigated in further studies so far and, therefore, the proteomic approaches have to be regarded as a collection of data that can be consulted for further studies of the nervous system in the context of neuropathies.

Five-year viewOver the next few years, several problems associated with pro-teomic ana lysis that hamper the ana lysis of the complete protein spectrum should be solved. In particular, the investigation of the CNS has been fraught with problems since sample generation of brain or spinal cord delivers a complex and heterogenous mix of cells, such as neurons and astroglia cells. Furthermore, a number of proteins are either expressed at low level or in a small percent-age of neurons only. The neurons themselves consist of a number of subcellular compartments comprising axons, dendrites, spines and synaptic terminals that perform distinct functions. The different cell types and the neuronal compartment could not be discriminated on 2D gels. Similarly, high- and low-molecu-lar weight proteins as well as hydrophobic membrane proteins are difficult to separate by 2D gel electrophoresis. However, there are a number of advances in neuroproteomic research that should improve the investigations. In the meantime, it is already possible to study isolated subcellular structures, such as the post-synaptic density in neurons, and also the ana lysis of high- and low-molecular weight proteins has been ameliorated. Therefore, proteomic ana lysis will go on delivering a substantial volume of data that will then serve as a basis for further research approaches and finally the development of novel efficient analgesics.

Key issues

Treatment of neuropathic pain is insufficient at present and therefore constitutes an unmet medical need.•

Neuropathic pain is associated with a number of protein modifications in the central and peripheral nervous systems.•

Animal models of neuropathic pain can reflect human conditions after nerve injury.•

Human cerebrospinal fluid can be used to find biomarkers for diseases associated with neuropathies.•

Nociceptive transmission involves different proteins in different nerve tissues.•

Inflammation and neuropathy involve different proteins in their pathophysiological mechanism.•

The majority of regulated proteins are associated with cellular homeostasis and metabolism.•

A proteomic network to investigate protein changes in neuropathy might improve the progress in proteomic neuropathy research • owing to standardized methods.

Clinical proteomics might provide novel biomarkers for different diseases.•



Financial & competing interests disclosureSupport was provided from the Ministry of Science and Technology (Bundesministerium für Bildung und Forschung [BMBF] 01EM0511), Hannoversche Straße 28–30, 10115 Berlin. The authors have no other rele-vant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

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AffiliationsEllen Niederberger, PhD • Pharmazentrum Frankfurt/ZAFES, Institut für Klinische Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universität Frankfurt, Theodor Stern Kai 7, 60590 Frankfurt am Main, Germany Tel.: +49 696 301 7616 Fax: +49 696 301 7636 [email protected]

Hilmar Kühlein • PhD student, pharmazentrum frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität, Theodor Stern Kai 7, 60590 Frankfurt am Main, Germany [email protected]

Gerd Geisslinger, MD, PhD • Professor, Director of the Institute, pharmazentrum frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität, Theodor Stern Kai 7, 60590 Frankfurt am Main, Germany Tel.: +49 696 301 7620 Fax: +49 696 301 7617 [email protected]

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