Address correspondence and reprint requests to Toshihide Yamashita, Department of Anatomy and Neuroscience, Osaka University Graduate School of Medicine, 2–2 Yamadaoka, Suita, Osaka 565–0871, Japan. E-mail: email@example.com
One of the most striking features of neurons in the mature peripheral nervous system is their ability to survive and to regenerate their axons following axonal injury. To perform a comprehensive survey of the molecular mechanisms that underlie peripheral nerve regeneration, we analyzed a cDNA library derived from the distal stumps of post-injured sciatic nerve which was enriched in non-myelinating Schwann cells using cDNA microarrays. The number of up- and down-regulated genes in the transected sciatic nerve was 370 and 157, respectively, of the 9596 spotted genes. In the up-regulated group, the number of known genes was 216 and the number of expressed sequence tag (EST) sequences was 154. In the down-regulated group, the number of known genes was 103 and that of EST sequences was 54. We obtained several genes that were previously reported to be involved in regeneration of the injured neurons, such as cathepsin D, ninjurin 1, tenascin C, and co-receptor for glial cell line-derived neurotrophic factor family of trophic factors. In addition to unknown genes, there seemed to be a lot of annotated genes whose role in nerve regeneration remains unknown.
co-receptor for glial cell line-derived neurotrophic factor family of trophic factors
insulin-like growth factor-1
One of the most striking features of neurons in the mature PNS is their ability to survive and to regenerate their axons following axonal injury. It has been increasingly evident that the success of axonal regeneration is dependent on the intrinsic as well as extrinsic growth properties of the axotomized neuron. The environment in which PNS axons regenerate consists of Schwann cells and their basal laminae, fibroblasts, collagen, degenerating myelin and phagocytic cells (Fawcett and Keynes 1990; Bunge and Griffin 1992; Araki and Milbrandt 1996). After nerve injury, the distal axonal and myelin segment undergoes dissolution and absorption by the surrounding cellular environment, a process called Wallerian degeneration. Then, the remaining Schwann cells divide and align longitudinally within basal lamina tubes. Growth cones from regenerating axons extend along the Schwann cell bands (the band of Büngner), growing along the Schwann cell membranes and basal laminae (Keynes 1987; Reichert et al. 1994; Grill and Tuszynske 1999). Such biological and morphologicalchanges of Schwann cells are thought to be controlled by injury-induced molecules that are expressed by neurons and Schwann cells themselves. Especially, at around 7 days after nerve injury, many nerve regeneration-related factors, such as p75NTR, co-receptor for glial cell line-derived neurotrophic factor family of trophic factors (GFRα1) and ninjurin 1 and 2, reach peak levels (Taniuchi et al. 1988; Araki and Milbrandt 1996; Baloh et al. 1997; Araki and Milbrandt 2000), and the growth cones of regenerating axons begin to move over the Schwann cell surface (Taniuchi et al. 1988; Goodrum et al. 1994). Successive large-scale screening, however, is required to reveal the spectrum of genes involved in this regeneration process. To perform a comprehensive survey of the molecular mechanisms that underlie the process, we analyzed a cDNA library derived from the distal stumps of sciatic nerve 7 days after injury using cDNA microarrays.
Complementary DNA microarrays contain cDNA probes for monitoring the expression of thousands or more genes in a single hybridization experiment (Schena et al. 1995; Shalon et al. 1996). Microarrays can identify the expression changes in different biological states, and provide a format for identifying genes as well as changes in their activity. In the present study, we monitored the up-regulated and down-regulated genes expressed in the distal segment of sciatic nerve 7 days after transection relative to those of intact sciatic nerve.
Materials and methods
Sixty-four adult Institute of Cancer Research (ICR) mice (male, body-weight 30 g) were anesthetized by intraperitoneal injection of 40 mg/kg pentobarbital. The right sciatic nerve was transected with microsurgical scissors at hip level. The contralateral nerve was exposed, but left uninjured (control). The wounds were closed with sutures. Seven days after transection, the operated animals were deeply anesthetized, and distal segments of the sciatic nerves from the transected and intact side were harvested.
Twenty sciatic nerve stumps were homogenized, at the same time, in Isogen RNA extraction reagent (Nippon Gene, Tokyo, Japan) with a Polytron homogenizer (Kinematica, Littau, Luzern, Switzerland) for 15 s, and total RNA was prepared from homogenized tissue using the Isogen reagent according to the manufacturer's instructions. The total RNA extraction was repeated four times, and extracts were pooled to gain a sufficient amount of the total RNA for cDNA microarrays. mRNA was isolated from total RNA preparations using oligo(dT) columns and the standard Oligotex (Takara, Shiga, Japan) protocol. The quality of extracted total RNA and mRNA was confirmed with an Agilent Technologies 2100 Bioanalyzer-Bio Sizing (Agilent Technologies, Palo Alto, CA, USA). A LifeArray chip (LifeArray System; Incyte Genomics, Palo Alto, CA, USA) was utilized to perform the cDNA microarray procedure. Briefly, there were 9596 mouse genes spotted on the chip. It contained both unique annotated genes and expressed sequence tag (EST) clusters. Information relating to these genes is available through the UniGene Mus musculus website of the National Center for Biotechnology Information (http://www.ncbi. nlm.nih.gov/UniGene/Mm.Home.html). cDNA from transected sciatic nerve was cyanine 3 (Cy3)-labeled by reverse transcription from 200 ng mRNA by means of a LifeArray Probe Labeling Kit (Incyte Genomics) according to the manufacturer's instructions, and cDNA from intact sciatic nerve was cyanine 5 (Cy5)-labeled. Two different dye-labeled cDNA probes were hybridized simultaneously with one cDNA chip at 60°C for 6 h using a LifeArray hybridization chamber, hardware kit, and wash solutions (Incyte Genomics) according to the manufacturer's instructions. After hybridization, scanning of the two fluorescent intensities of the cDNA chip was performed by a standard two-color microarray scanner (GenePix 4000A DNA Microarray Scanner; Axon Instruments, Union City, CA, USA). Differential gene expression was profiled with GemTools software (Incyte Genomics). The experiments were performed twice independently.
To confirm the accuracy of cDNA microarrays, RT-PCR was perfomed. Total RNA (5 µg) was reverse transcribed using oligo(dT) by reverse transcriptase from Moloney murine leukemia virus (Invitrogen, Carlsbad, CA, USA). For PCR amplification, specific oligonucleotide primer pairs (10 pmol each) were incubated with 1 µL of cDNA template in a 20-µL PCR reaction mixture containing 1.5 mm MgCl2, 25 mm KCl, 10 mm Tris, pH 9.2, mixed deoxynucleotides (1 mm each) and 1 unit of Taq polymerase. Dilutions of the cDNAs were amplified for 25 cycles. The amplified PCR products were analyzed by 1.2% agarose gel electrophoresis and ethidium bromide staining. The product of constitutively expressed β-actin mRNA served as an internal standard. All products were assayed in the linear response range of the RT-PCR amplification process; the cycle number used was determined by finding the midpoint of linear amplification on a sigmoid curve for amplification products with cycle numbers of 24–40 plotted against band density.
We compiled both up-regulated and down-regulated cDNA sequences in the transected sciatic nerve that showed greater than twofold changes in expression relative to those of intact sciatic nerve. The number of up- and down-regulated genes in the transected sciatic nerve was 370 and 157, respectively, of the 9596 spotted genes. In the up-regulated group, the number of known genes was 216 (58%) of 370 up-regulated genes, and the number of EST sequences was 154 (42%) (Fig. 1a). In the down-regulated group, the number of known genes was 103 (66%) of 157 down-regulated genes and that of EST sequences was 54 (34%) (Fig. 2a). Furthermore, we classified known genes and genes similar to them into 12 functional categories (Figs 1b and 2b). cDNA sequences spotted on the cDNA chip have already been categorized into functional groups, and our classification is based on them. Some genes are categorized into two or three functional groups; for example, fibroblast growth factor 1 is classified into the signal transduction and regulation group, and the secreted and extracellular group. Therefore, the sum of the percentage of each group exceeds 100%. Examples of up-regulated known genes in transected sciatic nerve are listed in Table 1 and all of the up-regulated ESTs in this study in Table 2. In addition, a complete list of the differentially expressed genes is available online at http://www.med.osaka-u.ac.jp/pub/anat2/shokai/ichiran.htm as supplementary information.
Table 1. Examples of known up-regulated genes in injured nerve genes detected by cDNA microarrays
SRY, Sex-determining region of the Y chromosome.
Signal transduction and regulation
Endothelial monocyte activating polypeptide 2
Interferon activated gene 204
Small inducible cytokine A8
Small inducible cytokine subfamily A17
Regulator of G-protein signaling 16
Tumor necrosis factor (ligand) superfamily, member 11
Small inducible cytokine B subfamily, member 5
Small inducible cytokine A2
Potassium channel, subfamily K, member 2
Chloride channel calcium activated 1
ATPase, H + transporting, lysosomal I
ATPase, H + transporting lysosomal (vacuolar proton pump), 9.2 kDa
Protein modification and maintenance
Extracellular proteinase inhibitor
Tissue inhibitor of metalloproteinase
Proteasome (prosome, macropain) subunit, alpha type 3
Proteasome (prosome, macropain) subunit, alpha type 2
There is a good correlation between the present results and previous observations, substantiating the accuracy of our data. For example, cathepsin D, ninjurin 1, tenascin C and GFRα1 were up-regulated in the microarray analysis (Fig. 3), consistent with previous reports (Sahenk et al. 1990; Araki and Milbrandt 1996; Baloh et al. 1997; Xiao et al. 1997). In addition, almost all myelin-associated proteins like peripheral myelin protein 22 kDa, myelin basic protein, protein zero and periaxin, whose expression was reported to be suppressed during axonal regeneration (Scherer 1997), were also down-regulated in this study (Fig. 3). Furthermore, the differential expression of several sequences was confirmed by RT-PCR (Fig. 3).
After peripheral nerve injury, Wallerian degeneration occurs in the distal segment of the injured site, where Schwann cells proliferate and form the band of Büngner (Reichert et al. 1994). Even in the transected nerve, Schwann cells migrate away from the two separated nerve ends to form a continuous tissue cable across the gap (Le Beau et al. 1988; Guenard et al. 1992; Levi et al. 1994; Torigoe et al. 1996), and lead the growth cones of regenerating axons into the band of Büngner. The importance of Schwann cells during axonal regeneration is also evidenced by the reduction in axonal growth when live Schwann cells are removed from an injured site (Hall 1986). On the other hand, when cultured Schwann cells and their associated extracellular matrix are transplanted into a lesion in the central nervous system, axonal regeneration is facilitated (Benfey and Aguayo 1982). Accordingly, to identify the genes that are involved in Schwann cell activation after nerve injury is invaluable not only for understanding pathways implicated in nerve regeneration but also for possible clinical applications. Such injury-induced genes that control biological and morphological changes of Schwann cells are thought to be expressed by neurons and Schwann cells themselves. Previous reports have demonstrated that Schwann cells in the distal segment of injured peripheral nerve express various neurotrophic factors including nerve growth factor, brain-derived nerve growth factor (BDNF), insulin-like growth factor-1 (IGF-1), and glial cell line-derived neurotrophic factor, and adhesion molecules that promote neurite outgrowth by regulating contact between axons and Schwann cells, such as L1, neural cell adhesion molecule, and N-cadherin (Bixby et al. 1988; Martini and Schachner 1988; Rieger et al. 1988). Furthermore, at around 7 days after nerve injury, many nerve regeneration-related factors, such as p75NTR., GFRα1 and ninjurin 1 and 2 reach their peak levels (Taniuchi et al. 1988; Araki and Milbrandt 1996; Baloh et al. 1997; Araki and Milbrandt 2000), and the growth cones of regenerating axons begin to move over the Schwann cell surface (Taniuchi et al. 1988; Goodrum et al. 1994). Araki et al. (2001) clustered the gene expression induced in injured peripheral nerve into four groups according to time-course expression patterns by differential screening of a subtractive library enriched for cDNAs expressed in injured sciatic nerve. The majority of the genes induced after injury had their peak level of expression at around 7 days after injury. On the basis of these data we examined expression patterns at 7 days after sciatic nerve transection, to identify the genes implicated in nerve regeneration.
We classified the differentially expressed genes into functional categories (Table 1). Cyclin D1 belongs to the signal transduction and regulation group, and the mitotic response of Schwann cells is completely inhibited during Wallerian degeneration after peripheral nerve injury in cyclin D1 knockout mice (Kim et al. 2000). However, axonal regrowth into the distal zone of a crushed nerve was not markedly impaired in cyclin D1 knockout mice, indicating that neuronal responses to nerve injury are independent of Schwann cell mitotic responses.
In the membrane transport group, potassium channel, subfamily K, member 2 was up-regulated. It was reported previously that K+ channel-blocking quaternary ammonium ions, but not a Na+ channel blocker, reduced Schwann cell proliferation in a dose-dependent fashion, offering further evidence for a role of K+ channels in Schwann cell proliferation (Wilson and Chiu 1993).
Cathepsin D has been shown to be present in the cytoplasm of Schwann cells but not in axons of intact nerves by immunohistochemistry. However, when the nerve was ligated or transected, intra-axonal cathepsin D appeared as granular or elongated particles and was increased with time at the cut end of the distal stump. In the proximal stump of transected nerves, cathepsin D was detected in the nascent axon tips, as well as in a length of axon extending up to the first node of Ranvier (Sahenk et al. 1990).
Lumican is a keratan sulfate proteoglycan, belonging to the small leucine-rich proteoglycan family. Following a stab wound in the cerebral cortex of the late postnatal rat, reactive gliosis was consistently observed along with an up-regulation of keratan sulfate proteoglycan (Geisert et al. 1996). However, up-regulation of keratan sulfate proteoglycan has not been reported in peripheral nerve injury.
Previous research has identified membrane proteins of the syntaxin family as a potential target for mediating the propagation of synaptic plasticity through neural networks. Syntaxins 3A and 3B are generated from the same gene by alternative splicing, and their expression was confirmed in adult rat brain. Their levels are increased in dentate granule cells 6 h after the induction of long-term potentiation (Rodger et al. 1988). However, the role of the syntaxin family in the peripheral nervous system remains unknown.
Drebrin has been shown to act on actin filaments at dendritic spines to cause morphological change, and might be related to the plasticity of synaptic transmission. Although the relevance of induction of this gene in Schwann cells remains unknown, a recent study revealed that drebrin immunoreactivity was up-regulated in the spinal motoneurons of rats following unilateral sciatic nerve transection (Kobayashi et al. 2001). Interestingly, drebrin-homology sequences are found in SH3P7 originally isolated by cloning of SH3 domain ligand targets from a mouse embryo cDNA library (Yamazaki et al. 2001). SH3 domain interacts with Rho GTPases, which are a family of signaling intermediates shown to be a pivotal regulator of the microfilament (Richnau and Aspenstrom 2001). Recent progress in the analysis of the role of the Rho family has demonstrated that neurotrophin binding to p75NTR modulates axonal outgrowth by regulating Rho activity (Yamashita et al. 1999). It is possible that the migration behavior of Schwann cells after injury may be altered by drebrin which is related to Rho function.
In addition to genes annotated already, the cDNA microarrays utilized in this study (LifeArray chip, Incyte Genomics) include EST sequences. Hughes et al. (2000) noted that proteins participating in similar biological pathways often had similar expression profiles. Based on this concept, use of a cDNA microarray that includes EST sequences has enabled us to extract interesting functional insights from the temporal expression profiles of newly detected genes. In this study, 208 EST sequences showed greater than twofold changes in expression relative to intact peripheral nerve (Figs 1 and 2). To analyze them functionally might lead to the discovery of novel genes that contribute to nerve regeneration.
The accuracy of the results from cDNA microarrays has been demonstrated (Schena et al. 1995; Shalon et al. 1996). In this study, consistent with previously established observations, for instance, the expression of cathepsin D, ninjurin 1, tenascin C and GFRα1 was up-regulated. In addition, the data are consistent with the results derived from a similar large screening of a subtractive library reported by Araki et al. (2001). Almost all myelin-associated proteins, including peripheral myelin protein 22 kDa, myelin basic protein, protein zero and periaxin, whose expression was reported to be suppressed during axonal regeneration (Scherer 1997), were also down-regulated in this study. Furthermore, we confirmed the differential expression of several sequences by RT-PCR as shown in Fig. 3. Taken together, we conclude that our results are reliable and could be used for functional analysis.
In summary, we have described the detection of genes induced in peripheral nerve after injury using cDNA microarrays. Differential expression of more than 500 genes in injured peripheral nerve was observed. Future studies will address the functional properties of these genes, which will enable us to understand the pathways involved in nerve regeneration, and to apply for therapies to treat intractable central nervous system injury.
The authors thank Masahiro Ogihara, Yoshinori Ohsima, Hiroomi Ipposhi, and Naoyuki Sato (Kurabo Industries Ltd.) for participating in this work.