Acta Neuropathol (2007) 113:451–459 DOI 10.1007/s00401-007-0198-5
O RI G I NAL PAPE R
Increased expression of neuregulin-1 in diVerentiating muscle satellite cells and in motoneurons during muscle regeneration Miyuki Hirata · Kunihiro Sakuma · Seiichiro Okajima · Hiroyoshi Fujiwara · Shuichiro Inashima · Masahiro Yasuhara · Toshikazu Kubo
Received: 14 June 2006 / Revised: 15 January 2007 / Accepted: 15 January 2007 / Published online: 9 February 2007 © Springer-Verlag 2007
Abstract Neuregulins belong to a family of multipotent growth-promoting proteins, and have been shown to have a crucial role in accumulating acetylcholine receptor at neuromuscular junctions. A functional role of neuregulins in muscle regeneration has not yet been identiWed. Using reverse transcription (RT)-PCR, Western blot and immunoXuorescence analysis following bupivacaine injection into rat muscle, we investigated the expression pattern of neuregulin-1 (NRG-1) in normal and regenerating tibialis anterior (TA) muscle. In addition, we examined changes in NRG-1 expression in the spinal cord following muscle damage. Western blotting showed that muscle NRG-1 protein decreased soon after the damage, and increased gradually after the 4th day following the damage. The amount of NRG-1 mRNA in the muscle increased from the 2nd to 6th post-surgical day. The amount of NRG-1 protein, but not mRNA, increased gradually in the spinal cord after muscle damage. ImmunoXuorescence revealed NRG-1 protein in some quiescent
M. Hirata · S. Okajima · H. Fujiwara · T. Kubo Department of Orthopedics, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi-hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan S. Inashima · M. Yasuhara Department of Legal Medicine, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi-hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan K. Sakuma (&) Health Science Center, Toyohashi University of Technology, 1-1 Hibarigaoka, Tenpaku-cho, Toyohashi 441-8580, Japan e-mail:
[email protected]
satellite cells identiWed by c-Met. After 6 and 10 days, clear co-localization between NRG-1 and myogenin was noted in diVerentiating satellite cells. Thus, NRG-1 may play an important role in the diVerentiation of satellite cells in muscle regeneration, while increased NRG-1 expression in motoneurons may enhance the remodeling of partially damaged axons. Keywords Neuregulin-1 · Muscle regeneration · DiVerentiation · Spinal cord · Satellite cell
Introduction Neuregulins (NRGs) (also known as heregulin or neu diVerentiation factor) belong to a complex family of proteins, and were identiWed as glial growth factors. They are mainly present at neuromuscular junctions (NMJs) and have been shown to accumulate the AChR molecule at NMJs [8, 14, 17]. Four related NRG genes (NRG-1, NRG-2, NRG-3 and NRG-4) have been identiWed and, in particular, the function of NRG-1 has been studied in detail. NRG has been shown to participate in the myelination of Schwann cells and oligodendrocytes, and in the specialization of astrocytes [1]. NRG promotes embryonic myogenesis [4, 10] and the glucose transport of skeletal muscles [2, 22]. Recently, Lebrasseur et al. [12] showed that the amount of NRG and ErbB proteins increased in several hindlimb muscles of rats after electrical stimulation and treadmill running. In myogenesis in vitro, Ford et al. [5] demonstrated that NRG-1 potently induces mitogenesis in L6 myoblasts, but inhibits myogenic diVerentiation by inhibiting myogenin expression. In contrast, Florini et al. [4]
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and Kim et al. [10] suggested a diVerentiation-promoting role of neuregulin using L6 rat myotubes. However, all of these studies investigating the functional role of NRG-1 utilized an in vitro culture model. This model did not necessarily reXect the adaptive changes in satellite cells in in vivo muscle regeneration. In vitro, myogenesis of L6 or C2C12 cells ultimately ends up in the formation of myotubes but not myoWbers, while in an in vivo muscle regeneration model, quiescent satellite cells migrate to the damaged site where they proliferate, diVerentiate and fuse with the damaged Wbers or form new Wbers [7]. Neurotrophic factors produced by regenerating muscle Wbers are carried into the neuronal cell body by retrograde transport [13, 23]. Kami et al. [9] found, using in situ hybridization, that leukemia inhibitory factor (LIF) and glial cell-line derived neurotrophic factor (GDNF) were upregulated in intramuscular nerves and damaged muscles, and their receptors in motoneurons were also up-regulated following muscle contusion. Loeb et al. [16] showed that the amount of NRG mRNA and proteins increased after adding several neurotrophic factors, such as GDNF, and brainderived neurotrophic factor (BDNF), to cultured motoneurons. Therefore, NRG-1 production may be inXuenced by the upregulation and/or downregulation of these factors in motoneurons or damaged muscle. The purpose of this study was to investigate the changes in NRG-1 expression in regenerating processes after muscle damage using RT-PCR and immunoblotting. Furthermore, using immunoXuorescence, we clariWed the expression of NRG-1 in skeletal muscles at the stages of proliferation and diVerentiation following muscle damage, as well as in motoneurons.
Materials and methods Experimental animals Adult male Wistar rats (10 weeks of age) were used. The rats were housed in a temperature- (22 § 2°C) and humidity-controlled (60 § 5%) room regulated to provide an alternating 12-h period of light and darkness. Animals were allowed to feed (commercial rat chow) and drink ad libitum. This experimental procedure was approved by the Committee for Animal Research, Kyoto Prefectural University of Medicine. Bupivacaine treatment Muscle regeneration was induced in the left TA muscles of each rat (n = 40) by an intramuscular injection
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of 0.5 ml of 0.5% bupivacaine hydrochloride prepared in 0.9% saline solution. Rats were euthanized with an overdose of pentobarbital, and the TA muscles of the injected legs and spinal cords from the 4th lumbar vertebra (L4) to the 1st sacral vertebra (S1) were dissected at 0 (n = 10), 1 (n = 3), 2 (n = 4), 4 (n = 4), 6 (n = 11), 10 (n = 4) and 14 (n = 4) days after surgery. We used nonoperated TA muscles and the spinal cords of animals sacriWced at 0 days after surgery as controls. Primary antibodies The antibodies employed in the present study were as follows: aYnity-puriWed rabbit polyclonal antibody to neuregulin-1 (1:100, SC-348, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and myogenin (1:100, Santa Cruz), aYnity-puriWed mouse monoclonal antibody to heregulin (1:100, Sigma), PCNA (1:100, Pharmingen, Becton Dickinson Company, USA), myogenin (1:100, NeoMarkers), bromodeoxyuridine (1:400, Pharmingen), dystrophin (1:100, Sigma) and c-Met (1:100, Santa Cruz) or aYnity-puriWed goat polyclonal antibody to microtubule-associated protein-2 (MAP-2; 1:100, Santa Cruz). Tissue preparation, gel electrophoresis and immunoblotting Each tissue was homogenized in 10–20 vols of 50 mM Tris–HCl pH 7.4, 5 mM EDTA, 10 g/ml phenylmethylsulfonylXuoride, 0.5 g/ml leupeptin, 0.2 g/ml aprotinin, 0.2% NP-40, 0.1% Triton X-100, 0.05% mercaptoethanol, and 1 mM Na3VO4 using a polytron (PCU-2, Kinematica, Steinhofhalde, Switzerland) for 30 s. The homogenized tissues were centrifuged for 25 min at 15,000g at 4°C, and the protein concentration of the supernatant was determined colorimetrically (Bio-Rad protein determination kit, Bio-Rad Laboratories, Richmond, CA, USA). Sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis were performed according to the methods of Laemmli [11]. Proteins separated by SDSPAGE were transferred electrophoretically onto nitrocellulose membranes (Hybond-ECL Western, Amersham, Arlington Heights, IL, USA). The blots were incubated with a blocking buVer of 0.1% Tween-20 and 1% gelatin in 10 mM Tris-buVered saline (TBS, 10 mM Tris, 135 mM NaCl, 1 mM KCl, 0.02 % NaN3, pH 7.4) for 30 min. The blots were incubated with primary antibodies in a cold room overnight and with alkaline phosphatase conjugated anti-rabbit IgG (1:300, Promega Co. Madison, WI, USA) for 30 min. They were visualized with Western Blue™ Stabilized Substrate for
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Alkaline Phosphatase (Promega Co.). Densitometric analysis of each blot was performed with a computerized image processing system (NIH image 1.59). RNA isolation and RT-PCR Total RNA was isolated from the TA muscles and spinal cords using Sepasol-RNA II Super (Nacalai Tesque, Inc., Kyoto, Japan), according to the manufacturer’s protocols. First-strand cDNA synthesis was performed with 1 g RNA according to a 1st Strand cDNA Synthesis Kit for RT-PCR (AMV; Takara Bio Inc., Otsu, Japan). Complementary DNA was then used as a template for PCR in a 20 l-reaction volume including 1 M of each primer, 20 M MgSO4, 20 M dNTP, 4 M KOD plus-BuVer and 0.2 l of KOD plus (KOD- Plus-PCR kit, Toyobo Co., Ltd.). The primers (SIGMA GENOSYS, Inc.) used for ampliWcation were 5⬘-ACTCCTCACTCAGATTCAAA-3⬘ and 5⬘-AGG CTGTCTCAGTCGAGGCT-3⬘ corresponding to 532–551 and 783–764 base pairs, respectively, of rat HRG (sequence with total identity to all neuregulin isoforms), 5⬘-TGCCATCCAGTACATTGAGCG-3⬘ and 5⬘-CCACCGTCCATTCACATAAGACT-3⬘ corresponding to 406–426 and 846–869 base pairs, respectively, of rat myogenin and 5⬘-TCCTGCACACCAA CTGCTTAGCC-3⬘ and 5⬘-CCCACTAAAGGGCAT CCTGGGCTA-3⬘, corresponding to 1294–1317 and 1647–1670 base pairs, respectively, of rat glyceraldehyde phosphate dehydrogenase (GAPDH). PCR conditions for the analysis of the expression of each gene were designed to avoid PCR saturation and to enable semi-quantitative determination. The amplicons were resolved by electrophoresis on 1.5% agarose gels and visualized with ethidium bromide staining. Each sample was run in duplicate, normalized to each data point against GAPDH, averaged and statistically compared. The densitometric analysis was performed with NIH image 1.59. Double-labeling immunoXuorescence As described previously [20], 5-bromo-2⬘-deoxyuridine (BrdU; Roche Diagnostics), a non-radioactive marker for DNA synthesis [4], was injected (100 mg/kg, intraperitoneally) 1 h before sampling for the labeling of proliferating satellite cells. Several 7 and 10 m transverse sections of TA and spinal cord, respectively, made with a cryostat (CM-502 Coldtome, Sakura seiki Co., Ltd., Tokyo, Japan) were mounted on silanized slides (Dako Japan, Tokyo). TA muscles and spinal cords were isolated and frozen in isopentane. Cryosections were incubated in blocking solution (10% normal
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goat serum in PBS) for 10 min at room temperature. Sections were incubated with primary antibodies or -bungarotoxin-tetramethylrhodamine (1:100, Sigma), overnight in the cold room. After washing in PBS, sections were incubated with anti-mouse or anti-goat FITC-conjugated (1:100 Wnal dilution; Rockland Immunochemicals, Inc., USA) and anti-rabbit Rhodamine-conjugated (1:100 Wnal dilution; Chemicon International Inc., USA) secondary antibodies. The sections were mounted using the slowfade antifade kit (Molecular Probes, Eugene, Oregon, USA). To increase the reliability of our data on NRG-1 expression in normal and regenerating muscle, we carried out several experiments using similar procedures. We investigated NRG1 immunoreactivity in normal and regenerating muscle without primary antibody, with another NRG-1 antibody (anti-heregulin, Sigma), and after treatment with recombinant human NRG1-1/HRG1-1 EGF domain (R&D systems) using three diVerent amounts (0.5, 1.0, 2.0 g) of serum along with the primary antibody. Images were acquired on an Olympus IX70 inverted microscope with a Xuorescent attachment (Olympus) and a Photonic Science CCD camera (Olympus DP50), controlled by the ViewWnder Lite (version 1.0) software (Pixera Corporation, Kawasaki, Japan). Statistical analysis All values were expressed as means § SEM. Unpaired t tests were used to evaluate the signiWcance of diVerences in each NRG-1 mRNA and protein levels between control and bupivacaine-treated rats. P < 0.05 was considered signiWcant.
Results The immunoreactivity of c-Met and dystrophin or NRG-1 were visualized using FITC- or Rhodamineconjugated secondary antibodies, respectively. The expression of NRG-1 was detected in some of the quiescent satellite cells expressing c-Met (Fig. 1a, b). Some c-Met negative mononuclear cells located near the membrane of muscle Wbers also possessed NRG-1 immunoreactivity in control TA muscles of rats (Fig. 1a, b). NRG-1 positive cells were detected in within (myonuclei) and outside (satellite cells) of the plasma membrane labeled with dystrophin (Fig. 1c, d). Previous studies showed the existence of multiple bands of NRG-1 at molecular weights of 183, 115, 81, 64, 50, 37 and 26 kDa using extensor digitorum longus (EDL) and soleus muscles of rats [12]. We observed major bands of NRG-1 at 81, 50 and 26 kDa (Fig. 2 a)
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Fig. 1 In the control TA muscle of rats, the expression of NRG1 was detected in some quiescent satellite cells expressing c-Met (a, b). Several c-Met negative mononuclear cells (myonucleus) existing near the membrane of muscle Wbers also possessed NRG1 immunoreactivity (b). These NRG-1 positive cells existed in the interior (myonuclei) and exterior (satellite cells) of the plasma membrane labeled with anti-dystrophin (c, d). C-Met (a) and dystrophin (c) immunoreactivity was visualized with FITC-, and
NRG-1 (b, d) was visualized with Rhodamine-conjugated secondary antibodies. White circles and squares indicate the same Wbers on diVerent immunoimages. White arrows denote the satellite cells that expressed both c-Met and NRG-1 or NRG-1 at the exterior of the plasma membrane labeled with dystrophin, and yellow arrows denote the myonuclei expressing NRG-1 but not c-Met or NRG-1 at the interior of the plasma membrane. Bar = 50 m
in the TA muscle of adult Wistar rats. When the muscle was damaged, Western blot analysis showed that all the NRG-1 isoforms in the regenerating muscles decreased on the 1st day after damage, which was most pronounced on the 4th day. It increased gradually after the 4th day up to 2 weeks following damage (Fig. 2a). On the other hand, NRG-1 and myogenin mRNA in the muscles similarly increased from 2 to 6 post-surgical days (Fig. 2b). Densitometric analysis at 6 days after bupivacaine treatment revealed a signiWcant increase in NRG-1 mRNA levels compared with the control TA muscle (Fig. 2c). In normal TA muscle of the rat, we observed -bungarotoxin immunoreactivity at neuromuscular junctions (Fig. 3a). In the regenerating muscle at 1 day post-surgery, loss of selective accumulation of -bungarotoxin occurred at neuromuscular junctions of all muscle Wbers (Fig. 3b). In order to investigate whether the proliferating satellite cells have expressed NRG-1 protein in regenerating muscles, we performed immunoXuorescence using anti-BrdU, which identiWes these cells. BrdU-positive proliferating satellite cells did not possess NRG-1 protein in the regenerating muscle at 2 days postsurgery (Fig. 3c, d). At 4 days post-surgery, we did not observe clear co-localization between
NRG-1 and myogenin in the regenerating muscle (Fig. 3e, f). NRG-1 expression was detected on myogenin-positive satellite cells at 6 (Fig. 3g, h) and 10 (Fig. 3i, j) days post-bupivacaine injection. To conWrm our data on NRG-1 expression in normal and regenerating muscle, we carried out several control experiments [19]. Another NRG-1 antibody (anti-heregulin, Sigma) made using a non-overlapping epitope of SC-348 (Santa Cruz) detected co-localization of NRG-1 and myogenin in the diVerentiating satellite cells (Fig. 4c, d). Moreover, NRG-1 immunoreactivity was lost after the treatment of recombinant human NRG-1 using diVerent amounts (0.5, 1.0, 2.0 g) of serum along with the primary antibody (Fig. 4e, f). To clarify changes in NRG-1 expression in the spinal cord following muscle damage, we conducted immunoblotting and RT-PCR at 0, 1, 2, 4, 6, 10 and 14 days postsurgery. In the spinal cord, we observed several bands of NRG-1 at 115, 81, 50 and 26 kDa, with bands at 115 and 26 kDa being decreased and bands at 81 and 50 kDa being increased after the Wrst and second day (Fig. 5a). Densitometric analysis at 6 days after injection showed a signiWcant increase in the amount of NRG-1 protein at 50 kDa compared with the controls (Fig. 5b).
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Acta Neuropathol (2007) 113:451–459 Fig. 2 Western blot (n = 1/ each timepoint) shows that NRG-1 proteins in the regenerating muscles decrease on the 1st day and increase after the 4th day up to 2 weeks (a). Lower lane is an -actin band. Equal amounts of protein were loaded in each lane. NRG-1 and myogenin mRNA in the muscle (n = 1/each timepoint) increases from 2 to 6 post-surgical days (b). Densitometric analysis showed a signiWcant increase in NRG1 mRNA in the regenerating muscles at 6 days after bupivacaine treatment compared to those of the controls (c). The integrated optical density (IOD) of NRG-1 normalized against the IOD of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (arbitrary units). Values are means § SEM (n = 5/ group). Asterisks denote signiWcant diVerences with the bupivacaine-treated rats
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a C
1
Post -bupivacaine injection (days) 2 4 6 10
C
1
Post-bupivacaine injection (days) 2 4 6 10
14
NRG-1 (81 kDa) NRG-1 (50 kDa) NRG-1 (26 kDa) a-Actin (45 kDa)
b
14
NRG-1 myogenin GAPDH
*
c 2.5 2 NRG-1 mRNA IOD/ GAPDH mRNA IOD (arbitrary units)
1.5 1 0.5 0
On the other hand, the amount of NRG-1 mRNA in the spinal cord did not signiWcantly change after muscle damage (Fig. 5c). To determine whether NRG-1 protein is expressed in the spinal motoneurons after muscle damage, we used an antibody for microtubule associated protein-2 (MAP-2). MAP-2 is associated with dendrite stability in mature neurons and normally exists in nerve cell bodies and dendrites. In the normal rat, NRG-1 expression was not detected in many of the MAP-2 positive motoneurons (Fig. 5d, e). In contrast, NRG-1 protein was abundantly expressed in the cell bodies of many motoneurons innervating the TA muscle subjected to bupivacaine injection (Fig. 5f, g).
Discussion Two main conclusions can be drawn from the present study. First, NRG-1 immunoreactivity was observed in diVerentiating satellite cells in regenerating muscle. Second, NRG-1 protein was expressed in several motoneurons in the spinal cord innervating the regenerating muscle.
Control
Bupivacaine
We showed that NRG-1 protein was expressed in some quiescent satellite cells expressing c-Met. It is well known that satellite cells play important roles in the regeneration of muscle Wbers; however, in the quiescent stage, satellite cells have no proliferating activity. It is possible that NRG-1 plays an important role in the diVerentiation of satellite cells in the regenerating TA muscle of rats. Indeed, we did not detect NRG-1 expression in the same nuclei of proliferating satellite cells possessing BrdU at the 2nd day postinjection. Ford et al. [5] demonstrated that NRG-1 promotes the proliferation of myoblasts and inhibits myoblast diVerentiation in early myogenesis in vitro. Additionally, our observation of the NRG-1 immunoreactivity in the many myogenin-positive diVerentiating satellite cells may coincide with previous studies that NRG stimulates the diVerentiation of L6 rat myotubes in culture [4, 10]. Since myogenin induces myoblasts to withdraw from the cell cycle and fuse to form multinucleated myotubes, our Wnding further supports the diVerentiation-enhancing role in NRG-1 against satellite cells in the regenerating muscle in vivo.
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Fig. 3 In normal TA muscle of the rat, we observed -bungarotoxin immunoreactivity at neuromuscular junctions (a). In the regenerating muscle at 1 day post-surgery, loss of selective accumulation of -bungarotoxin occurred at neuromuscular junctions of the muscle Wber (b). BrdUpositive satellite cells (c) did not possess NRG-1 protein in the regenerating muscle at 2 days post-surgery (d). At 4 days post-surgery, we did not observe clear co-localization between NRG-1 and myogenin in the regenerating muscle (e, f). NRG-1 expression was detected on the myogenin-positive satellite cells at 6 (g, h) and 10 days (i, j) post-bupivacaine injection. BrdU (c) and myogenin (e , g and i) immunoreactivity were visualized by FITC-, and bungarotoxin (a, b) and NRG1 (d, f, h and j) was visualized by Rhodamine-conjugated secondary antibodies. White circles and squares indicate the same Wbers on diVerent immunoimages. White arrows denote the same satellite cells. Bar = 50 m
Our results did not diVer from those in the report on the L6 myoblasts, which demonstrated that NRG-1 inhibits the formation of myotubes by downregulating the myogenin expression [5]. Although NRG-1 may
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inhibit early muscle diVerentiation (2–3 days after muscle damage in vivo) as suggested by Ford et al. [5], 6 days following damage seems to be a late period of muscle diVerentiation. Therefore, it is unlikely that
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Fig. 4 In the regenerating TA muscle of rats, there was no primary antibody that showed clearly NRG-1 immunoreactivity in myogenin-positive satellite cells 10 days after damage (a, b). Another NRG1 antibody (mouse anti-heregulin, Sigma) detected the colocalization between NRG-1 (c) and myogenin (d) in the diVerentiating satellite cells at 10 days. In many satellite cells possessing myogenin at 10 days (e), NRG-1 immunoreactivity was lost after treatment with recombinant human NRG-1 of 1.0 g (f), 0.5 or 2.0 g (data not shown) of serum along with the primary antibody. Myogenin (a, e) and NRG-1 (c) immunoreactivity was visualized by FITC-, and NRG-1 (b, f) and myogenin (d) were visualized by Rhodamine-conjugated secondary antibodies. White circles and squares indicate the same Wbers on diVerent immunoimages. White arrows denote the same satellite cells expressing myogenin. Bar = 50 m
NRG-1 co-localized with myogenin at this stage interferes with the potent muscle diVerentiation-promoting activity of myogenin. Although the molecular mechanism has not been understood in the regulation of myogenin expression by NRG-1 at fusing myotubes, this regulation was suggested using several neutralizing antibodies for NRG and its receptor, ErbB3 [10]. Using immunoXuorescence, we clearly observed NRG-1 immunoreactivity in c-Met negative mononuclear cells near the Wber membrane or in the interior of the plasma membrane in normal TA muscle. Several previous studies have shown a localization of NRG-1 at the position of nucleus in various cells of humans or rats [6, 15, 18]. For example, immunoXuorescence and Western blot of subcellular fractionated samples demonstrated an immunoreactivity of NRG-1, NRG–1, ErbB2 and ErbB3 in the nucleus of Schwan cells, as well as in the cytoplasm [18]. The exact mechanisms by which NRGs or ErbB receptors are localized to the nucleus are not known. However, the translocation of NRG-1 isoforms and/or ErbB receptors to the nucleus may be to initiate transcription of genes for a speciWc cellular response, such as glucose transport. This nuclear localization of NRG-1, a transmembrane
protein, resembled the distributing pattern of another membrane protein, presenilin-1, in the denervated tibialis anterior muscles of rats [21]. Western blot analysis in our study showed increased levels of NRG-1 protein at 81 and 50 kDa in the spinal cord with time following muscle damage. In addition, we showed abundant expression of NRG-1 protein in the cell bodies of many motoneurons on the damaged side. It is possible that NRG protein is associated with the innervating damaged muscle Wbers but not with the motoneurons themselves, because there were no signiWcant changes in NRG1 mRNA in the spinal cord after muscle damage. Indeed, loss of -bungarotoxin accumulation at NMJ in the damaged Wbers (Fig. 3b) indicates that some of the neuromuscular junctions were damaged after bupivacaine injection for the TA muscle of the rat. Various neurotrophic factors produced in the muscles are transported retrogradely to neural cell bodies of motoneurons after peripheral damage to skeletal muscle [13, 23], although there are diVerences in the muscle regeneration model. In contrast, muscle contusion increases the amount of LIF and GDNF in intramuscular nerves and damaged muscle, and the
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458 Fig. 5 Western blot analysis showing the gradual increase in NRG-1 proteins in the spinal cord after the Wrst day following damage (a). Ponceau staining (lower lane) showed an equal amount of protein loaded to each lane. Densitometric analysis 6 days after bupivacaine treatment shows a signiWcant increase in the NRG-1 protein levels compared with the controls (b). Values are expressed as percentages of the immunostaining intensity of NRG-1 in the spinal cord in age-matched normal rats, which is taken as 100 %, and are given as means § SEM (n = 8/group). Asterisks denote signiWcant diVerences with the bupivacaine-treated rats. NRG-1 mRNA in the spinal cord was not inXuenced after muscle damage (c). In the normal rat, NRG-1 expression was not detected in many motoneurons identiWed by MAP-2 immunoreactivity (d, e). In contrast, NRG protein was abundantly expressed in cell bodies of many of the motoneurons innervating the TA muscle subjected to bupivacaine injection (f, g) NRG-1 (d, f) immunoreactivity was visualized by Rhodamine-, and MAP-2 (e, g) was visualized by FITC-conjugated secondary antibodies. White arrows denote the MAP-2 positive spinal motoneurons. Bar = 100 m
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a C
Post-bupivacaine injection (days) 2 4 6 10
14
NRG-1 (115 kDa) NRG-1 (81 kDa) NRG-1 (50 kDa) NRG-1 (26 kDa) 83kDa
47.5kDa *
b (%) 200 NRG-1 (50 kDa)
150
(arbitrary units) 100 50 0
c
C
Control 1
2
Bupivacaine 4
6
10
14
NRG-1 GAPDH
d
e
f
g
amount of these receptors in motoneurons, respectively [9]. Since the amount of NRG mRNA and protein increased in cultured motoneurons following
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1
the addition of various neurotrophic factors (GDNF, BDNF, NT-3 and NT-4) in vitro [16], the NRG-1 level in motoneurons or muscle may be upregulated by the
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GDNF expression in these positions after muscle damage. These lines of evidence raise the possibility that the increased NRG-1 protein in motoneurons is made within the regenerating muscles and would be transported retrogradely from muscles to motoneurons. NRG-1 in motoneurons of the spinal cord may promote the reinnervation of muscles by intramuscular sprouting of motor axonal terminals and/or proliferation and migration of Schwann cells. In summary, the present study showed that the expression of NRG-1 increased during the regeneration of skeletal muscles and spinal cord. We suggest that NRG-1 promotes the diVerentiation of satellite cells, and that NRG-1 is transported retrogradely from muscles to motoneurons in vivo. Acknowledgments This work was supported by a research Grant-in-Aid for Young Scientists B (No. 17700500) and ScientiWc Research C (No. 16591505) from the Ministry of Education, Science, Sports and Culture of Japan, and by the grant for young researcher’s project of Research Center for Future Technology, Toyohashi University of Technology.
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