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The non-synaptic expression of transforming growth factor-beta 2 is neurally regulated and varies between skeletal muscle fibre types
Pergamon
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Neuroscience Vol. 87, No. 4, pp. 845–853, 1998 Copyright 1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(98)00180-8
THE NON-SYNAPTIC EXPRESSION OF TRANSFORMING GROWTH FACTOR-BETA 2 IS NEURALLY REGULATED AND VARIES BETWEEN SKELETAL MUSCLE FIBRE TYPES I. S. MLENNAN,* K. KOISHI, M. ZHANG and N. MURAKAMI Department of Anatomy and Structural Biology, University of Otago, Dunedin, New Zealand Abstract––In adult skeletal muscles, transforming growth factor-beta 2 is restricted to the postsynaptic domain of the neuromuscular junction. The various putative functions of this transforming growth factor-beta 2 predict different patterns of transforming growth factor-beta 2 expression in denervated muscles. We therefore denervated rat tibialis anterior, extensor digitorum longus and soleus muscles and examined the expression of transforming growth factor-beta 2 using semi-quantitative reverse-trascription polymerase chain reaction and immunohistochemistry. Denervation up-regulated transforming growth factor-beta 2 expression extrasynaptically with little or no effect on synaptic expression. The up-regulation was detectable by one day, had become significant by three days and remained elevated for at least two weeks. This proves that the transforming growth factor-beta 2 associated with the neuromuscular junction is not under neural control and is consistent with transforming growth factor-beta 2 being a trophic factor for motoneurons. This pattern of transforming growth factor-beta 2 expression is similar to that described for other proteins associated with the neuromuscular junction, notably the acetylcholine receptor subunit genes. However, in contrast to the acetylcholine receptor subunit genes, the extent of up-regulation of transforming growth factor-beta 2 varied between fibre types, with the glycolytic IIB fibres being less affected than other fibre types. 1998 IBRO. Published by Elsevier Science Ltd. Key words: muscles, neuromuscular junction, transforming growth factors, denervation, motoneuron.
Synapses involve an interplay between the nerve terminal, the effector cell and associated nonneuronal cells. Different synapses have different arrangements of these elements, and this is a major determinant of their function. With sympathetic synapses, the pre- and postsynaptic elements are separated by a wide junction, permitting diffusion of the neurotransmitter and action distant from the synapse.7 Neuromuscular junctions (nmj), in contrast, contain an intimate association between the synaptic bouton and the post-domain (muscle fibre) with the synapse being encased by a specialized terminal Schwann cell.11 This helps generate reliable activation of the postsynaptic fibre, with negligible effects on adjacent fibres. The molecular mechanisms that underlie the establishment and maintenance of the nmj have been extensively studied and partially elucidated, particularly with respect to the acetylcholine receptor *To whom correspondence should be addressed. Abbreviations: AChR, acetylcholine receptor; EDL, extensor digitorum longus; GAPDH, glyceraldehyde-3phosphate dehydrogenase; HRP, horseradish peroxidase; LAP, latency-associated peptide; mAb, monoclonal antibody; nmj, neuromuscular junction; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; RT, reverse transcription; SDS, sodium dodecyl sulphate; SSC, standard saline citrate; TGF-â2, transforming growth factor-beta 2.
(AChR). The presynaptic motoneuron affects the synthesis and aggregation of the AChR through the release of putative regulator peptides and as a consequence of the electrical activity associated with contraction of the muscle fibre.8,24 Signals originating from the muscle fibre are also important to the integrity of the nmj but the molecular identity of these signals and their functions are largely unknown. One of the characteristics of a muscle-derived signal is that its production should be elevated in the synaptic versus non-synaptic portions of a muscle fibre. To date, only two regulatory substances are known to be specifically associated with the postsynaptic domain of the nmj: transforming growth factor-beta 2 (TGF-â2)17 and nitric oxide synthase.16 TGF-â2 and nitric oxide are therefore prime candidates for regulators of nmj structure and/or function. In this paper, we report the effect of denervation on the production of TGF-â2 in the postsynaptic and extrasynaptic domains of muscle fibres. These experiments were done for several reasons. First, TGF-â2 is present in motoneurons6 and is anterogradely transported down the axon towards the nerve terminal (Jiang and McLennan, unpublished observations). Some cell types amplify their response to exogenous TGF-â1 by synthesizing TGF-â1.20 By analogy, we hypothesized that TGF-â2 is an autocrine regulator of the nmj. That is, motoneurons
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release TGF-â2 to regulate some aspect of the postsynaptic domain and that the muscle fibres amplify this signal by producing TGF-â2.17 If this hypothesis is correct, denervation should lead to a loss of TGF-â2 from the synaptic domain of muscle fibres. Second, muscle fibres maintain motoneurons in a functional mode by up-regulating the production of proteins associated with neurotransmitter function and by down-regulating the proteins associated with axon elongation.1 TGF-â2 is an obvious candidate for an anti-axon growth signal as it only becomes abundant at the nmj once axon growth has ceased and the nmj has matured.17 If TGF-â2 is an antigrowth signal, its production should be suppressed within denervated muscle, so as to permit the reinnervation of the muscle. Third, the production of the á, â, ä and ã subunits of the acetylcholine receptor and several other synapse-specific proteins occur along the length of immature muscle fibres, with the mature distribution of proteins being partially caused by down-regulation of their synthesis in the extrasynaptic domain. Denervation leads to the resumption of the synthesis in the extrasynaptic domains.8 High levels of TGF-â2 are present in the extrasynaptic domains of immature muscle fibres17 and we were therefore interested to determine whether denervation also up-regulates the production of TGF-â2 in the extrasynaptic domains of mature muscle fibres.
operations on the tibialis anterior and EDL muscles were equivalent. Muscles from the contralateral limb and from unoperated rats were used as controls. Northern blot hybridizations Total RNA was isolated with TRIzol reagent (GIBCOBRL, Gaithersburg, U.S.A.) according to the manufacturer’s instructions. Twenty micrograms of each RNA sample was size-fractionated by electrophoresis using 0.8% agarose gels and 4-molpholine-ethanesulphonic acid buffer and transferred to nylon membranes. The membranes were hybridized overnight at 55C in a solution containing 50% formamide, 0.5 M sodium phosphate, pH 7.2, 1% bovine serum albumin, 7% sodium dodecyl sulphate (SDS), 1% PEG6000 and a 32P-labelled probe prepared from a 1.1 kb Hind III-Xba I DNA fragment of a mouse TGF-â2 cDNA clone (pMTGFbeta2, ATCC no. 6319818). The membranes were then washed in a series of solutions, including a final wash with 0.1standard saline citrate (SSC) containing 0.1% SDS at 65C. The hybridization signals were detected by exposing the membranes to X-ray films with two intensifying screens at 80C for various lengths of time.23
Denervation of muscles
Detection of transforming growth factor-beta 2, acetylcholine receptor-å and glyceraldehyde-3-phosphate dehydrogenase messenger RNA by reverse transcription–polymerase chain reaction Total RNA was isolated as described above and genomic DNA removed by incubating each RNA fraction with RNase-free DNase (Promega, Madison, U.S.A.). The DNase-treated RNAs were converted to cDNAs using SuperScript II RNase H reverse transcriptase (GIBCOBRL) and oligo d(T)15 as the primer. The reaction was performed with 100 units of reverse transcriptase per 1 µg of RNA at 42C for 50 min using the buffer recommended by the manufacturer. Various amounts of cDNA were subsequently used as templates for polymerase chain reaction (PCR) in a 10 µl reaction mixture containing 0.5 units of Taq DNA polymerase (GIBCO-BRL), 1.0 µM each of primers, 200 µM each of dATP, dCTP, dGTP and dTTP, 20 mM Tris–HCl (pH 8.4), 50 mM KCl and an appropriate concentration of MgCl2 [2.5 mM for TGF-â2, and 1.5 mM for AChR-å subunit or glyceraldehyde-3-phosphate dehydrogenase (GAPDH)]. The PCR was performed in a thermal cycler (MJ Research, Watertown, U.S.A.) with programmes specific to the combination of primers. The denaturation, annealing and elongation steps were, respectively, 94C 15 s, 58C 10 s and 72C 20 s for TGF-â2; 95C 15 s, 66C 15 s and 72C 20 s for the AChR-å subunit; 94C 15 s, 50C 15 s and 72C 20 s for GAPDH. For all the primers, the initial denaturation step was 94–95C for 2 min and the final extension period was 72C for 3 min, followed by cooling at 4C for 10 min. The nucleotide sequences of the primers used were: TGF-â2, 5 -TCCTACAGACTGGAGTCACAAC AG-3 and 5 -ATCATATTGGAAAGCTGTTCGATC3 ;15 AChR-å subunit, 5 -TAGGAGACCTGAGGACA CTGTCA-3 and 5 -ATTGCCGTCGTCATCCACGGC AAAGA-3 ;4,29 GAPDH, 5 -TTCACACCCATCACAA AG-3 and 5 -CTTCATTGACCTCAACTA-3 .12
The tibialis anterior, extensor digitorum longus (EDL) and soleus muscles of nine rats were denervated by sectioning the sciatic nerve in one of their hindlimbs. The sciatic nerve was approached posteriorly between the biceps femoris, the semimembranosus and the semitendinosus. A 5–10 mm piece of the nerve was removed and the cut ends attached to adjacent muscles to prevent re-innervation of the target muscles. The rats were killed either one, three, five or seven days after the operation. In a further nine rats, the tibialis anterior and EDL were denervated by transectioning the common peroneal nerve, and the rats being killed between one and 35 days later. The effects of the two
Quantitation of the reverse transcription–polymerase chain reaction products The cycle numbers of the PCR were reduced to the levels where the amounts of PCR products were relative to those of the starting materials (22 cycles for TGF-â2 and AChR-å subunit and 20 cycles for GAPDH). These conditions were established using plasmid clones of murine TGF-â2 cDNA (ATCC, Maryland, U.S.A.) and rat AChR-å subunit (generous gift of Dr V. Witzemann). Once the PCR were completed, the products were size-fractionated by electrophoresis using 1.5% agarose gels, transferred to nylon
EXPERIMENTAL PROCEDURES
Animals All experiments were approved by the University of Otago’s Committee on Ethics in the Care and Use of Laboratory Animals. All efforts were made to minimize animal suffering and to reduce the number of animals used. There are no suitable alternatives to the in vivo techniques used in this study. All animals were bred by the Department of Laboratory Animal Science, University of Otago Mature Wistar rats (15- to 20-weeks-old) were killed by cervical dislocation and selected muscles removed, immersed at their resting length in melting isopentane and stored under liquid nitrogen until required. Rats which underwent surgical procedures were anaesthetized by an intraperitoneal injection of Ketamine (90 mg/kg) and Xylazine (3 mg/kg) (Parnell Laboratories, Auckland, New Zealand). All operations were done using sterile working conditions and the incisions closed with sutures.
Muscle TGF-â2 membranes (Hybond N+, Amersham, Buckinghamshire, U.K.) and hybridized with 32P-labelled oligonucleotides as probes. The probes were derived from specific parts of the internal sequences of amplicons and labelled by a kinase reaction with T4 polynucleotide kinase and [ã-32P]ATP:9 TGF-â2: 5 -TCCTACAGACTGG AGTCACAACAG-3 , AChR-å subunit: 5 -ACTA TCGG CTCAAC TTCAG CAAGGACGA-3 and GAPDH: 5 -ACTCAGCACCAG CATCACCCCATTTGA-3 . The membrane hybridizations were performed in a solution containing 0.5 M sodium phosphate (pH 7.0) and 7% SDS at 48C overnight.5 The membranes were then washed in 6SSC–0.1% SDS for 15 min and finally in 2SSC–0.1% SDS for 2 min at 48C. The images of hybridization signals were captured and quantified by a phosphoimager (Fuji film) using the analytical software supplied by the manufacturer. When higher resolution was required, the hybridized membranes were exposed to X-ray films (Kodak, Rochester, NY, U.S.A. Omat X-AR) with two intensifying screens at 80C for various lengths of time. Immunohistochemistry Five-micrometre sections of the muscles were cut in a cryostat and stained as previously described,17 with minor modifications: milk powder was omitted from the wash buffer and cold-fish gelatin was not included in the antibody diluting buffers. Briefly, the sections stained with either anti-TGF-â2 or anti-neurofilament antibodies were fixed in neutral buffered 4% paraformaldehyde at 4C for 30 min and incubated overnight at 4C with 1 µg/ml of antibody. The immunoreactivity was then developed using biotinylated anti-rabbit-Ig (Amersham), streptavidin biotinylated horseradish peroxidase complex (HRP, Amersham) and 3-amino-carbamide (AEC) as the chromogen. The sections stained with the various anti-myosin monoclonal antibody (mAb) were not fixed and biotinylated–antimouse-Ig (Amersham) was used as the secondary antibody. The slides were not counterstained and were examined using bright-field and phase-contrast microscopy with a Zeiss Axioplan photomicroscope. The specificity of the anti-TGF-â2 immunoreactivity was checked by absorbing it with a 25-fold excess by weight of the antigenic peptide and by comparing the stain produced by non-immune rabbit IgG. The anti-TGF-â2 was an affinity-purified rabbit antibody made to a peptides corresponding to the mature parts of TGF-â2 (Santa Cruz Biotechnology Inc., Santa Cruz, U.S.A.). The anti-neurofilament was a rabbit polyclonal antibody purchased from Sigma, St Louis, U.S.A. (N-4142) whereas the various anti-myosin mAbs were generous gifts either Dr Fitzsimons (NOQ7.1.1A, anti-type-I myosin) or Dr Schiaffino and Regeneron Pharmaceuticals (New York, U.S.A.) SC-71 (anti-IIa myosin), BF-F3 (anti-IIb myosin) and BF-35 (recognizes type I, IIa and IIb myosins but not IIx myosin).25 Quantitation of transforming growth factor-beta 2 immunoreactivity Sections of muscles were stained as described above except that (i) a fluorescent tag (streptavidin-BODIPY-Fl, Molecular Probes) was used instead of the streptavidin– biotinylated–HRP complex and (ii) higher concentrations of reagents (4 µg/ml of primary antibody) were used to ensure saturation and hence linearity between the amount of TGF-â2 in the section and the amount of immunoreactivity. The sections were examined in a MRC600 confocal laser scanning microscope using the 488 nm line of its krypton argon laser and the photon counting mode. In this mode, the amount of light detected is linearly related to the amount of fluorescence emitted. The total amount of fluorescence emitted from various fibres was then measured. A
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measure of the amount of TGF-â2 protein in various muscle fibres was made by subtracting the intensity of the staining present in sections reacted with non-immune IgG from that present in anti-TGF-â2-stained sections. The types of the fibres were determined by examining adjacent sections which had been stained with myosin-isoformspecific antibodies. Identification of synaptic sites Selected sections were double-labelled with anti-TGF-â2 and rhodamine-conjugated á-bungarotoxin (Molecular Probes) using the immunohistochemical procedure described above, except that rhodamine-á-bungarotoxin (1:500 dilution) was included with the secondary antibody incubation. The slides were mounted with Slow Fade (Molecular Probes, Eugene, U.S.A.). Western blots The TGF-â2 molecules present in innervated, one-week denervated and three-week denervated tibialis anterior and EDL muscles were concentrated by immunopreciptiation prior to western blotting. The muscles were homogenized (10% w/v) in a mixture containing 150 mM NaCl, 1% NP40, a cocktail of protease inhibitors (one tablet per 7 ml; Boehringer Mannheim, Mannheim, Germany, no. 1 697 498) and 10 mM Tris–HCl, pH 7.4. The homogenates were spun and the supernatants collected. An aliquot of the innervated muscle was spiked with 2 ng of recombinant human TGF-â2 (Genzyme, Cambridge, U.S.A.). One microgram of either anti-TGF-â2 or non-immune IgG was added to 4 ml aliquots of the muscles and after a 1 h incubation at 4C, 2 mg of protein-A sepharose beads (Sigma) were added. After 2 h further incubation, the beads were precipitated and the protein bound to them disolved in loading buffer containing mercaptoethanol and run on 4–20% gradient SDS–polyacrylamide gel electrophoresis (PAGE) gels. The proteins were then transferred to polyvinylidene difluoride membrane (Immobilon-P, Millipore, Bedford, U.S.A.) and stained with anti-TGF-â2 using a chemiluminescence detection system (ECL-Plus, Amersham). RESULTS
The efficacy of the denervation was checked by staining sections with an anti-neurofilament antibody and by measuring the levels of AChR-å mRNA, which are known to be increased after denervation.28 Intact axons were not observed in the intramuscular nerves, indicating that denervation was complete and that no re-innervation had occurred. Similarly, the levels of AChR-å mRNA increased three-fold by three days after the operation and remained elevated (Fig. 1c). Increased transforming growth factor-beta 2 expression in denervated muscles The expression of TGF-â2 in the denervated muscles was examined by northern blotting. The amount of TGF-â2 mRNA in innervated muscles was below the level of detection (Fig. 2). This is consistent with prior immunohistochemical studies which indicate that significant amounts of TGF-â2 are only associated with the subsynaptic nuclei, which constitutes fewer than 1% of muscle nuclei.17 The amount of TGF-â2 mRNA was elevated in the
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Fig. 1. Semi-quantitative RT–PCR estimates of the amounts of TGF-â2 and AChR-å subunit mRNAs in innervated and denervated skeletal muscles. Total RNAs were isolated, reverse transcribed and the prepared cDNAs amplified with primers specific to the target genes, as described in the Experimental Procedures. (a) cDNAs derived from 20 ng of total RNA were used for each PCR reaction when TGF-â2 and GAPDH mRNAs were amplified whereas a 10-fold higher concentration was used for each estimate of the AChR-å subunit mRNAs. (b) The intensities of the hybridization signals for the PCR products for TGF-â2 and AChR-å subunit mRNAs were standardized relative to those of the GAPDH. The values were then normalized relative to those of innervated muscles.
Fig. 2. Northern analysis of TGF-â2 transcripts in innervated and denervated skeletal muscles. Twenty microgram of each total RNA fraction was electrophoresed through a 0.8% agarose gel, transferred to a nylon membrane and hybridized with a 32P-labelled probe prepared from a murine TGF-â2 cDNA. The signals were detected by exposing the membrane to an X-ray film at 80C for two weeks, with two intensifying screens. (a) TGF-â2 hybridization signals and (b) agarose-gel stained with ethidium bromide to show loading of the total RNA.
denervated muscles, becoming first detectable around three days after the operation. Four mRNA species were detected, with estimated sizes of 3.4, 4.2, 4.8 and 6.3 kb. The signal from the 6.3 kb band was barely detectable (Fig. 2). All four messages were similarly increased after denervation. Multiple species of
TGF-â2 mRNA have been previously reported, but the biological importance of the various bands is presently unknown.18,27 Semi-quantitative reverse transcription (RT)–PCR was then used to obtain a more sensitive estimate of the effect of denervation on TGF-â2 expression. The amplified PCR fragments were examined by Southern blotting and the amounts of TGF-â2 mRNA were standardized relative to the expression of GAPDH messages. Plasmid clones of murine TGF-â2 cDNA and rat AChR-å subunit were used to verify that the intensity of the signals were linearly related to the cDNA copy number. The amount of TGF-â2 mRNA was not significantly changed oneday post-denervation. However, by three days after the operation, an approximately eight-fold increase in TGF-â2 mRNA had occurred, with the levels of message remaining high in the subsequent days (Fig. 1a) Denervation increases transforming growth factor-beta 2 protein in extrasynaptic portions of muscles The amount of TGF-â2 protein in the extrasynaptic portions of muscle fibres increased with time after denervation, in parallel with the change in TGF-â2 mRNA. TGF-â2 immunoreactivity in the nonsynaptic regions of denervated tibialis anterior and EDL muscle fibres was slightly elevated one day after cutting the common peroneal nerve and had become intense by four days (Fig. 3c). The intensity of TGF-â2 immunoreactivity continued to increase, reaching high levels in the muscles which had been denervated for longer than one week (Fig. 3d).
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Fig. 3. TGF-â2 immunoreactivity associated with normal and denervated tibialis anterior. (a) Innervated muscle; (b) the anti-TGF-â2 was absorbed with antigen prior to the immunohistochemical procedure; (c) four-day denervated muscle; (d) 14-day denervated muscle. The arrows point to the postsynaptic domains of nmj, which have elevated levels of immunoreactivity in normal and denervated muscle (see also Fig. 5). The larger (IIB) fibres are stained with lower intensity than the smaller oxidative fibres. Panel d is at slightly higher magnification to show the intracellular staining observed in the denervated fibres. Scale bars (a–c)=50 µm; (d)=25 µm.
Similar results were obtained with soleus muscle fibres after transection of the sciatic nerve, although the increase in TGF-â2 expression was slightly slower than occurred in the tibialis anterior or EDL. This slower rate may be a consequence of transection of the sciatic nerve leaving a longer distal stump than transection of the common peroneal nerve. The increase in TGF-â2 immunoreactivity was initially perinuclear and associated with the circumference of fibres (Fig. 3c). However, with the passage of time after denervation, the immunoreactivity began to be distributed throughout the cytoplasm of fibres. By 14-days post denervation, the cytoplasm of all fibres contained TGF-â2, although the intensity of the stain varied from fibre to fibre (Fig. 3d). This pattern of staining was still present 35-days post denervation, which is the longest time we examined. TGF-â2 immunoreactivity was not detected in either the basal lamina of muscle fibres or in the various connective tissues (Fig. 3). The Schwann cells associated with the intramuscular nerves also expressed increasing levels of TGF-â2 (Fig. 4). The TGF-â2 immunoreactivity was specific. A similar pattern of stain was not observed with various other primary antibodies and the stain produced with the anti-TGF-â2 antibody was abolished by prior incubation with the antigenic peptide (Fig. 3b).
Fig. 4. TGF-â2 immunoreactivity associated with the intramuscular nerves of a 14-day denervated skeletal muscle. Scale bar=20 µm.
Synaptic transforming growth factor-beta 2 is unaffected by denervation The nmj were identified by the use of rhodaminealpha-bungarotoxin (Fig. 5b), which binds to the nicotinic acetylcholine receptor and is a classical
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Fig. 5. TGF-â2 immunoreactivity associated with neuromuscular junctions of a three-day denervated muscle. Transverse sections of a muscle were stained with anti-TGF-â2 linked to BODIPY-FL and rhodamine-conjugated á-bungarotoxin and examined on a confocal laser scanning microscope (see Experimental Procedures). (a) TGF-â2 immunoreactivity; (b) á-bungarotoxin. The arrows point to nmj, all of which show elevated levels of TGF-â2 immunoreactivity. Scale bar=30 µm.
marker for the nmj.10 TGF-â2 immunoreactivity was associated with the nuclei lying subjacent to the alpha-bungarotoxin binding sites of denervated muscle: that is the subsynaptic nuclei of denervated fibres (Figs 3, 5). High levels of TGF-â2 were also associated with the subsynaptic nuclei of innervated nmj (Fig. 3a and see Ref. 17). This made it difficult to determine whether the intensity of stain at the nmj was unchanged or increased by denervation. However, with respect to our original objective of determining whether motoneurons up-regulate the production of TGF-â2 at nmj, the key observation is that levels of synaptic TGF-â2 were not diminished after denervation. Innervated and denervated IIB fibres have low expression of transforming growth factor-beta 2 The intensities of TGF-â2 expression in the extrasynaptic portions of the muscle fibres appeared to be correlated with the size of the muscle fibre, suggesting that expression of extrasynaptic TGF-â2 may vary with fibre type. This was confirmed by staining adjacent sections of five-day denervated muscles with either a myosin-isoform-specific antibody or with the anti-TGF-â2 antibody. Denervated type I, type IIA and IIX fibres all contained elevated TGF-â2 immunoreactivity, whereas all of the 35 type IIB fibres examined were minimally stained. The amounts of TGF-â2 immunoreactivity in the various fibre types were then quantified using an MRC 600 confocal microscope in its photoncounting mode. The intensities of stain of IIA and IIX fibres in seven-day denervated tibialis anterior were approximately three times that of the IIB fibres (Fig. 6, Table 1). The tibialis anterior contains only a small number of slow fibres. The soleus was therefore examined to obtain quantitative data on the type I fibres. The intensity of staining of the type I fibres was similar to that of the IIA fibres (Table 1). We have previously reported that the extrasynaptic expression of TGF-â2 in innervated muscle fibres is
very low.17 However, this conclusion was based on the analysis of muscle regions which predominantly consist of type IIB fibres. The expression of TGF-â2 in a range of muscles was therefore examined to determine whether TGF-â2 expression varies between muscles. The muscles examined included typical limb muscles (EDL and tibialis anterior), an anti-gravity limb muscle (soleus), an axial muscle (erector spinae), a respiratory muscle (diaphragm), a facial muscle (masseter), rectus abdominis and the cremaster. The expression of TGF-â2 in the subsynaptic portion of the fibres was uniformly high in all of the muscles examined. Similarly, the amount of extrasynaptic TGF-â2 was always low, although the absolute level of expression varied slightly from muscle to muscle. Transforming growth factor-beta 2 is cleaved in denervated muscles TGF-â2 is intially synthesized as a precursor molecule which is then cleaved to form the mature peptide and a latency-associated peptide (LAP). In some cells, TGF-â1 can be retained within cells as an inactive complex containing the uncleaved precursor molecule.19 As noted above, TGF-â2 accumulates in denervated fibres and we therefore analysed the molecular size of the TGF-â2 to determine whether the precursor molecule had been cleaved. Elevated levels of mature TGF-â2 were detected in the denervated muscles (Fig. 7), indicating that the TGF-â2 precursor molecule had been appropriately processed. DISCUSSION
Neuromuscular junction-associated transforming growth factor-beta 2 is independent of innervation High levels of TGF-â2-immunoreactivity were detected at denervated synaptic sites. Growth factors have short biological half-lives. Therefore, the persistence of TGF-â2 at denervated nmj strongly
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Fig. 6. TGF-â2 immunoreactivity in a seven-day denervated tibialis anterior muscle: correlation with fibre type. (a) A montage of the TGF-â2 immunoreactivity was made by examining neighbouring fields at high magnification, using the photon-counting mode of a BioRad MRC600 confocal laser scanning microscope (see Experimental Procedures). This ensures that the relationship between the amounts of antigen and immunoreactivity are linear: this is optimal for quantitation but gives rise to an image of lower contrast (cf. Figs 5a and 6a with Fig. 3c,d). The type of each fibre was identified from low magnification images of adjacent sections stained with either an anti-MHCIIa antibody (b), an anti-MHCIIb antibody (c) or an antibody which binds to all MHCs, except IIb (d). The arrowheads in b–d point to the fibres labelled in (a). Scale bar (a)=100 µm; (b–d)=300 µm. Table 1. Variation in the amount of transforming growth factor-beta 2 immunoreactivity in denervated skeletal muscles Fibre type
Area (µm2)
Amount of TGF-â2/unit area arbitrary units
Five-day denervated tibialis anterior IIA 1800400 10032(33) IIB 4100100 3312(64)* IIX 1900800 8725(34)# Seven-day denervated soleus I 2100200 IIA 2000400
9416(81) 10021(19)
Sections of denervated muscles were stained with an antiTGF-â2 antibody and the intensity of the resulting immunoreactivity measured using a confocal microscope (see Experimental Procedures). The data are the meanS.D., with the number of fibres examined indicated in parenthesis. *Significantly different to IIA and IIX fibres, P<0.001 (Student’s t-test). # Not significantly different to IIA fibres, P>0.05.
suggests that its production by subsynaptic myonuclei does not require the continued presence of the nerve. This disproves our postulate that muscle fibres produce TGF-â2 as an autocrine amplification of a signal from motoneurons (see Introduction). When muscle fibres are first formed, the á-, â-, ãand ä-AChR subunit mRNAs are produced along their entire length. A series of nerve-dependent changes then occurs: the production of the ã_subunit
Fig. 7. Size of TGF-â2 in normal and denervated muscles. TGF-â2 in normal and denervated muscles were concentrated by immunoprecipitation and run on 4–20% gradient SDS–PAGE gels. The proteins were then transferred to a membrane and stained with anti-TGF-â2. Lanes 1 and 5, innervated muscles; lanes 2 and 6, one-week postdenervation; lanes 3 and 7, three-weeks post-denervation; lane 8, innervated muscle spiked with 2 ng of recombinant TGF-â2; lane 9, 10 ng of recombinant TGF-â2. Lanes 1, 2, 3 and 8 were immunoprecipitated with anti-TGF-â2. Lanes 5–7 were immunopreciptiated with non-immune IgG and consequently lack TGF-â2. A size marker was run in lane 4 (not shown). The major bands, marked with an arrowhead, are 12,000–13,000 mol. wt which is consistent with the bands being the TGF-â2 monomer.
is repressed; the synapse-specific å-subunit is induced; and the extrasynaptic production of the AChR subunits is decreased.8,24 However, once the system is mature, removal of the neural influence leads to resumption of the extrasynaptic production of the AChR subunits but the production of the å-subunit and other AChR subunits continues to be elevated at the nmj. This has led to the suggestion that subsynaptic nuclei are permanently imprinted during the maturation of the nmj.3,28 Our results are consistent
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with this hypothesis and suggest that the imprinting involves a range of synapse-associated genes, and not just the various AChR subunit genes. Does transforming growth factor-beta 2 suppress axon outgrowth? Immature motoneurons are primed for axon outgrowth, thus enabling them to project to their target muscles and to compete for occupancy of synaptic sites. Once established, the pattern of innervation of a muscle is stable and the ability of motoneurons to sprout is restricted, except when the muscle is denervated or paralysed. TGF-â2 is initially dispersed within developing muscle fibres.17 The time at which it becomes concentrated at the nmj has not been precisely determined, but corresponds approximately to the establishment of the mature pattern of innervation. Thus, we postulated that TGF-â2 may switch motoneurons from a growth mode to a functional mode: to up-regulate the production of choline acetyltransferase and to down-regulate the proteins associated with growth. In apparent contradiction of the above hypothesis, the levels of TGF-â2 mRNAs were increased six- to eight fold in denervated muscles. Immunohistochemical studies proved that the levels of TGF-â2 protein were similarly increased and demonstrated that the TGF-â2 protein was associated with the Schwann cells of the intramuscular nerves and the entire length of the muscle fibres. Denervated nerve stumps are favourable sites for motor axon growth26 and the presence of high levels of TGF-â2 in Schwann cells would thus not be expected if one of the functions of TGF-â2 is to repress the ability of motoneurons to grow. Furthermore, exogenous TGF-â2 does not prevent axotomized hypoglossal motoneurons switching their pattern of gene expression from a functional to a growth mode (Zhang, Jiang and McLennan, unpublished observations). Is transforming growth factor-beta 2 a motoneuron survival factor? The location of TGF-â2 at the nmj,17 coupled with evidence that TGF-â2 has neurotrophic actions on various types of neurons,2,21 makes TGF-â2 a putative survival factor for motoneurons. Denervated muscles produce enhanced levels of motoneuron survival factor to support their reinnervation.13 The pattern of expression of TGF-â2 in denervated muscles, reported here, is thus consistent with it being a motoneuron survival factor. Proof of this, however, requires additional work, which is currently being undertaken in our laboratory. Comparison with other neuromuscular junction-specific genes Denervation leads to the re-expression of AChR subunit genes in the extrasynaptic portions of muscle fibres. Similarly, denervation also led to the up-
regulation of extrasynaptic production of TGF-â2, which persists for at least 35 days. However, in contrast to the AChR subunits the extent to which this occurred varied significantly between fibre types, with type IIB fibres producing little TGF-â2 outside of their synaptic zones. This is surprising since the extrasynaptic expression of TGF-â2 in immature type IIB fibres is similar to that of other immature fibres.17 Over 40 molecules are known to be concentrated at the nmj.10,11 The extrasynaptic expressions of many of these molecules are up-regulated by denervation. However, it is unclear whether any of the synapseassociated molecules are like TGF-â2 and show fibre-type specific differences after denervation. To our knowledge, fibre type-specific differences have not been previously reported but this may merely be because investigators have not systematically searched for such differences. The presence of molecular differences between denervated muscle fibre types is, however, to be expected as the speed of denervation-induced atrophy varies with fibre type.14 In particular, IIB fibres are preferentially affected in bed-rest patients and patients with CNS diseases or various congenital myopathies. Is muscle transforming growth factor-beta 2 biologically active? The TGF-âs are secreted as a latent high molecular weight complex consisting of the mature peptide, the LAP and a latent TGF-â binding protein.22 The mature peptide and the LAP are synthesized as a common precursor, which must be cleaved for biological activity to occur. TGF-â1 can be retained within cells as an inactive complex containing the uncleaved precursor molecule.19 As noted above, TGF-â2 accumulates in denervated fibres and we therefore analysed the molecular size of the TGF-â2 to determine whether the precursor molecule had been cleaved. Cleaved TGF-â2 was detected in both innervated and denervated muscles, indicating that appropriate post-translation modification of the precursor had occurred. It is thus likely that the TGF-â2 associated with muscle is active, although this point requires further verification. CONCLUSIONS
The expression of TGF-â2 at the nmj does not require continued innervation of muscle, proving that skeletal muscles are not producing TGF-â2 as an autocrine amplification of signals from motoneurons. In contrast, the extrasynaptic expression of TGF-â2 is neurally regulated. This is typical of proteins associated with the nmj, although the extrasynaptic expression of TGF-â2 is unusual in that the amount of TGF-â2 varies between muscle fibre types. The function of the TGF-â2 produced by skeletal muscle fibres remains to be elucidated, although the pattern of expression is consistent with TGF-â2 having a neurotrophic function.
Muscle TGF-â2 Acknowledgements—The authors thank Mr Rod McCall and Ms Sarah Booth for their excellent technical assistance. The Health Research Council (New Zealand) and The
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Otago Research Council are thanked for their financial support of this work. KK is supported by the Marsden Fund of New Zealand.
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
Armstrong D. M., Brady R., Hersh L. B., Hayes R. C. and Wiley R. G. (1991) Expression of choline acetyltransferase and nerve growth factor receptor witin hypoglossal motoneurones following nerve injury. J. comp. Neurol. 304, 596–607. Blottner D., Wolf N., Lachmund A., Flanders K. C. and Unsicker K. (1996) TGF-beta rescues target-deprived preganglionic sympathetic neurons in the spinal cord. Eur. J. Neurosci. 8, 202–210. Brenner H.-R., Witzemann V. and Sakmann B. (1990) Imprinting of acetylcholine receptor messenger RNA accumulation in mammalian neuromuscular synapses. Nature 344, 544–547. Buonanno A., Mudd J. and Merlie J. P. (1989) Isolation and characterization of the b and e subunit genes of mouse muscle acetylcholine receptor. J. biol. Chem. 264, 7611–7616. Church G. M. and Gilbert W. (1984) Genomic sequencing. Proc. natn. Acad. Sci. U.S.A. 81, 1991–1995. Colosetti P., Olsson T., Miyazono K. and Funa K. (1995) Axotomy of rat facial nerve induces TGF-beta and latent TGF-beta binding protein. Brain Res. Bull. 37, 561–567. Cottee L. J., Lavidis N. A. and Bennett M. R. (1996) Spatial relationships between sympathetic varicosities and smooth muscle cells in the longitudinal layer of the mouse vas deferens. J. Neurocytol. 25, 413–425. Duclert A. and Changeux J.-P. (1995) Acetylcholine receptor gene expression at the developing neuromuscular junction. Physiol. Rev. 75, 339–368. Gause W. C. and Adamovicz J. (1995) Use of PCR to quantitate relative differences in gene expression. In PCR Primer. A Laboratory Manual (eds Dieffenbach C. W. and Dveksler G. S.), pp. 293–311. Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, U.S.A. Grinnell A. D. (1995) Dynamics of nerve–muscle interaction in developing and mature neuromuscular junctions. Physiol. Rev. 75, 789–834. Hall Z. W. and Sanes J. R. (1993) Synaptic structure and development: the neuromuscular junction. Cell 72 Suppl., 99–121. Henderson C. E., Phillips H. S., Pollock R. A., Davies A. M., Lemeulle C., Armanini M., Simmons L. C., Moffet B., Vandlen R. A., Koliatsos V. E. and Rosenthal A. (1994) GDNF: a potent survival factor for motoneurones present in peripheral nerve and muscle. Science 266, 1062–1064. Houenou L. J., McManaman J. L., Prevette D. and Oppenheim R. W. (1991) Regulation of putative muscle-derived neurotrophic factors by muscle activity and innervation: in vivo and in vitro studies. J. Neurosci. 11, 2829–2837. Jennekens F. G. I. (1982) Neurogenic disorders of muscle. In Skeletal Muscle Pathology (eds Mastaglia F. L. and Walton J.), pp. 204–234. Churchill Livingstone, Edinburgh. Krieglstein K. and Unsicker K. (1994) Transforming growth factor-b promotes survival of midbrain dopaminergic neurons and protects them against n-methyl-4-phenylpyridium ion toxicity. Neuroscience 63, 1189–1196. Kusner L. L. and Kaminski H. J. (1996) Nitric oxide synthase is concentrated at the skeletal muscle endplate. Brain Res. 730, 238–242. McLennan I. S. and Koishi K. (1994) Transforming growth factor-beta-2 (TGF-b2) is associated with mature rat neuromuscular junctions. Neurosci. Lett. 177, 151–154. Miller D. A., Lee A., Pelton R. W., Chen E. Y., Moses H. L. and Derynck R. (1989) Murine transforming growth factor-b2 cDNA sequence and expression in adult tissues and embryos. Molec. Endocr. 3, 1108–1114. Miyazono K., Thyberg J. and Heldin C. H. (1992) Retention of the transforming growth factor-beta 1 precursor in the Golgi complex in a latent endoglycosidase H-sensitive form. J. biol. Chem. 267, 5668–5675. Obberghen-Schilling E. V., Roche N. S., Flanders K. C., Sporn M. B. and Roberts A. B. (1988) Transforming growth factor b1 positively regulates its own expression in normal and transformed cells. J. biol. Chem. 263, 7741–7746. Poulsen K. T., Armanini M. P., Klein R. D., Hynes M. A., Phillips H. S. and Rosenthal A. (1994) TGF beta 2 and TGF beta 3 are potent survival factors for midbrain dopaminergic neurons. Neuron 13, 1245–1252. Roberts A. B. and Sporn M. B. (1990) The transforming growth factor-âs. In Handbook of Experimental Pharmacology. Peptide Growth Factors and Their Receptors (eds Sporn M. B. and Roberts A. B.), pp. 419–472. Springer–Verlag, Berlin. Sambrook J., Fritsch E. F. and Maniatis T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, U.S.A. Sanes J. R. (1997) Genetic analysis of postsynaptic differentiation at the vertebrate neuromuscular junction. Curr. Opin. Neurobiol. 7, 93–100. Schiaffino S., Gorza L., Sartore S., Saggin L., Ausoni S., Vianello M., Gundersen K. and Lomo T. (1989) Three myosin heavy chain isoforms in type 2 skeletal muscle fibres. J. Muscle Res. Cell Motil. 10, 197–205. Tornqvist E. and Aldskogius H. (1994) Motoneurone survival is not affected by the proximo-distal level of axotomy but by the possibility of regenerating axons to gain access to the distal nerve stump. J. Neurosci. Res. 39, 159–165. Webb N. R., Madisen L., Rose T. M. and Purchio A. F. (1988) Structural and seuqnece analysis of TGF-b2 cDNA clones predicts two different precursor proteins produced by alternative mRNA splicing. DNA 7, 493–497. Witzemann V., Brenner H.-R. and Sakmann B. (1991) Neural factors regulate AChR subunits mRNAs at rat neuromuscular synapses. J. Cell Biol. 114, 125–141. Witzemann V., Stein E., Barg B., Konno T., Koenen M., Kues W., Criado M., Hofmann M. and Sakmann B. (1990) Primary structure and functional expression of a-, b-, g-, d- and e-subunits of the acetylcholine receptor from rat muscle. Eur. J. Biochem. 194, 437–448. (Accepted 23 March 1998)
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Neuroscience Vol. 87, No. 4, pp. 845–853, 1998 Copyright 1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(98)00180-8
THE NON-SYNAPTIC EXPRESSION OF TRANSFORMING GROWTH FACTOR-BETA 2 IS NEURALLY REGULATED AND VARIES BETWEEN SKELETAL MUSCLE FIBRE TYPES I. S. MLENNAN,* K. KOISHI, M. ZHANG and N. MURAKAMI Department of Anatomy and Structural Biology, University of Otago, Dunedin, New Zealand Abstract––In adult skeletal muscles, transforming growth factor-beta 2 is restricted to the postsynaptic domain of the neuromuscular junction. The various putative functions of this transforming growth factor-beta 2 predict different patterns of transforming growth factor-beta 2 expression in denervated muscles. We therefore denervated rat tibialis anterior, extensor digitorum longus and soleus muscles and examined the expression of transforming growth factor-beta 2 using semi-quantitative reverse-trascription polymerase chain reaction and immunohistochemistry. Denervation up-regulated transforming growth factor-beta 2 expression extrasynaptically with little or no effect on synaptic expression. The up-regulation was detectable by one day, had become significant by three days and remained elevated for at least two weeks. This proves that the transforming growth factor-beta 2 associated with the neuromuscular junction is not under neural control and is consistent with transforming growth factor-beta 2 being a trophic factor for motoneurons. This pattern of transforming growth factor-beta 2 expression is similar to that described for other proteins associated with the neuromuscular junction, notably the acetylcholine receptor subunit genes. However, in contrast to the acetylcholine receptor subunit genes, the extent of up-regulation of transforming growth factor-beta 2 varied between fibre types, with the glycolytic IIB fibres being less affected than other fibre types. 1998 IBRO. Published by Elsevier Science Ltd. Key words: muscles, neuromuscular junction, transforming growth factors, denervation, motoneuron.
Synapses involve an interplay between the nerve terminal, the effector cell and associated nonneuronal cells. Different synapses have different arrangements of these elements, and this is a major determinant of their function. With sympathetic synapses, the pre- and postsynaptic elements are separated by a wide junction, permitting diffusion of the neurotransmitter and action distant from the synapse.7 Neuromuscular junctions (nmj), in contrast, contain an intimate association between the synaptic bouton and the post-domain (muscle fibre) with the synapse being encased by a specialized terminal Schwann cell.11 This helps generate reliable activation of the postsynaptic fibre, with negligible effects on adjacent fibres. The molecular mechanisms that underlie the establishment and maintenance of the nmj have been extensively studied and partially elucidated, particularly with respect to the acetylcholine receptor *To whom correspondence should be addressed. Abbreviations: AChR, acetylcholine receptor; EDL, extensor digitorum longus; GAPDH, glyceraldehyde-3phosphate dehydrogenase; HRP, horseradish peroxidase; LAP, latency-associated peptide; mAb, monoclonal antibody; nmj, neuromuscular junction; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; RT, reverse transcription; SDS, sodium dodecyl sulphate; SSC, standard saline citrate; TGF-â2, transforming growth factor-beta 2.
(AChR). The presynaptic motoneuron affects the synthesis and aggregation of the AChR through the release of putative regulator peptides and as a consequence of the electrical activity associated with contraction of the muscle fibre.8,24 Signals originating from the muscle fibre are also important to the integrity of the nmj but the molecular identity of these signals and their functions are largely unknown. One of the characteristics of a muscle-derived signal is that its production should be elevated in the synaptic versus non-synaptic portions of a muscle fibre. To date, only two regulatory substances are known to be specifically associated with the postsynaptic domain of the nmj: transforming growth factor-beta 2 (TGF-â2)17 and nitric oxide synthase.16 TGF-â2 and nitric oxide are therefore prime candidates for regulators of nmj structure and/or function. In this paper, we report the effect of denervation on the production of TGF-â2 in the postsynaptic and extrasynaptic domains of muscle fibres. These experiments were done for several reasons. First, TGF-â2 is present in motoneurons6 and is anterogradely transported down the axon towards the nerve terminal (Jiang and McLennan, unpublished observations). Some cell types amplify their response to exogenous TGF-â1 by synthesizing TGF-â1.20 By analogy, we hypothesized that TGF-â2 is an autocrine regulator of the nmj. That is, motoneurons
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release TGF-â2 to regulate some aspect of the postsynaptic domain and that the muscle fibres amplify this signal by producing TGF-â2.17 If this hypothesis is correct, denervation should lead to a loss of TGF-â2 from the synaptic domain of muscle fibres. Second, muscle fibres maintain motoneurons in a functional mode by up-regulating the production of proteins associated with neurotransmitter function and by down-regulating the proteins associated with axon elongation.1 TGF-â2 is an obvious candidate for an anti-axon growth signal as it only becomes abundant at the nmj once axon growth has ceased and the nmj has matured.17 If TGF-â2 is an antigrowth signal, its production should be suppressed within denervated muscle, so as to permit the reinnervation of the muscle. Third, the production of the á, â, ä and ã subunits of the acetylcholine receptor and several other synapse-specific proteins occur along the length of immature muscle fibres, with the mature distribution of proteins being partially caused by down-regulation of their synthesis in the extrasynaptic domain. Denervation leads to the resumption of the synthesis in the extrasynaptic domains.8 High levels of TGF-â2 are present in the extrasynaptic domains of immature muscle fibres17 and we were therefore interested to determine whether denervation also up-regulates the production of TGF-â2 in the extrasynaptic domains of mature muscle fibres.
operations on the tibialis anterior and EDL muscles were equivalent. Muscles from the contralateral limb and from unoperated rats were used as controls. Northern blot hybridizations Total RNA was isolated with TRIzol reagent (GIBCOBRL, Gaithersburg, U.S.A.) according to the manufacturer’s instructions. Twenty micrograms of each RNA sample was size-fractionated by electrophoresis using 0.8% agarose gels and 4-molpholine-ethanesulphonic acid buffer and transferred to nylon membranes. The membranes were hybridized overnight at 55C in a solution containing 50% formamide, 0.5 M sodium phosphate, pH 7.2, 1% bovine serum albumin, 7% sodium dodecyl sulphate (SDS), 1% PEG6000 and a 32P-labelled probe prepared from a 1.1 kb Hind III-Xba I DNA fragment of a mouse TGF-â2 cDNA clone (pMTGFbeta2, ATCC no. 6319818). The membranes were then washed in a series of solutions, including a final wash with 0.1standard saline citrate (SSC) containing 0.1% SDS at 65C. The hybridization signals were detected by exposing the membranes to X-ray films with two intensifying screens at 80C for various lengths of time.23
Denervation of muscles
Detection of transforming growth factor-beta 2, acetylcholine receptor-å and glyceraldehyde-3-phosphate dehydrogenase messenger RNA by reverse transcription–polymerase chain reaction Total RNA was isolated as described above and genomic DNA removed by incubating each RNA fraction with RNase-free DNase (Promega, Madison, U.S.A.). The DNase-treated RNAs were converted to cDNAs using SuperScript II RNase H reverse transcriptase (GIBCOBRL) and oligo d(T)15 as the primer. The reaction was performed with 100 units of reverse transcriptase per 1 µg of RNA at 42C for 50 min using the buffer recommended by the manufacturer. Various amounts of cDNA were subsequently used as templates for polymerase chain reaction (PCR) in a 10 µl reaction mixture containing 0.5 units of Taq DNA polymerase (GIBCO-BRL), 1.0 µM each of primers, 200 µM each of dATP, dCTP, dGTP and dTTP, 20 mM Tris–HCl (pH 8.4), 50 mM KCl and an appropriate concentration of MgCl2 [2.5 mM for TGF-â2, and 1.5 mM for AChR-å subunit or glyceraldehyde-3-phosphate dehydrogenase (GAPDH)]. The PCR was performed in a thermal cycler (MJ Research, Watertown, U.S.A.) with programmes specific to the combination of primers. The denaturation, annealing and elongation steps were, respectively, 94C 15 s, 58C 10 s and 72C 20 s for TGF-â2; 95C 15 s, 66C 15 s and 72C 20 s for the AChR-å subunit; 94C 15 s, 50C 15 s and 72C 20 s for GAPDH. For all the primers, the initial denaturation step was 94–95C for 2 min and the final extension period was 72C for 3 min, followed by cooling at 4C for 10 min. The nucleotide sequences of the primers used were: TGF-â2, 5 -TCCTACAGACTGGAGTCACAAC AG-3 and 5 -ATCATATTGGAAAGCTGTTCGATC3 ;15 AChR-å subunit, 5 -TAGGAGACCTGAGGACA CTGTCA-3 and 5 -ATTGCCGTCGTCATCCACGGC AAAGA-3 ;4,29 GAPDH, 5 -TTCACACCCATCACAA AG-3 and 5 -CTTCATTGACCTCAACTA-3 .12
The tibialis anterior, extensor digitorum longus (EDL) and soleus muscles of nine rats were denervated by sectioning the sciatic nerve in one of their hindlimbs. The sciatic nerve was approached posteriorly between the biceps femoris, the semimembranosus and the semitendinosus. A 5–10 mm piece of the nerve was removed and the cut ends attached to adjacent muscles to prevent re-innervation of the target muscles. The rats were killed either one, three, five or seven days after the operation. In a further nine rats, the tibialis anterior and EDL were denervated by transectioning the common peroneal nerve, and the rats being killed between one and 35 days later. The effects of the two
Quantitation of the reverse transcription–polymerase chain reaction products The cycle numbers of the PCR were reduced to the levels where the amounts of PCR products were relative to those of the starting materials (22 cycles for TGF-â2 and AChR-å subunit and 20 cycles for GAPDH). These conditions were established using plasmid clones of murine TGF-â2 cDNA (ATCC, Maryland, U.S.A.) and rat AChR-å subunit (generous gift of Dr V. Witzemann). Once the PCR were completed, the products were size-fractionated by electrophoresis using 1.5% agarose gels, transferred to nylon
EXPERIMENTAL PROCEDURES
Animals All experiments were approved by the University of Otago’s Committee on Ethics in the Care and Use of Laboratory Animals. All efforts were made to minimize animal suffering and to reduce the number of animals used. There are no suitable alternatives to the in vivo techniques used in this study. All animals were bred by the Department of Laboratory Animal Science, University of Otago Mature Wistar rats (15- to 20-weeks-old) were killed by cervical dislocation and selected muscles removed, immersed at their resting length in melting isopentane and stored under liquid nitrogen until required. Rats which underwent surgical procedures were anaesthetized by an intraperitoneal injection of Ketamine (90 mg/kg) and Xylazine (3 mg/kg) (Parnell Laboratories, Auckland, New Zealand). All operations were done using sterile working conditions and the incisions closed with sutures.
Muscle TGF-â2 membranes (Hybond N+, Amersham, Buckinghamshire, U.K.) and hybridized with 32P-labelled oligonucleotides as probes. The probes were derived from specific parts of the internal sequences of amplicons and labelled by a kinase reaction with T4 polynucleotide kinase and [ã-32P]ATP:9 TGF-â2: 5 -TCCTACAGACTGG AGTCACAACAG-3 , AChR-å subunit: 5 -ACTA TCGG CTCAAC TTCAG CAAGGACGA-3 and GAPDH: 5 -ACTCAGCACCAG CATCACCCCATTTGA-3 . The membrane hybridizations were performed in a solution containing 0.5 M sodium phosphate (pH 7.0) and 7% SDS at 48C overnight.5 The membranes were then washed in 6SSC–0.1% SDS for 15 min and finally in 2SSC–0.1% SDS for 2 min at 48C. The images of hybridization signals were captured and quantified by a phosphoimager (Fuji film) using the analytical software supplied by the manufacturer. When higher resolution was required, the hybridized membranes were exposed to X-ray films (Kodak, Rochester, NY, U.S.A. Omat X-AR) with two intensifying screens at 80C for various lengths of time. Immunohistochemistry Five-micrometre sections of the muscles were cut in a cryostat and stained as previously described,17 with minor modifications: milk powder was omitted from the wash buffer and cold-fish gelatin was not included in the antibody diluting buffers. Briefly, the sections stained with either anti-TGF-â2 or anti-neurofilament antibodies were fixed in neutral buffered 4% paraformaldehyde at 4C for 30 min and incubated overnight at 4C with 1 µg/ml of antibody. The immunoreactivity was then developed using biotinylated anti-rabbit-Ig (Amersham), streptavidin biotinylated horseradish peroxidase complex (HRP, Amersham) and 3-amino-carbamide (AEC) as the chromogen. The sections stained with the various anti-myosin monoclonal antibody (mAb) were not fixed and biotinylated–antimouse-Ig (Amersham) was used as the secondary antibody. The slides were not counterstained and were examined using bright-field and phase-contrast microscopy with a Zeiss Axioplan photomicroscope. The specificity of the anti-TGF-â2 immunoreactivity was checked by absorbing it with a 25-fold excess by weight of the antigenic peptide and by comparing the stain produced by non-immune rabbit IgG. The anti-TGF-â2 was an affinity-purified rabbit antibody made to a peptides corresponding to the mature parts of TGF-â2 (Santa Cruz Biotechnology Inc., Santa Cruz, U.S.A.). The anti-neurofilament was a rabbit polyclonal antibody purchased from Sigma, St Louis, U.S.A. (N-4142) whereas the various anti-myosin mAbs were generous gifts either Dr Fitzsimons (NOQ7.1.1A, anti-type-I myosin) or Dr Schiaffino and Regeneron Pharmaceuticals (New York, U.S.A.) SC-71 (anti-IIa myosin), BF-F3 (anti-IIb myosin) and BF-35 (recognizes type I, IIa and IIb myosins but not IIx myosin).25 Quantitation of transforming growth factor-beta 2 immunoreactivity Sections of muscles were stained as described above except that (i) a fluorescent tag (streptavidin-BODIPY-Fl, Molecular Probes) was used instead of the streptavidin– biotinylated–HRP complex and (ii) higher concentrations of reagents (4 µg/ml of primary antibody) were used to ensure saturation and hence linearity between the amount of TGF-â2 in the section and the amount of immunoreactivity. The sections were examined in a MRC600 confocal laser scanning microscope using the 488 nm line of its krypton argon laser and the photon counting mode. In this mode, the amount of light detected is linearly related to the amount of fluorescence emitted. The total amount of fluorescence emitted from various fibres was then measured. A
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measure of the amount of TGF-â2 protein in various muscle fibres was made by subtracting the intensity of the staining present in sections reacted with non-immune IgG from that present in anti-TGF-â2-stained sections. The types of the fibres were determined by examining adjacent sections which had been stained with myosin-isoformspecific antibodies. Identification of synaptic sites Selected sections were double-labelled with anti-TGF-â2 and rhodamine-conjugated á-bungarotoxin (Molecular Probes) using the immunohistochemical procedure described above, except that rhodamine-á-bungarotoxin (1:500 dilution) was included with the secondary antibody incubation. The slides were mounted with Slow Fade (Molecular Probes, Eugene, U.S.A.). Western blots The TGF-â2 molecules present in innervated, one-week denervated and three-week denervated tibialis anterior and EDL muscles were concentrated by immunopreciptiation prior to western blotting. The muscles were homogenized (10% w/v) in a mixture containing 150 mM NaCl, 1% NP40, a cocktail of protease inhibitors (one tablet per 7 ml; Boehringer Mannheim, Mannheim, Germany, no. 1 697 498) and 10 mM Tris–HCl, pH 7.4. The homogenates were spun and the supernatants collected. An aliquot of the innervated muscle was spiked with 2 ng of recombinant human TGF-â2 (Genzyme, Cambridge, U.S.A.). One microgram of either anti-TGF-â2 or non-immune IgG was added to 4 ml aliquots of the muscles and after a 1 h incubation at 4C, 2 mg of protein-A sepharose beads (Sigma) were added. After 2 h further incubation, the beads were precipitated and the protein bound to them disolved in loading buffer containing mercaptoethanol and run on 4–20% gradient SDS–polyacrylamide gel electrophoresis (PAGE) gels. The proteins were then transferred to polyvinylidene difluoride membrane (Immobilon-P, Millipore, Bedford, U.S.A.) and stained with anti-TGF-â2 using a chemiluminescence detection system (ECL-Plus, Amersham). RESULTS
The efficacy of the denervation was checked by staining sections with an anti-neurofilament antibody and by measuring the levels of AChR-å mRNA, which are known to be increased after denervation.28 Intact axons were not observed in the intramuscular nerves, indicating that denervation was complete and that no re-innervation had occurred. Similarly, the levels of AChR-å mRNA increased three-fold by three days after the operation and remained elevated (Fig. 1c). Increased transforming growth factor-beta 2 expression in denervated muscles The expression of TGF-â2 in the denervated muscles was examined by northern blotting. The amount of TGF-â2 mRNA in innervated muscles was below the level of detection (Fig. 2). This is consistent with prior immunohistochemical studies which indicate that significant amounts of TGF-â2 are only associated with the subsynaptic nuclei, which constitutes fewer than 1% of muscle nuclei.17 The amount of TGF-â2 mRNA was elevated in the
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Fig. 1. Semi-quantitative RT–PCR estimates of the amounts of TGF-â2 and AChR-å subunit mRNAs in innervated and denervated skeletal muscles. Total RNAs were isolated, reverse transcribed and the prepared cDNAs amplified with primers specific to the target genes, as described in the Experimental Procedures. (a) cDNAs derived from 20 ng of total RNA were used for each PCR reaction when TGF-â2 and GAPDH mRNAs were amplified whereas a 10-fold higher concentration was used for each estimate of the AChR-å subunit mRNAs. (b) The intensities of the hybridization signals for the PCR products for TGF-â2 and AChR-å subunit mRNAs were standardized relative to those of the GAPDH. The values were then normalized relative to those of innervated muscles.
Fig. 2. Northern analysis of TGF-â2 transcripts in innervated and denervated skeletal muscles. Twenty microgram of each total RNA fraction was electrophoresed through a 0.8% agarose gel, transferred to a nylon membrane and hybridized with a 32P-labelled probe prepared from a murine TGF-â2 cDNA. The signals were detected by exposing the membrane to an X-ray film at 80C for two weeks, with two intensifying screens. (a) TGF-â2 hybridization signals and (b) agarose-gel stained with ethidium bromide to show loading of the total RNA.
denervated muscles, becoming first detectable around three days after the operation. Four mRNA species were detected, with estimated sizes of 3.4, 4.2, 4.8 and 6.3 kb. The signal from the 6.3 kb band was barely detectable (Fig. 2). All four messages were similarly increased after denervation. Multiple species of
TGF-â2 mRNA have been previously reported, but the biological importance of the various bands is presently unknown.18,27 Semi-quantitative reverse transcription (RT)–PCR was then used to obtain a more sensitive estimate of the effect of denervation on TGF-â2 expression. The amplified PCR fragments were examined by Southern blotting and the amounts of TGF-â2 mRNA were standardized relative to the expression of GAPDH messages. Plasmid clones of murine TGF-â2 cDNA and rat AChR-å subunit were used to verify that the intensity of the signals were linearly related to the cDNA copy number. The amount of TGF-â2 mRNA was not significantly changed oneday post-denervation. However, by three days after the operation, an approximately eight-fold increase in TGF-â2 mRNA had occurred, with the levels of message remaining high in the subsequent days (Fig. 1a) Denervation increases transforming growth factor-beta 2 protein in extrasynaptic portions of muscles The amount of TGF-â2 protein in the extrasynaptic portions of muscle fibres increased with time after denervation, in parallel with the change in TGF-â2 mRNA. TGF-â2 immunoreactivity in the nonsynaptic regions of denervated tibialis anterior and EDL muscle fibres was slightly elevated one day after cutting the common peroneal nerve and had become intense by four days (Fig. 3c). The intensity of TGF-â2 immunoreactivity continued to increase, reaching high levels in the muscles which had been denervated for longer than one week (Fig. 3d).
Muscle TGF-â2
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Fig. 3. TGF-â2 immunoreactivity associated with normal and denervated tibialis anterior. (a) Innervated muscle; (b) the anti-TGF-â2 was absorbed with antigen prior to the immunohistochemical procedure; (c) four-day denervated muscle; (d) 14-day denervated muscle. The arrows point to the postsynaptic domains of nmj, which have elevated levels of immunoreactivity in normal and denervated muscle (see also Fig. 5). The larger (IIB) fibres are stained with lower intensity than the smaller oxidative fibres. Panel d is at slightly higher magnification to show the intracellular staining observed in the denervated fibres. Scale bars (a–c)=50 µm; (d)=25 µm.
Similar results were obtained with soleus muscle fibres after transection of the sciatic nerve, although the increase in TGF-â2 expression was slightly slower than occurred in the tibialis anterior or EDL. This slower rate may be a consequence of transection of the sciatic nerve leaving a longer distal stump than transection of the common peroneal nerve. The increase in TGF-â2 immunoreactivity was initially perinuclear and associated with the circumference of fibres (Fig. 3c). However, with the passage of time after denervation, the immunoreactivity began to be distributed throughout the cytoplasm of fibres. By 14-days post denervation, the cytoplasm of all fibres contained TGF-â2, although the intensity of the stain varied from fibre to fibre (Fig. 3d). This pattern of staining was still present 35-days post denervation, which is the longest time we examined. TGF-â2 immunoreactivity was not detected in either the basal lamina of muscle fibres or in the various connective tissues (Fig. 3). The Schwann cells associated with the intramuscular nerves also expressed increasing levels of TGF-â2 (Fig. 4). The TGF-â2 immunoreactivity was specific. A similar pattern of stain was not observed with various other primary antibodies and the stain produced with the anti-TGF-â2 antibody was abolished by prior incubation with the antigenic peptide (Fig. 3b).
Fig. 4. TGF-â2 immunoreactivity associated with the intramuscular nerves of a 14-day denervated skeletal muscle. Scale bar=20 µm.
Synaptic transforming growth factor-beta 2 is unaffected by denervation The nmj were identified by the use of rhodaminealpha-bungarotoxin (Fig. 5b), which binds to the nicotinic acetylcholine receptor and is a classical
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Fig. 5. TGF-â2 immunoreactivity associated with neuromuscular junctions of a three-day denervated muscle. Transverse sections of a muscle were stained with anti-TGF-â2 linked to BODIPY-FL and rhodamine-conjugated á-bungarotoxin and examined on a confocal laser scanning microscope (see Experimental Procedures). (a) TGF-â2 immunoreactivity; (b) á-bungarotoxin. The arrows point to nmj, all of which show elevated levels of TGF-â2 immunoreactivity. Scale bar=30 µm.
marker for the nmj.10 TGF-â2 immunoreactivity was associated with the nuclei lying subjacent to the alpha-bungarotoxin binding sites of denervated muscle: that is the subsynaptic nuclei of denervated fibres (Figs 3, 5). High levels of TGF-â2 were also associated with the subsynaptic nuclei of innervated nmj (Fig. 3a and see Ref. 17). This made it difficult to determine whether the intensity of stain at the nmj was unchanged or increased by denervation. However, with respect to our original objective of determining whether motoneurons up-regulate the production of TGF-â2 at nmj, the key observation is that levels of synaptic TGF-â2 were not diminished after denervation. Innervated and denervated IIB fibres have low expression of transforming growth factor-beta 2 The intensities of TGF-â2 expression in the extrasynaptic portions of the muscle fibres appeared to be correlated with the size of the muscle fibre, suggesting that expression of extrasynaptic TGF-â2 may vary with fibre type. This was confirmed by staining adjacent sections of five-day denervated muscles with either a myosin-isoform-specific antibody or with the anti-TGF-â2 antibody. Denervated type I, type IIA and IIX fibres all contained elevated TGF-â2 immunoreactivity, whereas all of the 35 type IIB fibres examined were minimally stained. The amounts of TGF-â2 immunoreactivity in the various fibre types were then quantified using an MRC 600 confocal microscope in its photoncounting mode. The intensities of stain of IIA and IIX fibres in seven-day denervated tibialis anterior were approximately three times that of the IIB fibres (Fig. 6, Table 1). The tibialis anterior contains only a small number of slow fibres. The soleus was therefore examined to obtain quantitative data on the type I fibres. The intensity of staining of the type I fibres was similar to that of the IIA fibres (Table 1). We have previously reported that the extrasynaptic expression of TGF-â2 in innervated muscle fibres is
very low.17 However, this conclusion was based on the analysis of muscle regions which predominantly consist of type IIB fibres. The expression of TGF-â2 in a range of muscles was therefore examined to determine whether TGF-â2 expression varies between muscles. The muscles examined included typical limb muscles (EDL and tibialis anterior), an anti-gravity limb muscle (soleus), an axial muscle (erector spinae), a respiratory muscle (diaphragm), a facial muscle (masseter), rectus abdominis and the cremaster. The expression of TGF-â2 in the subsynaptic portion of the fibres was uniformly high in all of the muscles examined. Similarly, the amount of extrasynaptic TGF-â2 was always low, although the absolute level of expression varied slightly from muscle to muscle. Transforming growth factor-beta 2 is cleaved in denervated muscles TGF-â2 is intially synthesized as a precursor molecule which is then cleaved to form the mature peptide and a latency-associated peptide (LAP). In some cells, TGF-â1 can be retained within cells as an inactive complex containing the uncleaved precursor molecule.19 As noted above, TGF-â2 accumulates in denervated fibres and we therefore analysed the molecular size of the TGF-â2 to determine whether the precursor molecule had been cleaved. Elevated levels of mature TGF-â2 were detected in the denervated muscles (Fig. 7), indicating that the TGF-â2 precursor molecule had been appropriately processed. DISCUSSION
Neuromuscular junction-associated transforming growth factor-beta 2 is independent of innervation High levels of TGF-â2-immunoreactivity were detected at denervated synaptic sites. Growth factors have short biological half-lives. Therefore, the persistence of TGF-â2 at denervated nmj strongly
Muscle TGF-â2
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Fig. 6. TGF-â2 immunoreactivity in a seven-day denervated tibialis anterior muscle: correlation with fibre type. (a) A montage of the TGF-â2 immunoreactivity was made by examining neighbouring fields at high magnification, using the photon-counting mode of a BioRad MRC600 confocal laser scanning microscope (see Experimental Procedures). This ensures that the relationship between the amounts of antigen and immunoreactivity are linear: this is optimal for quantitation but gives rise to an image of lower contrast (cf. Figs 5a and 6a with Fig. 3c,d). The type of each fibre was identified from low magnification images of adjacent sections stained with either an anti-MHCIIa antibody (b), an anti-MHCIIb antibody (c) or an antibody which binds to all MHCs, except IIb (d). The arrowheads in b–d point to the fibres labelled in (a). Scale bar (a)=100 µm; (b–d)=300 µm. Table 1. Variation in the amount of transforming growth factor-beta 2 immunoreactivity in denervated skeletal muscles Fibre type
Area (µm2)
Amount of TGF-â2/unit area arbitrary units
Five-day denervated tibialis anterior IIA 1800400 10032(33) IIB 4100100 3312(64)* IIX 1900800 8725(34)# Seven-day denervated soleus I 2100200 IIA 2000400
9416(81) 10021(19)
Sections of denervated muscles were stained with an antiTGF-â2 antibody and the intensity of the resulting immunoreactivity measured using a confocal microscope (see Experimental Procedures). The data are the meanS.D., with the number of fibres examined indicated in parenthesis. *Significantly different to IIA and IIX fibres, P<0.001 (Student’s t-test). # Not significantly different to IIA fibres, P>0.05.
suggests that its production by subsynaptic myonuclei does not require the continued presence of the nerve. This disproves our postulate that muscle fibres produce TGF-â2 as an autocrine amplification of a signal from motoneurons (see Introduction). When muscle fibres are first formed, the á-, â-, ãand ä-AChR subunit mRNAs are produced along their entire length. A series of nerve-dependent changes then occurs: the production of the ã_subunit
Fig. 7. Size of TGF-â2 in normal and denervated muscles. TGF-â2 in normal and denervated muscles were concentrated by immunoprecipitation and run on 4–20% gradient SDS–PAGE gels. The proteins were then transferred to a membrane and stained with anti-TGF-â2. Lanes 1 and 5, innervated muscles; lanes 2 and 6, one-week postdenervation; lanes 3 and 7, three-weeks post-denervation; lane 8, innervated muscle spiked with 2 ng of recombinant TGF-â2; lane 9, 10 ng of recombinant TGF-â2. Lanes 1, 2, 3 and 8 were immunoprecipitated with anti-TGF-â2. Lanes 5–7 were immunopreciptiated with non-immune IgG and consequently lack TGF-â2. A size marker was run in lane 4 (not shown). The major bands, marked with an arrowhead, are 12,000–13,000 mol. wt which is consistent with the bands being the TGF-â2 monomer.
is repressed; the synapse-specific å-subunit is induced; and the extrasynaptic production of the AChR subunits is decreased.8,24 However, once the system is mature, removal of the neural influence leads to resumption of the extrasynaptic production of the AChR subunits but the production of the å-subunit and other AChR subunits continues to be elevated at the nmj. This has led to the suggestion that subsynaptic nuclei are permanently imprinted during the maturation of the nmj.3,28 Our results are consistent
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with this hypothesis and suggest that the imprinting involves a range of synapse-associated genes, and not just the various AChR subunit genes. Does transforming growth factor-beta 2 suppress axon outgrowth? Immature motoneurons are primed for axon outgrowth, thus enabling them to project to their target muscles and to compete for occupancy of synaptic sites. Once established, the pattern of innervation of a muscle is stable and the ability of motoneurons to sprout is restricted, except when the muscle is denervated or paralysed. TGF-â2 is initially dispersed within developing muscle fibres.17 The time at which it becomes concentrated at the nmj has not been precisely determined, but corresponds approximately to the establishment of the mature pattern of innervation. Thus, we postulated that TGF-â2 may switch motoneurons from a growth mode to a functional mode: to up-regulate the production of choline acetyltransferase and to down-regulate the proteins associated with growth. In apparent contradiction of the above hypothesis, the levels of TGF-â2 mRNAs were increased six- to eight fold in denervated muscles. Immunohistochemical studies proved that the levels of TGF-â2 protein were similarly increased and demonstrated that the TGF-â2 protein was associated with the Schwann cells of the intramuscular nerves and the entire length of the muscle fibres. Denervated nerve stumps are favourable sites for motor axon growth26 and the presence of high levels of TGF-â2 in Schwann cells would thus not be expected if one of the functions of TGF-â2 is to repress the ability of motoneurons to grow. Furthermore, exogenous TGF-â2 does not prevent axotomized hypoglossal motoneurons switching their pattern of gene expression from a functional to a growth mode (Zhang, Jiang and McLennan, unpublished observations). Is transforming growth factor-beta 2 a motoneuron survival factor? The location of TGF-â2 at the nmj,17 coupled with evidence that TGF-â2 has neurotrophic actions on various types of neurons,2,21 makes TGF-â2 a putative survival factor for motoneurons. Denervated muscles produce enhanced levels of motoneuron survival factor to support their reinnervation.13 The pattern of expression of TGF-â2 in denervated muscles, reported here, is thus consistent with it being a motoneuron survival factor. Proof of this, however, requires additional work, which is currently being undertaken in our laboratory. Comparison with other neuromuscular junction-specific genes Denervation leads to the re-expression of AChR subunit genes in the extrasynaptic portions of muscle fibres. Similarly, denervation also led to the up-
regulation of extrasynaptic production of TGF-â2, which persists for at least 35 days. However, in contrast to the AChR subunits the extent to which this occurred varied significantly between fibre types, with type IIB fibres producing little TGF-â2 outside of their synaptic zones. This is surprising since the extrasynaptic expression of TGF-â2 in immature type IIB fibres is similar to that of other immature fibres.17 Over 40 molecules are known to be concentrated at the nmj.10,11 The extrasynaptic expressions of many of these molecules are up-regulated by denervation. However, it is unclear whether any of the synapseassociated molecules are like TGF-â2 and show fibre-type specific differences after denervation. To our knowledge, fibre type-specific differences have not been previously reported but this may merely be because investigators have not systematically searched for such differences. The presence of molecular differences between denervated muscle fibre types is, however, to be expected as the speed of denervation-induced atrophy varies with fibre type.14 In particular, IIB fibres are preferentially affected in bed-rest patients and patients with CNS diseases or various congenital myopathies. Is muscle transforming growth factor-beta 2 biologically active? The TGF-âs are secreted as a latent high molecular weight complex consisting of the mature peptide, the LAP and a latent TGF-â binding protein.22 The mature peptide and the LAP are synthesized as a common precursor, which must be cleaved for biological activity to occur. TGF-â1 can be retained within cells as an inactive complex containing the uncleaved precursor molecule.19 As noted above, TGF-â2 accumulates in denervated fibres and we therefore analysed the molecular size of the TGF-â2 to determine whether the precursor molecule had been cleaved. Cleaved TGF-â2 was detected in both innervated and denervated muscles, indicating that appropriate post-translation modification of the precursor had occurred. It is thus likely that the TGF-â2 associated with muscle is active, although this point requires further verification. CONCLUSIONS
The expression of TGF-â2 at the nmj does not require continued innervation of muscle, proving that skeletal muscles are not producing TGF-â2 as an autocrine amplification of signals from motoneurons. In contrast, the extrasynaptic expression of TGF-â2 is neurally regulated. This is typical of proteins associated with the nmj, although the extrasynaptic expression of TGF-â2 is unusual in that the amount of TGF-â2 varies between muscle fibre types. The function of the TGF-â2 produced by skeletal muscle fibres remains to be elucidated, although the pattern of expression is consistent with TGF-â2 having a neurotrophic function.
Muscle TGF-â2 Acknowledgements—The authors thank Mr Rod McCall and Ms Sarah Booth for their excellent technical assistance. The Health Research Council (New Zealand) and The
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Otago Research Council are thanked for their financial support of this work. KK is supported by the Marsden Fund of New Zealand.
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