Motor neurone targeting of IGF-1 prevents specific force decline in ageing mouse muscle
1 Department of Physiology and Pharmacology
2 Department of Neurobiology & Anatomy
3 Department of Internal Medicine, Section on Gerontology
4 Neuroscience Program, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA
Abstract
IGF-1 is a potent growth factor for both motor neurones and skeletal muscle. Muscle IGF-1 is known to provide target-derived trophic effects on motor neurones. Therefore, IGF-1 overexpression in muscle is effective in delaying or preventing deleterious effects of ageing in both tissues. Since age-related decline in muscle function stems partly from motor neurone loss, a tetanus toxin fragment-C (TTC) fusion protein was created to target IGF-1 to motor neurones. IGF-1–TTC retains IGF-1 activity as indicated by [3H]thymidine incorporation into L6 myoblasts. Spinal cord motor neurones effectively bound and internalized the IGF-1–TTC in vitro. Similarly, IGF-1–TTC injected into skeletal muscles was taken up and retrogradely transported to the spinal cord in vivo, a process prevented by denervation of injected muscles. Three monthly IGF-1–TTC injections into muscles of ageing mice did not increase muscle weight or muscle fibre size, but significantly increased single fibre specific force over aged controls injected with saline, IGF-1, or TTC. None of the injections changed muscle fibre type composition, but neuromuscular junction post-terminals were larger and more complex in muscle fibres injected with IGF-1–TTC, compared to the other groups, suggesting preservation of muscle fibre innervation. This work demonstrates that induced overexpression of IGF-1 in spinal cord motor neurones of ageing mice prevents muscle fibre specific force decline, a hallmark of ageing skeletal muscle.
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Introduction
Ageing leads to decreased strength in the elderly that can impair daily function. This decline in strength is due to decline in both muscle mass (Lexell, 1995) and muscle fibre specific force (Brooks & Faulkner, 1988; González et al. 2000). An age-related denervation–re-innervation process (Larsson & Ansved, 1995; Kadhiresan et al. 1996) is thought to underlie muscle mass decline (Lexell, 1995), muscle fibre-type shift (Einsiedel & Luff, 1992; Lexell, 1995), and muscle specific force decline (Delbono, 2003; Payne & Delbono, 2004).
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Insulin-like growth factor-1 (IGF-1) is a potent trophic factor for motor neurones (Caroni & Grandes, 1990), and IGF-1 administration prevents motor neurone cell death and enhances re-innervation after nerve injury (Kanje et al. 1989; Sjoberg & Kanje, 1989; Li et al. 1994). IGF-1 from skeletal muscle exerts a target-derived trophic effect on survival of embryonic motor neurones (Neff et al. 1993) and on muscle innervation in aged animals (Messi & Delbono, 2003); consequently, overexpression of IGF-1 exclusively in skeletal muscle enhances motor neurone re-innervation after nerve injury (Rabinovsky et al. 2003).
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IGF-1 is also critical for growth and hypertrophy of skeletal muscle (Florini et al. 1996). Consequently, overexpression of IGF-1 in muscle produces muscle hypertrophy in transgenic mice (Coleman et al. 1995; Musaro et al. 2001). Therefore, treatment strategies for the loss of muscle mass and function with age may be directed toward this system. Indeed, overexpression of IGF-1 in skeletal muscle prevents age-related loss in muscle mass (Barton-Davis et al. 1998; Musaro et al. 2001), particularly among fast (Type IIb) fibre type muscles. Overexpression of IGF-1 in muscle also prevents excitation–contraction (EC) uncoupling (Renganathan et al. 1998; Wang et al. 2002) and maintains skeletal muscle fibre specific force (Renganathan et al. 1998; González et al. 2003) in aged mice. These findings may be due to the direct trophic effects of elevated IGF-1 on skeletal muscle dihydropyridine receptor (DHPR) expression (Zheng et al. 2002) or to target-derived trophic effects of muscle IGF-1 on motor neurones in aged mice via maintenance of muscle fibre innervation. Target-derived trophic effects of elevated muscle IGF-1 on mature muscle innervation has been suggested (Barton-Davis et al. 1998; Messi & Delbono, 2003), but whether elevated IGF-1 in spinal cord motor neurones prevents age-related declines in skeletal muscle force is not known.
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To probe this possibility, we targeted human IGF-1 (hIGF-1) delivery to motor neurones through injection of a hIGF-1–tetanus toxin fragment C fusion protein (hIGF-1–TTC) into hindlimb skeletal muscles of aged mice. The carboxy-terminal 451 amino acid segment, termed tetanus toxin fragment C (TTC), is capable of neuronal binding, uptake, and retrograde axonal transport in similar fashion to the native tetanus toxin, but is non-toxic itself (Bizzini et al. 1977). Therefore, we hypothesized that intramuscular injection of a hIGF-1–TTC fusion protein would (1) target delivery of IGF-1 to motor neurones of ageing mice and (2) prevent age-related decrease in skeletal muscle specific force.
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Methods
hIGF-1–TTC fusion protein production and purification
Plasmid design. Full-length hIGF-1 cDNA fragment was generated by PCR using hIGF-1 inserted in the pBSKS plasmid (provided by P. Rotwein, Oregon Health and Science University, Portland, OR, USA) and the following primers: 5'-GTCGGGATCCATGGGAAAAATCAGCAGTC -3' and 5'-CAGAGATCTCATCCTGTAGTTCTTGTTTC-3'. The fragment was inserted into the pGEM-T easy vector (Promega, Madison, WI, USA) and the orientation was confirmed by DNA sequencing. Full length TTC cDNA (nucleotides 1–1356) was generated by PCR from genomic DNA of Clostridium tetani CN655 (provided by M. R. Popoff, Institute Pasteur, Paris). Primers used were: 5'-GGAAGATCTTATTCTAAAAATCTGGATTGTTGGGT-3' and 5'- CGCGTCGACTCTAGATAATCATTTGTCCATCCTTCATCTGTA-3'. The TTC PCR fragment and pGEM-T easy–hIGF-1 plasmid were digested by BglII and SalI. The purified TTC cDNA fragment was inserted into the digested plasmid. The new plasmid pGEM-T easy–hIGF-1–TTC was used as template for next PCR. The hIGF-1–TTC cDNA fragment was generated by PCR using the primers: 5'-GGAATTCCATATGGTTAATCATTTGTCCATCCTTCATC-3' and 5'-GGAATTCCATCTGGGAAAAATCAGCAGTCTTC-3'. After NdeI digestion, the hIGF-1–TTC PCR fragment was inserted into the pET28a(+) vector (Novagen, San Diego, CA, USA) and the orientation was confirmed by DNA sequencing. The pET28a(+)–hIGF-1–TTC construct was transformed into BL21 DE3 strain of E. coli (Stratagene, La Jolla, CA, USA).
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Protein production and purification. Bacteria were cultured in 2x YT medium (16 g l–1 tryptone, 10 g l–1 yeast extract, 5 g l–1 NaCl, pH 7.0) containing 25 μg ml–1 kanamycin at 32°C for 2–3 h. Recombinant protein expression was induced by addition of 0.1 mM IPTG to the medium for 3–4 h. Cultures were centrifuged at 4000 g for 20 min. Pelleted bacteria were resuspended into 4 volumes (4 ml g–1) of lysis buffer containing (mM) 50 NaH2PO4, 300 NaCl, 10 imidazole, pH 8.0 with NaOH, and were lysed by French Press. Lysate was centrifuged at 10 000 g for 20–30 min. Supernatant was removed and saved for recombinant protein purification.
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The hIGF-1–TTC (with N-terminal His Tag) was purified with Ni-NTA agarose beads (Qiagen, Valencia, CA, USA). Cleared lysate was mixed with 50% Ni-NTA agarose bead slurry (4: 1), and shaken at 200 r.p.m. at 4°C for 60 min. Samples were then centrifuged for 10 s at 1000 g to pellet the beads. Supernatant was removed and saved. Beads were washed in buffer containing (mM) 50 NaH2PO4, 300 NaCl, 50 imidazole, pH 8.0 with NaOH, centrifuged 10 s at 1000 g, and supernatant saved. Wash step was repeated 4 times. Beads were resuspended in the same volume of elution buffer containing (mM) 50 NaH2PO4, 300 NaCl, 500 imidazole, pH 8.0 with NaOH, pelleted by centrifugation for 10 s at 1000 g, and supernatant containing purified recombinant hIGF-1–TTC was removed and saved.
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The protein was further purified by dialysis in SnakeSkin Pleated Dialysis Tubing (3500 MWCO thickness; Pierce, Rockford, IL, USA), placed into 1 litre of dialysis buffer containing 10 mM Tris, pH 7.0 with HCl and slowly shaken at 4°C for 36 h. Buffer was changed every 6 h. Purified protein was lyophilized and stored at –80°C. When needed, the protein was reconstituted into 5 ml phosphate-buffered saline (PBS) for dialysis in PBS. Final protein concentration was determined to be 1 μg ml–1.
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All Ni-NTA agarose bead wash and elution supernatant samples were subjected to SDS-PAGE on 10% acrylamide gels at 77 V for 1.5 h, followed by Coomassie Blue staining. Elution samples and native hIGF-1 were also immunoblotted by loading 5 μg of protein onto lanes of 10% acrylamide gels and run at 77 V for 1.5 h. Proteins were transferred onto nitrocellulose membranes at 100 V for 1 h. Protein bands were probed with either mouse anti-IGF-1 (Su et al. 1997) (Glaxo, Research Triangle Park, NC, USA) or mouse anti–TTC (Roche, Indianapolis, IN, USA) at 1: 1000 dilution. Secondary antibody was antimouse IgG conjugated with HRP (Amersham Biosciences, Piscataway, NJ, USA) in a 1: 3000 dilution.
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IGF-1 activity assay
IGF-1 activity was determined by [3H]thymidine incorporation into L6 skeletal muscle cell cultures, as reported (Sasaoka et al. 2001). L6 skeletal muscle cells were obtained from American Type Culture Collection (Manassas, VA, USA) and were maintained in -MEM supplemented with 10% fetal calf serum. Cells were grown to confluence in 24 multiwell culture plates (Corning Inc., Corning, NY, USA), and then serum deprived for 24 h. Cells were stimulated with hIGF-1 (20 ng ml–1; GroPep, Adelaide, Australia) or hIGF-1–TTC (200 ng ml–1) for 20 h. The difference in size of the proteins (hIGF-1, 7.6 kDa; hIGF-1–TTC, 67 kDa) requires a different amount of protein added to cultures to achieve the same concentration. Following hIGF-1 or hIGF-1–TTC stimulation, 1 μCi of [3H]thymidine was added for 4 h. Control cells received 1 μCi of [3H]thymidine immediately following the serum deprivation. Cells were washed twice with ice-cold PBS, twice with ice-cold 10% trichloroacetic acid, and once with 95% ethanol. The cells were then dissolved in 1 M NaOH, neutralized with 1 M HCl, and counted in a liquid scintillation counter.
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Motor neurone culture
Embryonic mouse motor neurones were purified following previously published procedures (Henderson et al. 1995; Arce et al. 1999), with some modifications. Briefly, spinal cords were dissected from E12 mouse embryos, treated with trypsin (2.5% w/v; final concentration 0.05%) for 10 min at 37°C, and then dissociated. The largest cells were isolated by centrifugation for 15 min at 830 g over a 5.2% Optiprep cushion (Sigma, St Louis, MO, USA), followed by centrifugation for 10 min at 470 g through a 4% BSA cushion. Purified motor neurones were plated inside 35-mm Petri dishes on 12-mm coverslips previously coated with polyornithine/laminin as described (Henderson et al. 1995; Arce et al. 1999), and grown 2–3 days in L15 medium with sodium bicarbonate (625 μg ml–1), glucose (20 mM), progesterone (2 x 10–8M), sodium selenite (3 x 10–8M), conalbumin (0.1 mg ml–1), putricine (10–4M), insulin (5 μg ml–1) and penicillin–streptomycin. CT-1 (1 ng ml–1), GDNF (1 ng ml–1) and CNTF (10 ng ml–1), and 2% horse serum were also added to the medium.
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hIGF-1–TTC binding and internalization assays
Binding and internalization assays for hIGF-1–TTC followed previously published methods (Francis et al. 1995; Coen et al. 1997; Bordet et al. 2001) with modifications. The cells were incubated for 2 h at either 0°C (binding) or 37°C (internalization) in binding buffer (20 mM Tris-HCl, pH 8, 1 mM CaCl2, 1 mM MgCl2, 0.25% BSA in PBS, pH 7.3) with diluted hIGF-1–TTC recombinant protein (5 μg ml–1). After incubation, the fusion protein-containing buffer was removed and cultured cells were washed 4 times for 10 min with PBS. Cells were fixed with 4% paraformaldehyde in PBS for 10 min, and then washed twice for 10 min in PBS. For immunofluorescence detection, cells were blocked with 5% donkey serum and 0.02% Triton X-100 in PBS for 30 min. The primary antibodies were diluted in 2% donkey serum in PBS. The respective primary antibodies used were: mouse anti-IGF-1 (Su et al. 1997) (Glaxo; 1: 25 dilution) and mouse anti–TTC (Roche; 1: 50 dilution). Cells were incubated with respective primary antibodies for 2 h at room temperature then washed 4 times for 10 min with PBS. Cells were then incubated 1 h at room temperature with the secondary antibody, donkey antimouse IgG conjugated with fluorescein isothiocyanate (FITC), in a 1: 100 dilution in 1% donkey serum in PBS, then were washed again 4 times for 10 min with PBS. Immunofluorescence was visualized in a thin plane of focus with a high NA (1.30) 100x oil-immersion objective (Fluar, Zeiss, Oberkochen, Germany) on an Axioskop 2 microscope (Zeiss) and a PXL-EEV-37 CCD camera (Photometrics, Tucson, AZ, USA) based imageing system. Isee software (Inovision, Durham, NC, USA) running in a Silicon Graphics (Mountain View, CA, USA) O2 workstation was used for data acquisition and image analysis.
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Immunocytochemical detection of hIGF-1–TTC in spinal cord
DBA (National Institute on Ageing/Harlan Sprague-Dawley, Indianapolis, IN, USA) and FVB (our colony) mice were housed at Wake Forest University School of Medicine and all procedures were approved by the Animal Care and Use Committee. Young adult (3–4 months) mice were injected with hIGF-1–TTC dissolved in PBS (1 μg ml–1, 10 μg protein per injection) into several hindlimb muscles (quadriceps, gastrocnemius, TA/EDL). Two animals underwent similar injections, while still under anaesthesia, immediately following transection of the sciatic nerve. Briefly, animals were anaesthetized with I.P. injections of ketamine and xylazine (100 mg kg–1 and 20 mg kg–1, respectively), a small incision was made at the sciatic notch and a 3–4 mm segment of the sciatic nerve was cut and removed to prevent re-innervation. The incision was stitched closed, and gastrocnemius and TA muscles were injected with several 10 μl injections of hIGF-1–TTC (1 μg ml–1, 10 μg protein per injection). Two spinal cords were collected at each postinjection time point to examine fusion protein uptake from the muscle and transport to the spinal cord motor neurone cell bodies, as well as longevity of the protein in spinal cord motor neurones. Animals were killed by cervical dislocation and lumbar enlargements of spinal cords were collected at 7, 14, 21 and 28 days postinjection after transcardiac perfusion of mice with 4% paraformaldehyde in PBS. Spinal cords from denervated mice were collected at 7 days post-injection. All samples were stored in 4% paraformaldehyde in PBS overnight at 4°C, followed by storage in 20% sucrose in PBS for 36–48 h at 4°C. Samples were then embedded in OCT medium and frozen in liquid nitrogen. All samples were stored at –80°C until use.
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For immunofluorescence detection of hIGF-1–TTC fusion protein in spinal cords, frozen samples were cut on a cryostat microtome cooled to –20°C (12 μm). Samples were blocked with 5% donkey serum and 0.02% Triton X-100 in PBS for 30 min. Mouse anti-hIGF-1 (Su et al. 1997) (Glaxo) was diluted 1: 25 in 2% donkey serum in PBS. Samples were incubated for 2 h at room temperature, and then washed 4 times for 10 min in PBS. Samples were then incubated in donkey antimouse IgG conjugated with FITC diluted 1: 100 in 1% donkey serum in PBS for 1 h at room temperature and washed 4 times for 10 min in PBS. Samples were then imaged using an Axioskop 2 microscope and a CCD camera based imaging system (see above).
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Single intact muscle fibre contraction
DBA mice aged 17–18 months were injected with 10 μl of saline, hIGF-1 (1 μg; 0.1 μg ml–1 in PBS), TTC (10 μg; 1 μg ml–1 in PBS), or hIGF-1–TTC (10 μg; 1 μg ml–1 in PBS) into the anterior compartment of the right lower leg (tibialis anterior, TA; extensor digitorum longus, EDL) every 28 days for three injections. Animals were killed by cervical dislocation 14–28 days past the third and final injection, rendering animals 20–21 months old. At time of kill, injected EDL muscles were carefully dissected and pinned into a Petri dish lined with Sylgard (Dow Corning, Auburn, MI, USA) containing recording solution, consisting of (mM): 121 NaCl, 5 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.4 NaH2PO4, 24 NaHCO3 and 5.5 glucose. The recording solution was bubbled continuously with a mixture of 5% CO2 and 95% O2 to achieve a pH of 7.4.
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During initial data collection, EDL muscles from saline and hIGF-1–TTC injected mice were trimmed of excess tendon, nerve and fat, and were weighed. In the second set of experiments, saline, IGF-1, TTC and hIGF-1–TTC injected EDL muscles were removed, and single intact fibres were dissected and mounted in a small flow-through chamber (150 μl) perfused with bubbled recording solution on the stage of an inverted microscope following procedures published previously (Lannergren & Westerblad, 1987; González et al. 2000).
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Muscle fibres were stimulated by an electrical field generated between two parallel silver electrodes connected to a Grass S48 stimulator (Astro-Medical, Inc., West Warwick, RI, USA). Fibre length was adjusted until maximum force was elicited by a single twitch contraction (LO) under isometric conditions. Suprathreshold square wave pulses of 0.5 ms duration were delivered to elicit twitch contractions. Tetanic contractions were elicited with 0.5 ms square wave pulses delivered in 200 ms trains. Frequency was increased until maximum force was attained. All subsequent tetanic contractions were elicited with the frequency that elicited maximal force (González et al. 2000; Payne et al. 2004). All experiments were carried out at room temperature (22–23°C). Once LO was attained, fibre diameter was measured at three to four randomly selected points along the length of the fibre and averaged. Cross-sectional area (CSA) was calculated as (d/2)2, where d is the average diameter of the fibre (Lannergren & Westerblad, 1987; González et al. 2000). Specific force (in kPa) was calculated as maximum tetanic force (kN)/CSA (m2).
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Myosin heavy chain isoform composition in TA muscles
Myosin heavy chain (MHC) composition of injected TA muscles was determined by SDS-PAGE, as previously described (Serrano et al. 1996) with some modifications. Briefly, TA muscles were minced on ice in 4 volumes of a high salt buffer containing (mM): 300 NaCl, 100 NaH2PO4, 50 Na2HPO4, 1 MgCl2, 10 Na4P2O7 and 10 EDTA, pH 6.5. Extracts were then centrifuged at 7500 g for 30 min at 2°C in a microcentrifuge. The supernatants were diluted in 9 volumes of 1 mM EDTA buffer (37% w/v EDTA and 0.01% (v/v) 2-mercaptoethanol), vortexed and allowed to precipitate overnight at 4°C. Centrifugation was repeated, and the resulting pellet dissolved in 0.5 M NaCl and 10 mM NaPO4 and denatured by immersion in boiling water for at least 2 min. Samples were diluted 1: 100 in SDS buffer (62.5 mM Tris-HCl, 2% w/v SDS, 10% v/v glycerol, and 0.001% w/v bromophenol blue, pH 6.8). Electrophoresis was performed in 6% acrylamide (37.5: 1 acrylamide: bis-acrylamide; Bio-Rad, Hercules, CA, USA) separating gel with 30% v/v glycerol and 3% stacking gel with no glycerol. Aliquots of 10-μl of diluted myosin were subject to electrophoresis for 6 h at 50 V and 12 h at 120 V at 4°C. Gels were silver stained according to published protocols (Giulian et al. 1983) using the silver stain plus kit and Silver Stain SDS-PAGE Standards, high range (Bio-Rad). As a reference for the four MHC isoforms (I, IIa, IIx and IIb), a mixture of mouse soleus, flexor digitorum brevis, and gastrocnemius muscles was used. After staining, gels were photographed with a digital camera. The digitized image was analysed with Kodak 1D software (Eastman Kodak, Rochester, NY, USA) to determine optical density of each stained band.
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Neuromuscular junction post-terminal staining
Postsynaptic acetylcholine receptor staining was performed as described (Messi & Delbono, 2003). TA muscles were removed, pinned at a slightly stretched length, embedded in OCT tissue medium, and frozen in isopentane cooled in liquid nitrogen. Longitudinal sections (30 μm) were cut on a cryostat microtome cooled to –20°C and sections were fixed in methanol–acetone (50: 50 v/v) for 10 min at room temperature. After washing in Tris-buffered saline (TBS), sections were blocked in 10% rabbit serum in TBS for 30 min and washed again in TBS. Sections were incubated 3 h with tetramethylrhodamine-conjugated -bungarotoxin (Molecular Probes, Eugene, OR, USA), diluted 1: 200 in TBS. Stained junctions were visualized using a Zeiss Axioskop 2 microscope equipped with a CCD-EEV37 camera. Images were analysed using Isee software (Inovision) (see above). The post-terminal area was calculated in pixels by tracing the perimeter of individual labelled regions on digitized images, and the area was converted into square micrometers.
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Data analysis
Data are presented as mean ±S.E.M. Data were analysed by ANOVA followed by the Holm-Sidak multiple comparisons test applied post hoc.
Results hIGF-1–TTC fusion protein expression, purification, and activity
The genetic fusion protein hIGF-1–TTC was produced in transfected bacterial cultures, and purified by His Tag Ni-NTA affinity beads. The protein contains hIGF-1 in the N-terminal position relative to TTC. This decision was made based on the findings that a TTC–cardiotrophin-1 fusion protein with cardiotrophin-1 in the C-terminal position often produced incomplete fragments along with the full-length fusion protein, whereas a construct with cardiotrophin-1 in the N-terminal position produced only the full-length fusion protein (Bordet et al. 2001). Figure 1A shows the end product of several steps in the purification of the hIGF-1–TTC fusion protein from BL21 DE3 bacteria cultures. Cleared bacterial lysate (lane 2) and lysate that has been through the Ni-NTA binding procedure (lane 3) both show myriad stained proteins on the gel. Following successive wash steps of the Ni-NTA agarose beads, very few to no proteins are visible on the stained gel (data not shown). After elution of the fusion protein from the Ni-NTA agarose beads, a single band of protein of approximately 67 kDa size is stained (lane 4), an appropriate size for the full length hIGF-1–TTC protein. The absence of any other bands of protein suggests little to no contamination by bacterial proteins. Figure 1B shows immunoblots using antibodies against IGF-1 (lanes 1 and 2) and TTC (lane 3). Purified hIGF-1 was loaded in lane 1 as a positive control, while the Ni-NTA bead elution was loaded in lanes 2 and 3. Lanes 2 and 3 show a 67 kDa protein product, indicating that the 67 kDa protein on the stained gel is the hIGF-1–TTC fusion protein. No incomplete protein fragments are present following purification procedures as indicated by the single band in lane 4 of Fig. 1A and lanes 2–3 of Fig. 1B.
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A, following transformation of cultures and protein synthesis induction, bacteria were lysed for purification of hIGF-1–TTC (see Methods). Lanes M to 4 illustrate a Coomassie Blue stained gel. Lane M, high range molecular weight marker. Lane 1, cleared bacterial lysate. Lane 2, supernatant of bacterial lysate following Ni-NTA affinity binding and centrifugation. Lane 3, elution from Ni-NTA affinity beads exhibiting one band of stained protein at 67 kDa. Lane 4, immunoblot of elution against hIGF-1. B, immunoblots following elution of the fusion protein from Ni-NTA affinity beads against IGF-1 (Lane 2) and against TTC (Lane 3). Immunoblots both display one band of protein at 67 kDa. Purified hIGF-1 was probed with anti-IGF-1 for a positive control (Lane 1).
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To assess mitogenic activity of the fusion protein, L6 myoblasts were incubated with equivalent molar amounts of either hIGF-1 or hIGF-1–TTC, followed by measurement of [3H]thymidine uptake. L6 myoblasts were used to determine IGF-1 activity since they express IGF-1 receptors but do not express IGF-1 (Musaro & Rosenthal, 1999). Figure 2 shows [3H]thymidine incorporation into L6 muscle cells stimulated with hIGF-1 or hIGF-1–TTC following 20 h of serum deprivation. Stimulation by both proteins increased radioactive uptake compared to control cells, which received no growth factor stimulation following serum deprivation. [3H]thymidine uptake in cells stimulated by hIGF-1–TTC was similar to cells that received an equivalent amount of hIGF-1 stimulation, indicating that the IGF-1 moiety of the hIGF-1–TTC fusion protein retains its biological activity. Data were acquired from 5, 9 and 9 experiments from control, hIGF-1 and hIGF-1–TTC, respectively.
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L6 muscle cells were serum deprived for 24 h, exposed to hIGF-1 (20 ng ml–1) or hIGF-1–TTC (200 ng ml–1) for 20 h, followed by addition of 1 μCi of [3H]thymidine for 4 h. Control cells received [3H]thymidine immediately following serum deprivation. [3H]thymidine incorporation was determined by a scintillation counter. Both hIGF-1 and hIGF-1–TTC increased [3H]thymidine uptake compared to control (P= 0.002). hIGF-1–TTC stimulation increased [3H]thymidine uptake to a similar amount as equivalent stimulation by hIGF-1.
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Binding and internalization of hIGF-1–TTC in cultured motor neurones
Following incubation at 0°C for 2 h, binding of the fusion protein to motor neurone surfaces was assessed with anti-IGF-1 or anti-TTC antibodies (Fig. 3, left). Immunofluorescence labelling showed that hIGF-1–TTC binding occurred on the surface of neurone cell bodies and processes, and that labelling was not different using antibodies against either TTC (Fig. 3, top left; n= 14 cells) or IGF-1 (Fig. 3, bottom left; n= 15 cells), suggesting binding of the full-length fusion protein rather than some degradation product. Following incubation at 37°C for 2 h, internalization of hIGF-1–TTC was also assessed using anti-TTC (Fig. 3, top right; n= 22 cells) or anti-IGF-1 (Fig. 3, bottom right; n= 25 cells). Immunofluorescence labelling was detected in neuronal cell bodies and central processes, and the protein was labelled by both primary antibodies. Labelling with both antibodies is located in distinct clusters, suggesting the protein was internalized into vesicular structures, similar to previous reports (Bordet et al. 2001; Miana-Mena et al. 2002). These findings indicate that hIGF-1–TTC is internalized by spinal cord motor neurones in a manner similar to other TTC-coupled proteins (Francis et al. 1995; Coen et al. 1997; Bordet et al. 2001; Miana-Mena et al. 2002).
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Cultured mouse spinal cord motor neurones were exposed to hIGF-1–TTC for 2 h at 0°C for binding assays (left) or for 2 h at 37°C for internalization assays (right). Immunodetection of hIGF-1–TTC was performed with antibodies against TTC (top) and IGF-1 (bottom). Binding assays (left) reveal immunofluorescence on surfaces of cell bodies and processes of cultured motor neurones. Internalization assays (right) reveal immunofluorescence located in clusters resembling vesicular structures in cell bodies and central processes of cultured motor neurones.
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In vivo retrograde transport of hIGF-1–TTC and stability in spinal cord motor neurones
To study kinetics of motor neurone uptake and retrograde transport of hIGF-1–TTC, several hindlimb muscles were injected with 10 μg each of the protein, and lumbar spinal cords were removed for immunocytochemical detection. Labelling was detected in ventral horn cell bodies at 7, 14 and 21 days following skeletal muscle injection (Fig. 4A–C), but could not be detected at 28 days postinjection (Fig. 4D). Figure 4E shows an enlarged view of the ventro-lateral lumbar spinal cord 7 days post-injection. Fluorescence is clearly visible in and around large cell bodies in the ventro-lateral motor column near the ventral root (between arrows), corresponding to motor neurones. No positive immunolabelling for hIGF-1 was detected in spinal cords of denervated animals at 7 days post-injection (Fig. 4F). Fluorescence was detected only ipsilateral relative to injection, and no fluorescence was detected at levels above or below the lumbar enlargement (data not shown). These findings indicate that hIGF-1–TTC is, indeed, delivered to spinal cord motor neurones by uptake and retrograde transport to the cell bodies in the ventral spinal cord, and are in agreement with previous reports of other TTC-coupled proteins undergoing similar uptake and retrograde transport (Coen et al. 1997; Miana-Mena et al. 2002).
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hIGF-1–TTC was injected into several hindlimb muscles of one leg of mice. Immunofluorescence was detectable in ventral lumbar spinal cord sections at 7 days (A), 14 days (B), and 21 days (C) post-injection, but was not detectable at 28 days (D) (n= 2 spinal cords per time point). Fluorescence was detectable only on one side of the spinal cord, in correspondence with hIGF-1–TTC intramuscular injections. E, enlarged view of the ventro-lateral spinal cord at 7 days postinjection. Arrows indicate the ventral root adjacent to fluorescent motor neurone cell bodies. F, hIGF-1–TTC was not detected in spinal cords of mice injected immediately following sciatic nerve transection (n= 2 cords).
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hIGF-1–TTC treatment prevents EDL fibre specific force decline in ageing mice
To study the effects of targeted delivery of hIGF-1–TTC to spinal cord motor neurones on skeletal muscle mass and function in ageing animals, hindlimb muscles were injected with the fusion protein over a 3- to 4-month time span near the end of the DBA and FVB mouse expected lifespan (23–24 month lifespan; see Methods for injection protocol). In an initial experiment, EDL muscles were removed and weighed following injection of saline or hIGF-1–TTC. EDL muscle weights were not different between groups (hIGF-1–TTC, 7.95 ± 0.29 g, n= 8; Saline, 8.55 ± 0.13 g, n= 4; P > 0.05). With the difficulties of accurately measuring specific force in whole muscle experiments (Sugi & Tsuchiya, 1998), single intact muscle fibres were dissected from EDL muscles following injection of saline, hIGF-1, TTC, or hIGF-1–TTC (n= 16, 15, 11 and 12 fibres from 7, 6, 4 and 5 mice, respectively) and used for in vitro contraction experiments. Single fibre absolute force was not different among groups (Fig. 5A). Fibre diameters of IGF-1, TTC and hIGF-1–TTC muscles were not different compared to control saline injected (Figs 5B, P > 0.05). However, EDL fibres from muscles injected with hIGF-1–TTC exhibited greater specific force than saline, hIGF-1 and TTC injected EDL fibres (Figs 5C, P= 0.006). Specific force was not different among saline, hIGF-1 and TTC injected fibres, indicating the introduction of hIGF-1 or TTC alone by this injection protocol is not beneficial to muscle and/or motor neurones in ageing mice. Figure 5D illustrates representative contraction traces from single intact EDL fibres, scaled to specific force.
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Single fibre maximal tetanic force (A) and diameter (B) were not different among groups. Maximum tetanic specific force was higher in fibres from muscles injected with hIGF-1–TTC than fibres injected with saline, hIGF-1, or TTC (C, P= 0.006; n= 16, 14, 11 and 12 fibres from 7, 6, 4 and 5 mice in saline, hIGF-1, TTC and hIGF-1–TTC groups, respectively). D, representative tetanic contraction traces from single intact EDL fibres from mice injected with saline (a), hIGF-1 (b), TTC (c) and hIGF-1–TTC (d).
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Muscle fibre type composition is unchanged by hIGF-1–TTC
Previous reports indicate that viral-mediated and transgenic overexpression of IGF-1 in skeletal muscle preserves Type IIb muscle fibres in ageing rats (Barton-Davis et al. 1998; Messi & Delbono, 2003). To determine if treatment of aged animals with hIGF-1–TTC prevents muscle fibre type changes, muscles were removed and subjected to SDS-PAGE to determine MHC composition of saline, hIGF-1, TTC, and hIGF-1–TTC injected muscles (n= 7, 6, 4 and 4 muscles, respectively; 1 muscle per mouse). TA muscles were used for this analysis because EDL muscles were used for single fibre contraction experiments. Furthermore, MHC analysis of EDL single fibres is not reliable using the single intact fibre contraction technique due to contamination by cellular debris of surrounding dead fibres (González et al. 2000). Figure 6A illustrates the pattern of MHC distribution on 6% acrylamide gels. All TA muscles from all groups display the presence of Type IIx and Type IIb MHC. However, no difference exists among groups in percentage of Type IIx and Type IIb MHC (Fig. 6B), indicating that injection of hIGF-1, TTC, or hIGF-1–TTC does not change fast-twitch muscle fibre type composition relative to saline-injected controls.
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TA muscles were removed from 20-month-old DBA mice following monthly with saline, hIGF-1, TTC, or hIGF-1–TTC injections. A, TA muscle proteins were subjected to SDS-PAGE on 6% acrylamide gels for detection of MHC isoforms. All TA muscles expressed detectable levels of Type IIx and IIb MHC. The far left lane shows all four adult MHC isoforms, indicated as IIa, IIx, IIb and I. B, no differences were found among the groups in percentage of Type IIx and IIb MHC (P > 0.05; n= 7, 6, 4 and 4 muscles, 1 muscle per mouse).
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hIGF-1–TTC increases post-terminal size in ageing muscle
Overexpression of hIGF-1 in muscle preserves neuromuscular junction (NMJ) size and morphology in ageing mice, suggesting a target-derived trophic effect of muscle IGF-1 on motor neurones (Messi & Delbono, 2003). To probe this further, NMJ post-terminals from aged mouse TA muscles injected with saline, hIGF-1, TTC and hIGF-1–TTC were examined (n= 2 muscles per group, 1 muscle per mouse; n= 16 sections per muscle, 32 sections per group; n= 86, 105, 97 and 101 NMJs from saline, hIGF-1, TTC and hIGF-1–TTC, respectively). NMJ post-terminals undergo atrophy and fragmentation in aged mammalian muscles. Atrophy is characterized by smaller, less complex stained areas (fewer acetylcholine receptors) (Courtney & Steinbach, 1981), and fragmentation by a dispersion of stained post-terminals in some fibres (Oda, 1984; Robbins & Fahim, 1985). Post-terminal atrophy (Fig. 7A–CversusFig. 7D) and fragmentation (Fig. 7C) were found in muscles from control animals, and these data agree with previous findings (Messi & Delbono, 2003). While the post-terminals from hIGF-1–TTC injected muscles are not as large as post-terminals from young mouse muscles (1700 μm2) (Messi & Delbono, 2003), treatment with the fusion protein does increase the size (Fig. 7E; P= 0.002) and morphological complexity of the post-terminal compared with saline, IGF-1 and TTC injected counterparts.
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NMJ post-terminal acetylcholine receptors in TA muscles from old mice were labelled with rhodamine-conjugated -bungarotoxin. A–D, representative NMJ post-terminals from saline (A), hIGF-1 (B), TTC (C) and hIGF-1–TTC (D) injected muscles. NMJ post-terminals from hIGF-1–TTC injected muscles display more complex morphology compared to saline, hIGF-1, and TTC injected muscles. In addition, fragmentation of NMJ post-terminals in ageing muscles (as seen in C) is eliminated by hIGF-1–TTC injection. Scale bar, 30 μm. E, muscles injected with hIGF-1–TTC exhibit increased NMJ post-terminal area, compared to saline, hIGF-1, and TTC injected muscles. (P= 0.002; n= 2 muscle per group, 1 muscle per mouse; n= 16 sections per muscle, 32 sections per group; n= 86, 105, 97 and 101 NMJs from saline, hIGF-1, TTC and hIGF-1–TTC groups, respectively).
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Discussion
This work reports: (1) the design, expression, production and purification of the genetic fusion protein hIGF-1–TTC, which contains equivalent mitogenic activity compared to hIGF-1; (2) in vitro binding and internalization of hIGF-1–TTC by cultured spinal cord motor neurones; (3) in vivo uptake and retrograde transport of hIGF-1–TTC by spinal cord motor neurones following injection into hindlimb skeletal muscles; (4) increased specific force in single intact muscle fibres from old mice injected with hIGF-1–TTC, compared to old mice injected with saline, hIGF-1, or TTC; and (5) increased NMJ post-terminal size and morphological complexity in hIGF-1–TTC injected compared to saline, hIGF-1 and TTC injected muscles.
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hIGF-1–TTC properties and actions in neurones hIGF-1–TTC in vitro interaction with cultured motor neurones
In this work, the motor neurone neurotrophic factor human IGF-1 was genetically fused to TTC to create a 67 kDa protein product, hIGF-1–TTC. Tetanus toxin is an extremely potent neurotoxin. Since the C-fragment (TTC) is capable of binding, uptake and retrograde axonal transport (Bizzini et al. 1977; Evinger & Erichsen, 1986) without toxic effects, it was proposed that TTC could be used to target and deliver proteins to motor neurones (Bizzini et al. 1980; Simpson, 1985). Indeed, conjugation and genetic fusion of TTC to proteins increases their uptake into neurones (Francis et al. 1995; Bordet et al. 2001; Francis et al. 2004). Similar to previous reports, hIGF-1–TTC bound to cultured spinal cord motor neurones at 0°C. Binding of hIGF-1–TTC to motor neurone membranes may be explained by ganglioside binding (Rogers & Snyder, 1981), but some binding may be explained by IGF-1 receptors on cultured motor neurone membranes (Vincent et al. 2004). Further experiments at 37°C show that hIGF-1–TTC is internalized into cultured spinal cord motor neurones. Although internalization of IGF-1 receptors and their ligand occurs (Dore et al. 1997), the visualization of hIGF-1–TTC in clusters resembling vesicular structures suggests internalization was accomplished primarily via TTC binding to gangliosides (Bordet et al. 2001; Miana-Mena et al. 2002). Co-labelling of bound and internalized hIGF-1–TTC by antibodies against IGF-1 and TTC ensures that the fusion protein is still intact and not degraded upon receptor binding or internalization.
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hIGF-1–TTC in vivo interactions with spinal cord motor neurones
To examine the in vivo uptake and retrograde transport properties of hIGF-1–TTC in motor neurones young adult mice were injected with hIGF-1–TTC into hindlimb muscles, and spinal cords were removed at various time points following injections. Immunofluorescence labelling was detected in and surrounding ventral horn large cell bodies up to 21 days post-injection, indicating uptake and retrograde transport of hIGF-1–TTC by spinal cord motor neurones. Injections were performed in only one hindlimb; therefore, detection with anti-hIGF-1 in only the ipsilateral spinal cord and only in the lumbar enlargement ensured that the antibody was detecting hIGF-1–TTC, not endogenous IGF-1 in the spinal cord. This finding, and the failure to detect hIGF-1–TTC in the spinal cords of denervated animals, indicates that entry of the protein into the spinal cord was by uptake from skeletal muscle and retrograde axonal transport. Detection in spinal cord up to 21 days also indicates that the protein is rather stable, possibly allowing the hIGF-1 portion of the protein to exert prolonged effects on motor neurones. The stability of the fusion protein in the spinal cord and the quick removal of TTC-coupled proteins from muscle (i.e. 24–48 h; Miana-Mena et al. 2002) allowed the long injection interval of 28 days, minimizing the direct effects on muscle IGF-1 receptors.
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Effects of hIGF-1–TTC injection on skeletal muscle in aged mice
While uptake and retrograde transport of hIGF-1–TTC in spinal cord motor neurones is evident in these findings, potential treatments for diseases or ailments of ageing must improve function, whether preventing decline or returning lost function. Ageing leads to many changes in skeletal muscle structure and function, the primary concern being decline in muscle strength accounted for by loss of muscle mass (Lexell, 1995) and by muscle fibre specific force decline (Brooks & Faulkner, 1988; González et al. 2000). Age-related denervation is more prominent that previously thought (Wang et al. 2005), and a denervation–re-innervation process (Larsson & Ansved, 1995; Lexell, 1995; Kadhiresan et al. 1996) is thought to underlie age-related changes in muscle structure and function (Lexell, 1995; Delbono, 2003; Payne & Delbono, 2004). We have previously shown that motor neurones in aged rodents retain sensitivity to IGF-1 (Shan et al. 2003) and that IGF-1 overexpression in muscle maintains skeletal muscle innervation in aged mice (Messi & Delbono, 2003). Therefore, we studied the effects of targeting IGF-1 to motor neurones on muscle function.
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To assess whether hIGF-1–TTC exerted beneficial effects on skeletal muscle innervation, we examined MHC composition of injected TA muscles. No differences in either Type IIx or IIb MHC were found among muscles injected with saline, hIGF-1, TTC, or hIGF-1–TTC. Ageing muscles primarily lose the fastest (Type IIb) fibres as a result of loss of the largest, fastest motor units (Einsiedel & Luff, 1992; Lexell, 1995; Kadhiresan et al. 1996). Previous reports have shown preservation of Type IIb MHC composition in aged muscles overexpressing IGF-1 (Barton-Davis et al. 1998; Musaro et al. 2001; Messi & Delbono, 2003), presumably, by preservation of innervation in these muscles (Messi & Delbono, 2003). Therefore, if the hIGF-1–TTC fusion protein preserves innervation, it should preserve MHC composition of injected muscles. However, injections were started at 17 months of age, so it is possible that loss of Type IIb MHC had already occurred to a large extent prior to treatment. Earlier onset of injections would allow for this to be examined more clearly, as would transgenic overexpression of IGF-1 in the central nervous system/spinal cord.
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To further investigate the effects of hIGF-1–TTC on muscle innervation, NMJ post-terminal size and morphological complexity were examined in injected muscles. NMJ post-terminals were larger and more complex in hIGF-1–TTC injected muscles compared to saline injected controls and hIGF-1 or TTC injected muscles. This finding agrees with previous data on the benefits of muscle-derived IGF-1 on innervation in aged muscle (Messi & Delbono, 2003). While NMJ post-terminals in the hIGF-1–TTC injected group were not as large as those in aged mice overexpressing IGF-1 in muscle, the difference in size probably arises from the difference in the level and duration of exposure of the motor neurones to IGF-1 arising from the muscle.
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In this study, monthly hIGF-1–TTC injections into aged mouse muscle resulted in no differences in EDL muscle weight, EDL fibre diameter, or absolute force compared to saline, IGF-1, or TTC injected groups. Age-related denervation, resulting in fibre atrophy and death, underlies muscle mass decline with age (Einsiedel & Luff, 1992; Lexell, 1995; Kadhiresan et al. 1996). IGF-1 overexpression in muscle prevents atrophy in aged muscle (Coleman et al. 1995; Barton-Davis et al. 1998; Musaro et al. 2001). Is this effect via direct stimulation of muscle IGF-1 receptors or by maintenance of innervation Monthly hIGF-1–TTC injections allow this question to be studied by presumably exerting minimal effects on the muscle IGF-1 receptors while having potentially prolonged effects on the motor neurone IGF-1 receptors. The fact that hIGF-1–TTC injections did not induce hypertrophy/prevent atrophy while preserving NMJ post-terminal size and complexity suggests (1) hIGF-1–TTC, indeed, had very little direct effect on muscle fibre IGF-1 receptors, and (2) IGF-1 overexpression in skeletal muscle prevents age-related atrophy primarily through constitutive muscle IGF-1 receptor signalling, rather than maintenance of innervation.
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Preservation of skeletal muscle function is illustrated by the increase in EDL single fibre specific force in muscles injected with hIGF-1–TTC, compared to fibres from saline, hIGF-1, or TTC injected muscles. We have previously identified excitation–contraction (EC) uncoupling as a primary culprit underlying age-related specific force decline; specifically, decline in functional DHPRs with age (Delbono et al. 1995; Renganathan et al. 1997; Wang et al. 2000) diminishes the intracellular Ca2+ transient and force production in aged fibres (González et al. 2003). Denervation in ageing is also thought to underlie EC uncoupling due to findings that experimental denervation reduces DHPR charge movement (Dulhunty & Gage, 1985; Delbono, 1992) and inward Ca2+ current (Delbono, 1992). Transgenic overexpression of hIGF-1 in skeletal muscle prevents EC uncoupling in aged mice (Renganathan et al. 1998; Wang et al. 2002), increasing the intracellular Ca2+ transient (Wang et al. 2002; González et al. 2003), and consequently, specific force in fast fibre type muscles (Barton-Davis et al. 1998; Renganathan et al. 1998; González et al. 2003). IGF-1 directly drives the expression of the skeletal muscle DHPR 1S subunit (Zheng et al. 2002). Similarly innervation state of a muscle fibre regulates functional DHPR expression (Dulhunty & Gage, 1985; Delbono, 1992). Therefore, the question remains: Does IGF-1 overexpression in muscle prevent age-related EC uncoupling by maintaining innervation or by constitutively stimulating muscle IGF-1 receptors The hIGF-1–TTC fusion protein allows examination of this question by acting as a target-derived trophic factor to motor neurones while having very little direct effect on muscle. The findings that hIGF-1–TTC injection into fast muscles increases single fibre specific force in aged mice in concert with increased NMJ size without increase in muscle fibre size suggest that preservation of specific force in aged animals overexpressing IGF-1 in muscle is achieved, in part, by maintenance of the motor neurone. Additionally, these findings provide further evidence for the role of muscle-derived IGF-1 as a neurotrophic factor in ageing skeletal muscle.
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It will be interesting to further study whether hIGF-1–TTC treatment induces satellite cell replication and muscle repair in aged mice in a similar manner as muscle-derived IGF-1 (Musaro et al. 2001, 2004). It will also be interesting to further study effect(s) of hIGF-1–TTC on excitation–SR Ca2+ release coupling in muscle fibres. These studies would help to further clarify the respective roles of muscle-derived IGF-1 in muscle and in motor neurones, and the role of motor neurone innervation on muscle EC coupling.
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Footnotes
A. M. Payne and Z. Zheng contributed equally to this work.
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2 Department of Neurobiology & Anatomy
3 Department of Internal Medicine, Section on Gerontology
4 Neuroscience Program, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA
Abstract
IGF-1 is a potent growth factor for both motor neurones and skeletal muscle. Muscle IGF-1 is known to provide target-derived trophic effects on motor neurones. Therefore, IGF-1 overexpression in muscle is effective in delaying or preventing deleterious effects of ageing in both tissues. Since age-related decline in muscle function stems partly from motor neurone loss, a tetanus toxin fragment-C (TTC) fusion protein was created to target IGF-1 to motor neurones. IGF-1–TTC retains IGF-1 activity as indicated by [3H]thymidine incorporation into L6 myoblasts. Spinal cord motor neurones effectively bound and internalized the IGF-1–TTC in vitro. Similarly, IGF-1–TTC injected into skeletal muscles was taken up and retrogradely transported to the spinal cord in vivo, a process prevented by denervation of injected muscles. Three monthly IGF-1–TTC injections into muscles of ageing mice did not increase muscle weight or muscle fibre size, but significantly increased single fibre specific force over aged controls injected with saline, IGF-1, or TTC. None of the injections changed muscle fibre type composition, but neuromuscular junction post-terminals were larger and more complex in muscle fibres injected with IGF-1–TTC, compared to the other groups, suggesting preservation of muscle fibre innervation. This work demonstrates that induced overexpression of IGF-1 in spinal cord motor neurones of ageing mice prevents muscle fibre specific force decline, a hallmark of ageing skeletal muscle.
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Introduction
Ageing leads to decreased strength in the elderly that can impair daily function. This decline in strength is due to decline in both muscle mass (Lexell, 1995) and muscle fibre specific force (Brooks & Faulkner, 1988; González et al. 2000). An age-related denervation–re-innervation process (Larsson & Ansved, 1995; Kadhiresan et al. 1996) is thought to underlie muscle mass decline (Lexell, 1995), muscle fibre-type shift (Einsiedel & Luff, 1992; Lexell, 1995), and muscle specific force decline (Delbono, 2003; Payne & Delbono, 2004).
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Insulin-like growth factor-1 (IGF-1) is a potent trophic factor for motor neurones (Caroni & Grandes, 1990), and IGF-1 administration prevents motor neurone cell death and enhances re-innervation after nerve injury (Kanje et al. 1989; Sjoberg & Kanje, 1989; Li et al. 1994). IGF-1 from skeletal muscle exerts a target-derived trophic effect on survival of embryonic motor neurones (Neff et al. 1993) and on muscle innervation in aged animals (Messi & Delbono, 2003); consequently, overexpression of IGF-1 exclusively in skeletal muscle enhances motor neurone re-innervation after nerve injury (Rabinovsky et al. 2003).
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IGF-1 is also critical for growth and hypertrophy of skeletal muscle (Florini et al. 1996). Consequently, overexpression of IGF-1 in muscle produces muscle hypertrophy in transgenic mice (Coleman et al. 1995; Musaro et al. 2001). Therefore, treatment strategies for the loss of muscle mass and function with age may be directed toward this system. Indeed, overexpression of IGF-1 in skeletal muscle prevents age-related loss in muscle mass (Barton-Davis et al. 1998; Musaro et al. 2001), particularly among fast (Type IIb) fibre type muscles. Overexpression of IGF-1 in muscle also prevents excitation–contraction (EC) uncoupling (Renganathan et al. 1998; Wang et al. 2002) and maintains skeletal muscle fibre specific force (Renganathan et al. 1998; González et al. 2003) in aged mice. These findings may be due to the direct trophic effects of elevated IGF-1 on skeletal muscle dihydropyridine receptor (DHPR) expression (Zheng et al. 2002) or to target-derived trophic effects of muscle IGF-1 on motor neurones in aged mice via maintenance of muscle fibre innervation. Target-derived trophic effects of elevated muscle IGF-1 on mature muscle innervation has been suggested (Barton-Davis et al. 1998; Messi & Delbono, 2003), but whether elevated IGF-1 in spinal cord motor neurones prevents age-related declines in skeletal muscle force is not known.
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To probe this possibility, we targeted human IGF-1 (hIGF-1) delivery to motor neurones through injection of a hIGF-1–tetanus toxin fragment C fusion protein (hIGF-1–TTC) into hindlimb skeletal muscles of aged mice. The carboxy-terminal 451 amino acid segment, termed tetanus toxin fragment C (TTC), is capable of neuronal binding, uptake, and retrograde axonal transport in similar fashion to the native tetanus toxin, but is non-toxic itself (Bizzini et al. 1977). Therefore, we hypothesized that intramuscular injection of a hIGF-1–TTC fusion protein would (1) target delivery of IGF-1 to motor neurones of ageing mice and (2) prevent age-related decrease in skeletal muscle specific force.
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Methods
hIGF-1–TTC fusion protein production and purification
Plasmid design. Full-length hIGF-1 cDNA fragment was generated by PCR using hIGF-1 inserted in the pBSKS plasmid (provided by P. Rotwein, Oregon Health and Science University, Portland, OR, USA) and the following primers: 5'-GTCGGGATCCATGGGAAAAATCAGCAGTC -3' and 5'-CAGAGATCTCATCCTGTAGTTCTTGTTTC-3'. The fragment was inserted into the pGEM-T easy vector (Promega, Madison, WI, USA) and the orientation was confirmed by DNA sequencing. Full length TTC cDNA (nucleotides 1–1356) was generated by PCR from genomic DNA of Clostridium tetani CN655 (provided by M. R. Popoff, Institute Pasteur, Paris). Primers used were: 5'-GGAAGATCTTATTCTAAAAATCTGGATTGTTGGGT-3' and 5'- CGCGTCGACTCTAGATAATCATTTGTCCATCCTTCATCTGTA-3'. The TTC PCR fragment and pGEM-T easy–hIGF-1 plasmid were digested by BglII and SalI. The purified TTC cDNA fragment was inserted into the digested plasmid. The new plasmid pGEM-T easy–hIGF-1–TTC was used as template for next PCR. The hIGF-1–TTC cDNA fragment was generated by PCR using the primers: 5'-GGAATTCCATATGGTTAATCATTTGTCCATCCTTCATC-3' and 5'-GGAATTCCATCTGGGAAAAATCAGCAGTCTTC-3'. After NdeI digestion, the hIGF-1–TTC PCR fragment was inserted into the pET28a(+) vector (Novagen, San Diego, CA, USA) and the orientation was confirmed by DNA sequencing. The pET28a(+)–hIGF-1–TTC construct was transformed into BL21 DE3 strain of E. coli (Stratagene, La Jolla, CA, USA).
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Protein production and purification. Bacteria were cultured in 2x YT medium (16 g l–1 tryptone, 10 g l–1 yeast extract, 5 g l–1 NaCl, pH 7.0) containing 25 μg ml–1 kanamycin at 32°C for 2–3 h. Recombinant protein expression was induced by addition of 0.1 mM IPTG to the medium for 3–4 h. Cultures were centrifuged at 4000 g for 20 min. Pelleted bacteria were resuspended into 4 volumes (4 ml g–1) of lysis buffer containing (mM) 50 NaH2PO4, 300 NaCl, 10 imidazole, pH 8.0 with NaOH, and were lysed by French Press. Lysate was centrifuged at 10 000 g for 20–30 min. Supernatant was removed and saved for recombinant protein purification.
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The hIGF-1–TTC (with N-terminal His Tag) was purified with Ni-NTA agarose beads (Qiagen, Valencia, CA, USA). Cleared lysate was mixed with 50% Ni-NTA agarose bead slurry (4: 1), and shaken at 200 r.p.m. at 4°C for 60 min. Samples were then centrifuged for 10 s at 1000 g to pellet the beads. Supernatant was removed and saved. Beads were washed in buffer containing (mM) 50 NaH2PO4, 300 NaCl, 50 imidazole, pH 8.0 with NaOH, centrifuged 10 s at 1000 g, and supernatant saved. Wash step was repeated 4 times. Beads were resuspended in the same volume of elution buffer containing (mM) 50 NaH2PO4, 300 NaCl, 500 imidazole, pH 8.0 with NaOH, pelleted by centrifugation for 10 s at 1000 g, and supernatant containing purified recombinant hIGF-1–TTC was removed and saved.
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The protein was further purified by dialysis in SnakeSkin Pleated Dialysis Tubing (3500 MWCO thickness; Pierce, Rockford, IL, USA), placed into 1 litre of dialysis buffer containing 10 mM Tris, pH 7.0 with HCl and slowly shaken at 4°C for 36 h. Buffer was changed every 6 h. Purified protein was lyophilized and stored at –80°C. When needed, the protein was reconstituted into 5 ml phosphate-buffered saline (PBS) for dialysis in PBS. Final protein concentration was determined to be 1 μg ml–1.
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All Ni-NTA agarose bead wash and elution supernatant samples were subjected to SDS-PAGE on 10% acrylamide gels at 77 V for 1.5 h, followed by Coomassie Blue staining. Elution samples and native hIGF-1 were also immunoblotted by loading 5 μg of protein onto lanes of 10% acrylamide gels and run at 77 V for 1.5 h. Proteins were transferred onto nitrocellulose membranes at 100 V for 1 h. Protein bands were probed with either mouse anti-IGF-1 (Su et al. 1997) (Glaxo, Research Triangle Park, NC, USA) or mouse anti–TTC (Roche, Indianapolis, IN, USA) at 1: 1000 dilution. Secondary antibody was antimouse IgG conjugated with HRP (Amersham Biosciences, Piscataway, NJ, USA) in a 1: 3000 dilution.
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IGF-1 activity assay
IGF-1 activity was determined by [3H]thymidine incorporation into L6 skeletal muscle cell cultures, as reported (Sasaoka et al. 2001). L6 skeletal muscle cells were obtained from American Type Culture Collection (Manassas, VA, USA) and were maintained in -MEM supplemented with 10% fetal calf serum. Cells were grown to confluence in 24 multiwell culture plates (Corning Inc., Corning, NY, USA), and then serum deprived for 24 h. Cells were stimulated with hIGF-1 (20 ng ml–1; GroPep, Adelaide, Australia) or hIGF-1–TTC (200 ng ml–1) for 20 h. The difference in size of the proteins (hIGF-1, 7.6 kDa; hIGF-1–TTC, 67 kDa) requires a different amount of protein added to cultures to achieve the same concentration. Following hIGF-1 or hIGF-1–TTC stimulation, 1 μCi of [3H]thymidine was added for 4 h. Control cells received 1 μCi of [3H]thymidine immediately following the serum deprivation. Cells were washed twice with ice-cold PBS, twice with ice-cold 10% trichloroacetic acid, and once with 95% ethanol. The cells were then dissolved in 1 M NaOH, neutralized with 1 M HCl, and counted in a liquid scintillation counter.
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Motor neurone culture
Embryonic mouse motor neurones were purified following previously published procedures (Henderson et al. 1995; Arce et al. 1999), with some modifications. Briefly, spinal cords were dissected from E12 mouse embryos, treated with trypsin (2.5% w/v; final concentration 0.05%) for 10 min at 37°C, and then dissociated. The largest cells were isolated by centrifugation for 15 min at 830 g over a 5.2% Optiprep cushion (Sigma, St Louis, MO, USA), followed by centrifugation for 10 min at 470 g through a 4% BSA cushion. Purified motor neurones were plated inside 35-mm Petri dishes on 12-mm coverslips previously coated with polyornithine/laminin as described (Henderson et al. 1995; Arce et al. 1999), and grown 2–3 days in L15 medium with sodium bicarbonate (625 μg ml–1), glucose (20 mM), progesterone (2 x 10–8M), sodium selenite (3 x 10–8M), conalbumin (0.1 mg ml–1), putricine (10–4M), insulin (5 μg ml–1) and penicillin–streptomycin. CT-1 (1 ng ml–1), GDNF (1 ng ml–1) and CNTF (10 ng ml–1), and 2% horse serum were also added to the medium.
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hIGF-1–TTC binding and internalization assays
Binding and internalization assays for hIGF-1–TTC followed previously published methods (Francis et al. 1995; Coen et al. 1997; Bordet et al. 2001) with modifications. The cells were incubated for 2 h at either 0°C (binding) or 37°C (internalization) in binding buffer (20 mM Tris-HCl, pH 8, 1 mM CaCl2, 1 mM MgCl2, 0.25% BSA in PBS, pH 7.3) with diluted hIGF-1–TTC recombinant protein (5 μg ml–1). After incubation, the fusion protein-containing buffer was removed and cultured cells were washed 4 times for 10 min with PBS. Cells were fixed with 4% paraformaldehyde in PBS for 10 min, and then washed twice for 10 min in PBS. For immunofluorescence detection, cells were blocked with 5% donkey serum and 0.02% Triton X-100 in PBS for 30 min. The primary antibodies were diluted in 2% donkey serum in PBS. The respective primary antibodies used were: mouse anti-IGF-1 (Su et al. 1997) (Glaxo; 1: 25 dilution) and mouse anti–TTC (Roche; 1: 50 dilution). Cells were incubated with respective primary antibodies for 2 h at room temperature then washed 4 times for 10 min with PBS. Cells were then incubated 1 h at room temperature with the secondary antibody, donkey antimouse IgG conjugated with fluorescein isothiocyanate (FITC), in a 1: 100 dilution in 1% donkey serum in PBS, then were washed again 4 times for 10 min with PBS. Immunofluorescence was visualized in a thin plane of focus with a high NA (1.30) 100x oil-immersion objective (Fluar, Zeiss, Oberkochen, Germany) on an Axioskop 2 microscope (Zeiss) and a PXL-EEV-37 CCD camera (Photometrics, Tucson, AZ, USA) based imageing system. Isee software (Inovision, Durham, NC, USA) running in a Silicon Graphics (Mountain View, CA, USA) O2 workstation was used for data acquisition and image analysis.
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Immunocytochemical detection of hIGF-1–TTC in spinal cord
DBA (National Institute on Ageing/Harlan Sprague-Dawley, Indianapolis, IN, USA) and FVB (our colony) mice were housed at Wake Forest University School of Medicine and all procedures were approved by the Animal Care and Use Committee. Young adult (3–4 months) mice were injected with hIGF-1–TTC dissolved in PBS (1 μg ml–1, 10 μg protein per injection) into several hindlimb muscles (quadriceps, gastrocnemius, TA/EDL). Two animals underwent similar injections, while still under anaesthesia, immediately following transection of the sciatic nerve. Briefly, animals were anaesthetized with I.P. injections of ketamine and xylazine (100 mg kg–1 and 20 mg kg–1, respectively), a small incision was made at the sciatic notch and a 3–4 mm segment of the sciatic nerve was cut and removed to prevent re-innervation. The incision was stitched closed, and gastrocnemius and TA muscles were injected with several 10 μl injections of hIGF-1–TTC (1 μg ml–1, 10 μg protein per injection). Two spinal cords were collected at each postinjection time point to examine fusion protein uptake from the muscle and transport to the spinal cord motor neurone cell bodies, as well as longevity of the protein in spinal cord motor neurones. Animals were killed by cervical dislocation and lumbar enlargements of spinal cords were collected at 7, 14, 21 and 28 days postinjection after transcardiac perfusion of mice with 4% paraformaldehyde in PBS. Spinal cords from denervated mice were collected at 7 days post-injection. All samples were stored in 4% paraformaldehyde in PBS overnight at 4°C, followed by storage in 20% sucrose in PBS for 36–48 h at 4°C. Samples were then embedded in OCT medium and frozen in liquid nitrogen. All samples were stored at –80°C until use.
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For immunofluorescence detection of hIGF-1–TTC fusion protein in spinal cords, frozen samples were cut on a cryostat microtome cooled to –20°C (12 μm). Samples were blocked with 5% donkey serum and 0.02% Triton X-100 in PBS for 30 min. Mouse anti-hIGF-1 (Su et al. 1997) (Glaxo) was diluted 1: 25 in 2% donkey serum in PBS. Samples were incubated for 2 h at room temperature, and then washed 4 times for 10 min in PBS. Samples were then incubated in donkey antimouse IgG conjugated with FITC diluted 1: 100 in 1% donkey serum in PBS for 1 h at room temperature and washed 4 times for 10 min in PBS. Samples were then imaged using an Axioskop 2 microscope and a CCD camera based imaging system (see above).
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Single intact muscle fibre contraction
DBA mice aged 17–18 months were injected with 10 μl of saline, hIGF-1 (1 μg; 0.1 μg ml–1 in PBS), TTC (10 μg; 1 μg ml–1 in PBS), or hIGF-1–TTC (10 μg; 1 μg ml–1 in PBS) into the anterior compartment of the right lower leg (tibialis anterior, TA; extensor digitorum longus, EDL) every 28 days for three injections. Animals were killed by cervical dislocation 14–28 days past the third and final injection, rendering animals 20–21 months old. At time of kill, injected EDL muscles were carefully dissected and pinned into a Petri dish lined with Sylgard (Dow Corning, Auburn, MI, USA) containing recording solution, consisting of (mM): 121 NaCl, 5 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.4 NaH2PO4, 24 NaHCO3 and 5.5 glucose. The recording solution was bubbled continuously with a mixture of 5% CO2 and 95% O2 to achieve a pH of 7.4.
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During initial data collection, EDL muscles from saline and hIGF-1–TTC injected mice were trimmed of excess tendon, nerve and fat, and were weighed. In the second set of experiments, saline, IGF-1, TTC and hIGF-1–TTC injected EDL muscles were removed, and single intact fibres were dissected and mounted in a small flow-through chamber (150 μl) perfused with bubbled recording solution on the stage of an inverted microscope following procedures published previously (Lannergren & Westerblad, 1987; González et al. 2000).
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Muscle fibres were stimulated by an electrical field generated between two parallel silver electrodes connected to a Grass S48 stimulator (Astro-Medical, Inc., West Warwick, RI, USA). Fibre length was adjusted until maximum force was elicited by a single twitch contraction (LO) under isometric conditions. Suprathreshold square wave pulses of 0.5 ms duration were delivered to elicit twitch contractions. Tetanic contractions were elicited with 0.5 ms square wave pulses delivered in 200 ms trains. Frequency was increased until maximum force was attained. All subsequent tetanic contractions were elicited with the frequency that elicited maximal force (González et al. 2000; Payne et al. 2004). All experiments were carried out at room temperature (22–23°C). Once LO was attained, fibre diameter was measured at three to four randomly selected points along the length of the fibre and averaged. Cross-sectional area (CSA) was calculated as (d/2)2, where d is the average diameter of the fibre (Lannergren & Westerblad, 1987; González et al. 2000). Specific force (in kPa) was calculated as maximum tetanic force (kN)/CSA (m2).
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Myosin heavy chain isoform composition in TA muscles
Myosin heavy chain (MHC) composition of injected TA muscles was determined by SDS-PAGE, as previously described (Serrano et al. 1996) with some modifications. Briefly, TA muscles were minced on ice in 4 volumes of a high salt buffer containing (mM): 300 NaCl, 100 NaH2PO4, 50 Na2HPO4, 1 MgCl2, 10 Na4P2O7 and 10 EDTA, pH 6.5. Extracts were then centrifuged at 7500 g for 30 min at 2°C in a microcentrifuge. The supernatants were diluted in 9 volumes of 1 mM EDTA buffer (37% w/v EDTA and 0.01% (v/v) 2-mercaptoethanol), vortexed and allowed to precipitate overnight at 4°C. Centrifugation was repeated, and the resulting pellet dissolved in 0.5 M NaCl and 10 mM NaPO4 and denatured by immersion in boiling water for at least 2 min. Samples were diluted 1: 100 in SDS buffer (62.5 mM Tris-HCl, 2% w/v SDS, 10% v/v glycerol, and 0.001% w/v bromophenol blue, pH 6.8). Electrophoresis was performed in 6% acrylamide (37.5: 1 acrylamide: bis-acrylamide; Bio-Rad, Hercules, CA, USA) separating gel with 30% v/v glycerol and 3% stacking gel with no glycerol. Aliquots of 10-μl of diluted myosin were subject to electrophoresis for 6 h at 50 V and 12 h at 120 V at 4°C. Gels were silver stained according to published protocols (Giulian et al. 1983) using the silver stain plus kit and Silver Stain SDS-PAGE Standards, high range (Bio-Rad). As a reference for the four MHC isoforms (I, IIa, IIx and IIb), a mixture of mouse soleus, flexor digitorum brevis, and gastrocnemius muscles was used. After staining, gels were photographed with a digital camera. The digitized image was analysed with Kodak 1D software (Eastman Kodak, Rochester, NY, USA) to determine optical density of each stained band.
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Neuromuscular junction post-terminal staining
Postsynaptic acetylcholine receptor staining was performed as described (Messi & Delbono, 2003). TA muscles were removed, pinned at a slightly stretched length, embedded in OCT tissue medium, and frozen in isopentane cooled in liquid nitrogen. Longitudinal sections (30 μm) were cut on a cryostat microtome cooled to –20°C and sections were fixed in methanol–acetone (50: 50 v/v) for 10 min at room temperature. After washing in Tris-buffered saline (TBS), sections were blocked in 10% rabbit serum in TBS for 30 min and washed again in TBS. Sections were incubated 3 h with tetramethylrhodamine-conjugated -bungarotoxin (Molecular Probes, Eugene, OR, USA), diluted 1: 200 in TBS. Stained junctions were visualized using a Zeiss Axioskop 2 microscope equipped with a CCD-EEV37 camera. Images were analysed using Isee software (Inovision) (see above). The post-terminal area was calculated in pixels by tracing the perimeter of individual labelled regions on digitized images, and the area was converted into square micrometers.
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Data analysis
Data are presented as mean ±S.E.M. Data were analysed by ANOVA followed by the Holm-Sidak multiple comparisons test applied post hoc.
Results hIGF-1–TTC fusion protein expression, purification, and activity
The genetic fusion protein hIGF-1–TTC was produced in transfected bacterial cultures, and purified by His Tag Ni-NTA affinity beads. The protein contains hIGF-1 in the N-terminal position relative to TTC. This decision was made based on the findings that a TTC–cardiotrophin-1 fusion protein with cardiotrophin-1 in the C-terminal position often produced incomplete fragments along with the full-length fusion protein, whereas a construct with cardiotrophin-1 in the N-terminal position produced only the full-length fusion protein (Bordet et al. 2001). Figure 1A shows the end product of several steps in the purification of the hIGF-1–TTC fusion protein from BL21 DE3 bacteria cultures. Cleared bacterial lysate (lane 2) and lysate that has been through the Ni-NTA binding procedure (lane 3) both show myriad stained proteins on the gel. Following successive wash steps of the Ni-NTA agarose beads, very few to no proteins are visible on the stained gel (data not shown). After elution of the fusion protein from the Ni-NTA agarose beads, a single band of protein of approximately 67 kDa size is stained (lane 4), an appropriate size for the full length hIGF-1–TTC protein. The absence of any other bands of protein suggests little to no contamination by bacterial proteins. Figure 1B shows immunoblots using antibodies against IGF-1 (lanes 1 and 2) and TTC (lane 3). Purified hIGF-1 was loaded in lane 1 as a positive control, while the Ni-NTA bead elution was loaded in lanes 2 and 3. Lanes 2 and 3 show a 67 kDa protein product, indicating that the 67 kDa protein on the stained gel is the hIGF-1–TTC fusion protein. No incomplete protein fragments are present following purification procedures as indicated by the single band in lane 4 of Fig. 1A and lanes 2–3 of Fig. 1B.
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A, following transformation of cultures and protein synthesis induction, bacteria were lysed for purification of hIGF-1–TTC (see Methods). Lanes M to 4 illustrate a Coomassie Blue stained gel. Lane M, high range molecular weight marker. Lane 1, cleared bacterial lysate. Lane 2, supernatant of bacterial lysate following Ni-NTA affinity binding and centrifugation. Lane 3, elution from Ni-NTA affinity beads exhibiting one band of stained protein at 67 kDa. Lane 4, immunoblot of elution against hIGF-1. B, immunoblots following elution of the fusion protein from Ni-NTA affinity beads against IGF-1 (Lane 2) and against TTC (Lane 3). Immunoblots both display one band of protein at 67 kDa. Purified hIGF-1 was probed with anti-IGF-1 for a positive control (Lane 1).
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To assess mitogenic activity of the fusion protein, L6 myoblasts were incubated with equivalent molar amounts of either hIGF-1 or hIGF-1–TTC, followed by measurement of [3H]thymidine uptake. L6 myoblasts were used to determine IGF-1 activity since they express IGF-1 receptors but do not express IGF-1 (Musaro & Rosenthal, 1999). Figure 2 shows [3H]thymidine incorporation into L6 muscle cells stimulated with hIGF-1 or hIGF-1–TTC following 20 h of serum deprivation. Stimulation by both proteins increased radioactive uptake compared to control cells, which received no growth factor stimulation following serum deprivation. [3H]thymidine uptake in cells stimulated by hIGF-1–TTC was similar to cells that received an equivalent amount of hIGF-1 stimulation, indicating that the IGF-1 moiety of the hIGF-1–TTC fusion protein retains its biological activity. Data were acquired from 5, 9 and 9 experiments from control, hIGF-1 and hIGF-1–TTC, respectively.
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L6 muscle cells were serum deprived for 24 h, exposed to hIGF-1 (20 ng ml–1) or hIGF-1–TTC (200 ng ml–1) for 20 h, followed by addition of 1 μCi of [3H]thymidine for 4 h. Control cells received [3H]thymidine immediately following serum deprivation. [3H]thymidine incorporation was determined by a scintillation counter. Both hIGF-1 and hIGF-1–TTC increased [3H]thymidine uptake compared to control (P= 0.002). hIGF-1–TTC stimulation increased [3H]thymidine uptake to a similar amount as equivalent stimulation by hIGF-1.
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Binding and internalization of hIGF-1–TTC in cultured motor neurones
Following incubation at 0°C for 2 h, binding of the fusion protein to motor neurone surfaces was assessed with anti-IGF-1 or anti-TTC antibodies (Fig. 3, left). Immunofluorescence labelling showed that hIGF-1–TTC binding occurred on the surface of neurone cell bodies and processes, and that labelling was not different using antibodies against either TTC (Fig. 3, top left; n= 14 cells) or IGF-1 (Fig. 3, bottom left; n= 15 cells), suggesting binding of the full-length fusion protein rather than some degradation product. Following incubation at 37°C for 2 h, internalization of hIGF-1–TTC was also assessed using anti-TTC (Fig. 3, top right; n= 22 cells) or anti-IGF-1 (Fig. 3, bottom right; n= 25 cells). Immunofluorescence labelling was detected in neuronal cell bodies and central processes, and the protein was labelled by both primary antibodies. Labelling with both antibodies is located in distinct clusters, suggesting the protein was internalized into vesicular structures, similar to previous reports (Bordet et al. 2001; Miana-Mena et al. 2002). These findings indicate that hIGF-1–TTC is internalized by spinal cord motor neurones in a manner similar to other TTC-coupled proteins (Francis et al. 1995; Coen et al. 1997; Bordet et al. 2001; Miana-Mena et al. 2002).
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Cultured mouse spinal cord motor neurones were exposed to hIGF-1–TTC for 2 h at 0°C for binding assays (left) or for 2 h at 37°C for internalization assays (right). Immunodetection of hIGF-1–TTC was performed with antibodies against TTC (top) and IGF-1 (bottom). Binding assays (left) reveal immunofluorescence on surfaces of cell bodies and processes of cultured motor neurones. Internalization assays (right) reveal immunofluorescence located in clusters resembling vesicular structures in cell bodies and central processes of cultured motor neurones.
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In vivo retrograde transport of hIGF-1–TTC and stability in spinal cord motor neurones
To study kinetics of motor neurone uptake and retrograde transport of hIGF-1–TTC, several hindlimb muscles were injected with 10 μg each of the protein, and lumbar spinal cords were removed for immunocytochemical detection. Labelling was detected in ventral horn cell bodies at 7, 14 and 21 days following skeletal muscle injection (Fig. 4A–C), but could not be detected at 28 days postinjection (Fig. 4D). Figure 4E shows an enlarged view of the ventro-lateral lumbar spinal cord 7 days post-injection. Fluorescence is clearly visible in and around large cell bodies in the ventro-lateral motor column near the ventral root (between arrows), corresponding to motor neurones. No positive immunolabelling for hIGF-1 was detected in spinal cords of denervated animals at 7 days post-injection (Fig. 4F). Fluorescence was detected only ipsilateral relative to injection, and no fluorescence was detected at levels above or below the lumbar enlargement (data not shown). These findings indicate that hIGF-1–TTC is, indeed, delivered to spinal cord motor neurones by uptake and retrograde transport to the cell bodies in the ventral spinal cord, and are in agreement with previous reports of other TTC-coupled proteins undergoing similar uptake and retrograde transport (Coen et al. 1997; Miana-Mena et al. 2002).
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hIGF-1–TTC was injected into several hindlimb muscles of one leg of mice. Immunofluorescence was detectable in ventral lumbar spinal cord sections at 7 days (A), 14 days (B), and 21 days (C) post-injection, but was not detectable at 28 days (D) (n= 2 spinal cords per time point). Fluorescence was detectable only on one side of the spinal cord, in correspondence with hIGF-1–TTC intramuscular injections. E, enlarged view of the ventro-lateral spinal cord at 7 days postinjection. Arrows indicate the ventral root adjacent to fluorescent motor neurone cell bodies. F, hIGF-1–TTC was not detected in spinal cords of mice injected immediately following sciatic nerve transection (n= 2 cords).
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hIGF-1–TTC treatment prevents EDL fibre specific force decline in ageing mice
To study the effects of targeted delivery of hIGF-1–TTC to spinal cord motor neurones on skeletal muscle mass and function in ageing animals, hindlimb muscles were injected with the fusion protein over a 3- to 4-month time span near the end of the DBA and FVB mouse expected lifespan (23–24 month lifespan; see Methods for injection protocol). In an initial experiment, EDL muscles were removed and weighed following injection of saline or hIGF-1–TTC. EDL muscle weights were not different between groups (hIGF-1–TTC, 7.95 ± 0.29 g, n= 8; Saline, 8.55 ± 0.13 g, n= 4; P > 0.05). With the difficulties of accurately measuring specific force in whole muscle experiments (Sugi & Tsuchiya, 1998), single intact muscle fibres were dissected from EDL muscles following injection of saline, hIGF-1, TTC, or hIGF-1–TTC (n= 16, 15, 11 and 12 fibres from 7, 6, 4 and 5 mice, respectively) and used for in vitro contraction experiments. Single fibre absolute force was not different among groups (Fig. 5A). Fibre diameters of IGF-1, TTC and hIGF-1–TTC muscles were not different compared to control saline injected (Figs 5B, P > 0.05). However, EDL fibres from muscles injected with hIGF-1–TTC exhibited greater specific force than saline, hIGF-1 and TTC injected EDL fibres (Figs 5C, P= 0.006). Specific force was not different among saline, hIGF-1 and TTC injected fibres, indicating the introduction of hIGF-1 or TTC alone by this injection protocol is not beneficial to muscle and/or motor neurones in ageing mice. Figure 5D illustrates representative contraction traces from single intact EDL fibres, scaled to specific force.
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Single fibre maximal tetanic force (A) and diameter (B) were not different among groups. Maximum tetanic specific force was higher in fibres from muscles injected with hIGF-1–TTC than fibres injected with saline, hIGF-1, or TTC (C, P= 0.006; n= 16, 14, 11 and 12 fibres from 7, 6, 4 and 5 mice in saline, hIGF-1, TTC and hIGF-1–TTC groups, respectively). D, representative tetanic contraction traces from single intact EDL fibres from mice injected with saline (a), hIGF-1 (b), TTC (c) and hIGF-1–TTC (d).
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Muscle fibre type composition is unchanged by hIGF-1–TTC
Previous reports indicate that viral-mediated and transgenic overexpression of IGF-1 in skeletal muscle preserves Type IIb muscle fibres in ageing rats (Barton-Davis et al. 1998; Messi & Delbono, 2003). To determine if treatment of aged animals with hIGF-1–TTC prevents muscle fibre type changes, muscles were removed and subjected to SDS-PAGE to determine MHC composition of saline, hIGF-1, TTC, and hIGF-1–TTC injected muscles (n= 7, 6, 4 and 4 muscles, respectively; 1 muscle per mouse). TA muscles were used for this analysis because EDL muscles were used for single fibre contraction experiments. Furthermore, MHC analysis of EDL single fibres is not reliable using the single intact fibre contraction technique due to contamination by cellular debris of surrounding dead fibres (González et al. 2000). Figure 6A illustrates the pattern of MHC distribution on 6% acrylamide gels. All TA muscles from all groups display the presence of Type IIx and Type IIb MHC. However, no difference exists among groups in percentage of Type IIx and Type IIb MHC (Fig. 6B), indicating that injection of hIGF-1, TTC, or hIGF-1–TTC does not change fast-twitch muscle fibre type composition relative to saline-injected controls.
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TA muscles were removed from 20-month-old DBA mice following monthly with saline, hIGF-1, TTC, or hIGF-1–TTC injections. A, TA muscle proteins were subjected to SDS-PAGE on 6% acrylamide gels for detection of MHC isoforms. All TA muscles expressed detectable levels of Type IIx and IIb MHC. The far left lane shows all four adult MHC isoforms, indicated as IIa, IIx, IIb and I. B, no differences were found among the groups in percentage of Type IIx and IIb MHC (P > 0.05; n= 7, 6, 4 and 4 muscles, 1 muscle per mouse).
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hIGF-1–TTC increases post-terminal size in ageing muscle
Overexpression of hIGF-1 in muscle preserves neuromuscular junction (NMJ) size and morphology in ageing mice, suggesting a target-derived trophic effect of muscle IGF-1 on motor neurones (Messi & Delbono, 2003). To probe this further, NMJ post-terminals from aged mouse TA muscles injected with saline, hIGF-1, TTC and hIGF-1–TTC were examined (n= 2 muscles per group, 1 muscle per mouse; n= 16 sections per muscle, 32 sections per group; n= 86, 105, 97 and 101 NMJs from saline, hIGF-1, TTC and hIGF-1–TTC, respectively). NMJ post-terminals undergo atrophy and fragmentation in aged mammalian muscles. Atrophy is characterized by smaller, less complex stained areas (fewer acetylcholine receptors) (Courtney & Steinbach, 1981), and fragmentation by a dispersion of stained post-terminals in some fibres (Oda, 1984; Robbins & Fahim, 1985). Post-terminal atrophy (Fig. 7A–CversusFig. 7D) and fragmentation (Fig. 7C) were found in muscles from control animals, and these data agree with previous findings (Messi & Delbono, 2003). While the post-terminals from hIGF-1–TTC injected muscles are not as large as post-terminals from young mouse muscles (1700 μm2) (Messi & Delbono, 2003), treatment with the fusion protein does increase the size (Fig. 7E; P= 0.002) and morphological complexity of the post-terminal compared with saline, IGF-1 and TTC injected counterparts.
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NMJ post-terminal acetylcholine receptors in TA muscles from old mice were labelled with rhodamine-conjugated -bungarotoxin. A–D, representative NMJ post-terminals from saline (A), hIGF-1 (B), TTC (C) and hIGF-1–TTC (D) injected muscles. NMJ post-terminals from hIGF-1–TTC injected muscles display more complex morphology compared to saline, hIGF-1, and TTC injected muscles. In addition, fragmentation of NMJ post-terminals in ageing muscles (as seen in C) is eliminated by hIGF-1–TTC injection. Scale bar, 30 μm. E, muscles injected with hIGF-1–TTC exhibit increased NMJ post-terminal area, compared to saline, hIGF-1, and TTC injected muscles. (P= 0.002; n= 2 muscle per group, 1 muscle per mouse; n= 16 sections per muscle, 32 sections per group; n= 86, 105, 97 and 101 NMJs from saline, hIGF-1, TTC and hIGF-1–TTC groups, respectively).
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Discussion
This work reports: (1) the design, expression, production and purification of the genetic fusion protein hIGF-1–TTC, which contains equivalent mitogenic activity compared to hIGF-1; (2) in vitro binding and internalization of hIGF-1–TTC by cultured spinal cord motor neurones; (3) in vivo uptake and retrograde transport of hIGF-1–TTC by spinal cord motor neurones following injection into hindlimb skeletal muscles; (4) increased specific force in single intact muscle fibres from old mice injected with hIGF-1–TTC, compared to old mice injected with saline, hIGF-1, or TTC; and (5) increased NMJ post-terminal size and morphological complexity in hIGF-1–TTC injected compared to saline, hIGF-1 and TTC injected muscles.
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hIGF-1–TTC properties and actions in neurones hIGF-1–TTC in vitro interaction with cultured motor neurones
In this work, the motor neurone neurotrophic factor human IGF-1 was genetically fused to TTC to create a 67 kDa protein product, hIGF-1–TTC. Tetanus toxin is an extremely potent neurotoxin. Since the C-fragment (TTC) is capable of binding, uptake and retrograde axonal transport (Bizzini et al. 1977; Evinger & Erichsen, 1986) without toxic effects, it was proposed that TTC could be used to target and deliver proteins to motor neurones (Bizzini et al. 1980; Simpson, 1985). Indeed, conjugation and genetic fusion of TTC to proteins increases their uptake into neurones (Francis et al. 1995; Bordet et al. 2001; Francis et al. 2004). Similar to previous reports, hIGF-1–TTC bound to cultured spinal cord motor neurones at 0°C. Binding of hIGF-1–TTC to motor neurone membranes may be explained by ganglioside binding (Rogers & Snyder, 1981), but some binding may be explained by IGF-1 receptors on cultured motor neurone membranes (Vincent et al. 2004). Further experiments at 37°C show that hIGF-1–TTC is internalized into cultured spinal cord motor neurones. Although internalization of IGF-1 receptors and their ligand occurs (Dore et al. 1997), the visualization of hIGF-1–TTC in clusters resembling vesicular structures suggests internalization was accomplished primarily via TTC binding to gangliosides (Bordet et al. 2001; Miana-Mena et al. 2002). Co-labelling of bound and internalized hIGF-1–TTC by antibodies against IGF-1 and TTC ensures that the fusion protein is still intact and not degraded upon receptor binding or internalization.
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hIGF-1–TTC in vivo interactions with spinal cord motor neurones
To examine the in vivo uptake and retrograde transport properties of hIGF-1–TTC in motor neurones young adult mice were injected with hIGF-1–TTC into hindlimb muscles, and spinal cords were removed at various time points following injections. Immunofluorescence labelling was detected in and surrounding ventral horn large cell bodies up to 21 days post-injection, indicating uptake and retrograde transport of hIGF-1–TTC by spinal cord motor neurones. Injections were performed in only one hindlimb; therefore, detection with anti-hIGF-1 in only the ipsilateral spinal cord and only in the lumbar enlargement ensured that the antibody was detecting hIGF-1–TTC, not endogenous IGF-1 in the spinal cord. This finding, and the failure to detect hIGF-1–TTC in the spinal cords of denervated animals, indicates that entry of the protein into the spinal cord was by uptake from skeletal muscle and retrograde axonal transport. Detection in spinal cord up to 21 days also indicates that the protein is rather stable, possibly allowing the hIGF-1 portion of the protein to exert prolonged effects on motor neurones. The stability of the fusion protein in the spinal cord and the quick removal of TTC-coupled proteins from muscle (i.e. 24–48 h; Miana-Mena et al. 2002) allowed the long injection interval of 28 days, minimizing the direct effects on muscle IGF-1 receptors.
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Effects of hIGF-1–TTC injection on skeletal muscle in aged mice
While uptake and retrograde transport of hIGF-1–TTC in spinal cord motor neurones is evident in these findings, potential treatments for diseases or ailments of ageing must improve function, whether preventing decline or returning lost function. Ageing leads to many changes in skeletal muscle structure and function, the primary concern being decline in muscle strength accounted for by loss of muscle mass (Lexell, 1995) and by muscle fibre specific force decline (Brooks & Faulkner, 1988; González et al. 2000). Age-related denervation is more prominent that previously thought (Wang et al. 2005), and a denervation–re-innervation process (Larsson & Ansved, 1995; Lexell, 1995; Kadhiresan et al. 1996) is thought to underlie age-related changes in muscle structure and function (Lexell, 1995; Delbono, 2003; Payne & Delbono, 2004). We have previously shown that motor neurones in aged rodents retain sensitivity to IGF-1 (Shan et al. 2003) and that IGF-1 overexpression in muscle maintains skeletal muscle innervation in aged mice (Messi & Delbono, 2003). Therefore, we studied the effects of targeting IGF-1 to motor neurones on muscle function.
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To assess whether hIGF-1–TTC exerted beneficial effects on skeletal muscle innervation, we examined MHC composition of injected TA muscles. No differences in either Type IIx or IIb MHC were found among muscles injected with saline, hIGF-1, TTC, or hIGF-1–TTC. Ageing muscles primarily lose the fastest (Type IIb) fibres as a result of loss of the largest, fastest motor units (Einsiedel & Luff, 1992; Lexell, 1995; Kadhiresan et al. 1996). Previous reports have shown preservation of Type IIb MHC composition in aged muscles overexpressing IGF-1 (Barton-Davis et al. 1998; Musaro et al. 2001; Messi & Delbono, 2003), presumably, by preservation of innervation in these muscles (Messi & Delbono, 2003). Therefore, if the hIGF-1–TTC fusion protein preserves innervation, it should preserve MHC composition of injected muscles. However, injections were started at 17 months of age, so it is possible that loss of Type IIb MHC had already occurred to a large extent prior to treatment. Earlier onset of injections would allow for this to be examined more clearly, as would transgenic overexpression of IGF-1 in the central nervous system/spinal cord.
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To further investigate the effects of hIGF-1–TTC on muscle innervation, NMJ post-terminal size and morphological complexity were examined in injected muscles. NMJ post-terminals were larger and more complex in hIGF-1–TTC injected muscles compared to saline injected controls and hIGF-1 or TTC injected muscles. This finding agrees with previous data on the benefits of muscle-derived IGF-1 on innervation in aged muscle (Messi & Delbono, 2003). While NMJ post-terminals in the hIGF-1–TTC injected group were not as large as those in aged mice overexpressing IGF-1 in muscle, the difference in size probably arises from the difference in the level and duration of exposure of the motor neurones to IGF-1 arising from the muscle.
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In this study, monthly hIGF-1–TTC injections into aged mouse muscle resulted in no differences in EDL muscle weight, EDL fibre diameter, or absolute force compared to saline, IGF-1, or TTC injected groups. Age-related denervation, resulting in fibre atrophy and death, underlies muscle mass decline with age (Einsiedel & Luff, 1992; Lexell, 1995; Kadhiresan et al. 1996). IGF-1 overexpression in muscle prevents atrophy in aged muscle (Coleman et al. 1995; Barton-Davis et al. 1998; Musaro et al. 2001). Is this effect via direct stimulation of muscle IGF-1 receptors or by maintenance of innervation Monthly hIGF-1–TTC injections allow this question to be studied by presumably exerting minimal effects on the muscle IGF-1 receptors while having potentially prolonged effects on the motor neurone IGF-1 receptors. The fact that hIGF-1–TTC injections did not induce hypertrophy/prevent atrophy while preserving NMJ post-terminal size and complexity suggests (1) hIGF-1–TTC, indeed, had very little direct effect on muscle fibre IGF-1 receptors, and (2) IGF-1 overexpression in skeletal muscle prevents age-related atrophy primarily through constitutive muscle IGF-1 receptor signalling, rather than maintenance of innervation.
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Preservation of skeletal muscle function is illustrated by the increase in EDL single fibre specific force in muscles injected with hIGF-1–TTC, compared to fibres from saline, hIGF-1, or TTC injected muscles. We have previously identified excitation–contraction (EC) uncoupling as a primary culprit underlying age-related specific force decline; specifically, decline in functional DHPRs with age (Delbono et al. 1995; Renganathan et al. 1997; Wang et al. 2000) diminishes the intracellular Ca2+ transient and force production in aged fibres (González et al. 2003). Denervation in ageing is also thought to underlie EC uncoupling due to findings that experimental denervation reduces DHPR charge movement (Dulhunty & Gage, 1985; Delbono, 1992) and inward Ca2+ current (Delbono, 1992). Transgenic overexpression of hIGF-1 in skeletal muscle prevents EC uncoupling in aged mice (Renganathan et al. 1998; Wang et al. 2002), increasing the intracellular Ca2+ transient (Wang et al. 2002; González et al. 2003), and consequently, specific force in fast fibre type muscles (Barton-Davis et al. 1998; Renganathan et al. 1998; González et al. 2003). IGF-1 directly drives the expression of the skeletal muscle DHPR 1S subunit (Zheng et al. 2002). Similarly innervation state of a muscle fibre regulates functional DHPR expression (Dulhunty & Gage, 1985; Delbono, 1992). Therefore, the question remains: Does IGF-1 overexpression in muscle prevent age-related EC uncoupling by maintaining innervation or by constitutively stimulating muscle IGF-1 receptors The hIGF-1–TTC fusion protein allows examination of this question by acting as a target-derived trophic factor to motor neurones while having very little direct effect on muscle. The findings that hIGF-1–TTC injection into fast muscles increases single fibre specific force in aged mice in concert with increased NMJ size without increase in muscle fibre size suggest that preservation of specific force in aged animals overexpressing IGF-1 in muscle is achieved, in part, by maintenance of the motor neurone. Additionally, these findings provide further evidence for the role of muscle-derived IGF-1 as a neurotrophic factor in ageing skeletal muscle.
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It will be interesting to further study whether hIGF-1–TTC treatment induces satellite cell replication and muscle repair in aged mice in a similar manner as muscle-derived IGF-1 (Musaro et al. 2001, 2004). It will also be interesting to further study effect(s) of hIGF-1–TTC on excitation–SR Ca2+ release coupling in muscle fibres. These studies would help to further clarify the respective roles of muscle-derived IGF-1 in muscle and in motor neurones, and the role of motor neurone innervation on muscle EC coupling.
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Footnotes
A. M. Payne and Z. Zheng contributed equally to this work.
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