Spinal muscular atrophy with respiratory distress type 1 is a neuromuscular disorder characterized by progressive weakness and atrophy of the diaphragm and skeletal muscles, leading to death in childhood. No effective treatment is available. The neuromuscular degeneration (Nmd2J) mouse shares a crucial mutation in the immunoglobulin mu-binding protein 2 gene (Ighmbp2) with spinal muscular atrophy with respiratory distress type 1 patients and also displays some basic features of the human disease. This model serves as a promising tool in understanding the complex mechanisms of the disease and in exploring novel treatment modalities such as insulin-like growth factor 1 (IGF1) which supports myogenic and neurogenic survival and stimulates differentiation during development. Here we investigated the treatment effects with polyethylene glycol-coupled IGF1 and its mechanisms of action in neurons and muscles. Polyethylene glycol-coupled IGF1 was applied subcutaneously every second day from post-natal Day 14 to post-natal Day 42 and the outcome was assessed by morphology, electromyography, and molecular studies. We found reduced IGF1 serum levels in Nmd2J mice 2 weeks after birth, which was normalized by polyethylene glycol-coupled IGF1 treatment. Nmd2J mice showed marked neurogenic muscle fibre atrophy in the gastrocnemius muscle and polyethylene glycol-coupled IGF1 treatment resulted in muscle fibre hypertrophy and slowed fibre degeneration along with significantly higher numbers of functionally active axonal sprouts. In the diaphragm with predominant myogenic changes a profound protection from muscle fibre degeneration was observed under treatment. No effects of polyethylene glycol-coupled IGF1 were monitored at the level of motor neuron survival. The beneficial effects of polyethylene glycol-coupled IGF1 corresponded to a marked activation of the IGF1 receptor, resulting in enhanced phosphorylation of Akt (protein kinase B) and the ribosomal protein S6 kinase in striated muscles and spinal cord from Nmd2J mice. Based on these findings, polyethylene glycol-coupled IGF1 may hold promise as a candidate for future treatment trials in human patients with spinal muscular atrophy with respiratory distress type 1.
Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a devastating neuromuscular disorder with childhood onset. Muscle weakness and atrophy primarily affect the diaphragm and distal limbs (Bertini et al., 1989; Grohmann et al., 2001, 2003). Therefore breathing difficulties are one striking symptom of SMARD1 leading to early respiratory failure and death (Bertini et al., 1989; Grohmann et al., 2001, 2003; Rudnik-Schoneborn et al., 2004b). Disease-causing mutations affect the gene encoding for a ribosome associated ATPase/Helicase termed immunoglobulin mu-binding protein 2 (IGHMBP2) (Grohmann et al., 2001; Guenther et al., 2009). To date no effective treatment for SMARD1 is available. One proof of principle approach is the administration of growth factors with their cell survival promoting abilities. The modifying effects of one such neurotrophic factor, CNTF, were demonstrated in mouse models for the proximal form of spinal muscular atrophy (Simon et al., 2010), amyotrophic lateral sclerosis (Giess et al., 2002), and in the progressive motor neuronopathy mouse model (pmn) (Sendtner et al., 1992). Another growth factor of interest is insulin-like growth factor 1 (IGF1). It is a potent survival factor that stimulates axon growth in motor neurons (Hughes et al., 1993; Neff et al., 1993), increases axonal sprouting in denervated or paralysed muscles (Caroni and Grandes, 1990; Caroni and Schneider, 1994; Caroni et al., 1994), and prevents injury-induced skeletal muscle damage and atrophy (Rommel et al., 2001; Martins et al., 2013). We previously described that a polyethylene glycol (PEG)-coupled variant of IGF1 (PEG-IGF1) with improved in vivo pharmacokinetic properties (Metzger et al., 2011; Saenger et al., 2011) indeed mediates beneficial effects in the pmn mouse (Jablonka et al., 2011), the SOD1 mouse—a model of familial ALS—(Saenger et al., 2012), and in the mdx mouse, a model for Duchenne muscular dystrophy (Metzger et al., 2011), where this treatment especially protected against diaphragmatic weakness and fatigue (Saenger et al., 2011; Gehrig et al., 2012). The Nmd2J (neuromuscular degeneration) mouse carries a point mutation in the Ighmbp2 gene, leading to a ubiquitously reduced IGHMBP2 full-length protein level (Cox et al., 1998). The pathological features of the Nmd2J mouse are comparable to juvenile patients with SMARD1 including weakness of hindlimbs and of the diaphragm. First signs of muscle atrophy in the Nmd2J mouse appear 2 weeks after birth (Cox et al., 1998; Grohmann et al., 2004; Krieger et al., 2013). Motor neuron loss in Nmd2J mice of ∼30% is detectable 10 days post-natally, a time point when the mice are not yet phenotypically afflicted. At later disease stages (5 to 12 weeks after birth), motor neuron loss does not markedly progress any further (Grohmann et al., 2004). In contrast with other motor neuron disorders, motor neuron degeneration does not primarily affect the neuromuscular junction (Krieger et al., 2013). Muscle fibre degeneration in the diaphragm starts ∼6–8 weeks after birth and does not correlate with motor axon loss in the phrenic nerve (Grohmann et al., 2004; Krieger et al., 2013). In addition, Nmd2J mice display a cardiomyopathy (Maddatu et al., 2004). The survival rate is highly variable ranging from 6 to 10 weeks up to 7 months depending on unknown modifying polymorphisms on chromosome 13 (Cox et al., 1998).
Here, we identified a systemic IGF1 deficit in the blood serum of Nmd2J mice. Our treatment study aimed at testing the efficacy of an IGF1 derivative (PEG-IGF1) with a prolonged lifetime and reduced side effects in the Nmd2J mouse, the model for the juvenile form of SMARD1. IGF1 is a global player in developmental processes of neurons, such as axon growth (Ozdinler and Macklis, 2006), promoting peripheral neuron survival (Gao et al., 1999; Leinninger et al., 2004; Willaime-Morawek et al., 2005), and inducing regeneration (Tiangco et al., 2001). Lack of IGF1 delays survival and maturation of the CNS in mice (Ye et al., 2002). The high impact of IGF1 on axonal sprouting and muscle fibre orchestration encouraged us to investigate more precisely its cellular targets. We found that PEG-IGF1 application restores endogenous IGF1 levels and ameliorates core disease features in the Nmd2J mouse by two mechanisms, (i) by acting on nerve terminal sprouts; and (ii) by protection against muscle fibre degeneration which corresponds to increased phosphorylation of Akt (protein kinase B) and ribosomal protein S6 kinase.
Materials and methods
Transgenic mouse lines
The Nmd2J mouse line (Cox et al., 1998) and the thy1-YFP-Htg mice (YFP-12-H) (Feng et al., 2000) on a C57Bl/6 background were housed in the central animal facility of the University of Würzburg. All described procedures and experiments were approved by the animal care and ethics committees of our institution and by the Bavarian state authorities. Genotyping of Nmd2J and thy1-YFP-Htg mice was performed as described previously (Cox et al., 1998; Feng et al., 2000). For all the experiments Nmd2J/thy1-YFP-Htg mice were used and termed thereafter Nmd2J.
Nmd2J and control mice were treated with multiple subcutaneous injections of 0.05 mg/kg PEG-IGF1 [synthesized by F. Hoffmann-La Roche Ltd. as described (Metzger et al., 2011)] diluted in PBS every second day, from post-natal Day 14 (disease onset) until post-natal Day 42 (marked disease). This systemic dose (0.05 mg/kg) had no effects on weight and size of the Nmd2J mice (Supplementary Fig. 2A and B). As stated by others, only doses >0.3 mg/kg seem to induce a significant increase of the body weight in wild-type mice as well as in mouse models for motor neuron degeneration (Saenger et al., 2012). Some mice received only one single injection of PEG-IGF1 on post-natal Days 13 or 41. Mice were sacrificed 24 ± 1 h after the last injection, when serum levels were peaking after subcutaneous injection (Metzger et al., 2011). As PBS injections had no effect either on control or on Nmd2J mice untreated mice were used as controls.
Rotarod and grip strength
Grip strength and Rotarod tests were used as validated motor performance tests. The tail suspension method was used to visualize the limb contractures. For more detailed information see the Supplementary material.
All studies were done on a digital Neurosoft-Evidence 3102 electromyograph (Schreiber & Tholen Medizintechnik). Motor and compound motor-sensory nerve conduction studies were done in the sciatic nerve and EMGs were performed in the gastrocnemius muscle of post-natal Day 21, 28 and 42 PEG-IGF1 treated and untreated Nmd2J and control mice using proven techniques described previously in numerous mutant mouse systems (Zielasek et al., 1996; Kimura 2001a, b; Berghoff et al., 2005; Sommer et al., 2005; Bremer et al., 2010). For details see Supplementary material.
Histology, immunohistochemistry and morphometry of spinal cord, ventral roots, sciatic nerves and whole mount preparation of the gastrocnemius muscle
For the histological analyses of spinal cord, ventral roots, and sciatic nerves, post-natal Day 42 untreated and PEG-IGF1 treated Nmd2J and Nmd+/+ mice were perfused with 4% paraformaldehyde and processed as described by Krieger et al. (2013). For further details see the online Supplementary material and Ey et al., 2007; Krieger et al., 2013.
Morphological analysis of the heart
Hearts from untreated and PEG-IGF1 treated control and Nmd2J mice at post-natal Day 42 were snap frozen for the biochemical and molecular measurements and fixed material was embedded for paraffin sections. For further details see the online Supplementary material and Hofmann et al., 2012a,b.
Primary motor neuron cultures
The procedure of the enrichment of primary mouse motor neurons was carried out as described by Wiese et al. (2010). Isolated and enriched mouse motor neurons were plated on laminin-111 and cultured with 10 ng/ml CNTF for 3 days (using B27 supplement free from insulin and IGF1; Invitrogen) that was followed by withdrawing CNTF for 24 h. On Day 5 in vitro, one PEG-IGF1 (10 ng/ml) pulse was applied for 1 h, and subsequently motor neurons were harvested for western blot.
Antibodies used for western blot analysis
For the western blot technique see the Supplementary material.
The following antibodies were used: rabbit anti-IGF1 receptor β subunit (1:1000, Cell Signaling), rabbit anti-Phospho-IGF1 receptor β subunit (1:1000, Cell Signaling), rabbit anti-Phospho-AktSer473 (1:1000, Cell Signaling), rabbit anti-Phospho-AktThr308 (1:1000, Cell Signaling), rabbit anti-Phospho-p70 S6 KinaseThr389 (1:1000, Cell Signaling), rabbit anti-p70 S6 Kinase [1:2000, Cell Signaling; this antibody recognizes three ribosomal protein S6 kinase 1 isoforms at 60 kDa, 70 kDa, and 85 kDa, similar as described by Kim et al. (2009)], rabbit anti-IGHMBP2 peptide antiserum (1:100; Grohmann et al., 2004), rabbit anti-Akt (1:20 000, Cell Signaling), and mouse anti-GAPDH (1:10 000, Calbiochem).
ELISA and western ligand blot
Control and Nmd2J mice treated with single or multiple doses of PEG-IGF1 were deeply anaesthetized 24 ± 1 h after the last injection. Blood was extracted from the heart and incubated for 15 min at room temperature. For further details see the online Supplementary material.
The statistical significance of differences between the four groups (untreated and accordingly PEG-IGF1 treated control and Nmd2J mice) was calculated using one-way ANOVA and Bonferroni multiple comparison post hoc test; statistical significance between two groups was analysed using one-sample t-test and Student’s t-test. Densitometric band intensities were normalized to the loading control. Values from untreated conditions in each experiment were standardized as 100%. One-sample t-test was used to compare the means to this reference value. Analyses were done with GraphPad Prism software; significance levels were set at P < 0.05. For the electromyographic data a non-parametric test (Mann-Whitney) was used, with a significance level of P < 0.05, one-sided. Bars represent means ± standard error of the mean (SEM), means ± standard deviation (SD) or median ± SD; significance levels are indicated throughout as non-significant and *P < 0.05, **P < 0.01, ***P < 0.001.
IGF1 serum levels in young Nmd2J mice are reduced and can be compensated by PEG-IGF1 treatment
We analysed endogenous IGF1 and PEG-IGF1 levels using ELISA in Nmd2J and control mice at post-natal Days 14 and 42 covering the treatment period of 4 weeks with PEG-IGF1 at a dose of 0.05 mg/kg subcutaneous every second day. At disease onset (post-natal Day 14) we observed a ∼50% decline of endogenous serum IGF1 levels in Nmd2J mice whereas liver Igf1 transcript levels were preserved (Fig. 1A). This systemic IGF1 deficit was still detectable at post-natal Day 42 and PEG-IGF1 raised total IGF1 levels in both controls and Nmd2J mutants alike (Fig. 1B). In addition, the IGF binding proteins IGFBP2 and IGFBP3 were increased in Nmd2J mice as early as at post-natal Day 14 compared with age-matched controls whereas messenger RNA production in the liver was unchanged (Supplementary Fig. 1A and C). However, at post-natal Day 42, levels for IGFBP2 and IGFBP3 were balanced and after PEG-IGF1 injections increased both in controls and Nmd2J mice (Supplementary Fig. 1B and D). Full-length IGHMBP2 as indicated by the 110 kDa band in spinal cord tissue of post-natal Day 14 Nmd2J mice was unaffected by PEG-IGF1 application (Supplementary Fig. 1E). Our data suggest that the pathology in young Nmd2J mice might be associated with an early post-natal impairment in peripheral IGF1 homeostasis, thus supporting the PEG-IGF1 treatment paradigm.
PEG-IGF1 treatment reduces motor deficits in Nmd2J mice
To study whether external IGF1 application could functionally compensate for impaired motor function in Nmd2J mice, we performed Rotarod and grip strength tests after treatment with PEG-IGF1 from post-natal Days 14 to 42. We found that 4 weeks of treatment with PEG-IGF1 resulted in improved grip strength and Rotarod performance at the post-natal time points Days 21, 28, 35 and—in addition—at post-natal Day 42 for Rotarod performance (Fig. 2A and B). During PEG-IGF1 treatment, grip strength values peaked at post-natal Day 21 and declined thereafter until post-natal Day 42 (Fig. 2A). PEG-IGF1 also induced a small but significant increase in muscle strength in healthy controls (Fig. 2A). The Rotarod performance was significantly increased ∼3.6-fold in Nmd2J mice under PEG-IGF1 treatment and still remained elevated at post-natal Day 42 (Fig. 2B). In Rotarod and grip strength experiments no gender-specific differences were observed at post-natal Day 21 (Supplementary material). Body weight and body length mildly increased in controls and were also elevated but blunted in Nmd2J mutants after PEG-IGF1 treatment (Supplementary Fig. 2A and B). Nmd2J mice at post-natal Day 42 were not able to spread their hindlimbs as compared to control mice (Supplementary Fig. 2C and D). The observed hindlimb-contractures of Nmd2J mice (Supplementary Fig. 2D–F) likely may have increased the apparent motor impairment. Several studies have described neurogenic muscle atrophy of the gastrocnemius muscle as a result of IGHMBP2 deficiency (Grohmann et al., 2004; Ruiz et al., 2005; Krieger et al., 2013). To functionally define the degree of the neurogenic component in skeletal muscle of the model disorder and to evaluate any treatment effects, we performed an EMG study in the gastrocnemius muscle at post-natal Days 21, 28, and 42. At post-natal Day 42 all Nmd2J mice exhibited moderate to marked signs of denervation as indicated by increased spontaneous activity (Fig. 2D), in contrast to control mice (Fig. 2C). Despite a large variability between individual mice, there was a significant reduction of spontaneous activity (mean reduction 84%) in PEG-IGF1 treated Nmd2J mice as compared to untreated mutants (Fig. 2D–F), but no gender-specific discrepancies appeared (81% for Nmd2J female mice and 82% for Nmd2J male mice).
No spontaneous activity was noted in Nmd2J mice at post-natal Day 21 despite motor deficits. At post-natal Day 28, spontaneous activity was present in six of seven Nmd2J mutant mice yet with high variability between individual mice. There was a trend towards less pronounced spontaneous activity in the PEG-IGF1 treated mutants (untreated Nmd2J mice median 79 ± 57 events/s, n = 7, versus PEG-IGF1 treated Nmd2J mice median 36 ± 48 events/s, n = 6), albeit these apparent differences did not reach statistical significance (P = 0.3).
Moreover, electrophysiological testing of sciatic nerve conduction in the hindlimbs of Nmd2J mice revealed reduced compound muscle action potential amplitudes when compared with controls as evidence of muscle atrophy, which may be a sign of denervation or of muscle fibre degeneration or both as part of the complex phenotype of Nmd2J mice (Grohmann et al., 2004; Ruiz et al., 2005). Median compound muscle action potential amplitudes in post-natal Day 42 Nmd2J mice were significantly reduced of ∼34% when compared to control mice, and of about 21% after PEG-IGF1 treatment, but because of the high interindividual variability the difference between treated and untreated Nmd2J mutants did not reach significance (Fig. 2G and Supplementary Fig. 3A–C). There was neither a relevant difference in shape and duration of the compound muscle action potentials nor in conduction velocities. The late potentials of low amplitude were comprised of superimposed H-reflexes and F-waves that could better be discerned at higher stimulation frequency (Nowicki et al., 2013). F-wave persistence rates and latencies were all within the normal range. These results in Nmd2J mice are in agreement with those from other mouse models mimicking degenerative neuronal disorders (Holtmann et al., 1999; Lindberg et al., 1999). Testing for a neuromuscular transmission defect in the foot muscle by repetitive tibial nerve stimulation (3 and 10 per second) revealed a mildly abnormal decremental response as a sign of synaptic dysfunction in only 2 of 23 Nmd2J mice tested (9%), indicating that an additional transmission disorder is rather the exception than the rule in Nmd2J mice. Studying the compound motor-sensory nerve action potential amplitudes revealed no difference between Nmd2J mutants and control animals, PEG-IGF1 treated as well as untreated (Supplementary Fig. 4A–C).
PEG-IGF1 treatment protects against muscle fibre atrophy in Nmd2J mice
Nmd2J mice typically exhibit muscle fibre atrophy in gastrocnemius and diaphragm muscles mimicking the human situation in SMARD1 (Bertini et al., 1989; Grohmann et al., 2001; Eckart et al., 2012). We discovered that PEG-IGF1 treatment of Nmd2J mutants had a positive effect on muscle fibre calibre in the diaphragm (Fig. 3A–F and M) without affecting the relative contribution of type I and II fibre distribution or the degree of fibre type grouping (Fig. 3D–F). Nmd2J mice showed perimysial and central cell nuclei in the diaphragm (Fig. 3B and Supplementary Fig. 5B) combined with scattered loss or reduction in the myofibrillar network (Fig. 3B, white arrowheads), but no fibre type grouping (Fig. 3E). All these abnormalities suggest a predominant myopathic pattern of pathology which was markedly ameliorated or even prevented by PEG-IGF1 treatment (Fig. 3C and F and Supplementary Fig. 5C).
In the gastrocnemius muscle (Fig. 3G–L and N) grouped muscle fibre atrophy associated with abundant perimysial cell nuclei and rows of central muscle nuclei was present in Nmd2J mice (Fig. 3H). As shown by F4/80 immunohistochemistry, only a small fraction of the perimysial nuclei were activated macrophages (Supplementary Fig. 5E and F), whereas in the diaphragm no F4/80-positive macrophages were encountered (Supplementary Fig. 5A–C). Moreover, central nuclei (Fig. 3H) were pronounced in areas of denervation atrophy as indicated by isolated or grouped small angulated fibres and fibre splitting (Fig. 3H). Some fibres showed basophilic alterations as an early indicator of fibre degeneration (Fig. 3H). Neither of these abnormalities was seen in control muscles (Fig. 3G and Supplementary Fig. 5D). PEG-IGF1 treatment significantly reduced the number of central nuclei and activated F4/80-positive macrophages (Fig. 3H and I and Supplementary Fig. 5E–G). This was paralleled by the appearance of some hypertrophic fibres often arranged in small groups (Fig. 3I and L). In Nmd2J mutants we found a significant fibre calibre reduction in the gastrocnemius muscle (Fig. 3N and Table 1) with a shift towards type I fibres as compared to control animals (Fig. 3J and K). PEG-IGF1 treatment induced a significant amelioration of reduced fibre calibre in Nmd2J mice as compared to untreated mutants, almost reaching control values (Fig. 3N). ATPase pH 4.6 stainings revealed areas of type I fibre hypertrophy after PEG-IGF1 treatment (Table 1 and Fig. 3I and L) and a significant fibre type grouping (Fig. 3L).
|Post-natal Day 42, Nmd2J mice (n = 3)||Post-natal Day 42, PEG-IGF1 treated Nmd2J mice (n = 3)|
|Atrophic %||Hypertrophic %||Atrophic %||Hypertrophic %|
|Type I fibres||Type II fibres||Type I fibres||Type II fibres||Type I fibres||Type II fibres||Type I fibres||Type II fibres|
|Post-natal Day 42, Nmd2J mice (n = 3)||Post-natal Day 42, PEG-IGF1 treated Nmd2J mice (n = 3)|
|Atrophic %||Hypertrophic %||Atrophic %||Hypertrophic %|
|Type I fibres||Type II fibres||Type I fibres||Type II fibres||Type I fibres||Type II fibres||Type I fibres||Type II fibres|
As in a previous study signs of cardiomyopathy were shown in Nmd2J mutants (Maddatu et al., 2004), we investigated the heart by weight measurements, morphology, and molecular analyses. Nmd2J mice showed mild cardiac hypertrophy at post-natal Day 42 as indicated by a significantly increased ratio of heart weight to body weight, which tended to decline with PEG-IGF1 treatment (Supplementary Fig. 6A). The total collagen content, measured by picrosirius red and Masson’s trichrome (blue) staining, was not altered in hearts from Nmd2J mice compared with control mice (Supplementary Fig. 6B–E). We observed a large variability between mice for the ratio of lung weight and body weight, and atrial natriuretic peptide RNA, but the obtained data did not differ significantly between Nmd2J mutants and control mice (data not shown).
PEG-IGF1 treatment does not compensate for motor neuron and motor axon loss, but stabilizes additional axonal sprouts in Nmd2J mice
We then questioned whether PEG-IGF1 may ameliorate loss of motor neurons and their axons in Nmd2J mice at post-natal Day 42. In accordance to our recent study (Krieger et al., 2013) we used Nmd2J mice crossbred with thy1-YFP-Htg mice to track motor axons into the periphery. We found that PEG-IGF1 treatment did not protect against loss of motor neuron cell bodies in the spinal cord (Fig. 4A), or against loss of motor axons from ventral roots and sciatic nerves of Nmd2J mice (Fig. 4B and C). However, confocal microscopy of nerve terminals in the gastrocnemius muscle from PEG-IGF1 treated Nmd2J mice revealed an increased number of terminal sprouts in comparison with untreated Nmd2J mutants (Table 2) indicating a direct effect of PEG-IGF1 (Fig. 4D and Supplementary Fig. 7A–D). Although the degenerative process in motor axon terminals was apparently progressive over time in both untreated and PEG-IGF1 treated Nmd2J mice (Fig. 4D and Supplementary Fig. 7A–D), the relative increase in small axonal sprouts (Fig. 4D, Supplementary Fig. 7A–D and Table 2) indicated that PEG-IGF1 may ameliorate or at least partially compensate for axonal degeneration, slow down the evolution of progression in motor axon terminals or may contribute to motor axonal sprouting, respectively. All of these morphological findings are in line with the electrophysiological observations.
|Neuromuscular junctions innervated by one single axon||Terminal sprouts per axon at post-natal Day 42 (%)|
|Nmd2J mouse (n = 13)||PEG-IGFI treated Nmd2J mouse (n = 10)|
|Neuromuscular junctions innervated by one single axon||Terminal sprouts per axon at post-natal Day 42 (%)|
|Nmd2J mouse (n = 13)||PEG-IGFI treated Nmd2J mouse (n = 10)|
Multiple PEG-IGF1 injections restore normal IGF1 receptor levels in affected muscles of Nmd2J mice
The reduced IGF1 serum level in Nmd2J mice and the compensatory effect of external PEG-IGF1 application suggested a mechanistic defect of IGF1 signalling. Therefore we investigated tissue expression of the IGF1 receptor (IGF1R) in PEG-IGF1 treated and untreated control and Nmd2J mice, respectively. Spinal cord, gastrocnemius muscle, and diaphragm tissues were prepared at post-natal Days 14 and 42 for western blot analyses. At post-natal Day 14 the IGF1 receptor was detectable and no differences in signal intensities between control and Nmd2J tissue could be observed (Fig. 5A). In contrast, the IGF1 receptor in gastrocnemius and diaphragm muscles of 6-week-old Nmd2J mice was markedly increased when compared with controls (Fig. 5B and C), and PEG-IGF1 treatment over 4 weeks restored the IGF1 receptor protein levels in Nmd2J mutants almost to control levels in both muscles (Fig. 5B and C). The effects were entirely due to post-transcriptional regulation, as no change in IGF1 receptor messenger RNA was detectable in either muscle under any condition (Supplementary Fig. 8A and B). No increased IGF1 receptor levels were detectable in spinal cord of Nmd2J mutants at post-natal Day 42, and PEG-IGF1 did not have any impact on IGF1 receptor levels in the spinal cord (Fig. 5D).
One single application of PEG-IGF1 stimulates phosphorylation of Akt in Nmd2J mutant and control mice
The previous results raised the question about the signalling of PEG-IGF1 in muscle and spinal cord. We therefore investigated the phosphorylation of the IGF1 receptor (IGF1R) and Akt as one of its downstream targets in 2-week-old Nmd2J and control mice 24 h after just one single PEG-IGF1 injection, i.e. at the peak of the signalling response (Metzger et al., 2011), in the gastrocnemius and diaphragm muscle and in the spinal cord by western blot analysis. A single subcutaneous application of PEG-IGF1 increased IGF1 receptor phosphorylation in control and mutant mice at post-natal Day 14, both in the gastrocnemius muscle and the diaphragm (Fig. 6A and B), but not in spinal cord tissue (Fig. 6C) where the phosphorylated IGF1 receptor was not detectable. PEG-IGF1 treatment further stimulated Akt phosphorylation at Ser473 and Thr308. Interestingly, the dynamic increase of phospho-Akt at Thr308 was much higher in muscle tissue of Nmd2J mutants than in controls (Fig. 6A–C). Although no IGF1 receptor phosphorylation signal was detectable in the spinal cord, PEG-IGF1 application led to an increase in Akt phosphorylation at both Ser473 and Thr308 in Nmd2J mutants but not in controls (Fig. 6C). Based on these findings, it remains to be shown whether Akt phosphorylation may still be inducible at later stages during disease progression. At post-natal Day 42, Akt phosphorylation was similarly but more modestly elevated (Fig. 7). One single PEG-IGF1 injection restored the IGF1 level both in controls and in Nmd2J mutants (Fig. 7A), but as expected, did not revert the increased IGF1 receptor levels in striated muscles from Nmd2J mice (Fig. 7B). A strong Akt phosphorylation was still detectable at post-natal Day 42 in the diaphragm and gastrocnemius muscle from Nmd2J mutants by single and multiple PEG-IGF1 applications alike (Fig. 7C), but only moderately in the spinal cord and exclusively after multiple PEG-IGF1 doses (Fig. 7C). In control mice, no Akt phosphorylation was detectable at post-natal Day 42 in gastrocnemius muscle and spinal cord (data not shown).
One single PEG-IGF1 injection stimulates phosphorylation of different ribosomal protein S6 kinase isoforms
As we observed an upregulation of phospho-Akt at Thr308 in post-natal Day 14 striated muscles and in spinal cord after one single PEG-IGF1 injection, the question still remained if further signalling down-stream from Akt is likewise detectable in muscles as well as in spinal cord and motor neurons. Akt phosphorylation is associated with ribosomal protein S6 kinase activation, a positive regulator of protein translation (von Manteuffel et al., 1996; Glass, 2003). To address this question, we investigated whether phosphorylation of ribosomal protein S6 kinase is inducible in striated muscle and spinal cord at post-natal Day 14 by a single PEG-IGF1 application. In the diaphragm and the gastrocnemius muscle, one single PEG-IGF1 injection stimulated the phosphorylation of the 70 kDa isoform of ribosomal protein S6 kinase (p70S6K) in Nmd2J mice (Fig. 8A and B), while markedly stimulated phosphorylation of the 85 kDa isoform (p85S6K) was monitored exclusively in the spinal cord (Fig. 8C). In control mice no phosphorylation of ribosomal protein S6 kinase was observed (Fig. 8A–C). The total protein amount of ribosomal protein S6 kinase was unchanged (Fig. 8A–C). In enriched isolated motor neurons from embryonic Day 13.5 mouse embryos, one PEG-IGF1 pulse resulted in enhanced phosphorylation of Akt, p70S6K, and p85S6K both in control and Nmd2J motor neurons (Fig. 8D).
The Nmd2J mouse serves as a mouse model for SMARD1 caused by a mutation of an ATPase/Helicase. The principal finding of our study is that Nmd2J mice suffer from a muscle IGF1 signalling deficit associated with peripheral IGF1 deficiency, and that they profit in their motor function defects from repeated systemic applications of PEG-IGF1 in a time period when the model disorder progresses rapidly (post-natal Days 14 until 42). Within this 4-week time interval, we did not observe differences in survival as no Nmd2J mouse died from the disease. At the cellular level PEG-IGF1 application stimulated the phosphorylation of the IGF1 receptor in striated muscle fibres and in turn activated the Akt/p70S6K signalling pathway.
PEG-IGF1 acts beneficially on motor nerve fibres and striated muscles in Nmd2J mice
As evidenced by morphological and functional analyses, Nmd2J mice showed profound denervation and muscle degeneration with rapid progression between post-natal Days 14 and 42, including progressive contractures of the lower limbs adding up to a complex motor impairment. Because the Nmd2J mouse is characterized by a highly variable lifespan ranging from 6 to 10 weeks up to 7 months (Cox et al., 1998) or—in our mouse facility—from 4 to 7 months (unpublished data) we decided not to perform a survival study over several months, but focused on a time period when the natural course shows clear progression of disease signs and when our applied parameters are still reliably quantifiable in untreated Nmd2J mice to get reliable results for the PEG-IGF1 effects in treated Nmd2J mice.
The ameliorated motor function in Nmd2J mice treated with PEG-IGF1 was most likely brought about by two different mechanisms in the gastrocnemius muscle acting in a complementary way. First, PEG-IGF1 led to a partial preservation of axonal sprouts of motor axons at the NMJ; and second, it acted by decreasing the degree of muscle fibre degeneration. Our electrophysiological analyses corroborated mainly the first of the two mechanisms in that the profoundly reduced denervation activity (spontaneous activity) in the gastrocnemius muscle EMG following treatment is in line with an increase of functional nerve terminals/sprouts in PEG-IGF1 treated Nmd2J mice. We also found a reduction of compound muscle action potential amplitudes in Nmd2J mice, a non-specific indicator of muscle atrophy and denervation that tended to be slightly higher after PEG-IGF1 treatment. The degree of compound muscle action potential reduction was less pronounced than described in a previous report (Grohmann et al., 2004), presumably due to the higher numbers of analysed animals in the present study and the higher variability that may have diminished the apparent effects. Moreover, in PEG-IGF1-treated Nmd2J mice we observed a much lesser decline in muscle fibre calibre and even found groups of hypertrophic fibres and some enlargement of motor unit territories in the gastrocnemius muscle. This is best interpreted as a consequence of still functional neuromuscular junctions because motor nerve terminals were either preserved by the treatment or allowed to regenerate better as indicated by enhanced and/or stabilized axonal sprouting. This is in agreement with recent findings (Ruiz et al., 2005; Krieger et al., 2013). The improved mobility of PEG-IGF1 treated Nmd2J mice is thus best explained as a result of stabilized axonal sprouts. This interpretation is corroborated by reports showing that IGF1 is a target-derived growth factor influencing sprouting and plasticity of axonal nerve terminals (Neff et al., 1993; Caroni and Schneider, 1994; Iwasaki and Ikeda 1999; Kaspar et al., 2003). However, the question still remains why motor neuron survival in PEG-IGF1 treated animals is not beneficially affected. A potential reason for these negative results may be that the serum concentrations with our systemic dose (0.05 mg/kg) may not have built up a sufficient gradient to penetrate through an intact blood–brain barrier and in turn to reach an effective local tissue concentration. This assumption is in line with recent data showing positive effects on spinal motor neuron survival in the pmn mouse only if a 3-fold higher dose was applied than that used here with Nmd2J mice (Jablonka et al., 2011). Unfortunately, our Nmd2J mutants did not tolerate higher PEG-IGF1 doses (unpublished data), probably due to hypersensitivity of IGF1 signalling pathways in these mutants, as indicated by the high IGF1 receptor levels in diaphragm and gastrocnemius muscle tissue. As systemic PEG-IGF1 in the chosen tolerable dose did not affect motor neuron survival, one might consider local application of PEG-IGF1 into the CSF compartment. Combined treatments with other neurotrophic factors such as CNTF could also be envisaged. As CNTF already showed beneficial effects in other models for motor neuron diseases (Sendtner et al., 1992; Giess et al., 2002; Simon et al., 2010), it may have additional potency on motor neuron survival with respect to IGHMBP2 deficiency, a hypothesis that needs to be tested. In terms of clinical feasibility, a systemic mode of application would be preferable. The issue of dose-finding will require special consideration once a long-term treatment trial is planned.
Various rescue effects by IGF1 were induced in multiple experimental models (Hantai et al., 1995; Gregorevic et al., 2002; Kaspar et al., 2003; Nagano et al., 2005; Narai et al., 2005; Schertzer et al., 2006; Lepore et al., 2007; Dodge et al., 2008; Franz et al., 2009; Kumar et al., 2011; Jablonka et al., 2011; Gehrig et al., 2012; Rinaldi et al., 2012) including improvement in muscle function, delay of disease-associated progression, and prolonged survival. Application of recombinant IGF1 both systemically or through an adeno-associated viral vector did not lead to relevant beneficial effects in the proximal form of spinal muscular atrophy (Bosch-Marce et al., 2011; Shababi et al., 2011), which in part may be because of unfavourable drug properties and insufficient drug concentration in spinal cord and muscle, demanding the use of modified IGF1 variants (e.g. PEG-IGF1) with a more homeostatic exposure and markedly reduced immunogenicity (Metzger et al., 2011; Saenger et al., 2011). Derailment of the IGF1 homeostasis has been made responsible for the side effects of systemic IGF1 application, e.g. hypoglycaemia and growth hormone suppression (Chen et al., 2005). These side effects are strongly reduced with PEG-IGF1 in mice (Metzger et al., 2011; Saenger et al., 2012), and are one of the reasons why PEG-IGF1 may be a better candidate for treatment trials.
We propose that a dual and potentially independent mechanism of PEG-IGF1 action may have taken place in Nmd2J mice. In addition to the positive effects on nerve terminal sprouts there is an important second mechanism based on the muscle pathology. Predominantly in the diaphragm we detected abnormal patterns typical of a myopathy, characterized by degeneration of single muscle fibres with scattered basophilia and loss of myofibrillar structures up to occasional ghost fibres. This view is corroborated by the published observation that the degeneration of the diaphragm in Nmd2J mice does not correlate with motor axon loss of the phrenic nerve (Grohmann et al., 2004). In the gastrocnemius muscle denervation was the leading sign but the increase in activated muscle cell nuclei and modestly increased macrophage numbers are more likely due to myogenic pathology than to motor neuron degeneration. It is likely that under these conditions macrophages may serve as phagocytes scavenging degenerating muscle fibre profiles, or else may promote pathology. Such a disease promoting role of low number of macrophages has been shown to occur in mouse models for Charcot–Marie–Tooth neuropathies where macrophages phagocytize genetically abnormal, but morphologically intact myelin (Martini et al., 2013). In a number of disease models macrophages can play detrimental as well as protective roles depending on the investigated model (Gordon, 2003; Ydens et al., 2012, 2013). Of particular relevance for the PEG-IGF1 treatment effects in Nmd2J mice is a study showing that macrophage-borne IGF1 can promote repair of muscle fibres after experimental damage (Lu et al., 2011). And it is not only in atrophic mouse models that PEG-IGF1 alleviates myogenic deficits. Indeed, a protective effect against fatigue of the diaphragm was shown in mdx mice, a model with pure muscular pathology (Gehrig et al., 2012). Moreover, in a model of myotoxic muscle injury it was shown that intramuscular application of PEG-IGF1 caused a beneficial effect on muscle fibre regeneration whereas systemic treatment did not (Martins et al., 2013). Higher local doses may have been needed for the therapeutic effects because scar formation may have reduced drug penetration to the sites of injury.
For the diaphragm, the protective effect of PEG-IGF1 was impressive and nearly complete. After 4 weeks of PEG-IGF1 treatment features of myopathy were only rare in the diaphragm of Nmd2J mice. Moreover, there was reduced and slowed fibre size reduction and the number of muscle cell nuclei was decreased. We suggest that PEG-IGF1 also acts by inducing a myogenic signalling pathway corresponding to ribosomal protein S6 kinase activation which may compensate for muscle fibre loss in the diaphragm. In translational terms, the myopathic aspects might be the culprit for the fast progression of diaphragmatic weakness in the natural course of Nmd2J mice and in the human disease SMARD1 (Bertini et al., 1989; Grohmann et al., 2001, 2003; Krieger et al., 2013), and PEG-IGF1 application would then be a potential therapeutic tool to treat the progressive diaphragmatic weakness.
Two other features could have had an impact on motor performance. First, Nmd2J mutants develop moderate to severe contractures of the proximal hindlimbs between post-natal Days 14 and 42 that are poorly understood. These contractures were not amenable to treatment with PEG-IGF1. As treated and untreated Nmd2J mice were equally affected these contractures unlikely became a confounding factor for the treatment effects. Early contractures of all major joints have also been described in children with spinal muscular atrophy type I and II (Rudnik-Schoneborn et al., 2004a).
Second, Nmd2J mice may develop severe cardiomyopathy (Maddatu et al., 2004). In our Nmd2J mice at post-natal Day 42 we found only mild cardiac hypertrophy but no signs indicating overt heart failure as suggested by normal lung weight, atrial natriuretic peptide, and absence of collagen depositions. Different to the previous report on end-stage heart failure (Maddatu et al., 2004), interstitial fibrosis was absent at post-natal Day 42. This may be because of the highly variable phenotype of this mouse line (Cox et al., 1998) and does not exclude congestive cardiomyopathy developing at later time points in our Nmd2J mice. Based on the present findings it seems unlikely that a cardiomyopathy had an influence on motor performance during the treatment period.
PEG-IGF1 application compensates for IGF1 signalling deficits in Nmd2J mice
The still preserved high level of detectable IGF1 receptor in striated muscles of Nmd2J mice 6 weeks after birth indicated a high unmet demand of IGF1 signalling in muscle tissue, and prompted us to do a detailed analysis of the IGF1 signalling pathway. Endogenous serum IGF1 levels from post-natal Day 14 and Day 42 Nmd2J mice were significantly reduced whereas messenger RNA levels in the liver as the major IGF1 producing tissue remained unchanged (Fig. 1). In addition, IGFBP2 and IGFBP3 levels were transiently elevated at post-natal Day 14 (Supplementary Fig. 1A and C), suggesting an impairment of the entire peripheral IGF1 signalling system in Nmd2J mice. The fact that IGHMBP2 is a ribosome-associated helicase (Guenther et al., 2007, 2009) brings IGHMBP2 into a context with protein biosynthesis. The reduced serum level of IGF1 with unchanged liver IGF1 messenger RNA supports the idea that IGHMBP2 deficiency leads to altered cellular mechanisms potentially associated with the protein translation machinery and thus may be affecting the total amount of IGF1 protein. This peripheral IGF1 deficit parallels the situation found in different spinal muscular atrophy mouse models (Hua et al., 2011; Murdocca et al., 2012). In terms of striated muscles, IGF1 controls myoblast and satellite cell proliferation and differentiation (Florini et al., 1993). The myogenic activity of IGF1 is mediated through the IGF1 receptor as IGF1 receptor deficient mice die at birth for respiratory distress due to a lack of the diaphragm and a general deficiency in skeletal muscles (Powell-Braxton et al., 1993; Coolican et al., 1997). In Nmd2J mice, IGF1 receptor levels in both diaphragm and gastrocnemius muscle were strongly increased at post-natal Day 42, presumably due to high demand of IGF1 signalling in those muscles, and 4 weeks of PEG-IGF1 treatment nearly completely prevented this higher IGF1 receptor presence. Here we showed that one single PEG-IGF1 application stimulated Akt phosphorylation (Ser473 and Thr308) and the phosphorylation of the 70 kDa isoform of the ribosomal protein S6 kinase (p70S6K) in striated muscles of Nmd2J mutants at post-natal Day 14. Muscle differentiation and protein biosynthesis mechanisms are induced through the PI3 kinase/p70S6K/mTOR pathway (von Manteuffel et al., 1996; Coolican et al., 1997) and thus promote muscle hypertrophy and fibre growth (Glass, 2003), postulating that the beneficial effect of PEG-IGF1 in striated muscle is mediated through the stimulated Akt/p70S6K signalling pathway. However, the situation is different to spinal cord where we exclusively detected the phosphorylation of p85S6K, the isoform of the ribosomal protein S6 kinase, which is predominantly localized to the nucleus (Reinhard et al., 1994). In contrast, in primary motor neurons from control and Nmd2J mouse embryos we found that phosphorylation of both ribosomal protein S6 kinase isoforms (p70S6K and p85S6K) was inducible. This observation leads us to suggest that either the activated signalling pathways from whole spinal cord only partially represents the specific situation in motor neurons in vivo or p70S6K phosphorylation only occurs at embryonic and very early post-natal stages in the spinal cord.
One indicator for treatment-related altered IGF1 levels is a concomitant increase of IGF binding proteins, such as IGFBP2 and IGFBP3 at post-natal Day 14. As it was found in IGFBP2 transgenic mice (Hoeflich et al., 1999), muscle tissue represents the most sensitive tissue for the negative growth effects of IGFBP2, and muscle mass was disproportionally reduced as compared to body mass. A microarray study from Corti et al. (2006) revealed that Igfbp5 gene expression was upregulated in spinal cord tissue from Nmd2J mutants. Thus, reduced levels of IGF1 in association with higher levels of IGF binding proteins may bear on the phenotype of Nmd2J mice. It remains to be established in future treatment trials whether the PEG-IGF1 treatment paradigm demonstrated in our study may show sustained efficacy in Nmd2J mice over several months.
If the dual disease mechanism for the ultimate muscle paralysis described in this report holds true for patients with SMARD1 and the diaphragm is the main target of treatment efforts, PEG-IGF1 may become a reasonable candidate as a therapeutic compound in afflicted patients.
We are grateful to Hiltrud Klüpfel for providing expert technical assistance on muscle histology and Helga Wagner for performing the heart morphology, and Regine Sendtner and Viktor Buterus for the stock breeding. We would like to acknowledge Dennis Klein for advice and help with the macrophage analysis and Benjamin Dombert for cell culture assistance; we thank Michael Sendtner and Rudolf Martini for helpful discussions concerning the manuscript, and Utz Fischer for providing IGHMBP2-overexpressing HEK293 and siRNA HeLa cells.
This work was funded by the Deutsche Forschungsgemeinschaft (SFB 581, subproject B24). K.V.T. is the recipient of a senior professorship by the Würzburg University Medical School and is financially supported by intramural research funds.
Supplementary material is available at Brain online.
protein kinase B
spinal muscular atrophy with respiratory distress type 1