Fasudil improves survival and promotes skeletal muscle development in a mouse model of spinal muscular atrophy
© Bowerman et al; licensee BioMed Central Ltd. 2012
Received: 8 December 2011
Accepted: 7 March 2012
Published: 7 March 2012
Spinal muscular atrophy (SMA) is the leading genetic cause of infant death. It is caused by mutations/deletions of the survival motor neuron 1 (SMN1) gene and is typified by the loss of spinal cord motor neurons, muscular atrophy, and in severe cases, death. The SMN protein is ubiquitously expressed and various cellular- and tissue-specific functions have been investigated to explain the specific motor neuron loss in SMA. We have previously shown that the RhoA/Rho kinase (ROCK) pathway is misregulated in cellular and animal SMA models, and that inhibition of ROCK with the chemical Y-27632 significantly increased the lifespan of a mouse model of SMA. In the present study, we evaluated the therapeutic potential of the clinically approved ROCK inhibitor fasudil.
Fasudil was administered by oral gavage from post-natal day 3 to 21 at a concentration of 30 mg/kg twice daily. The effects of fasudil on lifespan and SMA pathological hallmarks of the SMA mice were assessed and compared to vehicle-treated mice. For the Kaplan-Meier survival analysis, the log-rank test was used and survival curves were considered significantly different at P < 0.05. For the remaining analyses, the Student's two-tail t test for paired variables and one-way analysis of variance (ANOVA) were used to test for differences between samples and data were considered significantly different at P < 0.05.
Fasudil significantly improves survival of SMA mice. This dramatic phenotypic improvement is not mediated by an up-regulation of Smn protein or via preservation of motor neurons. However, fasudil administration results in a significant increase in muscle fiber and postsynaptic endplate size, and restores normal expression of markers of skeletal muscle development, suggesting that the beneficial effects of fasudil could be muscle-specific.
Our work underscores the importance of muscle as a therapeutic target in SMA and highlights the beneficial potential of ROCK inhibitors as a therapeutic strategy for SMA and for other degenerative diseases characterized by muscular atrophy and postsynaptic immaturity.
Keywordsspinal muscular atrophy fasudil survival motor neuron protein NMJ muscle
As the leading genetic cause of infant deaths, spinal muscular atrophy (SMA) is a devastating and incurable neuromuscular disorder [1, 2]. SMA affects 1 in 6,000 to 10,000 births and results from deletions or mutations in the survival motor neuron 1 (SMN1) gene [1–3]. The primary pathological hallmark of SMA is the loss of lower motor neurons from the spinal cord and corresponding muscular atrophy with subsequent paralysis and in most severe cases, death [1, 2].
The complete loss of the SMN protein is embryonic lethal . In humans however, a recent duplication event in chromosome 5 has given rise to the centromeric SMN2 gene . While both SMN1 and SMN2 genes differ by only a few nucleotides, a critical C to T substitution lies within position 6 of SMN2 exon 7 [5, 6]. This silent mutation results in the aberrant splicing of exon 7, giving rise to the biologically unstable SMNΔ7 protein [3, 5]. Although the SMN2 gene produces predominantly the SMNΔ7 protein, a small amount of full-length SMN is still produced . Thus, the number of SMN2 gene copies in SMA patients is a key modifier of disease severity [3, 7, 8].
One of the major hurdles in SMA is to understand how the loss of a ubiquitously expressed protein leads to the specific loss of spinal cord motor neurons. Work from various research groups has identified distinct roles for SMN in neurodevelopment, neuromaintenance, RNA metabolism, at the neuromuscular junction (NMJ) and in skeletal muscle (reviewed in ). As of yet however, none of these various functions of the SMN protein have been recognized as being solely responsible for SMA pathogenesis.
Work from our laboratory has shown that Smn depletion in cellular and mouse models results in altered expression and localization of a number of regulators of actin cytoskeletal dynamics [10–12]. Indeed, analysis of spinal cords from SMA mice revealed a significant increase in active RhoA (RhoA-GTP) , a major upstream regulator of the actin cytoskeleton . RhoA-GTP signaling in neuronal cells modulates various cellular functions such as growth, neurite formation, polarization, regeneration, branching, pathfinding, guidance and retraction (reviewed in [14, 15]). Our previous work demonstrated that administration of the RhoA/Rho kinase (ROCK) inhibitor Y-27632  leads to a dramatic increase in survival in an intermediate mouse model of SMA . Recently Nölle et al. demonstrated that knockdown of Smn in PC12 cells affects the phosphorylation state of downstream effectors of ROCK, supporting the value of the ROCK pathway as a therapeutic target for SMA pathogenesis .
In the present work, we have treated SMA mice with fasudil, a ROCK inhibitor approved for US clinical trials. We show that fasudil dramatically improves the lifespan and increases muscle fiber size in Smn 2B/- SMA mice. Furthermore, we report for the first time that ROCK inhibition restores normal expression of markers of skeletal muscle development in SMA mice. Our study highlights the beneficial effects of ROCK inhibition not only for SMA pathogenesis but also for any degenerative disease that has NMJ and skeletal muscle development defects. Importantly, as fasudil is currently being used in US Food and Drug Administration (FDA)-approved clinical trials for other disorders, re-purposing it is an exciting and feasible therapeutic approach for the treatment of SMA.
The Smn 2B/- mice were established in our laboratory and maintained in our animal facility on a C57BL/6 × CD1 hybrid background. The 2B mutation consists of a substitution of three nucleotides in the exon splicing enhancer of exon 7 [18, 19]. The Smn knock-out allele was previously described by Schrank et al.  and Smn +/- mice were obtained from The Jackson Laboratory (Bar Harbor, Maine, USA). All animal procedures were performed in accordance with institutional guidelines (Animal Care and Veterinary Services, University of Ottawa).
Fasudil (LC Laboratories; Woburn, Massachusetts, USA) was diluted in water and administered by a modified oral gavage procedure  to Smn 2B/- and Smn 2B/+ mice from post-natal day (P)3 to P21. The three fasudil dosage regimens were as follows: low dose (30 mg/kg once daily), medium dose (30 mg/kg twice daily), and high dose (30 mg/kg twice daily from P3 to P6; 50 mg/kg twice daily from P7 to P13; 75 mg/kg twice daily from P14 to P21). Vehicle-treated animals received water. Survival and weight were monitored daily.
The primary antibodies used were as follows: mouse anti-actin (1:800; Fitzgerald; Acton, Massachusetts, USA), mouse anti-Smn (1:5000; BD Transduction Laboratories; Mississauga, Ontario, Canada), rabbit anti-phosphorylated cofilin (1:250; Chemicon; Billerica, Massachusetts, USA), rabbit anti-cofilin (1:500; Millipore; Billerica, Massachusetts, USA), rabbit anti-phosphorylated cofilin 2 (1:500; Cell Signaling; Danvers, Massachusetts, USA), rabbit anti-cofilin 2 (1:500; Millipore), mouse anti-myogenin (1:250; BD Transduction Laboratories), rabbit anti-HB9 (1:50; Abcam; Cambridge, Massachusetts, USA), mouse anti-2H3 (1:250; Developmental Studies Hybridoma Bank; Iowa City, Iowa, USA) and mouse anti-SV2 (1:250; Developmental Studies Hybridoma Bank). The secondary antibodies used were as follows: horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5000; Bio-Rad; Mississauga, Ontario, Canada), HRP-conjugated goat anti-rabbit IgG (1:5000; Bio-Rad), DyLight goat anti-mouse (1:250; Jackson ImmunoResearch; West Grove, Pennsylvania, USA), goat anti-rabbit biotin-SP-conjugated (1:200; Dako; Burlington, Ontario, Canada)), streptavidin-Cy3-conjugated (1:600; Jackson ImmunoResearch) and Alexa Fluor 680 goat anti-mouse (1:5000; Molecular Probes; Burlington, Ontario, Canada). The α-bungarotoxin (BTX) conjugated to tetramethylrhodamine isothiocyanate was from Molecular Probes (5 μg/mL).
Equal amounts of spinal cord and tibialis anterior (TA) muscle tissue extracts were separated by electrophoresis on 10% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes (Amersham; Baie d'Urfe, Quebec, Canada). The membranes were blocked in 5% non-fat milk in TBST (10 mM Tris-HCl pH 8.0, 150 mM NaCl, and 0.1% Tween 20 (Sigma; Oakville, Ontario, Canada)). Membranes were incubated overnight at 4°C with primary antibody, followed by a one-hour incubation with the secondary antibody. All washes were performed with TBST. Signals were visualized using the ECL or the ECL plus detection kit (Amersham). Exposure times were chosen based on the saturation of the highest amounts of protein.
Hematoxylin and eosin staining
Spinal cord (L1-L2 lumbar regions) and TA muscle sections (5 μm) were deparaffinized in xylene and fixed in 100% ethanol. Following a rinse in water, samples were stained in hematoxylin (Fisher; El Paso, Texas, USA) for three minutes, rinsed in water, dipped 40 times in a solution of 0.02% HCl in 70% ethanol and rinsed in water again. The sections were stained in a 1% eosin solution (BDH; Billerica, Massachusetts, USA) for one minute, dehydrated in ethanol, cleared in xylene and mounted with Permount (Fisher). Images were taken with a Zeiss Axioplan2 microscope, with a 20× objective.
Quantitative assays were performed on three mice for each genotype and five sections per mouse. Motor neurons were identified by their shape and size (> 10 μm in diameter) in the same designated area of the ventral horn region of the spinal cord. Every fifth section was analyzed and the subsequent totals were multiplied by five to give an estimate of total motor neuron number. Only motor neurons with visible nuclei were counted so as to prevent double-counting. For TA quantitative assays, the area of muscle fiber within designated regions of the TA muscle sections was measured using the Zeiss AxioVision software.
For immunohistochemistry, spinal cord sections were first deparaffinized in xylene (3 × 10 minutes), fixed in 100% ethanol (2 × 10 minutes), rehydrated in 95% and 75% ethanol (5 seconds each) and placed for 5 minutes in 1 M Tris-HCl pH 7.5. Sections were then placed in boiling sodium citrate antigen retrieval buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for 20 minutes in the microwave. The sections were rinsed for 10 minutes under running cold tap water and incubated for 2 hours at room temperature (RT) in blocking solution (TBLS (10% NaN3), 20% goat serum, 0.3% Triton X-100). This was followed by an overnight incubation at 4°C with the primary antibody. Subsequently, sections were incubated for 1 hour at RT with the biotinylated rabbit antibody followed by a 1 hour incubation at RT with streptavidin-Cy3. All washes were done with PBS. Hoechst (1:1000) was added to the last PBS wash followed by the slides being mounted in fluorescent mounting medium (Dako). Images were taken with a Zeiss confocal microscope, with a 20× objective, equipped with filters suitable for Cy3/Hoechst fluorescence.
Neuromuscular junction immunohistochemistry
Transversus abdominis (TVA) and TA muscle sections were labeled by immunohistochemistry to allow quantification of neuromuscular innervation as described previously [12, 22]. Briefly, TVA muscles were immediately dissected from recently sacrificed mice and fixed in 4% paraformaldehyde (Electron Microscopy Science; Hatfield, Pennsylvania, USA) in PBS for 15 minutes. Post-synaptic acetylcholine receptors were labeled with αBTX for 10 minutes. Muscles were then permeabilized in 2% TritonX for 30 minutes and blocked in 4% bovine serum albumin/1% TritonX in PBS for 30 minutes before incubation overnight in primary antibodies and visualized with DyLight-conjugated secondary antibodies. Whole TA muscles were dissected and fixed in 4% paraformaldehyde. Following the removal of connective tissue, the TA muscles were incubated with αBTX Alexa Fluor 555 conjugate for 20 minutes at RT. Whole TVA muscle and a thin filet of TA muscle were mounted in Dako fluorescent mounting media. Images were taken with a Zeiss confocal microscope equipped with filters suitable for fluorescein isothiocyanate (FITC)/Cy3/fluorescence. Categorization of pre- and postsynaptic morphologies was performed as previously reported .
Balance and strength were assessed using the pen test as described . Mice were placed on a suspended pen at different time-points (P12, 14, 17 and 21). The latency to fall from the pen was measured with a plateau of 30 seconds. At each time-point, individual mice were assessed three consecutive times.
All statistical analyses were performed using the GraphPad Prism software. For the Kaplan-Meier survival analysis, the log-rank test was used and survival curves were considered significantly different at P < 0.05. When appropriate, the Student's two-tail t test for paired variables and one-way ANOVA were used to test for differences between samples and data were considered significantly different at P < 0.05.
Fasudil increases lifespan of Smn2B/-mice
The Smn 2B allele harbors a sequence change in the exon splicing enhancer of exon 7 of the murine Smn gene, leading to the predominant production of the SmnΔ7 protein [18, 19]. The Smn 2B allele in combination with the knockout allele results in an intermediate SMA mouse model (Smn 2B/- ) with a median lifespan of 30 days that displays motor neuron loss, neuromuscular defects and immature NMJs . We have previously shown that the ROCK inhibitor Y-27632 dramatically improved the lifespan of Smn 2B/- mice . Since Y-27632 has not been approved for clinical use, we set out to determine if the ROCK inhibitor, fasudil, which has been approved for US clinical trials , would have similar beneficial effects on the Smn 2B/- mice.
Additional file 2: Smn 2B/- mice treated with fasudil show improved gait and movement. A movie of aging fasudil- (left mouse) and vehicle-treated (right mouse) Smn 2B/- mice (two months of age). The fasudil-treated mouse displays the agility and ability to freely walk and move around the cage while still displaying a minor neurological phenotype. The vehicle-treated Smn 2B/- mouse displays a severe neurological phenotype and reduced motor functions. (M4V 8 MB)
Fasudil activity in the spinal cord does not prevent motor neuron loss in Smn2B/-mice
To investigate if the beneficial effects of fasudil administration are mediated through an increase in Smn expression, we compared Smn protein levels in P21 spinal cords of wild type, vehicle-treated and fasudil-treated Smn 2B/- mice. This comparison shows that fasudil does not lead to a significant upregulation of Smn expression (Figure 2B) and further suggests that fasudil acts via an Smn-independent pathway to improve the survival of Smn 2B/- mice.
As a major hallmark of SMA is loss of lower motor neuron cell bodies from the spinal cord, we assessed the effect of fasudil on the motor neuron loss previously characterized in the Smn 2B/- mouse model [11, 23]. Quantification of the number of motor neuron cell bodies in the ventral horn region of L1-L2 lumbar spinal cord sections revealed similar significant reductions in both vehicle- and fasudil-treated Smn 2B/- mice compared to wild type controls (Figure 2C, D). This implies that the beneficial effects observed following fasudil administration are not mediated via a preservation of motor neuron cell bodies. It is therefore possible that fasudil acts on other SMA-afflicted tissues and/or compartments that subsequently influence the functionality of the surviving motor neurons.
Fasudil increases skeletal muscle fiber size
Fasudil-treated muscles display restored myogenin expression
Recent work from our laboratory suggests that hindlimb muscles from P21 Smn 2B/- mice display defects in muscle development, as evidenced by the misregulation of myogenin (JGB, unpublished data), a transcription factor that plays a well-characterized role in myogenesis . We thus investigated whether fasudil had any impact on myogenin levels. Analysis of TA muscles from P21 mice confirms the increased levels of myogenin in skeletal muscle of Smn 2B/- mice compared to wild type controls (Figure 4D, E). Importantly, fasudil administration leads to a significant decrease in myogenin levels in Smn 2B/- mice (Figure 4D, E). In fact, myogenin levels in fasudil-treated TA muscles are restored to wild type levels. Thus, fasudil returns the expression level of myogenin to normal, suggesting that fasudil may increase muscle size by restoring the typical development of skeletal muscle.
Fasudil does not ameliorate the pre-synaptic phenotype of NMJs from Smn2B/-mice
Fasudil increases endplate area of Smn2B/-NMJs
Increased NMJ maturation is observed in aging fasudil-treated Smn2B/-mice
Previous work has implicated the RhoA/ROCK pathway in SMA pathogenesis [10, 12, 17]. In the present study, we demonstrate that targeting the ROCK pathway with the inhibitor fasudil significantly increases the lifespan of the Smn 2B/- SMA mice. The increased survival is independent of Smn expression, weight gain, pen test performance and pre-synaptic NMJ phenotype. We find, however, that fasudil benefits post-synaptic pathology and muscle development. Importantly, the results obtained from other fasudil clinical trials are proof-of-principle of its feasibility and availability as a therapeutic approach for the treatment of SMA. Future SMA clinical endeavors should therefore consider assessing the beneficial potential of ROCK inhibitors.
Smn protein levels remained significantly low in both fasudil-treated spinal cord and muscle samples of SMA mice. These findings are important when considering therapeutic avenues for SMA. There are presently many strategies being developed to increase the expression of SMN, such as gene therapy, modulation of transcription and splicing of SMN2, and the use of various histone deacetylase (HDAC) inhibitors (reviewed in [34–36]). Although these therapeutic approaches show promising results, they remain in pre-clinical stages and may not be as efficient if administered to mid- to late-symptomatic patients . It is therefore crucial to understand the pathological molecular pathways that are affected upon SMN loss and how these can be modulated to attenuate their degenerative effects. Along with other research groups, we have shown that the RhoA/ROCK pathway is indeed perturbed in SMA cellular and animal models and that its targeting leads to a significant beneficial outcome [10, 12, 17].
We had previously identified the upregulation of RhoA-GTP in the spinal cords of Smn 2B/- mice . The misregulated RhoA/ROCK pathway in the spinal cord was, therefore, the primary target of our Fasudil therapeutic strategy . Interestingly, we have observed that fasudil does not prevent the motor neuron loss that occurs in the Smn 2B/- mice. In fact, the most apparent effects of fasudil appear to be the restoration of normal skeletal muscle growth and development, as well as increased post-synaptic EP area. A number of recent reports suggest that the SMN protein may have a muscle-intrinsic role that influences SMA pathology ([28–30] and JGB, unpublished data). Active RhoA has previously been shown to positively regulate the expression of myogenin [38, 39]. Furthermore, work performed in avian and murine myoblasts shows that inhibition of ROCK promotes exit from the cell cycle and subsequent terminal differentiation . Indeed, myoblasts treated with the ROCK inhibitor Y-27632 display increased differentiation, cell fusion and myotube formation . Fasudil's inhibition of the RhoA/ROCK pathway most likely restores the normal skeletal muscle developmental program of Smn 2B/- mice via modulation of myoblast differentiation and fusion, as well as myogenin expression. The fasudil-dependent increase in myofiber size could lead to the subsequent increase in EP size. Indeed, a positive correlation has previously been established between myofiber size and motor EP size . Furthermore, various reports suggest that post-synaptic differentiation and formation is initially muscle-dependent and motor axon-independent [42, 43]. Our study, therefore, highlights two important points. Firstly, therapeutic strategies that improve skeletal muscle and EP growth should be considered when developing therapies for SMA. Secondly, ROCK inhibition may have positive outcomes in other pre-clinical disease models characterized by muscle atrophy and NMJ pathology.
Intriguingly, the dramatic increase in skeletal muscle myofiber size of fasudil-treated Smn 2B/- mice is not accompanied by changes in weight or strength, when compared to vehicle-treated Smn 2B/- mice. Previous studies have reported this phenomenon, providing a variety of potential explanations. In cases of sarcoplasmic hypertrophy, the non-contractile myofiber components expand while muscular strength remains unchanged . Further, the characterization of a post-natal myogenin knockout mouse model revealed normal skeletal muscle size albeit with a 30% weight loss compared to control littermates . The authors suggest that this phenotype is caused by a slower growth rate and perturbed energy homeostasis . Finally, Rehfeldt et al. showed that mice homozygous for the Compact myostatin mutation (C/C) display muscular hyperplasia and increased muscle weight but with a reduction in overall body weight . The authors also identify a reduction in the number of capillaries per muscle in the C/C mice, subsequently impacting oxidative metabolism . Interestingly, recent work in the severe SMA mouse model demonstrated a significant decrease in the capillary bed density within skeletal muscle . Thus, the findings mentioned above highlight the fact that an increase in muscle size and or weight does not necessarily positively correlate with an increase in body weight. Regardless, the restoration of myofiber growth and skeletal muscle development by fasudil, in the absence of weight gain, appears to be sufficient to provide therapeutic benefits to the Smn 2B/- mice.
In recent years, it has been postulated that SMA may be a die-back neuropathy, where the motor axons initially reach the EP but subsequently retract as disease progresses [48–50]. This hypothesis suggests that synapses are selectively vulnerable in SMA, with synapse loss preceding cell body degeneration. In addition, it has been suggested that neurons undergo compartmental degeneration, where the soma, axons and synapses of neurons possess specific and compartmentalized mechanisms of degeneration [51–53]. It therefore follows that therapeutics which target distal compartments of the cell, such as the synapse or axon, can be protective to the cell body. In our study, we show that while fasudil administration has little impact upon the initial loss of motor neurons, it dramatically increases myofiber and EP size in SMA mice. We therefore suggest that this improvement in post-synaptic parameters stabilizes the synaptic connections and subsequently protects the remaining motor neurons. Consistent with this observation, the surviving synapses constitute NMJs that will eventually develop and mature normally. Given the tight correlation between EP maturation and neuromuscular activity (reviewed in ), fasudil may indirectly improve NMJ transmission, subsequently ameliorating motor EP maturation. Alternatively, considering the crucial role of the actin cytoskeleton in the redistribution of acetylcholine receptors (AChRs) during post-synaptic remodeling [55, 56], fasudil's modulation of actin dynamics could directly restore normal AChR clustering. Clearly, the understanding and identification of fasudil's influence on NMJ maturation in SMA mice requires further investigation. Nevertheless, our work highlights the applicability of the compartmental degeneration hypothesis to SMA pathogenesis and the potential of therapies aimed at preventing synaptic degeneration.
ROCK has evolved as an important therapeutic target in various models of cardiovascular disease, spinal cord injury and glaucoma (reviewed in [57–59]). Furthermore, the ROCK inhibitor fasudil, which has been approved in US clinical trials, has shown beneficial effects in patients with vasospastic angina , stable effort angina , general heart failure  and pulmonary hypertension . It has now become evident that the pathogenic misregulation of the RhoA/ROCK pathway in various Smn-depleted cellular and animal models can also be modulated by the ROCK inhibitors Y-27632 and fasudil, leading to significant positive outcomes [10, 12, 17].
The administration of fasudil to SMA mice significantly increases their lifespan without an obvious increase in Smn expression or preservation of spinal cord motor neurons. In fact, fasudil improves post-synaptic and skeletal muscle development. Our work underscores the importance of muscle as a therapeutic target in SMA and highlights the beneficial potential of ROCK inhibitors as a therapeutic strategy for SMA and for other degenerative diseases characterized by muscular atrophy and postsynaptic immaturity.
central nervous system
Food and Drug Administration
spinal muscular atrophy
survival motor neuron
synaptic vesicle 2
We are grateful to the Kothary laboratory for helpful discussions. This project was funded by a grant from the Canadian Institutes of Health Research (CIHR), and The Muscular Dystrophy Association (USA) to RK. MB and JGB are recipients of a Frederick Banting and Charles Best CIHR Doctoral Research Award, LMM is a recipient of a Multiple Sclerosis Society of Canada Postdoctoral Fellowship, and RK is a recipient of a University Health Research Chair from the University of Ottawa.
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