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
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
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
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reports suggest that the SMN protein may have a muscle-intrinsic role that influences SMA pathology ([28–30] and JGB, unpublished data).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
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
mice is not accompanied by changes in weight or strength, when compared to vehicle-treated Smn
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
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].