Intraperitoneal administration of the proteasome inhibitor, bortezomib, resulted in a 2-fold increase in SMN protein levels in peripheral tissues of SMA model mice. Affected mice treated with bortezomib alone showed improved righting times but no significant change in lifespan. The lack of a survival benefit in the absence of CNS availability of bortezomib highlights the critical role of SMN in the CNS. Nevertheless, we observed increased myofiber size and number in bortezomib-treated mice and, surprisingly, an increased number of AHCs in the spinal cord. Immunohistochemical examination of the NMJ of bortezomib-treated mice showed increased NMJ size compared with vehicle-treated mice. This improvement in NMJ size could explain the increase in AHC number and the improvement in motor function we observed in treated animals despite the lack of increased SMN levels in the CNS with bortezomib treatment and suggests that treatments that target peripheral tissues may contribute to improving the disease phenotype.
It remains unclear whether SMA is cell autonomous, i.e. caused by the effects of reduced SMN in multiple tissues or solely in motor neurons. In Drosophila
, a null mutation in Smn is partially rescued by maternal SMN expression that allows development to the larval stage (11
). The eventual depletion of SMN in all tissues results in death and can only be rescued by providing SMN to both muscles and neurons. Depletion of SMN in muscle in flies and mice results in muscle degeneration, indicating that SMN is an essential protein in muscle, as in other tissues (12
). Recent work with transgenic mice expressing SMN in muscle under the control of the human skeletal muscle actin promoter showed that SMN restoration in skeletal muscle alone has no appreciable impact on the SMA phenotype (13
). However, this does not rule out the possibility that increasing SMN levels in muscle may enhance whatever beneficial effect repletion of motor neuron SMN may have. Two studies examined inhibitors of the myostatin pathway in SMA mice with contrasting results, one showing a modest extension in lifespan and gross motor function, with delivery of recombinant follistatin, the other detecting no phenotypic improvement in the SMA mice with ActRIIB-Fc treatment or transgenic overexpression of follistatin (14
). The basis for this discrepancy is unclear; however, the possibility remains that motor neurons may require additional support in peripheral tissues to respond optimally to SMN-based therapeutics. In this study, we found that a 2-fold increase in SMN in peripheral tissues had no effect on the survival of SMA mice, but a similar increase in SMN in animals treated with the CNS penetrant drug, TSA, was sufficient to significantly improve survival. Taken together, these studies would indicate the need for adequate levels of SMN in the CNS for survival.
While this study was ongoing, it was reported that the hydroxamic acid-derived HDAC inhibitor LBH589 markedly increases SMN levels in SMA patient-derived fibroblasts by increasing SMN2
gene expression and blocking SMN protein degradation (16
). We show here that TSA and bortezomib delivered together synergistically increased SMN protein levels in cultured cells and tissues of SMA mice. Mice treated with both drugs lived longer and showed increased body weight. Immunohistochemical analysis of NMJs in mice treated with both drugs showed a cumulative effect on their maturation. Although TSA alone increases SMN protein levels, mitigates muscle and nerve pathophysiology and extends the lifespan of SMA model mice (9
), co-administering TSA with bortezomib approximately doubled SMN levels in affected mice compared with TSA alone and further extended survival from 3 to 6 days.
Of particular interest in this study was the finding that bortezomib alone significantly improved the motor function of SMA mice. We observed an increase in SMN protein levels in muscle tissue of treated mice and increases in the number of myofibers and in myofiber diameters. AHC loss was also delayed in bortezomib-treated animals, indicating a central effect of this peripherally acting drug. These SMA mice show motor deficits as early as postnatal day 2, although they do not exhibit significant spinal motor neuron loss at this stage and there is evidence to suggest that defects at the NMJ precede motor neuron loss (17
). We found that the NMJs of bortezomib-treated mice were larger than those of vehicle-treated animals. We presume that increased SMN in muscle can positively influence the maturation of the NMJ and survival of motor neurons. It is also possible that inhibition of the proteasome may contribute to muscle improvement by another mechanism independent of SMN. Muscle is a source of neurotrophic factors that are protective of motor neurons (18
). Muscle-derived NT-4 is an activity-dependent neurotrophic signal for growth and remodeling of adult motor nerve terminals, and muscle-derived neurotrophic factors are retrogradely transported by motor neurons with effects at the level of the cell body (19
). Recently, muscle-specific insulin-like growth factor 1 expression was shown to reduce spinal cord and muscle pathology in spinal and bulbar muscle atrophy demonstrating that muscle cell signaling can have an effect on motor neuron survival (20
). These data indicate that drugs acting peripherally may ameliorate SMA.
The UPS is involved in key events in neuronal development such as neuronal migration and synaptogenesis (21
), operating at both the pre-synaptic and at the post-synaptic level. Alteration of the ubiquitination of specific substrates due to mutations of ubiquitin conjugating enzymes and ligases may be associated with neurological disease (24
). Recently, X-linked infantile SMA has been linked to changes in the E1 ubiquitin-activating enzyme (27
), suggesting that altered ubiquitination in motor neurons could cause defective development of the motor unit. Maturation of the NMJ involves the pruning of axons from muscle fibers that are innervated by multiple motor neurons, the formation of a highly branched motor axon terminal and the coordinated expansion of the nerve terminal and muscle fiber (28
). These events occur during the first 2 weeks of postnatal development in mice with effects on both neuronal development and the growth and maturation of muscle fibers. Interestingly, the proteasome-associated deubiquitinating enzyme Usp14 has been reported to be crucial for the postnatal maturation of the peripheral nervous system by regulating ubiquitin pools at the nerve terminal (29
). These and other findings are consistent with a critical role for ubiquitin homeostasis and proteasome activity at the NMJ and could, along with increased SMN levels in peripheral tissues, explain the improvement we observed in the motor unit of SMA mice treated with bortezomib.
Proteasome inhibitors have potential as treatment for a variety of diseases, including immunologic, inflammatory, metabolic, and neurological disorders, viral diseases, muscular dystrophies and tuberculosis (30
). Dystrophin and other proteins of the dystrophin glycoprotein complex are degraded by the UPS in the muscle of Duchenne muscular dystrophy patients. Treatment with the proteasome inhibitor MG–132 restores the presence and cellular localization of dystrophin and associated proteins in mdx mice and in skeletal muscle explants from Duchenne muscular dystrophy patients (32
). Similar results were obtained with bortezomib, a more specific proteasome inhibitor (34
). Bortezomib has been approved for clinical use in multiple myeloma and is currently being tested in phase II clinical trials as a possible therapeutic agent for other malignancies (35
). Two phase I studies involving bortezomib were conducted in children affected by refractory leukemia and solid tumors (39
). Considerable efforts have been made over the past few years to identify and optimize structurally different proteasome inhibitors using medicinal chemistry or isolating natural compounds with the long-term goal of increasing the beneficial effects and reducing side-effects and toxicity.
Our results provide preclinical support for the UPS as a potential therapeutic target for SMA therapy. Strategies to inhibit SMN degradation can be used in combination with stimulation of SMN gene expression and may enable the use of lower doses of the latter, possibly increasing efficacy and reducing toxicity. Further validation of the role of the UPS in SMA would provide the opportunity to rationally design drugs that target this pathway. Studies elucidating the E3 ligases for SMN and the regulatory events upstream of the proteasome would help to further define the pathway and allow the identification of more specific targets for therapeutic intervention.