Improved therapeutic approach for spinal muscular atrophy via ubiquitination-resistant survival motor neuron variant.

BACKGROUND: Zolgensma is a gene-replacement therapy that has led to a promising treatment for spinal muscular atrophy (SMA). However, clinical trials of Zolgensma have raised two major concerns: insufficient therapeutic effects and adverse events. In a recent clinical trial, 30% of patients failed to achieve motor milestones despite pre-symptomatic treatment. In addition, more than 20% of patients showed hepatotoxicity due to excessive virus dosage, even after the administration of an immunosuppressant. Here, we aimed to test whether a ubiquitination-resistant variant of survival motor neuron (SMN), SMNK186R, has improved therapeutic effects for SMA compared with wild-type SMN (SMNWT). METHODS: A severe SMA mouse model, SMA type 1.5 (Smn-/-; SMN2+/+; SMN∆7+/-) mice, was used to compare the differences in therapeutic efficacy between AAV9-SMNWT and AAV9-SMNK186R. All animals were injected within Postnatal Day (P) 1 through a facial vein or cerebral ventricle. RESULTS: AAV9-SMNK186R-treated mice showed increased lifespan, body weight, motor neuron number, muscle weight and functional improvement in motor functions as compared with AAV9-SMNWT-treated mice. Lifespan increased by more than 10-fold in AAV9-SMNK186R-treated mice (144.8 ± 26.11 days) as compared with AAV9-SMNWT-treated mice (26.8 ± 1.41 days). AAV9-SMNK186R-treated mice showed an ascending weight pattern, unlike AAV9-SMNWT-treated mice, which only gained weight until P20 up to 5 g on average. Several motor function tests showed the improved therapeutic efficacy of SMNK186R. In the negative geotaxis test, AAV9-SMNK186R-treated mice turned their bodies in an upward direction successfully, unlike AAV9-SMNWT-treated mice, which failed to turn upwards from around P23. Hind limb clasping phenotype was rarely observed in AAV9-SMNK186R-treated mice, unlike AAV9-SMNWT-treated mice that showed clasping phenotype for more than 20 out of 30 s. At this point, the number of motor neurons (1.5-fold) and the size of myofibers (2.1-fold) were significantly increased in AAV9-SMNK186R-treated mice compared with AAV9-SMNWT-treated mice without prominent neurotoxicity. AAV9-SMNK186R had fewer liver defects compared with AAV9-SMNWT, as judged by increased proliferation of hepatocytes (P < 0.0001) and insulin-like growth factor-1 production (P < 0.0001). Especially, low-dose AAV9-SMNK186R (nine-fold) also reduced clasping time compared with SMNWT. CONCLUSIONS: SMNK186R will provide improved therapeutic efficacy in patients with severe SMA with insufficient therapeutic efficacy. Low-dose treatment of SMA patients with AAV9-SMNK186R can reduce the adverse events of Zolgensma. Collectively, SMNK186R has value as a new treatment for SMA that improves treatment effectiveness and reduces adverse events simultaneously.

Baf155 regulates skeletal muscle metabolism via HIF-1a signaling.

During exercise, skeletal muscle is exposed to a low oxygen condition, hypoxia. Under hypoxia, the transcription factor hypoxia-inducible factor-1α (HIF-1α) is stabilized and induces expressions of its target genes regulating glycolytic metabolism. Here, using a skeletal muscle-specific gene ablation mouse model, we show that Brg1/Brm-associated factor 155 (Baf155), a core subunit of the switch/sucrose non-fermentable (SWI/SNF) complex, is essential for HIF-1α signaling in skeletal muscle. Muscle-specific ablation of Baf155 increases oxidative metabolism by reducing HIF-1α function, which accompanies the decreased lactate production during exercise. Furthermore, the augmented oxidation leads to high intramuscular adenosine triphosphate (ATP) level and results in the enhancement of endurance exercise capacity. Mechanistically, our chromatin immunoprecipitation (ChIP) analysis reveals that Baf155 modulates DNA-binding activity of HIF-1α to the promoters of its target genes. In addition, for this regulatory function, Baf155 requires a phospho-signal transducer and activator of transcription 3 (pSTAT3), which forms a coactivator complex with HIF-1α, to activate HIF-1α signaling. Our findings reveal the crucial role of Baf155 in energy metabolism of skeletal muscle and the interaction between Baf155 and hypoxia signaling.

Bap1/SMN axis in Dpp4+ skeletal muscle mesenchymal cells regulates the neuromuscular system.

The survival of motor neuron (SMN) protein is a major component of the pre-mRNA splicing machinery and is required for RNA metabolism. Although SMN has been considered a fundamental gene for the central nervous system, due to its relationship with neuromuscular diseases, such as spinal muscular atrophy, recent studies have also revealed the requirement of SMN in non-neuronal cells in the peripheral regions. Here, we report that the fibro-adipogenic progenitor subpopulation expressing Dpp4 (Dpp4+ FAPs) is required for the neuromuscular system. Furthermore, we also reveal that BRCA1-associated protein-1 (Bap1) is crucial for the stabilization of SMN in FAPs by preventing its ubiquitination-dependent degradation. Inactivation of Bap1 in FAPs decreased SMN levels and accompanied degeneration of the neuromuscular junction, leading to loss of motor neurons and muscle atrophy. Overexpression of the ubiquitination-resistant SMN variant, SMNK186R, in Bap1-null FAPs completely prevented neuromuscular degeneration. In addition, transplantation of Dpp4+ FAPs, but not Dpp4- FAPs, completely rescued neuromuscular defects. Our data reveal the crucial role of Bap1-mediated SMN stabilization in Dpp4+ FAPs for the neuromuscular system and provide the possibility of cell-based therapeutics to treat neuromuscular diseases.

Maintenance of type 2 glycolytic myofibers with age by Mib1-Actn3 axis.

Age-associated muscle atrophy is a debilitating condition associated with loss of muscle mass and function with age that contributes to limitation of mobility and locomotion. However, the underlying mechanisms of how intrinsic muscle changes with age are largely unknown. Here we report that, with age, Mind bomb-1 (Mib1) plays important role in skeletal muscle maintenance via proteasomal degradation-dependent regulation of α-actinin 3 (Actn3). The disruption of Mib1 in myofibers (Mib1ΔMF) results in alteration of type 2 glycolytic myofibers, muscle atrophy, impaired muscle function, and Actn3 accumulation. After chronic exercise, Mib1ΔMF mice show muscle atrophy even at young age. However, when Actn3 level is downregulated, chronic exercise-induced muscle atrophy is ameliorated. Importantly, the Mib1 and Actn3 levels show clinical relevance in human skeletal muscles accompanied by decrease in skeletal muscle function with age. Together, these findings reveal the significance of the Mib1-Actn3 axis in skeletal muscle maintenance with age and suggest the therapeutic potential for the treatment or amelioration of age-related muscle atrophy.

The hypothalamic-pituitary-gonadal axis controls muscle stem cell senescence through autophagosome clearance.

BACKGROUND: With organismal aging, the hypothalamic-pituitary-gonadal (HPG) activity gradually decreases, resulting in the systemic functional declines of the target tissues including skeletal muscles. Although the HPG axis plays an important role in health span, how the HPG axis systemically prevents functional aging is largely unknown. METHODS: We generated muscle stem cell (MuSC)-specific androgen receptor (Ar) and oestrogen receptor 2 (Esr2) double knockout (dKO) mice and pharmacologically inhibited (Antide) the HPG axis to mimic decreased serum levels of sex steroid hormones in aged mice. After short-term and long-term sex hormone signalling ablation, the MuSCs were functionally analysed, and their aging phenotypes were compared with those of geriatric mice (30-month-old). To investigate pathways associated with sex hormone signalling disruption, RNA sequencing and bioinformatic analyses were performed. RESULTS: Disrupting the HPG axis results in impaired muscle regeneration [wild-type (WT) vs. dKO, P < 0.0001; Veh vs. Antide, P = 0.004]. The expression of DNA damage marker (in WT = 7.0 ± 1.6%, dKO = 32.5 ± 2.6%, P < 0.01; in Veh = 13.4 ± 4.5%, Antide = 29.7 ± 5.5%, P = 0.028) and senescence-associated ß-galactosidase activity (in WT = 3.8 ± 1.2%, dKO = 10.3 ± 1.6%, P < 0.01; in Veh = 2.1 ± 0.4%, Antide = 9.6 ± 0.8%, P = 0.005), as well as the expression levels of senescence-associated genes, p16Ink4a and p21Cip1 , was significantly increased in the MuSCs, indicating that genetic and pharmacological inhibition of the HPG axis recapitulates the progressive aging process of MuSCs. Mechanistically, the ablation of sex hormone signalling reduced the expression of transcription factor EB (Tfeb) and Tfeb target gene in MuSCs, suggesting that sex hormones directly induce the expression of Tfeb, a master regulator of the autophagy-lysosome pathway, and consequently autophagosome clearance. Transduction of the Tfeb in naturally aged MuSCs increased muscle mass [control geriatric MuSC transplanted tibialis anterior (TA) muscle = 34.3 ± 2.9 mg, Tfeb-transducing geriatric MuSC transplanted TA muscle = 44.7 ± 6.7 mg, P = 0.015] and regenerating myofibre size [eMyHC+ tdTomato+ myofibre cross-section area (CSA) in control vs. Tfeb, P = 0.002] after muscle injury. CONCLUSIONS: Our data show that the HPG axis systemically controls autophagosome clearance in MuSCs through Tfeb and prevents MuSCs from senescence, suggesting that sustained HPG activity throughout life regulates autophagosome clearance to maintain the quiescence of MuSCs by preventing senescence until advanced age.