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Salbutamol inhibits ubiquitin-mediated survival motor neuron protein degradation in spinal muscular atrophy cells.

ABSTRACT: Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder that is currently incurable. SMA is caused by decreased levels of the survival motor neuron protein (SMN), as a result of loss or mutation of SMN1. Although the SMN1 homolog SMN2 also produces some SMN protein, it does not fully compensate for the loss or dysfunction of SMN1. Salbutamol, a β2-adrenergic receptor agonist and well-known bronchodilator used in asthma patients, has recently been shown to ameliorate symptoms in SMA patients. However, the precise mechanism of salbutamol action is unclear. We treated SMA fibroblast cells lacking SMN1 and HeLa cells with salbutamol and analyzed SMN2 mRNA and SMN protein levels in SMA fibroblasts, and changes in SMN protein ubiquitination in HeLa cells. Salbutamol increased SMN protein levels in a dose-dependent manner in SMA fibroblast cells lacking SMN1, though no significant changes in SMN2 mRNA levels were observed. Notably, the salbutamol-induced increase in SMN was blocked by a protein kinase A (PKA) inhibitor and deubiquitinase inhibitor, respectively. Co-immunoprecipitation assay using HeLa cells showed that ubiquitinated SMN levels decreased in the presence of salbutamol, suggesting that salbutamol inhibited ubiquitination. The results of this study suggest that salbutamol may increase SMN protein levels in SMA by inhibiting ubiquitin-mediated SMN degradation via activating β2-adrenergic receptor-PKA pathways.

SUBMITTER: Harahap NIF

PROVIDER: S-EPMC5669398 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Salbutamol inhibits ubiquitin-mediated survival motor neuron protein degradation in spinal muscular atrophy cells.

Harahap Nur Imma Fatimah NIF Nurputra Dian Kesumapramudya DK Ar Rochmah Mawaddah M Shima Ai A Morisada Naoya N Takarada Toru T Takeuchi Atsuko A Tohyama Yumi Y Yanagisawa Shinichiro S Nishio Hisahide H

Biochemistry and biophysics reports 20151028

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder that is currently incurable. SMA is caused by decreased levels of the survival motor neuron protein (SMN), as a result of loss or mutation of SMN1. Although the SMN1 homolog SMN2 also produces some SMN protein, it does not fully compensate for the loss or dysfunction of SMN1. Salbutamol, a β2-adrenergic receptor agonist and well-known bronchodilator used in asthma patients, has recent ...[more]

PMID: 29124224

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Project description:BackgroundZolgensma 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).MethodsA 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.ResultsAAV9-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.ConclusionsSMNK186R 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.

Project description:Spinal muscular atrophy (SMA) is a progressive neuromuscular disorder caused by a loss of the survival of motor neuron 1 (SMN1) gene, resulting in a loss of spinal motor neurons (MNs), leading to muscle weakness and wasting. The pathogenesis of MN loss in SMA and the selective vulnerability in different cellular populations are not fully understood. To investigate the role of spinal astrocytes in the pathogenesis of late-onset SMA, we used a mouse model in addition to in vitro approaches. Immunostaining, Western blot analysis, small interfering ribonucleic acid (siRNA) transfections, functional assays, enzyme-linked immunosorbent assay (ELISA), behavioral tests, and electrophysiological measurements were performed. Early activation of spinal astrocytes and a reduction of the excitatory amino acid transporter 1 (EAAT1) on postnatal day (P) 20 preceded the loss of spinal MNs in SMA mice occurring on P42. EAAT1 reduction resulted in elevated glutamate levels in the spinal cord of SMA mice at P20 and P42. SMA-like astrocytes generated by siRNA and an ex vivo model of glutamate excitotoxicity involving organotypic spinal cord slice cultures revealed the critical role of glutamate homeostasis in the degeneration of MNs. The pre-emptive administration of arundic acid (AA), as an inhibitor of astrocyte activation, to SMA mice prior to the loss of motor neurons (P28) resulted in elevated EAAT1 protein levels compared to vehicle-treated SMA mice and prevented the increase of glutamate in the spinal cord and the loss of spinal MNs. Furthermore, AA preserved motor functions during behavioral experiments, the electrophysiological properties, and muscle alteration of SMA mice. In a translational approach, we transfected healthy human fibroblasts with SMN1 siRNA, resulting in reduced EAAT1 expression and reduced uptake but increased glutamate release. These findings were verified by detecting elevated glutamate levels and reduced levels of EAAT1 in cerebrospinal fluid of untreated SMA type 2 and 3 patients. In addition, glutamate was elevated in serum samples, while EAAT1 was not detectable. Our data give evidence for the crucial role of spinal astrocytes in the pathogenesis of late-onset SMA, a potential driving force for MN loss by glutamate excitotoxicity caused by EAAT1 reduction as an early pathophysiological event. Furthermore, our study introduces EAAT1 as a potential therapeutic target for additional SMN-independent therapy strategies to complement SMN-enhancing drugs.

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Salbutamol inhibits ubiquitin-mediated survival motor neuron protein degradation in spinal muscular atrophy cells.

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Salbutamol inhibits ubiquitin-mediated survival motor neuron protein degradation in spinal muscular atrophy cells.

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