Small-molecule flunarizine increases SMN protein in nuclear Cajal bodies and motor function in a mouse model of spinal muscular atrophy

Abstract

The hereditary neurodegenerative disorder spinal muscular atrophy (SMA) is characterized by the loss of spinal cord motor neurons and skeletal muscle atrophy. SMA is caused by mutations of the survival motor neuron (SMN) gene leading to a decrease in SMN protein levels. The SMN deficiency alters nuclear body formation and whether it can contribute to the disease remains unclear. Here we screen a series of small-molecules on SMA patient fibroblasts and identify flunarizine that accumulates SMN into Cajal bodies, the nuclear bodies important for the spliceosomal small nuclear RNA (snRNA)-ribonucleoprotein biogenesis. Using histochemistry, real-time RT-PCR and behavioural analyses in a mouse model of SMA, we show that along with the accumulation of SMN into Cajal bodies of spinal cord motor neurons, flunarizine treatment modulates the relative abundance of specific spliceosomal snRNAs in a tissue-dependent manner and can improve the synaptic connections and survival of spinal cord motor neurons. The treatment also protects skeletal muscles from cell death and atrophy, raises the neuromuscular junction maturation and prolongs life span by as much as 40 percent (p < 0.001). Our findings provide a functional link between flunarizine and SMA pathology, highlighting the potential benefits of flunarizine in a novel therapeutic perspective against neurodegenerative diseases.

The Small-Molecule Flunarizine in Spinal Muscular Atrophy Patient Fibroblasts Impacts on the Gemin Components of the SMN Complex and TDP43, an RNA-Binding Protein Relevant to Motor Neuron Diseases

“…The immortalized Type I SMA fibroblast cell line was grown in Dulbecco's modified Eagle's medium (DMEM)-Glutamax supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 mg/ml) at 37 • C with 5% CO2 (Lefebvre et al, 2002). The flunarizine treatment was carried out as previously described (Sapaly et al, 2018). Briefly, cells are grown at a density of 10 000 cells/cm 2 and treated with flunarizine or control DMSO (0.1%) for either 4 or 16 h.…”

“…The normalized expression levels were calculated according to the Ct method. The snRNA, 5 S, 5.8 S, RPL13a and SDHA primers have been reported (Sapaly et al, 2018). The RNA sequencing approach was carried out at the Genom'ic Core Facility at the Institut Cochin, University of Paris Descartes.…”

“…Interestingly, Gemin5 regulates internal ribosome entry sites in the 5 untranslated region of TXNIP mRNA (Lampe et al, 2018). Given that flunarizine affects in vivo the relative snRNA levels in brain and spinal cord of SMA mice (Sapaly et al, 2018), we hypothesized that even small changes in protein levels of the SMN complex components can lead to larger effects on their target RNAs. Therefore, we evaluated by RT-qPCR the levels of the spliceosomal snRNAs in total RNA preparations.…”

“…Reduced SMN protein levels alter the snRNP repertoire in SMA patient cells and mouse models. Indeed, a reduction of the minor snRNAs (U11, U12, U4atac, and U6atac) is detected (Wan et al, 2005;Gabanella et al, 2007;Zhang et al, 2008;Lotti et al, 2012;Sapaly et al, 2018). Although widespread splicing alterations have been observed in tissues of SMA mice, the correction of the expression of Stasimon, encoded by a gene possessing a minor-intron, markedly improves motor circuit function in disease models (Imlach et al, 2012;Lotti et al, 2012).…”

“…To address this at the cellular level, we previously used SMA patient fibroblast cells to screen small chemical libraries for hits that could replenish nuclear Cajal bodies with SMN protein (Lefebvre et al, 2014). Flunarizine is one of the hits selected for in vivo studies (Sapaly et al, 2018). Interestingly, no increases of SMN protein levels were found in flunarizinetreated patient cells.…”