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. 2018 Feb 1;8(1):2075.
doi: 10.1038/s41598-018-20219-1.

Small-molecule Flunarizine Increases SMN Protein in Nuclear Cajal Bodies and Motor Function in a Mouse Model of Spinal Muscular Atrophy

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Free PMC article

Small-molecule Flunarizine Increases SMN Protein in Nuclear Cajal Bodies and Motor Function in a Mouse Model of Spinal Muscular Atrophy

Delphine Sapaly et al. Sci Rep. .
Free PMC article

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.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Microscopy cell-based assay to screen for SMN protein in nuclear-body Cajal bodies (CBs) of SMA patient-derived fibroblasts. (A) Immunofluorescent staining of SMN and CB-marker coilin protein in flunarizine treated (2 μg/ml) and untreated (DMSO) immortalized Type I SMA fibroblasts using rabbit polyclonal anti-SMN and mouse monoclonal anti-coilin antibodies. (B) Analysis of the immunolocalization of SMN and snRNPs (TMG-capped snRNAs) into CBs of immortalized Type I SMA fibroblasts for seven molecules. (C) Total protein extracts from immortalized type I SMA fibroblasts treated with 2 μg/ml of each molecule were separated by SDS-PAGE and SMN protein was detected by western blotting using tubulin as loading control. Odd and even numbers correspond to 5 and 15 μg of proteins, respectively. (D) The ability of the molecule to recruit SMN to CBs in immortalized SMA fibroblasts was tested in the presence of decreasing concentrations of each molecule, the 1:1 dilution being 2 μg/ml. (E) Analysis of the immunostaining of SMN in CBs of drug-treated primary fibroblast cultures from patients affected with the three forms of SMA disease. See Supplemental Table 3 for statistical analyses. (F) HeLa cancer cells were treated with 4 (2 μg/ml) and 20 μM flunarizine for 4 h and compared to 1 or 5 μl/ml DMSO. An increase in the proportion of cells with 3 or 4 CBs per nucleus is observed with both concentrations of flunarizine. Data represent the mean values ± SEM. Chi2 test, ***p < 0.001. Scale bar, 10 μm.
Figure 2
Figure 2
Treatment with flunarizine improves the phenotype of SMA mice. (A) Kaplan-Meier survival curves for vehicle (V)- (n = 19) and flunarizine (Flz)-treated SMA mice (n = 12); (B) Body weight of vehicle- (n = 67) and flunarizine-treated controls (n = 68) and vehicle- (n = 19) and flunarizine-treated SMA mice (n = 12); (C) antigravity hanging performance of vehicle- (n = 35) and flunarizine-treated controls (n = 40) and vehicle- (n = 18) and flunarizine-treated SMA mice (n = 10); (D) Number of crossings during 5 min for vehicle- (n = 35) and flunarizine-treated controls (n = 40) and vehicle- (n = 19) and flunarizine-treated SMA mice (n = 10). The drug is administrated at a daily dose of 0.5 mg/kg. Mean ± SEM.
Figure 3
Figure 3
Treatment with flunarizine accumulates SMN into nuclear-body Cajal bodies in spinal cord motor neurons in SMA mice. Immunofluorescent staining was to detect SMN and Cajal bodies in lumbar spinal cord motor neurons of 11 day-old mice using rabbit monoclonal anti-SMN and mouse monoclonal anti-coilin antibodies. The confocal microscope was focused on coilin-positive Cajal bodies. The laser was adjusted to obtain similar labeling intensities. Scale bar, 10 μm.
Figure 4
Figure 4
Effects of flunarizine on the expression and splicing profiles of candidate genes in SMA mice. (A) The snRNP-specific reduction of snRNAs in brain and spinal cord of SMA mice is modulated by flunarizine. The snRNA levels are determined by RT-qPCR and the relative amount is presented as percent of the vehicle-treated controls. 5 S and 5.8 S were used as controls for normalization as described. (B) RT-qPCR analysis of different genes shows that flunarizine reduces the retention of SMN2 intron3 in brain of SMA mice. Four genes were used as controls for normalization in brain (RPL13A, PPIA, HPRT1, SDHA) and spinal cord (RPL13A, PPIA, HPRT1, ACTB). (CF) The expression ratio between exons or isoforms of SMN2, TTYH3, AGRN and SNAP25 genes. SMN2 and TTYH3 were analysed by RT-qPCR whereas AGRN and SNAP25 were by RT-PCR experiments. No significant differences of SMN2 exon 7/ex 4 ratios were found between flunarizine- and vehicle -treated SMA mice in panel 4 C (p = 0.007). Total RNA was prepared from tissues of flunarizine (Flz)- and vehicle (V)-treated controls and SMA mice at P11. Three mice per group. Data represent the mean values ± SD (errors bars). Statistical test used is the two-way Anova followed by the Tukey’s multiple comparisons test (GraphPad).
Figure 5
Figure 5
Treatment with flunarizine elicits protective effects on spinal cord motor neurons of SMA mice. (A) Co-labeling of the glutamatergic synapse marker vGlut1 (green, arrowheads) and the motor neuron marker ChaT (red) to assess the central excitatory glutamatergic synapses on spinal cord motor neurons of controls and SMA mice in response to flunarizine. Scale bar, 10 μm. (B) Analysis of vGlut1 appositions per motor neuron of flunarizine (Flz)- and vehicle (V)-treated control mice. Each motor neuron is represented with a dot and the number of vGlut1 boutons is plotted as a function of the somata size. There is no difference between the two control groups. (C) Distribution of the spinal cord motor neurons of flunarizine- (bleue dots) and vehicle-treated SMA mice (red dots) compared to all controls from panel B (black dots). The treatment increases both the frequency of motor neurons with 24 to 30 vGlut1 boutons and the number of larger motor neurons (≥1000 μm) in SMA mice.
Figure 6
Figure 6
Differential changes in slow and fast muscle fibers of SMA mice treated with flunarizine. Type I and type IIa muscle fibers compose the slow and fast fatigable motor units, respectively. Myofiber composition is determined by the presence of specific myosin heavy chain (MyHC) isoforms: type I being coded by the MYH7 gene and type IIa by MYH2 gene. The fiber types are revealed by immunofluorence experiments using specific anti-MyHC antibodies. The graphs show the number of immunolabeled type I or type IIa fibers (y axis) versus unlabeled fibers or body weight (x axis) for the soleus, plantaris and tibialis of 10-day-old mice for n ≥ 5 mice per experimental group excepted for the tibialis from control mice (n = 3). Each mouse is presented with a symbol. A fiber type shift takes place in SMA mice treated with flunarizine (Flz), as detected by a shift of type IIa positive fibers in the soleus and plantaris of SMA mice. See also the Supplemental Fig. 1 for frequency histograms of type I, II, IIa, neonatal and embryonic myofibers.
Figure 7
Figure 7
Muscle morphology reveals muscle-specific responses to treatment with flunarizine in SMA mice. (A) Hematoxylin and eosin staining of the soleus from 11-day-old vehice-treated control mouse compared to vehicle- and flunarizine-treated SMA mice. Scale bar, 50 μm. (B) Analyses of the fiber size in the soleus, plantaris and tibialis of flunarizine- and vehicle-treated control and SMA mice. Data represent fiber area ± SEM (errors bars) of three (5-day-old, in grey) to ≥5 mice (11-day-old, in black) per experimental group. The flunarizine treatment corrects the atrophy in the plantaris and reduces it in the soleus of SMA mice. The statistical analysis was performed using Kruskal-Wallis test followed by Dunn’s multiple comparison rank test. Wiskers are calculated by the Tukey’s method. The * and # between 5- and 11-day-old mice, respectively. One to three symbols represent p < 0.1, 0.01 and 0.001, respectively. (C) Analyses of the number of fibers for the three muscles for the same number of mice per group. The treatment prevents the loss of fibers in the soleus of SMA mice. (D) The snRNP-specific reduction of snRNAs is observed in the soleus, plantaris and tibialis of SMA mice. Total RNA was prepared from tissues of flunarizine- and vehicle-treated controls and SMA mice at 11 days of age (3 mice per group). The snRNA levels are determined by RT-qPCR and the relative amount is presented as percent of the vehicle-treated controls. Statistical analyses are performed as in Fig. 3. The treatment with flunarizine has modest effects on snRNA levels in muscles of SMA mice.
Figure 8
Figure 8
Flunarizine mitigates the defects at the neuromuscular junctions of SMA mice. (A) Immunofluorescent analyses of the neuromuscular junctions (NMJs) in the tibialis using anti-SNAP25 and anti-neurofilament light chain antibodies in red and labeled alpha-bungarotoxin (BGTX) staining for the acetylcholine receptors (AChRs) in green for the pre- and postsynaptic NMJs, respectively. Scale bar, 100 μm. (B) Frequency histograms depict the proportion of neuromuscular junctions in three stages (plaques, perforated structures and pretzels) in three muscles, namely soleus, plantaris and tibialis. The analysis shows that endplates are more elaborated in the soleus and tibialis of SMA mice upon the flunarizine treatment. Statistical test is two-way ANOVA followed by Tukey’s multiple comparisons test. Three mice per group. (C) Representative motor endplates from the tibialis of controls and SMA mice. Scale bar, 15 μm. (D) Quantification of the areas of the AChR clusters reveals a significant increase in endplate size in control and SMA mice upon flunarizine treatment. Kruskal-Wallis followed by Dunn’s multiple comparison rank test. Wiskers are calculated by the Tukey ‘s method. Three mice per group.

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References

    1. Liu Q, Dreyfuss G. A novel nuclear structure containing the survival of motor neurons protein. EMBO J. 1996;15:3555–3565. - PMC - PubMed
    1. Lefebvre S, et al. Correlation between severity and SMN protein level in spinal muscular atrophy. Nat Genet. 1997;16:265–269. doi: 10.1038/ng0797-265. - DOI - PubMed
    1. Carvalho T, et al. The spinal muscular atrophy disease gene product, SMN: A link between snRNP biogenesis and the Cajal (coiled) body. J Cell Biol. 1999;147:715–28. doi: 10.1083/jcb.147.4.715. - DOI - PMC - PubMed
    1. Hebert MD, Szymczyk PW, Shpargel KB, Matera AG. Coilin forms the bridge between Cajal bodies and SMN, the spinal muscular atrophy protein. Genes Dev. 2001;15:2720–2729. doi: 10.1101/gad.908401. - DOI - PMC - PubMed
    1. Morris GE. The Cajal body. Biochim Biophys Acta. 2008;1783:2108–2115. doi: 10.1016/j.bbamcr.2008.07.016. - DOI - PubMed

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