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. 2015 Feb 1;29(3):288-97.
doi: 10.1101/gad.256644.114. Epub 2015 Jan 12.

Motor neuron cell-nonautonomous rescue of spinal muscular atrophy phenotypes in mild and severe transgenic mouse models

Yimin Hua  1 , Ying Hsiu Liu  2 , Kentaro Sahashi  2 , Frank Rigo  3 , C Frank Bennett  3 , Adrian R Krainer  4
Affiliations

Motor neuron cell-nonautonomous rescue of spinal muscular atrophy phenotypes in mild and severe transgenic mouse models

Yimin Hua et al. Genes Dev. .

Abstract

Survival of motor neuron (SMN) deficiency causes spinal muscular atrophy (SMA), but the pathogenesis mechanisms remain elusive. Restoring SMN in motor neurons only partially rescues SMA in mouse models, although it is thought to be therapeutically essential. Here, we address the relative importance of SMN restoration in the central nervous system (CNS) versus peripheral tissues in mouse models using a therapeutic splice-switching antisense oligonucleotide to restore SMN and a complementary decoy oligonucleotide to neutralize its effects in the CNS. Increasing SMN exclusively in peripheral tissues completely rescued necrosis in mild SMA mice and robustly extended survival in severe SMA mice, with significant improvements in vulnerable tissues and motor function. Our data demonstrate a critical role of peripheral pathology in the mortality of SMA mice and indicate that peripheral SMN restoration compensates for its deficiency in the CNS and preserves motor neurons. Thus, SMA is not a cell-autonomous defect of motor neurons in SMA mice.

Keywords: SMN; SMN2; antisense oligonucleotide; mouse models; spinal muscular atrophy.

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Figures

Figure 1.
Figure 1.
Systemic delivery of ASO10–27 promotes tail growth and prevents necrosis in mild SMA mice. (A) Dose-dependent tail growth and complete prevention of tail necrosis were achieved by two SC injections of ASO10–27 at 0 mg/kg (SC0, n = 13), 40 mg/kg (SC40, n = 13), 80 mg/kg (SC80, n = 14), or 120 mg/kg (SC120, n = 18) on P0 and P2, one injection per day. Untreated heterozygous mice (Het, n = 13) were used as normal controls. Tail length was measured weekly for up to 3 mo. (B) Pictures of 3-mo-old mice treated as in A. (C) Prevention of tail necrosis was achieved by two late SC injections at 120 mg/kg. Injections were performed on P0 and P2 (P0P2, n = 13), on P7 and P9 (P7P9, n = 20), on P14 and P16 (P14P16, n = 12), or between P23 and P31 (P23–31, n = 11) for each mouse. For the P23–P31 group, necrosis was just starting on the tail tip. (D) Mice in the P23–P31 group at the time of the first injection of ASO10–27 (left) and 5 mo after treatment (right). (E) Analysis of SMN2 exon 7 inclusion in the liver, brain, and spinal cord of P23–P31 mice by radioactive RT–PCR. Saline treatment was used as a control. Tissues were collected 5 d after the second injection. (FL) Full-length transcript; (Δ7) exon 7-skipped transcript; [Incl (%)] 100 × FL/(FL + Δ7). (F) Histogram of exon 7 inclusion data from E (n = 3). (*) P < 0.001; (#) P > 0.05.
Figure 2.
Figure 2.
Intracerebroventricularly injected decoy oligonucleotide neutralized the CNS effect of subcutaneously injected ASO10–27 but had no negative impact on tail growth and prevention of necrosis in mild SMA mice. (A) RT–PCR analysis of SMN2 exon 7 splicing in the spinal cord, brain, and liver of mice that received two SC injections of ASO10–27 at 120 mg/kg per injection on P0 and P2, one injection per day, together with ICV delivery of 0 μg of decoy (decoy-0) or 10 μg of decoy (decoy-10) on P0 or 20 μg of decoy (decoy-20) on P0 and P2 (10 μg per day). The control group was treated with saline for both SC and ICV injections (untreated). Tissues were collected on P7. (B) Histogram of exon 7 inclusion data from A (n = 3). (*) P < 0.001 versus the untreated group; (#) P > 0.05 versus untreated mice. (C) Tail growth of decoy-0 (n = 18) and decoy-20 (n = 15) groups as described in A. Tail length was measured weekly for up to 3 mo; no necrosis occurred in decoy-20 mice throughout their lives. Heterozygous littermates (Het, n = 13) were used as normal controls.
Figure 3.
Figure 3.
ICV delivery of decoy abrogated the CNS effect of subcutaneously administered ASO10–27 in severe SMA mice. (A) RT–PCR analysis of SMN2 exon 7 splicing in the spinal cord, brain, and liver of mice treated with three SC injections of ASO10–27 at 120 mg/kg per injection between P0 and P2, one injection per day, together with ICV injection of saline (SC-alone) or 30 μg of decoy (SC+decoy) on P0 (10 μg) and P2 (20 μg). The control group was treated with saline for both SC and ICV injections (untreated). Tissues were collected on P7. (B) Histogram of exon 7 inclusion data from A (n = 3). (**) P < 0.01 versus the other two groups; (*) P < 0.01 versus untreated mice; (Δ) P < 0.05 versus untreated mice. (C) Western blotting analysis of SMN levels in the spinal cord, brain, and liver of mice as described in A. Tissues were collected on P7. Tubulin was used as a loading control. (D) Histogram of SMN levels relative to tubulin levels from C (n = 3). Data were normalized to the untreated group. (**) P < 0.05 versus the other two groups; (*) P < 0.01 versus untreated mice; (#) P > 0.05 versus untreated mice.
Figure 4.
Figure 4.
Immunohistochemical staining of ASO and SMN in spinal cord cross-sections of severe SMA mice. Mice were treated with either SC and ICV injections of saline (A–C; untreated), three SC injections (one injection per day between P0 and P2) of ASO10–27 alone at 120 mg/kg (D–F; SC-alone), or both SC injections of ASO10–27 as in the SC-alone group and ICV injection of 30 μg of decoy oligonucleotide, on P0 (10 μg) and P2 (20 μg) (G–I; SC+decoy). Tissues were collected on P7 (n = 3 for all groups). A, D, and G were stained with hematoxylin and eosin (H&E). B, E, and H were probed with a rabbit polyclonal antibody that recognizes the phosphorothioate backbone of both ASO10–27 and the decoy oligonucleotide. C, F, and I were probed with a monoclonal human-specific anti-SMN antibody, SMN-KH. Nuclei were counterstained with DAPI.
Figure 5.
Figure 5.
Gem counts in spinal cord L1–L2 motor neurons of mice. Mice were treated with either SC and ICV injections of saline (A–C; untreated, n = 3), three SC injections (one injection per day between P0 and P2) of ASO10–27 alone at 120 mg/kg (D–F; SC-alone, n = 3), or both SC injections of ASO10–27 as in the SC-alone group and ICV injection of 30 μg of decoy oligonucleotide on P0 (10 μg) and P2 (20 μg) (G–I; SC+decoy, n = 3). Tissues were collected on P7. Immunofluorescence of paraffin-embedded sections was carried out to detect SMN and ChAT; nuclei were counterstained with DAPI. Gems per 100 motor neurons (J) and percentage of motor neurons containing zero, one, two, three, or four or more gems (K) in the above three groups were calculated. (*) P < 0.01 versus the other two groups; (Δ) P < 0.05 versus the other two groups; (#) P > 0.05 versus untreated mice.
Figure 6.
Figure 6.
Increase of SMN in peripheral tissues alone robustly rescued the phenotype of severe SMA mice. The SC-alone group was treated with three SC injections of ASO10–27 at 120 mg/kg at P0–P2, the SC+decoy group was treated as in the SC-alone group plus ICV delivery of 30 μg decoy at P0–P2, and mice that received SC and ICV injections of saline were used as controls (untreated). (A) Survival curves. P < 0.001 for either the SC-alone (n = 19) or the SC+decoy (n = 27) group versus untreated mice (n = 18). P = 0.5496 for the SC-alone versus the SC+decoy group. (B) Body weight curves were similar for the SC-alone and SC+decoy groups. Untreated heterozygous littermates (Het-Con, n = 12) were used as positive controls. (C) Immunofluorescence of spinal cord L1–L2 sections with primary antibody against ChAT and Alexa fluor 568 secondary antibody. Nuclei were counterstained with DAPI. Tissues were collected on P7. (D) Motor neurons in the spinal cord L1–L2 ventral horn labeled as in C were counted (n = 6). (*) P < 0.01 versus all three groups; (#) P > 0.05 versus the SC-alone group. (E) NMJ staining of the longissimus capitus of SMA mice (untreated) and heterozygous littermates (Het-Con) reveals no denervation of this muscle. Tissues were collected on P9. Representative pictures are shown. (Red) Endplates; (blue) synaptic vesicles; (green) neurofilaments. (F) Quantitation of AChR cluster shapes of the longissimus capitus (n = 3) NMJs (n > 100) as stained in E. Mature AChR clusters include branched and pretzel-like clusters. (*) P < 0.05 versus heterozygous controls. (G) Quantitation of AChR cluster shapes of the longissimus capitus NMJs (n > 100) in P21 mice (n = 3). (#) P > 0.05 compared with the other two groups. (H) Grip strength and rotarod tests of rescued SMA mice (n = 10) at 2–3 mo of age. (*) P < 0.05 versus the other two groups; (#) P > 0.05 versus the SC-alone group.
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