Increasing expression and decreasing degradation of SMN ameliorate the spinal muscular atrophy phenotype in mice

Abstract

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by reduced levels of the survival motor neuron (SMN) protein. Here we show that the proteasome inhibitor, bortezomib, increases SMN in cultured cells and in peripheral tissues of SMA model mice. Bortezomib-treated animals had improved motor function, which was associated with reduced spinal cord and muscle pathology and improved neuromuscular junction size, but no change in survival. Combining bortezomib with the histone deacetylase inhibitor trichostatin A (TSA) resulted in a synergistic increase in SMN protein levels in mouse tissue and extended survival of SMA mice more than TSA alone. Our results demonstrate that a combined regimen of drugs that decrease SMN protein degradation and increase SMN gene transcription synergistically increases SMN levels and improves the lifespan of SMA model mice. Moreover, this study indicates that while increasing SMN levels in the central nervous system may help extend survival, peripheral tissues can also be targeted to improve the SMA disease phenotype.

Figure 1.

SMN is increased in bortezomib-treated cells. (A) SMA patient fibroblasts treated for 16 h with different doses (1, 10, 50 and 100 µm, respectively) of the proteasome inhibitor bortezomib showed dose-dependent increases in SMN protein levels. Quantification is shown in lower panel. (B) HEK 293T cells treated with bortezomib have increased SMN ubiquitination in a dose-dependent manner. Values represent mean ± SEM of three independent experiments. **P< 0.01.

Figure 2.

Bortezomib increases SMN protein levels in vivo. Mice at P5 were treated with either bortezomib (0.15 mg/kg) or vehicle (water) every other day and sacrificed at P13. The brain, spinal cord, liver, kidney and muscle tissues were removed, and protein lysates were isolated for biochemical analysis. A densitometry analysis was performed on the resulting western blots to ascertain relative SMN protein levels in each tissue. Values represent mean ± SEM. **P< 0.01.

Figure 3.

Bortezomib improves motor function; does not affect survival. SMA mice were treated with i.p. injections of bortezomib (0.15 mg/kg) or vehicle (water) every other day starting on P5. (A) Survival curves of SMA mice treated with bortezomib or vehicle (log-rank test, P= 0.76; n= 12 bortezomib; n= 10 vehicle). (B) Righting time in SMA mice treated with bortezomib (n= 12) or vehicle (n= 10).

Figure 4.

Combining trichostatin A and bortezomib increases SMN in vivo. SMA mice aged P5 were treated with TSA (4 mg/kg) and bortezomib (0.075 mg/kg) or with vehicle (DMSO and water). Mice were sacrificed for biochemical analysis at P13. The brain, spinal cord, liver, kidney and muscle tissues were removed and protein lysates from these tissues were isolated to examine SMN protein levels. The ratio of SMN to actin protein levels was determined by a densitometry analysis. Values represent mean ± SEM. *P< 0.05; **P< 0.01; ***P< 0.001.

Figure 5.

TSA with bortezomib extends survival in SMA mice and improves the SMA phenotype in transgenic mice. SMA mice were treated with daily i.p. injections of TSA (4 mg/kg) and bortezomib (0.075 mg/kg) or vehicle (equal amounts of DMSO and water). A control group was treated with vehicle (DMSO) and TSA alone (4 mg/kg) in the same manner. (A) Kaplan–Meier survival curves of SMA mice treated with TSA alone (n= 12; median survival = 16 days) or vehicle (n= 10; median survival = 18.5 days; log-rank test, P= 0.007). (B) Survival curves of SMA mice treated with TSA with bortezomib (n = 21; median survival = 20 days) or vehicle (DMSO and water; n= 21; median survival = 14 days; log-rank test, P< 0.0001). (C) Righting times of SMA mice treated with TSA and bortezomib, TSA alone and vehicle. (D) Body weights of SMA mice treated with TSA with bortezomib gained more weight than mice treated with either TSA alone (P< 0.05) or vehicle (P< 0.0001).

Figure 6.

TSA plus bortezomib ameliorates muscle pathology in SMA model mice. SMA mice were treated with vehicle (n= 3), bortezomib (n= 3), TSA (n= 3), TSA + bortezomib (n= 3) from P5 to P13. (A) Hematoxylin and eosin staining of quadriceps muscles from vehicle- and drug-treated mice. Scale bars: 50 μm. (B) Average myofiber diameter was increased with bortezomib treatment (P< 0.05), TSA treatment (P< 0.01) and TSA + bortezomib (P< 0.01). The total TA myofiber number increased with individual bortezomib (P< 0.01) and TSA treatments (P< 0.05) and increased further with TSA + bortezomib (P< 0.001). Values represent mean ± SEM. *P< 0.05; **P< 0.01; ***P< 0.001.

Figure 7.

TSA plus bortezomib delays motor neuron death in lumbar spinal cord tissues of SMA model mice. SMA mice treated with vehicle (n= 3), bortezomib alone (0.15 mg/kg; n= 3), TSA alone (4 mg/kg) or bortezomib (0.075 mg/kg) plus TSA (4 mg/kg; n= 3) were sacrificed at P13. (A) Nissl-stained sections of ventral spinal cords. Quantitative analysis showed an increase in motor neurons/section in mice treated with bortezomib alone (P< 0.01) and a combination of TSA and bortezomib (P< 0.01) but not with TSA alone. (B) Western blot analysis showed an increase in the ChAT protein levels in spinal cord of bortezomib (P= 0.056) and TSA + bortezomib-treated mice compared with vehicle-treated mice (P < 0.05). Values represent mean ± SEM. *P< 0.05; **P< 0.01; ***P< 0.001.

Figure 8.

TSA plus bortezomib improves the NMJ. NMJs were isolated from the TA muscles of vehicle- and drug-treated animals at P13. While TSA and bortezomib (n = 3) both independently increased NMJ surface area, combining both TSA and bortezomib further improved NMJ size in SMA mice (P< 0.01). Values represent mean ± SEM. **P< 0.01.