Treatment of spinal muscular atrophy by sodium butyrate

Abstract

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by degeneration of the anterior horn cells of the spinal cord, leading to muscular paralysis with muscular atrophy. No effective treatment of this disorder is presently available. Studies of the correlation between disease severity and the amount of survival motor neuron (SMN) protein have shown an inverse relationship. We report that sodium butyrate effectively increases the amount of exon 7-containing SMN protein in SMA lymphoid cell lines by changing the alternative splicing pattern of exon 7 in the SMN2 gene. In vivo, sodium butyrate treatment of SMA-like mice resulted in increased expression of SMN protein in motor neurons of the spinal cord and resulted in significant improvement of SMA clinical symptoms. Oral administration of sodium butyrate to intercrosses of heterozygous pregnant knockout-transgenic SMA-like mice decreased the birth rate of severe types of SMA-like mice, and SMA symptoms were ameliorated for all three types of SMA-like mice. These results suggest that sodium butyrate may be an effective drug for the treatment of human SMA patients.

Figures

Figure 1

Effects of sodium butyrate on the expression of the human SMN2 gene in lymphoid cell lines of SMA patients with SMN1 deletion. (a) RT-PCR analyses of exons 6–8 of the SMN2 gene showed that the exon 7-containing transcript was increased with 5 ng/ml to 500 μg/ml sodium butyrate treatment. Quantitative analysis of the exon 7-containing transcript is shown on the right, in which the ratio of exon 7 inclusion to exon 7 exclusion is indicated (mean ± SD; n = 3). M: 100 bp-ladder marker. E7 in.: exon 7 inclusion; E7 ex.: exon 7 exclusion. (b) The SMA lymphoid cell lines were treated with sodium butyrate for 1, 2, 3, 4, 8, 22, and 32 h. RT-PCR analyses of exons 6–8 of the SMN2 gene showed that the exon 7-containing transcript was increased after 4-h treatment with 500 ng/ml sodium butyrate. A quantitative analysis of the exon 7-containing transcript is shown on the right (mean ± SD; n = 3). (c) RT-PCR amplification of whole cDNAs (exons 1–8) of SMN2 genes from different types of SMA lymphoid cell lines showed that the full-length transcript of the SMN2 gene is very similar to the transcript of the SMN1 gene after sodium butyrate treatment. (I, II, and III represent different types of SMA lymphoid cell lines; −, untreated; +, treated; C, normal). (d–f) RT-PCR analyses of exons 1–4 (d), 4–6 (e), and 6–8 (f) for SMN2 gene expression showed that only the transcript pattern of exon 7 was changed due to alternative splicing. There was no change for exons 3 and 5.

Figure 2

Effects of sodium butyrate on the expression of the human SMN protein in lymphoid cell line of a representative SMA type I patient. (Upper) Western blot analysis showed a gradual increase in exon 7-containing SMN protein expression in nuclear fraction after 0.5 ng/ml to 500 μg/ml sodium butyrate treatment. The amount in the cytosolic fraction decreased with increasing doses (5, 50, and 500 μg) of sodium butyrate. (Lower) Quantitative analysis of SMN and tubulin ratios are shown. The control represents an untreated SMA control.

Figure 3

Effects of sodium butyrate on the expression of human SR proteins in the lymphoid cell line of a representative SMA type I patient. (a) Western blot analysis showed two SR proteins (*) were induced after 500 ng and 5 μg sodium butyrate stimulation, which were detected by mouse anti-SR protein mAb (6H3), but no difference was found by using mouse anti-SRp20 mAb. (b) The induced SR proteins (*) disappeared after adding mitogen-activated protein kinase inhibitor PD98059 or phosphate inhibitor (okadaic acid), and the expression pattern of SMN2 also changed to untreated status. The control represents an untreated SMA control.

Figure 4

Survival time of type 2 SMA-like mice after sodium butyrate treatment shown in a Kaplan–Meier survival curve. Sodium butyrate was added to the drinking water after diagnosis of type 2 SMA-like mice that showed poor activity after postnatal day 10; 15 mice were left untreated (red), and 15 mice were treated with sodium butyrate (green) from diagnosis to death. The type 2 SMA-like mice treated with sodium butyrate lived significantly longer than those in the untreated SMA control group (P = 0.0004). Treated group: mean = 21.7 days, range = 14–30 days; untreated group: mean = 15.5 days, range = 13–20 days.

Figure 5

Histological analysis of a type 3 SMA-like mouse after sodium butyrate treatment. (a1–a3) Normal control. (b1–b3) Type 3 SMA-like representative mouse after sodium butyrate treatment. (c1–c3) Type 3 SMA-like representative mouse with no treatment. Cross sections of tails were stained with hematoxylin and eosin (×40 for a1, b1, and c1; ×100 for a2 and b2; ×400 for c2, a3, b3, and c3). The tails of sodium butyrate-treated mice had a few regions of muscular bundle atrophy but no subcutaneous edema (b1–b3). The tails of untreated mice had severe muscular bundle atrophy (c1 and c3), thrombus in the vessel walls due to muscular atrophy, resulting in venous blood stasis (c2, arrow), and mild subcutaneous edema (c1) compared with normal controls (a1–a3).

Figure 6

Expression of human exon 7-containing SMN protein in types 2 and 3 SMA mice after sodium butyrate treatment. (a) Western blot analysis of exon 7-containing SMN protein (detected by SMN H7 antibody) in cytosolic (C) and nuclear (N) fractions from different tissues. The exon 7-containing SMN protein in treated mice was increased in skeletal muscle (SK. Muscle) and spinal cord (SP. Cord) after sodium butyrate treatment, especially in the nuclear fraction, compared with untreated types 2 and 3 mice. (b) Control incubation was performed with an anti-α-tubulin antibody to determine the relative amount of SMN protein (Tubulin). (c–f) Immunohistochemical staining of anterior horn cells of the spinal cord showed an increase of SMN protein in motor neurons of type 2 SMA-like mice after sodium butyrate treatment. (c) Untreated (H2 antibody). (d) Treated (H2 antibody). (e) Untreated (H7 antibody). (f) Treated (H7 antibody). Arrow: motor neuron.