Triptolide increases transcript and protein levels of survival motor neurons in human SMA fibroblasts and improves survival in SMA-like mice

Abstract

Background and purpose: Spinal muscular atrophy (SMA) is a progressive neuromuscular disease. Since disease severity is related to the amount of survival motor neuron (SMN) protein, up-regulated functional SMN protein levels from the SMN2 gene are considered a major SMA drug-discovery strategy. In this study, we investigated the possible effects of triptolide, a diterpene triepoxide purified from Tripterygium wilfordii Hook. F., as a new compound for increasing SMN protein.

Experimental approach: The effects and mechanisms of triptolide on the production of SMA protein were determined by cell-based assays using the motor neuronal cell line NSC34 and skin fibroblasts from SMA patients. Wild-type (Smn(+/+) SMN2(-/-) , C57BL/6) and SMA-like (Smn(-/-) SMN2) mice were injected with triptolide (0.01 or 0.1 mg·kg(-1) ·day(-1) , i.p.) and their survival rate and level of change in SMN protein in neurons and muscle tissue measured.

Key results: In NSC34 cells and human SMA fibroblasts, pM concentrations of triptolide significantly increased SMN protein expression and the levels of SMN complex component (Gemin2 and Gemin3). In human SMA fibroblasts, triptolide increased SMN-containing nuclear gems and the ratio of full-length transcripts (FL-SMN2) to SMN2 transcripts lacking exon 7 (SMN2Δ7). Furthermore, in SMA-like mice, triptolide significantly increased SMN protein levels in the brain, spinal cord and gastrocnemius muscle. Furthermore, triptolide treatment increased survival and reduced weight loss in SMA-like mice.

Conclusion and implications: Triptolide enhanced SMN protein production by promoting SMN2 activation, exon 7 inclusion and increasing nuclear gems, and increased survival in SMA mice, which suggests triptolide might be a potential candidate for SMA therapy.

Figure 1

The chemical structure of triptolide, the biologically active diterpene triepoxide derivatives from the Chinese herb Tripterygium wilfordii Hook. F.

Figure 2

Effects of triptolide on (A) SMN protein levels and (B) SMN complex components in NSC34 cells. Cells were treated with vehicle (0.001% DMSO in distilled water) and triptolide (0.01, 0.1 and 1 pM) for 24 h. Protein extractions were then subjected to SDS-PAGE and protein expression of SMN, Gemin2, Gemin3 and β-actin were analysed by Western blotting. Changes in SMN, Gemin2 and Gemin3 levels normalized to β-actin were quantified and represented as percentages of vehicle group. Columns represent the mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. vehicle group. anova followed by Dunnett's test.

Figure 3

Up-regulation of SMN protein levels by triptolide in human SMA fibroblasts derived from different types of SMA patients. Fibroblasts were treated with triptolide (0.01, 0.1 and 1 pM) for 24 h, respectively. Proteins were extracted and subjected to SDS-PAGE. Then protein expression of SMN and β-actin were measured by Western blotting. Changes in SMN protein levels normalized to β-actin were quantified and represented as percentages of vehicle group. Columns represent the mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. vehicle group. anova followed by Dunnett's test.

Figure 4

Effects of triptolide on SMN complex components Gemin2 and Gemin3 protein expression in human SMA fibroblasts derived from different types of SMA patients. Fibroblasts were treated with triptolide (0.01, 0.1, 1 pM) for 24 h. Protein expression of Gemin2, Gemin3 and β-actin were analysed by Western blotting. Changes in Gemin2 and Gemin3 protein levels normalized to β-actin were quantified. Columns represent the mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. vehicle group. anova followed by Dunnett's test.

Figure 5

Effect of triptolide on (A) SMN-containing gems in the nucleus and (B) the number of nuclear gems in human SMA type III fibroblast cell lines. Fibroblasts were treated with triptolide (1 pM) for 24 h. The total number of SMN-containing nuclear gems/100 cells was observed by confocal microscopy with anti-SMN- and anti-Gemin2-specific antibodies. Alexa Fluor 555 goat anti-mouse IgG (red) and Alexa Fluor 488 goat anti-rabbit IgG (green) were used as secondary antibodies. 4′,6-diamidino-2-phenylindole (DAPI) (blue) was used for nuclei staining. Columns represent the mean ± SEM from three independent experiments. *P < 0.05 vs. vehicle group. anova followed by Dunnett's test.

Figure 6

Quantitative analysis of SMN transcripts. FL-SMN2 and SMN2Δ7 transcripts levels were determined by quantitative real time-PCR in (A, B) SMA type III and (C, D) type I fibroblasts. Cells were treated with triptolide (0.01, 0.1 and 1 pM) for 24 h. The FL-SMN2/SMN2Δ7 ratio normalized to GAPDH was quantified. All data are expressed as the mean ± SEM in arbitrary units relative to GAPDH from three independent experiments. *P < 0.05 and **P < 0.01 vs. vehicle group; anova followed by Dunnett's test.

Figure 7

(A) Lack of cytotoxic effect of triptolide on human SMA type III fibroblasts. SMA fibroblasts were treated with vehicle and triptolide (0.01, 0.1, 1, 10 and 100 pM) for 24 h. Cell viability was determined by the MTT assay. Changes in survival rate are presented as percentages of the vehicle group. (B) Apoptosis was analysed by flow cytometry using Annexin V/PI staining. The apoptosis of each group is shown as an apoptosis index, evaluated by counting the percentage of apoptotic cells (Annexin V-positive cells). Cycloheximide 10 µg·mL⁻¹ (CHX) was used as a positive control for apoptosis induction. Columns represent the mean ± SEM from three independent experiments. ***P < 0.001 vs. vehicle group. anova followed by Dunnett's test.

Figure 8

Up-regulation of SMN protein levels in neuronal and muscular tissues of triptolide-treated SMA-like mice. Mice (Smn^−/−SMN2) were injected with vehicle or triptolide (0.01 or 0.1 mg·kg⁻¹·day⁻¹i.p.) for 1 week. Brain, spinal cord and gastrocnemius muscle were isolated from wild-type (WT, Smn^+/+SMN2^−/−) and vehicle- or triptolide-treated groups. (A) Total protein from multiple tissues were extracted and the SMN protein levels were determined by Western blotting. (B) Quantification of SMN protein in the brain, spinal cord and gastrocnemius muscle showed a significant elevation after triptolide treatment. β-actin (for brain and spinal cord) or GADPH (for gastrocnemius muscle) were used as loading controls. *P < 0.05 and **P < 0.01 vs. vehicle group; ^###P < 0.001 vs. WT group; anova followed by Dunnett's test.

Figure 9

Triptolide increases survival rate and attenuates weight loss of SMA-like mice. SMA-like mice (Smn^−/−SMN2) and wild-type mice (WT, Smn^+/+SMN2^−/−) were treated with daily i.p. injections of triptolide (0.1 mg·kg⁻¹) or vehicle on days P5–P18. (A) Kaplan–Meier survival curves of mice treated with triptolide (n= 15) or vehicle (n= 13). P < 0.001, log-rank test. (B) Weights of SMA mice treated with triptolide (n= 15) or vehicle (n= 13), and WT mice treated with triptolide (n= 20) or vehicle (n= 20). *P < 0.05 and**P < 0.01 vs. SMA mice treated with vehicle group; anova followed by Tukey's test.