Trichostatin A increases SMN expression and survival in a mouse model of spinal muscular atrophy (original) (raw)
TSA activates SMN2 gene expression in vitro. TSA was previously shown to increase activation of an SMN2 promoter reporter by approximately 2-fold with an EC50 of 17 nM in a transgenic motor neuronal cell line (21). In order to confirm activation of the endogenous human SMN2 gene in vitro, a fibroblast cell line derived from a type III SMA patient (cell line no. 2906; see Methods) was treated for 4 hours with a range of doses of TSA (2.5–50 nM). All TSA doses tested led to increased mRNA levels of SMN containing exon 7 (SMN+7) and SMN lacking exon 7 (SMNΔ7) above vehicle-treated levels (Figure 1A). In this cell line, SMN+7 mRNA levels increased to a greater degree than did SMNΔ7 mRNA levels. This may indicate that TSA has dual effects, activation of the SMN2 promoter and promotion of exon 7 inclusion in SMN transcripts, as has been described for other HDAC inhibitors and SMN (25, 26). A fibroblast cell line derived from a type I SMA patient (cell line no. 232; see Methods) was also treated for 4 hours with a larger range of TSA doses (0.05–100 nM). These experiments showed a dose-dependent increase in both SMN+7 and SMNΔ7 mRNA levels, with a maximum induction of 1.7-fold at 50 nM (data not shown). A third fibroblast cell line derived from another type I SMA patient (cell line no. 3813; see Methods) was treated for various time periods with 50 nM TSA. Again, an approximately 2-fold increase over baseline in SMN+7 and SMNΔ7 mRNA levels was observed. This effect was rapid in onset and transient, with increased levels evident at 1 hour and a return to baseline by 24 hours (Figure 1B). The increase in SMNΔ7 mRNA appeared to be shorter lived than that of SMN+7 mRNA, which may indicate that the SMNΔ7 mRNA species is more unstable.
TSA activates the SMN2 gene in vitro. (A) The SMA fibroblast cell line 2906 was treated with 0–50 nM TSA, and SMN+7 and SMNΔ7 mRNA was measured after 4 hours. Values represent mean ± SEM of 5 separate experiments. (B) The SMA fibroblast cell line 3813 was treated with 50 nM TSA, and SMN+7 and SMNΔ7 mRNA levels were determined over a period of 0–72 hours. Values represent mean ± SEM of 2 experiments for the 2-, 4-, 17-, and 24-hour time points and 1 experiment for the 0.5-, 1-, 5-, 48-, and 72-hour time points.
A single dose of TSA increases histone acetylation and SMN gene expression in mice. In order to determine what dose of TSA inhibits HDACs and modifies gene expression in different tissue compartments, nontransgenic mice were treated with single intraperitoneal doses of 2, 5, or 10 mg/kg TSA, doses that have previously been used in mice (31–33). Liver and brain tissues were isolated after 2 hours. Dose-dependent increases in acetylated H3 and H4 histones were evident in both brain and liver, with an approximately 15-fold induction in the brain (Figure 2, A and B). Quantitative RT-PCR (qRT-PCR) was used to measure the mRNA levels of mouse SMN and follistatin. Follistatin, which interacts with members of the TGF-β family, was measured as a positive control because it has been previously shown to be activated by TSA (34). We observed increases in follistatin mRNA in both brain and liver and an increase of mouse SMN mRNA in liver (Figure 2C). Because the 10-mg/kg TSA dose produced the greatest increase in acetylated histone levels and in gene expression, this dose was used for all subsequent experiments.
TSA treatment increases histone acetylation and gene expression in nontransgenic mice. (A) Representative Western blot showing acetylated H3 (Ac H3) histones compared with H1 histone levels in the brains of nontransgenic mice 2 hours after treatment with vehicle (Veh) or with 2, 5, or 10 mg/kg TSA. Each lane represents an individual animal. (B) Quantification of acetylated H3 and H4 histones in the brains and livers of nontransgenic mice treated with vehicle or with 2, 5, or 10 mg/kg TSA. (C) Mouse Smn and follistatin (Foll) mRNA levels after treatment with vehicle or with 2, 5, or 10 mg/kg TSA. Values represent mean ± SEM of 3 mice per group. *P < 0.01; #P < 0.05.
TSA has been previously shown to have rapid pharmacokinetics in the mouse: mice dosed by intraperitoneal injection had absorption into the plasma within 2 minutes and a plasma t1/2 of approximately 5–10 minutes (31). In order to investigate the time course of gene activation by TSA, nontransgenic mice were treated with single doses of 10 mg/kg TSA, and tissues were harvested at 2, 4, 6, and 16 hours after dosing. These studies revealed maximal gene activation at approximately 2–4 hours, with a return to baseline by 16 hours (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI29562DS1). No changes in SMN protein levels were observed at any time point (data not shown). The 2- or 4-hour time points were selected for all subsequent experiments.
We next treated a cohort of nontransgenic mice with a single dose of 10 mg/kg TSA (n = 8) or vehicle (n = 7), and brain, liver, spinal, and muscle tissues were harvested at 2 hours for determination of mRNA and protein levels. Follistatin mRNA increased in brain and liver, as expected, but did not change in spinal cord and muscle tissues (Figure 3A). This result in muscle was consistent with previous observations that follistatin is not induced in healthy muscle, only in injured muscle (34). In order to verify that TSA was modulating gene expression in these tissues, qRT-PCR was performed for brain-derived neurotrophic factor (BDNF), another gene that has been shown to be activated by HDAC inhibitors (35). Increases in BDNF mRNA were evident in spinal cord and muscle, verifying the biological effect of TSA in these tissues (Figure 3A). TSA treatment also resulted in a modest, but significant, increase in mouse SMN mRNA in the liver (P < 0.001) and muscle (P < 0.05; Figure 3A). No change in SMN or BDNF protein expression was found in these tissues (Figure 3B).
A single dose of TSA increases Smn gene expression but not SMN protein levels in nontransgenic mice. (A) Smn, follistatin, and BDNF mRNA levels and (B) SMN and BDNF protein levels were determined in brains, livers, spinal cords, and muscle tissues 2 hours after nontransgenic mice were treated with a single dose of TSA (10 mg/kg; n = 8) or vehicle (n = 7). BDNF mRNA levels were too low in liver to be reliably measured. Values represent mean ± SEM. #P < 0.05; *P < 0.01; **P < 0.001.
In order to verify the HDAC-inhibitory effects of TSA in SMA model mice, P10 SMA mice (Smn–/–SMN2+/+SMN_Δ_7+/+; ref. 20) were treated with a single dose of 10 mg/kg TSA or vehicle, and brain and liver tissues were harvested at 2 hours. Acetylated H3 and H4 levels increased in the brain and to a lesser extent in the liver (Supplemental Figure 2). A cohort of P10 SMA mice was next treated with a single dose of 10 mg/kg TSA or vehicle (n = 7 per group), and liver, brain, spinal cord, and muscle tissues were harvested after 4 hours. No changes in SMN+7 mRNA were observed, and small increases in SMNΔ7 mRNA were seen in all tissues (data not shown). No changes in SMN protein levels were observed in any of these tissues (data not shown). Together, these results indicate that single doses of TSA cause dose-dependent and time-limited inhibition of HDACs and a modest increase in SMN gene expression, but no change in SMN protein expression.
Repeated doses of TSA increase SMN2 gene expression, SMN protein levels, and snRNP assembly activity in SMA mice. Given the lack of change in SMN protein levels after a single dose of TSA, we postulated that multiple doses might be required. Consequently, we treated a cohort of mice beginning on P5 with daily injections of TSA 10 mg/kg and harvested tissues 4 hours after the last dose on P13 (Figure 4). As expected based on the single-dosing experiments in nontransgenic mice, BDNF mRNA increased approximately 2-fold in spinal cord and muscle (Figure 4A). Follistatin no longer served as a good marker of gene activation in brain and liver in chronically treated mice, as levels of this mRNA either did not change or decreased, suggesting that this gene may be autoregulated and become silenced with chronic stimulation. Follistatin levels significantly decreased in SMA mouse muscle, in contrast to a recently reported increase of follistatin in mouse muscular dystrophy models (36). SMN+7 mRNA levels increased in the brain, liver, and spinal cord, although these changes did not reach statistical significance, whereas SMN+7 levels increased approximately 5-fold (P < 0.01), and SMNΔ7 levels increased approximately 2-fold (P < 0.01), in muscle. Western blot analysis of protein isolated from these tissues revealed the presence of both the full-length SMN protein and the SMNΔ7 protein, as has been previously described in these mice (20). Full-length SMN and SMNΔ7 protein levels increased by approximately 1.5- to 2-fold in the brain, liver, and spinal cord with TSA treatment (Figure 4B). This increase was apparent for both SMN isoforms. No obvious change in the ratio of full-length to SMNΔ7 protein was evident. Western blot of muscle tissues showed that SMN protein bands were very faint or undetectable in vehicle-treated samples but were visible in the TSA-treated mice, indicating an increase (Figure 4B). Despite increases in BDNF mRNA, mature BDNF protein as measured by ELISA showed no change in response to TSA treatment (Figure 4C). No differences in SMN mRNA or protein levels were observed between TSA and vehicle-treated heterozygous littermate mice (data not shown).
Repeated doses of TSA increase SMN+7 mRNA levels and SMN protein levels in SMA mice. (A) SMN+7, SMNΔ7, follistatin, and BDNF mRNA levels and (B and C) SMN and BDNF protein levels were determined in brain, liver, spinal cord, and muscle isolated 4 hours after the last dose from SMA mice treated daily from P5 through P13. (B) Western blots, with each lane representing 1 animal. In the upper panels, the upper bands show full-length SMN protein; the lower bands show SMNΔ7 protein. (C) Quantification of Western blots shown in B. Lanes denoted with arrowheads were excluded as underloaded relative to the other visualized lanes. SMN protein bands were not visible in the muscle of untreated SMA mice, although they were evident in treated mice; therefore, quantification of the changes in muscle SMN protein levels was limited. Values represent mean ± SEM. #P < 0.05; *P < 0.01; **P < 0.001.
Spliceosomal snRNPs are composed of 1 small nuclear RNA (snRNA) molecule, a common core of 7 Sm proteins, and additional proteins specific for each snRNP. To date, the best characterized function of the SMN complex is to mediate the efficient and accurate assembly of Sm proteins and snRNAs into snRNPs (12). In order to investigate the activity of the SMN complex in vehicle- and TSA-treated SMA mice and heterozygous littermates, snRNP assembly activity was measured in brain extracts prepared from P13 mice using a previously described in vitro assay (37). Only brain tissues could be analyzed at this time point because of the strong physiological downregulation of SMN activity that occurs postnatally in several tissues, including the spinal cord (38). Western blot analysis of the prepared extracts confirmed an increase in SMN protein levels similar to those demonstrated in Figure 4B (data not shown). In vitro, transcribed radioactive U1 snRNAs were incubated with the extracts, anti-Sm antibodies were used to immunoprecipitate assembled snRNPs, and U1 snRNA levels were quantified to determine the relative snRNP assembly efficiency. Heterozygous vehicle-treated mice showed approximately 5-fold greater snRNP assembly activity than did vehicle-treated SMA mice (data not shown). A detailed analysis of the impairment of SMN activity in snRNP assembly in mouse models of SMA will be reported elsewhere (F. Gabanella, M.E.R. Butchbach, A.H. Burghes, and L. Pellizzoni, unpublished observations). TSA treatment improved snRNP assembly activity by approximately 1.8-fold (P < 0.0017), comparable to the improvement in SMN protein levels (Figure 5, A and B). In contrast, no differences in snRNP assembly were observed in brain extracts derived from vehicle- and TSA-treated heterozygous littermates (Supplemental Figure 3). These data indicate that increased SMN protein levels are associated with increased SMN activity in snRNP assembly in brain extracts of TSA-treated SMA mice.
TSA treatment increases snRNP assembly activity in brains of SMA mice. (A) snRNP assembly reactions were carried out using in vitro transcribed radioactive U1 snRNA and 25 mg of brain extracts from either vehicle- or TSA-treated SMA mice. Following immunoprecipitation with anti-Sm antibodies, input (2.5%) and immunoprecipitated U1 snRNAs were analyzed by electrophoresis on 10% polyacrylamide, 8M urea denaturing gels and autoradiography. (B) Quantification of relative snRNP assembly activity in brain extracts from vehicle- and TSA-treated SMA mice. Brain extracts from either vehicle-treated (n = 5) or TSA-treated (n = 7) SMA mice were prepared and analyzed by snRNP assembly and immunoprecipitation experiments as in A, and the amount of immunoprecipitated U1 snRNA was quantified as described in Methods. Values represent mean ± SEM. *P < 0.01.
TSA treatment improves survival of SMA mice when started after disease onset. Having observed that repeated daily doses of TSA increased SMN protein levels and snRNP assembly activity, we next examined whether TSA treatment ameliorates the disease phenotype in SMA mice. The Smn–/–SMN2+/+SMNΔ7+/+ SMA mice have been reported to have severe muscle weakness, approximately 20% loss of anterior horn cells (AHCs), diffusely small myofibers, and a median survival of approximately 2 weeks (20). In addition, by P5 the mice show clear manifestations of disease: they are significantly underweight and have a markedly impaired righting reflex (20). In order to test whether HDAC inhibitors are beneficial when delivered after disease onset, we administered once-daily injections starting at P5 and continued until P20. All the pups in litters derived from heterozygous Smn+/–SMN2+/+SMNΔ7+/+ breeding pairs (including 48 SMA, 101 heterozygous, and 60 WT littermates (Figure 6A) were randomly assigned to receive TSA or vehicle, and pups were monitored daily by recording weight and performing behavioral tests. Of the heterozygous and WT littermates, 5 TSA-treated and 3 vehicle-treated mice died during the treatment period, most of them presumably from injection injury. As expected, before the start of treatment P5 SMA mice were significantly underweight (2.85 ± 0.09 g versus 3.32 ± 0.06 g; P < 0.0001) and had significantly impaired righting time (24.9 ± 1.5 s versus 5.1 ± 0.7 s; P < 0.0001) compared with WT and heterozygous littermates. The cohorts of SMA mice receiving TSA or vehicle were equally matched for weight (2.82 ± 0.12 g versus 2.87 ± 0.14 g) and litter size (8.1 ± 0.4 versus 8.2 ± 0.5) at P5. Of the 48 SMA mice treated with TSA, 3 pups were lost during cage maintenance and were therefore excluded from the survival analysis. TSA-treated SMA mice showed significantly improved survival compared with vehicle-treated mice (P = 0.0003, log-rank test; Figure 6B). The median difference in survival was 3 days, or 19% (19 versus 16 days), with one-quarter of the mice showing no improvement in survival and one-quarter of the mice showing an improvement of 30% or better. The pups with increased survival of 30% or greater tended to have less weight loss compared with WT and heterozygous littermates at P5 (0.35 ± 0.12 g versus 0.55 ± 0.22 g) and smaller litter size (7.3 ± 0.9 versus 8.8 ± 0.1), although these differences did not reach statistical significance. No statistically significant difference in survival between male and female mice was observed (data not shown).
TSA increases survival, attenuates weight loss, and enhances motor behavior of SMA mice. SMA mice and their WT and heterozygous littermates were treated with daily intraperitoneal injections of TSA (10 mg/kg) or vehicle on days P5–P20. (A) Four mice from the same litter at P13, showing the gross appearance of a TSA-treated SMA mouse, a vehicle-treated SMA mouse, a heterozygous (Het) mouse, and a WT mouse. (B) Kaplan-Meier survival curves of mice treated with TSA (n = 23) or vehicle (n = 22). P < 0.0003, log-rank test. (C) Weights of SMA mice treated with TSA (n = 26) or vehicle (n = 22), heterozygous mice treated with TSA (n = 52) or vehicle (n = 49), and WT mice treated with TSA (n = 30) or vehicle (n = 30). (D) Righting time in SMA mice treated with TSA (n = 20) or vehicle (n = 15), heterozygous mice treated with TSA (n = 29) or vehicle (n = 37), and WT mice treated with TSA (n = 12) or vehicle (n = 19).
SMA mice treated with TSA also showed an increased maximal weight and reduced weight loss compared with vehicle-treated mice that was evident starting at P13 (P = 0.02; Figure 6C). This occurred even though TSA caused some decrease in weight gain in the treated WT and heterozygous littermates (Figure 6C). Mice treated with TSA were observed to have some diarrhea. This was further investigated with blood chemistry and hematology panels and gross and microscopic examinations at necropsy of 5 TSA-treated and 2 vehicle-treated WT and heterozygous littermates. These studies showed no abnormalities (data not shown). After termination of drug treatment, the weight difference between TSA- and vehicle-treated WT and heterozygous mice decreased by P30 (18.93 ± 0.66 g versus 19.53 ± 0.71 g) and was no longer apparent at P60 (23.64 ± 0.67 g versus 23.29 ± 0.73 g). SMA mice also showed improved motor function, as assessed by timed righting (Figure 6D). Improvement in righting was evident starting at P9 (P = 0.03), well before the attenuation of weight loss became apparent at P13. TSA-treated mice also showed improved ambulation (Supplemental Video) as well as improved forelimb grip strength (Supplemental Figure 3).
TSA improves the morphology of the motor unit. In order to investigate the pathological correlate of this amelioration in the SMA disease phenotype, we treated a cohort of mice (4 SMA vehicle-treated mice, 4 SMA TSA-treated mice, and 3 vehicle-treated heterozygous littermates) beginning on P5 and sacrificed them on P13 for examination of muscle and spinal cord tissues. The weight of one of the mice in the vehicle-treated SMA group was nearly 3 standard deviations above the mean weight of P13 vehicle-treated SMA mice in the survival analysis, perhaps because this mouse was derived from an unusually small litter of only 2 pups. We considered this mouse an outlier, and the pathological data was analyzed both including and excluding this mouse.
We analyzed the diameter and number of neurons greater than 25 μm in the ventral horn of the lumbar spinal cord at 15 levels. The median number of ventral horn neurons per level in heterozygous mice was 29, compared with 22 in both SMA vehicle-treated and SMA TSA-treated mice (Figure 7, A and B). These neurons had a median diameter of 35.9 μm in heterozygous littermates, 33.0 μm in vehicle-treated SMA mice, and 34.3 μm in TSA-treated SMA mice (Figure 7, A and C). These data show that SMA caused some decrease in AHC number and size and that TSA treatment increased AHC size (P = 0.003), but not AHC number. This difference in AHC size between and vehicle- and TSA-treated SMA mice diminished (34.0 μm versus 34.3 μm) and was no longer statistically significant with inclusion of the outlier mouse (P = 0.16). We next measured the mRNA levels of the acetylcholine synthesis enzyme choline acetyltransferase (ChAT) as alterations of this marker have been described in different experimental conditions associated with axonal damage (39, 40). Vehicle-treated SMA mouse spinal cords showed reduced ChAT expression compared with heterozygous littermates, whereas TSA treatment resulted in increased ChAT levels in SMA mouse spinal cords (Figure 7D).
TSA increases AHC size but not AHC number. SMA mice were treated with vehicle (n = 3) or TSA (n = 3) and heterozygous littermates were treated with vehicle (n = 3) on days P5–P13. (A) Nissl-stained cross-sections of lumbar spinal cord showing 1 ventral horn. Scale bars: 10 μm. (B) Median ventral horn neuron number was reduced in SMA mice compared with heterozygous littermates (P < 0.0001) and was not changed by TSA treatment. Lines represent median values, boxes represents the twenty-fifth and seventy-fifth percentiles, whiskers represent values within 1.5 times the interquartile range, and dots represent outliers. (C) Median ventral horn neuron size was increased in heterozygous mice compared with SMA mice (P < 0.0001) and was increased by TSA treatment (P = 0.003). (D) ChAT mRNA levels were determined in spinal cord isolated from SMA mice treated daily with vehicle (n = 5) or TSA (n = 5) and heterozygous littermates treated with vehicle (n = 6) on days P5–P13.
The main pathological abnormality of SMA mouse muscles compared with normal heterozygous littermates was globally small myofibers. These fibers had a small cytoplasm without a change in the number of nuclei per fiber. There was no evidence of fibrosis, inflammatory infiltrate, or abundant degeneration. Only very rare fibers showed centralized nuclei, the hallmark feature of regenerating myofibers. The median total cross-sectional area, diameter of myofibers, and number of myofibers in the tibialis anterior (TA) muscle were significantly reduced in SMA mice compared with heterozygous littermates (Figure 8, A–E). TSA treatment resulted in a statistically significant improvement in total muscle area (P = 0.03) and median myofiber diameter (P < 0.0001; Figure 8, A–D) and a trend toward improvement in median myofiber number (P = 0.08) when the outlier mouse was excluded (Figure 8E). With the outlier included, the increase in myofiber diameter was still statistically significant (P < 0.0001), but the increases in total cross-sectional area and total myofiber number were no longer significant (P = 0.25 and P = 0.39, respectively). TSA treatment resulted in no change in the number of nuclei per myofiber, nor did it cause an increase in the number of myofibers with centralized nuclei (data not shown), indicating that there were no histological features of regeneration.
TSA increases myofiber size and number in SMA mice. SMA mice were treated with vehicle (n = 3) or TSA (n = 3) and heterozygous littermates were treated with vehicle (n = 3) on days P5–P13. (A) H&E-stained cross-sections of TA muscle. Scale bars: 10 μm. (B) Histograms of myofiber diameters. (C) Median TA muscle cross-sectional area was reduced in SMA mice compared with heterozygous littermates (P = 0.05) and increased with TSA treatment (P = 0.03). Lines represent median values, boxes represents the twenty-fifth and seventy-fifth percentiles, whiskers represent values within 1.5 times the interquartile range, and dots represent outliers. (D) Median myofiber diameter in the TA muscle was reduced in SMA mice compared with heterozygous littermates (P < 0.0001) and increased with TSA treatment (P < 0.0001). (E) Median TA muscle total myofiber number was reduced in SMA mice compared with heterozygous littermates (P = 0.05) and increased with TSA treatment (P = 0.08).
In order to further explore the mechanism of improvement in the neuromuscular pathology, we next analyzed the expression pattern of markers associated with muscle regeneration and differentiation. Postnatal myofiber size is determined by 2 distinct mechanisms: (a) regulation of cytoplasmic volume associated with individual myonuclei, a process dependant on rates of protein synthesis and degradation (41), and (b) control of the number of myonuclei within an individual myofiber, a process involving fusion of activated satellite cells with existing fibers or with themselves to form a new myofiber (42). Compared with heterozygous mouse muscle, vehicle-treated SMA mouse muscle showed slightly increased expression of Pax7, desmin, and MyoD, markers associated with early satellite cell activation, and reduced expression of myogenin, a marker of terminal differentiation of muscle cells (Figure 9A). In addition, vehicle-treated SMA mice showed increased expression of perinatal myosin heavy chain (MyHC) and reduced expression of adult MyHC compared with heterozygous littermates (Figure 9B). Together these results indicate immaturity of SMA muscle, as has been described in previous ultrastructural and biochemical studies (43, 44). TSA treatment of SMA mice did not result in an increase in early satellite cell activation markers, but did result in increased expression of myogenin and all of the MyHC isoforms. These results, together with our histological findings, suggest that the increased size of myofibers in TSA-treated SMA mice is caused by enhanced synthesis of contractile filaments and increased maturity of muscle cells rather than by activation of new satellite cells and regeneration.
TSA increases maturity of SMA myofibers. (A) Pax7, desmin, MyoD, and myogenin and (B) embryonic, perinatal, and adult MyHC mRNA levels were determined in muscle isolated from SMA mice treated daily with vehicle (n = 5) or TSA (n = 6) and heterozygous littermates treated with vehicle (n = 5) on days P5–P13. Values represent mean ± SEM.