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Abstract

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Objective

Spinal muscular atrophy (SMA) is a common inherited neuromuscular disorder caused by homozygous loss of function of the survival motor neuron 1 (SMN1) gene. All SMA patients carry at least one copy of a nearly identical SMN2 gene. However, a critical nucleotide change in SMN2 results in alternative splicing and exclusion of exon 7 in the majority of SMN2 messenger RNA (mRNA), thus producing a low level of functional SMN protein. Increasing SMN protein production by promoting SMN2 exon 7 inclusion could be a therapeutic approach for SMA. It has been shown that cellular pH microenvironment can modulate pre-mRNA alternative splicing in vivo. In this study, we tested whether inhibitors of the Na+/H+ exchanger can modulate the exon 7 splicing of SMN2 mRNA

Methods

We treated SMA lymphoid cell lines with Na+/H+ exchanger inhibitors and then measured SMN2 exon 7 splicing by reverse transcriptase polymerase chain reaction and SMN protein production by Western blotting and immunofluorescence

Results

We found that treatment with an Na+/H+ exchanger inhibitor, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), significantly enhances SMN2 exon 7 inclusion and SMN protein production in SMA cells. In addition, EIPA increases the number of nuclear gems in SMA cells. We further explored the underlying mechanism, and our results suggest that EIPA may promote SMN2 exon 7 inclusion through upregulation of the splicing factor SRp20 in the nucleus

Interpretation

Our finding that EIPA, an inhibitor of the Na+/H+ exchanger, can increase SMN protein expression in SMA cells provides a new direction for the development of drugs for SMA treatment. However, further translational studies are needed to determine whether this finding is applicable for SMA treatment or just a proof of cellular pH effect on SMN splicing. Ann Neurol 2007

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by degeneration of motor neurons in the anterior horn of spinal cord, leading to muscular paralysis and atrophy. Clinically, according to the age of onset and its severity, SMA is traditionally categorized into three types with onset during childhood (type I: severe; type II: intermediate; type III: mild) and two additional types (type IV: adult onset of mild symptom; type 0: prenatal onset of severe symptom and early neonatal death).1–3 SMA occurs in approximately 1 in 6,000 to 10,000 live births and has a carrier frequency of 1 in 50. It is the second most common autosomal recessive inherited disorder in humans, and yet the most common genetic cause of infant mortality.4

SMA is caused by the homozygous deletion or mutations of the telomeric copy of the survival motor neuron (SMN1) gene on chromosome 5q13, which encodes the survival motor neuron (SMN) protein.5, 6 A second centromeric copy of the SMN gene (SMN2) also locates at the same chromosomal region.7 However, SMN2 expresses only limited amount of functional full-length SMN protein. A single nucleotide change (C to T) at the 6th position of exon 7 in SMN2 results in about 80% of SMN2 messenger RNA (mRNA) lacking exon 7, in comparison with the SMN1 mRNA that typically includes exon 7.8, 9 The absence of exon 7 in SMN transcripts results in a defective SMN protein with reduced ability for self-oligomerization, leading to protein instability and degradation.10 The SMN protein level in SMA patients is low and insufficient for normal functions in motor neurons.11–13 The C-to-T transition in the exon 7 of SMN2 is thought to disrupt an exonic splicing enhancer recognized by the serine/arginine-rich (SR) splicing factor SF2/ASF14 or to create a novel splicing silencer site bound by heterogeneous nuclear ribonucleoprotein (hnRNP) A1.15 A recent study supports the enhancer-loss model and suggests that hnRNP A1 inhibits exon 7 inclusion by a mechanism that is independent of the C-to-T transition and is, therefore, common to both SMN1 and SMN2.16

The full-length SMN protein is ubiquitously expressed and localizes to both cytoplasm and nucleus. In the nucleus, it appears concentrated in dotlike structures known as gems.17 The SMN protein exists as a component of multiprotein complex, which contains at least seven other proteins, named Gemins2-8, and plays an essential role in the assembly of spliceosomal small nuclear ribonucleoproteins.18, 19 In addition, SMN may also have neuron-specific function in axonal transport of RNA.20, 21

SMN2 gene is a modifier for SMA disease severity. SMA is caused by the loss of SMN1 gene, but the disease severity correlates with the copy number of SMN2 gene and the corresponding amount of full-length SMN protein from these genes.12, 13, 22–24 Thus, increasing the expression of full-length SMN protein by enhancing SMN2 gene transcription and/or promoting SMN2 exon 7 inclusion may reduce the clinical severity of SMA.

Several studies have shown that cellular pH microenvironment may be an important element for the regulation of pre-mRNA alternative splicing in vivo.25, 26 Because the intracellular pH (pHi) homeostasis of many mammalian cell types is controlled by the plasma membrane Na+/H+ exchanger,27, 28 we hypothesized that inhibitors of the Na+/H+ exchanger might be able to modulate the exon 7 splicing of SMN2 mRNA. In this study, we found that an Na+/H+ exchanger inhibitor, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), can enhance the exon 7 inclusion in SMN2 mRNA and the production of SMN protein in SMA cells. In addition, our results also suggest that EIPA may modulate SMN2 exon 7 splicing through upregulation of the SR splicing factor SRp20 expression.

Subjects and Methods

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Spinal Muscular Atrophy Cell Lines and Cell Culture

Human lymphoblast cell lines were established from SMA patients with homozygous SMN1 deletion in our laboratory by Epstein–Barr virus transformation as described elsewhere.29 The cells were grown in RPMI-1640 medium supplemented with 2mM L-glutamine, 10% fetal bovine serum, 100 units/ml penicillin, and 100μg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Human primary fibroblasts were established from skin biopsies of SMA patients with informed consent. The skin biopsies were digested by collagenase, and fibroblasts were isolated by standard procedures. After isolation, the cells were grown in Dulbecco's minimum essential medium/F12 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100μg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2.

Drug Treatment

SMA lymphoid cell lines were plated at 5 × 105 cells/well of a 24-well tissue culture plate and grown overnight before treatment. The cells were treated with various concentrations of EIPA for 24 hours to determine the dosage effect. To determine the time-course effect, we exposed the cells to 10μM EIPA and harvested them at different time intervals (2–24 hours).

Reverse Transcriptase Polymerase Chain Reaction Analysis

After drug treatment, mRNA was extracted from cells using TurboCapture 8 mRNA kit (Qiagen, Chatsworth, CA) according to the manufacturer's instructions. Reverse transcription of the mRNA was performed by using a mixture of oligo(dT) and random primers and Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). Polymerase chain reaction (PCR) was then used to amplify the SMN complementary DNA from exon 6 to exon 8 by using the following pair of primers: forward primer, 5′-CTCCCATATGTCCAGATTCTCTTGATGATGC-3′; reverse primer, 5′-ACTGCCTCACCACCGTGCTGG-3′. The PCR product derived from exon 7–containing SMN2 mRNA (421bp) and that derived from exon 7–lacking SMN2 mRNA (367bp) were separated by agarose gel electrophoresis. The PCR and band intensity quantification were performed as described previously.29

Western Blot Analysis

Protein extract preparation and Western blot analysis were performed essentially as described previously.29 In brief, proteins were resolved by SDS-PAGE and transferred to polyvinyl difluoride membrane (Millipore, Bedford, MA). The membrane was blocked with a 5% skim milk solution and then exposed to the appropriate concentrations of primary antibodies for 1 hour at room temperature. The following primary antibodies were used: mouse monoclonal anti-SMN (1:5,000; BD Biosciences, San Jose, CA), mouse monoclonal anti-SR 16H3 (1:500; Zymed Laboratory, San Francisco, CA), mouse monoclonal anti-SRp20 (1:500; Zymed Laboratory), mouse monoclonal anti-hnRNP A1 (1:1,000; Acris, Hiddenhauser, Germany), mouse monoclonal anti-hnRNP A2/B1 (1:1,000, Aeris), mouse monoclonal anti-Sam68 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-SF2/ASF (1:2,000; Zymed Laboratory), mouse monoclonal anti-Akt (1:1,000; Cell Signaling Technology, Beverly, MA), mouse monoclonal anti-phosphorylated Akt (1:1,000; Cell Signaling Technology), mouse monoclonal anti–α-tubulin (1:5,000; Abcam, Cambridge, MA), and mouse monoclonal anti–β-tubulin (1:2,000; Santa Cruz Biotechnology). After washing with TBST (0.05% Tween 20 in Tris-buffered saline), the membrane was incubated for 1 hour with secondary antibody at a 1:2,500 dilution and then detected by enzyme chemiluminescence kit (Amersham, Buckinghamshire, United Kingdom). Intensity of the signals was measured using the LabWorks software (UVP, Upland, CA).

Subcellular Fractionation

The cytoplasmic and nuclear fractions of cells were isolated by using NE-PER reagent (Pierce, Rockford, IL). In brief, 100μl of ice-cold cytoplasmic extraction reagent (CER) I solution was added to 20mg of cell pellet and vortexed for 15 seconds to fully suspend the pellet. The sample was incubated on ice for 10 minutes; then 5.5μl ice-cold CER II solution was added and vortexed for 5 seconds. The sample was further incubated on ice for 1 minute and then centrifuged at 16,000g for 5 minutes. The supernatant (cytoplasmic fraction) was transferred to a clean, prechilled tube immediately. The pellet that contains nuclei was resuspended in 50μl ice-cold nuclear extraction reagent and vortexed for 15 seconds. The sample was chilled on ice for 10 minutes followed by a 15-second vortexing. After repeating the chilling and vortexing step 4 times, the sample was centrifuged at 16,000g for 10 minutes. The supernatant (nuclear fraction) was immediately transferred to a clean, prechilled tube. All the extracts were stored at −80°C until use.

Nuclear Gem Counting

SMA fibroblast cells were grown on glass coverslips and treated with 10μM EIPA for 24 or 48 hours. After the treatment, cells were fixed with paraformaldehyde and permeabilized with 0.1% Triton X-100 (Sigma) in phosphate-buffered saline (PBS). After blocking with 1% bovine serum albumin in PBS, the cells were treated with a mouse monoclonal anti-SMN antibody (BD Biosciences) for 1 hour, followed by a rhodamine-conjugated secondary antibody for another hour. The cells were also stained with 4′,6-diamidino-2-phenylindole (DAPI) and then visualized by a confocal fluorescence microscope (FluoView; Olympus, Center Valley, PA). At least 100 cells per slide were examined, and the total number of stained gems per 100 cells was counted.

Measurement of Intracellular pH

The procedures of cytofluorometric measurement were modified from a published method.30 In brief, 3 million SMA lymphoid cells were treated with 0, 2, or 10μM EIPA in 3ml of growth medium for 4 hours in a 37°C CO2 incubator, washed in PBS, and then incubated in PBS containing 5μM of carboxy seminaphthorhodafluor-1 (SNARF-1) AM acetate (Molecular Probes, Eugene, OR) for 30 minutes at 37°C, also in the absence or presence of 2 or 10μM EIPA. The SNARF-1 loaded cells collected by centrifugation at 200g for 10 minutes were divided into 7 aliquots, 5 for the calibration standard curves and 2 for sample reading. For the pHi calibration curves, each of the 5 aliquots suspended in high potassium balanced salt solution (K+-BSS = 140mM KCl, 1mM MgCl2, 2mM CaCl2, 5mM glucose) was adjusted to pH 6.8, pH 7.1, pH 7.4, pH 7.7, or pH 8.0 with a 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (Hepes) or bicine buffer, and subsequently added 2μg/ml nigericin (Sigma) and incubated at room temperature for 20 minutes, to equalize the pHi and the buffered pH of the extracellular K+-BSS medium. The aliquots for sample reading were suspended in Hepes-buffered pH 7.1 and pH 7.4 K+-BSS, without or with EIPA addition, but in the absence of nigericin. After standing in the darkness at room temperature for 20 minutes, the SNARF-1 fluorescence measurement was performed in a cytofluorometer (FACSAria; BD Biosciences) by excitation at 488nm and fluorescent emissions at 580 (FL-2) and 640nm (FL-3). The cytofluorometric data were analyzed by using FACSDiVa and CellQuest softwares (Tampa, FL) to gate the major viable cell populations for producing a standard curve of the 640/580nm fluorescence ratios (y-axis) versus the high-K BSS buffer pHs (x-axis) of nigericin-treated cells. The pHi of the SMA lymphoid cells with or without EIPA treatment were determined by using the 640/580nm ratio of the sample aliquots to find the corresponding pH in the standard curve.

Cell Viability and Growth Assays

To determine the cytotoxic effect of EIPA, we exposed 2.5 × 105 lymphoid cells/0.5ml/well in a 12-well dish to 0, 2, 5, 10, 20, or 50μM EIPA. Viability was determined by trypan blue dye exclusion at 24 hours. To determine the cell growth effect of EIPA, we exposed 2.5 × 105 lymphoid cells/0.5ml/well in a 12-well dish to 0, 2, 5, 10, 20, or 50μM EIPA. A total of 0.5ml of fresh medium (without or with the same EIPA addition) was added at 72 hours, and cell counts were made at 120 hours.

Statistical Analysis

Statistical analysis of data was performed using Microsoft Excel 2003 software (Microsoft, Redmond, WA). Student's t test was conducted to evaluate differences between groups. A probability of less than 0.05 was considered to be statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

5-(N-ethyl-N-isopropyl)-amiloride Promotes the Exon 7 Inclusion in SMN2 Messenger RNA in Spinal Muscular Atrophy Cells

To determine whether inhibitors of the Na+/H+ exchanger can modulate the exon 7 splicing of SMN2 mRNA, we treated human SMA lymphoid cell lines with several Na+/H+ exchanger inhibitors and then measured the relative levels of SMN2 exon 7–containing versus exon 7–lacking transcripts by reverse transcriptase PCR and quantitative densitometry. We found that one Na+/H+ exchanger inhibitor, EIPA, can significantly increase the ratio of SMN2 exon 7–containing to exon 7–lacking transcripts at concentrations greater than 2μM (Fig 1A). In addition, time-course experiments showed that the ratio of SMN2 exon 7–containing to exon 7–lacking transcripts was significantly increased 4 hours after treatment with 10μM EIPA (see Fig 1B). We have tested EIPA on six SMA lymphoid cell lines derived from six SMA patients including two type I, two type II, and two type III, and similar results were observed. In summary, our results indicate that EIPA can promote SMN2 exon 7 inclusion in a dose-dependent and time-related manner in SMA cells.

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Figure 1. 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) promotes survival motor neuron 2 (SMN2) exon 7 inclusion in a dose-dependent and time-related manner in spinal muscular atrophy (SMA) cells. (A) Human SMA lymphoid cell lines were treated with increasing concentrations of EIPA (2, 10, and 50μM) for 24 hours. RNA was then isolated and subjected to the reverse transcriptase polymerase chain reaction (RT-PCR) analysis to amplify both the exon 7–containing (exon7+) and the exon 7–lacking (exon7−) SMN2 transcripts. (top) A representative RT-PCR result is shown. (bottom) Bar graph shows the quantitative data of the exon7+/exon7− ratio summarized from three independent RT-PCR experiments. Significant differences between treated and untreated cells were shown (*p < 0.05; **p < 0.01; ***p < 0.001). (B) Human SMA lymphoid cell lines were treated with 10μM EIPA for various time periods (2, 4, 8, and 24 hours). RT-PCR and quantitative analyses were performed and shown as described in (A).

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5-(N-ethyl-N-isopropyl)-amiloride Enhances the Expression of SMN Protein in Spinal Muscular Atrophy Cells

To determine whether EIPA can also increase the production of SMN protein, we treated SMA lymphoid cell lines with various concentrations of EIPA for 24 hours and then measured the level of SMN protein by Western blotting and quantitative densitometry. To gain additional information about subcellular distribution, we separated the cell extracts into cytoplasmic and nuclear fractions, and measured the protein level separately. We found that EIPA at concentrations of 2, 10, and 50μM increased the SMN protein level by 1.8-, 1.4-, and 2.6-fold in the cytoplasm and 2.2-, 2.0-, and 3.3-fold in the nucleus of SMA cells (Fig 2). Thus, EIPA can indeed enhance the expression of SMN protein in SMA cells.

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Figure 2. 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) enhances the survival motor neuron (SMN) protein expression in spinal muscular atrophy (SMA) cells. Human SMA lymphoid cell lines were treated with various concentrations of EIPA (2, 10, and 50μM) for 24 hours. After the treatment, cells were lysed and separated into cytoplasmic and nuclear fractions. Proteins extracted from both fractions were subjected to Western blot analysis to detect the SMN protein and also α-tubulin as an endogenous reference. After quantitative densitometry, the level of SMN protein was first normalized to the level of α-tubulin. The fold of change in the SMN protein level compared with the untreated cells is indicated below the photograph.

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5-(N-ethyl-N-isopropyl)-amiloride Increases the Number of Nuclear Gems in Spinal Muscular Atrophy Cells

Both the level of SMN protein and the number of nuclear gems are markedly reduced in SMA patients. To further confirm the effect of EIPA on the production of SMN protein, we measured the number of nuclear gems in human SMA fibroblast cells with or without EIPA treatment by immunofluorescence and confocal microscopy. The results showed that the total number of nuclear gems per 100 cells increased significantly after treatment with 10μM EIPA for 24 (p = 0.027) or 48 hours (p = 0.013) (Table). Thus, EIPA can increase not only the level of SMN protein but also the number of nuclear gems in SMA cells.

Table  . Increase in the Total Number of Nuclear Gems per 100 Cells after 5-(N-ethyl-N-isopropyl)-amiloride Treatment
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5-(N-ethyl-N-isopropyl)-amiloride Increases the Level of SRp20 in the Cell Nucleus

Several studies have shown that the alternative splicing of SMN exon 7 is controlled by some SR and hnRNP proteins.14, 15, 31, 32 To explore the mechanism of the EIPA effect, we treated SMA lymphoid cell lines with the drug and then examined the change in the levels of several SR and hnRNP proteins by Western blotting and quantitative densitometry. The protein levels in cytoplasmic and nuclear fractions were also measured separately to gain information about subcellular distribution. We found that among the SR and hnRNP proteins examined, EIPA markedly increased the level of SRp20 in the nucleus by approximately fourfold (Fig 3). Our results suggest that EIPA may modulate SMN2 exon 7 splicing through upregulation of SRp20 in the cell nucleus.

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Figure 3. 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) increases the level of SRp20 in the cell nucleus. Human spinal muscular atrophy (SMA) lymphoid cell lines were treated with 10μM EIPA for 24 hours. After the treatment, cells were lysed and separated into cytoplasmic and nuclear fractions. (top) Proteins extracted from both fractions were subjected to Western blot analysis to detect several serine/arginine-rich (SR) and heterogeneous nuclear ribonucleoprotein (hnRNP) proteins as shown and also β-tubulin as an endogenous reference. After quantitative densitometry, the levels of SR and hnRNP proteins were first normalized to the level of β-tubulin. (bottom) Fold of change in the protein levels compared with the untreated cells. Light gray bars indicate cytoplasmic fractions; dark gray bars indicate nuclear fractions.

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5-(N-ethyl-N-isopropyl)-amiloride Does Not Affect the Level of Akt and Its Phosphorylation

The phosphatidylinositol 3-kinase/Akt signaling pathway has been shown to modify SR protein activity in response to certain extracellular cues.33, 34 To determine whether Akt is involved in the effect of EIPA, we treated SMA lymphoid cell lines with various concentrations of EIPA and then measured the levels of total Akt and phosphorylated Akt by Western blotting and quantitative densitometry. We found that EIPA did not cause significant change in the levels of Akt and phosphorylated Akt (Fig 4). Thus, our results suggest that the effect of EIPA may not involve the phosphatidylinositol 3-kinase/Akt signaling pathway.

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Figure 4. 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) does not affect the level of Akt and its phosphorylation. Human spinal muscular atrophy (SMA) lymphoid cell lines were treated with various concentrations of EIPA (2, 10, and 50μM) for 24 hours. After the treatment, cells were lysed and separated into cytoplasmic and nuclear fractions. Proteins extracted from both fractions were subjected to Western blot analysis to detect the Akt protein and its phosphorylated form, as well as the endogenous reference α-tubulin. After quantitative densitometry, the Akt protein levels were first normalized to the level of α-tubulin. The fold of change in the Akt protein levels compared with the untreated cells is shown below the traces.

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Intracellular pH Lowering and Cytotoxic Effects of 5-(N-ethyl-N-isopropyl)-amiloride on Spinal Muscular Atrophy Lymphoid Cells

As a Na+/H+ exchanger inhibitor, EIPA is expected to reduce pHi and may possess some cytotoxicity. Thus, we determined the pHi lowering and cytotoxic effects of EIPA on SMA lymphoid cells. We found that the normal pHi, approximately 7.40, of untreated control cells was reduced to 7.15 ± 0.10 and 7.03 ± 0.15, respectively, in 4 hours by addition of 2μM and 10μM EIPA (Fig 5). The cell viability remained at greater than 90% in 20μM (or lower) EIPA-containing medium after 24 hours but was down to 60% in 50μM EIPA medium (Fig 6A). From a dose–response curve of 120-hour in vitro cultures, it was estimated that 8μM EIPA could cause 50% growth inhibition of SMA lymphoid cells (see Fig 6B).

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Figure 5. Effect of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) on intracellular pH of spinal muscular atrophy (SMA) lymphoid cells. Human SMA lymphoid cells were treated with 0, 2, or 10μM EIPA for 4 hours and then incubated with carboxy SNARF-1 AM. For the calibration standard curve, aliquots of the SNARF-1 loaded cells were suspended in high potassium buffer adjusted to pH 6.8, pH 7.1, pH 7.4, pH 7.7, or pH 8.0 with the subsequent addition of nigericin. For the sample measurement, aliquots of the SNARF-1 loaded cells were suspended in high potassium buffer at pH 7.1 or pH 7.4 without nigericin addition. The SNARF-1 fluorescent emissions at 580 and 640nm were then acquired by flow cytometry. The cytofluorometric data were analyzed as described in Subjects and Methods. The standard curve of the 640/580nm fluorescence ratios (y-axis) versus the high potassium buffer pHs (x-axis) and the corresponding curve-fitting equation are shown. (insets) Intracellular pH of the samples determined by using the 640/580nm ratio to find the corresponding pH in the standard curve are shown.

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Figure 6. Cytotoxic effect of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) on spinal muscular atrophy (SMA) lymphoid cells. Human SMA lymphoid cells were treated with 0, 2, 5, 10, 20, or 50μM of EIPA. (A) Percentages of viable cells were determined by trypan blue dye exclusion at 24 hours. (B) To determine the cell growth effect, we made cell counts at 120 hours.

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Discussion

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Most of SMA patients have SMN2 gene only, and more than 80% of SMN2 transcripts lack exon 7. The amount of exon 7–containing full-length SMN protein has been shown to be an inverse indicator of disease severity in SMA patients and mice.12, 13, 22–24 Therefore, drugs that can increase the expression of full-length SMN protein may have clinically therapeutic value for SMA patients. In this study, we found that EIPA, an inhibitor of the Na+/H+ exchanger, can enhance the exon 7 inclusion in SMN2 mRNA and also the expression of SMN protein in SMA cells. Several compounds were previously reported to increase SMN protein level in SMA fibroblasts or lymphoid cell lines, or both. These compounds can be divided into several categories. The largest category belongs to the histone deacetylase inhibitors, including sodium butyrate,29 valproic acid,35, 36 phenylbutyrate,37 M344,38 and SAHA.39 Compounds in other categories include aclarubicin,40 an anthracycline antibiotic that interacts with DNA and topoisomerases; sodium vanadate,41 a phosphatase inhibitor; indoprofen,42 a nonsteroidal antiinflammatory drug and cyclooxygenase inhibitor; tobramycin and amikacin,43 aminoglycosides that promote read-through of termination codons; and hydroxyurea,44 a ribonucleotide reductase and cell-cycle inhibitor. Although some of these compounds have been tested in pilot human trials,45, 46 definite proof that they are beneficial to SMA patients is still lacking. Thus, there is continuing need to identify more compounds whose efficacy in SMA animals and patients can be evaluated. In this study, our finding that EIPA, an inhibitor of the Na+/H+ exchanger, can increase the SMN protein level in SMA cells adds new category to the list of effective compounds and provides a novel direction for the development of drugs for SMA treatment. However, further translational studies are needed to determine whether this finding is applicable for SMA treatment or just a proof of cellular pH effect on SMN splicing.

It has been reported that changes in extracellular pH can modulate the alternative splicing pattern of tenascin-C pre-mRNA25, 26 and human ATP synthase γ subunit pre-mRNA.47 Because EIPA inhibits the functioning of the Na+/H+ exchanger, this inhibition may cause a change in pHi, which, in turn, modulates the exon 7 splicing of SMN2 mRNA. However, among the several Na+/H+ exchanger inhibitors we have tested, only EIPA can enhance the exon 7 inclusion in SMN2 mRNA, as well as the production of SMN protein. We have determined the pHi lowering and cytotoxic effects of EIPA on the SMA lymphoid cells, which were not significantly different from those reported for lymphoid cells of normal or hypertensive patients.48 Thus, whether the specific effect of EIPA is due to the potencies of these different Na+/H+ exchanger inhibitors or some other feature distinct to EIPA remains to be determined.

Several studies have shown that the alternative splicing of SMN exon 7 is controlled by some SR and hnRNP proteins.14, 15, 31, 32 The C-to-T transition in the exon 7 of SMN2 is thought to disrupt an exonic splicing enhancer recognized by the SR splicing factor SF2/ASF14 or to create a novel splicing silencer site bound by hnRNP A1.15 Although a recent study suggested that the inhibitory effect of hnRNP A1 on exon 7 inclusion is independent of the C-to-T transition, knockdown of hnRNP A1 by small interfering RNA nonetheless increased SMN2 exon 7 inclusion.16 In addition, overexpression of splicing factors such as Tra2-β1, SRp30c, and hnRNP G also promotes SMN2 exon 7 inclusion via a splicing enhancer element located in the middle of exon 7.31, 32, 49 Additional regulatory elements, both in exon 750 and in flanking introns,51 also influence exon 7 recognition, but their associated factors are not well defined. Thus, many regulatory elements and splicing factors can modulate the exon 7 splicing efficiency, even if they are not directly involved in the SMN2 exon 7 splicing defect. In this study, we found that EIPA can increase the expression of the SR splicing factor SRp20. Although SRp20 has not been shown to interact with the exon 7 of SMN2, it binds to the pre-mRNA sequences of Drosophila doublesex gene,52 CD44 alternative exon v9,53 SRp20 exon 4,54 and calcitonin/calcitonin gene–related peptide gene,55 and modulates their splicing. SRp20 also regulates the exon 10 skipping in the tau transcript.56 Interestingly, M344, an histone deacetylase inhibitor that promotes SMN2 exon 7 inclusion and SMN protein production, also increases the expression level of SRp20.38 Thus, an increase in the level and/or activity of SRp20 may promote the SMN2 exon 7 inclusion by a mechanism that remains to be characterized.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by the National Science Council of Taiwan (NSC-95-2320-B-037-049, J.-G. C.; NSC-96-2314-B-037-036-MY3, J.-G. C., C. Y. Y.).

We thank T. S. Lin for technical assistance in the early phase of this project.

References

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References