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Research ArticleArticle

Effects of Inhibitors of SLC9A-Type Sodium-Proton Exchangers on Survival Motor Neuron 2 (SMN2) mRNA Splicing and Expression

Sambee Kanda, Emily Moulton and Matthew E.R. Butchbach
Molecular Pharmacology August 2022, 102 (2) 92-105; DOI: https://doi.org/10.1124/molpharm.122.000529

Abstract

Spinal muscular atrophy (SMA) is an autosomal recessive, pediatric-onset disorder caused by the loss of spinal motor neurons, thereby leading to muscle atrophy. SMA is caused by the loss of or mutations in the survival motor neuron 1 (SMN1) gene. SMN1 is duplicated in humans to give rise to the paralogous survival motor neuron 2 (SMN2) gene. This paralog is nearly identical except for a cytosine to thymine transition within an exonic splicing enhancer element within exon 7. As a result, the majority of SMN2 transcripts lack exon 7 (SMNΔ7), which produces a truncated and unstable SMN protein. Since SMN2 copy number is inversely related to disease severity, it is a well established target for SMA therapeutics development. 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of sodium/proton exchangers (NHEs), has previously been shown to increase exon 7 inclusion and SMN protein levels in SMA cells. In this study, NHE inhibitors were evaluated for their ability to modulate SMN2 expression. EIPA as well as 5-(N,N-hexamethylene)amiloride (HMA) increase exon 7 inclusion in SMN2 splicing reporter lines as well as in SMA fibroblasts. The EIPA-induced exon 7 inclusion occurs via a unique mechanism that does not involve previously identified splicing factors. Transcriptome analysis identified novel targets, including TIA1 and FABP3, for further characterization. EIPA and HMA are more selective at inhibiting the NHE5 isoform, which is expressed in fibroblasts as well as in neuronal cells. These results show that NHE5 inhibition increases SMN2 expression and may be a novel target for therapeutics development.

SIGNIFICANCE STATEMENT This study demonstrates a molecular mechanism by which inhibitors of the sodium-protein exchanger increase the alternative splicing of SMN2 in spinal muscular atrophy cells. NHE5 selective inhibitors increase the inclusion of full-length SMN2 mRNAs by targeting TIA1 and FABP3 expression, which is distinct from other small molecule regulators of SMN2 alternative splicing. This study provides a novel means to increase full-length SMN2 expression and a novel target for therapeutics development.

Introduction

Proximal spinal muscular atrophy (SMA) is an early-onset neurodegenerative disease characterized by the loss of α-motor neurons in the anterior horn of the spinal cord, which leads to muscle weakness and atrophy (Crawford and Pardo, 1996; Tisdale and Pellizzoni, 2015). SMA is an autosomal recessive disease with an incidence of 1 in 6000–10,000 births (Pearn, 1978; Cuscó et al., 2002). SMA results from the loss or mutation of survival motor neuron 1 (SMN1) con chromosome 5q13 (Lefebvre et al., 1995). Uniquely in humans, a large tandem chromosomal duplication has led to a second copy of this gene known as survival motor neuron 2 (SMN2) (Lorson et al., 1999; Monani et al., 1999). SMN2 is functionally distinguishable from SMN1 by a single nucleotide difference (SMN2 c.850C>T) in exon 7 that disrupts an exonic splice enhancer. As a result, about 80%–90% of SMN2 mRNAs lack exon 7 (SMNΔ7) and produce a protein that is both unstable and not fully functional (Lorson and Androphy, 2000; Burnett et al., 2009; Cho and Dreyfuss, 2010). Because 10%–20% of the SMN2 gene product is fully functional (Lorson et al., 1999; Monani et al., 1999), increased genomic copies of SMN2 inversely correlate with disease severity among individuals with SMA (Butchbach, 2016). Studies using transgenic mouse models for SMA have shown that increased SMN2 copy number lessens the phenotypic severity of disease (Hsieh-Li et al., 2000; Monani et al., 2000; Michaud et al., 2010).

As SMN2 is a major genetic modifier of SMA phenotype, it has become the primary target for the development of small molecule therapies for SMA (Cherry et al., 2014). SMN2 gene expression can be regulated by increasing promoter activation, increasing inclusion of exon 7 in SMN2 mRNA transcripts, and including translational readthrough of SMNΔ7 mRNAs (Calder et al., 2016). Although there is currently no cure for SMA, a splice modifying oligonucleotide that increases SMN2 exon 7 inclusion (nusinersen, Spinraza) recently received Food and Drug Administration (FDA) approval for SMA patients (Finkel et al., 2017; Mercuri et al., 2018). Splice-modifying oligonucleotides, however, have suboptimal properties, including not being able to cross the blood-brain barrier, not being orally bioavailable, potentially being toxic at high doses, and being expensive to manufacture (Sumner and Crawford, 2018). Despite these exciting advances, other therapies are needed, particularly if they are complementary to these current therapeutic options.

Small molecule inducers of SMN2 exon 7 inclusion have been identified. NVS-SM1 (branaplam) is a small molecule SMN2 exon 7 splicing modulator that is orally bioavailable and central nervous system penetrant (Palacino et al., 2015). The pyridopyrimidinone class of small molecule modulators of SMN2 exon 7, including RG7800 and RG7916 (risdiplam), have also shown efficacy in cell culture as well as in animal models for SMA (Naryshkin et al., 2014; Feng et al., 2016; Ratni et al., 2016; Woll et al., 2016; Sivaramakrishnan et al., 2017; Ratni et al., 2018; Wang et al., 2018). Risdiplam (Evrysdi; Genetech and Roche) was recently approved by the FDA for treating SMA patients (Baranello et al., 2021). Other classes of small molecules have been identified as modulators of SMN2 exon 7 splicing. 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of sodium/proton exchangers (NHEs, also known as SLC9A family), upregulates SMN2 expression in SMA lymphoblastoid cells by increasing the inclusion of exon 7 in SMN2 transcripts (Yuo et al., 2008). In this study, we examine the effects of other NHE inhibitors—both structurally related to EIPA as well as other classes of inhibitors—on SMN2 alternative splicing at exon 7 and SMN expression in SMA cells. The modulatory effects of EIPA and its analogs are also compared against RG7800, a well characterized SMN2 exon 7 splicing modulator.

Materials and Methods

Test Compounds

Amiloride, cariporide, EIPA, 5-(N,N dimethyl)amiloride (DMA), and zoniporide were obtained from Cayman Chemicals (Ann Arbor, MI), and 5-(N, N-hexamethylene)amiloride (HMA) was purchased from Sigma-Aldrich (St. Louis, MO). The structures of the amiloride test compounds are shown in Fig. 1. RG7800 was obtained from MedKoo Biosciences, Inc (Morrisville, NC). All stock solutions were made by dissolving the compound in DMSO (Sigma-Aldrich).

Fig. 1.
Fig. 1.

Chemical structures of the SLC9A-type NHE inhibitors tested.

Cell Culture

Fibroblast cells derived from type II SMA patients (GM03813, GM22592, and AIDHC-SP22) have a homozygous deletion of SMN1 and three copies of SMN2 (Stabley et al., 2015; Stabley et al., 2017). GM03814 fibroblasts (Scudiero et al., 1986) were derived from the carrier mother of GM03813, with one copy of SMN1 and five copies of SMN2 (Stabley et al., 2015). GM03813, GM22592, and GM03814 fibroblasts were obtained from Coriell Cell Repositories (Camden, NJ), and the other fibroblast lines were generated at Nemours Children’s Hospital Delaware (Stabley et al., 2017). All fibroblast lines were authenticated using short tandem repeat profiling and digital polymerase chain reaction (PCR) (Stabley et al., 2017). The SMN2 exon 7 splicing reporter cell line NSC-34:SMN2:Mg2:bla5.3 (Andreassi et al., 2001) was obtained from Vertex Pharmaceuticals (Boston, MA).

Fibroblast lines as well as NSC-34:SMN2:Mg2:bla5.3 cells were maintained in Dulbecco’s modified essential medium (Life Technologies, Grand Island, NY) containing 10% EquaFETAL (Atlas Biologicals; Fort Collins, CO), 2 mM L-glutamine (Life Technologies), and 1% penicillin-streptomycin (Life Technologies). All cell lines were maintained in a humidified chamber at 37°C with 5% CO2.

SMN2 Exon 7 Splicing Reporter Assay

NSC-34:SMN2:Mg2:bla5.3 cells (Andreassi et al., 2001) were seeded onto black-walled, clear-bottom 96-well tissue culture plates (Santa Cruz Biotechnology, Dallas, TX) at a density of 5 × 104 cells per cm2. Drug compounds (n = 4 per dose) were added to serum-free medium at a dilution of 1:500. One-hundred microliters of drug-containing medium was then added to maintenance medium over the seeded NSC-34 cells. After incubation for 19 hours, media containing drug compounds was aspirated, and 100 µl of fresh maintenance medium was added to each well. Twenty microliters of 6X CCF2-AM loading solution (GeneBlazer In Vivo Detection Kit, Life Technologies; containing 6 μm CCF2-AM and 12 mM probenecid) were added to each of the assay wells, and plates were incubated at room temperature for 2 hours before the plates were read on a Victor ×4 (Perkin Elmer, Waltham, MA) fluorescence plate reader (λex = 405 nm, λem = 530 nm, and λem = 460 nm). The 460-nm:530-nm fluorescence ratios were then calculated for each sample.

Drug Treatment of Cells

Fibroblasts were seeded 24 hours prior to drug treatment at a density of 3.2 × 104 cells per well in a 6-well plate for RNA analysis and of 4.0 × 105 cells per dish in a 10-cm dish for protein analysis. Test compounds were added to each sample at a 1:1000 dilution, and compound-containing medium was replaced every 24 hours during the 5-day treatment period. Cells were then harvested 24 hours after last drug compound treatment.

RNA Isolation

Total RNA was extracted from cell pellets using the RNeasy Mini kit (QIAGEN; Germantown, MD) per the manufacturer’s instructions. RNA quality was assessed using a 2100 Bioanalyzer (Applied Biosystems).

SMN Exon 7 Inclusion Assay

First-strand cDNA was synthesized from total RNA (500 ng) using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) as described previously (Gentillon et al., 2017). PCRs were run for sample cDNAs using GoTaq Green Polymerase Mix (Promega; Madison, WI) with the following primer sets (Integrated DNA Technologies; Coralville, IA): SMN, SMNex6 (F) 5′-cccatatgtccagattctcttgat-3′; SMNex8(R) 5′-ctacaacacccttctcacag-3′; and human collagen-IIIA (COL3A), COL3A (F) 5′-gctctgcttcatcccactatt-3′; COL3A(R) 5′-ggaataccagggtcaccattt-3′. PCR products were resolved through a 2% agarose gels via electrophoresis. Images were captured with an AlphaImager gel documentation station (ProteinSimple, San Jose, CA), and band intensities were quantified using Image J 1.45s (National Institutes of Health, Bethesda, MD).

Quantitative Reverse Transcriptase-Polymerase Chain Reaction (qPCR)

qPCR was completed for each treated sample using the iScript cDNA Synthesis Kit and the Bullseye EvaGreen qPCR MasterMix (Midsci, Valley Park, MO) as described previously (Gentillon et al., 2017). Each sample was assayed in triplicate. The following primer sets were used: full-length SMN (FL-SMN) (SMNex6F) 5′-ccatatgtccagattctcttgatga-3′, (SMNex78R) 5′-atgccagcatttctccttaattta-3′; SMNΔ7 (SMNex6F), (SMNex68R) 5′-atgccagcatttccatataatagc-3′; full-length striatin-3 (FL-STRN3) (STRN3F) 5′-ggaagaaaggggtgaagagg-3′, (STRN3R) 5′-tgattcctgaagggatgtgg-3′; STRN3 lacking exons 8 and 9 (STRN3Δ89) (STRN3D89F) 5′-cagaatgggctgaaccaataa-3′, (STRN3D89R) 5′-accgtcaagtctgcaaggtc-3′; forkhead box protein M1A (FOXM1A) (FOXM1AF) 5′-gaacatgaccatcaaaaccgaactc-3′, (FOXM1AR) 5′-aaattaaacaagctggtgatgggtg-3′; (FOXM1B) (FOXM1BF) 5′-ggaccaggtgtttaagcagcag-3′, (FOXM1BR) 5′-caatgcggactcgcttgctat-3′; (FOXM1C) (FOXM1CF) 5′-ttgcccgagcagttggaatca-3′, (FOXM1CR) 5′-tcctcagctagcagcaccttg-3′; heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) (hnRNPA1-F) 5′-agggcgaaggtaggctggca-3′, (hnRNPA1-R) 5′-gcttcctcagctcttcgggct-3′; transformer 2 β homolog (hTra2β1) (hTRA2bF) 5′-cacatcgaccggcgacagca-3′ (hTRA2bR) 5′-ccccgatccgtgagcacttcc-3′; splicing factor-2 homolog/alternative-splicing factor (SF2/ASF) (hSF2ASF-F) 5′-cagagtggttgtctctg-3′, (hSF2ASF-R) 5′-ctccacgacaccagtgcc-3′; Src-associated in mitosis 68 kDa (SaM68) (hSAM68F) 5′-atctctgtaattgggaaagggc-3′, (hSAM68R) 5′-agagcataagcctcacatgg-3′; serine/arginine-rich splicing factor 20 kDa (SRp20) (hSRp20F) 5′-atgcatcgtgattcctg-3′, (hSRp20R) 5′-ctgcgacgaggtggagg-3′; T-cell–restricted intracellular antigen-1 (TIA1) (TIA1-F) 5′-cagcgttcacaagatcatttcc-3′, (TIA1-R) 5′-tcccttagactttcctgttgc-3′; fatty acid binding protein 3 (FABP3) (FABP3-F) 5′-aaatgggacgggcaagag-3′, (FABP3-R) 5′-tgcctctttctcataagtgcg-3′; 7-dehydrocholesterol reductase (DHCR7) (DHCR7-F) 5′-gcaacccaacattcccaaag-3′, (DHCR7-R) 5′-agtgaaaaccagtccacctc-3′; transient receptor potential cation channel subfamily V member 4 (TRPV4) (TRPV4-F) 5′-accttttccgattcctgctc-3′, (TRPV4-R) 5′-tcctcattgcacaccttcatg-3′; ataxin-1 (ATXN1) (ATXN1-F) 5′-catccagagtgcagagataagc-3′, (ATXN1-R) 5′-ctctaccaaaacttcaacgctg-3′; and protein kinase cAMP-dependent type II regulatory subunit β (PRKAR2B) (PRKAR2B-F) 5′-tgatcaaggtgacgatggtg-3′, (PRKAR2B-R) 5′-tgtacattaaggccagttcgc-3′. Primers for the human reference transcripts β-actin, large ribosomal protein P0, and glyceraldehyde-3-phosphate dehydrogenase were purchased from Real Time Primers LLC (Elkins Park, PA).

The relative transcript levels were calculated using the efficiency-adjusted 2-ΔΔCt method (Schmittgen and Livak, 2008; Yuan et al., 2008). The PCR efficiency (E) for each primer set was calculated from the slope of a cycle threshold (Ct) versus log10(cDNA serial dilution) curve (E = 10[-1/slope]) (Pfaffl, 2001). ΔCt,adjusted is the difference between the adjusted Ct (Ct,measured × E) for the target transcript and the geometric mean of the adjusted Ct values for the three reference genes, and ΔΔCt is defined as the difference between the ΔCt for the SMA sample and the ΔCt for the control sample.

Immunoblot

Protein extracts were generated from cell pellets as described previously (Gentillon et al., 2017). Protein extracts from treated cells (15 μg protein/lane) were resolved from miniPROTEAN TGX gradient precast acrylamide gels (BioRad) via electrophoresis as described previously (Gentillon et al., 2017). The resolved proteins were then transferred onto polyvinylidene difluoride membranes via electroblotting. Immunoblotting was completed as described in (Gentillon et al., 2017). The following antibodies were used in this study: mouse anti-SMN monoclonal antibody (1:2000; clone 8, BD Biosciences), mouse anti–β-actin monoclonal antibody (1:10,000; clone AC15, Sigma-Aldrich, St. Louis, MO), horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies (1:5000; Rockland Immunochemicals, Inc., Gilbertsville, PA). After extensive washing, the bound antibody was detected by chemiluminescence using either Western Sure ECL Substrate (LiCor, Lincoln, NE) or SuperSignal West Femto (Thermo Scientific) kits and captured with the C-DiGit Blot Scanner (LiCor). Band intensities, defined as the band signal divided by the band area, were measured using the Image StudioTM Lite software (LiCor). The measured band areas were the same for each sample on a blot. Band intensities for the target protein (SMN) were divided by those for the reference protein (β-actin) to obtain normalized band intensities. To measure the relative protein level for a sample, the normalized band intensity for the drug-treated sample was divided by the normalized band intensity for the control sample (DMSO-treated cells).

Microarray

cDNAs from treated RNA samples—with RNA integrity numbers greater than 9.0—were prepared using the GeneChip WT PLUS Reagent Kit (Applied Biosystems, Foster City, CA). Double-stranded cDNA was synthesized from 100 ng total RNA using a random primer that incorporated a T7 promoter. This double-stranded cDNA was then used as a template to generate cRNA via a 16-hour in vitro transcription reaction followed by purification with magnetic beads. Single-stranded cDNA was regenerated from this cRNA through a random primed reverse transcription using a deoxynucleoside triphosphate mix containing deoxyuridine triphosphate. After RNA hydrolysis with RNase H, the cDNA was purified using magnetic beads and then enzymatically fragmented with a mixture of uracil-DNA glycosylase and apyrinic/apyrimidinic endonuclease 1. This fragmented cDNA was then end labeled with a biotinylated dideoxynucleotide using terminal transferase. Fragmented, biotinylated cDNA was added to a hybridization cocktail, denatured, loaded on a Clariom D human GeneChips, and hybridized for 16 hours at 45°C and 60 rpm. Following hybridization, the chips were washed and stained using the preprogrammed FS450_0001 protocol. The stained chips were scanned at 532 nm with a GeneChip Scanner 3000 (Applied Biosystems).

The resultant data were analyzed with the TAC 4.0 software (Applied Biosystems). The raw data have been deposited into the National Center for Biotechnology Information Gene Expression Omnibus (Barrett et al., 2013) under the accession number GSE179861. Identification of biologic pathways and upstream regulators was completed using Ingenuity Pathway Analysis (version 21901358; QIAGEN Redwood City, Inc., Redwood City, CA) as described previously (Maeda et al., 2014). Biologic function and canonical pathways were determined to be over-represented using the Fisher exact test with a false discovery rate correction (P ≤ 0.05). Upstream regulators were considered as being activated if their z-scores were greater than or equal to 2.0 or inhibited if they were less than or equal to −2.0 (Krämer et al., 2014).

Statistical Analysis

Data are expressed as mean plus or minus standard error. Parametric data were analyzed by ANOVA with a Holm-Sidak (expression analysis) post hoc test. Statistical significance was set at P ≤ 0.05. Comparisons between data were performed with Sigma Plot v.12.0 or SPSS v.22.0.

Results

Effects of NHE Inhibitors on SMN2 Exon 7 Alternative Splicing

To determine the effect of NHE inhibitors on increasing SMN2 expression, we first examined their effects on the inclusion of SMN2 exon 7. Using a SMN2 exon 7 splicing reporter assay established in motor neuron–like NSC-34 cells (Andreassi et al., 2001), we measured the effects of amiloride, DMA, EIPA, HMA, cariporide, and zoniporide (Fig. 1) on β-lactamase (BLA) activity—a marker for SMN2 exon 7 inclusion. The EIPA and HMA significantly increased exon 7 inclusion, as shown by an increase in the λ460nm to λ530nm fluorescence ratio, in these reporter cells (Fig. 2A). Amiloride, cariporide, and zoniporide, on the other hand, significantly reduced BLA activity.

Fig. 2.
Fig. 2.

Effects of NHE inhibitors on SMN2 alternative splicing. (A) SMN2 exon 7 inclusion reporter cells (NSC-34:SMN2:Mg2:bla5.3) were treated with varying concentrations of the NHE inhibitors amiloride, DMA, EIPA, HMA, cariporide, or zoniporide (1 nM to 10 µM; n = 4 per dose) or DMSO for 19 hours. BLA activity was measured fluorimetrically. (B and C) Effect of NHE inhibitors on SMN2 exon 7 inclusion in type II SMA fibroblasts. Type II SMA fibroblasts (GM03813) were treated with varying concentrations (100 nM to 10 μM; n = 3 per group) of NHE inhibitors or DMSO for 5 days (n = 3 per treatment group). After total RNA isolation, samples were analyzed for relative amounts of FL-SMN and SMNΔ7 transcripts by RT-PCR and agarose gel electrophoresis. COL3A served as a loading control in this assay. The relative amounts of FL-SMN and SMNΔ7 transcripts were also measured on carrier fibroblasts (GM03814). The asterisk (*) denotes a statistically significant (P < 0.05) difference between NHE inhibitor- and vehicle-treated cells.

We also examined the effect of NHE inhibitors on the inclusion of exon 7 in SMN2 mRNAs in a SMA intracellular environment by using patient-derived fibroblasts. GM03813 type II SMA fibroblasts (Scudiero et al., 1986) were treated with different concentrations of amiloride, DMA, EIPA, HMA, zoniporide, or cariporide for 5 days. After treatment, SMN2 exon 7 inclusion was measured via reverse-transcription polymerase chain reaction (RT-PCR) using primers spanning exons 6 through 8 of SMN2. COL3A transcript levels were used as a loading control as it is highly and constitutively expressed in fibroblast cells (Heier et al., 2007). As shown in Fig. 2B, the proportion of FL-SMN (top band) relative to SMNΔ7 (bottom band) transcripts was increased in SMA fibroblasts treated with EIPA and HMA but not in any of the other NHE inhibitors. HMA was more potent at increasing SMN2 exon 7 inclusion than EIPA (Fig. 2C).

There are 5 different isoforms of SLC9A-type Na+/H+ antiporters (NHE1, NHE2, NHE3, NHE4, and NHE5) that are present on the plasma membrane (Masereel et al., 2003). Using RT-PCR, we determined the SLC9A isoform expression profiles for NSC-34 cells as well as for GM03813 and GM03814 fibroblasts. NSC-34 cells as well as fibroblast cell lines express predominantly NHE1 and NHE5 (Supplemental Fig. 1).

Effects of NHE Inhibitors on SMN2 Expression in Type II SMA Fibroblasts

We treated GM03813 type II SMA fibroblasts with different concentrations (0.1–10 µM) of the NHE inhibitors for 5 days. FL-SMN and SMNΔ7 transcript levels were measured by qPCR. EIPA and HMA significantly increase FL-SMN mRNA levels in GM03813 cells to about 80% of the amount of FL-SMN seen in carrier fibroblasts (GM03814) (Fig. 3A). Amiloride, cariporide, and zoniporide, however, reduced the abundance of FL-SMN transcripts in SMA fibroblasts. EIPA was the only NHE inhibitor that significantly reduced the levels of SMNΔ7 mRNA in treated cells (Fig. 3B). To demonstrate that these observations were not unique to a single SMA cell line, we measured the effects of the NHE inhibitors on FL-SMN and SMNΔ7 transcript levels in two other type II SMA fibroblast lines— GM22592 and AIDHC-SP22. EIPA and HMA also increased FL-SMN transcripts in GM22592 and AIDHC-SP22 cells, indicating that their effects on SMN2 mRNA regulation are cell-line independent (Fig. 3C). SMNΔ7 transcript levels were also reduced in AIDHC-SP22 and GM22592 fibroblasts treated with EIPA or HMA (Fig. 3D).

Fig. 3.
Fig. 3.

Effects of NHE inhibitors on expression of FL-SMN and SMNΔ7 mRNA transcripts in SMA patient-derived fibroblasts. GM03813 type II SMA fibroblasts were treated with different concentrations of NHE inhibitors (100 nM to 10 μM; n = 3 per dose) or DMSO for 5 days. Changes in FL-SMN (A) or SMNΔ7 (B) transcript levels were measured via quantitative RT-PCR with β-actin, glyceraldehyde-3-phosphate dehydrogenase, and large ribosomal protein P0 serving as reference transcripts. FL-SMN and SMNΔ7 transcript levels were also measured in GM03814 carrier fibroblasts. Changes in FL-SMN (C) or SMNΔ7 (D) transcript levels were measured in two other type II SMA fibroblast lines (GM22592 and AIDHC-SP22) treated with NHE inhibitors (10 μM; n = 3 per inhibitor) for 5 days. All transcript levels were expressed relative to DMSO-treated GM03813 cells (dashed line). The asterisk (*) denotes a statistically significant (P < 0.05) difference between NHE inhibitor- and vehicle-treated cells.

We measured SMN protein levels of GM03813 type II SMA fibroblasts treated with the aforementioned NHE inhibitors for 5 days (Fig. 4). Amiloride, cariporide, and zoniporide had marginal effects on SMN protein levels in these cells. EIPA and HMA increased SMN protein levels in GM03813 fibroblasts, with EIPA showing a maximal effect at 10 μM, whereas the maximal effect of HMA was observed at 1 μM. Interestingly, DMA also increased SMA protein levels in SMA fibroblasts even though it had no effect on FL-SMN mRNA levels nor on exon 7 inclusion. This observation suggests that the DMA affects SMN gene regulation at a different level from EIPA and HMA.

Fig. 4.
Fig. 4.

Effects of NHE inhibitors on SMN protein levels in SMA fibroblasts. GM03813 type II SMA fibroblasts were treated with different concentrations of NHE inhibitors (100 nM to 10 μM; n = 3 per dose) or DMSO for 5 days. Changes in SMN protein levels were measured via immunoblot using β-actin as a reference protein. SMN protein levels were also measured in GM03814 carrier fibroblasts.

Effects of NHE Inhibitors on Alternative Splicing of STRN3 and FOXM1 in SMA Fibroblasts

We measured the effects of the NHE inhibitors on the alternative splicing of other transcripts—aside from SMN2—that are affected by the pyridopyridinone RG7800 (Ratni et al., 2016; Woll et al., 2016) to determine if EIPA and HMA operate via a similar mechanism to promote exon 7 inclusion. STRN3 has a similar pre-mRNA structure to the SMN2 exon 7:intron 7 junction, and RG7800 increases the inclusion of STRN3 exons 8 and 9 (Naryshkin et al., 2014; Sivaramakrishnan et al., 2017). There are 3 isoforms of FOXM1 generated by alternative splicing of exons Va and VIIa: FOXM1A (which contains both exons Va and VIIa), FOXM1B (which contains neither exon), and FOXM1C (which contains only exon Va) (Liao et al., 2018). RG7800 increased the abundance of FOXM1A while reducing FOXM1C levels (Ratni et al., 2018).

The levels of STRN3 and FOXM1 splice variants were measured in GM03813 type II SMA fibroblasts treated with amiloride, EIPA, HMA, or RG7800 for 5 days. EIPA and HMA had no effect on the amount of FL-STRN3 mRNA, whereas amiloride significantly increased FL-STRN3 transcript levels (Fig. 5A). RG7800 also increased the abundance of FL-STRN3 transcripts in treated SMA fibroblasts. Interestingly, all NHE inhibitors tested increased STRN3Δ89 mRNA levels (Fig. 5B), whereas RG7800 reduced the amount of STRN3Δ89 transcripts. None of the NHE inhibitors increased the levels of FOXM1A in SMA fibroblasts (Fig. 5C). EIPA but not amiloride nor HMA significantly decreased FOXM1C transcript levels in SMA fibroblasts (Fig. 5D). FOXM1B transcripts could not be detected in fibroblast samples (data not shown). Predictably, RG7800 increased relative FOXM1A levels and reduced the amount of FOXM1C transcript levels in treated cells (Fig. 5, C and D). The mechanism of action of EIPA and HMA on the alternative splicing of SMN2 exon 7, therefore, is distinct from that used by RG7800.

Fig. 5.
Fig. 5.

Effects of the NHE inhibitors on alternative splicing of STRN3 and FOXM1 in type II SMA fibroblasts. GM03813 type II SMA fibroblasts were treated with 10 μM amiloride, 10 μM EIPA, 10 μM HMA, 1 μM RG7800, or DMSO (n = 3 per group) for 5 days. The levels of FL-STRN3 (A), STRN3Δ89 (B), FOXM1 containing exons Va and VIIa [FOXM1A (C)], and FOXM1 containing exon Va [FOXM1C; (D)] transcripts were measured in total RNA extracted from treated cells by quantitative RT-PCR. All transcript levels were expressed relative to DMSO-treated GM03813 cells (dashed line). The asterisk (*) denotes a statistically significant (P < 0.05) difference between drug- and vehicle-treated cells.

Effects of NHE Inhibitors on Expression of Regulators of SMN2 Exon 7 Splicing

To understand the mechanism of action for the increased inclusion of exon 7 in SMN2 transcripts induced by EIPA and HMA, we first examined the effects of NHE inhibitors on the expression of previously identified proteins that modulate the splicing of SMN2 at exon 7. We focused on the following splicing regulators: hnRNP-A1 (Kashima et al., 2007a; Kashima et al., 2007b; Doktor et al., 2011; Harahap et al., 2012), SF2/ASF (SRSF1) (Cartegni and Krainer, 2002; Cartegni et al., 2006; Wee et al., 2014), hTra2β1 (SRSF10) (Helmken and Wirth, 2000; Hofmann et al., 2000; Hofmann and Wirth, 2002; Chen et al., 2015), SaM68 (KHDRBS1) (Pedrotti et al., 2010; Pagliarini et al., 2015), and SRp20 (SRSF3) (Helmken et al., 2003). The transcript levels of these splicing factors were measured in type II SMA fibroblasts treated with NHE inhibitors or DMSO for 5 days. hnRNP-A1 transcript levels were significantly reduced in GM03813 cells treated with DMA, EIPA, HMA, and zoniporide (Fig. 6A). DMA, EIPA, HMA, and cariporide reduced SF2/ASF levels in SMA fibroblasts (Fig. 6B). Cariporide was the only NHE inhibitor to increase hTra2β1 mRNA levels (Fig. 6C). HMA significantly reduced SaM68 transcript levels in SMA fibroblasts (Fig. 6D). EIPA, HMA, cariporide, and zoniporide decreased SRp20 mRNA levels, whereas DMA increased SRp20 transcript levels (Fig. 6E). Interestingly, hrRNP-A1, hTra2β1, and SRp20 mRNA levels are significantly elevated in GM03813 type II SMA fibroblasts relative to GM03814 carrier fibroblasts (Fig. 6, A, C, and E). Although the NHE inhibitors differentially regulate the expression of splicing factors that regulate SMN2 exon 7 inclusion, there was no correlation between the differential expression of any of these splicing factors and the enhanced inclusion of SMN2 exon 7 induced by EIPA or HMA in SMA fibroblasts.

Fig. 6.
Fig. 6.

Effects of NHE inhibitors on the expression of splicing regulators involved in SMN2 exon 7 alternative splicing. GM03813 type II SMA fibroblasts were treated with 10 μM NHE inhibitors (amiloride, DMA, EIPA, HMA, cariporide, or zoniporide) or DMSO (n = 3 per group) for 5 days. hnRNP-A1 (A), SF2/ASF (B), hTRA2β1 (C), SaM68 (D), and SRp20 (E) transcript levels were measured in total RNA extracted from treated cells by quantitative RT-PCR. Transcript levels were also measured in GM03814 carrier fibroblasts. All transcript levels were expressed relative to DMSO-treated GM03813 cells (dashed line). The asterisk (*) denotes a statistically significant (P < 0.05) difference between drug- and vehicle-treated cells.

Identification of Differentially Expressed Transcripts in SMA Fibroblasts Treated with EIPA

To understand the molecular mechanisms by which EIPA enhances SMN2 exon 7 inclusion, we compared the transcriptomes of GM03813 type II SMA fibroblasts treated with 10 μM EIPA against those treated with DMSO as well as against those treated with 10 μM amiloride, which did not increase SMN2 exon 7 inclusion. Principal component analysis correctly distributed each of the samples within their treatment groups (Fig. 7A). Hierarchical clustering of the identified transcripts from amiloride-treated (Fig. 7B) and EIPA-treated (Fig. 7C) fibroblasts showed consistent differential expression between each treatment group. Amiloride treatment of GM03813 SMA fibroblasts altered the levels of 1269 transcripts when compared against DMSO-treated cells (Fig. 7D; Supplemental Table 1A). There were 999 differentially expressed transcripts in SMA fibroblasts treated with 10 μM EIPA when compared against those cells exposed to DMSO (Fig. 7E; Supplemental Table 1B). To identify those differentially expressed transcripts that may be relevant to SMN2 alternative splicing, we compared the EIPA transcriptome against the amiloride transcriptome and identified 839 EIPA-unique differentially expressed transcripts (Fig. 7F; Supplemental Table 1C).

Fig. 7.
Fig. 7.

Identification of differentially expressed transcripts in type II SMA fibroblasts treated with amiloride or EIPA. GM03813 type II SMA fibroblasts were treated with 10 μM amiloride, 10 μM EIPA, or DMSO (n = 3 per group) for 5 days, and their RNA pools were analyzed for differential transcript expression using Clariom D human transcriptome arrays. (A) Principal component analysis of samples treated with amiloride (purple), EIPA (red), or DMSO (blue). Hierarchical clustering analysis of amiloride versus DMSO (B) and EIPA versus DMSO (C). Volcano plots of amiloride versus DMSO (D) and EIPA versus DMSO (E) type II SMA fibroblast transcriptomes. Significantly upregulated transcripts are shown in red, and significantly downregulated transcripts are shown in blue. (F) Venn diagram showing the similarities and differences between the amiloride versus DMSO (red) and EIPA versus DMSO (blue) transcriptomes. The overlap between these two transcriptomes is shown in purple. (G) The top dozen canonical pathways—out of 165—that were significantly over-represented in the EIPA-unique transcriptome. The numbers next to the pathway lines represent the number of differentially expressed molecules for each pathway. (H) The upstream regulators that are significantly and uniquely differentially regulated in EIPA-treated type II SMA fibroblasts. (I) Distributions of the categorized differential splicing events between amiloride versus DMSO and EIPA versus DMSO transcriptomes.

Ingenuity Pathways Analysis (Krämer et al., 2014) uses a manually curated literature database to determine the biologic relevance of differentially expressed transcripts. There were 165 canonical pathways that were significantly overrepresented (Fisher’s exact test P value ≤ 0.05) in EIPA-treated SMA fibroblasts relative to amiloride-treated cells (Supplemental Table 2), with the top 12 overrepresented pathways shown in Fig. 7G. Most of the top 12 overrepresented pathways contained Ras-family GTPases (RAP2A, RAP1A, and MRAS) and subunits of the phosphatidylinositol-4-phosphate 3-kinase (PIK3R1, PIK3C2G, and PIK3CB). Upstream regulator analysis (Krämer et al., 2014) can identify potential upstream molecules that may be responsible for EIPA-mediated differential gene expression. UPA identified 19 potential upregulators (10 of which were activated and 9 were inhibited) in EIPA-treated SMA fibroblasts relative to amiloride-treated fibroblasts (Fig. 7H; Supplemental Table 3). Thrombospondin-1 (THBS1) and DHCR7 are overrepresented target molecules in this analysis.

Transcriptome arrays can also provide important information about differential splicing in response to drug treatment. There were 10,307 splicing events that were differentially expressed in amiloride-treated GM03813 SMA fibroblasts, whereas EIPA treatment showed 8307 differentially expressed splicing events. Of those events, only 352 were classifiable as either intron retention, alternative 5′ donor, alternative 3′ acceptor, or cassette exon events in amiloride-treated cells and 251 in EIPA-treated cells (Fig. 7I). The splicing index (SI) is a measure of exon expression that is normalized to the expression level of that gene (Clark et al., 2007). In EIPA-treated cells, there was an increase in the SMN2 exons 6 and 7 splice junction (JUC0500051219; SI = +2.34, P = 0.0031) and a decrease in SMN2 exons 6 and 8 splice junction (JUC0500051223; SI = -2.68; P = 0.0086). Amiloride treatment, however, did not significantly alter the abundance of either splice junction. The amount of SMN2 exon 7 inclusion was, therefore, increased in EIPA-treated SMA fibroblasts.

To validate our microarray analysis, we used biologic replicates of type II SMA fibroblasts (Fang and Cui, 2011) that were treated with either 10 μM EIPA, 10 μM HMA, 10 μM amiloride, 1 μM RG7800, or DMSO. We focused on the following transcripts: TIA1 (Fig. 8A; 2.06-fold decrease), FABP3 (Fig. 8B; 6.44-fold increase), DHCR7 (Fig. 8C; 2.07-fold decrease), TRPV4 (Fig. 8D; 2.13-fold decrease), ATXN1 (Fig. 8E; 2.58-fold decrease), and PRKAR2B (Fig. 8F; 2.15-fold increase). The differential expressions of these transcripts, with respect to direction, in response to EIPA treatment were validated in the biologic replicates. Transcripts that are differentially expressed only in EIPA- and HMA-treated SMA fibroblasts would potentially provide insights into the molecular mechanisms underlying EIPA- and HMA-induced SMN2 exon 7 inclusion. FABP3 (Fig. 8B) transcript levels were markedly increased in cells treated with EIPA and HMA but not with amiloride or RG7800. EIPA and HMA as well as RG7800 reduced TIA1 transcript levels (Fig. 8A) in SMA fibroblasts. For the remaining transcripts, the direction of change in response to EIPA treatment was different from that to HMA, i.e., increased in EIPA-treated cells but decreased in HMA-treated cells.

Fig. 8.
Fig. 8.

Validation of EIPA- and HMA-responsive transcripts in type II SMA fibroblasts. GM03813 type II SMA fibroblasts were treated with 10 μM amiloride, 10 μM EIPA, 10 μM HMA, 1 μM RG7800, or DMSO (n = 3 per group) for 5 days. TIA1 (A), FABP3 (B), DHCR7 (C), TRPV4 (D), ATXN1 (E), and PRKARB2 (F) transcript levels were measured in total RNA extracted from treated cells by quantitative RT-PCR. All transcript levels were expressed relative to DMSO-treated GM03813 cells (dashed line). The asterisk (*) denotes a statistically significant (P < 0.05) difference between drug- and vehicle-treated cells.

Discussion

Because of the inverse relationship between SMA severity and SMN2 copy number, SMN2 is a primary target of SMA treatment and drug discovery through multiple mechanisms including promoter activation and increased exon 7 inclusion of SMN2 pre-mRNA transcripts (Cherry et al., 2014). Many structurally distinct small molecules such as EIPA (Yuo et al., 2008), the pyridopyrimidinones RG7800 and RG7916 (Naryshkin et al., 2014; Feng et al., 2016; Ratni et al., 2016; Woll et al., 2016; Sivaramakrishnan et al., 2017; Ratni et al., 2018; Wang et al., 2018), and NVS-SM1 (Palacino et al., 2015) increase SMN2 expression by enhancing exon 7 inclusion. EIPA is a derivative of amiloride and inhibits the activity of the SLC9A family of Na+/H+ antiporters (Kleyman and Cragoe, 1988). In this study, we examined the effects of amiloride derivatives like EIPA and HMA as well as other NHE inhibitors on SMN2 alternative splicing of exon 7. EIPA and HMA but none of the other SCL9A inhibitors tested increase SMN2 exon 7 inclusion via a novel mechanism not involving previously identified regulators of SMN2 exon 7 splicing.

There are 5 different isoforms of SLC9A-type Na+/H+ antiporters that are localized to the plasma membrane in mammalian cells (Masereel et al., 2003). NHE1 is ubiquitously expressed in most mammalian cell types, whereas NHE5 is primarily expressed in neurons and skeletal muscle (Donowitz et al., 2013). Tissue distribution profiles of NHE1 and NHE5 in humans and mice show strong expression in the tissues from which the cell lines used in this study were derived, i.e., brain and skin (Fagerberg et al., 2014; Yue et al., 2014; Cheng et al., 2019). EIPA and the related amiloride analog HMA are potent inhibitors of the NHE1 and NHE5 isoforms (Kleyman and Cragoe, 1988; Szabó et al., 2000; Masereel et al., 2003). Cariporide and zoniporide, on the other hand, are selective inhibitors of NHE1 (Masereel et al., 2003).

EIPA modulates neuronal plasticity and long-term potentiation in mice (Rönicke et al., 2009). NHE5 has been shown to be involved in neuronal excitation and long-term potentiation by negatively regulating dendrite spine growth in an activity-dependent manner (Diering et al., 2011). NHE5 knockout mice display enhanced learning and memory and increased BDNF/TrkB-mediated signaling (Chen et al., 2017). NHE5 also regulates the membrane trafficking of the receptor tyrosine kinase Met and β1 integrins in glioma cells (Fan et al., 2016; Kurata et al., 2019). NHE5 is positively regulated by AMP-activated protein kinase (AMPK) in neuronal as well as nonneuronal cells (Jinadasa et al., 2014). NHE5 membrane localization is regulated by phosphatidylinositol 3-kinase (PI3K) activity and the actin cytoskeleton (Szászi et al., 2002). In addition to these roles in neuronal signaling, NHE5 regulates autophagy in neuronal cells (Togashi et al., 2013). Based on our observations, the selectivity of EIPA and HMA in increasing SMN2 exon 7 inclusion may result from inhibition of a specific NHE isoform, in this case NHE5. The regulation of alternative splicing by NHE5 has not been previously reported; future studies using gene knockdown approaches in SMA model systems will further elucidate the role of NHE5 in SMN2 exon 7 splicing. It is possible that selective inhibition of NHE5 may not be sufficient to increase SMN2 exon 7 inclusion. To address this possibility, future studies would determine if inhibition of other NHE isoforms, like NHE1, would be necessary for or would augment SMN2 alternative splicing resulting from inhibition of NHE5.

The pyridopyridinone RG7800—which is undergoing clinical trials with SMA patients—increases SMN2 exon 7 inclusion by binding to an exonic splice enhancer (ESE2) element present on the 5′ splice site of the exon 7:intron 7 junction (Ratni et al., 2016; Woll et al., 2016; Sivaramakrishnan et al., 2017). Binding at these sites facilitates the binding of U1 small nuclear ribonucleoproteins by dissociation of the inhibitory splicing factor hnRNP-G. STRN3 has a similar pre-mRNA structure to the SMN2 exon 7:intron 7 junction, and RG7800 increases the inclusion of STRN3 exons 8 and 9 (Naryshkin et al., 2014; Sivaramakrishnan et al., 2017). We show that EIPA and HMA have no effect on exon 8 and 9 inclusion in STRN3 transcripts. Furthermore, EIPA and HMA do not modulate the alternative splicing of FOXM1, another transcript whose splicing is modulated by RG7800 (Ratni et al., 2018). These data suggest that EIPA and HMA modulate SMN2 exon 7 alternative splicing via a mechanism that is distinct from the pyridopyridinones.

NHE antiporters regulate the cellular pH in mammalian cells (Putney et al., 2002; Masereel et al., 2003). Alterations in pH have been shown to affect the splicing of multiple mRNA transcripts including tenascin C and SMN2 (Borsi et al., 1995; Chen et al., 2008). Low extracellular pH increases SMN2 exon 7 skipping, whereas a high extracellular pH promotes exon 7 inclusion (Chen et al., 2008). The decrease in exon 7 inclusion at low pH may be the result of diminished nuclear localization of hnRNP-A1, a splicing factor that prevents exon 7 inclusion via binding to an exonic enhancer element (Chen et al., 2008). EIPA (Yuo et al., 2008) and elevating extracellular pH (Chen et al., 2008) increase the nuclear localization of the splicing factor SRp20. In this study, we did not identify any relationship between the differential expression of any of these splicing factors and the enhanced inclusion of SMN2 exon 7 induced by EIPA or HMA in SMA fibroblasts. Furthermore, the effects of EIPA and HMA on SMN2 exon 7 alternative splicing may not be linked with regulation of cellular pH as other NHE1-selective inhibitors like cariporide and zoniporide do not alter SMN2 exon 7 splicing. The effects of EIPA and HMA on SMN2 alternative splicing may be mediated by a novel mechanism.

TIA1 is a splicing factor that has been shown to increase SMN2 exon 7 levels (Singh et al., 2011). Loss of Tia1 worsens disease progression in female, but not male, SMA-like mice (Howell et al., 2017). We show here that EIPA and HMA decrease TIA1 mRNA levels in SMA fibroblasts even though these compounds increase SMN2 exon 7 inclusion. Muscle biopsies from patients with Welander distal myopathy that harbor a point mutation in TIA1 [TIA1(E384K)] have reduced FL-SMN2 transcript levels but elevated SMNΔ7 transcript levels (Klar et al., 2013); however, a recent report (Carrascoso et al., 2018) has shown that mutant TIA1 only modestly affects SMN2 exon 7 alternative splicing in different cell types. Future studies will elucidate the role of NHE5 inhibition by EIPA and HMA on TIA1 expression and the regulation of TIA1 expression on the modulation SMN2 exon 7 alternative splicing.

FABP3 transcript levels were markedly elevated in SMA fibroblasts treated with EIPA or HMA but not by other NHE inhibitors. FABP3 is robustly expressed in neurons, as well as other nonneural tissues, and is responsible for intracellular transport of long-chain polyunsaturated fatty acids (Liu et al., 2010; Falomir-Lockhart et al., 2019). FABP3 increases the aggregation of α-synuclein within dopaminergic neurons of the substantia nigra pars compacta, which leads to cell death and neurodegeneration (Shioda et al., 2014). In murine GABAergic neurons within the anterior cingulate cortex, FABP3 modulates the expression of glutamic acid decarboxylase (Gad67) by differential promoter methylation (Yamamoto et al., 2018). Further studies examining the effect of increased FABP3 expression on SMN2 alternative splicing would provide important insights into a novel mechanism of gene regulation.

Although EIPA and HMA are potent inhibitors of NHE-type antiporters (Masereel et al., 2003), it is possible their mode of action with respect to SMN2 exon 7 inclusion may be separate from NHE inhibition. Certain amilorides can also inhibit different types of Ca2+-activated nonspecific cation channels like acid-sensing ion channel 1A or transient receptor potential P3, also known as polycystin-2 (Dai et al., 2007; Leng et al., 2016). It is possible that the effects of EIPA and HMA on SMN2 alternative splicing may be mediated by inhibition of these other channels. To address this possibility, SMA fibroblasts and other cellular models can be treated with more specific acid-sensing ion channel 1A or transient receptor potential P3 inhibitors, like phenamil and benzamil (Dai et al., 2007; Leng et al., 2016), to monitor their effects on SMN2 exon 7 inclusion.

SMA can now be considered an actionable disease since there are currently 3 therapies approved by the FDA for SMA patients: nusinersen (Finkel et al., 2017; Mercuri et al., 2018), risdiplam (Baranello et al., 2021), and onasemnogene abeparvovec (Mendell et al., 2017). Despite these exciting advances, other therapies are needed, particularly if they are complementary to current therapeutic options. Traditional small-molecule therapies have been the mainstay of the pharmaceutical industry for several important reasons. Small molecule inducers of SMN2 expression could serve as complementary therapies for SMA patients who are either not good candidates or poor responders to biologic therapies. NHE5 inhibitors like EIPA and HMA may be able to serve this complementary role, but they will need to be tested in animal models for SMA. The identification of more precise targets for therapeutic development will ultimately lead to additional drug candidates for the treatment of SMA, which can be used either alone or in combination with existing SMA therapies.

Acknowledgments

The authors would like to thank Dr. Norman Gerry and the staff at Advanced BioMedical Laboratories, LLC (Cinnaminson, NJ) for completing the microarray experiments; Vertex Pharmaceuticals, Inc. for generously providing the NSC34:SMN2.Mg2.bla5.3 reporter cell line; and the Nemours Biomolecular Core Laboratory for access to the 7900HT Fast Real-Time PCR system.

Note Added in Proof: The title of this article was changed from that used in the Fast Forward version that appeared online June 6, 2022.

Authorship Contributions

Participated in research design: Kanda, Butchbach.

Conducted experiments: Kanda, Moulton.

Contributed new reagents or analytic tools: Butchbach.

Performed data analysis: Kanda, Moulton, Butchbach.

Wrote or contributed to the writing of the manuscript: Kanda, Moulton, Butchbach.

Footnotes

    • Received March 15, 2022.
    • Accepted May 9, 2022.

  • This work was supported by National Institutes of Health Institute of General Medical Sciences [Grant P20-GM103446] (M.E.R.B.) and [Grant P20-GM103464] (M.E.R.B.), Nemours Foundation, and University of Delaware Graduate Research Fellowship.

  • No author has an actual or perceived conflict of interest with the contents of this article.

  • https://doi.org/10.1124/molpharm.122.000529.

  • ↵Embedded ImageThis article has supplemental material available at molpharm.aspetjournals.org.

Abbreviations

ATXN1
ataxin-1
BLA
β-lactamase
COL3A
collagen IIIA
Ct
cycle threshold
DHCR7
7-dehydrocholesterol reductase
DMA
5-(N,N-dimethyl)amiloride
E
efficiency
EIPA
5-(N-ethyl-N-isopropyl)amiloride
FABP3
fatty acid binding protein 3
FDA
Food and Drug Administration
FL-SMN
full-length SMN
FL-STRN3
full-length striatin-3
FOXM1
forkhead box protein M1
HMA
5-(N,N-hexamethylene)amiloride
hnRNPA1
heterogeneous nuclear ribonucleoprotein A1
hTra2β1
transformer 2 β homolog
NHE
sodium/proton exchanger
PCR
polymerase chain reaction
PRKAR2B
protein kinase cAMP-dependent type II regulatory subunit β
RT-PCR
reverse-transcription polymerase chain reaction
SaM68
Src-associated in mitosis 68 kDa
SF2/ASF
splicing factor 2 homolog/alternative-splicing factor
SI
splicing factor
SMA
spinal muscular atrophy
SMN1
survival motor neuron 1
SMN2
survival motor neuron 2
SRp20
serine/arginine-rich splicing factor 20 kDa
STRN3
striatin 3
STRN3Δ89
STRN3 lacking exons 8 and 9
TIA1
T-cell–restricted intracellular antigen 1
TRPV4
transient receptor potential cation channel subfamily V member 4

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EIPA, HMA, and SMN2 Gene Regulation

Sambee Kanda, Emily Moulton and Matthew E.R. Butchbach
Molecular Pharmacology August 1, 2022, 102 (2) 92-105; DOI: https://doi.org/10.1124/molpharm.122.000529
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