Skip Navigation


Human Molecular Genetics Advance Access originally published online on March 24, 2005
Human Molecular Genetics 2005 14(9):1199-1210; doi:10.1093/hmg/ddi131
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
14/9/1199    most recent
ddi131v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (24)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Wolstencroft, E. C.
Right arrow Articles by Lorson, C. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wolstencroft, E. C.
Right arrow Articles by Lorson, C. L.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

A non-sequence-specific requirement for SMN protein activity: the role of aminoglycosides in inducing elevated SMN protein levels

Elizabeth C. Wolstencroft, Virginia Mattis, Anna A. Bajer, Philip J. Young{dagger} and Christian L. Lorson*

Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211-7310, USA

* To whom correspondence should be addressed at: Department of Veterinary Pathobiology Life Sciences Center, 471G, 1201 Rollins Road, University of Missouri, Columbia, MO 65211-7310, USA. Tel: +1 5738842219; Fax: +1 5738849395; Email: lorsonc{at}missouri.edu

Received February 1, 2005; Revised March 13, 2005; Accepted March 13, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Spinal muscular atrophy (SMA) is caused by homozygous loss of the survival motor neuron (SMN1) gene. In virtually all SMA patients, a nearly identical copy gene is present, SMN2. SMN2 cannot fully compensate for the loss of SMN1 because the majority of transcripts derived from SMN2 lack a critical exon (exon 7), resulting in a dysfunctional SMN protein. Therefore, the critical distinction between a functional and a dysfunctional SMN protein is the inclusion or the exclusion of the exon 7 encoded peptide. To determine the role of the 16 amino acids encoded by SMN exon 7, a panel of synthetic mutations were transiently expressed in SMA patient fibroblasts and HeLa cells. Consistent with previous reports, the protein encoded by SMN exons 1–6 was primarily restricted to the nucleus. However, a variety of heterologous sequences fused to the C-terminus of SMN exons 1–6 allowed mutant SMN proteins to properly distribute to the cytoplasm and to the nuclear gems. These data demonstrate that the SMN exon 7 sequence is not specifically required, rather this region functions as a non-specific ‘tail’ that facilitates proper localization. Therefore, a possible means to restore additional activity to the SMN{Delta}7 protein could be to induce a longer C-terminus by suppressing recognition of the native stop codon. To address this possibility, aminoglycosides were examined for their ability to restore detectable levels of SMN protein in SMA patient fibroblasts. Aminoglycosides can suppress the accurate identification of translation termination codons in eukaryotic cells. Consistent with this, treatment of SMA patient fibroblasts with tobramycin and amikacin resulted in a quantitative increase in SMN-positive gems and an overall increase in detectable SMN protein. Taken together, this work describes the role of the critical exon 7 region and identifies a possible alternative approach for therapeutic intervention.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Spinal muscular atrophy (SMA) is a neurodegenerative disorder characterized by loss of the {alpha}-motor neurons of the spinal cord, resulting in progressive atrophy of the limb and trunk muscles (1Go). SMA can be divided into three clinical subgroups, on the basis of age of onset and severity of the symptoms (1Go,2Go). In all instances, SMA is caused by mutations affecting the survival motor neuron (SMN) gene (3Go,4Go). Two nearly identical copies of SMN are present on chromosome 5q13 : SMN1 and SMN2 (3Go). SMN2 cannot fully compensate for the loss of SMN1 because the majority of SMN2-derived transcripts are alternatively spliced and encode a truncated protein that is unstable and only partially functional. The SMN1 allele produces high levels of a full-length SMN transcript of nine exons encoding 294 amino acids. A translation termination codon is present at the 3' end of exon 7; therefore, in the full-length transcript, exon 8 is non-coding (3Go). In contrast, SMN2 produces low levels of the full-length protein and high levels of an aberrantly spliced form that lacks exon 7 (SMN{Delta}7) (3Go,5Go). This alternative splicing event is caused by a silent ‘C’ to ‘T’ transition, which disrupts an exon splicing enhancer site six nucleotides into SMN exon 7 (5Go,6Go). Owing to the exclusion of exon 7, translation of SMN{Delta}7 is terminated at a stop codon four amino acids into exon 8 (3Go).

Humans are the only species that contain the SMN2 gene (7Go,8Go); however, SMN2 performs an essential role in the development of disease and is an important modifier of disease severity. Accordingly, the number of SMN2 copies generally correlates with disease severity: increased SMN2 copies result in a decrease in disease severity (9Go–11Go). Although essentially all SMA patients retain at least one copy of SMN2 (3Go,4Go), the native SMN2 gene and its transcripts have been perceived as ideal candidates for therapeutic intervention.

The 38 kDa SMN protein is ubiquitously expressed and localizes to the cell cytoplasm, the nucleolus and to the punctate nuclear structures, called gems (gemini of coiled bodies) or Cajal bodies (12Go–14Go). In cells and tissues from SMA patients, the number of gems is reduced such that in tissue derived from severe patients, fewer than five gems per 100 nuclei are observed relative to unaffected tissues in which ~75–100 gems per observed in 100 nuclei (11Go,15Go,16Go).

The SMN protein has been shown to be involved in U snRNP biogenesis and pre-mRNA splicing (17Go–19Go), transcription (20Go,21Go), nucleocytoplasmic transportation (22Go), apoptosis (23Go–25Go) and ribosomal assembly (14Go,26Go). The SMN{Delta}7 protein interacts less efficiently with essentially all SMN-interacting factors and is defective in most functional assays. Although SMN exon 7 does not directly mediate the multitude of SMN activities, the requirement for exon 7 is likely indirect and based on its role in SMN self-association and protein stability. A number of groups have demonstrated that SMN functions at least as a dimer; therefore, self-association is a prerequisite for SMN activities. In the absence of efficient self-association, essentially all downstream activities are disrupted to a degree that reflects the defect in self-association (27Go,28Go). It remains unclear why removal of the small 16 amino acid sequence encoded by exon 7 destabilizes the protein to such an extent. It has been suggested that a five amino acid sequence in exon 7 (QNQKE) is responsible for SMN cytoplasmic localization (29Go), although it is currently unclear whether this region is necessary for SMN localization in the context of the endogenous SMN protein (30Go).

Here, we demonstrate that a variety of native and synthetic C-terminal amino acids can restore wild-type SMN subcellular localization patterns when fused to SMN exons 1–6. Substitution of the native SMN exon 7 with a variety of heterologous sequences that varied in length and amino acid composition, demonstrate that the SMN protein requires a non-specific C-terminal peptide, which allows proper targeting of the SMN protein to the cytoplasm and nuclear gems. In addition, fusion of amino acids derived from exon 8 onto the C-terminus of SMN exons 1–6 functionally substitutes for the excised exon 7 sequences. On the basis of these observations, a panel of aminoglycosides were tested for their ability to induce SMN protein levels in SMA patient fibroblasts. These drugs were chosen on the basis of their ability to suppress stop codon identification (31Go–39Go), which in the case of SMN{Delta}7, would result in a protein with an additional five amino acids. Although some drugs were highly toxic, tobramycin and amikacin resulted in significantly higher levels of SMN-positive gems in SMA type I fibroblasts and total SMN protein levels. Taken together, these experiments demonstrate a critical function for the SMN C-terminus and highlight a novel therapeutic approach for the treatment of SMA.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Distribution of SMN mutants in HeLa cells and SMA type 1 patient fibroblasts
To investigate the role of the peptide encoded by SMN exon 7 in the subcellular distribution of the SMN protein, a series of SMN exon 7 mutant constructs were generated (Fig. 1A). These included a construct that consisted exclusively of SMN exons 1–6 (SMN 1–6); the primary product from SMN2, SMN{Delta}7, which encodes SMN exons 1–6 and the first four amino acids from exon 8 (SMN{Delta}7) and three SMN constructs with premature stop codons introduced to create an exon 7 of four (SMN 7–4), nine (SMN 7–9) or 14 (SMN 7–14) amino acids (Fig. 1A). In each of these three instances, the remaining exon 7 sequences were native exon 7 sequences.





View larger version (163K):
[in this window]
[in a new window]
  		Figure 1. Expression of transiently transfected SMN mutant constructs (A) Schematic of SMN exon 7 and exon 8 mutant constructs. Exons 1–6- (hatched), exon 7- (solid) and exon 8-derived (spotted) coding regions are indicated. White region indicates alanine substitutions for the QNQKE motif in exon 7. (B) Distribution of SMN mutants in type I SMA patient fibroblasts. HA-tagged SMN (and derivatives) was visualized by immunofluorescence with an anti-HA primary antibody and an FITC-conjugated secondary antibody. Nuclei were stained with DAPI. (C) Distribution of SMN mutants in HeLa cells. HA-tagged SMN (and derivatives) were visualized by immunofluorecence with an anti-HA primary antibody and a FITC-conjugated secondary antibody. Nuclei were stained with DAPI.

 
Each of the epitope-tagged (HA) SMN cDNAs were transiently transfected in HeLa cells and SMA type I patient fibroblasts (3813) (Fig. 1B and C). The 3813 cells do not contain SMN1 but do contain at least two copies of SMN2; therefore, endogenous SMN protein levels are exceptionally low and gems occur infrequently (approximately five gems per 100 nuclei) (12Go,15Go). As expected, transiently expressed full-length SMN localized to punctate nuclear bodies and also resulted in diffuse cytoplasmic staining (Fig. 1B and C). Consistent with previous findings, SMN 1–6 displayed a predominantly nuclear localization in HeLa cells, with the majority of the HA-tagged SMN protein found in large nuclear aggregates (Fig. 1C) (29Go). In contrast, the SMN2-derived SMN{Delta}7 construct and each of the constructs with a truncated exon 7 displayed patterns similar to wild-type SMN, with high levels of diffuse cytoplasmic protein and numerous nuclear gems (Fig. 1C). Similarly, a construct in which the reported exon 7 nuclear export sequence QNQKE had been replaced by a motif of five alanine residues ({Delta}QNQKE) was tested. In 3813 (Fig. 1B) and HeLa cells (Fig. 1C), this construct displayed a subcellular distribution similar to full-length SMN, resulting in high levels of cytoplasmic SMN and abundant nuclear foci. This suggests the cytoplasmic targeting of SMN is mediated by the C-terminal peptide in a non-sequence-specific manner and does not specifically require the QNQKE sequence for cytoplasmic localization.

To further test the hypothesis that the requirement for SMN exon 7 was non-sequence-specific, we produced a panel of SMN mutant constructs derived from SMN{Delta}7. This series of mutations created synthetic fusions at the C-terminus of SMN{Delta}7 by fusing sequences derived from the non-coding sequence of SMN exon 8. The first mutant, named SMN 8–16, contains an 18 bp deletion that removes the first stop codon of exon 8 and causes a shift in the reading frame. This frame shift results in the translation of a 16 amino acid peptide, derived from exon 8 sequences, equivalent in size to the deleted exon 7 (Fig. 1A). This construct has been further altered by the incorporation of stop codons, to produce mutants containing the first seven (8Go–7Go), 10Go (8Go–10Go) and 14Go (8Go–14Go) amino acids of exon 8 (Fig. 1A). These constructs were transiently expressed in HeLa (data not shown) and 3813 cells (Fig. 1B). All mutant constructs examined produced a pattern similar to full-length SMN (Fig. 1B). In contrast to SMN 1–6 (Fig. 1B and C) which was retained exclusively in the nucleus, constructs comprising SMN exons 1–6 fused to a variety of C-terminal sequences derived from exon 7 or 8 are present in the nuclear gems and diffusely throughout the cytoplasm (Fig. 1B). These results demonstrate that proper SMN subcellular distribution can be achieved by a variety of heterologous C-terminal peptides and that SMN cytoplasmic localization does not specifically require SMN exon 7 sequences.

Aminoglycoside treatment of 3813 type I SMA patient fibroblasts
Although tethering a four amino acid peptide to the C-terminus of the 1–6 appeared to restore the proper cellular distribution of the SMN protein, the addition of four amino acids must not confer complete functionality to the SMN truncated protein because SMN{Delta}7 is unstable and essentially no SMN protein can be detected in 3813 cells (skin fibroblasts from an SMA type I patient). The 5' end of exon 8 contains two stop codons: the first stop codon occurs after the fourth codon and is utilized in the production of SMN{Delta}7; the second stop codon, which is within the 3' untranslated region of exon 8, is present an additional 18 nt downstream from the native stop codon. Therefore, if the native stop codon of SMN{Delta}7 could be experimentally induced to be ‘read-through’, the resulting SMN{Delta}7-derived protein would consist of exons 1–6 and nine amino acids derived from exon 8. This artificially lengthened C-terminal peptide could confer higher levels of functionality and stability when compared with the SMN{Delta}7 protein.

To test whether the SMN{Delta}7 read-through product resulted in subcellular distributions similar to wild-type SMN, a read-through SMN{Delta}7 product (SMNKO) was generated (Fig. 1A). To create the SMNKO construct, the SMN 1–6 coding sequence was fused to the first 30 nt derived from SMN exon 8, with the exception that the native stop codon for SMN{Delta}7 transcripts was mutated to encode for an alanine residue. The SMN exon 8 sequence encoded nine amino acids in total, followed by a stop codon. Transiently expressed SMNKO displayed a similar distribution pattern to full-length SMN in HeLa cells and 3813 fibroblasts, as evidenced by the abundant levels of cytoplasmic SMN and SMN-positive nuclear foci (Fig. 1B and C). Therefore, we hypothesized that treatment of SMA patient fibroblasts with compounds that suppressed the accurate identification of the SMN{Delta}7 stop codon would allow the incorporation of additional amino acids derived from exon 8 that could further stabilize the SMN{Delta}7-derived protein.

Aminoglycosides are a group of drugs that comprise a number of FDA approved antibiotics that can alter protein synthesis in mammalian cells by disrupting translational fidelity, resulting in the incorporation of an amino acid at stop codon positions (31Go–39Go). To determine whether aminoglycoside administration could increase gem numbers and total SMN protein levels in SMA patient fibroblasts, 3813 cells (human type I SMA patient fibroblasts) were treated with a select group of aminoglycosides. Several hundred aminoglycosides are commercially available; however, in this study, we selected drugs that had been previously characterized regarding the ability to induce the misincorporation of stop codons or premature termination codons (31Go–39Go). The effect of aminoglycoside treatment on 3813 patient fibroblasts was determined using a panel of antibiotics in a range of concentrations (75 and 150 µg/ml) previously shown to alter translational fidelity in eukaryotic cells: amikacin, geneticin, gentamicin, lividomycin, streptomycin and tobramycin. Fibroblasts were treated with two drug concentrations (75 or 150 µg/ml) and the effect on SMN protein distribution was initially examined by indirect immunofluorescence analysis of nuclear gem levels following 48 h of drug treatment (Fig. 2A). Nuclear gem levels have previously been used as a marker to determine a compound's (such as valproic acid, phenyl butyrate and aclarubicin) ability to elevate SMN protein levels in SMA patient fibroblasts compounds (15Go,40Go,41Go).





View larger version (154K):
[in this window]
[in a new window]
  		Figure 2. Aminoglycoside treatment of SMA type I patient fibroblasts. (A) SMN distribution in aminoglycoside treated SMN type I patient fibroblasts. Cells were treated with a panel of aminoglycoside antibiotics at 75 or 150 µg/ml. FITC indicates endogenous SMN; DAPI was used to identify nuclei. (B) SMN distribution in valproic acid treated SMN type I patient fibroblasts. Cells were treated with 150 µg/ml valproic acid. FITC indicates endogenous SMN, and DAPI was used to counterstain nuclei. (C) Aminoglycoside treatment does not result in large-scale nuclear re-organization. Sm core protein and p80 coilin were visualized in IGCs and Cajal bodies, respectively, by using monoclonal antibodies (anti-Sm, Y12; anti-p80 coilin, A3) against each factor and an FITC conjugated secondary antibody. The 3813 cells were untreated (top panel) or treated for 48 h with 150 µg/ml tobramycin or amikacin. Localization patterns were similar in carrier SMA fibroblasts, 3814 cells (data not shown). DAPI was used to identify nuclei.

 
Treatment of 3813 fibroblasts with gentamycin had no detectable effect on SMN distribution at any concentration examined (Fig. 2A). Cells exposed to low concentrations (75 µg/ml) of geneticin, lividomycin and streptomycin resulted in a small increase in gem numbers (Fig. 2A). However, higher concentrations (150 µg/ml) of geneticin and lividomycin were toxic, triggering high levels of cell death (data not shown). Similarly, higher concentrations (150 µg/ml) of streptomycin were cytotoxic (data not shown) and resulted in a redistribution of SMN to the nucleolus (Fig. 2A). In parallel control experiments, treatment of 3813 cells with valproic acid resulted in higher levels of gems as previously reported (Fig. 2B) and did not result in increased levels of cell death when compared with untreated controls (data not shown).

Valproic acid is an histone-deacetylase inhibitor that increases SMN protein levels by activating the SMN2 promoter and suppressing the alternative splicing of SMN2 exon 7 (40Go,42Go). Although valproic acid serves as a positive control for gem formation in these assays, it is likely that mechanisms for SMN induction are different for valproic acid and the aminoglycosides. Similar drug treatments on HeLa cells or an SMA carrier fibroblasts cell line (3814 cells) did not result in detectable increases in gem numbers above untreated controls (data not shown).

Fibroblasts treated with 75 or 150 µg/ml of tobramycin or amikacin resulted in an increase in the number of nuclear gems without the toxicity associated with the previous aminoglycosides (Fig. 2A). The elevated gem numbers were detectable 48 h after exposure to either aminoglycoside (Fig. 2A). The increased number of gems observed following tobramycin or amikacin treatment was not due to a general cellular response because the frequency of other nuclear bodies such as Cajal bodies and interchromatin granules (as identified by p80 coilin and Sm core protein staining, respectively) appeared normal following amikacin or tobramycin treatment in 3813 cells (Fig. 2C). Cytoplasmic levels of Gemin2, an SMN-interacting protein (17Go,18Go,43Go), were qualitatively elevated following treatment with amikacin or tobramycin, although Gemin2 did not localize within gems during the time points that were analyzed (data not shown).

Following a 48 h treatment with amikacin or tobramycin, elevated gem levels were maintained even after growth in drug-free media for 48 h, although gem counts were not as high as in cells that were examined immediately following the 48 h drug treatment (Fig. 3). Following treatment with amikacin, time points >48 h in drug-free media resulted in low gem numbers similar to untreated levels (Fig. 3). When cells were treated for 48 h with tobramycin, and then re-fed with drug-free media, fewer fibroblasts displayed nuclear gems; however, some cells contained more than 10 gems per nuclei (Fig. 3). The induction of high gem numbers was maintained for up to 96 h after a 48 h treatment with tobramycin (Fig. 3).



View larger version (37K):
[in this window]
[in a new window]
  		Figure 3. Lasting effect of aminoglycoside treatment of SMA type I patient fibroblasts. Fibroblasts were cultured for 48 h in the presence of tobramycin (150 µg/ml) or amikacin (150 µg/ml) or in the absence of aminoglycoside antibiotic. SMN distribution was detected at 0, 24 and 48 h post-treatment using an anti-SMN antibody (BD Translabs) (1 : 50) and cells counterstained with DAPI.

 
Although geneticin, gentamicin, lividomycin and streptomycin resulted in only modest increases in gem levels or were highly toxic, the following experiments focused upon amikacin and tobramycin. Consistent with an elevation in gem numbers, treatment of 3813 cells with tobramycin and amikacin for 48 h resulted in higher total cellular levels of SMN protein as determined by western blot (Fig. 4). The steady-state levels of SMN protein following tobramycin and amikacin treatment were similar to those achieved with the treatment of valproic acid (Fig. 4) (40Go,42Go).



View larger version (50K):
[in this window]
[in a new window]
  		Figure 4. SMN protein levels are elevated in 3813 cells following tobramycin and amikacin treatments. Extracts from 3813 cells treated for 48 h with 150 µg/ml of tobramycin, amikacin or valproic acid (lanes 4–6) or untreated extracts from untreated HeLa, 3814 or 3813 cells (lanes 1, 2 and 3, respectively) were analyzed by western blot with an anti-SMN mouse monoclonal antibody (4F7) and visualized by chemiluminescence. The blot was stripped and re-probed with an anti-actin rabbit polyclonal antibody as a loading control.

 
To quantify the effect of amikacin and tobramycin on increasing gem numbers in 3813 cells, gem counts were carried out in the presence and absence of each aminoglycoside. Cells were cultured for 48 h in the presence of amikacin or tobramycin, then the drug was removed and gem counts were evaluated at 24 h time points up to 96 h. At each time point, gem counts were carried out in excess of 100 cells. Each experiment was performed in triplicate and the average number of gems per 100 nuclei at each time point was compared (Fig. 5). As expected and consistent with previously published work (11Go,13Go), fewer than 10 gems per 100 nuclei were observed in the untreated 3813 cells. After 48 h in the presence of amikacin (150 µg/ml), ~120 gems per 100 nuclei were recorded, consistent with gem numbers observed in normal unaffected fibroblasts (43Go) (data not shown). Gem counts were still significantly elevated even 24 h after the removal of amikacin, after which time the number of gems observed decreased (Fig. 5).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 5. Gems counts per 100 nuclei of aminoglycoside treated SMA type I patient fibroblasts. Fibroblasts were cultured for 48 h in the presence of tobramycin (150 µg/ml) (black) or amikacin (hatched) (150 µg/ml) or in the absence of aminoglycoside antibiotic (white). SMN distribution was detected at 0, 24, 48, 72 and 96 h post-treatment using an anti-SMN antibody (1 : 50) and average gem count per 100 nuclei was calculated at each time point.

 
Exposure to tobramycin for 48 h elevated gem numbers to ~120 gems per 100 nuclei (Fig. 5), similar to gem numbers observed in normal unaffected fibroblasts (43Go) (data not shown). Elevated gem levels were also observed 48 h after the removal of tobramycin (~100 gems per 100 nuclei). Gem totals were still elevated but reduced to ~70 gems per 100 nuclei, 72 h after removal of tobramycin. Surprisingly, 96 h after removal of tobramycin, gem totals recovered markedly, resulting in an average of ~110 gems per 100 nuclei (Fig. 5). On studying the effects of these two drugs on gem numbers, a clear distinction was detected: amikacin increased the total gem counts by mainly increasing the number of cells expressing relatively normal gem levels (one to five gems per nuclei), whereas tobramycin also appeared to increase the number of nuclei expressing abnormally high gem numbers (10 or more gems per nuclei). In a parallel dose-curve of amikacin and tobramycin (1, 10, 25, 75 and 150 µg/ml), SMN was increased in the cytoplasm as low as at 10 µg/ml, whereas increased cytoplasmic SMN and low levels of nuclear gems were detected at 25 µg/ml for each drug (data not shown).

In the previous experiments, average gem counts were calculated based on total gem numbers in 100 nuclei, therefore in tobramycin-treated cells, the relatively few cells expressing high levels of gems would increase the total gem number for a population of cells examined. To obtain a more thorough analysis, gem counts were determined by calculating the number of gems in individual cells at each time point (Fig. 6).







View larger version (54K):
[in this window]
[in a new window]
  		Figure 6. Gem counts of aminoglycoside treated SMA type I patient fibroblasts. Fibroblasts were cultured for 48 h in the presence of tobramycin (150 µg/ml) (black), amikacin (150 µg/ml) (hatched) or in the absence of aminoglycoside antibiotic (white). SMN was detected post-treatment using an anti-SMN antibody (1 : 50) at (A) 0 h, (B) 24 h, (C) 48 h, (D) 72 h and (E) 96 h.

 
As expected, <10% of untreated 3813 cells contained gems. In contrast, treatment with amikacin or tobramycin for 48 h resulted in ~50% of cells with at least one gem (Fig. 6A). In amikacin-treated 3813 cells, this effect was maintained for up to 48 h culture in the absence of aminoglycoside (Fig. 6B and C). After the 48 h time point in drug-free media, <15% of amikacin treated cells contained detectable gems. By 96 h in drug-free media, the effect of amikacin had diminished and 94% cells did not contain gems, and of those that did, none contained more than two gems per nuclei (Fig. 6D and E). Treatment with tobramycin, however, resulted in a quantitatively higher number of gems that was sustained for a longer period of time even after tobramycin was removed. After 48 h culture in the absence of tobramycin, 30% of cells examined still contained at least one gem, and after 96 h this number had decreased to 25% cells (Fig. 6B–E), suggesting that tobramycin treatment had a more prolonged effect on the population of treated cells as a whole.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
SMA develops because the primary transcript from the SMN2 gene encodes the SMN{Delta}7 protein, a defective and unstable protein that is not capable of compensating for the reduced levels of full-length SMN (5Go). Although a motor neuron-specific role for SMN exon 7 has not yet been defined, its deletion results in a protein defective in nearly every biochemical property ascribed to the full-length protein (19Go,23Go,25Go,28Go,29Go,44Go). A five amino acid motif within exon 7, QNQKE, has been shown to act as a cytoplasmic targeting sequence (29Go). Here, we conclusively demonstrate that this sequence is not required for the cytoplasmic targeting of the SMN protein. We also demonstrate that the non-functional nature of the SMN{Delta}7 protein is not due to nuclear retention, as the four amino acids encoded by exon 8 present in the SMN2-derived SMN{Delta}7 protein facilitates cytoplasmic and nuclear localization. Additionally, either the size or the amino acid composition of C-terminal domain fused to the 3' end of exon 6 altered the distribution of the mutant SMN constructs in HeLa or 3813 cells: all SMN{Delta}7 derivatives were enriched in the cytoplasm and present in the numerous nuclear gems. Collectively, these results imply that the physical presence of the C-terminal peptide stabilizes the exon 6 domain, enabling correct protein folding, and therefore, resulting in the formation of functional SMN oligomers. Although it is clear that the QNQKE motif present within SMN exon 7 can function as a heterologous cytoplasmic targeting motif (29Go), our results and recently published results demonstrate that QNQKE is sufficient, but not necessary, in mediating SMN subcellular distribution patterns (30Go). Because SMN missense mutations have been identified at the extreme 5' end of exon 7 (amino acid 279) (45Go), it is possible that there are some SMN-related functions that require specific exon 7 sequences or that the missense mutations result gross SMN protein defects.

Because essentially all SMA patients retain at least one copy of SMN2, the native SMN2 gene and its transcripts have been perceived as ideal candidates for therapeutic intervention. At present, high throughput screens are focused towards two different mechanisms: (1) increasing total expression from SMN2 by elevating SMN2 promoter activity; and (2) increasing the SMN-FL/SMN{Delta}7 ratio from SMN2 by suppressing the alternative splicing event that excises SMN exon 7 from the majority of SMN2-derived transcripts (46Go). Recently, a number of compounds, including several histone-deacetylase inhibitors, have been identified that increase SMN protein levels in cultured cells (15Go,40Go,41Go,47Go). Presumably, many of these compounds are functioning through similar mechanistic pathways; however, the specific mode of action for these compounds regarding SMN activation is currently unknown. Other compounds have also been identified, including indoprofen, a non-steroidal anti-inflammatory drug; however, the mode of action is currently unclear (46Go).

Aminoglycosides are a class of antibiotics that are widely used in the treatment of bacterial infections. Their mode of action is based on interfering with the prokaryotic ribosomes. In eukaryotes, several members of the aminoglycoside family have also been shown to disrupt the fidelity of the translation machinery by allowing the misincorporation of an amino acid at a stop codon. These compounds have been able to suppress termination of various reporter constructs and endogenous genes (31Go,32Go,34Go,36Go–39Go,48Go–50Go). Notably, functionality was partially restored in the mdx mouse—a mouse model for Duchenne muscular dystrophy—and in a cystic fibrosis murine model (31Go,39Go). In these instances, a premature termination codon within the dystrophin or CFTR gene was suppressed by treatment with gentamicin and levels of each respective full-length protein were increased in a dose-dependent manner. To date, no clinical trials in humans have achieved this level of success (32Go).

In vitro studies have indicated that different aminoglycosides have varied capacities to suppress stop codon incorporation (34Go). This is based on the specific sequence of the stop codon (UAA versus UAG versus UGA) and the sequence context surrounding the stop codon. These results are not exhaustive; however, it is clear that not all aminoglycosides will suppress all stop codons equivalently, and these differences can be partially attributed to sequence preferences (34Go).

This class of drugs was initially envisioned as an ideal treatment for diseases, such as cystic fibrosis and Duchenne muscular dystrophy, because a large number of these patients have premature stop codons in the respective disease-causing genes; however, success has largely been limited to tissue culture models and in vitro experiments. One significant caveat to consider in these experiments is that the premature termination codons not only interfere with protein expression, but also likely reduce the half-life of the RNA target by evoking regulatory responses such as nonsense-mediated decay (51Go,52Go). Nonsense-mediated decay is believed to be a surveillance mechanism that results in rapid turnover of RNA transcripts that contain premature termination codons. Therefore, in diseases such as cystic fibrosis, the aminoglycoside will likely need to suppress the stop codon, but it may also need to overcome a rapidly degrading mRNA template.

In contrast, the transcripts generated from SMN2 should not be considered aberrant RNA species. Probably, SMN1 and SMN2 should be viewed as completely separate genes that have their own unique splicing patterns and regulatory mechanisms. Although the C/T exchange is not an actual de novo point mutation, the C/T difference is a non-polymorphic nucleotide difference between two independent genes, and it is therefore not surprising that the C/T transition in SMN exon 7 does not alter the stability of the mRNA (5Go,6Go). Therefore, in the SMN context, there is a distinct advantage when compared with previously examined gene products, regarding the potential effectiveness for read-through of the normal SMN{Delta}7 stop codon.

Here, we identify an alternative approach to increasing SMN protein levels that likely involves altering the fidelity of the translational machinery to misidentify the initial stop codon in exon 8 and produce an elongated SMN2 protein containing the nine amino acids encoded by exon 8. An alternative hypothesis to the previously described read-through-inducing activity of aminoglycosides is that SMN{Delta}7 protein may accumulate in SMA patient fibroblasts due to an indirect effect of aminoglycoside treatment, potentially by inhibiting the protein degradation process. A recent report demonstrated that treatment with the proteosome inhibitor MG132 resulted in an increase in SMN levels in SMA patient fibroblasts (53Go). Experiments are currently being performed to differentiate between a read-through event and an indirect effect such as interference with the general proteosome pathway.

The read-through effect of aminoglycoside antibiotics has been shown to be sequence and context specific (34Go,37Go,54Go), therefore we tested the effect of a panel of aminoglycosides on SMN distribution in type I SMA patient fibroblasts (3813 cells). From this panel of antibiotics, amikacin and tobramycin quantitatively increased gem numbers and total SMN protein levels in 3813 cells. Previous studies have used an increase in gem count in 3813 cells in response to drug treatment as an assay of the presence of functional SMN (15Go,40Go,41Go). Importantly, amikacin and tobramycin did not induce detectable levels of cytotoxicity at the concentrations examined. Additionally, even after the removal of the drug, gem levels in some cells remained significantly elevated for up to 4 days. As expected, the increase in SMN protein observed in these studies was not due to an alteration in splicing patterns as determined by RT–PCR analysis (data not shown). Therefore, aminoglycoside treatment likely causes the read-through of the SMN{Delta}7 stop codon in exon 8, conferring additional stability to the unstable SMN{Delta}7 product. The possibility exists, however, that SMN has multiple functions and that stabilization of the C-terminus only restores a subset of these activities. For example, restoration of the nuclear function of SMN may allow for the accumulation of SMN protein in gems; however, an axon-specific activity that is directly relevant to SMA may not be restored by the introduction of an artificial C-terminus. Comparison of the chemical structure of gentamicin and tobramycin highlights their similarities (Fig. 7). Current experiments are being performed to determine the domains of these and related compounds that are specifically required for inducing elevated SMN levels. Because amikacin and tobramycin do not alter SMN exon 7 pre-mRNA inclusion/exclusion ratios, this raises the possibility of combining treatments that elevate SMN levels through distinct mechanisms. The ability of members of this family of FDA-approved antibiotics to potentially confer function to the SMN2 primary product, SMN{Delta}7, provides an alternative mechanism to consider in the search for SMA therapy.



View larger version (7K):
[in this window]
[in a new window]
  		Figure 7. Schematic of (A) amikacin and (B) tobramycin. Variations are highlighted in bold.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of SMN constructs
Full-length SMN cDNA was cloned into a modified pCI (Promega) vector containing an N-terminal HA-tag as previously described (27Go). A panel of SMN C-terminal mutants were generated from this template using QuickChange Site Directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions. Each mutant construct was confirmed by sequencing.

Production of antibodies
Full-length his-tagged SMN or the C-terminal 40 KDa of p80 coilin recombinant protein was expressed from pET32A in Escherichia coli strain Rosetta and purified on a Ni-NTA column as previously described (43Go). Monoclonal antibodies were produced by immunization of BALB/c mice and fusion of spleen cells with Sp2/0 myeloma cells. Positive hybridoma supernatants were screened by ELISA, western blot and immunofluorescence microscopy (nuclear Cajal body staining on HeLa cell lines). Hybridoma cell lines were cloned to homogeneity by limiting dilution. Clone 4B7 was used in western blot analysis at a dilution of 1 : 100.

Cell culture and immunohistochemistry
HeLa cells and SMA type I patient fibroblasts (3813) (11Go) were plated on coverslips and grown in Dulbecco's modified Eagle's medium (DMEM) medium (Invitrogen) containing 10% (v/v) fetal calf serum and antibiotics. After 4 h, cells were transfected with 0.5 µg cDNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Following an additional 24 h incubation, transfected cells were washed three times with phosphate-buffered saline (PBS), before fixing with 1 : 1 acetone/methanol. Immunohistochemistry staining was carried out as previously described (44Go) using either anti-HA antibody (Santa Cruz) diluted 1 : 50, MANSMA1 (43Go) diluted 1 : 10 or a commercially available anti-SMN antibody (BD Translabs) diluted 1 : 50. Anti-SMN antibody 4B7 was also used to confirm the results (data not shown). Target proteins were visualized using FITC-conjugated secondary antibody (Sigma). Cell nuclei were counterstained with 4',6'-diamidino-2-phenlindole (DAPI).

Aminoglycoside treatment
SMA type I fibroblasts (3813) were plated on cover slips and grown in DMEM medium (Gibco) containing 10% (v/v) fetal calf serum and antibiotics for 24 h. Cells were washed with PBS and re-fed with DMSO containing aminoglycoside antibiotics (Sigma) diluted to the indicated concentration (75 or 150 µg/ml). In prolonged treatment experiments, the medium containing freshly diluted aminoglycoside was changed every 24 h. For cells used in western blot analysis, cells were plated at ~80% confluency in six-well dishes and treated for 48 h. Fresh drug-containing media was replaced every 24 h. An aliquot of 150 µg/ml of valproic acid, amikacin and tobramycin correspond to 0.902, 0.191 and 0.320 M, respectively.

Gem count values presented represent three separate experiments. In each instance, cells were initially identified by DAPI staining not by the presence or the absence of SMN and gems. Only, after a field of view was obtained was the SMN/FITC channel observed. The DAPI field was done randomly across a large number of treated cells, providing an unbiased assessment of gem numbers throughout the cell population.

Western blot analysis
HeLa, 3814 or 3813 cells (treated or untreated) were plated at ~80% confluency in three wells from a six-well plate. Cells were washed and fresh media was added with freshly prepared amikacin or tobramycin (or untreated, as indicated), 12 h later and incubated for 24 h at which time fresh drug-containing media was replaced and cells were incubated an additional 24 h. Cells were harvested in lysis buffer 48 h after initiation of drug treatment (27Go) and the three wells were pooled and run in a single lane on a SDS–PAGE gel. Anti-SMN antibody 4B7 was used in western blot analysis at a dilution of 1 : 100 to identify SMN in cellular extracts from treated and untreated cells and the membranes were subsequently stripped and re-probed with an anti-actin (Santa Cruz) polyclonal rabbit antibody at 1 : 300 dilution. Probes were visualized by chemiluminescence using the Pierce SuperSignal Pico reagents.


   ACKNOWLEDGEMENTS
 
P.J.Y. was supported by a fellowship from Andrews' Buddies/FightSMA. This work was funded by grants from Andrew's Buddies/FightSMA, Muscular Dystrophy Association (C.L.L.) and the National Institutes of Health (C.L.L., R01 NS41584-01).


   FOOTNOTES
 
{dagger} Present address: Peninsula Medical School, University of Exeter, St Luke's Campus, Exeter EX1 2LU, UK. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Crawford, T.O. and Pardo, C.A. (1996) The neurobiology of childhood spinal muscular atrophy. Neurobiol. Dis., 3, 97–110.[CrossRef][ISI][Medline]

  2. Munsat, T.L., Skerry, L., Korf, B., Pober, B., Schapira, Y., Gascon, G.G., Al-Rajeh, S.M., Dubowitz, V., Davies, K., Brzustowicz, L.M. et al. (1990) Phenotypic heterogeneity of spinal muscular atrophy mapping to chromosome 5q11.2–13.3 (SMA 5q). Neurology, 40, 1831–1836.[Abstract/Free Full Text]

  3. Lefebvre, S., Burglen, L., Reboullet, S., Clermont, O., Burlet, P., Viollet, L., Benichou, B., Cruaud, C., Millasseau, P., Zeviani, M. et al. (1995) Identification and characterization of a spinal muscular atrophy- determining gene. Cell, 80, 155–165.[CrossRef][ISI][Medline]

  4. Lefebvre, S., Burglen, L., Frezal, J., Munnich, A. and Melki, J. (1998) The role of the SMN gene in proximal spinal muscular atrophy. Hum. Mol. Genet., 7, 1531–1536.[Abstract/Free Full Text]

  5. Lorson, C.L., Hahnen, E., Androphy, E.J. and Wirth, B. (1999) A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Proc. Natl Acad. Sci. USA, 96, 6307–6311.[Abstract/Free Full Text]

  6. Monani, U.R., Lorson, C.L., Parsons, D.W., Prior, T.W., Androphy, E.J., Burghes, A.H. and McPherson, J.D. (1999) A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2. Hum. Mol. Genet., 8, 1177–1183.[Abstract/Free Full Text]

  7. Rochette, C.F., Gilbert, N. and Simard, L.R. (2001) SMN gene duplication and the emergence of the SMN2 gene occurred in distinct hominids: SMN2 is unique to Homo sapiens. Hum. Genet., 108, 255–266.[CrossRef][ISI][Medline]

  8. DiDonato, C.J., Chen, X.N., Noya, D., Korenberg, J.R., Nadeau, J.H. and Simard, L.R. (1997) Cloning, characterization, and copy number of the murine survival motor neuron gene: homolog of the spinal muscular atrophy-determining gene. Genome Res., 7, 339–352.[Abstract/Free Full Text]

  9. Monani, U.R., Coovert, D.D. and Burghes, A.H. (2000) Animal models of spinal muscular atrophy. Hum. Mol. Genet., 9, 2451–2457.[Abstract/Free Full Text]

  10. Monani, U.R., Sendtner, M., Coovert, D.D., Parsons, D.W., Andreassi, C., Le, T.T., Jablonka, S., Schrank, B., Rossol, W., Prior, T.W. et al. (2000) The human centromeric survival motor neuron gene (SMN2) rescues embryonic lethality in Smn(–/–) mice and results in a mouse with spinal muscular atrophy. Hum. Mol. Genet., 9, 333–339.[Abstract/Free Full Text]

  11. Coovert, D.D., Le, T.T., McAndrew, P.E., Strasswimmer, J., Crawford, T.O., Mendell, J.R., Coulson, S.E., Androphy, E.J., Prior, T.W. and Burghes, A.H. (1997) The survival motor neuron protein in spinal muscular atrophy. Hum. Mol. Genet., 6, 1205–1214.[Abstract/Free Full Text]

  12. Young, P.J., Le, T.T., thi Man, N., Burghes, A.H. and Morris, G.E. (2000) The relationship between SMN, the spinal muscular atrophy protein, and nuclear coiled bodies in differentiated tissues and cultured cells. Exp. Cell Res., 256, 365–374.[CrossRef][ISI][Medline]

  13. Young, P.J., Le, T.T., Dunckley, M., Nguyen, T.M., Burghes, A.H. and Morris, G.E. (2001) Nuclear gems and Cajal (coiled) bodies in fetal tissues: nucleolar distribution of the spinal muscular atrophy protein, SMN. Exp. Cell Res., 265, 252–261.[CrossRef][ISI][Medline]

  14. Wehner, K.A., Ayala, L., Kim, Y., Young, P.J., Hosler, B.A., Lorson, C.L., Baserga, S.J. and Francis, J.W. (2002) Survival motor neuron protein in the nucleolus of mammalian neurons. Brain Res., 945, 160–173.[CrossRef][ISI][Medline]

  15. Andreassi, C., Jarecki, J., Zhou, J., Coovert, D.D., Monani, U.R., Chen, X., Whitney, M., Pollok, B., Zhang, M., Androphy, E. et al. (2001) Aclarubicin treatment restores SMN levels to cells derived from type I spinal muscular atrophy patients. Hum. Mol. Genet., 10, 2841–2849.[Abstract/Free Full Text]

  16. Andreassi, C., Patrizi, A.L., Monani, U.R., Burghes, A.H., Brahe, C. and Eboli, M.L. (2002) Expression of the survival of motor neuron (SMN) gene in primary neurons and increase in SMN levels by activation of the N-methyl-D- aspartate glutamate receptor. Neurogenetics, 4, 29–36.[CrossRef][ISI][Medline]

  17. Fischer, U., Liu, Q. and Dreyfuss, G. (1997) The SMN-SIP1 complex has an essential role in spliceosomal snRNP biogenesis. Cell, 90, 1023–1029.[CrossRef][ISI][Medline]

  18. Liu, Q., Fischer, U., Wang, F. and Dreyfuss, G. (1997) The spinal muscular atrophy disease gene product, SMN, and its associated protein SIP1 are in a complex with spliceosomal snRNP proteins. Cell, 90, 1013–1021.[CrossRef][ISI][Medline]

  19. Pellizzoni, L., Kataoka, N., Charroux, B. and Dreyfuss, G. (1998) A novel function for SMN, the spinal muscular atrophy disease gene product, in pre-mRNA splicing. Cell, 95, 615–624.[CrossRef][ISI][Medline]

  20. Williams, B.Y., Hamilton, S.L. and Sarkar, H.K. (2000) The survival motor neuron protein interacts with the transactivator FUSE binding protein from human fetal brain. FEBS Lett., 470, 207–210.[CrossRef][ISI][Medline]

  21. Strasswimmer, J., Lorson, C.L., Breiding, D.E., Chen, J.J., Le, T., Burghes, A.H. and Androphy, E.J. (1999) Identification of survival motor neuron as a transcriptional activator- binding protein. Hum. Mol. Genet., 8, 1219–1226.[Abstract/Free Full Text]

  22. Rossoll, W., Jablonka, S., Andreassi, C., Kroning, A.K., Karle, K., Monani, U.R. and Sendtner, M. (2003) Smn, the spinal muscular atrophy-determining gene product, modulates axon growth and localization of beta-actin mRNA in growth cones of motoneurons. J. Cell Biol., 163, 801–812.[Abstract/Free Full Text]

  23. Kerr, D.A., Nery, J.P., Traystman, R.J., Chau, B.N. and Hardwick, J.M. (2000) Survival motor neuron protein modulates neuron-specific apoptosis. Proc. Natl Acad. Sci. USA, 97, 13312–13317.[Abstract/Free Full Text]

  24. Young, P.J., Day, P.M., Zhou, J., Androphy, E.J., Morris, G.E. and Lorson, C.L. (2002) A direct interaction between the survival motor neuron protein and p53 and its relationship to spinal muscular atrophy. J. Biol. Chem., 277, 2852–2859.[Abstract/Free Full Text]

  25. Vyas, S., Bechade, C., Riveau, B., Downward, J. and Triller, A. (2002) Involvement of survival motor neuron (SMN) protein in cell death. Hum. Mol. Genet., 11, 2751–2764.[Abstract/Free Full Text]

  26. Lefebvre, S., Burlet, P., Viollet, L., Bertrandy, S., Huber, C., Belser, C. and Munnich, A. (2002) A novel association of the SMN protein with two major non-ribosomal nucleolar proteins and its implication in spinal muscular atrophy. Hum. Mol. Genet., 11, 1017–1027.[Abstract/Free Full Text]

  27. Lorson, C.L., Strasswimmer, J., Yao, J.M., Baleja, J.D., Hahnen, E., Wirth, B., Le, T., Burghes, A.H. and Androphy, E.J. (1998) SMN oligomerization defect correlates with spinal muscular atrophy severity. Nat. Genet., 19, 63–66.[ISI][Medline]

  28. Paushkin, S., Gubitz, A.K., Massenet, S. and Dreyfuss, G. (2002) The SMN complex, an assemblyosome of ribonucleoproteins. Curr. Opin. Cell Biol., 14, 305–312.[CrossRef][ISI][Medline]

  29. Zhang, H.L., Pan, F., Hong, D., Shenoy, S.M., Singer, R.H. and Bassell, G.J. (2003) Active transport of the survival motor neuron protein and the role of exon-7 in cytoplasmic localization. J. Neurosci., 23, 6627–6637.[Abstract/Free Full Text]

  30. Hua, Y. and Zhou, J. (2004) Modulation of SMN nuclear foci and cytoplasmic localization by its C-terminus. Cell. Mol. Life Sci., 61, 2658–2663.[CrossRef][ISI][Medline]

  31. Barton-Davis, E.R., Cordier, L., Shoturma, D.I., Leland, S.E. and Sweeney, H.L. (1999) Aminoglycoside antibiotics restore dystrophin function to skeletal muscles of mdx mice. J. Clin. Invest., 104, 375–381.[ISI][Medline]

  32. Wagner, K.R., Hamed, S., Hadley, D.W., Gropman, A.L., Burstein, A.H., Escolar, D.M., Hoffman, E.P. and Fischbeck, K.H. (2001) Gentamicin treatment of Duchenne and Becker muscular dystrophy due to nonsense mutations. Ann. Neurol., 49, 706–711.[CrossRef][ISI][Medline]

  33. Sleat, D.E., Sohar, I., Gin, R.M. and Lobel, P. (2001) Aminoglycoside-mediated suppression of nonsense mutations in late infantile neuronal ceroid lipofuscinosis. Eur. J. Paediatr. Neurol., 5(Suppl. A), 57–62.[Medline]

  34. Manuvakhova, M., Keeling, K. and Bedwell, D.M. (2000) Aminoglycoside antibiotics mediate context-dependent suppression of termination codons in a mammalian translation system. RNA, 6, 1044–1055.[Abstract]

  35. Lai, C.H., Chun, H.H., Nahas, S.A., Mitui, M., Gamo, K.M., Du, L. and Gatti, R.A. (2004) Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons. Proc. Natl Acad. Sci. USA, 101, 15676–15681.[Abstract/Free Full Text]

  36. Keeling, K.M., Brooks, D.A., Hopwood, J.J., Li, P., Thompson, J.N. and Bedwell, D.M. (2001) Gentamicin-mediated suppression of Hurler syndrome stop mutations restores a low level of alpha-L-iduronidase activity and reduces lysosomal glycosaminoglycan accumulation. Hum. Mol. Genet., 10, 291–299.[Abstract/Free Full Text]

  37. Howard, M.T., Anderson, C.B., Fass, U., Khatri, S., Gesteland, R.F., Atkins, J.F. and Flanigan, K.M. (2004) Readthrough of dystrophin stop codon mutations induced by aminoglycosides. Ann. Neurol, 55, 422–426.[CrossRef][ISI][Medline]

  38. Howard, M., Frizzell, R.A. and Bedwell, D.M. (1996) Aminoglycoside antibiotics restore CFTR function by overcoming premature stop mutations. Nat. Med., 2, 467–469.[CrossRef][ISI][Medline]

  39. Du, M., Jones, J.R., Lanier, J., Keeling, K.M., Lindsey, J.R., Tousson, A., Bebok, Z., Whitsett, J.A., Dey, C.R., Colledge, W.H. et al. (2002) Aminoglycoside suppression of a premature stop mutation in a Cftr–/– mouse carrying a human CFTR-G542X transgene. J. Mol. Med., 80, 595–604.[CrossRef][ISI][Medline]

  40. Sumner, C.J., Huynh, T.N., Markowitz, J.A., Perhac, J.S., Hill, B., Coovert, D.D., Schussler, K., Chen, X., Jarecki, J., Burghes, A.H. et al. (2003) Valproic acid increases SMN levels in spinal muscular atrophy patient cells. Ann. Neurol., 54, 647–654.[CrossRef][ISI][Medline]

  41. Andreassi, C., Angelozzi, C., Tiziano, F.D., Vitali, T., De Vincenzi, E., Boninsegna, A., Villanova, M., Bertini, E., Pini, A., Neri, G. et al. (2004) Phenylbutyrate increases SMN expression in vitro: relevance for treatment of spinal muscular atrophy. Eur. J. Hum. Genet., 12, 59–65.[CrossRef][ISI][Medline]

  42. Brichta, L., Hofmann, Y., Hahnen, E., Siebzehnrubl, F.A., Raschke, H., Blumcke, I., Eyupoglu, I.Y. and Wirth, B. (2003) Valproic acid increases the SMN2 protein level: a well-known drug as a potential therapy for spinal muscular atrophy. Hum. Mol. Genet., 12, 2481–2489.[Abstract/Free Full Text]

  43. Young, P.J., Man, N.T., Lorson, C.L., Le, T.T., Androphy, E.J., Burghes, A.H. and Morris, G.E. (2000) The exon 2b region of the spinal muscular atrophy protein, SMN, is involved in self-association and SIP1 binding. Hum. Mol. Genet., 9, 2869–2877.[Abstract/Free Full Text]

  44. Young, P.J., Francis, J.W., Lince, D., Coon, K., Androphy, E.J. and Lorson, C.L. (2003) The Ewing's sarcoma protein interacts with the Tudor domain of the survival motor neuron protein. Brain Res. Mol. Brain Res., 119, 37–49.[Medline]

  45. Wirth, B. (2000) An update of the mutation spectrum of the survival motor neuron gene (SMN1) in autosomal recessive spinal muscular atrophy (SMA). Hum. Mutat., 15, 228–237.[CrossRef][ISI][Medline]

  46. Lunn, M.R., Root, D.E., Martino, A.M., Flaherty, S.P., Kelley, B.P., Coovert, D.D., Burghes, A.H., Man, N.T., Morris, G.E., Zhou, J. et al. (2004) Indoprofen upregulates the survival motor neuron protein through a cyclooxygenase-independent mechanism. Chem. Biol., 11, 1489–1493.[CrossRef][ISI][Medline]

  47. Chang, J.G., Hsieh-Li, H.M., Jong, Y.J., Wang, N.M., Tsai, C.H. and Li, H. (2001) Treatment of spinal muscular atrophy by sodium butyrate. Proc. Natl Acad. Sci. USA, 98, 9808–9813.[Abstract/Free Full Text]

  48. Bedwell, D.M., Kaenjak, A., Benos, D.J., Bebok, Z., Bubien, J.K., Hong, J., Tousson, A., Clancy, J.P. and Sorscher, E.J. (1997) Suppression of a CFTR premature stop mutation in a bronchial epithelial cell line. Nat. Med., 3, 1280–1284.[CrossRef][ISI][Medline]

  49. Grayson, C., Chapple, J.P., Willison, K.R., Webster, A.R.,