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Figure.
Agarose gel electrophoresis of SOD1 messenger RNA. The first amplified product of 423 base pairs (bp) for the control and the 11 TARDBP or FUS mutants is shown on top. The second product of 528 bp is shown under the first product for the same samples.

Agarose gel electrophoresis of SOD1 messenger RNA. The first amplified product of 423 base pairs (bp) for the control and the 11 TARDBP or FUS mutants is shown on top. The second product of 528 bp is shown under the first product for the same samples.

1.
Dion  PADaoud  HRouleau  GA Genetics of motor neuron disorders: new insights into pathogenic mechanisms. Nat Rev Genet 2009;10 (11) 769- 782
PubMedArticle
2.
Kabashi  EValdmanis  PNDion  P  et al.  TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nat Genet 2008;40 (5) 572- 574
PubMedArticle
3.
Kwiatkowski  TJ  JrBosco  DALeclerc  AL  et al.  Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science 2009;323 (5918) 1205- 1208
PubMedArticle
4.
Belzil  VVValdmanis  PNDion  PA  et al. S2D team, Mutations in FUS cause FALS and SALS in French and French Canadian populations. Neurology 2009;73 (15) 1176- 1179
PubMedArticle
5.
Lagier-Tourenne  CPolymenidou  MCleveland  DW TDP-43 and FUS/TLS: emerging roles in RNA processing and neurodegeneration. Hum Mol Genet 2010;19 (R1) R46- R64
PubMedArticle
6.
Ling  SCAlbuquerque  CPHan  JS  et al.  ALS-associated mutations in TDP-43 increase its stability and promote TDP-43 complexes with FUS/TLS. Proc Natl Acad Sci U S A 2010;107 (30) 13318- 13323
PubMedArticle
Research Letters
March 2011

No Effect on SOD1 Splicing by TARDP or FUS Mutations

Author Affiliations

Author Affiliations: Research Center, Centre of Excellence in Neuromics of Université de Montréal Centre Hospitalier de l’Université de Montréal (CHUM) (Ms Belzil and Drs Daoud, Dion, and Rouleau); Departments of Pathology and Cellular Biology (Dr Dion) and Medicine, Faculty of Medicine (Dr Rouleau), Université de Montreal; and the Research Center, Centre Hospitalier Universitaire Sainte-Justine (Dr Rouleau), Montreal, Quebec, Canada.

Arch Neurol. 2011;68(3):395-399. doi:10.1001/archneurol.2011.1

Amyotrophic lateral sclerosis (ALS) is characterized by a neuronal loss in the motor cortex, brainstem, and spinal cord that progresses over a 3 to 5 year period. Approximately 90% of cases are considered sporadic, while the other 10% of cases are familial.1 Extensive research has been conducted on the SOD1 (RefSeq NM_000454.4) gene after mutations were identified more than 17 years ago in 15% to 20% of familial ALS cases, as well as a small number of sporadic ALS cases in later reports.1 In the last 2 years, mutations in TARDBP, which encodes TAR DNA-binding protein 43 (TDP-43), and in FUS have been identified in both familial and sporadic ALS cases.24 Both genes encode for RNA/DNA binding proteins, are mainly localized in the nucleus, and are implicated in the regulation of RNA processing.5 Specifically, TDP-43 and FUS have been shown to be associated with other splicing factors and are believed to play a role in splicing regulation, as variation in their expression level influences the splicing of certain targets.5 Interestingly, mutant TDP-43 proteins identified in patients with ALS were recently reported to be more stable than wild-type TDP-43 and to display an enhanced interaction with fused in sarcoma (FUS) polypeptides.6 Particularly, it was reported that the FUS protein interacts more predominantly with mutant TDP-43, which display an increased half-life compared with wild-type TDP-43. The authors concluded on a note about the efforts needed to determine whether the increased association affects the RNA targets for TDP-43, FUS/translocated in liposarcoma protein, or both.6 Considering that abnormal RNA processing and splicing patterns are involved in neurodegenerative diseases1 and that variation in the RNA splicing of SOD1 can cause familial ALS by destabilizing the resulting protein,1 our aim was to assess if TARDBP and/or FUS ALS-predisposing mutations that are known to lead to the accumulation of TDP-43 or FUS aggregations in the cytoplasm also lead to aberrant SOD1 RNA splicing events, and to determine if SOD1 could be an RNA target for TDP-43, FUS, or both.

Methods

We studied total RNA prepared from immortalized lymphoblastoid cells of 1 healthy control individual as well as 7 participants with different TARDBP mutations (p.D169G, p.G287S, p.G348C, p.R361S, p.Y374X, p.A382T, p.N390D) and 4 participants with different FUS mutations (p.P18S, p.G174del, c.1542-2A>C, p.R521H). Protocols were approved by the ethics committee and the institutional review board of the University of Montreal. All patients gave written informed consent, after which patient information and blood were collected. The RNA was extracted using standard conditions. Reverse-transcription polymerase chain reactions were conducted using prepared complement DNA and the Moloney murine leukemia virus reverse transcriptase enzyme (Invitrogen, Carlsbad, California) per the manufacturer's instructions. The amplifications were performed using 2 sets of primer pairs that covered the entire messenger RNA of SOD1. Polymerase chain reaction products were sequenced at the Genome Quebec Innovation Centre (Montréal, Québec, Canada) using a 3730XL DNA analyzer (Applied Biosystems, Carlsbad, California). The first set amplified a region of 423 base pairs (bp) (c.1-439), starting at the beginning of the 5′ untranslated region and ending at the beginning of exon 4. The second set of primers amplified a product of 528 bp (c.398-462), starting at the beginning of exon 4 and ending at the end of the 3′ untranslated region.

Results

Agarose gel electrophoresis showed that only 1 product was amplified for each of the complement DNA amplified (Figure). In addition, no variation was found in the sequence traces of the SOD1 complement DNA products.

Comment

We can conclude that TARDBP or FUS ALS-predisposing mutations do not affect the splicing of SOD1 and that, while it can't be excluded that there may be a common ALS pathogenic pathway, it appears that mutant TARDBP and FUS do not act by affecting the splicing of the most frequently mutated gene, SOD1, in ALS.

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Article Information

Correspondence: Dr Rouleau, CHUM Research Centre, Notre-Dame Hospital, 1560 Sherbrooke E, Y-3633, Montreal, QC H2L 4M1, Canada (guy.rouleau@umontreal.ca).

Author Contributions:Study concept and design: Belzil, Dion, and Rouleau. Acquisition of data: Daoud. Analysis and interpretation of data: Belzil, Daoud, and Dion. Drafting of the manuscript: Belzil. Critical revision of the manuscript for important intellectual content: Belzil, Daoud, Dion, and Rouleau. Obtained funding: Rouleau. Administrative, technical, and material support: Belzil, Daoud, and Dion. Study supervision: Dion and Rouleau.

Financial Disclosure: None reported.

Funding/Support: This study was supported by the Canadian Institutes of Health Research (Ms Belzil and Drs Daoud and Rouleau).

Additional Contributions: We would like to thank the patients involved in this study.

References
1.
Dion  PADaoud  HRouleau  GA Genetics of motor neuron disorders: new insights into pathogenic mechanisms. Nat Rev Genet 2009;10 (11) 769- 782
PubMedArticle
2.
Kabashi  EValdmanis  PNDion  P  et al.  TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nat Genet 2008;40 (5) 572- 574
PubMedArticle
3.
Kwiatkowski  TJ  JrBosco  DALeclerc  AL  et al.  Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science 2009;323 (5918) 1205- 1208
PubMedArticle
4.
Belzil  VVValdmanis  PNDion  PA  et al. S2D team, Mutations in FUS cause FALS and SALS in French and French Canadian populations. Neurology 2009;73 (15) 1176- 1179
PubMedArticle
5.
Lagier-Tourenne  CPolymenidou  MCleveland  DW TDP-43 and FUS/TLS: emerging roles in RNA processing and neurodegeneration. Hum Mol Genet 2010;19 (R1) R46- R64
PubMedArticle
6.
Ling  SCAlbuquerque  CPHan  JS  et al.  ALS-associated mutations in TDP-43 increase its stability and promote TDP-43 complexes with FUS/TLS. Proc Natl Acad Sci U S A 2010;107 (30) 13318- 13323
PubMedArticle
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