title: Loss of nuclear TDP-43 in amyotrophic lateral sclerosis (ALS) causes altered expression of splicing machinery and widespread dysregulation of RNA splicing in motor neurones.
abstract: OBJECTIVE
  Loss of nuclear TDP-43 characterizes sporadic and most familial forms of amyotrophic lateral sclerosis (ALS). TDP-43 (encoded by TARDBP) has multiple roles in RNA processing. We aimed to determine whether (1) RNA splicing dysregulation is present in lower motor neurones in ALS and in a motor neurone-like cell model; and (2) TARDBP mutations (mtTARDBP) are associated with aberrant RNA splicing using patient-derived fibroblasts.
  
  
  METHODS
  Affymetrix exon arrays were used to study mRNA expression and splicing in lower motor neurones obtained by laser capture microdissection of autopsy tissue from individuals with sporadic ALS and TDP-43 proteinopathy. Findings were confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in NSC34 motor neuronal cells following shRNA-mediated TDP-43 depletion. Exon arrays and immunohistochemistry were used to study mRNA splicing and TDP-43 expression in fibroblasts from patients with mtTARDBP-associated, sporadic and mutant SOD1-associated ALS.
  
  
  RESULTS
  We found altered expression of spliceosome components in motor neurones and widespread aberrations of mRNA splicing that specifically affected genes involved in ribonucleotide binding. This was confirmed in TDP-43-depleted NSC34 cells. Fibroblasts with mtTARDBP showed loss of nuclear TDP-43 protein and demonstrated similar changes in splicing and gene expression, which were not present in fibroblasts from patients with sporadic or SOD1-related ALS.
  
  
  CONCLUSIONS
  Loss of nuclear TDP-43 is associated with RNA processing abnormalities in ALS motor neurones, patient-derived cells with mtTARDBP, and following artificial TDP-43 depletion, suggesting that splicing dysregulation directly contributes to disease pathogenesis. Key functional pathways affected include those central to RNA metabolism.
authors.affiliation: Sheffield Institute for Translational Neuroscience (SITraN), University of Sheffield, Sheffield, UK.
authors.forename: J Robin
authors.initials: JR
authors.lastname: Highley
authors.forename: Janine
authors.initials: J
authors.lastname: Kirby
authors.forename: Joeri A
authors.initials: JA
authors.lastname: Jansweijer
authors.forename: Philip S
authors.initials: PS
authors.lastname: Webb
authors.forename: Channa A
authors.initials: CA
authors.lastname: Hewamadduma
authors.forename: Paul R
authors.initials: PR
authors.lastname: Heath
authors.forename: Adrian
authors.initials: A
authors.lastname: Higginbottom
authors.forename: Rohini
authors.initials: R
authors.lastname: Raman
authors.forename: Laura
authors.initials: L
authors.lastname: Ferraiuolo
authors.forename: Johnathan
authors.initials: J
authors.lastname: Cooper-Knock
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: McDermott
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Wharton
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Shaw
authors.forename: Paul G
authors.initials: PG
authors.lastname: Ince
mesh_terms: D000368:Aged; D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics*; D000818:Animals; D004268:DNA-Binding Proteins / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D006801:Humans; D008297:Male; D051379:Mice; D008875:Middle Aged; D009046:Motor Neurons / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics; D012326:RNA Splicing; D013116:Spinal Cord / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins
keywords: TDP-43; amyotrophic lateral sclerosis; mRNA; microarray; post mortem; splicing