title: Dipeptide repeat protein inclusions are rare in the spinal cord and almost absent from motor neurons in C9ORF72 mutant amyotrophic lateral sclerosis and are unlikely to cause their degeneration.
abstract: BACKGROUND
  Cytoplasmic TDP-43 inclusions are the pathological hallmark of amyotrophic lateral sclerosis (ALS) and tau-negative frontotemporal lobar dementia (FTLD). The G4C2 repeat mutation in C9ORF72 is the most common cause of ALS and FTLD in which, in addition to TDP-43 inclusions, five different di-peptide repeat (DPR) proteins have been identified. Di-peptide repeat proteins are translated in a non-canonical fashion from sense and antisense transcripts of the G4C2 repeat (GP, GA, GR, PA, PR). DPR inclusions are abundant in the cerebellum, as well as in the frontal and temporal lobes of ALS and FTLD patients and some are neurotoxic in a range of cellular and animal models, implying that DPR aggregation directly contributes to disease pathogenesis. Here we sought to quantify inclusions for each DPR and TDP-43 in ALS cases with and without the C9ORF72 mutation. We characterised the abundance of DPRs and their cellular location and compared this to cytoplasmic TDP-43 inclusions in order to explore the role of each inclusion in lower motor neuron degeneration.
  
  
  RESULTS
  Spinal cord sections from ten cases positive for the C9ORF72 repeat expansion (ALS-C9+ve) and five cases that were not were probed by double immunofluorescence staining for individual DPRs and TDP-43. Inclusions immunoreactive for each of the DPRs were present in the spinal cord but they were rare or very rare in abundance (in descending order of frequency: GA, GP, GR, PA and PR). TDP-43 cytoplasmic inclusions were 45- to 750-fold more frequent than any DPR, and fewer than 4 % of DPR inclusions colocalized with TDP-43 inclusions. In motor neurons, a single cytoplasmic DPR inclusion was detected (0.1 %) in contrast to the 34 % of motor neurons that contained cytoplasmic TDP-43 inclusions. Furthermore, the number of TDP-43 inclusions in ALS cases with and without the C9ORF72 mutation was nearly identical.
  
  
  CONCLUSIONS
  For all other neurodegenerative diseases, the neurotoxic protein aggregates are detected in the affected population of neurons. TDP-43 cytoplasmic aggregation is the dominant feature of ALS spinal cord pathology irrespective of C9ORF72 mutation status. The near absence of DPR inclusions in spinal cord motor neurons challenges their contribution to lower motor neuron degeneration in ALS-C9+ve cases.
authors.affiliation: Department of Basic and Clinical Neurosciences, Maurice Wohl Clinical Neurosciences Institute, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, SE5 8AF, UK.
authors.forename: Jorge
authors.initials: J
authors.lastname: Gomez-Deza
authors.affiliation: Department of Basic and Clinical Neurosciences, Maurice Wohl Clinical Neurosciences Institute, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, SE5 8AF, UK.
authors.forename: Youn-Bok
authors.initials: YB
authors.lastname: Lee
authors.affiliation: Department of Basic and Clinical Neurosciences, Maurice Wohl Clinical Neurosciences Institute, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, SE5 8AF, UK.|London Neurodegenerative Disease Brain Bank, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, SE5 8AF, UK.
authors.forename: Claire
authors.initials: C
authors.lastname: Troakes
authors.affiliation: London Neurodegenerative Disease Brain Bank, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, SE5 8AF, UK.
authors.forename: Matthew
authors.initials: M
authors.lastname: Nolan
authors.affiliation: London Neurodegenerative Disease Brain Bank, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, SE5 8AF, UK.
authors.forename: Safa
authors.initials: S
authors.lastname: Al-Sarraj
authors.affiliation: Department of Basic and Clinical Neurosciences, Maurice Wohl Clinical Neurosciences Institute, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, SE5 8AF, UK.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Gallo
authors.affiliation: Department of Basic and Clinical Neurosciences, Maurice Wohl Clinical Neurosciences Institute, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London, SE5 8AF, UK. Christopher.shaw@kcl.ac.uk.
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Shaw
mesh_terms: D000328:Adult; D000368:Aged; D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics* / Q000473:pathology; D000073885:C9orf72 Protein; D042622:DNA Repeat Expansion / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004151:Dipeptides / Q000235:genetics* / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009046:Motor Neurons / Q000378:metabolism* / Q000473:pathology; D011506:Proteins / Q000235:genetics*; D013116:Spinal Cord / Q000378:metabolism / Q000473:pathology*
chemical_list: D000073885:C9orf72 Protein; C568651:C9orf72 protein, human; D004268:DNA-Binding Proteins; D004151:Dipeptides; D011506:Proteins