Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Resource
  • Published:

Distinct brain transcriptome profiles in C9orf72-associated and sporadic ALS

Abstract

Increasing evidence suggests that defective RNA processing contributes to the development of amyotrophic lateral sclerosis (ALS). This may be especially true for ALS caused by a repeat expansion in C9orf72 (c9ALS), in which the accumulation of RNA foci and dipeptide-repeat proteins are expected to modify RNA metabolism. We report extensive alternative splicing (AS) and alternative polyadenylation (APA) defects in the cerebellum of c9ALS subjects (8,224 AS and 1,437 APA), including changes in ALS-associated genes (for example, ATXN2 and FUS), and in subjects with sporadic ALS (sALS; 2,229 AS and 716 APA). Furthermore, heterogeneous nuclear ribonucleoprotein H (hnRNPH) and other RNA-binding proteins are predicted to be potential regulators of cassette exon AS events in both c9ALS and sALS. Co-expression and gene-association network analyses of gene expression and AS data revealed divergent pathways associated with c9ALS and sALS.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Differential regulation of gene expression in c9ALS and sALS.
Figure 2: WGCNA of top identified modules in cerebellum and frontal cortex of individuals with c9ALS.
Figure 3: Widespread AS defects are found in individuals with c9ALS and sALS.
Figure 4: Extensive misregulation of CE splicing occurs in the c9ALS cerebellum.
Figure 5: Misregulation of CE splicing in c9ALS affects transcripts with roles in diverse molecular pathways.
Figure 6: APA changes are prominent in the cerebellum of individuals with c9ALS and sALS.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Geser, F. et al. Evidence of multisystem disorder in whole-brain map of pathological TDP-43 in amyotrophic lateral sclerosis. Arch. Neurol. 65, 636–641 (2008).

    Article  PubMed  Google Scholar 

  2. DeJesus-Hernandez, M. et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72, 245–256 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Renton, A.E. et al. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72, 257–268 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. van Blitterswijk, M., DeJesus-Hernandez, M. & Rademakers, R. How do C9ORF72 repeat expansions cause amyotrophic lateral sclerosis and frontotemporal dementia: can we learn from other noncoding repeat expansion disorders? Curr. Opin. Neurol. 25, 689–700 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Ash, P.E. et al. Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS. Neuron 77, 639–646 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Mori, K. et al. Bidirectional transcripts of the expanded C9orf72 hexanucleotide repeat are translated into aggregating dipeptide repeat proteins. Acta Neuropathol. 126, 881–893 (2013).

    Article  CAS  PubMed  Google Scholar 

  7. Mori, K. et al. The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS. Science 339, 1335–1338 (2013).

    Article  CAS  PubMed  Google Scholar 

  8. Gendron, T.F. et al. Antisense transcripts of the expanded C9ORF72 hexanucleotide repeat form nuclear RNA foci and undergo repeat-associated non-ATG translation in c9FTD/ALS. Acta Neuropathol. 126, 829–844 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Mizielinska, S. et al. C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins. Science 345, 1192–1194 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. May, S. et al. C9orf72 FTLD/ALS-associated Gly-Ala dipeptide repeat proteins cause neuronal toxicity and Unc119 sequestration. Acta Neuropathol. 128, 485–503 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zhang, Y.J. et al. Aggregation-prone c9FTD/ALS poly(GA) RAN-translated proteins cause neurotoxicity by inducing ER stress. Acta Neuropathol. 128, 505–524 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kwon, I. et al. Poly-dipeptides encoded by the C9ORF72 repeats bind nucleoli, impede RNA biogenesis, and kill cells. Science 345, 1139–1145 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Yamakawa, M. et al. Characterization of the dipeptide repeat protein in the molecular pathogenesis of c9FTD/ALS. Hum. Mol. Genet. 24, 1630–1645 (2015).

    Article  CAS  PubMed  Google Scholar 

  14. Wen, X. et al. Antisense proline-arginine RAN dipeptides linked to C9ORF72-ALS/FTD form toxic nuclear aggregates that initiate in vitro and in vivo neuronal death. Neuron 84, 1213–1225 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Tao, Z. et al. Nucleolar stress and impaired stress granule formation contribute to C9orf72 RAN translation-induced cytotoxicity. Hum. Mol. Genet. 24, 2426–2441 (2015).

    Article  CAS  PubMed  Google Scholar 

  16. Donnelly, C.J. et al. RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention. Neuron 80, 415–428 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Lagier-Tourenne, C. et al. Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration. Proc. Natl. Acad. Sci. U.S.A. 110, E4530–E4539 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Sareen, D. et al. Targeting RNA foci in iPSC-derived motor neurons from ALS patients with a C9ORF72 repeat expansion. Sci. Transl. Med. 5, 208ra149 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Lee, Y.B. et al. Hexanucleotide repeats in ALS/FTD form length-dependent RNA foci, sequester RNA binding proteins, and are neurotoxic. Cell Rep. 5, 1178–1186 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Cooper-Knock, J. et al. Sequestration of multiple RNA recognition motif-containing proteins by C9orf72 repeat expansions. Brain 137, 2040–2051 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Neumann, M. et al. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314, 130–133 (2006).

    Article  CAS  PubMed  Google Scholar 

  22. Gendron, T.F., Josephs, K.A. & Petrucelli, L. Review: transactive response DNA-binding protein 43 (TDP-43): mechanisms of neurodegeneration. Neuropathol. Appl. Neurobiol. 36, 97–112 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Sreedharan, J. et al. TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science 319, 1668–1672 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kwiatkowski, T.J. Jr. et al. Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science 323, 1205–1208 (2009).

    Article  CAS  PubMed  Google Scholar 

  25. Vance, C. et al. Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science 323, 1208–1211 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Elden, A.C. et al. Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS. Nature 466, 1069–1075 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Couthouis, J. et al. Evaluating the role of the FUS/TLS-related gene EWSR1 in amyotrophic lateral sclerosis. Hum. Mol. Genet. 21, 2899–2911 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Ticozzi, N. et al. Mutational analysis reveals the FUS homolog TAF15 as a candidate gene for familial amyotrophic lateral sclerosis. Am. J. Med. Genet. B Neuropsychiatr. Genet. 156B, 285–290 (2011).

    Article  CAS  PubMed  Google Scholar 

  29. Kim, H.J. et al. Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS. Nature 495, 467–473 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Wu, J., Anczukow, O., Krainer, A.R., Zhang, M.Q. & Zhang, C. OLego: fast and sensitive mapping of spliced mRNA-Seq reads using small seeds. Nucleic Acids Res. 41, 5149–5163 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Affaitati, A., de Cristofaro, T., Feliciello, A. & Varrone, S. Identification of alternative splicing of spinocerebellar ataxia type 2 gene. Gene 267, 89–93 (2001).

    Article  CAS  PubMed  Google Scholar 

  32. Yen, S.H., Hutton, M., DeTure, M., Ko, L.W. & Nacharaju, P. Fibrillogenesis of tau: insights from tau missense mutations in FTDP-17. Brain Pathol. 9, 695–705 (1999).

    Article  CAS  PubMed  Google Scholar 

  33. Kremerskothen, J. et al. Brain-specific splicing of α-actinin 1 (ACTN1) mRNA. Biochem. Biophys. Res. Commun. 295, 678–681 (2002).

    Article  CAS  PubMed  Google Scholar 

  34. Tuncel, H. et al. PARP6, a mono(ADP-ribosyl) transferase and a negative regulator of cell proliferation, is involved in colorectal cancer development. Int. J. Oncol. 41, 2079–2086 (2012).

    Article  CAS  PubMed  Google Scholar 

  35. Gothié, E., Richard, D.E., Berra, E., Pages, G. & Pouyssegur, J. Identification of alternative spliced variants of human hypoxia-inducible factor-1α. J. Biol. Chem. 275, 6922–6927 (2000).

    Article  PubMed  Google Scholar 

  36. Ray, D. et al. A compendium of RNA-binding motifs for decoding gene regulation. Nature 499, 172–177 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Johnson, J.O. et al. Mutations in the Matrin 3 gene cause familial amyotrophic lateral sclerosis. Nat. Neurosci. 17, 664–666 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Xia, Z. et al. Dynamic analyses of alternative polyadenylation from RNA-seq reveal a 3′-UTR landscape across seven tumour types. Nat. Commun. 5, 5274 (2014).

    Article  CAS  PubMed  Google Scholar 

  39. van Blitterswijk, M. et al. Ataxin-2 as potential disease modifier in C9ORF72 expansion carriers. Neurobiol. Aging 35, 2421.e13–2421.e17 (2014).

    Article  CAS  Google Scholar 

  40. Mori, K. et al. hnRNP A3 binds to GGGGCC repeats and is a constituent of p62-positive/TDP43-negative inclusions in the hippocampus of patients with C9orf72 mutations. Acta Neuropathol. 125, 413–423 (2013).

    Article  CAS  PubMed  Google Scholar 

  41. Shi, Y. Alternative polyadenylation: new insights from global analyses. RNA 18, 2105–2117 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Batra, R. et al. Loss of MBNL leads to disruption of developmentally regulated alternative polyadenylation in RNA-mediated disease. Mol. Cell 56, 311–322 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Jenal, M. et al. The poly(A)-binding protein nuclear 1 suppresses alternative cleavage and polyadenylation sites. Cell 149, 538–553 (2012).

    Article  CAS  PubMed  Google Scholar 

  44. Thivard, L. et al. Diffusion tensor imaging and voxel based morphometry study in amyotrophic lateral sclerosis: relationships with motor disability. J. Neurol. Neurosurg. Psychiatry 78, 889–892 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Seeley, W.W. et al. Frontal paralimbic network atrophy in very mild behavioral variant frontotemporal dementia. Arch. Neurol. 65, 249–255 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Tan, R.H. et al. Cerebellar integrity in the amyotrophic lateral sclerosis-frontotemporal dementia continuum. PLoS One 9, e105632 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Bede, P. et al. Patterns of cerebral and cerebellar white matter degeneration in ALS. J. Neurol. Neurosurg. Psychiatry 86, 468–470 (2015).

    Article  CAS  PubMed  Google Scholar 

  48. Irwin, D.J. et al. Cognitive decline and reduced survival in C9orf72 expansion frontotemporal degeneration and amyotrophic lateral sclerosis. J. Neurol. Neurosurg. Psychiatry 84, 163–169 (2013).

    Article  PubMed  Google Scholar 

  49. Mahoney, C.J. et al. Longitudinal neuroimaging and neuropsychological profiles of frontotemporal dementia with C9ORF72 expansions. Alzheimers Res. Ther. 4, 41 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Boeve, B.F. et al. Characterization of frontotemporal dementia and/or amyotrophic lateral sclerosis associated with the GGGGCC repeat expansion in C9ORF72. Brain 135, 765–783 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  51. Brooks, B.R. El Escorial World Federation of Neurology criteria for the diagnosis of amyotrophic lateral sclerosis. Subcommittee on Motor Neuron Diseases/Amyotrophic Lateral Sclerosis of the World Federation of Neurology Research Group on Neuromuscular Diseases and the El Escorial “Clinical limits of amyotrophic lateral sclerosis” workshop contributors. J. Neurol. Sci. 124, 96–107 (1994).

    Article  PubMed  Google Scholar 

  52. Brooks, B.R., Miller, R.G., Swash, M. & Munsat, T.L. El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotroph. Lateral Scler. Other Motor Neuron Disord. 1, 293–299 (2000).

    Article  CAS  PubMed  Google Scholar 

  53. Braak, H. & Braak, E. Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol. 82, 239–259 (1991).

    Article  CAS  PubMed  Google Scholar 

  54. Murray, M.E. et al. Clinical and neuropathologic heterogeneity of c9FTD/ALS associated with hexanucleotide repeat expansion in C9ORF72. Acta Neuropathol. 122, 673–690 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Murray, M.E. et al. Neuropathologically defined subtypes of Alzheimer's disease with distinct clinical characteristics: a retrospective study. Lancet Neurol. 10, 785–796 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  56. Zhang, Y.J. et al. Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity. Proc. Natl. Acad. Sci. USA 106, 7607–7612 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Kalari, K.R. et al. MAP-RSeq: Mayo Analysis Pipeline for RNA sequencing. BMC Bioinformatics 15, 224 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  58. Charizanis, K. et al. Muscleblind-like 2-mediated alternative splicing in the developing brain and dysregulation in myotonic dystrophy. Neuron 75, 437–450 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Robinson, M.D., McCarthy, D.J. & Smyth, G.K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

    Article  CAS  PubMed  Google Scholar 

  60. Huang, D.W., Sherman, B.T. & Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

    Article  CAS  Google Scholar 

  61. de Hoon, M.J., Imoto, S., Nolan, J. & Miyano, S. Open source clustering software. Bioinformatics 20, 1453–1454 (2004).

    Article  CAS  PubMed  Google Scholar 

  62. Langfelder, P. & Horvath, S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 9, 559 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  63. Cline, M.S. et al. Integration of biological networks and gene expression data using Cytoscape. Nat. Protoc. 2, 2366–2382 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Emig, D. et al. AltAnalyze and DomainGraph: analyzing and visualizing exon expression data. Nucleic Acids Res. 38, W755–W762 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Franceschini, A. et al. STRING v9.1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res. 41, D808–D815 (2013).

    Article  CAS  PubMed  Google Scholar 

  66. Machanick, P. & Bailey, T.L. MEME-ChIP: motif analysis of large DNA datasets. Bioinformatics 27, 1696–1697 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Huelga, S.C. et al. Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins. Cell Rep. 1, 167–178 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Masamha, C.P. et al. CFIm25 links alternative polyadenylation to glioblastoma tumour suppression. Nature 510, 412–416 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are extremely grateful to all individuals who agreed to donate their brains to research. This work was supported by the National Institutes of Health/National Institute on Aging (R01AG026251 and P50AG016574 to L.P.), the National Institutes of Health/National Institute of Neurological Disorders and Stroke (R21NS089979 to T.F.G. and K.B.B.; R21NS084528, R01NS088689 and R01NS077402 to L.P.; R01NS063964 to L.P. and C.D.L.; P01NS084974 to L.P., D.W.D., R.R. and K.B.B.), the National Institute of Environmental Health Sciences (R01ES20395 to L.P.), the Department of Defense (ALSRP AL130125 to L.P.), the Mayo Clinic Foundation (L.P.), the Mayo Clinic Center for Individualized Medicine (L.P. and K.B.B.), the ALS Association (K.B.B., L.P., M.P. and T.F.G.), the Robert Packard Center for ALS Research at Johns Hopkins (L.P.), Target ALS (L.P.), the ALS Association (Milton Safenowitz postdoctoral fellowships to V.V.B. and M.P.), the Canadian Institutes of Health Research (postdoctoral fellowship to V.V.B.), the Siragusa Foundation (Career Development Award for Young Investigators to V.V.B.), and the Robert and Clarice Smith & Abigail Van Buren Alzheimer's Disease Research Foundation (postdoctoral fellowship to V.V.B.). H.L. and M.E.M. are supported by the Mayo Clinic Center for Individualized Medicine and the Donors Cure Foundation.

Author information

Authors and Affiliations

Authors

Contributions

M.P., V.V.B., R.B. and C.A.R. contributed equally to this work. M.P., V.V.B. and L.P. contributed to the conception and design of the study. M.P., V.V.B., L.J.P., M.E.M., K.K.O., A.E.P.-J., P.D., M.D., M.D.D., M.C.B., R.B.P., K.B.B. and D.W.D. contributed to tissue selection and collection. M.P., L.J.P. and M.D.D. performed RNA extractions. M.P., V.V.B. and M.D.D. made cDNA. M.P. ran qRT-PCRs for expression and AS validation. R.B., C.A.R. and H.L. performed expression and WGCNA bioinformatics analyses. R.B. conducted AS, APA and system network analyses. M.P. and R.B. carried out GO analyses. H.L. supervised the bioinformatics analyses. T.F.G. and K.B. performed histological analyses. M.P., V.V.B., R.B., C.A.R., T.F.G., C.D.L., H.L. and L.P. interpreted the data and prepared the manuscript. All authors contributed to critical revision of the manuscript for important intellectual content and approved the final version for publication.

Corresponding authors

Correspondence to Hu Li or Leonard Petrucelli.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Principal-component analyses reveal divergence between c9ALS and sALS.

Principal-component analysis was performed using Log2(n+1) transformed RPKM data of the top 5,000 differentially expressed c9ALS genes from (a) 26 cerebellar and (b) 27 frontal cortex tissues. Green circles represent controls, purple circles represent sALS cases, and black circles represent c9ALS cases. Numbers for each case were randomly assigned and correspond to the cases described in Supplementary Table 1.

Supplementary Figure 2 Hierarchical clustering of differentially expressed genes in c9ALS cerebellum with clinical features shown for each case.

Hierarchical clustering representation of differentially expressed c9ALS transcripts in cerebellum with clinical features shown for each case. Each row of the heat map corresponds to a control (N = 8), sALS case (N = 10) or c9ALS case (N = 8), as designated by the color-coded bar to the left of the heat maps. Clinical features of each case, also color-coded, are shown to the right of the heat maps. Note that clinical information on the second column represents family history of neurodegeneration. Complete clinical information can be found in Supplementary Table 1.

Supplementary Figure 3 Hierarchical clustering of differentially expressed genes in c9ALS frontal cortex with clinical features shown for each case.

Hierarchical clustering representation of differentially expressed c9ALS transcripts in frontal cortex with clinical features shown for each case. Each row of the heat map corresponds to a control (N = 9), sALS case (N = 10) or c9ALS case (N = 9), as designated by the color-coded bar to the left of the heat maps. Clinical features of each case, also color-coded, are shown to the right of the heat maps. Note that clinical information on the second column represents family history of neurodegeneration. Complete clinical information can be found in Supplementary Table 1.

Supplementary Figure 4 Validation of top differentially expressed genes in c9ALS cerebellum and frontal cortex.

Bar graphs (mean ± s.e.m.) showing qRT-PCR validations, and the corresponding P values when comparing c9ALS (N = 9) and sALS (N = 10) to controls (N = 9 cerebellum, N = 8 frontal cortex) below, using RNA from cerebellum (a,b) and frontal cortex (c,d) for top differentially expressed genes in c9ALS (P < 0.05, │log2FC│ ≥ 2). Relative mRNA levels are normalized to the endogenous control, RPLP0, and the control group (mean value set to 1). Statistical differences were calculated by one-way ANOVA with Bonferroni post-hoc test (*P < 0.05, **P < 0.01, ***P < 0.005). The full list of oligonucleotides used in this study can be found in Supplementary Table 9.

Supplementary Figure 5 Intron retention is a common AS event in c9ALS.

Venn diagrams and bar graphs depicting the number of unique and common introns that are more (red) or less (blue) retained in c9ALS and sALS cerebellum (a) or frontal cortex (b) (FDR < 0.05). The black section of bar graph (a) shows common intron retention events but going in opposite directions. c9ALS (N = 8), sALS (N = 10), controls (N = 8 cerebellum, N = 9 frontal cortex).

Supplementary Figure 6 Hierarchical clustering of AS cassette exon variants in c9ALS cerebellum and clinical features for all cases.

Hierarchical clustering of down- or up-regulated c9ALS AS cassette exon-inclusion events in cerebellum from c9ALS (N = 8), sALS (N = 10) and controls (N = 8) with corresponding clinical features. Each row of the heat map corresponds to either a control, sALS or c9ALS case, as designated by the color-coded bar to the left of the heat map. Clinical features for all cases, also color-coded, are shown to the right of the heat maps. Note that clinical information on the second column represents family history of neurodegeneration. Complete clinical information can be found in Supplementary Table 1.

Supplementary Figure 7 Hierarchical clustering of AS cassette exon variants in c9ALS frontal cortex and clinical features for all cases.

Hierarchical clustering of down- or up-regulated c9ALS AS cassette exon-inclusion events in frontal cortex tissues from c9ALS (N = 8), sALS (N = 10) and controls (N = 9) with corresponding clinical features. Each row of the heat map corresponds to either a control, sALS or c9ALS case, as designated by the color-coded bar to the left of the heat map. Clinical features for all cases, also color-coded, are shown to the right of the heat maps. Note that clinical information on the second column represents family history of neurodegeneration. Complete clinical information can be found in Supplementary Table 1.

Supplementary Figure 8 Additional validation of AS cassette exon events in c9ALS cerebellum.

(a) RNA-Seq wiggle plots and bar graphs (mean ± s.e.m.) showing additional qRT-PCR validations in the cerebellum (see Fig. 5a in the main text for other validations). Shown is an example of the three technical qRT-PCR replicates performed for each AS event. Relative mRNA levels were normalized to the endogenous control, RPLP0, and controls (mean value set to 1). Note that inclusion of the cassette exon (of a mutually exclusive CE event) in U2AF1 is indicated on the wiggle plot by the arrow. The full list of primers used in this study can be found in Supplementary Table 9. (b,c) P values when comparing the levels of cassette exon inclusion, from qRT-PCR validations, in c9ALS (N = 9) and sALS (N = 10) to either non-neurological disease controls (controls, N = 9), (b) or to other neurological disease (PSP, N = 13) group (c). Statistical differences were calculated by one-way ANOVA with Bonferroni post-hoc test (*P < 0.05, **P < 0.01, #P < 0.0001). ND, not determined.

Supplementary Figure 9 Validation of AS cassette exon events in c9ALS frontal cortex.

(a) RNA-Seq wiggle plots and bar graphs (mean ± s.e.m.) showing qRT-PCR validations in the frontal cortex. Relative mRNA levels were normalized to the endogenous control, RPLP0, and controls (mean value set to 1). Note that inclusion of the cassette exon (of a mutually exclusive CE event) in U2AF1 is indicated on the wiggle plot by the arrow. The full list of primers used in this study can be found in Supplementary Table 9. (b) P values when comparing the levels of cassette exon inclusion, from qRT-PCR validations, in c9ALS (N = 9) and sALS (N = 10) to non-neurological disease controls (controls, N = 8). Statistical differences were calculated by one-way ANOVA with Bonferroni post-hoc test (*P < 0.05, #P < 0.0001).

Supplementary Figure 10 Validation of AS cassette exon events in c9ALS motor cortex.

(a) Bar graphs (mean ± s.e.m.) showing qRT-PCR validations of significantly misregulated cassette exons in the motor cortex. The full list of primers used in this study can be found in Supplementary Table 9. (b) P values when comparing the levels of cassette exon inclusion, from qRT-PCR validations, in c9ALS (N = 10) and sALS (N = 9) to non-neurological disease controls (controls, N = 10). Relative mRNA levels were normalized to the endogenous control, RPLP0, and controls (mean value set to 1). Statistical differences were calculated by one-way ANOVA with Bonferroni post-hoc test (*P < 0.05, ***P < 0.005).

Supplementary Figure 11 RNA-binding protein motif enrichment analysis of AS cassette exons highlights hnRNPH binding motifs.

RNA-binding protein (RBP) motif enrichment analysis was performed using AS cassette exon sequences and their flanking intronic regions of misspliced cassette exon events occurring in cerebellum and frontal cortex of c9ALS or sALS cases (c9ALS, N = 8; sALS, N = 10; controls, N = 8 cerebellum and N = 9 frontal cortex. RBP motif enrichment P values, shown in parentheses, were calculated using MEME-ChIP software. The resulting enriched motifs were cross-referenced with known RBP motifs from Ray and colleagues (2013)1 and an RBP database (http://rbpdb.ccbr.utoronto.ca).

1Ray, D. et al. A compendium of RNA-binding motifs for decoding gene regulation. Nature 499, 172–177 (2013).

Supplementary Figure 12 Gene-association network of top 1,000 genes of mis-spliced cassette exons in c9ALS cerebellum.

Genes with the top most significant AS cassette exon events in the c9ALS cerebellum (FDR < 0.05, │dl│ ≥ 0.1) were selected to construct a gene-association network using String v9.11 and Cytoscape 3.1.12 software. dl: differential index value. c9ALS (N = 8), sALS (N = 10), controls (N = 8). Note that this is a more complete version of Figure 5c shown in the main text. Genes are represented by nodes of different colors, which vary according to degree. The size of the node denotes neighborhood connectivity: nodes are bigger if they are connected to other nodes with higher connectivity. Edges are colored according to edge betweenness to indicate the proximity to other nodes, with low betweeness (closer proximity) meaning larger influence to other nodes. 1Franceschini, A. et al. STRING v9.1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res. 41, D808–D815 (2013). 2Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504 (2003).

Supplementary Figure 13 Gene-association network for all mis-spliced cassette exons in sALS cerebellum.

All genes presenting AS cassette exons in sALS cerebellum (FDR < 0.05, │dl│ ≥ 0.1) were used to construct a gene-association network using String v9.11 and Cytoscape 3.1.12 software. dl: differential index value. c9ALS (N = 8), sALS (N = 10), controls (N = 9). Genes are represented by nodes of different colors, which vary according to degree. The size of the node denotes neighborhood connectivity: nodes are bigger if they are connected to other nodes with higher connectivity. Edges are colored according to edge between-ness to indicate the proximity to other nodes, with low between-ness (closer proximity) meaning larger influence to other nodes. Gene ontology terms of different interconnected cellular pathways are indicated.

1Franceschini, A. et al. STRING v9.1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res. 41, D808–D815 (2013).

2Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504 (2003).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13 and Supplementary Tables 1–11 (PDF 2794 kb)

Supplementary Checklist

(PDF 383 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Prudencio, M., Belzil, V., Batra, R. et al. Distinct brain transcriptome profiles in C9orf72-associated and sporadic ALS. Nat Neurosci 18, 1175–1182 (2015). https://doi.org/10.1038/nn.4065

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn.4065

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing