Acta Neuropathologica

, Volume 123, Issue 6, pp 825–839

Pattern of ubiquilin pathology in ALS and FTLD indicates presence of C9ORF72 hexanucleotide expansion

Authors

  • Johannes Brettschneider
    • Center for Neurodegenerative Disease Research (CNDR)University of Pennsylvania School of Medicine
    • Department of NeurologyUniversity of Ulm
  • Vivianna M. Van Deerlin
    • Center for Neurodegenerative Disease Research (CNDR)University of Pennsylvania School of Medicine
    • Department of Pathology and Laboratory MedicineUniversity of Pennsylvania School of Medicine
  • John L. Robinson
    • Center for Neurodegenerative Disease Research (CNDR)University of Pennsylvania School of Medicine
  • Linda Kwong
    • Center for Neurodegenerative Disease Research (CNDR)University of Pennsylvania School of Medicine
  • Edward B. Lee
    • Department of Pathology and Laboratory MedicineUniversity of Pennsylvania School of Medicine
  • Yousuf O. Ali
    • Center for Neurodegenerative Disease Research (CNDR)University of Pennsylvania School of Medicine
  • Nathaniel Safren
    • Center for Biomedical Engineering and Technology and Department of Anatomy and NeurobiologyUniversity of Maryland
  • Mervyn J. Monteiro
    • Center for Biomedical Engineering and Technology and Department of Anatomy and NeurobiologyUniversity of Maryland
  • Jon B. Toledo
    • Center for Neurodegenerative Disease Research (CNDR)University of Pennsylvania School of Medicine
  • Lauren Elman
    • Department of NeurologyUniversity of Pennsylvania School of Medicine
  • Leo McCluskey
    • Department of NeurologyUniversity of Pennsylvania School of Medicine
  • David J. Irwin
    • Center for Neurodegenerative Disease Research (CNDR)University of Pennsylvania School of Medicine
    • Department of NeurologyUniversity of Pennsylvania School of Medicine
  • Murray Grossman
    • Department of NeurologyUniversity of Pennsylvania School of Medicine
  • Laura Molina-Porcel
    • Center for Neurodegenerative Disease Research (CNDR)University of Pennsylvania School of Medicine
  • Virginia M.-Y. Lee
    • Center for Neurodegenerative Disease Research (CNDR)University of Pennsylvania School of Medicine
    • Department of Pathology and Laboratory MedicineUniversity of Pennsylvania School of Medicine
    • Center for Neurodegenerative Disease Research (CNDR)University of Pennsylvania School of Medicine
    • Department of Pathology and Laboratory MedicineUniversity of Pennsylvania School of Medicine
Original Paper

DOI: 10.1007/s00401-012-0970-z

Cite this article as:
Brettschneider, J., Van Deerlin, V.M., Robinson, J.L. et al. Acta Neuropathol (2012) 123: 825. doi:10.1007/s00401-012-0970-z

Abstract

C9ORF72-hexanucleotide repeat expansions and ubiquilin-2 (UBQLN2) mutations are recently identified genetic markers in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We investigate the relationship between C9ORF72 expansions and the clinical phenotype and neuropathology of ALS and FTLD. Genetic analysis and immunohistochemistry (IHC) were performed on autopsy-confirmed ALS (N = 75), FTLD-TDP (N = 30), AD (N = 14), and controls (N = 11). IHC for neurodegenerative disease pathology consisted of C9ORF72, UBQLN, p62, and TDP-43. A C9ORF72 expansion was identified in 19.4 % of ALS and 31 % of FTLD-TDP cases. ALS cases with C9ORF72 expansions frequently showed a bulbar onset of disease (57 %) and more rapid disease progression to death compared to non-expansion cases. Staining with C9ORF72 antibodies did not yield specific pathology. UBQLN pathology showed a highly distinct pattern in ALS and FTLD-TDP cases with the C9ORF72 expansion, with UBQLN-positive cytoplasmic inclusions in the cerebellar granular layer and extensive UBQLN-positive aggregates and dystrophic neurites in the hippocampal molecular layer and CA regions. These UBQLN pathologies were sufficiently unique to allow correct prediction of cases that were later confirmed to have C9ORF72 expansions by genetic analysis. UBQLN pathology partially co-localized with p62, and to a minor extent with TDP-43 positive dystrophic neurites and spinal cord skein-like inclusions. Our data indicate a pathophysiological link between C9ORF72 expansions and UBQLN proteins in ALS and FTLD-TDP that is associated with a highly characteristic pattern of UBQLN pathology. Our study indicates that this pathology is associated with alterations in clinical phenotype, and suggests that the presence of C9ORF72 repeat expansions may indicate a worse prognosis in ALS.

Keywords

Amyotrophic lateral sclerosisFrontotemporal lobar degenerationC9ORF72UBQLN2UBQLN1

Supplementary material

401_2012_970_MOESM1_ESM.tif (4 mb)
Supplementary figure 1 CORF72 antibody detects both short and long isoforms of the translated protein. 293T cells were transfected with vectors overexpressing either GFP or the C9ORF72 long (DDK-Myc-T1) or short isoforms (DDK-Myc-T2) tagged at the C-terminal with DDK- and myc-tags, using Lipofectamine-2000 (Invitrogen). At 48 h post-transfection, the cells were lysed with RIPA buffer and proteins concentrations were measured using the Bradford protein assay (BioRad). Sample lysates were resolved by SDS-PAGE and probed with: anti-DDK 1:2000 (Origene Technologies) or anti-C9ORF72 1:1000 (Sigma). Western blot analysis was performed by incubating with the infrared dye-conjugated secondary antibodies, IR700 and IR800 (LI-COR Biosciences) for 1 h at room temperature; blots were imaged and processed on an Odyssey® Infrared Imaging System. The DDK antibody (left panel, red) detected the overexpressed proteins: around 56 kDa for the long isoform and around 26 kDa for the short isoform. The commercial C9ORF72 antibody (middle panel, green) was able to detect both the overexpressed isoforms as well (see overlap between the 700 and 800 channels in right panel). In addition, the commercial antibody was also able to detect the endogenous C9ORF72 long isoform, which is abundantly present in 293T cells. (TIFF 4053 kb)
401_2012_970_MOESM2_ESM.jpg (414 kb)
Supplementary figure 2 C9ORF72 pathology in ALS and CTRL as seen by IHC. Coarse punctate staining of synaptic terminals in the CA4 region of the hippocampus (a, shown in higher resolution in b and c) that were observed in ALS, FTLD-TDP and CTRL without any difference in extent or regional distribution between the groups. Scale bars: a 200 µm, b, c 20 µm. (JPEG 414 kb)
401_2012_970_MOESM3_ESM.tif (864 kb)
Supplementary figure 3 Testing UBQLN antibody specificity. (a-c) Either human embryonic 293T or murine 3T3 fibroblasts were transfected with GFP-UBQLN1 or control GFP vectors, using Lipofectamine-2000 (Invitrogen). 293T cells were used to test if the antibodies could detect endogenous human UBQLN proteins; mouse 3T3 cells allowed for testing the specificity of the antibodies for human, and not endogenous mouse UBQLN proteins. At 48 h post-transfection, the cells were lysed with RIPA buffer and proteins concentrations were measured using the Bradford protein assay (BioRad). Both sample lysates and purified recombinant GST-tagged UBQLN1 protein were resolved by SDS-PAGE and probed with: anti-UBQLN1, 1:500 or anti-UBQLN2 1:1000 (Abnova, Walnut, CA). Western blot analysis was performed by incubating with the infrared dye-conjugated secondary antibodies, IR700 and IR800 (LI-COR Biosciences) for 1 h at room temperature; blots were imaged and processed on an Odyssey® Infrared Imaging System. The UBQLN1 antibody (a) specifically detected the recombinant GST-UBQLN1 (~98 kDa) as well as GFP-UBQLN1, and endogenous human UBQLN1 (3rd lane). The commercial monoclonal UBQLN 2 antibody recognized both UBQLN1 and 2 band in b. (c) GFP-specific antibody detected the GFP-UBQLN1. (TIFF 864 kb)

Copyright information

© Springer-Verlag 2012