Research ArticleAmyotrophic Lateral Sclerosis

Poly(GP) proteins are a useful pharmacodynamic marker for C9ORF72-associated amyotrophic lateral sclerosis

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Science Translational Medicine  29 Mar 2017:
Vol. 9, Issue 383, eaai7866
DOI: 10.1126/scitranslmed.aai7866
  • Fig. 1. Poly(GP) is detected in CSF from asymptomatic and symptomatic C9ORF72 repeat expansion carriers.

    (A) Poly(GP) in CSF from C9ORF72 repeat expansion carriers (C9+; n = 134) and noncarriers (C9−; n = 120). ****P < 0.0001, as assessed by van Elteren stratified Wilcoxon rank sum test. (B) CSF poly(GP) concentrations in asymptomatic C9ORF72 mutation carriers (ASX; n = 27) and symptomatic c9ALS patients with or without comorbid FTD (SX; n = 83). No significant difference in poly(GP) between ASX and SX subjects was observed using a linear regression model adjusted for gender and age at CSF collection. Red lines denote the median.

  • Fig. 2. Longitudinal trajectory of poly(GP) in CSF.

    Poly(GP) in CSF collected longitudinally from 33 C9ORF72 repeat expansion carriers who either were asymptomatic or had c9ALS or c9ALS-FTD. Twenty-four subjects had two measurements, 6 had three measurements, 2 had four measurements, and 1 had five measurements. One patient (denoted by red circles) converted from a clinical diagnosis of ALS to ALS-FTD between the first and second CSF collection.

  • Fig. 3. Poly(GP) is detected in PBMCs from C9ORF72 mutation carriers, and c9ASO-1 treatment decreases poly(GP) in lymphoblastoid cell lines.

    (A to C) Poly(GP) in lysates from PBMCs from C9ORF72 mutation carriers (C9+; n = 36) or noncarriers (C9−; n = 34) (A) or in lysates and media from lymphoblastoid cell lines (LCLs) from C9+ (n = 12) or C9− (n = 7) subjects. For (A) and (B), the red line indicates the median. **P < 0.01, ****P < 0.0001, Wilcoxon rank sum test. For (C), a significant correlation was observed between extracellular and intracellular poly(GP) (Spearman’s r = 0.77, P = 0.004, n = 12). (D to F) Two C9+ LCLs were treated with 5 μM nontargeting control ASO (CTL ASO) or one that targets G4C2 repeat–containing RNA (c9ASO-1) for 10 days. After treatment, RNA was extracted from cells to measure mRNA of C9ORF72 variants (D), sister cells were subjected to RNA fluorescence in situ hybridization for the detection of G4C2 RNA–positive foci (E), or cell lysates were prepared for analysis of poly(GP) (F). For (D) to (F), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unpaired, two-tailed t test. Error bars represent SEM for the three replicates for each cell line. a.u., arbitrary units. Scale bar, 10 μm.

  • Fig. 4. c9ASO-2 treatment decreases intracellular and extracellular poly(GP) in c9ALS iPSC neurons.

    (A) Poly(GP) in lysates and media from cultured iPSNs derived from C9ORF72 repeat expansion carriers (C9+; n = 7) and noncarriers (C9−; n = 3). Red horizontal lines indicate the median. *P < 0.05, Wilcoxon rank sum test. (B) A significant correlation was observed between extracellular and intracellular poly(GP) in the c9ALS iPSN lines (Spearman’s r = 0.86, P = 0.02, n = 7). (C to E) At day 45 of differentiation, three c9ALS iPSN lines were treated with a control ASO or an ASO targeting intron 1 of C9ORF72 (c9ASO-2) at the indicated concentrations. Media were collected before treatment (day 0) and every 5 days thereafter. On day 20, RNA or protein was prepared from cells. (C) Total C9ORF72 or repeat-containing C9ORF72 mRNA transcripts. *P < 0.05, ***P < 0.001, one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. (D) Poly(GP) in c9ALS iPSN media at different time points after treatment initiation (**P < 0.01, ***P < 0.001, ****P < 0.0001, two-way ANOVA) or in cell lysates after the 20-day exposure to c9ASO-2 (**P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA followed by Tukey’s multiple comparisons test). (E) Intracellular and extracellular poly(GP) in c9ALS iPSNs treated with ASO was significantly correlated (Spearman’s r = 0.99, P < 0.0001, n = 12).

  • Fig. 5. Decreased CSF and brain poly(GP) in (G4C2)66 mice treated with c9ASO-1.

    At 4 to 4.5 months of age, (G4C2)2 mice and (G4C2)66 mice were treated with a single intracerebroventricular bolus injection of PBS or c9ASO-1. Eight weeks later, CSF and tissues were harvested from mice for biochemical and immunohistochemical analyses. (A and B) Amount of repeat-containing mRNA or endogenous mouse C9orf72 mRNA in brain tissue from (G4C2)66 mice treated with PBS (n = 11) or c9ASO-1 (n = 9). (C and D) Representative images of RNA foci in the motor cortex of (G4C2)66 mice and quantitative analysis of the percentage of foci-positive cells (n = 6 per group). (E and F) Immunohistochemical analysis and quantification of poly(GA), poly(GP), or poly(GR) pathology in the motor cortex of (G4C2)66 mice treated with PBS (n = 6) or c9ASO-1 (n = 5). Scale bar, 10 μm. (G and H) Poly(GA) or poly(GP) in brain homogenates or CSF of (G4C2)66 mice treated with PBS (n = 11) or c9ASO-1 (n = 9). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unpaired, two-tailed t test. Red horizontal lines indicate the median. See also related figs. S3 and S4.

  • Table 1. Subject characteristics according to C9ORF72 repeat expansion status and disease group.

    The sample median (minimum, 25th percentile, 75th percentile, and maximum) is given for continuous variables. The 15 subjects without a C9ORF72 repeat expansion in the “Other diseases” subgroup comprised 4 patients with FTD, 10 with Alzheimer’s disease, and 1 with primary lateral sclerosis. The 24 subjects with a C9ORF72 repeat expansion in the “Other diseases” subgroup comprised 20 patients with FTD, 2 with Alzheimer’s disease, 1 with dementia with Lewy bodies, and 1 diagnosed with bipolar disease. Information was unavailable regarding age of disease onset [n = 12; 11 non-C9ORF72 repeat expansion carriers (n = 1 with ALS and n = 10 with a disease other than ALS or ALS-FTD) and 1 C9ORF72 repeat expansion carrier with FTD], disease onset to CSF collection (n = 11; 10 non-C9ORF72 repeat expansion carriers with a disease other than ALS or ALS-FTD and 1 C9ORF72 repeat expansion carrier with FTD), and onset site (n = 3; one c9ALS and two c9ALS-FTD). For ALSFRS-R scores, data were missing from 10 ALS or ALS-FTD patients without the C9ORF72 repeat expansion, 19 asymptomatic subjects with the expansion, and 23 patients with c9ALS or c9ALS-FTD. N/A, not applicable.

    CharacteristicNon-C9ORF72 repeat expansion carriersC9ORF72 repeat expansion carriers
    Healthy controls
    (n = 48)
    ALS (n = 57)Other diseases
    (n = 15)
    Asymptomatic
    (n = 27)
    ALS or ALS-FTD
    (n = 83)
    Other diseases
    (n = 24)
    Age at disease
    onset (years)
    N/A54 (24, 43, 60, 78)62 (39, 51, 74, 76)N/A56 (33, 52, 62, 74)61 (20, 54, 64, 75)
    Age at CSF
    collection (years)
    57 (23, 41, 64, 85)57 (25, 47, 64, 79)60 (51, 53, 64, 77)46 (28, 32, 56, 63)59 (35, 54, 63, 76)63 (33, 57, 67, 77)
    Gender (male)17 (35.4%)39 (68.4%)9 (60.0%)8 (29.6%)51 (61.4%)12 (50.0%)
    Disease onset to
    CSF collection (months)
    N/A26 (0, 12, 51, 204)12 (0, 12, 12, 240)N/A24 (0, 12, 31, 132)47 (0, 25, 63, 160)
    Onset site
    —BulbarN/A11 (19.3%)N/AN/A25 (31.3%)N/A
    —LimbN/A45 (78.9%)N/AN/A51 (63.8%)N/A
    —OtherN/A1 (1.8%)N/AN/A4 (5.0%)N/A
    ALSFRS-R scoreN/A36 (14, 28, 40, 47)N/A48 (40, 45, 48, 48)36 (7, 28, 42, 46)N/A
    Poly(GP) (ng/ml)0.0 (0.0, 0.0,
    0.0, 0.0)
    0.0 (0.0, 0.0,
    0.0, 0.1)
    0.0 (0.0, 0.0,
    0.0, 0.0)
    0.5 (0.0, 0.2,
    1.0, 4.0)
    0.8 (0.0, 0.5,
    1.6, 5.2)
    1.1 (0.0, 0.4,
    1.9, 5.3)
  • Table 2. Associations of brain or CSF poly(GP) with c9ALS-like pathological features in (G4C2)66 mice.

    Spearman’s r correlation coefficients, 95% CIs, and P values are presented.

    CharacteristicBrain poly(GP)CSF poly(GP)
    Spearman’s r (95% CI)PSpearman’s r (95% CI)P
    Brain poly(GP) (n = 20)0.74 (0.42–0.89)0.0002
    Brain poly(GA) (n = 20)0.92 (0.80–0.97)<0.00010.75 (0.44–0.90)0.0002
    G4C2 repeat–containing mRNA (n = 20)0.87 (0.69–0.95)<0.00010.64 (0.26–0.85)0.0025
    Cells with foci in motor cortex (%) (n = 12)0.78 (0.35–0.94)0.0040.82 (0.45–0.95)0.0017

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/383/eaai7866/DC1

    Materials and Methods

    Fig. S1. Validation of poly(GP) detection in CSF.

    Fig. S2. Relationships between CSF poly(GP) and ALSFRS-R score, behavioral impairment, and cognitive impairment.

    Fig. S3. Decreases in RNA foci and c9RAN protein pathology are observed in (G4C2)66 mice treated with c9ASO-1.

    Fig. S4. Decreases in brain and CSF poly(GP) are observed in (G4C2)66 mice treated with c9ASO-1.

    Fig. S5. Validation of poly(GA) Meso Scale Discovery sandwich immunoassay.

    Table S1. Distribution of the 254 subjects among the sites that provided CSF.

    Table S2. Poly(GP) in CSF from C9ORF72 repeat expansion carriers and noncarriers.

    Table S3. Associations of poly(GP) with C9ORF72 repeat expansion and disease status.

    Table S4. Characteristics according to C9ORF72 repeat expansion carrier status.

    Table S5. Characteristics for asymptomatic C9ORF72 repeat expansion carriers and patients with c9ALS or c9ALS-FTD.

    Table S6. Poly(GP) in CSF collected longitudinally from C9ORF72 repeat expansion carriers.

    Table S7. Secondary comparisons of CSF poly(GP) in patients with c9ALS or c9ALS-FTD.

    Table S8. Neuropsychological test battery for Mayo Clinic and NIH.

    Table S9. Characteristics according to behavioral impairment in patients with c9ALS or c9ALS-FTD.

    Table S10. Characteristics according to cognitive impairment in patients with c9ALS or c9ALS-FTD.

    Table S11. Subject characteristics for PBMCs.

    Table S12. Subject characteristics for lymphoblastoid cell lines.

    Table S13. Subject characteristics for iPSC lines.

  • Supplementary Material for:

    Poly(GP) proteins are a useful pharmacodynamic marker for C9ORF72-associated amyotrophic lateral sclerosis

    Tania F. Gendron, Jeannie Chew, Jeannette N. Stankowski, Lindsey R. Hayes, Yong-Jie Zhang, Mercedes Prudencio, Yari Carlomagno, Lillian M. Daughrity, Karen Jansen-West, Emilie A. Perkerson, Aliesha O'Raw, Casey Cook, Luc Pregent, Veronique Belzil, Marka van Blitterswijk, Lilia J. Tabassian, Chris W. Lee, Mei Yue, Jimei Tong, Yuping Song, Monica Castanedes-Casey, Linda Rousseau, Virginia Phillips, Dennis W. Dickson, Rosa Rademakers, John D. Fryer, Beth K. Rush, Otto Pedraza, Ana M. Caputo, Pamela Desaro, Carla Palmucci, Amelia Robertson, Michael G. Heckman, Nancy N. Dieh, Edythe Wiggs, Michael Tierney, Laura Braun, Jennifer Farren, David Lacomis, Shafeeq Ladha, Christina N. Fournier, Leo F. McCluskey, Lauren B. Elman, Jon B. Toledo, Jennifer D. McBride, Cinzia Tiloca, Claudia Morelli, Barbara Poletti, Federica Solca, Alessandro Prelle, Joanne Wuu, Jennifer Jockel-Balsarotti, Frank Rigo, Christine Ambrose, Abhishek Datta, Weixing Yang, Denitza Raitcheva, Giovanna Antognetti, Alexander McCampbell, John C. Van Swieten, Bruce L. Miller, Adam L. Boxer, Robert H. Brown, Robert Bowser, Timothy M. Miller, John Q. Trojanowski, Murray Grossman, James D. Berry, William T. Hu, Antonia Ratti, Bryan J. Traynor, Matthew D. Disney, Michael Benatar, Vincenzo Silani, Jonathan D. Glass, Mary Kay Floeter, Jeffrey D. Rothstein, Kevin B. Boylan, Leonard Petrucelli*

    *Corresponding author. Email: petrucelli.leonard{at}mayo.edu

    Published 29 March 2017, Sci. Transl. Med. 9, eaai7866 (2017)
    DOI: 10.1126/scitranslmed.aai7866

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Validation of poly(GP) detection in CSF.
    • Fig. S2. Relationships between CSF poly(GP) and ALSFRS-R score, behavioral impairment, and cognitive impairment.
    • Fig. S3. Decreases in RNA foci and c9RAN protein pathology are observed in (G4C2)66 mice treated with c9ASO-1.
    • Fig. S4. Decreases in brain and CSF poly(GP) are observed in (G4C2)66 mice treated with c9ASO-1.
    • Fig. S5. Validation of poly(GA) Meso Scale Discovery sandwich immunoassay.
    • Table S1. Distribution of the 254 subjects among the sites that provided CSF.
    • Table S2. Poly(GP) in CSF from C9ORF72 repeat expansion carriers and noncarriers.
    • Table S3. Associations of poly(GP) with C9ORF72 repeat expansion and disease status.
    • Table S4. Characteristics according to C9ORF72 repeat expansion carrier status.
    • Table S5. Characteristics for asymptomatic C9ORF72 repeat expansion carriers and patients with c9ALS or c9ALS-FTD.
    • Table S6. Poly(GP) in CSF collected longitudinally from C9ORF72 repeat expansion carriers.
    • Table S7. Secondary comparisons of CSF poly(GP) in patients with c9ALS or c9ALS-FTD.
    • Table S8. Neuropsychological test battery for Mayo Clinic and NIH.
    • Table S9. Characteristics according to behavioral impairment in patients with c9ALS or c9ALS-FTD.
    • Table S10. Characteristics according to cognitive impairment in patients with c9ALS or c9ALS-FTD.
    • Table S11. Subject characteristics for PBMCs.
    • Table S12. Subject characteristics for lymphoblastoid cell lines.
    • Table S13. Subject characteristics for iPSC lines.

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