Poly(GR) and poly(GA) in cerebrospinal fluid as potential biomarkers for C9ORF72-ALS/FTD

Gopinath Krishnan, Denitza Raitcheva, Daniel Bartlett, Mercedes Prudencio, Diane M McKenna-Yasek, Catherine Douthwright, Björn E Oskarsson, Shafeeq Ladha, Oliver D King, Sami J Barmada, Timothy M Miller, Robert Bowser, Jonathan K Watts, Leonard Petrucelli, Robert H Brown, Mark W Kankel, Fen-Biao Gao, Gopinath Krishnan, Denitza Raitcheva, Daniel Bartlett, Mercedes Prudencio, Diane M McKenna-Yasek, Catherine Douthwright, Björn E Oskarsson, Shafeeq Ladha, Oliver D King, Sami J Barmada, Timothy M Miller, Robert Bowser, Jonathan K Watts, Leonard Petrucelli, Robert H Brown, Mark W Kankel, Fen-Biao Gao

Abstract

GGGGCC repeat expansion in C9ORF72, which can be translated in both sense and antisense directions into five dipeptide repeat (DPR) proteins, including poly(GP), poly(GR), and poly(GA), is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we developed sensitive assays that can detect poly(GA) and poly(GR) in the cerebrospinal fluid (CSF) of patients with C9ORF72 mutations. CSF poly(GA) and poly(GR) levels did not correlate with age at disease onset, disease duration, or rate of decline of ALS Functional Rating Scale, and the average levels of these DPR proteins were similar in symptomatic and pre-symptomatic patients with C9ORF72 mutations. However, in a patient with C9ORF72-ALS who was treated with antisense oligonucleotide (ASO) targeting the aberrant C9ORF72 transcript, CSF poly(GA) and poly(GR) levels decreased approximately 50% within 6 weeks, indicating they may serve as sensitive fluid-based biomarkers in studies directed against the production of GGGGCC repeat RNAs or DPR proteins.

Trial registration: ClinicalTrials.gov NCT01925196.

Conflict of interest statement

D.R., D.B., and M.W.K. are employees and shareholders of Biogen. S.L. received research support from Biogen (Cambridge, MA), Sanofi (Paris, France), Amylyx Pharmaceuticals (Cambridge, MA), Mitsubishi Tanabe Pharma (Osaka, Japan), and consulting from Biogen (Cambridge, MA). B.O. received research support from Biogen (Cambridge, MA), Mitsubishi Tanabe Pharma America (Jersey City, NJ), MediciNova (La Jolla, CA), AZ Therapeutics (Cambridge, MA), Eisai (Tokyo, Japan), and consulting fees from Biogen (Cambridge, MA), Amylyx Pharmaceuticals, (Cambridge, MA), Mitsubishi Tanabe Pharma America (Jersey City, NJ), MediciNova (La Jolla, CA), and Tsumura (Tokyo, Japan). S.J.B. consults for Exicure (Chicago, IL), Clene (Salt Lake City, UT), KorroBio (Cambridge, MA), Faze Medicine (Cambridge, MA), and NeuroCures Foundation. T.M.M. received consulting fees from Ionis, Disarm Therapeutics, Cytokinetics and has licensing agreement with Ionis and C2N, and serves on the advisory boards of Biogen and UCB. R.B. is the founder of Iron Horse Diagnostics (Phoenix, AZ) and consults for Mitsubishi Tanabe Pharma America (Jersey City, NJ), Takeda (Tokyo, Japan), Aural Analytics (Scottsdale, AZ), and NeuroCures Foundation. J.K.W. is an ad hoc consultant for BridgeBio and Flagship Pioneering, and is on the Scientific Advisory Board of PepGen. L.P. is a consultant for Expansion therapeutics. R.H.B. consulted for Wave Life Sciences and is co-founder of Apic Bio. F.-B.G. received an honorarium from Alkermes company. The remaining authors declare no competing interests.

© 2022. The Author(s).

Figures

Fig. 1. Blinded measurement of poly(GR) levels…
Fig. 1. Blinded measurement of poly(GR) levels in CSF in multiple cohorts of patients with C9ORF72 mutations using the MSD-based poly(GR) immunoassay.
a In spike-and-recovery experiments, recombinant (GR)8 peptide was added to CSF from healthy individuals and subsequently serially diluted into a separate tube containing CSF from healthy individuals to create a 5-point series. The average recovery rate in two different samples was 81–88%. b The assay can detect purified (GR)8 peptide at concentrations as low as 3 pg/ml. Replicates of 8-point series standard curve run on different dates are shown. A four-parameter logistic curve was used to fit the dose-response with GraphPad Prism 9.1. c Across the dynamic range of the assay, the coefficient of variation is less than 16% for all points except one concentration (100 pg/ml), below the 20% cutoff recommended by the FDA in standard variability for liquid-binding assays. d Dilution linearity was evaluated by measuring duplicates of 5 dilutions of 2 patient CSF samples spiked with 200 ng/ml (GR)8 peptide. e Poly(GR) immunoassay specifically detects (GR)8 peptide in control CSF and did not cross react with even up to 100 ng of (GP)8 peptide in control CSF, n = 3 independent experiments, values are mean ± s.e.m. f CSF samples from 4 healthy and 4 patients with C9ORF72 mutations from the Mayo Clinic, Florida, values are mean ± s.e.m (p = 0.0017, Unpaired t-test with Welch’s correction). g CSF samples from 3 healthy people from the NEALS cohort and 5 CSF samples from two patients with C9ORF72 mutations from the Barrow Institute, Arizona, values are mean ± s.e.m (p = 0.0012, Unpaired t-test with Welch’s correction). h 19 healthy people, 19 patients with ALS but without C9ORF72 mutations (3 patients with duplicate samples, 16 patients with single samples), and 10 patients with C9ORF72 mutations (8 patients with duplicate samples, 2 patients with single samples) from the NEALS Consortium. Values from duplicate samples are presented as mean ± s.e.m (p = 0.9460 control vs non-C9-ALS, p < 0.0001 non-C9-ALS vs C9-ALS, p < 0.0001 control vs C9-ALS Ordinary one-way ANOVA-Tukey’s multiple comparisons test). Source data are provided as a Source Data file.
Fig. 2. Poly(GR) levels in CSF do…
Fig. 2. Poly(GR) levels in CSF do not correlate with clinical features of patients with C9ORF72 mutations.
a–c Correlations between poly(GR) levels and rate of ALSFRS-R score decline (a), age at disease onset (b), and disease duration (c), determined by two-tailed, Spearman’s rank-correlation analysis. d Poly(GR) levels in CSF of symptomatic and asymptomatic patients with C9ORF72 mutations are similar (p = 0.114 by two-tailed unpaired t-test with Mann–Whitney test). Poly(GR) concentrations are presented as values equivalent to (GR)8 peptide and not as an absolute poly(GR) concentration since the size of poly(GR) in CSF is unknown. 25 out of 41 symptomatic CSF samples and 10 out of 14 pre-symptomatic CSF samples had poly(GR) levels within the linear range of our standard curves and are presented here. ALS Functional Rating Scale revised (ALSFRS-R). Source data are provided as a Source Data file.
Fig. 3. Poly(GA) levels in CSF do…
Fig. 3. Poly(GA) levels in CSF do not correlate with clinical features of patients with C9ORF72 mutations.
a–c Correlation between poly(GA) levels and the rate of ALSFRS-R score decline (a), age at disease onset (b), and disease duration (c), determined by two-tailed Spearman’s rank-correlation analysis. d Poly(GA) levels in CSF from symptomatic and asymptomatic patients with C9ORF72 mutations are similar by two-tailed unpaired t-test with Mann–Whitney test. Poly(GA) concentrations are presented as values equivalent to (GA)60 and not as an absolute poly(GA) concentration since the size of poly(GA) in CSF is unknown. The samples assessed in Figs. 2, 3 are overlapping but not identical and include 17 samples for which both poly(GA) and poly(GR) were detected. Source data are provided as a Source Data file.
Fig. 4. ASO treatment in a patient…
Fig. 4. ASO treatment in a patient with C9ORF72-ALS decreases CSF DPR levels.
a Poly(GR) levels in CSF were generally stable over time in two symptomatic and two asymptomatic patients with C9ORF72 mutations. b Longitudinal trend of poly(GR) levels in CSF from a larger cohort of patients with C9ORF72 mutations. c Longitudinal trend of poly(GA) levels in CSF from patients with C9ORF72 mutations. d After ASO treatment, CSF poly(GP), poly(GA), and poly(GR) levels in a patient with C9ORF72-ALS decreased over time. Note: colors in panels b, c do not indicate the same patients. The fifth time point value for symptomatic patient 2 in panel a and the last time point for poly(GR) value in panel b were above background but below the lower limit of detection and were not included. Source data are provided as a Source Data file.

References

    1. Ling SC, Polymenidou M, Cleveland DW. Converging mechanisms in ALS and FTD: disrupted RNA and protein homeostasis. Neuron. 2013;79:416–438. doi: 10.1016/j.neuron.2013.07.033.
    1. Abramzon YA, et al. The overlapping genetics of amyotrophic lateral sclerosis and frontotemporal dementia. Front. Neurosci. 2020;14:42. doi: 10.3389/fnins.2020.00042.
    1. Gao F-B, Almeida S, Lopez-Gonzalez R. Dysregulated molecular pathways in amyotrophic lateral sclerosis–frontotemporal dementia spectrum disorder. EMBO J. 2017;36:2931–2950.
    1. DeJesus-Hernandez M, et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron. 2011;72:245–256. doi: 10.1016/j.neuron.2011.09.011.
    1. Renton AE, et al. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron. 2011;72:257–268. doi: 10.1016/j.neuron.2011.09.010.
    1. Mori K, et al. The C9orf72GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS. Science. 2013;339:1335–1338. doi: 10.1126/science.1232927.
    1. Ash PEA, et al. Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS. Neuron. 2013;77:639–646. doi: 10.1016/j.neuron.2013.02.004.
    1. Zu T, et al. RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia. Proc. Natl Acad. Sci. USA. 2013;110:E4968–E4977. doi: 10.1073/pnas.1315438110.
    1. Saberi S, et al. Sense-encoded poly-GR dipeptide repeat proteins correlate to neurodegeneration and uniquely co-localize with TDP-43 in dendrites of repeat-expanded C9orf72 amyotrophic lateral sclerosis. Acta Neuropathol. 2018;135:459–474. doi: 10.1007/s00401-017-1793-8.
    1. Sakae N, et al. Poly-GR dipeptide repeat polymers correlate with neurodegeneration and clinicopathological subtypes in C9ORF72-related brain disease. Acta Neuropathol. Commun. 2018;6:63. doi: 10.1186/s40478-018-0564-7.
    1. Quaegebeurn A, et al. Soluble and insoluble dipeptide repeat protein measurements in C9orf72-frontotemporal dementia brains show regional differential solubility and correlation of poly-GR with clinical severity. Acta Neuropathol. Commun. 2020;8:184. doi: 10.1186/s40478-020-01036-y.
    1. Gitler AD, Tsuji H. There has been an awakening: emerging mechanisms of C9orf72 mutations in ALS/FTD. Brain Res. 2016;1647:19–29. doi: 10.1016/j.brainres.2016.04.004.
    1. Schmitz A, et al. Emerging perspectives on dipeptide repeat proteins in C9ORF72 ALS/FTD. Front. Cell Neurosci. 2021;15:637548. doi: 10.3389/fncel.2021.637548.
    1. Choi S, et al. C9ORF72-ALS/FTD-associated poly(GR) binds ATP5alpha1 and compromises mitochondria function in vivo. Nat. Neurosci. 2019;22:851–862. doi: 10.1038/s41593-019-0397-0.
    1. Mackenzie IR, et al. Quantitative analysis and clinico-pathological correlations of different dipeptide repeat protein pathologies in C9ORF72 mutation carriers. Acta Neuropathol. 2015;130:845–861. doi: 10.1007/s00401-015-1476-2.
    1. Schludi MH, et al. Distribution of dipeptide repeat proteins in cellular models and C9orf72 mutation cases suggests link to transcriptional silencing. Acta Neuropathol. 2015;130:537–555. doi: 10.1007/s00401-015-1450-z.
    1. Zhang YJ, et al. C9ORF72 poly(GA) aggregates sequester and impair HR23 and nucleocytoplasmic transport proteins. Nat. Neurosci. 2016;19:668–677. doi: 10.1038/nn.4272.
    1. LaClair KD, et al. Congenic expression of poly-GA but not poly-PR in mice triggers selective neuron loss and interferon responses found in C9orf72 ALS. Acta Neuropathol. 2020;140:121–142. doi: 10.1007/s00401-020-02176-0.
    1. May S, et al. C9orf72 FTLD/ALS-associated Gly-Ala dipeptide repeat proteins cause neuronal toxicity and Unc119 sequestrationC9orf72 FTLD/ALS-associated Gly-Ala dipeptide repeat proteins cause neuronal toxicity and Unc119 sequestration. Acta Neuropathol. 2014;128:485–503. doi: 10.1007/s00401-014-1329-4.
    1. Zhang YJ, et al. Aggregation-prone c9FTD/ALS poly(GA) RAN-translated proteins cause neurotoxicity by inducing ER stress. Acta Neuropathol. 2014;128:505–524. doi: 10.1007/s00401-014-1336-5.
    1. Lee YB, et al. C9orf72 poly GA RAN-translated protein plays a key role in amyotrophic lateral sclerosis via aggregation and toxicity. Hum. Mol. Genet. 2017;26:4765–4777. doi: 10.1093/hmg/ddx350.
    1. Gendron TF, et al. Poly(GP) proteins are a useful pharmacodynamic marker for C9ORF72-associated amyotrophic lateral sclerosis. Sci. Transl. Med. 2017;9:eaai7866. doi: 10.1126/scitranslmed.aai7866.
    1. Lehmer C, et al. Poly-GP in cerebrospinal fluid links C9orf72-associated dipeptide repeat expression to the asymptomatic phase of ALS/FTD. EMBO Mol. Med. 2017;9:859–868. doi: 10.15252/emmm.201607486.
    1. Cammack AJ, et al. Prospective natural history study of C9orf72 ALS clinical characteristics and biomarkers. Neurology. 2019;93:e1605–e1617. doi: 10.1212/WNL.0000000000008359.
    1. Bennett CF, Kordasiewicz HB, Cleveland DW. Antisense drugs make sense for neurological diseases. Annu. Rev. Pharmacol. Toxicol. 2021;61:831–852. doi: 10.1146/annurev-pharmtox-010919-023738.
    1. Tran H, et al. Suppression of mutant C9orf72 expression by a potent mixed backbone antisense oligonucleotide. Nat. Med. 2022;28:117–124. doi: 10.1038/s41591-021-01557-6.
    1. Nguyen L, et al. Antibody therapy targeting RAN proteins rescues C9 ALS/FTD phenotypes in C9orf72 mouse model. Neuron. 2020;105:645–662.e11. doi: 10.1016/j.neuron.2019.11.007.
    1. Zhou Q, et al. Active poly-GA vaccination prevents microglia activation and motor deficits in a C9orf72 mouse model. EMBO Mol. Med. 2020;12:e10919.
    1. Lopez-Gonzalez R, et al. Poly(GR) in C9ORF72-related ALS/FTD compromises mitochondrial function and increases oxidative stress and DNA damage in iPSC-derived motor neurons. Neuron. 2016;92:383–391. doi: 10.1016/j.neuron.2016.09.015.
    1. Almeida S, et al. Production of poly(GA) in C9ORF72 patient motor neurons derived from induced pluripotent stem cells. Acta Neuropathol. 2019;138:1099–1101. doi: 10.1007/s00401-019-02083-z.
    1. Stefura WP, et al. Improved methods for quantifying human chemokine and cytokine biomarker responses: Ultrasensitive ELISA and meso scale electrochemiluminescence assays. Methods Mol. Biol. 2019;2020:91–114. doi: 10.1007/978-1-4939-9591-2_7.
    1. U.S. Food and Drug Administration. Guidance for industry: Bioanalytical method validation. (2013).
    1. Andreasson U, et al. A practical guide to immunoassay method validation. Front. Neurol. 2015;19:179.
    1. Meeter LHH, et al. Poly(GP), neurofilament and grey matter deficits in C9orf72 expansion carriers. Ann. Clin. Transl. Neurol. 2018;5:583–597. doi: 10.1002/acn3.559.
    1. Gendron TF, et al. Cerebellar c9RAN proteins associate with clinical and neuropathological characteristics of C9ORF72 repeat expansion carriers. Acta Neuropathol. 2015;130:559–573. doi: 10.1007/s00401-015-1474-4.
    1. van Blitterswijk M, et al. Novel clinical associations with specific C9ORF72 transcripts in patients with repeat expansions in C9ORF72. Acta Neuropathol. 2015;130:863–876. doi: 10.1007/s00401-015-1480-6.
    1. Vallabh SM, et al. Prion protein quantification in human cerebrospinal fluid as a tool for prion disease drug development. Proc. Natl Acad. Sci. USA. 2019;116:7793–7798. doi: 10.1073/pnas.1901947116.
    1. Shoukry RS, et al. Longitudinal changes in resting state networks in early presymptomatic carriers of C9orf72 expansions. Neuroimage Clin. 2020;28:102354. doi: 10.1016/j.nicl.2020.102354.
    1. Krishnan G, et al. CRISPR deletion of the C9ORF72 promoter in ALS/FTD patient motor neurons abolishes production of dipeptide repeat proteins and rescue neurodegeneration. Acta Neuropathol. 2020;140:81–84. doi: 10.1007/s00401-020-02154-6.
    1. Sonobe Y, et al. A C. elegans model of C9orf72-associatred ALS/FTD uncovers a conserved role for eIF2D in RAN translation. Nat. Commun. 2021;12:6025. doi: 10.1038/s41467-021-26303-x.

Source: PubMed

スポンサーと協力者

医学的状態

薬物療法

3
購読する