C9orf72 RNA foci: Much Ado About Nothing in ALS and FTD?

One-Two-Three? The production of C9orf72 repeat-containing RNAs, synthesis of dipeptide repeat proteins, loss of c9orf72 function, and other factors may work together to determine if, when, and how ALS, FTD or ALS/FTD appears in people harboring repeat expansions in the C9orf72 gene according to a new study (DeJesus-Hernandez et al., 2017). [Gitler and Tsuiji, 2016 under CC-BY 4.0 license.]

Clumps of RNA and associated proteins filling the nuclei of brain cells carrying an ALS-linked repeat expansion in the C9orf72 gene certainly look like they could mean trouble. But according to a large post-mortem brain tissue analysis of 63 expanded repeat carriers, published online May 15 in Acta Neuropathologica, there is very little correlation between the number or distribution of these nuclear RNA foci and if and when people develop ALS, frontotemporal dementia (FTD), or a combination of both.

“They likely are not the main determinants of whether somebody gets FTD or ALS,” concluded Rosa Rademakers of the Mayo Clinic in Jacksonville, Florida, senior author of the study. “But I don’t think that means that they’re not important, or they’re not contributing to the disease.” More likely, she suspects, the RNA foci are players in an ensemble cast, which may include C9orf72-associated dipeptide repeat proteins (DRPs). These RNAs and proteins could act in concert with a variety of factors such as a person’s lifestyle, genetic and epigenetic makeup, and loss of C9orf72 function to determine whether, when, and how these expanded repeats might manifest as disease.

Five Years’ FISHing

C9orf72 repeats result in a variety of disease phenotypes—or may not cause symptoms at all. That leaves people harboring the expansion in the dark about what to expect, notes Mayo Clinic’s Marka Van Blitterswijk in Florida, co-senior author of the study. She, Rademakers, and colleagues have already investigated how the size of these repeats, the presence of specific C9orf72 isoforms, and the DPRs line up with clinical and pathological features of the disease (see November 2013 conference news; Van Blitterswijk et al., 2013; Van Blitterswijk et al., 2015; Gendron et al., 2015). They found some significant correlations, such as a link between increased levels of one C9orf72 isoform and survival after onset (Van Blitterswijk et al., 2015), but the understanding of what underlies the clinical and pathological variability remains far from complete.

RNA foci, the researchers reasoned, might also be relevant to that variability. In 2012, first author Mariely DeJesus-Hernandez began to count RNA foci in the post-mortem brain tissue of people harboring expanded C9orf72 repeats. Upon completion, DeJesus-Hernandez had examined the frontal cortex and cerebellum of 63 expansion carriers, including people who had ALS, FTD, or a combination of both, and one who had Alzheimer’s disease.

To carry out this analysis, she used fluorescence in situ hybridization (FISH) to label RNA foci containing either sense-direction or antisense C9orf72 RNAs, since both directions can be transcribed and form foci, and thereby potentially could contribute to the disease (see November 2013 news; Zu et al., 2013; Gendron et al., 2013).

AI Artificial intelligence. Researchers designed a computer method to outline neuronal nuclei in red, but ignore glial nuclei, then count the green-labeled RNA foci within. [DeJesus-Hernandez et al., 2017 under CC BY 4.0 license.]

Others have assessed RNA foci patterns in the brain before (Mizielinska et al., 2013; Cooper-Knock et al., 2015), but DeJesus-Hernandez analyzed a much larger sample set. To assist with the monumental task of counting all those foci, Rademaker’s team developed and validated an automated computer pipeline to identify the cell nuclei and count the foci in each microscopic field. The computer’s results correlated well with those obtained by human eye, according to their analysis.

Using this approach, DeJesus-Hernandez found that in the frontal cortex of the brain, 32% of neurons contained sense RNA foci and 16% contained antisense RNA foci in the nucleus. In the cerebellum, 23% and 1% of granule cells contained sense and antisense RNA foci, respectively. In the same area, 70% of Purkinje cells had sense RNA foci and 74% of them had antisense RNA foci.

A Single Correlation

A toxic sponge? Repeat antisense RNAs form foci in Purkinje neurons from C9orf72 patients, sequestering RNA binding proteins such as hnRNP A1, hnRNP H/F, ALYREf, and hnRNP K. This could result in toxicity by disrupting RNA splicing or translation. [Cooper-Knock et al., 2015 under a CC-BY 4.0 license.]

Then, Van Blitterswijk and colleagues asked whether the pattern of RNA foci would correlate with an array of clinical and pathological features including disease type, age of onset and death, repeat length, number of total C9orf72 transcripts, and the presence of DPRs. They came up nearly empty. For example, there was no indication that people with more foci in the frontal cortex were more likely to have FTD than ALS. But there was one statistically significant hit: the older a person was when they first showed symptoms of FTD, the higher the number of antisense RNA foci present in the frontal cortex at death.

There are two possible interpretations, said Rademakers. It could be that the antisense RNA foci are somehow protective, keeping the people harboring C9orf72 expanded repeats healthy longer (see September 2015 news; Tran et al., 2015). But she and Van Blitterswijk think it’s more likely that the people who had a later age of onset were simply older when they died, and thus had more years to accumulate them.

Adrian Isaacs of University College London, who was not involved in the study, agreed that the idea of antisense RNA foci being protective seems unlikely. If that were true, he pointed out, one would expect to see the number and/or distribution of these foci to correlate with some of the other disease measures Van Blitterswijk examined.

The Protector? C9orf72 expansion carriers who were older when disease symptoms struck tended to have more antisense RNA foci in the frontal cortex, but researchers suspect this was merely a result of these foci accumulating in these cells over a lifetime [DeJesus-Hernandez et al., 2017 under a CC-BY 4.0 license.]

“There’s no consistency,” said Isaacs, adding that the paper confirms a general impression in the field that there are no obvious harmful effects of RNA foci.

But that does not mean that all C9orf72 RNA foci are absolved of any link to ALS and/or FTD, according to Isaacs. “The one thing the paper doesn’t address is cytoplasmic [RNA] foci.” These RNA foci are less frequent than the nuclear ones in the brain, according to a 2013 analysis led by Isaacs. But he and his colleagues did detect them in at least 1% of neurons in people with C9orf72-linked FTD (Mizielinksa et al., 2013). Therefore, he said, soluble C9orf72 RNA or RNA foci in the cytoplasm could still play a role in the disease.

But it’s clear, from this and previous studies, that there’s no starring role for RNA foci in C9orf72-based ALS, commented Robert Baloh of Cedars-Sinai Medical Center in Los Angeles, California who was not involved in the study. For example, he said, some mouse models carrying C9orf72 repeats develop RNA foci but not neurodegeneration (see December 2015 news; O’Rourke et al., 2015; Peters et al., 2015). Plus, RNA foci appear early in neurons derived from induced pluripotent stem cells from patients, Baloh said. That suggests to him that in people, they probably appear in young neurons, decades before any symptoms—making it hard to see a link between the foci and the onset of the disease (see July 2016 news; Almeida et al., 2013; Sareen et al., 2013; Ho et al., 2016). “This does not rule out that [RNA foci] cannot contribute in concert with other factors,” he added.

Sense & antisensibility. Some cells in the frontal cortex of the brain contained several RNA foci. While cells holding sense foci were more common, when antisense foci were present, they were more likely to be in greater abundance. [DeJesus-Hernandez et al., 2017 under CC-BY 4.0 license.]

One more piece of evidence supports that idea, said Van Blitterswijk: their single case with Alzheimer’s disease, who also had a secondary diagnosis of FTD. That woman had the highest percentage of antisense foci in the brain of all the subjects tested, 28% of her cells. And, her brain had the most sense RNA foci in the cerebellum, 54% of granule cells and 89% of Purkinje cells. She also exhibited some of the highest levels of C9orf72 transcripts and DPRs, but relatively few repeats, at about 8 kb. Perhaps, Van Blitterswijk and Rademakers hypothesize, the combination of these extreme characteristics may explain why she developed the unusual phenotype of Alzheimer’s disease with FTD, though they caution it’s only one case.

Clinical Implications

Meanwhile, research teams, in collaboration with Biogen in Massachusetts and Ionis Pharmaceuticals in California, are continuing to develop antisense oligonucleotides (ASOs) that would destroy C9orf72 RNAs (see May 2016 news; Lagier-Tourenne et al., 2013; Sareen et al., 2013). “They would, in theory, get rid of both RNA and DPRs,” said Rademakers. “It’s still, at this point, the best strategy.”

Van Blitterswijk added that tests to measure RNA focus load, along with those for DPR quantity, should still be useful to test target engagement in preclinical studies of anti-C9orf72 therapeutics (see February 2017 news). In human clinical trials, DPRs may have the advantage; there are already two tests to detect at least one version, poly-glycine-proline, in cerebrospinal fluid (see March 2017, April 2017 news; Gendron et al., 2017; Lehmer et al., 2017). It would likely be trickier to identify RNA foci themselves in blood or other easily accessible tissues from a living person, Rademakers said. However, she speculated, it might be possible to see transcriptional changes when RNA foci form and RNA-binding proteins are sequestered. Those changes could theoretically serve as a biomarker in developing a treatment.

Featured Paper

DeJesus-Hernandez M, Finch NA, Wang X, Gendron TF, Bieniek KF, Heckman MG, Vasilevich A, Murray ME, Rousseau L, Weesner R, Lucido A, Parsons M, Chew J, Josephs KA, Parisi JE, Knopman DS, Petersen RC, Boeve BF, Graff-Radford NR, de Boer J, Asmann YW, Petrucelli L, Boylan KB, Dickson DW, Van Blitterswijk M, Rademakers R. In-depth clinic-pathological examination of RNA foci in a large cohort of C9ORF72 expansion carriers. Acta Neuropathol. 2017 May 15. [Epub ahead of print] [PubMed]


Van Blitterswijk M, DeJesus-Hernandez M, Niemantsverdriet E, Murray ME, Heckman MG, Diehl NN, Brown PH, Baker MC, Finch NA, Bauer PO, Serrano G, Beach TG, Josephs KA, Knopman DS, Petersen RC, Boeve BF, Graff-Radford NR, Boylan KB, Petrucelli L, Dickson DW, Rademakers R. Association between repeat sizes and clinical and pathological characteristics in carriers of C9ORF72 repeat expansions (Xpansize-72): a cross-sectional cohort study. Lancet Neurol. 2013 Oct;12(10):978-88. [PubMed]

Van Blitterswijk M, Gendron TF, Baker MC, DeJesus-Hernandez M, Finch NA, Brown PH, Daughrity LM, Murray ME, Heckman MG, Jiang J, Lagier-Tourenne C, Edbauer D, Cleveland DW, Josephs KA, Parisi JE, Knopman DS, Petersen RC, Petrucelli L, Boeve BF, Graff-Radford NR, Boylan KB, Dickson DW, Rademakers R. Novel clinical associations with specific C9ORF72 transcripts in patients with repeat expansions in C9ORF72. Acta Neuropathol. 2015 Dec;130(6):863-76. [PubMed]

Gendron TF, Van Blitterswijk M, Bieniek KF, Daughrity LM, Jiang J, Rush BK, Pedraza O, Lucas JA, Murray ME, Desaro P, Robertson A, Overstreet K, Thomas CS, Crook JE, Castanedes-Casey M, Rousseau L, Josephs KA, Parisi JE, Knopman DS, Petersen RC, Boeve BF, Graff-Radford NR, Rademakers R, Lagier-Tourenne C, Edbauer D, Cleveland DW, Dickson DW, Petrucelli L, Boylan KB. Cerebellar c9RAN proteins associate with clinical and neuropathological characteristics of C9ORF72 repeat expansion carriers. Acta Neuropathol. 2015 Oct;130(4):559-73. [PubMed]

Zu T, Liu Y, Bañez-Coronel M, Reid T, Pletnikova O, Lewis J, Miller TM, Harms MB, Falchook AE, Subramony SH, Ostrow LW, Rothstein JD, Tronosco JC, Ranum LP. RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia. Proc Natl Acad Sci U S A. 2013 Dec 17;110(51):E4968-77. [PubMed]

Gendron TF, Bieniek KF, Zhang YJ, Jansen-West K, Ash PE, Caulfield T, Daughrity L, Dunmore JH, Castanedes-Casey M, Chew J, Cosio DM, Van Blitterswijk M, Lee WC, Rademakers R, Boylan KB, Dickson DW, Petrucelli L. Antisense transcripts of the expanded C9ORF72 hexanucleotide repeat form nuclear RNA foci and undergo repeat-associated non-ATG translation in c9FTD/ALS. Acta Neuropathol. 2013 Dec;126(6):829-44. [PubMed]

Tran H, Almeida S, Moore J, Gendron TF, Chalasani U, Lu Y, Du X, Nickerson JA, Petrucelli L, Weng Z, Gao FB. Differential Toxicity of Nuclear RNA Foci versus Dipeptide Repeat Proteins in a Drosophila Model of C9ORF72 FTD/ALS. Neuron. 2015 Sep 23;87(6):1207-1214. [PubMed].

Mizielinska S, Lashley T, Norona FE, Clayton EL, Ridler CE, Fratta P, Isaacs AM. C9orf72 frontotemporal lobar degeneration is characterised by frequent neuronal sense and antisense RNA foci. Acta Neuropathol. 2013 Dec;126(6):845-57. [PubMed]

Cooper-Knock J, Higginbottom A, Stopford MJ, Highley JR, ince PG, Wharton SB, Pickering-Brown S, Kirby J, Hautbergue GM, Shaw PJ. Antisense RNA foci in the motor neurons of C9ORF72-ALS patients are associated with TDP-43 proteinopathy. Acta Neuropathol. 2015 Jul;130(1):63-75. [PubMed]

O’Rourke JG, Bogdanik L, Muhammad AK, Gendron TF, Kim KJ, Austin A, Cady J, Liu EY, Zarrow J, Grant S, Ho R, Bell S, Carmona S, Simpkinson M, Lall D, Wu K, Daughrity L, Dickson DW, Harms MB, Petrucelli L, Lee EB, Lutz CM, Baloh RH. C9orf72 BAC transgenic mice display typical pathological features of ALS/FTD. Neuron. 2015 Dec 2;88(5):892-901. [PubMed]

Peters OM, Cabrera GT, Tran H, Gendron TF, McKeon JE, Metterville J, Weiss A, Wightman N, Salameh J, Ki J, Sun H, Boylan KB, Dickson D, Kennedy Z, Lin Z, Zhang YJ, Daughrity L, Jung C, Gao FB, Sapp PC, Horvitz HR, Bosco DA, Brown SP, de Jong P, Petrucelli L, Mueller C, Brown RH Jr. Human C9ORF72 hexanucleotide expansion reproduces RNA foci and dipeptide repeat proteins but not neurodegeneration in BAC transgenic mice. Neuron. 2015 Dec 2;88(5):902-9. [PubMed]

Almeida S, Gascon E, Tran H, Chou HJ, Gendron TF, Degroot S, Tapper AR, Sellier C, Charlet-Berguerand N, Karydas A, Seeley WW, Boxer AL, Petrucelli L, Miller GL, Gao FB. Modeling key pathological features of frontotemporal dementia with C9ORF72 repeat expansion in iPSC-derived human neurons. Acta Neuropathol. 2013 Sep;126(3):385-99. [PubMed]

Sareen D, O’Rourke JG, Meera P, Muhammad AK, Grant S, Simpkinson M, Bell S, Carmona S, Ornelas L, Sahabian A, Gendron T, Petrucelli L, Baughn M, Ravits J, Harms MB, Rigo F, Bennett CF, Otis TS, Svendsen CN, Baloh RH. Targeting RNA foci in iPSC-derived motor neurons from ALS patients with a C9ORF72 repeat expansion. Sci Tranls Med. 2013 Oct 23;5(208):208ra149. [PubMed]

Ho R, Sances S, Gowing G, Amoroso MW, O’Rourke JG, Sahabian A, Wichterle H, Baloh RH, Sareen D, Svendsen CN. ALS disrupts spinal motor neuron maturation and aging pathways within gene co-expression networks. Nat Neurosci. 2016 Sep;19(9):1256-67. [PubMed]

Lagier-Tourenne C, Baughn M, Rigo F, Sun S, Liu P, Li HR, Jiang J, Watt AT, Chun S, Katz M, Qiu J, Sun Y, Ling SC, Zhu Q, Polymenidou M, Drenner K, Artates JW, McAlonis-Downes M, Markmiller S, Hutt KR, Pizzo DP, Cady J, Harms MB, Baloh RH, Vandenberg SR, Yeo GW, Fu XD, Bennett CF, Cleveland DW, Ravits J. Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal dementia. Proc Natl Acad Sci U S A. 2013 Nov 19;110(47):E4530-9. [PubMed]

Gendron TF, Chew J, Stankowski JN, Hayes LR, Zhang YJ, Prudencio M, Carlomagno Y, Daughrity LM, Jansen-West K, Perkerson EA, O’Raw A, Cook C, Pregent L, Belzil V, Van Blitterswijk M, Tabassian LJ, Lee CW, Yue M, Tong J, Song Y, Castanedes-Casey M, Rousseau L, Philips V, Dickson DW, Rademakers R, Fryer JD, Rush BK, Pedraza O, Caputo AM, Desaro P, PAlmucci C, Robertson A, Heckman MG, Diehl NN, Wiggs E, Tierney M, Braun L, Farren J, Lacomis D, Ladha S, Fournier CN, McCluskey LF, Elman LB, Toledo JB, McBride JD, Tiloca C, Morelli C, Poletti B, Solca F, Prelle A, Wuu J, Jockel-Balsarotti J, Rigo F, Ambrose C, Datta A, Yang W, Raitcheva D, Antognetti G, McCampbell A, Van Sweiten JC, Miller BL, Boxer AL, Brown RH, Bowser R, Miller TM, Trojanowski JQ, Grossman M, Berry JD, Hu WT, Ratti A, Traynor BJ, Disney MD, Benatar M, Silani V, Glass JD, Floeter MK, Rothstein JD, Boylan KB, Petrucelli L. Poly(GP) proteins are a useful pharmacodynamics marker for C9ORF72-assocated amyotrophic lateral sclerosis. Sci Transl Med. 2017 Mar 29;9(383). [PubMed]

Lehmer C, Oeckl P, Weishaupt JH, Volk AE, Diehl-Schmid J, Schroeter ML, Lauer M, Kornhuber J, Levin J, Fassbender K, Landwehrmeyer B; German Consortium for Frontotemporal Lobar Degeneration., Schludi MH, Arzberger T, Kremmer E, Flatley A, Feederle R, Steinacker P, Weydt P, Ludolph AC, Edbauer D, Otto M.Poly-GP in cerebrospinal fluid links C9orf72-associated dipeptide repeat expression to the asymptomatic phase of ALS/FTD. EMBO Mol Med. 2017 Apr 13. pii: e201607486. [PubMed]

Further Reading

Gitler AD, Tsuiji H. There has been an awakening: Emerging mechanisms of C9orf72 mutations in FTD/ALS. Brain Res. 2016 Sep 15;1647:19-29. [PubMed]

antisense oligonucleotides ASOs c9orf72 disease-ad disease-als disease-ftd RNA foci
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