Tackling Mutant C9ORF72 Transcripts at the Source

Some patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) carry a long repeat expansion in the C9ORF72 gene. Those stretches of DNA can be transcribed in both sense and antisense directions, leading to the build-up of RNA foci and translated dipeptide repeat proteins. What if there was a way to nip transcription in the bud? That is the aim of a study published in the August 11 Science. Researchers led by Leonard Petrucelli of the Mayo Clinic in Jacksonville, Florida, and Aaron Gitler of Stanford University School of Medicine in California report that suppressing a transcription elongation factor called Spt4, which is required for transcribing repetitive sections of DNA, limits transcription of the toxic hexanucleotide repeat. This lengthens the lifespan of a variety of animal models. Alzforum covered the preliminary findings when they were presented at a Society for Neuroscience satellite conference last year (Nov 2015 conference news). The results could point to a novel treatment strategy for C9ORF72-related disease.

Poly’s Little Helper: Transcription factors SUPT4H1 and SUPT5H bind to RNA polymerase II and help it transcribe long-repeat DNA. [Courtesy of Science/AAAS.]

“It’s a stellar example of how basic science approaches can have direct application to human diseases,” said Benjamin Wolozin, Boston University, who was not involved in the work. Eukaryotic cells require the Spt4 enzyme to transcribe genes with extended repeats, hence it is plausible that a small molecule that blocks the enzyme could prevent disease phenotypes. “That would be applicable to multiple different diseases so is important for drug development,” Wolozin added.

A few years ago, scientists in the lab of co-author Stanley Cohen found that mutating Spt4 prevented yeast from transcribing the trinucleotide CAG repeat expansion responsible for Huntington’s disease (Feb 2012 news onLiu et al., 2012). Spt4 and its binding partner Spt5 help RNA polymerase II stay the course when transcribing repetitive DNA, especially sequences that assume complicated secondary structures such as hairpins and quadruplexes that might otherwise cause the enzyme to fall off (Malone et al., 1993).

Knocking down the mammalian ortholog, SUPT4H1, in mouse striatal neurons reduced expression of the expanded CAG repeat, delayed motor problems, and increased lifespan, but left transcription of short CAG regions alone. That led first authors Nicholas Kramer, Yari Carlomagno, and Yong-Jie Zhang of the current study to wonder whether Spt4 was required for expression of the hexanucleotide GGGGCC repeat in C9ORF72 that is responsible for some cases of ALS and FTD.

To find out, the researchers deleted SPT4 in yeast that harbor the GGGGCC expansion. Those yeast produced fewer repeat transcripts, fewer RNA foci, and little of the translated dipeptide repeat protein (DPR) poly(GP). Of the possible DPRs, the authors started with poly(GP) because it is easiest to detect and most abundant, but the group is also looking into the others. In nematodes with the hexanucleotide expansion, RNA interference (RNAi) that knocked down Spt4 also reduced the toxic RNA transcripts and poly(GP), while extending the worms’ lifespan. In Drosophila that express the hexanucleotide repeats in the eye, suppressing Spt4 reduced retinal degeneration and helped these animals live longer, as well.

Would the same strategy work in human cells? Kramer and colleagues used small interfering RNAs to knock down either SUPT4H1 or its binding partner SUPT5H in skin cells from ALS patients carrying a C9ORF72 repeat expansion. This treatment reduced the repeat mRNAs, their foci, and poly(GP). The siRNAs did not seem toxic to the cells; however, expression of 301 genes changed, 46 of them more than threefold. Similarly, suppressing SUPT4H1 in cortical neurons derived from induced pluripotent stem cells of C9ORF72 carriers reined in repeat mRNAs and poly(GP). Interestingly, levels of SUPT4H1 and SUPT5H correlated with C9ORF72 mRNA and DPRs in the cerebellum of autopsied brains from C9ORF72 carriers.

“Targeting SUPT4H1 might reduce some of the pathologies associated with transcription of the C9ORF72 gene,” Kramer told Alzforum. A therapeutic strategy that stops transcription of the repeat could have advantages over antisense oligonucleotides (ASOs) in development that take aim at the C9ORF72 mRNA transcripts, Kramer believes (Nov 2015 conference newsApr 2016 news). For one, targeting Spt4 could take care of both sense and antisense RNAs in one fell swoop, said Kramer. ASOs that bind Spt4 RNA instead are an alternative and are already being tested in Huntington’s models.

Kramer said he will next test whether suppressing SUPT4H1 rescues phenotypes in mice that express the C9ORF72 mutation (Oct 2015 conference news). He and colleagues will also closely examine how transcription of other genes is regulated by SUPT4H1, and whether these might lead to off-target effects.

“This is really exceptionally nice science,” Jeffrey Rothstein, Johns Hopkins University, Baltimore, wrote to Alzforum (see full comment below). It’s certainly possible to consider this a drug target, he added, but offered two caveats. ASOs being tried in Huntington’s mouse models have not been that effective in knocking down SUPT4H1, although perhaps a better ASO could be designed, he said. By contrast, ASOs that target the C9ORF72 expansion itself manage to hit more than 90 percent of the transcripts and reduce downstream toxicity, whether from RNA or dipeptide repeats.

The authors note that an effective SPT4-targeting therapy will only partially reduce SPT4 function, as knockout is lethal in mice. Wolozin noted that the new approach has yet to show its worth in higher animal models, though he predicts it will work there as well.

Primary Reference:
Kramer NJ, Carlomagno Y, Zhang YJ, Almeida S, Cook CN, Gendron TF, Prudencio M, Van Blitterswijk M, Belzil V, Couthouis J, Paul JW 3rd, Goodman LD, Daughrity L, Chew J, Garrett A, Pregent L, Jansen-West K, Tabassian LJ, Rademakers R, Boylan K, Graff-Radford NR, Josephs KA, Parisi JE, Knopman DS, Petersen RC, Boeve BF, Deng N, Feng Y, Cheng TH, Dickson DW, Cohen SN, Bonini NM, Link CD, Gao FB, Petrucelli L, Gitler AD. Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts. Science. 2016 Aug 12;353(6300):708-12. [Pubmed].

Mizielinska S, Isaacs AM. NEURODEGENERATION. One target for amyotrophic lateral sclerosis therapy?. Science. 2016 Aug 12;353(6300):647-8. [PubMed].

To view commentaries, primary articles and linked stories, go to the original posting on Alzforum.org here.

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c9orf72 disease-als disease-ftd topic-preclinical
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