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 on Liu 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.—Gwyneth Dickey Zakaib


  1. This is really exceptionally nice science. It’s certainly possible to consider this a drug target except for the following possible caveats:

    1) The antisense oligonucleotides are not that effective at knockdown of SUPT4H1, at least as compared to the ASO for the actual C9 expasion itself, which can very specifically knock down more than 90 percent of the transcript and the downstream toxicity, be it from RNA or dipeptide repeats. Of course that does not rule out the possibility that a better ASO for SUPT4H1 could be generated.

    2) The possibility that inhibiting this gene—which can handle many other expansion repeats—could prove to be neurotoxic or cytotoxic, because there could be so many “off-target” actions. My experience suggests most companies would shy away from a drug with real possible off-target toxicity. This problem is far, far less likely with the C9 ASOs.

    Could there be a therapeutic advantage to knocking down both sense and antisense transcripts? Certainly yes—if in fact the antisense strand contributes to real disease and not artificially generated models of antisense toxicity. But accumulating data from ASO to sense C9 suggests that the vast majority of actual DPRs originate from the sense strand, and as yet there is no real evidence for endogenous toxicity of the DPR synthesized from the antisense strand.

    After studying neurodegeneration for years, I learned the hard lesson that unfortunately, fine science does not equate to real drug therapies in humans. Mice, flies, and yeast go only so far in defining a real human therapy, but only future work will be able to address that. The present Gitler/Petrucelli work certainly suggests we should consider that additional course. It’s great to have the additional druggable targets this work provides.

Make a Comment

To make a comment you must login or register.


News Citations

  1. Listen Up, Gene Silencing Strikes a Chord at RNA Meeting
  2. Huntington’s Strategies Tap Transcription, Htt Phosphorylation
  3. Paper Alert: Could Antisense Therapy One Day Squelch Toxic Repeats in ALS or FTD?
  4. C9ORF72 Mice Point to Gain of Toxic Function in ALS, FTD

Paper Citations

  1. . Spt4 is selectively required for transcription of extended trinucleotide repeats. Cell. 2012 Feb 17;148(4):690-701. PubMed.
  2. . Molecular and genetic characterization of SPT4, a gene important for transcription initiation in Saccharomyces cerevisiae. Mol Gen Genet. 1993 Mar;237(3):449-59. PubMed.

Further Reading


  1. . Therapeutic progress in amyotrophic lateral sclerosis-beginning to learning. Eur J Med Chem. 2016 Oct 4;121:903-17. Epub 2016 Jun 14 PubMed.

Primary Papers

  1. . Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts. Science. 2016 Aug 12;353(6300):708-12. PubMed.
  2. . NEURODEGENERATION. One target for amyotrophic lateral sclerosis therapy?. Science. 2016 Aug 12;353(6300):647-8. PubMed.