C9ORF72 Killer Dipeptides Clog the Nucleolus

Proposing a potential mechanism for C9ORF72-based amyotrophic lateral sclerosis and frontotemporal dementia in Science online July 31, researchers report that repeat dipeptides made by the mutant gene target the nucleolus. In this organelle, the cell’s ribosomal RNA factory, the dipeptides appear to gum up the works, impeding production of both rRNA and mRNA. The peptides eventually killed cultured cells. “The toxicities driven by the [repeat dipeptides] may account for the pathophysiological deficits observed in nerve cell dysfunction in patients carrying repeat expansions in the C9ORF72 gene,” the authors conclude.

Experts who spoke with Alzforum called the idea provocative but noted that it remains to be seen if this phenomenon occurs in people. “This is a very good paper that will be highly cited for the next decade and more,” commented J. Paul Taylor of St. Jude Children’s Research Hospital in Memphis, Tennessee, adding that investigators with access to patient tissue will now want to check if the same process occurs there.

Senior author Steven McKnight of the University of Texas Southwestern Medical Center in Dallas studies proteins with so-called low-complexity domains, a category that includes ALS- and FTD-related FUS, EWS, TAF15, hnRNPA1, and hnRNPA2. Proteins with low-complexity domains often create amyloid-like structures that bind RNA in places like the nucleolus, spliceosome, nuclear pores, and RNA granules such as stress granules, Taylor said.

McKnight and first author Ilmin Kwon started out studying the loosely structured serine-arginine (SR) repeats found in many splicing factors. They found that the SR peptides targeted the nucleolus. There, the serines underwent phosphorylation, which caused the peptide to detach from the nucleolus and move into nuclear speckles, where pre-mRNA splicing occurs. A phosphorylation-resistant glycine-arginine (GR) dipeptide did not bind the nucleolus.

With that GR dipeptide, Kwon’s work intersected with a new line of thought in ALS and FTD research. The C9ORF72 expansion, a six-nucleotide intronic repeat that appears hundreds to thousands of times in many people with ALS or FTD, undergoes nonstandard translation to produce just this kind of unstructured peptide. Depending on where the translation starts and in which direction it goes, the C9ORF72 repeat makes five different dipeptides: glycine-alanine, glycine-proline, glycine-arginine, proline-alanine, and proline-arginine. One hypothesis for C9ORF72-based disease is that one or more of these dipeptides could be toxic (see Feb 2013 news story; Feb 2013 news story; Nov 2013 news story).

Kwon and McKnight focused on the GR and PR dipeptides because they suspected the arginines would direct them to the nucleolus. Indeed, synthetic peptides with 20 repeats of GR or PR entered cultured human bone cells and bound nucleoli (see image below). Within a day, the peptides caused the cells to shrivel and die.

PR repeat peptides target the nucleolus of bone cells. [Image courtesy of Science/AAAS.]

The authors hypothesized that GR and PR dipeptides clog the nucleolus. They cannot leave because, unlike SR peptides, they lack phosphorylation sites. By getting in the way, they could prevent SR-containing proteins and perhaps others from performing their proper RNA-processing tasks. Those tasks include mRNA maturation and rRNA synthesis, so Kwon investigated the RNAs of PR-treated human astrocyte cultures. Sequencing of mRNAs showed that the treated cells exhibited splicing abnormalities, such as exon skipping and intron inclusion. Some RNAs were present at higher-than-normal concentrations.

Kwon also used polymerase chain reaction to examine the production of ribosomal RNA. Normally, the nucleolus processes the 45S rRNA precursor into three subunits: 28S, 18S and 5.8S rRNA. In the PR-treated cells, the processing and production of these subunits was off; in particular, 5.8S levels were only 30 percent of normal. “These assays provide evidence of nucleolar dysfunction,” the authors concluded.

Philip Wong of the Johns Hopkins University School of Medicine in Baltimore noted that the authors included no control peptides, such as SR, in their toxicity or RNA-processing experiments. Without that, it remains possible that any arginine-containing dipeptide could be toxic to Kwon’s cultures. Wong added, “Most of this is in cell culture, so I think it would be important to validate in vivo.”

Kwon and colleagues attempted to link their finding to human disease by analyzing splicing of the excitatory amino acid transporter 2 (EAAT2). A previous study found that in people with ALS, this mRNA displays particular splicing mistakes: It picks up more than 1,000 extra residues from intronic DNA, and loses its ninth exon (Lin et al., 1998). Kwon reported that he saw these EAAT2 changes in the PR-treated astrocytes.

However, the original finding of EAAT2 missplicing being specific to ALS was questioned in later publications. Other researchers have found the same splice variant in people with dementia as well as in neurologically normal controls (Meyer et al., 1999; Honig et al., 2000; Flowers et al., 2001).

“It is provocative that he finds this abnormal splicing pattern for EAAT2,” said Taylor, who speculated that C9ORF72 expansions—unknown at the time of the earlier splicing work—might be present in only a subset of patients and explain the discrepancies in the different studies. In future work, Taylor said, “I would like to see if those dipeptide repeats are in nucleoli in the patients … and whether the patients are showing signs of splicing abnormalities that replicate what he has seen.”

McKnight declined to comment for Alzforum. Researchers who did speak with Alzforum found the cell culture experiments to be solid. “These peptides very convincingly enter the cells and enter the nucleus,” said Jiou Wang of Johns Hopkins University. “[Kwon et al.] have certainly demonstrated that these peptides have this specific affinity to the nucleolus.” Wang and colleagues recently reported another link between C9ORF72 and the nucleolus. They found that the RNA products of the C9ORF72 repeats bind nucleolar protein and disrupt normal rRNA synthesis (see Mar 2014 news story). Both RNA- and peptide-based toxic mechanisms could be at play in people, he said. Together, the studies may point to the nucleolus as a “hub for C9ORF72 pathogenic cascades,” Wang speculated (see full comment).


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