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A Novel Disease Pathway Suggested for C9ORF72-Linked ALS/Frontotemporal Dementia

Proteins that result from breaking the rules of biology have been discovered in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) who have the C9ORF72 gene mutation — chromosome 9 open reading frame 72 — and have scientists wondering if there may be a new pathogenic process at work in the disease. The discovery was announced in papers from two independent groups published online before print in February in Neuron and Science.

“It suggests there is going to be a new mechanism of disease,” according to Leonard Petrucelli, PhD, principal investigator on one of the studies and professor of neuroscience at the Mayo Clinic in Jacksonville, FL.

The protein is the result of “repeat-associated nonstandard” or RAN translation, in which the ribosome translates sections of RNA into protein despite the absence of any start signal on the RNA. Researchers found that the long CCCGG repeat that defines the C9ORF72 mutation was translated into proteins composed solely of two amino acids. These dipeptide proteins accumulated into inclusions.

According to all the textbooks, translation of messenger RNA at the ribosome always begins with the start codon sequence AUG. This codon sets the reading frame, and each successive triplet encodes a new amino acid for the growing polypeptide, until finally a stop codon is reached.

Laura Ranum, PhD, first described RAN translation in 2011 in spinocerebellar ataxia 8, due to a repeat expansion that encodes polyglutamine. Dr. Ranum — professor of molecular genetics and microbiology, and the director of the Center for Neurogenetics, at the University of Florida at Gainesville — found that even after mutating the start codon in the SCA8 gene (spinocerebellar ataxia type 8) cells continued to produce polyglutamine, apparently translating the long “CAG-CAG” repeats despite the absence of the usual start signal. And with nothing to set the reading frame, they also produced polyserine (from reading repeated “AGC-AGC”) and polyalanine (from “GCA-GCA”). She found the same phenomenon in myotonic dystrophy, also due to an expanded genetic repeat.

The C9ORF72 gene mutation, now recognized as the most common cause of familial ALS and FTD, contains hundreds to thousands of GGGGCC repeats. The repeat is known to be transcribed into RNA, and accumulation of RNA into foci has been described in patient neurons, suggesting one possible mechanism of disease.

But Dr. Petrucelli wondered if the repeat might also give rise to RAN translation (RANT) products. He reasoned that the repeat could be read in any one of three reading frames, suggesting possible RANT products of strings of glycine-alanine (from GGG-GCC); or glycine-proline (from GGG-CCG); or glycine-arginine (from GGC-CGG); or, as Dr. Ranum's work suggested, all three.

The investigators generated purified proteins made up of each dipeptide, then raised antibodies to them to use as a probe. Because the antibodies responded most strongly to poly-(glycine-proline), he interpreted further results as most strongly indicative of the presence of that protein, although the others were likely present as well.

Dr. Petrucelli then exposed tissue from ALS/FTD patients with C9ORF72 to the antibodies. He began with cerebellar tissue, because this group of patients has significant cerebellar pathology. The antibodies identified the target proteins in cerebellar tissue from these patients, but not in patients without the C9ORF72 expansion, or in patients with other neurodegenerative diseases.

The proteins were present in cytoplasmic and intranuclear inclusions in neurons, but not in astrocytes, smooth muscle, or vascular endothelia. They co-localized with the protein p62, which has been previously found in ALS/FTD inclusions, and were distinct from inclusions carrying TDP-43 (TAR-DNA binding protein 43). TDP-43 inclusions are found in virtually every type of ALS except that due to SOD1 (superoxide dismutase 1) mutations.

The inclusions were also found in hippocampus and neocortex, but only sparsely in the spinal cord, and not at all in the periphery.

A second group, led by Dieter Edbauer, PhD, professor of translational neurobiochemistry at Ludwig Maximilian University in Munich, Germany, found similar results. In his study in Science, poly-(glycine-alanine) reacted most strongly to the antibodies, and inclusions were most prominent in the cerebellum and hippocampus.

One patient with the C9ORF72 mutation in that study had no TDP-43 pathology, suggesting the RANT product-containing aggregates may play a role in pathogenesis in this case. The cerebellar atrophy seen in patients with C9ORF72 mutations — but not in non-C9 patients who as well don't have these inclusions — strengthens the case for a pathogenic role.

The possibility of a link between the distribution of RANT products and pathology “is the major question,” Dr. Petrucelli said. The number of cases is still small, but there does seem to be a correlation between location and symptoms. “That's what our results are suggesting early on. It is very interesting to see a cognitive correlation. The lack of a major burden in the spinal cord would suggest it is not having a major impact on motor neurons.”

“We can probably safely assume that this is going to add another layer of complexity to the disease mechanism,” Dr. Petrucelli said.

Whatever their effect on the disease, the presence of RANT products may offer researchers a valuable biomarker for expression of the C9ORF72 gene, potentially important in future clinical trials aimed at reducing its effects. The huge expansion makes the RNA itself difficult to handle quantitatively; there is, for example, no convenient way to even measure the exact length of a repeat. A decline in RANT products might serve as an easily quantifiable surrogate for a decline in that RNA, a likely goal of C9ORF72-directed therapeutics.

Dr. Ranum, who discovered the RAN translation, told Neurology Today there is still a great deal to learn about the mechanism of this unusual process, as well as about the effects of the proteins themselves. “The next step is to understand the contributions these proteins play, or don't play, in the disease.” Her own work suggests that they are contributing some measure of toxicity in both SCA8 and myotonic dystrophy.

Regarding the discovery in C9ORF72 patients, she said: “These are potentially new pathways, and I am hopeful they are going to help us understand the disease process in more detail than we have understood it before.” There may be other diseases in which RAN translation plays a part as well; all that seems to be needed is an RNA expansion long enough to trigger this unusual process. “There is a lot more we have to learn about it,” she said.

John Q. Trojanowski, MD, PhD, professor of geriatric medicine and co-director of the Center for Neurodegenerative Disease Research at the University of Pennsylvania School of Medicine in Philadelphia, concurred. “Anything more we can learn about the effects of these expansions is going to be important in understanding the mechanism of disease,” he said. “It will be of great interest to understand how the expansion triggers that kind of pathology.”

Two aspects of the new discovery are intriguing, he said. The first is that the new proteins are not mutated versions of normal proteins, unlike all other proteins found to date in neurodegenerative disease inclusions. “They are anomalous in that way,” he said. In addition, they inclusions they are found in remain separate from those containing TDP-43. Whether that suggests a second pathogenic process in the neuron remains to be elucidated, he said, “but now we have another important clue to pursue.”


•. Ash PE, Bieniek KF, Petrucelli L, et al.Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS. Neuron 2013;77(4):639–46. E-pub 2013 Feb. 12.
    •. Mori K, Weng SM, Edbauer D, et al.The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS. Science 2013; E-pub 2013 Feb 7.
      •. Zu T, Gibbens B, Ranum LP, et al.Non-ATG-initiated translation directed by microsatellite expansions. Proc Natl Acad Sci U S A 2011;108(1):260–265. E-pub 2010 Dec 20.
        •. Neurology Today archive on C9ORF72: