Ever since the 2011 discovery that an expanded repeat in the C9ORF72 gene underlies cases of amyotrophic lateral sclerosis and frontotemporal dementia, researchers have wondered how the mutation causes disease. Recent work suggested that small peptides made from the transcribed expansion could be to blame, but in the October 16 Neuron, researchers led by Jeffrey Rothstein at Johns Hopkins University, Baltimore, Maryland, instead spotlight the role of RNA aggregates. These deposits sequester at least one RNA-binding protein that seems to be essential for cell health, the authors found. C9ORF72 antisense oligonucleotides nearly eliminated the RNA aggregates and partially normalized cellular physiology. The findings do not preclude additional sources of toxicity, the authors note. Rothstein previously presented some of this data at the 23rd annual International Symposium on ALS/MND, held 5–7 December 2012 in Chicago (see ARF related news story).
Other researchers expressed enthusiasm for the findings. “The use of antisense oligonucleotides to prevent some of the aberrant phenotypes in C9ORF72 cells is really exciting and an important step forward for the field,” said Adrian Isaacs at University College London. The authors suggest that antisense RNA could have therapeutic potential. Researchers, including Rothstein, are already testing this strategy in clinical trials for other genetic forms of ALS, as well as planning trials for other repeat-expansion diseases such as Huntington’s and spinal muscular atrophy.
In another disease, myotonic dystrophy, deposits of RNA containing an expanded repeat seem to sequester crucial RNA-binding proteins, leading to aberrant editing of many gene transcripts and subsequent neurodegeneration (for a review, see Todd and Paulson, 2010). To investigate whether a similar mechanism occurs in C9ORF72 ALS, first author Christopher Donnelly derived induced pluripotent stem (iPS) cells from skin fibroblasts taken from people carrying the toxic C9ORF72 variant. He used the iPS cells to generate mixed neuronal cultures, which recapitulated the RNA aggregates and other pathological features seen in postmortem brain tissue from C9ORF72 ALS patients. These characteristics included increased sensitivity to glutamate excitotoxicity, abnormal patterns of gene expression, and deposits of dipeptide repeat (DPR) protein made from the expansion through a process known as repeat-associated, non-ATG-initiated (RAN) translation (see ARF related news story, ARF news story).
Next, the authors looked for proteins that might become trapped in the RNA deposits. They probed protein arrays using the C9ORF72 expanded repeat sequence, and turned up 19 candidates. Of these, they pursued one: the RNA-binding protein ADARB2. The ADAR protein family helps edit RNA transcripts, and some evidence indicates one member, ADAR2, is greatly suppressed in the spinal motor neurons of ALS patients (see Hideyama et al., 2012). The authors found ADARB2 in RNA foci in both the induced C9ORF72 neurons and in patient tissue.
To see if ADARB2 helps drive disease, the authors knocked down the gene in induced neurons from C9ORF72 patients. The number of neurons with RNA foci dropped by half, suggesting that ADARB2 helps form or maintain these deposits. Moreover, in induced neurons made from healthy controls, knocking down ADARB2 increased glutamate toxicity to levels seen in patient cells. These data support the idea that sequestering ADARB2 could cause pathology, the authors suggest. In future work, Rothstein plans to look for the normal targets of ADARB2 and examine how a lack of this protein damages cells.
To ameliorate pathology, the authors tested more than 250 different antisense oligonucleotides (ASOs) to C9ORF72. ASOs that targeted the expanded repeat nearly abolished RNA foci without suppressing levels of normal C9ORF72 RNA. Treatment helped to normalize gene expression and partially rescued glutamate toxicity, as well. Intriguingly, DPR peptide deposits were unchanged.
The results support using antisense approaches in ALS clinical trials, Rothstein said. He helped run the recent Phase 1 trial of ASOs in ALS patients carrying a mutation in the superoxide dismutase 1 (SOD1) gene (see ARF related story). Isis Pharmaceuticals of Carlsbad, California, supplied the ASOs for that trial as well as for the C9ORF72 study. Rothstein noted that other pharmaceutical companies have expressed interest in targeting C9ORF72. The main challenge in ASO therapy is how to deliver these large molecules to the brain. Companies are taking a variety of approaches (see ARF related news story).
Does this paper settle the question of how the C9ORF72 variant confers toxicity? Not yet, commentators agreed. The data strengthen the case for RNA foci as a key factor, but do not rule out additional effects from C9ORF72 protein loss or DPR toxicity. In the case of DPR proteins, attention may swing from aggregates to smaller, soluble species, suggested Boris Rogelj at the Jozef Stefan Institute in Ljubljana, Slovenia. He noted that in the October 6 Acta Neuropathologica, researchers at the University of British Columbia, Vancouver, Canada, reported little correlation between sites of DPR deposition and neurodegeneration in ALS brains (see Mackenzie et al., 2013). Together with the data from the Neuron paper, the results imply that DPR aggregates themselves may not be major players in disease and could even be protective, Rogelj speculated. Some scientists believe the same may be true for other protein aggregates, including Aβ plaques. As Peter Todd and Henry Paulson at the University of Michigan Medical Center, Ann Arbor, pointed out in an editorial accompanying the Neuron paper, “This result does not preclude a role for continually produced RAN products in ongoing neurotoxicity.” Rothstein concurred with this view, and noted that ASO therapy targeting the expanded repeat would suppress both RNA and newly-synthesized DPR oligomers, removing both potential sources of toxicity.
Notably, this paper presents the first published data from iPS cells made from C9ORF72 ALS patients, complementing a recent paper that used the strategy to study FTD patients with the C9ORF72 toxic variant (see Almeida et al., 2013). “These data show that you can use iPS cells to model this disease, understand its mechanisms, and discover a therapy,” Rothstein told Alzforum.—Madolyn Bowman Rogers
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