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Dec 1
2019

Packard researchers find new way to reduce C9orf72 toxicity

A team of Packard researchers have identified a protein that may help reduce the production of some of the toxic by-products of the C9ORF72 repeat expansion.

The disruption of a gene called C9ORF72 with a six-nucleotide sequence, repeated hundreds or thousands of times, is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Ever since the repeat expansion was discovered eight years ago, scientists from Packard and around the world have been working to figure out how the C9ORF72 mutation causes disease and how they might intervene to prevent the onset of ALS/FTD or slow its progression. Now, in a new study in Neuron, a team of Packard researchers led by Shuying Sun of the Brain Science Institute at the Johns Hopkins School of Medicine has identified a protein that may help reduce the production of some of the toxic by-products of the C9ORF72 repeat expansion. The results, scientists say, provide potential therapeutic targets for C9ALS.

Broadly speaking, C9ORF72 repeat expansion can cause disease through two different pathways. If the mutation makes the C9ORF72 protein unable to do its normal job (a job that scientists are currently trying to pin down), it would be considered a loss of function mutation. A gain of function mutation, on the other hand, would be caused if the repeat expansion did something toxic to cells. Researchers have identified two potential gain of function toxicities related to the ALS-linked C9ORF72 mutation. When the cell transcribes the C9ORF72 DNA into RNA on its way to making protein, it also transcribes the repeat sequences. These can create sticky RNA foci that attach to RNA-binding proteins and interfere with these proteins’ abilities to turn RNA into protein (one potential gain of function mechanism).

Another toxic mechanism occurs when the cell’s protein-making machinery tries to translate the repeat-filled C9ORF72 RNA into protein. The large number of repeats can make it hard for the machinery to know where and how to start reading the RNA. What results is an unconventional ‘reading’ of the mutant C9ORF72 RNA, leading to the creation of small, harmful proteins called dipeptide repeats (DPRs). Previous experiments showed that increasing DPR production increases toxicity, leading researchers to hypothesize that reducing DPR synthesis might reduce cellular injury.

Sun Lab, together with Jeff Rothstein, MD, PhD, Founder and Director of the Packard Center, Packard scientists James Shorter of the University of Pennsylvania, Fen-Biao Gao of the University of Massachusetts Medical School, and their colleagues searched across the genome for genes that could modify DPR protein production in human cells. After several rounds of screening, the researchers identified 76 genes that enhanced DPR amount and 145 that suppressed it. Further analysis revealed that a disproportionate number of these genes were involved in translation and RNA transport, two key steps that influence final protein levels in the cell. An independent confirmatory testing and validation identified 19 genes that specifically influenced the repeat RNA processing, including the abnormal protein translation process that creates the DPRs, known as RAN translation.

The researchers found that one group of genes that was overrepresented in the screen were helicases, which bind to and unwind DNA and RNA. The gene that showed the strongest links to DPR levels was an RNA helicase gene called DDX3X. Reducing levels of the DDX3X protein in cultured cells increased DPR production. The reverse was also true: increasing DDX3X levels reduced DPR proteins when the C9ORF72 repeat was being read in the forward direction. Subsequent work showed that DDX3X represses RAN translation by binding directly to the repeat RNA but did not affect the normal translation process.

Fruit fly models of C9ORF72 toxicity had previously shown that the toxic DPRs led to retinal damage in the flies. When the researchers studied the links between DDX3X and toxic DPRs in this same fruit fly model, they found that the partial loss of the fruit fly version of DDX3X increased retinal damage. In cells from ALS patients with the C9ORF72 repeat expansion, researchers found that levels of some DPRs jumped when they reduced levels of DDX3X. This also occurred in motor neurons derived from induced pluripotent stem cells from C9 ALS patients, confirming that DDX3X helps to stop RAN translation in people with ALS.

The scientists then took the experiments to the next step by showing that increasing DDX3X levels can reduce DPR production. Adding additional copies of DDX3X restored normal transport of proteins between the nucleus and the cytoplasm (a process disrupted by DPRs) and improved neuron survival in iPSC derived C9 patient motor neurons. The results provide not only more detail on the molecular impact of DPRs but also provides a potential therapeutic strategy for individuals with C9 ALS/FTD.