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Dec 1
2019

Decoding the process that makes abnormal ALS proteins

Two Packard Center researchers have identified a protein called RPS25 that helps to regulate the abnormal cellular processes caused by the C9ORF72 mutations.

Even as some Packard scientists have been scrambling to understand the precise molecular mechanisms by which the C9ORF72 repeat expansion causes ALS and FTD, others have begun to drill down into the molecular processes that lead to motor neuron degeneration and death. A team led by Packard researchers Aaron Gitler of Stanford University and Leonard Petrucelli of the Mayo Clinic identified a protein called RPS25 that helps to regulate the abnormal cellular processes caused by the C9ORF72 mutations, the most common genetic cause of ALS/FTD.

“It’s an interesting first step towards understanding why some of these abnormal cellular processes happen,” says Shizuka Yamada, a graduate student in the Gitler lab and first author on the paper, which appeared in Nature Neuroscience. “The results are really exciting but there is still a lot of follow-up to be done.”

One of the primary ways researchers believe the C9ORF72 mutation leads to disease is through the creation of small toxic proteins called dipeptide repeats (DPRs). The massive and repetitive sequence of DNA within the repeat expansion confuses the cellular machinery that turns DNA to RNA to protein. Instead of starting from an established signpost and producing the normal protein, protein synthesis machinery creates many different repetitive proteins, a process known as RAN translation. In C9 ALS/FTD, RAN translation creates five toxic DPRs.

Other Packard scientists had worked on identifying features of repeats that promote RAN translation, but Yamada was interested in the process itself, specifically the genes and proteins that regulate it. After the researchers discovered that RAN translation occurs in yeast, they began to use the single-celled organism to screen for genes that specifically affected RAN translation without altering levels of repeat-generated RNA or other forms of translation. One gene that stood out in their results was a gene called RPS25A, which encodes a protein that makes up part of the smaller half of the ribosome. Previous studies showed that RPS25 is known to play a role in unconventional translation processes, including those used by a variety of viruses.

When Yamada and colleagues deleted the RPS25A gene from yeast, they found a 50% drop in levels of one DPR called poly(GP). In human cells, the researchers found a 90% reduction of poly(GA), and a 30% reduction of poly(GR). These RPS25A knockout cells showed no changes to overall translation or cellular growth rate. Although these results indicated close links between C9 RAN translation and RPS25, they didn’t indicate whether the protein also played a role in RAN translation in other repeats.

Previous studies from Gitler and other researchers had connected a repeat expansion in the gene ATXN2 to ALS. Using human cells with RPS25 mutations, the researchers found that these cells showed a marked reduction in one peptide produced by ATXN2- and HTT-associated RAN translation.

The team repeated their experiments in cultured induced pluripotent stem cells to study the role of RPS25 in a more clinically relevant system. The reduction of RPS25 also lowered poly(GP) DPR levels without impacting RNA transcription of the repeat or overall levels of the C9ORF72 protein. Transgenic fruit flies that were engineered to carry 36 repeats of the six-nucleotide repeat sequence found in C9 ALS patients showed DPR accumulation and a shortened lifespan. Reducing levels of the Drosophila RPS25 protein also lowered poly(GP) and significantly increased the flies’ lifespan. Experiments in motor neurons derived from C9 ALS patient induced pluripotent stem cells used short sequences of nucleotides called antisense oligonucleotides (ASOs) to lower RPS25 protein levels. The ASO-treated induced motor neurons from C9 patients showed fewer poly(GR) and poly(PR) foci and had a larger proportion of surviving cells.

The findings are important because they suggest a potential therapeutic target not just for C9 ALS but also for other neurodegenerative diseases caused by repeat expansions, such as Huntington’s disease.