How Common ALS Mutation Damages Cells and Leads to Toxic Protein Being Produced Detailed in Study

The most common genetic cause of amyotrophic lateral sclerosis (ALS) disrupts the shape and workings of a compartment inside a cell’s nucleus, researchers report, detailing a discovery that helps to explain how this mutation prompts cell death in ALS.

Abnormalities resulting from this mutation, specifically the length of the damaged protein it produces, may also help in determining likely disease progression in patients, the scientists said.

The research, “C9orf72 Poly(PR) Dipeptide Repeats Disturb Biomolecular Phase Separation and Disrupt Nucleolar Function,” was published in Molecular Cell. It was led by scientists at St. Jude Children’s Research Hospital.

Repeat expansion in the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis. Repeat expansion refers to cases in which, because of the mutation, the number of nucleotide repeats — short DNA lettered sequences that repeat a number of times in a row — are repeated in greater numbers than those found in a healthy version of a gene.

In ALS, this type of mutation affects the GGGGCC segment of the gene. Although there’s still no consensus on how many repeats are disease-causing, it is believed that more than 30 or so repeats disrupt the gene’s function (normally, a segment of C9orf72 gene is repeated up to 20 to 30 times).

This mutation is linked to the formation of abnormal proteins of varying lengths (called dipeptide repeat polypeptides, or DPR), which tend to turn toxic and aggregate in the brain.

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The disease mechanism of ALS is not yet fully understood, but research have demonstrated that toxic DPRs, containing an amino acid called arginine, alter the shape and function of the nucleolus – a round, membrane-less structure inside the cell nucleus; it houses the cell’s protein factory. Longer DPRs are significantly more toxic to cells than shorter ones.

In biology, cells resemble liquid droplets that maintain membrane-less compartments, which are important for concentrating certain molecules and facilitating the regulation of cellular functions. Such structures originate via a process called active liquid-liquid phase separation, the process one that causes oil to generate droplets in water. This molecular process allows the nucleolus to maintain its form and function.

“We have identified the protein, nucleophosmin, as a site of DPR toxicity,” Richard Kriwacki, PhD, a study author and member of the St. Jude Department of Structural Biology, said in a press release.

Nucleophosmin (NPM1) is an abundant protein present in the nucleolus, which is where protein factories (ribosomes) — composed of protein and RNA — are produced.

Using an analytical chemistry technique called nuclear magnetic resonance (NMR), Kriwacki’s team found that toxic DPRs changed cell function by tightly binding to key regions of the nucleophosmin protein, displacing other binding partners that work to maintain the nucleolus and tis protein factory assembly.

“We show that poly(PR) DPRs bind tightly to a long acidic tract within the intrinsically disordered region of NPM1, altering its phase separation with nucleolar partners to the extreme of forming large, soluble complexes that cause droplet dissolution in vitro [or in a lab dish],” the study reported.

In other words, rapid disruption of the nucleolus’ shape and function was associated with a higher concentration of toxic DPRs; these abnormal proteins appear to bind to and insulate NMP1, leading to a partial loss of the membrane-less cell component, which could lead to cell death. DPRs were also found to bind to and isolate ribosomal RNAs (an important component of proteins’ factory) and alter how cells work.

“DPR toxicity is exquisitely length dependent,” Kriwacki said, adding that in “the future, DPR length may have prognostic value for people with a diagnosis of ALS.”

“The work also provides a new direction for thinking about possible therapies to target toxic DPRs and their sites of action in patient cells,” said Michael White, PhD, a postdoctoral fellow in Kriwacki’s laboratory and the study’s first author.