Research by scientists at the Spanish National Cancer Research Centre (CNIO) has uncovered a mechanism that may explain what causes neuronal cell death in cases of amyotrophic lateral sclerosis (ALS) that are associated with mutations in a gene known as C9ORF72. The findings suggest that the C9ORF72 gene mutations are toxic because they induce the cell to produce small proteins or peptides that are very rich in arginine, an amino acid that, due to its positive charge and chemical nature, binds very avidly to nucleic acids, including DNA and RNA. The resulting peptides stick to the DNA “like a kind of tar,” effectively blocking them from their normal interactions with other proteins, and disrupting fundamental cell processes.
Reporting on their findings in The EMBO Journal, the team, headed by Óscar Fernández-Capetillo, PhD, group leader, suggested that while the newly identified toxic mechanism is associated with mutations in C9ORF72 specifically, it is likely that other ALS-related mutations are acting in a similar way, i.e., by blocking the DNA and RNA of motor neurons.
Vanesa Lafarga, PhD, staff scientist and co-author of the study, explained, “… the vast majority of mutations found in ALS patients are in proteins that bind RNA, and what these mutations generally do is prevent the binding of these proteins to RNA. Moreover, the cells of these patients also have very general problems with their nucleic acids. That is why we believe that, although mutations in C9ORF72 only affect a fraction of ALS patients, the mechanism underlying the toxicity of neurons may not be fundamentally different from what happens in the rest of ALS patients. Trying to show whether this is the case is something we are working on now.”
Fernández-Capetillo added, “In all these decades of ALS research, neuroscience researchers have been publishing all sorts of problems in reactions using nucleic acids: translation, replication, etc. Nothing works! We think our model gives a simple answer to all these observations.”
The authors reported their findings in a paper titled, “Widespread displacement of DNA- and RNA-binding factors underlies toxicity of arginine-rich cell-penetrating peptides.”
ALS is a motor neuron disease that results in the progressive death of neurons that control body movement. The disorder leads to paralysis of muscles in the limbs, and progressively throughout the whole body, which ultimately makes it impossible to breathe. The disorder is currently untreatable, and its cause is unknown.
What is known is that there is a strong genetic component in 10% of affected individuals, which can lead to ALS in several members of a single family. In about half of these cases of familial ALS the origin lies in a gene called C9ORF72. But why mutations in the gene lead to motor neuron death hasn’t been understood.
ALS researchers had previously observed that many basic cellular processes that use nucleic acids fail in the neurons of affected ALS patients. The newly reported studies by Fernández-Capetillo’s team at the CNIO Genomic Instability Group provide a model that connects previous observations and might explain the defects that result from mutations in C9ORF72. Their research suggests that the C9ORF72 gene mutations are toxic because they induce the cell to produce small proteins or peptides that are very rich in arginine, an amino acid that, due to its positive charge and chemical nature, binds very avidly to nucleic acids, DNA and RNA.
DNA contains the instructions for the cell to make the proteins it needs for proper function. Hundreds of proteins need to anchor themselves to the DNA and RNA to read their instructions and eventually make new components for the cell. But “the presence of arginine-rich peptides hampers any reaction involving nucleic acids,” the authors of the new study added.
The CNIO study indicates that, by binding to nucleic acids with such high affinity, these arginine-rich proteins displace all cellular proteins that interact with DNA and RNA in a widespread manner, which blocks any cellular reaction that involves DNA or RNA. And with its nucleic acids effectively blocked, the cell will eventually die.
Fernández-Capetillo’s research usually focuses on cancer, but in 2014, he started working on ALS, convinced that a technique recently established in his group could help them understand the toxicity of mutations in the C9ORF72 gene. And it was a flash of insight, an idea that came up after having coffee at the CNIO with Nobel laureate Jack Szostak, which put him on the trail of arginine. As Fernández-Capetillo explained, “Szostak investigates the chemistry of the origin of life, and he told me that to stop reactions involving nucleic acids what they used in their experiments was precisely synthetic peptides with lots of arginines because of their high affinity for nucleic acids. So I thought, what if this is what is going on, what if the arginine-rich proteins in ALS patients are blocking DNA and RNA in a generalized way?”
This initial hypothesis was supported when the group decided to test whether similar problems were also seen when cells are exposed to a natural protein that has a lot of arginine. This protein, protamine, is normally only expressed transiently during the development of sperm cells.
Consistent with the model now published in The EMBO Journal, the biological function of protamine is to displace histones from the DNA. Histones are proteins that facilitate DNA compaction. “By exchanging histones for protamine, which is smaller, sperm DNA can become more compact,” explained Fernández-Capetillo. However, protamine is toxic to any cell that is not a sperm cell.
The newly reported research indicates that the cellular effects of protamine are identical to those of the arginine-rich peptides found in ALS patients. As Fernández-Capetillo commented, “What we have seen is that arginine-containing peptides are like a kind of tar that sticks to nucleic acids and decorates them, and in doing so they displace the proteins that are normally bound to the nucleic acids so that nothing that involves DNA or RNA works … We think that what happens in ALS patients is equivalent to what would happen if their motor neurons accidentally started to produce protamine … I think we have a pretty satisfactory model that helps us understand what is going on in the motor neurons of ALS patients, what is killing them.”
And once scientists begin to understand why arginine-rich peptides are toxic, the next step is to find ways to overcome this toxicity. Research along these lines has already begun in the CNIO group, alongside work to create animal models in which the problem—the production of toxic peptides—is reproduced to offer a platform for testing potential therapies. “We are excited, as the key to curing any disease is to understand first what is not working. Only then can you start looking for a treatment,” Fernández-Capetillo noted.
Learning how to alleviate the toxicity of these peptides may also be useful in addressing non-C9ORF72-associated ALS, that is, the disease as a whole. The authors of the paper believe that the widespread mechanism of nucleic acid blocking is probably what happens in ALS in general.
The team concluded, “We propose that the high affinity of arginine-rich peptides for RNA or DNA leads to a widespread coating of nucleic acids in cells, with a consequent generalized displacement of RNA- or DNA-binding factors from chromatin and RNA … Collectively, the experiments presented here provide a unifying mechanism that explains the widespread effects of arginine-rich CPPs [cell-penetrating peptides] in nucleic acid metabolism and provides some initial proof-of-principle ideas as to how this knowledge could be used to limit their toxicity in mammalian cells.”