Normally, U2-snRNP (red) is found in the nucleus of motor neurons (left), but it accumulates outside the nucleus in the neurons of patients with ALS and frontotemporal dementia who have a certain genetic mutation. [Reed Lab/Harvard Medical School]
Normally, U2-snRNP (red) is found in the nucleus of motor neurons (left), but it accumulates outside the nucleus in the neurons of patients with ALS and frontotemporal dementia who have a certain genetic mutation. [Reed Lab/Harvard Medical School]

Many cases of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are associated with the C9ORF72 gene, which is known to encode toxic molecules, dipeptide repeat proteins, that interfere with the proper splicing of mRNA. Exactly how these toxic molecules could gum up the splicing mechanism, however, has been unclear. The sticking point, new results indicate, is a small nuclear ribonucleoprotein particle, also known as a snRNP (“snurp”) that is needed for the proper functioning of the spliceosome, the cell’s mRNA-splicing machine.

A new paper that appeared June 13 in Cell Reports, in an article entitled “Evidence that C9ORF72 Dipeptide Repeat Proteins Associate with U2 snRNP to Cause Mis-splicing in ALS/FTD Patients,” describes how researchers from Harvard Medical School found a link between toxins expressed by C9ORF72, errors in RNA splicing, and an intermediary step for translating genetic instructions into functional proteins.

“We show that addition of proline-arginine (PR) and glycine-arginine (GR) toxic DPR peptides to nuclear extracts blocks spliceosome assembly and splicing, but not other types of RNA processing,” wrote the authors of the Cell Reports paper. “Proteomic and biochemical analyses identified the U2 snRNP as a major interactor of PR and GR peptides.”

Genes affected by the resulting splicing errors include those with mitochondrial, neuronal, and gene-expression functions. These processes have been previously linked to ALS and FTD, suggesting that restoring normal splicing activity may have potential as a therapeutic strategy for patients with ALS, FTD, or both.

“Our findings indicate that the most prevalent mutation found in inherited ALS and FTD creates errors in spliceosome assembly,” said senior study author Robin Reed, Ph.D., professor of cell biology at Harvard Medical School. “Since splicing is upstream of so many critical cellular functions, a better understanding of this mechanism could illuminate new approaches to help patients with these diseases, which currently have no effective treatments.”

A specific mutation to the C9ORF72 gene accounts for around 25% of cases of FTD, where gradual loss of nerve cells in the frontal lobe of the brain leads to profound behavioral and cognitive deficits. Such mutations are also believed to fuel 30–40% of inherited forms of ALS and Lou Gehrig's disease, a fatal disorder involving gradual loss of control over voluntary motor functions. Roughly one in five patients with ALS also develops FTD.

The C9ORF72 mutation causes the abnormal accumulation of many copies of a small, six-nucleotide long segment of DNA, which are processed by cells into mRNA—the molecules that carry instructions from DNA for the production of proteins. These extraneous mRNAs code for so-called dipeptide repeat proteins, two of which, GR and PR, have been found to be toxic in human, yeast, and fruit fly cells.

Exactly how these peptides cause toxicity was, up until now, unclear, but previous studies have shown that they significantly increase the number of errors in splicing, the cellular process for editing raw mRNAs, which contain unnecessary segments that must be removed to accurately code for a protein.

In their current study, Dr. Reed and colleagues found that these toxic peptides strongly and specifically associate with a component of the spliceosome, known as U2 snRNP. “In addition,” the authors of the current study noted, “U2 snRNP, but not other splicing factors, mislocalizes from the nucleus to the cytoplasm both in C9ORF72 patient induced pluripotent stem cell (iPSC)-derived motor neurons and in HeLa cells treated with the toxic peptides.”

“Bioinformatic studies support a specific role for U2-snRNP-dependent mis-splicing in C9ORF72 patient brains,” the study’s authors added. “Together, our data indicate that DPR-mediated dysfunction of U2 snRNP could account for as much as ∼44% of the mis-spliced cassette exons in C9ORF72 patient brains.”

Mis-splicing may affect the expression of genes involved in the function of mitochondria, the power generators of the cell, whose malfunction has been previously linked to ALS. The Harvard team also found affected genes involved in neuronal structure and growth, and others that play roles in gene expression—cellular functions that have also been previously associated with ALS and FTD.

“It was striking how these peptides are so specific to U2 snRNP. No other cellular processes appeared to be affected, whereas splicing was completely blocked,” Dr. Reed commented. “When these peptides are expressed at high levels, they are completely toxic to the cell, but if they are produced at a low enough level, they can inhibit the splicing of genes that are U2-dependent, which may have some role in the development of disease.”

It is currently unknown whether and how these mis-splicing events are involved in the development of ALS, FTD, or other motor neuron diseases in human patients. While C9ORF72 mutations account for the majority of inherited forms of ALS and FTD, several other genes have also been implicated. In addition, more than 90% of ALS cases are sporadic, with no known genetic cause.

Mis-splicing, however, has been previously implicated in both inherited and sporadic ALS, FTD, as well as in spinal muscular atrophy, another motor neuron disease that the Reed lab previously showed shares biochemical pathways with ALS, suggesting that the process is a promising target for future therapeutic development.

“What we are finding is that disruptions in RNA splicing appear to be a common thread linking these motor neuron disorders,” Dr. Reed concluded. “Much more research is needed, but if we could correct splicing errors with so-called splicing modulator compounds, we could prevent disruptions downstream at sites such as mitochondria, neuronal axons, or the neuromuscular junction, which may have efficacy for the treatment of ALS and FTD.”

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