Nuclear Pore Problems May Lead to ALS and Dementia

Neurons (red), created from ALS patients bearing the C9ORF72 expansion, show clumps of the RanGAP protein (yellow) on their nuclei (white). The nuclei of other cells are in blue.Jeffrey Rothstein laboratory, Johns Hopkins Medicine.

Tens of thousands of Americans are currently living with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both diseases are caused by the death of neurons in the central nervous system. In ALS, this leads to movement difficulty and eventual paralysis. In FTD, it causes personality changes as well as problems with language and decision-making.

Recent research has connected many inherited cases of both disorders—as well as some noninherited cases—to alterations in the C9ORF72 gene. This gene usually has less than 20 repeats of a 6-letter DNA sequence (GGGGCC, or G₄C₂). Disease-causing versions are expanded, containing hundreds to even thousands of these repeats. Exactly how this expansion leads to neuron death was unknown. The overly long RNA might alter the function of RNA-binding proteins or cause cells to produce toxic proteins.

Dr. Jeffrey Rothstein’s lab at Johns Hopkins University School of Medicine previously identified more than 400 proteins that bind particularly well to G₄C₂ repeats. To figure out whether the RNA could interact with any of these candidates to cause cell death, his lab collaborated with Dr. Thomas Lloyd’s team at Johns Hopkins. The researchers altered neurons in the eyes of fruit flies to express 30 G₄C₂ repeats, causing eye defects. Using these flies, the team screened mutant versions of the various candidate proteins for effects on fly eyes. The work was partially funded by NIH’s National Institute of Neurological Disorders and Stroke (NINDS) and National Cancer Institute (NCI). Results were published in Nature on September 3, 2015.

The scientists found less eye deterioration in flies with an overactive version of a protein called RanGAP. RanGAP is important for transporting material between the cell’s gel-like interior (cytoplasm) and the nucleus though a structure called the nuclear pore. Increasing production of RanGAP relieved movement problems in flies with the G₄C₂ repeats. This suggests that the C9ORF72 mutation may cause disease by disrupting this process, called nuclear or nucleocytoplasmic transport.

The team next examined brain tissue from ALS patients with the C9ORF72 expansion as well as neurons grown from patients’ cells. In both, the expanded RNA bound RanGAP to form abnormal clumps outside the nucleus. Other proteins involved in nuclear transport were also stuck outside. Treating neurons with compounds that prevented the RNA from interacting with RanGAP eliminated the transport problem, suggesting potential treatment strategies.

In an accompanying paper in Nature, another NIH-funded team, led by Dr. J. Paul Taylor of St. Jude Children’s Research Hospital, used a different genetic screen to look for genes that modify the effects of 58 G₄C₂ repeats in flies. The screen identified numerous genes that encode components of the nuclear pore and the nucleocytoplasmic transport machinery, providing strong evidence for this pathway as the mechanism of neurodegeneration.

A third study, published in Nature Neuroscience by a team led by Stanford University’s Dr. Aaron Gitler, focused on the potential toxic effects of proteins produced by the expanded G₄C₂ repeats. Experiments in yeast demonstrated toxic protein effects. Like the other studies, this approach also tied the expansions to nucleocytoplasmic transport defects.

Taken together, these studies don’t prove whether abnormal RNA-protein binding, toxic protein products, or both are responsible for diseases caused by these G₄C₂ repeats. The results do, however, provide important insights into the disease mechanisms and highlight potential therapeutic targets.

“We still don’t know every step between the C9ORF72 mutation and cellular death in the brain,” Rothstein says. “Now we have some information about what it is doing early on to damage brain and spinal cord cells.”

—by Brandon Levy and Harrison Wein, Ph.D.