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Aug 10

Research Bit: ALS-linked mutation leads to nuclear defects

New project by Packard Center scientist Alyssa Coyne provides new detail on the underlying and initiating events through which the C9orf72mutation causes disease.

ALS-linked mutation leads to nuclear defects

Scientists have identified problems with the movement of large molecules between the nucleus and the cytoplasm of the cell in many neurodegenerative diseases, including ALS/FTD caused by mutations in the C9orf72 gene. Despite a growing body of literature linking defects in nucleocytoplasmic transport to the onset and progression of ALS/FTD, researchers still don’t understand exactly what happens to the nuclear pore that would cause these deficiencies. Working in the lab of Packard Center founder and director Jeffrey Rothstein, MD, PhD, postdoc Alyssa Coyne and colleagues used super-resolution structured illumination microscopy to show that the abnormal RNA produced by the C9orf72 mutation causes a reduction in one of the nuclear pore components that sparks a cascade of events leading to motor neuron death -like the first domino to fall. The results, published in Neuron, provide new detail on the underlying and initiating events through which the C9orf72mutation causes disease.

Alyssa Coyne, PhD (pictured with Dr. Jeffrey Rothstein, MD, PhD) recently presented at the 20th Annual Packard Center ALS Research Symposium

In 2011, two international teams of scientists showed that the most common genetic cause of ALS and FTD was due to a repeat expansion in C9orf72. Since then, researchers have identified several ways through which the mutation could lead to disease. Emerging work over the last several years has shown changes to the transport of macromolecules between the nucleus and cytoplasm. This occurs via the nuclear pore, a large protein complex with multiple copies of 30 nucleoporins (Nups) that provide physical scaffolding and anchor the pore to the nuclear membrane. Most macromolecules need to be actively moved through the pore using import and export proteins, all of which is fueled by a different protein called Ran GTPase. Although some alterations to Nups and nucleocytoplasmic transport occur with normal aging, scientists have found marked problems with this transport in C9 ALS/FTD.

Coyne in the Rothstein lab, and colleagues used super-resolution structured illumination microscopy to study the nuclear pore complex in induced pluripotent stem cell-derived motor neurons carrying the C9 repeat expansion. The researchers combined the microscopy with immunofluorescent staining to count the number of nuclear pores and Nups on the nucleus. The researchers found that 8 of the 23 nucleoporins were reduced compared to controls. However, they didn’t find a reduction in the overall number of Nups or nuclear pores on the nucleus- demonstrating that the pore complexes themselves were deficient in these Nup building blocks. When the authors looked at postmortem samples from C9 ALS/FTD patients, they found alterations to the nuclear pore and Nups were identical to the iPSC neuron model, which shows that this human cell model accurately recapitulates the disease in patients.

Other studies on C9orf72 had shown that molecules called antisense oligonucleotides (ASOs) could target the abnormal repeat expansion in the gene, so Coyne et al. investigated whether treatment with ASOs could reduce changes to the Nups. ASO treatment did restore normal levels of the 8 Nups that were reduced by the C9 mutation. It also restored the normal location of Ran GTPase from the cytoplasm to the nucleus, allowing it to fuel normal nucleocytoplasmic transport. Overexpression of the one of the Nups, POM121, also returned Ran GTPase to its normal location and restored the nuclear expression of other Nups. Reducing POM121 expression in control neurons, on the other hand, caused a similar phenotype to the one seen in C9 neurons. Thus Pom12 appears to be the single Nup that starts the degradative injury.

These results, however, didn’t show how the C9orf72 repeat expansion led to changes in the nuclear pore and in Nups like POM121. Further experiments showed that it was the pathological G4C2 repeat RNA that led to decreased POM121 levels in iPSC neurons, and not the small, toxic dipeptide repeat proteins made by the expansion, or by the loss of normal C9 function. Together, this work shows that the reduction of POM121 in nuclear pore complexes sets off a pathological cascade of events that results in impaired nucleocytoplasmic transport and ends in motor neuron death. The findings that both ASOs and POM121 overexpression can reverse some of these changes hints at the potential for future therapeutics.