Changes to a critical structure within the cell nucleus of spinal nerve cells — the nucleolus — preceded the appearance of other disease markers in people with either familial or sporadic forms of amyotrophic lateral sclerosis (ALS), a study has discovered.
The findings suggest that nucleolar stress occurs early in both forms of ALS, the scientists said.
The study, “Nucleolar stress in C9orf72 and sporadic ALS spinal motor neurons precedes TDP-43 mislocalization,” was published in the journal Acta Neuropathologica Communications.
Mutations in the C9orf72 gene are the most common genetic defect in familial ALS (FALS) and also may cause a neurodegenerative condition known as frontotemporal dementia (FTD).
In both conditions, C9orf72 mutations are linked with changes and stress to the nucleolus, a structure within the nucleus of a cell that plays a critical role in building the ribosome — the molecular machinery that synthesizes proteins from a genetic message.
Smaller nucleoli have been found in brain cells isolated from FTD patients and in immune cells, collagen-producing cells, and nerve cells derived from people with C9orf72 mutations (C9-ALS).
Now, researchers at the University of California, San Diego, conducted a series of experiments to determine if changes to the size of the nucleolus occur in spinal cord motor neurons from C9-ALS patients and compared them to those from sporadic ALS cases, which arise without a familial connection. Of note, spinal motor neurons, which are damaged in ALS, transmit signals to and from the brain to control voluntary movement.
“We aimed to elucidate whether nucleolar stress is specific for C9-ALS or also occurs in SALS,” the team wrote.
Human tissues were obtained from postmortem samples, including spinal cords from 11 C9-ALS patients, 11 people who had sporadic ALS, and 11 controls.
First, the team isolated spinal motor neurons and measured the size of the nucleolus, the nucleus, as well as the cytoplasm (the area inside the cell, but outside the nucleus).
All three compartments were smaller in C9-ALS and sporadic ALS than in controls, with the ratio of nucleolar to the nuclear area significantly reduced in both C9-ALS and sporadic ALS neurons. This indicated “greater shrinkage of nucleoli compared to nuclei,” the scientists wrote.
Next, experiments showed that cell shrinkage in ALS samples was due to a decrease in the size of the larger alpha motor neurons, which are the main targets of neurodegeneration, and not due to an abundance or shrinkage of smaller gamma motor neurons.
In controls, the nucleus and cytoplasm area were significantly larger in men than women, but the nucleolar area was not significantly different. These sex differences were not seen in C9-ALS neurons, and there were no size correlations between disease onset or disease course.
Two correlated markers of ALS pathology are the toxic buildup and clumping (aggregation) of the protein TDP-43 in the cytoplasm and the presence of RNA foci — altered RNA that arises from C9orf72 mutations that aggregate and are retained in the nucleus, becoming toxic to the cells.
While reductions in all three cellular compartments were observed regardless of the presence of nucleolar RNA foci, nucleolar shrinkage was greatest in neurons without nucleolar RNA foci.
“Thus, it appears that nucleolar shrinkage may occur before pathological markers appear,” the researchers wrote.
Although normal TDP-43 is found in the nucleus, the disease-related TDP-43 aggregates are located in the cytoplasm (mislocalized). The researchers found that nucleolar area was reduced in both C9-ALS and sporadic ALS neurons regardless of TDP-43 location, but as with RNA foci, shrinkage in all three cell compartments was greatest in motor neurons without TDP-43 clumps.
“This also suggests that nucleolar stress as marked by nucleolar shrinkage may occur early in disease and precede pathology, either TDP-43 mislocalization and/or antisense RNA foci,” the scientists confirmed.
“In summary, we have demonstrated that nucleolar stress occurs in both C9-ALS and SALS [sporadic ALS] spinal motor neurons,” and likely precedes other disease-related mechanisms such as TDP-43 mislocalization and RNA foci, the investigators concluded.
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