Mutated ataxin-1 protein forms harmful, doughnut-shaped aggregates that are not easily destroyed

Written by Brenda Toscano Marquez   Edited by Marija Cvetanovic

Insoluble clumps of mutated ataxin-1 capture essential proteins and trigger the creation of toxic reactive oxygen species.

All proteins produced by our cells consist of long chains of amino acids that are coiled and bent into a particular 3D structure. Changes in that structure can cause serious issues in a cell’s function, sometimes resulting in disease. Spinocerebellar ataxia type 1 (SCA1) is thought to be the result of one such structural change. The cause of SCA1 is a mutation that makes a repeating section of the ATXIN1 gene abnormally long. This repeated genetic code, “CAG,” is what encodes the amino acid glutamine in the resulting ataxin-1 protein. Therefore, in the cells of patients with SCA1, the Ataxin-1 protein is produced with an expanded string of glutamines, one after the other. This polyglutamine expansion makes the mutated ataxin-1 protein form clumps in many different types of cells – most notably, though, in the cells most affected in SCA1: the brain’s Purkinje cells.

Recent research suggests that these clumps, or “aggregates,” not only take up space in the cell, but that the act of ataxin-1 proteins clustering together might even be beneficial in early stages of disease (it’s possible that the proteins wreak less havoc when they’re in large clumps, rather than all floating around individually). However, another line of research suggests that ataxin-1 aggregates might also be toxic, triggering signals that lead to the cell’s death. As such, how exactly these aggregates affect the deterioration of cells has remained an important question in SCA1 research.

n a search for answers, an international team led by Stamatia Laidou designed a unique cell model of SCA1 to track the development of ataxin-1 aggregates. Their study, published in a recent paper, made use of normal human mesenchymal stem cells that had been engineered to make a modified version of the ataxin-1 protein. In these cells, ataxin-1 was produced not only with the SCA1-causing expansion, but also with a marker protein attached to its end. This marker, known as “green fluorescent protein” (GFP), is used extensively in biological research because it glows under fluorescent light.

doughnut with white and pink sprinkles
Laidou and colleagues have observed mutated ataxin-1 clumps that cause cell stress. Photo by Tim Gouw on

Using this to their advantage, Laidou and her team used a fluorescent microscope to follow the formation of ataxin-1 aggregates over the course of 10 days. The abnormal protein first started accumulating in the nucleus as small dots. As time went on, these dots started blending together, increasing in size. By ten days, the ataxin-1 aggregates had grown even more, forming a doughnut-shaped structure that occupied most of the cell’s nucleus – a crucial structure that houses the cell’s genetic information. These results were intriguing, since the accumulation of normal, non-expanded Ataxin-1 protein does not result in an aggregate with a doughnut shape.

They hypothesized that this characteristic shape might be due to the expansion of the polyglutamine region in mutated Ataxin-1; specifically, they thought that this extra-long section of the protein might bend and coil into some abnormal structure that makes it interact differently with its fellow mutated ataxin-1 proteins. To investigate this, the researchers used a method called FTIR spectroscopy, which allowed them to capture a more detailed “fingerprint” of the structure of the aggregates. It showed that mutated ataxin-1 does indeed form different 3D shapes that tend to become more entangled with each other, making the aggregates harder to dissolve. As a consequence, the aggregates remain in the cell’s nucleus, slowly growing over time. In addition, they found that the doughnut-shaped aggregates are so “sticky” that they capture other proteins, including some that are critical for maintaining the health of the cell.

The research team’s next step was to find out if having a large aggregate crowding the nucleus and hijacking essential proteins was toxic to cells. Indeed, they found that the presence of even small aggregates in the early stages of formation increased the production of reactive oxygen species, which can harm cells through a process known as “oxidative stress.” Furthermore, as the aggregates started to become larger and less soluble, more reactive oxygen species were formed.

To see the specific way that the aggregates caused these effects, they then used a transcriptome analysis. This technique examines thousands of genes at once to create a global snapshot of what is happening in a group of cells. The authors performed this analysis on their SCA1 cell line at two different points in time: at an early stage (when aggregates are small), and at the doughnut-shaped stage (when aggregates are large and mostly insoluble). They found that mutated ataxin-1 altered the expression of multiple genes. More interestingly, though, it appeared that many of these expression changes were dependent on the size of aggregates (small aggregates vs. doughnut-shaped ones).

After this, a key question remained: how are all these changes relevant to the Purkinje cells in the cerebellum – the cells most affected in SCA1 patients? To test this, the team measured gene expression in cells from the cerebellum of a late-stage SCA1 patient. In these cells, they found expression changes in some of the same genes that were affected in their large aggregate cell model. These genes are mainly related to the production of new proteins, a vital process performed by the cell’s ribosomes.

Together, these results confirm previous SCA1 studies that seem to show that mutated ataxin-1 aggregates cause stress to cells, and that this stress gets worse as the disease progresses. In addition, we are left with a clearer picture of the structure of the ataxin-1 aggregates and the cellular processes affected by them. And, as an added bonus, we now have an additional SCA1 cell model that can be used to investigate the aggregation of ataxin-1 in future studies – studies that could open the door to the development of protective approaches against SCA1 at its different stages.

Key Terms

Human mesenchymal stem cells: cells that are able to differentiate into multiple types of cells. They originate from embryonic connective tissue.

FTIR spectroscopy (FTIR): a method of analysis that is used to determine the structure of individual molecules and/or the composition of molecular mixtures. FTIR involves shining infrared light on a sample, which is absorbed at specific frequencies depending on the type of atoms in the sample molecule and how they are connected. This absorption forms a characteristic pattern of bands, providing a fingerprint for the sample’s molecular structure. To decode this fingerprint, researchers measure the intensity of these bands and their position in the frequency spectrum.

Transcriptome analysis: a method of analysis that profiles a group of genetic material (usually mRNA) produced by a cell under specific circumstances. This creates a global snapshot of actively-expressed genes, and can therefore show changes in gene expression when compared to data from another sample.

Ribosome: the structure in the cell that is responsible for protein synthesis. Ribosomes convert the cell’s genetic code into proteins by reading a sequence of messenger RNA (mRNA) and translating it into a sequence of amino acids, which is then shaped into a protein.

Conflict of Interest Statement

The author and editor declare no conflict of interest.

Citation of Article Reviewed

Stamatia Laidou, et al. Nuclear inclusions of pathogenic ataxin-1 induce oxidative stress and perturb the protein synthesis machinery. RedoxBiol.2020.32 (1014583).  (