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.

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Spotlight: The Neuro-D lab Leiden

Principal Investigator: Dr. Willeke van Roon-Mom

Location: Leiden University Medical Centre, Leiden, The Netherlands

Year Founded: 1995

What disease areas do you research?

What models and techniques do you use?

A group photo of members of the Neuro-D lab Leiden standing outside on a patio.
This is a group picture taken during our brainstorm day last June. From left to right: Boyd Kenkhuis, Elena Daoutsali, Tom Metz, Ronald Buijsen, Willeke van Roon-Mom (PI), David Parfitt, Hannah Bakels, Barry Pepers, Linda van der Graaf and Elsa Kuijper. Image courtesy of Ronald Buijsen.

Research Focus

What is your research about?

The Neuro-D research group studies how diseases develop and progress at the molecular level in several neurodegenerative diseases. They focus on diseases that have protein aggregation, where the disease proteins clump up into bundles in the brain and don’t work correctly.

We focus strongly on translational research, meaning we try to bridge the gap between research happening in the laboratory to what is happening in medical clinics. To do this we use more “traditional” research models like animal and cell models. But we also use donated patient tissues and induced pluripotent stem cell (iPSC) models, which is closer to what is seen in medical clinics.

Our aim is to unravel what is going wrong in these diseases, then discover and test potential novel drug targets and therapies.

One thing we are doing to work towards this goal is identifying biomarkers to measure how diseases progress over time. To do this, we use sequencing technology and other techniques to look at new and past data from patients.

Why do you do this research?

So far there are no therapies to stop the progression of ataxia. If we can understand what is happening in diseases in individual cells, we can develop therapies that can halt or maybe even reverse disease progression.

Identifying biomarkers is also important, because it will help us figure out the best time to treat patients when we eventually have a therapy to test.

Stylized logo for the Dutch Center for RNA Therapeutics
The Neuro-D lab Leiden is part of the Dutch Center for RNA Therapeutics, which focuses on RNA therapies like antisense oligonucleotides. Logo designed by Justus Kuijer (VormMorgen), as 29 year old patient with Duchenne muscular dystrophy.

Are you recruiting human participants for research?

Yes, we are! We are looking for participants for a SCA1 natural history study and biomarker study. More information can be found here. Please note that information about this study is only available in Dutch.

Fun Fact

All our fridges and freezers have funny names like walrus, seal, snow grouse and snowflake.

For More Information, check out the Neuro-D lab Leiden website!

Written by Dr. Ronald Buijsen, Edited by Celeste Suart

Sunrise of Gene Therapy for Friedreich’s Ataxia

Written by Dr. Marija Cvetanovic   Edited by Dr. Ronald Buijsen

Researchers from the University of California show they can “edit” the Frataxin gene in human cells from Friedreich’s Ataxia and transplant them into mice. This lays the groundwork for this method to be tested for safety.

Friedreich’s ataxia is a progressive, neurodegenerative movement disorder. It is often associated with heart issues and diabetes. Symptoms first start to appear in patients when they are around 10 to 15 years old. Friedreich’s ataxia has the prevalence of approximately 1 in 40,000 people and is inherited in a recessive manner. This means that patients with Friedreich’s ataxia inherited a disease gene from both the father and mother. Friedreich’s ataxia is caused by an overexpansion of the GAA repeat in the Frataxin gene, all these extra repeats causes less Frataxin protein to be made.

Human hematopoietic stem and progenitor cells (HSPCs) are the stem cells that give make to other types of blood cells. You can find HSPCs in the blood all around the body.

HSPCs are ideal candidates for use in stem cell therapy because of a few reasons. First, you can easily get them out of the body through a blood donation (at least easier than some other types of cells!). Second, they can self-renew, meaning they will make more of themselves. Third, other folks have researched this type of cell before, so we know they are fairly safe. Researchers wanted to test if these cells could be used to help treat Friedreich’s ataxia.

CRISPR-Cas9 is a customizable tool that lets scientists cut and insert small pieces of DNA at precise areas along a DNA strand. The tool is composed of two basic parts: the Cas9 protein, which acts like the wrench, and the specific RNA guides, CRISPRs, which act as the set of different socket heads. These guides direct the Cas9 protein to the correct gene, or area on the DNA strand, that controls a particular trait. This lets scientists study our genes in a specific, targeted way and in real-time.
Researchers used CRISPR editing to fix the mutation causing Friedreich’s ataxia in patient blood cells. Photo Credit: Ernesto del Aguila III, National Human Genome Research Institute, National Institutes of Health
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Terapia gênica validada em celulas estaminais SCA3 humanas

Escrito por Dr. Marija Cvetanovic, Editado por Dr. Sriram Jayabal, Traduzido para Português por Guilherme Santos, Publicado inicialmente em: 20 de março de 2020.

Grupo de pesquisa em Michigan relata a criação do primeiro modelo de célula humana aprovado pelo NIH que reflete as características da doença SCA3 – defeitos celulares que, após terapia genética, mostram melhora

A ataxia espinocerebelar tipo 3 (SCA3) é uma doença genética de início tardio, de herança dominante, que afeta várias regiões do cérebro. Os indivíduos afetados sofrem de vários sintomas, sendo a coordenação do movimento a mais prejudicada e debilitante. A SCA3 é causada por uma mutação no gene Ataxin-3 (ATXN3). Em indivíduos não afetados, o gene ATXN3 geralmente tem de 12 a 44 repetições do código genético “CAG;” no entanto, no código genético de algumas pessoas, o número de repetições de CAG pode se tornar anormalmente alto. Se essa mutação de “expansão repetida” fizer com que o gene ATXN3 tenha mais de 56 repetições CAG, a pessoa desenvolverá ataxia SCA3. As células usam sequências CAG repetidas em seu genoma para produzir proteínas com longas extensões do aminoácido glutamina. Nas células SCA3, esses tratos de “poliglutamina” (polyQ) são anormalmente longos na proteína ATXN3, o que torna a proteína mais propensa a formar aglomerados (ou “agregados”) na célula. A presença desses aglomerados de proteínas nas células do cérebro é uma das características do SCA3.

Apesar de conhecer a causa genética da SCA3, ainda não se sabe como essa mutação afeta as células no nível molecular. Dito isto, vários modelos celulares e animais foram desenvolvidos nas últimas duas décadas para ajudar a estudar esses mecanismos subjacentes. Os modelos SCA3 não apenas ajudaram a nossa compreensão da progressão da doença em todos os níveis (molecular, celular, tecido e comportamental), mas também nos ajudaram a nos aproximar de intervenções terapêuticas. Por exemplo, estudos recentes usando modelos de camundongo SCA3 estabeleceram que direcionar o ATXN3 com uma forma de terapia genética conhecida como tratamento com oligonucleotídeo antisense (ASO) poderia muito bem ser uma estratégia eficaz para melhorar a vida dos pacientes. Os ASOs direcionados ao ATXN3 fazem com que as células do cérebro produzam menos proteína ATXN3 mutante e, quando administradas a camundongos SCA3, melhoram sua função motora. Esses resultados apoiam fortemente o uso potencial de ASOs no tratamento da SCA3. Ainda assim, é importante verificar se esse achado pode ser repetido em neurônios humanos (um passo necessário para nos aproximar dos ensaios clínicos da ASO).

Female scientist in a while lab coat busy at work, we are looking at her from behind through some glass bottles
Imagem de um cientista pesquisador trabalhando no laboratório. Imagem cortesia de pxfuel.

A experiência anterior de ensaios clínicos malsucedidos destaca a importância de determinar as semelhanças e diferenças entre humanos e camundongos quando se trata de doença. Por exemplo, a mutação SCA3 não ocorre naturalmente em camundongos; portanto, modelar SCA3 em camundongos geralmente requer manipulação genética adicional, o que poderia levar a efeitos inesperados que normalmente não vemos nos humanos. Além disso, podemos perder importantes determinantes da patologia de SCA3 devido às diferenças inerentes entre humanos e camundongos. Por exemplo, proteínas que ajudam a contribuir para a SCA3 em pacientes humanos podem simplesmente não estar presentes nos neurônios do rato (e vice-versa). Devido a essas diferenças de espécies, as intervenções terapêuticas eficazes em camundongos nem sempre são tão eficazes em humanos.

Os neurônios humanos SCA3 podem ajudar a preencher a lacuna entre modelos de roedores e pacientes humanos, atuando como uma ferramenta clinicamente relevante para examinar os mecanismos da doença e testar novas terapias. Como não podemos remover uma parte do cérebro de um paciente com SCA3 para estudar a doença, esses neurônios devem ser criados em laboratório. Os neurônios humanos podem ser gerados a partir de células estaminais pluripotentes induzidas (iPSCs) ou de células estiminais embrionárias humanas (hESCs). As células estaminais pluripotentes induzidas (iPSCs) são produzidas a partir de células adultas (geralmente células do sangue ou da pele) que são reprogramadas para retornar a uma forma semelhante a um embrião (conhecido como estado “pluripotente”). Assim como durante o desenvolvimento normal, as iPSCs podem criar muitos tipos diferentes de células, incluindo neurônios. Um problema com essa abordagem é que o processo de reprogramação pode potencialmente alterar essas células de maneira a afetar a forma como a doença se apresenta. Para evitar esse problema, os pesquisadores também podem criar neurônios humanos a partir de células estaminais embrionárias humanas (hESCs), derivadas de embriões e, portanto, naturalmente pluripotentes. Como os hESCs não requerem reprogramação, é mais provável que modelem com precisão a doença. No entanto, eles são mais difíceis de obter e trabalhar. Os pesquisadores deste estudo, liderados por Lauren Moore no laboratório do Dr. Hank Paulson na Universidade de Michigan, usaram hESCs para gerar o primeiro modelo SCA3 aprovado pelo Instituto Nacional de Saúde (Americano) – National Institutes of Health (NIH) usando células humanas.

Continue reading “Terapia gênica validada em celulas estaminais SCA3 humanas”

Gene Therapy Validated In Human SCA3 Stem Cells

Written by Dr. Marija Cvetanovic Edited by Dr. Sriram Jayabal

Research group in Michigan report the creation of the first NIH-approved human cell model that mirrors SCA3 disease features – cellular defects that, after gene therapy, show improvement

Spinocerebellar ataxia type 3 (SCA3) is a dominantly-inherited, late onset genetic disease that affects multiple brain regions. Affected individuals suffer from several symptoms, with impaired movement coordination being the most debilitating. SCA3 is caused by a mutation in the Ataxin-3 (ATXN3) gene. In unaffected individuals, ATXN3 typically has anywhere from 12 to 44 repeats of the genetic code “CAG;” however, in some people’s genetic code, the number of CAG repeats can become abnormally high. If this “repeat expansion” mutation causes the ATXN3 gene to have more than 56 CAG repeats, the person develops SCA3. Cells use repeating CAG sequences in their genome to make proteins with long tracts of the amino acid glutamine. In SCA3 cells, these “polyglutamine” (polyQ) tracts are abnormally long in the ATXN3 protein, which makes the protein more prone to form clumps (or “aggregates”) in the cell. The presence of these protein clumps in the cells of the brain is one of the hallmarks of SCA3.

Despite knowing the genetic cause of SCA3, it is still not known how this mutation affects cells on the molecular level. Having said that, several cellular and animal models have been developed in the past two decades to help study these underlying mechanisms. SCA3 models have not only helped to  our increased understanding of the disease’s progression at all levels – molecular, cellular, tissue, and behavioral – but also helped move us closer to therapeutic interventions. For instance, recent studies using SCA3 mouse models have established that targeting ATXN3 with a form of gene therapy known as antisense oligonucleotide (ASO) treatment could very well be an effective strategy for improving the lives of patients. ATXN3-targeting ASOs cause the cells of the brain to produce less of the mutant ATXN3 protein and, when given to SCA3 mice, improved their motor function. These results strongly support the potential use of ASOs to treat SCA3. Still, it is important to see if this finding can be repeated in human neurons (a step that is needed to bring us closer to ASO clinical trials).

Female scientist in a while lab coat busy at work, we are looking at her from behind through some glass bottles
Image of a research scientist hard at work in the lab. Image courtesy of pxfuel.

Previous experience from unsuccessful clinical trials highlight the importance of determining the similarities and differences between humans and mice when it comes to disease. For instance, the SCA3 mutation does not naturally occur in mice; therefore, modeling SCA3 with mice usually requires additional genetic manipulation, which could lead to unexpected effects that we do not typically see in patients. In addition, we may miss important determinants of SCA3 pathology due to the inherent differences between humans and mice. For example, proteins that help contribute to SCA3 in human patients may simply not be present in mouse neurons (and vice versa). Because of such species differences, the therapeutic interventions that are effective in mice are not always as effective in humans.

SCA3 human neurons can help bridge the gap between rodent models and human patients, acting as a clinically relevant tool for looking into disease mechanisms and testing new therapies. Because we cannot remove a portion of an SCA3 patient’s brain to study the disease, these neurons must be created in a lab. Human neurons can be generated from induced pluripotent stem cells (iPSCs) or from human embryonic stem cells (hESCs). Induced pluripotent stem cells (iPSCs) are made from adult cells (usually blood or skin cells) that are reprogrammed to return to an embryo-like form (known as the “pluripotent” state). Just like during normal development, iPSCs can create many different types of cells, including neurons. One problem with this approach is that the process of reprograming can potentially change these cells in way that could affect how the disease presents. To avoid this issue, researchers can also create human neurons from human embryonic stem cells (hESCs), which are derived from embryos and are, therefore, naturally pluripotent. Because hESCs do not require reprograming, they are more likely to accurately model disease. However, they are more difficult to obtain and work with. The researchers in this study, led by Lauren Moore in Dr. Hank Paulson’s lab at the University of Michigan, used hESCs to generate the first ever National Institutes of Health (NIH)-approved SCA3 model using human cells.

Continue reading “Gene Therapy Validated In Human SCA3 Stem Cells”