Using the gene scissors CRISPR and stem cells, researchers at Stockholm University and the UK Dementia Research Institute (UK DRI) at King’s College London have managed to identify a common denominator for different gene mutations that all cause the neurological disease ALS. The research shows that ALS-linked dysfunction occurs in the energy factories of nerve cells, the mitochondria, before the cells show other signs of disease, which was not previously known. The study was recently published in the scientific journal Nature Communications.

“We show that the nerve cells, termed motor neurons, that will eventually die in ALS have problems soon after they are formed. We saw the earliest sign of problems in the cell’s energy factories, the mitochondria*, and also in how they are transported out into the nerve cells’ long processes where there is a great need for them and the energy they produce,” says Dr Eva Hedlund at Stockholm University, head of the study together with Dr Marc-David Ruepp at the UK Dementia Research Institute at King’s College London.

The research team was able to establish that these problems were common to all ALS-caused mutations, which will be important for future treatments of the disease.

“This means that there are common factors that could be targeted with drugs, regardless of the cause of the disease,” says Dr Eva Hedlund.

Reprogrammed cells

The researchers used the gene scissors CRISPR/Cas9 to introduce various ALS-causing mutations into human stem cells, called iPS cells*. From these, motor neurons, the nerve cells that are lost in in ALS, and interneurons, nerve cells that are relatively resistant to the disease, were produced. These were then analyzed with single-cell RNA sequencing, a method that enables identification of all messenger molecules (mRNA) in each individual cell and with that understand how a particular cell works, how it talks to its neighbors and if it starts to have problems.

“In the data we obtained, we identified a common disease signature across all ALS-causing mutations, which was unique to motor neurons and thus did not arise in resistant neurons,” says Dr Christoph Schweingruber, first author of the study.

This happened very early and was completely independent of whether the disease-causing mutated proteins (FUS, or TDP-43) were in the wrong place in the cell or not.

“Until now, it has been believed that it is the change where the proteins are within the cells, called mislocalization*, that occurs first,” says Dr Marc-David Ruepp.

A groundbreaking discovery

In ALS, it is often said that some problems are caused by a loss of function in a protein that is mutated, while other problems arise due to the opposite, namely the emergence of a new toxic function that has been obtained through the mutation, called “gain-of-function”, but according to Eva Hedlund, it has not always been easy to clarify how it really works and much is still unknown.

“By making various CRISPR mutations in the ALS-causing FUS-gene*, we have now been able to show for the first time that most errors arising are caused by a new toxic property of the protein, not by a loss of function,” says Dr Christoph Schweingruber.

Affecting the cells' energy factories

A third discovery was that the transport of mitochondria out into the axons*, the extensions of the nerve cells where most mitochondria in nerve cells are needed, was radically affected in the ALS lines. This happened independently of whether the disease-causing proteins were in the wrong place in the cell or not.

“A fact that poses a problem because there is a great need for these energy factories in the extensions of the nerve cells. Without them the nerve cells do not have enough energy to communicate properly with other cells,” says Dr Eva Hedlund.

The new discoveries open up for early treatment methods, something that for the research team is a continuous work in progress.

“We are trying to understand how these early errors occur in the sensitive motor neurons in ALS, and how it affects energy levels in the cells and their communication and necessary contacts with muscle fibers. We believe that these are important keys to the understanding of why the synapses between motor neurons and muscles is broken in ALS and also to identify new targets for therapies,” says Dr Eva Hedlund.

Read more
Find the publication in Nature Communications:
"Single-cell RNA-sequencing reveals early mitochondrial dysfunction unique to motor neurons shared across FUS- and TARDBP-ALS".
DOI: https://doi.org/10.1101/2023.03.16.531876

About ALS
The fatal disease ALS is characterized by the death of nerve cells in our brain and spinal cord, so-called motor neurons, which control all our skeletal muscles in the body, and this leads to the breakdown of the muscles. In most cases of ALS, the disease occurs sporadically, but in 10–20 percent of cases, different gene mutations cause the disease. It has so far been unknown whether different mutations cause ALS in the same way. It has also been unknown why some nerve cells are sensitive while others are resistant.

Glossary to ALS research

• * iPSCs are reprogrammed cells that have been isolated from adult human tissues to be reverted in development to cells that resemble embryonic stem cells.
• *Mitochondria are small structures found in all our cells. They are often called the power plants of the cell; in them, sugar and fat are burned to water and carbon dioxide. The energy that is then released is stored in a molecule, ATP, which is used in various cellular processes.
• *Axons are the projections that nerve cells send out to contact other cells. In the case of motor neurons, it is muscle fibers that need to be contacted, which are sometimes over a meter away.
• *Mislocalization means that something ends up in the wrong place. In ALS and other neurodegenerative diseases, misfolded proteins are seen in places where the protein should not normally be.
• *FUS-gene, Fused in Sarcoma; mutations in this gene leads to ALS
• *TARDBP gene, TAR DNA binding protein (TDP-43); mutations in this gene leads to ALS