A lay summary written by Sophie Badger and reviewed by Dr Jon Wood and an MND lay panel
Background:
Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease, and frontotemporal dementia (FTD) are two neurodegenerative diseases that have the same genetic causes. Currently, the underlying changes that occur and cause neurodegeneration in these diseases are still being discovered. The most common genetic cause of ALS/FTD is a mutation in a gene called C9orf72. This mutation is called a ‘repeat expansion’ mutation, because a sequence of DNA made up of six letters is repeated several times. Most people only have a few repeats, but some people can have hundreds or thousands, which can cause ALS/FTD. One of the products of these long repeats is the production of five different proteins, called dipeptide repeat proteins (DPRs), which cluster together into protein inclusions in neurons (nerve cells). Inclusions are clumps of protein that stick together and can be toxic if not removed from the neurons.
Another genetic cause of ALS/FTD are mutations in a gene called TBK1 that produces a protein also called TBK1. In this case, the mutations result in a TBK1 protein that no longer performs its proper function. TBK1 is involved in the sorting and recycling of waste in cells. Therefore, reducing the activity of the TBK1 protein through a genetic mutation interferes with this system, potentially leading to detrimental effects as old proteins and other material are no longer recycled or degraded and start to build up inside cells.
On the rare occasion that the C9orf72 and TBK1 mutations occur together in the same person, that person exhibits an earlier age of ALS/FTD disease onset and a faster disease course. However, it is unknown how these two genes interact to cause this more severe disease.
Why is the study important?
Based on observations that people with both TBK1 and C9orf72 mutations develop earlier disease onset and a faster disease course, the authors wanted to investigate how these two mutations interact to cause neurodegeneration. By discovering the mechanisms and pathways by which neurodegeneration in ALS/FTD occurs, researchers can begin identifying targets and developing therapeutics to reduce or prevent it.
What did the authors do and how did they do it?
The authors used a mouse model of ALS/FTD with a C9orf72 mutation to observe how the presence of that mutation affected a normal TBK1 protein. After establishing that the C9orf72 mutation did adversely affect TBK1, the authors simulated a human that has both C9orf72 and TBK1 mutations by inserting a gene that produces a DPR protein into mice with a TBK1 mutation. They tested this C9orf72+TBK1 mouse model with various strength and coordination tests, such as how long they could hold onto a wire before letting go, to see whether the mouse developed a more severe disease like that seen in humans. They also examined the brains of these mice to look for evidence of neurodegeneration.
Lastly, the authors looked at whether a reduction in TBK1 protein levels in neurons interfered with the way waste products are transported around the neurons. In human motor neurons, loss of TBK1 protein interfered with this function, which means neurons cannot clear the protein aggregates formed by the DPR proteins. This also caused a protein called TDP-43 to accumulate inside them. TDP-43 is an important protein required for normal cell function but is thought to have a major role in causing neurodegeneration because it is known to build up in the brains of most people with ALS/FTD.
What are the results?
The initial findings showed that the presence of a C9orf72 repeat mutation resulted in altered TBK1 protein in the brains of mice that led to lower activity. The authors investigated a DPR protein and theorised that its presence reduced the activity of TBK1 and prevented it from clearing the cell of toxic protein aggregates produced by the C9orf72 mutation. Therefore, mutations that reduce TBK1 activity further would exacerbate this effect, providing an explanation for why people with both C9orf72 and TBK1 mutations exhibit a more severe disease. Logically, artificially increasing the levels of normal TBK1 protein would solve this issue, and when the authors did this in cells, they saw a decrease in the levels of toxic protein aggregates.
When the authors looked at a TBK1 mouse model, which already showed strength and coordination impairments, and inserted a C9orf72 mutation alongside it to create the C9orf72+TBK1 model, they found that those impairments became more severe—mirroring what is seen in humans that harbour both C9orf72 and TBK1 mutations.
Previous work has shown that loss of TBK1 protein causes defects in the transport system in human motor neurons, and the authors wanted to see if those defects occurred in their C9orf72+TBK1 mouse model. In short, they found that cells with DPR protein aggregates had an altered transport system, suggesting a link between the two. In humans, this altered transport system causes the TDP-43 protein to clump together into aggregates, which the authors found in their mouse model. This suggests that the presence of the C9orf72 mutation reduced the function of the TBK1 protein which led to defects in the transport system in cells. This caused TDP-43 to clump together, ultimately leading to neurodegeneration.
What do the findings mean going forward for people with the disease?
Previous work has suggested that parts of the sorting and recycling system in cells may be involved in ALS, and this paper has identified how two genetic causes of ALS/FTD lead to changes in TDP-43 seen in most ALS patients via this system. By doing this, the authors can go on to investigate other ALS-linked mutations that affect this system and explore what happens when it is affected in different ways. Additionally, this work has expanded knowledge about the mechanisms of neurodegeneration in ALS/FTD. The C9orf72+TBK1 mouse model created in this paper could be used in future studies to identify potential drugs to prevent the TDP-43 changes seen in motor neurons, thereby reducing or preventing neurodegeneration from occurring.