Research news

Drug hunters have been particularly interested in the repeating C-A-G letters of genetic code that lead to Huntington’s disease (HD). The number of CAG repeats gets bigger in vulnerable brain cells over time and may hold the key for slowing or stopping HD. Many scientists have been asking what happens to HD symptoms if we stop this expansion. Recent work from a group in London led by Dr. Gill Bates examined exactly this, seeking to define the threshold of CAG repeats needed to cause disease. Let’s discuss what her team found!

We’re all just alphabet soup

The genetic code of every living organism is made up of only 4 letters – C, A, G, and T. They’re combined in different ways to make every gene in our body. That’s a lot of diversity for just 4 letters!

Within the huntingtin gene that leads to HD is a stretch of repeating C-A-G letters. People with HD are born with 36 or more CAG repeats in the huntingtin gene. As a person grows older, we know the number of CAG repeats can shift and wobble in some cells, getting bigger over time.

This ongoing CAG expansion is called “somatic instability”. This specifically happens in brain cells damaged by HD. It’s important to note that the CAG repeat size is relatively stable in blood. So a blood test showing 42 CAGs at the age of 18 will very likely still show 42 CAGs at age 50. But the brain cells of that person could have more than 100 CAG repeats, and a few may even have 200 repeats or more.

Expansions may be the key

Some scientists think that preventing CAG repeats from increasing in the brain may be key to stopping HD altogether. But no one knows how many CAGs are too many in the brain, or at what age CAG increases start to happen.

Several important genetic studies in the past few years have suggested that longer CAG repeats could help explain why brain cells die in HD. For example, people who develop HD earlier or later than expected have changes in genes that impact somatic instability of the CAG repeat in huntingtin. These genes are called “modifiers” – they modify the age at which someone starts to show symptoms of HD.

What’s interesting is that modifier genes mostly participate in the same process in the body, called mismatch repair, which is known to affect somatic instability of the CAG repeat. Very suspicious! This suggests that somatic instability of the CAG repeat is pretty important in HD.

Since somatic instability in brain cells may contribute to how these cells die, and since mismatch repair genes impact somatic instability, HD researchers are now very interested in drugs that target mismatch repair genes. Perhaps by targeting the right mismatch repair gene, we can stop somatic instability of the CAG repeat in vulnerable brain cells. The hope is that a drug which does this could slow or stop HD.

A numbers game

It turns out that we can stop somatic instability in the brain! At least we can in mice, for right now. Several pharmaceutical companies are developing HD drugs targeting mismatch repair genes and somatic instability in HD (for example, LoQus23, Rgenta, and Voyager Pharmaceuticals).

But no one really knows how long a CAG repeat must be to damage brain cells, or how early you might need to stop somatic instability in people as a treatment for HD. Recent studies in HD mice have tried to help answer these questions by looking at the impact of stopping somatic instability in HD mice with different CAG repeat lengths.

What’s helpful about HD mice is that they are born with many more CAG repeats than people with HD – because HD researchers want mice to develop symptoms of HD much faster than people do. For example, a type of mouse that models HD called “Q111” has over 100 CAG repeats. Another HD mouse model called “Q175” has about 185 CAG repeats. Both the Q111 and Q175 HD mice show symptoms of HD in less than a year.

Defining the threshold

Researchers think this threshold of about 100 CAGs may be the number of repeats needed to kill brain cells in people with HD. So what happens if you stop somatic instability in these HD mice? Do the mice get better? The answer for mice born with 185 CAG repeats, surprisingly, is no. They still develop HD, even when somatic instability is halted.

In a newly published study from the lab of Dr. Gill Bates at University College London, Q175 mice having about 185 CAG repeats were altered so that they didn’t have the mismatch repair gene MSH3. MSH3 is a high priority target for HD drug hunters since somatic instability stops altogether when MSH3 is gone.

As expected, somatic instability stopped almost completely in the brains of Q175 mice when MSH3 was eliminated. But these mice still developed features of HD, even though MSH3 was eliminated and somatic instability of the CAG repeat was halted.

What could this mean? Shouldn’t stopping somatic instability prevent the mice from developing HD? Gill’s group reasons that mice born with 185 CAG repeats already have too many repeats in the brain, so stopping expansions below 185 CAG will probably be necessary to treat HD in people.

This parallels the conclusions of a previous study which eliminated MSH3 in Q111 mice that have 100 CAG repeats, fewer than the 185 CAG repeats studied by Gill. In this other study, Dr. Vanessa Wheeler showed that Q111 mice without MSH3 have no somatic instability and have improved cellular markers of HD. So stopping somatic instability in brain cells before they reach 100 CAG repeats may be necessary for this strategy to work in people.

When should we treat HD?

This begs the question many people are asking lately: when should we treat HD? How early would a person with HD need to be treated to stop their brain cells from expanding across the threshold of 100 CAG repeats? Some brain cells appear to have 100 CAG repeats before people start to show measurable symptoms of HD. So it may be necessary to treat people even before they start to develop symptoms.

Treating people before they develop symptoms of HD poses lots of difficult questions that no one quite has the answers to yet. However, many brilliant scientists are now looking at CAG repeats directly in brains of people with HD to find answers. These insights detailing the threshold of CAG toxicity will help scientists to design better drugs and upcoming clinical trials to target somatic instability as a potential HD therapy.

A recently published collaboration between academic researchers and pharmaceutical companies was successful at detecting huntingtin in tears. The scientists were looking for a new, easy way to track Huntington’s disease (HD). If you don’t mind shedding a tear or two, they found it!

Biomarkers - biological metrics in tune with disease progression

Tracking disease progression is not only medically important to ensure patients are living a healthy life, but it’s also important for developing medicines for diseases like HD. Biological metrics that are in tune with disease progression are called biomarkers. There are different kinds of biomarkers, from images of organs, to tests of metabolism, to measurements made in body fluids.

Biomarkers are tools that researchers can use to assess how well a potential medicine is working. If a drug slows or stops the progression of a disease according to one or more biomarkers, it could mean that drug is working!

HD researchers have been working to identify biomarkers that not only track with disease progression, but also change before someone ever starts to show symptoms. Having very early HD biomarkers would allow researchers to know if a medicine is helping someone before they ever start to show disease onset. Since lots of studies are starting to indicate that the earlier we treat HD, the better off someone will be, good biomarkers will be critical for future trials.

How do we currently track HD progression?

We’ve known for a long time that HD causes brain cells to die. So imaging, like MRIs, has been used to track brain cell loss as HD progresses. However, it’s not always easy and convenient (or cheap!) to jump in an MRI machine. There are big advantages to finding easier, more accessible ways to track HD progression.

The HD research field has been moving toward identifying biomarkers in biofluids, like blood and the cerebrospinal fluid (CSF) that bathes the brain and spine. The two most notable biofluid biomarkers for HD have been neurofilament light (NfL) and the huntingtin protein (HTT) itself.

NfL has been detected in both blood and CSF. NfL is released from brain cells when they die. So as HD progresses and more brain cells are lost, amounts of NfL rise. Researchers have shown that NfL is increased in people with HD up to 24 years before they even start to show clinical symptoms! This currently makes NfL our most sensitive biomarker to track HD progression.

Getting more specific

However, NfL isn’t specific for HD. It’s released from brain cells that are dying for any reason. This could make it tricky to precisely follow HD progression if there are other reasons someone might have lost brain cells, like an illness or a hard hit to the head. To specifically track HD, researchers have turned to HTT itself.

Detecting expanded HTT in blood and CSF has been difficult. Overall, expanded HTT isn’t produced by the body in large amounts, so there isn’t much there to begin with. This means ultra-sensitive techniques must be used. HTT is also inside the cell, making it hard to get to in blood. It can be accessed more easily in CSF, but that requires a lumbar puncture. Because of this, researchers are now turning to other biofluids, like tears!

It’s just something in (both my) eyes

No one prefers to get a jab in their vein or back, if other options are available. To see if biomarkers of HD progression can be obtained more easily, researchers from the Netherlands and Germany teamed up and looked at tear fluid.

To get the tears, a small strip of special paper is placed on the lower eyelid, just touching the eye. The tears are wicked onto the paper and the strip is removed after 5 minutes.

Tears contain a surprising number of proteins – close to 1,500! Biomarkers from tears are also being explored to track other diseases, like Alzheimer’s, Parkinson’s, and multiple sclerosis. Because of this, the researchers thought tears might be a good source for HD biomarkers.

They found that amounts of expanded HTT were higher in tears from people that carry the gene for HD, whether they currently had symptoms or not. While their data were quite accurate in determining if someone carried the gene for HD, this test doesn’t appear to be sensitive enough to determine years from symptom onset or distinguish those who are experiencing symptoms from those who aren’t.

A new tool for the box

Finding new and novel ways to identify biomarkers expands our toolbox and offers easier ways for people with HD to track disease progression. Using tears to look at expanded HTT means researchers now have a new tool to examine HD in a fluid that can be collected in a non-invasive way.

Researchers will continue to advance biomarkers that are easy to collect and track with HD progression very early. Having sensitive biomarkers that can be used to measure HD before someone ever shows symptoms will set us up for success when we start testing preventative treatments. When that day arrives, we’ll be ready with tears of joy.

Long repetitive sequences of C-A-G letters in the DNA code are associated with at least 12 genetic diseases, including Huntington’s disease (HD). A group of scientists in Massachusetts, USA, have recently developed a new genetic strategy to study how CAG repeats can lead to harmful proteins being made in cells, causing cells to become unhealthy. Their findings showed that expanded CAG repeats can interfere with a process called ‘splicing’, which chops up and organises genetic message molecules before they are turned into proteins.

CAG repetition

Our DNA is a genetic code that holds instructions for making thousands of different proteins, the molecular machines that run our cells. This code is made of four building blocks or ‘bases’: C, A, G, and T. DNA is arranged like a twisted ladder with two DNA strands bound together in a helix, each made of a string of bases. The bases on one DNA strand pair with bases on the opposite DNA strand to form the ‘rungs’ of the ladder.

HD is known as a ‘CAG repeat expansion disease’. Everyone has a repetitive sequence of C-A-G DNA letters in their huntingtin gene, but people who go on to develop HD have over 36 C-A-G repeats. The number of CAG repeats can increase over time, called repeat expansion, and this seems to happen mainly in cells that get the most unhealthy in HD such as brain cells.

If we can understand exactly how a longer CAG repeat itself makes cells sick, we may be able to keep brain cells healthy and delay when HD symptoms appear. There are also other diseases caused by expansions in CAG repeats, including spinocerebellar ataxias and myotonic dystrophies. Trying to find similarities between what happens in cells affected by these other diseases may help us learn more about what goes on in HD.

Cutting scenes in the genetic script

When a cell wants to make a protein coded by a certain gene, the two DNA strands unwind and separate from each other. Cellular machinery then reads the opened-up DNA base code and makes a copy of it, called an RNA message molecule, a bit like making a photocopy of a recipe from a cookery book.

However, before any RNA message molecules are read by the next set of cellular machinery to make the corresponding protein, an essential process needs to take place. Much like editing out unnecessary scenes from a film to make a final polished version, this process involves editing the RNA message to remove all of the waffly bits of genetic code copied from DNA which aren’t actually needed to make a protein. The process of going from the unedited RNA message molecule to a shorter more succinct message is called ‘splicing’. During splicing, non-essential sections of the unedited message are cut out and the important sections that remain are pasted together to produce what is known as ‘mature’ RNA. This final mature RNA product has only the necessary instructions that the cell needs to make proteins.

Expanded CAG repeats can cause genetic plot twists

In diseases caused by expanding CAGs, the CAG repeat in the DNA is copied into the RNA message, which can cause abnormal proteins to be made. In the case of HD, an extra-long version of the huntingtin protein is made. A group of scientists led by Dr Jain in Cambridge, Massachusetts, previously found that repeat-containing RNA messages, along with the proteins made from them, combine to form toxic clumps in cells which can cause serious damage.

To find out exactly how longer CAG repeats cause the production of harmful RNA and proteins, Rachel Anderson and colleagues within the Jain team recently developed a clever new method to look in detail at the precise genetic message in RNA molecules containing large CAG repeats. Interestingly, they found that CAG repeats in RNA cause mistakes to be made during splicing of that RNA message molecule. Expanded CAG repeats in RNA cause other sections of the message molecule, sometimes far away from the CAG repeat itself, to be cut and pasted into or next to the repeat during splicing.

Here, the expanded CAG repeat can act like the opening credits of a film, into which the final scenes of the film get mistakenly inserted out of order. When this happens, the plot of the film no longer makes sense. Similarly, the final RNA message doesn’t make much sense when other sections of genetic information are inserted into the CAG repeat during splicing. This leads to the creation of many different repeat-containing mature RNAs with unexpected sequences.

The researchers found that the longer the CAG repeat in the RNA message, the more faulty splicing events that occurred. This is interesting as the CAG number in HD tracks with the age at which symptoms start and the rate at which they progress. The researchers showed that when they stopped all splicing events in cells using a chemical, repeat-containing RNA messages did not form clumps in cells and so did not cause cell toxicity.

Protein production glitches

So far, these results explain how expanded CAG repeats lead to abnormal and incorrectly spliced mature RNA messages, but what happens when these messages are read to make proteins?
Any mature RNAs that are ready to be read by cellular machinery to make a protein contain a ‘start’ signal, like a green traffic light. The researchers found that sometimes when repeat-containing RNAs are incorrectly spliced, more of these start signals are found before the repeat, causing many different proteins to be made from a single RNA message than normal. The researchers altered these start signals in the CAG repeat-containing RNAs to turn them off and found that this stopped abnormal proteins from being made.

The researchers also studied the RNA messages containing CAGs that were copied from genes associated with CAG repeat expansion diseases, including spinocerebellar ataxia and myotonic dystrophy. The researchers showed that expanded CAGs copied from these genes also caused abnormal splicing into the repeat, which again contained more protein reading start signals which may cause more abnormal proteins to be made.

What does this mean for CAG repeat expansion diseases?

Understanding how important processes in cells are impacted by long CAG repeats can help researchers piece together exactly how cells become unhealthy in CAG repeat expansion diseases and point to which processes can be targeted with therapeutics. The findings from this study add another piece to the puzzle of what happens in cells, suggesting expanded CAG repeats in RNA interfere with splicing, which can lead to damaging proteins being made.

Importantly, these experiments were performed in cell types, such as kidney cells, which are easy to grow and manage in the lab but are not most affected by HD. Therefore, these cells may not accurately reflect what causes cells to become sick in HD. A lot more work is needed looking at how expanded repeats alter RNA splicing and protein production in cell and animal models of HD. Nonetheless, targeting splicing may be a potentially exciting avenue that researchers can pursue to develop medicines for HD and other repeat expansion diseases.