We were actually very surprised to find that the total amount of huntingtin protein stayed roughly the same throughout the disease, but levels of the mutant protein built up. That’s not something that has been described before; certainly not in HD, and not in peripheral blood cells.
Dr Ed Wild
Researchers from University College London, the Novartis Institutes for Biomedical Research and King's College London have used an ultra-sensitive light-based technique to measure the accumulation of the harmful protein which causes Huntington’s disease (HD).
The paper, which appeared this week in the Journal of Clinical Investigation
, describes the team’s use of Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) testing to detect mutant huntingtin in the white blood cells of patients in different stages of this neurodegenerative genetic disease. The effects of HD, which include brain atrophy, lack of muscle coordination and psychiatric symptoms, usually become apparent in adulthood. It is currently incurable and ultimately fatal.
Led by Professor Sarah Tabrizi of the UCL Institute of Neurology, the researchers found that fragments of the most toxic part of the mutant huntingtin protein are constantly building up in a patient’s body – particularly in the white blood cells – even before any symptoms show.
I spoke to Dr Ed Wild, NIHR Clinical Lecturer at the Institute and one of the co-authors of the study to find out more about the disease, clinical research into possible treatments, and how the TR-FRET test may prove very useful in the future. I began by asking just how much is known about the role played by mutant huntingtin in HD.
"It’s nearly 20 years since the huntingtin gene was discovered as the cause of Huntington’s disease, and we know with 100 per cent certainty that the gene and the protein are the absolute cause of all the problems in Huntington’s disease," Dr Wild explained. "However, even after studying the huntingtin protein for 20 years – in human tissues and the various non-human models of HD – we still know surprisingly little about what the protein does, how it behaves, and how the mutant form of the protein causes the death of brain cells."
Huntingtin is ubiquitous, and is produced in every cell of the body. It appears that, in HD patients, both the mutant and the normal protein are produced in every cell. Although it is common for HD researchers to say that the huntingtin protein is a large protein of unknown function, Dr Wild revealed that this is not quite the whole truth.
"While we certainly don’t understand its functions fully (and the more we study it the more functions we uncover), the most important functions of huntingtin seem to be related to helping the cell respond to stress, and the movement of energy-related metabolites around cells or up and down neurons.
"It’s more like a Winnebago than a Corvette; more like a campervan than a Ford Escort. It’s multi-functional and has various parts that are likely to play many roles, but huntingtin is mainly involved with structure, anchoring, signalling, shuttling and those sorts of things."
The properties and behaviour of the huntingtin protein and its mutant cousin make them extremely difficult to work with and to understand.
"It’s huge and very sticky; it’s intracellular and intranuclear," said Dr Wild. "It aggregates and is bound up with dozens of other proteins. Additionally, of course, the brain tissues that we want to study are very difficult to get at. It’s frustrating because it’s like looking through a frosted glass window; we know exactly what’s going on behind the window, but can’t quite bring it into focus."
While TR-FRET is not a brand new technique in that it has been used to study and quantify proteins before, this is the first time it has been applied in HD research. I asked Dr Wild how it is that the test can give such accurate results.
"The technique itself is accurate because it uses two antibodies, each of which binds to a different part of the huntingtin protein," he stated. "You only get the light signal if the two antibodies stick to the same molecule – they have to be that
close together. This is why it gives such a high degree of sensitivity.
"If you have decent, specific antibodies and good light-emitting and light-detecting add-ons to those antibodies in the form of fluorophores, you get a very accurate reading. A very specific and sensitive signal can be achieved because this doesn’t rely on enzyme activity or other ways of detecting the antibody – you simply shine the light and light comes back at you.
"The other advantage of the FRET technique is that different antibody pairs can be used to study different forms of the protein. This was how we discovered the build up of fragments of the protein in cells. We got a signal that looked like the total amount of mutant huntingtin was increasing. Based on the antibody pairs, however, it seems it is the very beginning of the protein – the first page, if you like, in the book of the protein – that is actually building up. That’s interesting because we know that this is the fragment which contains the mutation as well as the fragment that is poisonous to cells."
Dr Wild recalled that the team embarked on the project in the hope it would be possible to detect the mutant protein in blood cells, and that this in turn might reveal something about the way in which the protein causes damage. HD researchers have been occupied by trying to find out whether the neurodegenerative effects of the disease are due to a constant level of damage or whether there is a build up of harm within the body over time.
"We were actually very surprised to find that the total amount of huntingtin protein stayed roughly the same throughout the disease, but levels of the mutant protein built up. That’s not something that has been described before; certainly not in HD, and not in peripheral blood cells."
Aggregated ‘clumps’ of the protein are observed in the brain cells of post-mortem HD patients, but it is the soluble form of the protein on which the team has focused their efforts. Many believe the soluble protein has the potential to be even more harmful than the clumped form. Dr Wild explained that the way the new findings relate to previous work on the topic could answer some questions.
"A couple of years ago we looked at cytokines – inflammatory signalling molecules produced by white blood cells – and found that the amount of cytokines increases as the disease progresses. We had already found this progressive signature in blood cells, but we didn’t know what was causing it or what it might mean.
"Finding that, as the disease goes on, you get a build up of fragments of the mutant protein in exactly the same cells tells us, I think, one possible mechanism by which this progressive abnormality of white blood cells might be occurring. It may be that the mutant protein is building up in the white blood cells and that is causing them to produce these inflammatory cytokines. That may in turn be doing harm or altering the way the brain handles the fact that it has this disease."
The research may have implications for the development and testing of potentially life-changing treatments for Huntington’s patients.
"The most ground-breaking finding to come from this study is that the amount of mutant huntingtin in the white blood cells directly and independently mirrors the rate of brain shrinkage," Dr Wild said. "That has never been seen in any neurodegenerative illness before. It gives us an indication that we’re not just looking at something that is interesting; it is something potentially important and useful.
"For me the most exciting possibility is that we’re on the brink of an extremely exciting time in Huntington’s where gene silencing or huntingtin-lowering drugs will be entering clinical trials in human patients in the next year or two."
The TR-FRET test itself may have a significant contribution to make to future HD research and monitoring. While some of the drugs Dr Wild mentioned will be administered directly into the brain, others will be taken as treatments affecting the whole body, to help cells get rid of the harmful protein. In each case, it will be essential to have analytical techniques to determine the effectiveness of the drug.
"As I said, we can’t take samples of brain tissue for analysis. If you’re taking a pill that would be expected to affect the whole body, it would be of value to be able to directly measure the huntingtin level in the blood. This technique enables you to do that. If an injection into the brain is involved, the technique can be used to measure whether there’s any overflow into the rest of the body."