The Development of Therapeutics for Huntington’s Disease

 Gillian Bates, Medical and Molecular Genetics,
GKT School of Medicine, King’s College London.

 

The mutation that causes Huntington’s disease (HD) is an increase in the number of copies of the DNA sequence ‘CAG’ which occurs close to the beginning of the HD gene. People who have up to and including 35 CAGs in their HD gene remain unaffected whereas those with 40 and above will develop HD within a normal lifespan. Repeats of 36-39 confer an increasing risk of developing the disease.

These extra CAGs result in an abnormally long stretch of glutamines (a protein building block represented by the single letter Q) in the HD protein: huntingtin. This protein is found in all cell types in all tissues and is present before birth and throughout life.

The HD mutation has pronounced effects on the brain and causes brain cells (neurons) to malfunction and to die. It forms clumps, or aggregates of the huntingtin protein in all parts of neurons throughout many regions of the brain, causes a generalised shrinkage of the brain and results in the death of neurons in specific brain regions.

The extent to which the symptoms of HD are caused by neurons malfunctioning or by neuronal cell death is not known, although malfunction is now thought to play a major part in the presentation of disease 

How does the long glutamine tract cause HD? Any proposed mechanism must be able to explain a number of properties of the disease, namely why 35 repeats do not cause HD whereas an increase of just one repeat to 36 can, why this disease has a late age of onset when the mutation is present in all cells from before birth, why longer repeats generally result in an earlier onset of the disease and why it predominantly causes brain cells to malfunction and to die.

Simple experiments have shown that as the number of glutamines increases across the threshold from the unaffected to affected range, the speed with which they change their shape and form aggregates increases dramatically. It is thought that the extra glutamines cause the beginning of huntingtin to take on a new shape and structure that can form aggregates. This misfolded or aggregated protein interacts with itself and with other proteins in ways that are detrimental to neurons.

Many research groups are now trying to understand the very first molecular interactions that go wrong in HD. One of the first events is thought to be that the beginning of huntingtin, containing the glutamine tract, becomes separated from the rest of the protein. This may be a naturally occurring event, irrespective of how many glutamines are present in the protein. However, when it has a repeat of 36 or more glutamines, this small fragment is more likely to change structure and aggregate.

The consequences of this change in structure are being uncovered. They include: alterations in the levels to which many genes in a cell are switched on, an impairment in ability of the cell to break down proteins that are non-functional or no longer needed and an impairment in the ability of the cell to generate the energy that it needs, among others. Together these could result in neuronal malfunction. Neurons do not exist in isolation but communicate with each other through a complex network.

The effects described above could result in altered inputs and outputs which would result in a breakdown in communication and may even result in the death of some cells through for example, over stimulation or under nourishment

As the early molecular steps in HD become better understood, they become therapeutic targets and we can try to find compounds (drugs) that will interrupt or to some extent redress the impairment caused by the extra glutamines. There are two approaches to doing this. The first is to try to predict which if any drugs that are already available or under development might have the required effect. The second is to generate a screening assay capable of testing up to hundreds of thousands of chemical compounds for the required activity in the hope of developing an entirely new drug.

To date, the only event for which it has been possible to set up such large screens is the change in structure and aggregation of huntingtin. There are currently a number of these screens taking place in order to find drugs that will prevent changes in huntingtin structure and/or prevent cell death.

In order to determine whether compounds arising from either of these two approaches might lead to potential drugs for HD, a wide range and variety of disease models are needed. We are fortunate in the HD research field in that long CAG repeats have been inserted into the DNA of yeast, the worm: C. elegans, the fruit fly: Drosophila melanogaster, mammalian cells grown in culture dishes and mice and that all of these organisms can be used to model aspects of the disease. The yeast and cultured cell models can be manipulated very easily and turn over very quickly allowing them to be used to screen tens of thousands of compounds for their ability to, for example, prevent aggregation 

However, any promising drugs that arise from these screens must be able to prevent the malfunction and degeneration of neurons in a whole organism and to alleviate symptoms. The C. elegans and fruit fly models are very useful for the next stage in the screening process, as they each have life cycles of around two weeks and therefore it is still possible to screen several hundred potential drugs and obtain results very quickly. Finally, compounds must be tested in mouse models, which are the most accurate models of HD that can realistically be used for drug screening.

As compounds move through this drug development pipeline, the number of drugs that can be tested at each stage decreases considerably and the cost of testing increases dramatically. The most promising drugs to emerge will be taken forward into the clinic. As clinical trials are extremely expensive and very time consuming, it is essential that only the most promising drugs go on to be tested in the clinic and that the assessment of drugs at each stage in this process is as rigorous as possible 

How do we know that these models are going to predict which drugs will work in people affected with HD? At the moment we don’t, and in reality we shall only know how good our models are when a successful drug has been found. Are there any drugs that have been shown to work in a wide range of these models? Yes, a class of drugs known as histone deacetylase (HDAC) inhibitors.

These were not found through screening large numbers of compounds but were tested because they affect the way in which genes are switched on. The extra long glutamine tract in HD causes the level at which many genes are switched on to be turned down, which is predicted to affect the way in which neurons function and communicate with each other. Although the mechanism by which this happens is not really understood, it was thought that a drug that could turn genes back up again, might have beneficial effects. So far this type of drug has been shown to be beneficial in yeast, mammalian cell, fruit fly and mouse models.  

In our lab we have shown that the HDAC inhibitor, SAHA, dramatically improves the motor performance of the R6/2 mouse model of HD. The R6/2 mice are used extensively for drug screening as symptoms can first be detected when they are 5-6 weeks old and a drug screen can be completed by 14 weeks of age. SAHA was administered to the mice from five weeks and had the effect of delaying the progression of the movement disorder by one month, i.e. mice on drug at twelve weeks of age showed the same level of impairment as mice without drug aged 8 weeks. Since this work, Bob Ferrante’s group has shown that another HDAC inhibitor: sodium butyrate also shows beneficial effects in the R6/2 mice.

Why is SAHA not being tested in HD clinical trials? SAHA is a new drug that has not yet been approved for use in the clinic. It is being tested in cancer trials in the USA, and a lot of information about its safety will come out of that work. The HDAC inhibitors are a new class of drugs and because they are of interest for the cancer field there is a lot of research going into developing better versions.  

We expect that HDAC inhibitors will be tested in HD clinical trials but it is too soon to say which is the best drug or the best approach to take. Might HDAC inhibitors cure HD? This is unlikely as they are probably only going to target one aspect of the disease. However they might slow down disease onset and progression and it is feasible that if they fulfil

their promise that they will be used in combination with other drugs that target other aspects of the disease process.  

There is currently a large and co-ordinated research effort-taking place throughout the world to identify drugs that target various stages in the progression of HD. There is every reason to be optimistic that drugs that improve aspects of HD will be identified over the course of the next few years. 

Acknowledgement: Huntington’s Disease Association (England & Wales) Newsletter issue 64 March 2004

Reprinted:         “Gateway” AHDA (NSW) Inc. Volume 7 No 3 May/June 2004

“Gateway”  Australian Huntington’s Disease Association (NSW) Inc