Overview of HDSA Research - Christopher Ross, M.D.

Scientists are working on the problem of HD by using a variety of models from cells to to worms to mice to people. HD is a difficult problem to solve but fortunately researchers now have a tremendously powerful set of tools.

HD research has a broader implication because HD is a prototypical disease for:

• Triplet polyglutamine repeat disorders
• Neurodegenerative disorders
• Other diseases with cell death
• Genetic disorders.

Dr. Ross discussed the background of the Human Genome Project and it's connection to HD. Although the HD gene was discovered in 1993, there is more to learn. The Human Genome Project will

• Accelerate the discovery of other genes which modify the effect of the HD gene.
• Acclelerate the discovery of how the HD mutation could alter the transcription of other genes
• Provide the sequence of all the genes which codes for all the proteins within cells.
• Accelerate our understanding of HD, since some of these proteins are involved in HD cell toxicity.

One of the findings that have emerged from the human genome project is that our chromosomes are very close to other organisms. All life is close, thus it is useful to study lower organisms.

He urged advocacy on the area of stem cell research. This may prove to be very important to treating HD. SB 723 would counteract some of the restrictions that have been imposed. There is also presidential action that can further limit or further expand the use of this research.

He explained that "everyone has two copies of the huntingtin gene which makes the huntingtin protein. The normal function of the huntingtin protein may involve basic cellular processes and contribute to neuronal survival. The mutation may disrupt the normal function of huntingtin but probably more importantly, gives it novel toxic properties that lead to neuronal dysfunction and death."

Huntingtin normally has a DNA triplet repeat. It includes a stretch of amino acids that code for glutamine (CAGCAGCAG….) "This CAG repeat is expanded from the normal range of between 10 and 30 repeats coding from 10 to 30 glutamines to 36 or more glutamines. Thus HD is also known as a polyglutamine repeat expansion disease."

"The genetic mutation is dominantly inherited, so each child of a parent who has HD has a 50 percent chance of inheriting the defective gene."

We know a lot about what goes wrong in HD but we don't know what the first trigger is or how these problems interact with each other. The approach is to try to intervene at all of the points along the pathway because we may not even find what the 'first' event is that leads toward cell death.

We now have several different mouse models that replicate many different features of the disease. They are a powerful tool for understanding the disease and looking for therapy

We can also now grow cells in a dish and 'transfect' them -- introduce the mutated gene. In these kinds of experiments you can look at cell death. Introducing the mutated gene causes cell toxicity whereas introducing the normal gene does not. We can reproduce cell death in just a matter of days, which is a very powerful tool.

A major finding has come from cell research that Dr. Ross and his colleagues have done. Mutant HD interacts with CBP. However, when we coexpress a normal CBP which has NOT been interfered with, it rescues the cell. This won't lead directly to therapy but the understanding that has been gained will.

Normally the huntingtin protein has some key function in the cell but the mutation causes it to have an abnormal structure which makes a number of abnormal things happen in the cell, fragmentation, caspace activation, interaction with chaperones, aggregations in various parts of the cell, inclusion bodies in the nucleus, and the alteration of the transcription of other genes.

The better we understand the exact molecules that are involved in the pathway, the better we can design therapies. It's likely that the pathway is not linear but rather that there are multiple pathways. Probably there will be a cocktail of medications developed, several different drugs each targeted at a different part of the pathogenic process.

The drug discovery pipeline: We start with ideas, we use high throughput screening techniques, validation (different models, then the mouse), preclinical trials, clinical trials. We want to speed up the intermediate steps.

They also hope to discover something like insulin has been for diabetes, something that will profoundly delay onset and slow progression and change lives.

Rapid, automatic screening of compounds is being done without necessarily having a detailed understanding of what is going on. Researchers can take a library of compounds and just run them through testing.

Dr. Ross described the HDSA Therapeutics Initiative whose goal is to accelerate the path from basic research to clinical trials. Stage One is to refine laboratory models for screening. Stage Two is to screen many compounds for beneficial effects. Stage Three is to test compounds in mouse models.

There are two approaches to screening compounds and both are valuable. One is to "develop and screen a specific molecular target based on a detailed understanding of the mechanism of the disease. This yields compounds whose effects are well understood."

Another approach is to "develop a cell or animal model and screen for beneficial effects. This yields compounds whose effects are less well understood - but it may be more rapid."

The HDSA Therapeutics Initiative has already made good progress:

• Model development within the coalition
• Addition novel projects funded
• Partnership with NINDS to fund high throughput drug screening proposals for HD and other neurodegenerative disorders.
• Applications under review by NIMDS and HDSA

In the last five years researches began to understand the disease process based on the discovery of the HD gene. Cell and animal models were developed. In the next five years the focus will be on identifying target areas for therapies and testing them.

The research goals are to

• Find treatments which will have measurable effects to slow progression.
• Find treatments which will have measurable effect to delay onset
• Modify and improve effective treatments, using basic science.

Alterations in Gene Transcription in HD - Leslie Thompson, Ph.D.

Mutant huntingtin's protein alters normal transcription within the brain. Traditionally scientists have examined changes in protein levels in HD. Her work is targeted upward at the signals that direct proteins to be made. The huntingtin's protein is a large protein with a large polyglutamine stretch in the amino termiinus. Michael Hayden and others have been working on understanding how this stretch fragments and the damage that this causes.

Normally huntingtin's is found in the cytosol but mutant huntingtin's finds its way into the nucleus of the cell after fragmentation. In the nucleus there are a variety of proteins which act upon DNA transcription by forming a complex in a specifc, regulated way. The levels of each of these proteins are critical. They will bind to a complex of DNA and dictate how the DNA will express itself. The enzymes will 'read' a section of the DNA and form an RNA molecule.

We know that CBP, P53 as well as many transciption factors can bind with the mutant huntingtin's protein, interfering with its function. Further, within aggregates, you find the transcription factors sequestered. So there's less of these transcription factors both functionally and physically. This means that there's less RNA being made and less protein being made.

Gene expression profiling experiments are a very important advance. You can profile thousands of genes at one time and come up with ideas and theories that you might never have thought of.

The advantages of the new gene profiling techniques are:

• Accelerate HD research - thousands of genes can be tested at once
• Detection changes in gene expressed in only a few copies per cell
• High sensitivity
• Finding pathways for drug discovery previously uncharacterized

For example, by profiling the expression of over 6,000 genes in 12 week old HD mice as compared to normal mice, they found that inflammation of the brain was up, neurotransmitters and receptors were down as were nuclear hormone receptors and secondary messenger systems.

Dr. Thompson and her colleagues are studying drosophilia in their lab. Drosophila goes through a characterized life cycle. The model that they use expresses a polyglutamine repeat which causes extreme toxicity. The flies die during larval development. They feed the flies a variety of compounds to see how many more make it to adulthood. A couple of the compounds that they have tested have rescued neurons. Minocycline and creatine helps the flies and they'd discovered a third compound that rescues cell from the CBP binding so this is very promising..

Transcription is an early event so this is a promising line of research for treatment.

- published July 1, 2001
by Marsha Miller, Ph.D.