Clinical neurologist Dimitri Krainc sees patients and does research at Harvard-affiliated Massachusetts General Hospital in Boston. He recalls one of several similar case histories. "A teen-age patient," he related, "one of whose parents had Huntington's disease, was diagnosed as a positive. This young subject had abnormally expanded polyglutamine repeats, and he died in a car accident. The family donated the presymptomatic brain for research. Such brains are extremely rare, and hard to get. So people who have gene abnormalities but no symptoms die from some other cause."

HD's hallmark is a mutated protein in the brain's striatum called huntingtin. Full-blown disease begins when the number of polyglutamine repeats - also known as CAG repeats - exceeds 38 iterations. Normal, healthy huntingtin carries up to that 38-CAG cut-off score.

"All the cells in everybody's body," Krainc explained, "contain abnormal huntingtin genes, and the number of CAG [cytosine-adenosine-guanine] repeats can be sampled by a simple blood test. Less simple are the mechanisms by which loose-cannon, mutant huntingtin - over 38 repeats - wreaks havoc among the body's genes and proteins."

Krainc is senior author of a paper in the current Sciencexpress, released online May 2, 2002. Its title: "Interactions of mutant huntingtin with Sp1 and TAFII 130 leading to early transcriptional dysfunction in Huntington's disease."

"The most exciting finding in our article," Krainc told BioWorld Today, "is that in presymptomatic HD patients there is abnormality in gene transcription. So the people who are gene-positive but don't have any symptoms already have abnormal function of transcription factors. Those factors have to perform in a very regular manner for genes to be transcribed and expressed in an appropriate way. What we found is that huntingtin interferes with that assembly of transcription factors, by physically interacting with at least two of them - Sp1 and TAFII 130. Huntingtin is a protein that's abnormal in Huntington's disease patients. The abnormality consists of the huntingtin mutant protein physically moving into position between the two factors - in connection with its pathogenic CAG repeat process."

Three Complementary Tests Confirm Findings

"We studied the actual mechanism of those abnormalities in primary neurons and also in human and transgenic mouse brain," Krainc recounted. "The main finding is that abnormal huntingtin uncouples the activator-mediated transcription from the general transcription machinery. In practical terms," he continued, "it means that it disrupts transcription of essential genes, such as dopamine receptor genes, which are essential for the functioning of basal ganglia - the striatum. And HD directly interferes with the transcription of genes like that."

In this danse macabre there is a tango-like couple - Sp1 and TAFII 130 - on which mutant huntingtin is forever cutting in.

"Sp1 is an important activator and transcription factor of many different genes," Krainc observed. "TAFII 130 is a co-activator. Like closely coupled dancers, they have to be in sync. For example, they need to work together to transcribe dopamine2 receptor genes. If mutant huntingtin comes in between, that interaction between activator and co-activator is disrupted, along with the whole mechanism of gene transcription."

Out on the ballroom floor, enter dopamine - D2. Dopamine is a crucial neurotransmitter in the basal ganglia, located on neurons that die of HD. Dopamine receptors have been found decreased in presymptomatic and symptomatic HD patients; also in transgenic mice. So people have postulated that loss of dopamine 2 receptors is important in the pathogenesis of Huntington's disease.

"The D2 promoter is a regulatory part of the D2 receptor," Krainc pointed out, "where all the binding sites are present for transcription factor Sp1. So Sp1 and TAF would normally bind to the D2 promoter - and that binding is diminished in the HD brain. That's where the factors sit, and huntingtin comes right in between them - physically - and disrupts the promoter's function. That's the major finding of our paper," he added.

To assemble this data, he and his co-authors conducted a three-tier analysis: primary neurons, human HD brain neurons and transgenic mouse brains.

"Transgenic mice have expansions of huntingtin protein," Krainc recounted. "They are over-expressing the abnormal multirepeat mutant. Normal mice don't get Huntington's disease, but the HD mouse model carries that abnormal gene. All three tests yielded complementary results. The main result of the primary neuron assay was to analyze different parts of Sp1 and TAF 130 in huntingtin - and their interaction. The human brain studies aimed to see if those neuronal studies were happening in real-life human tissue. In human brain we found that binding of the transcription factor S21 to DNA was disrupted also, in the presence of mutant huntingtin.

"In transgenic mice," Krainc said, "we did similar experiments, but the main one was we took the cells from the striatum of those animals, studied their cells in culture, and analyzed this pathway of transcriptional dysregulation of dopamine receptors. We wanted to confirm our results in neuronal culture, in transgenic mice and in human brain - to have all the aspects of this disease. So that was the idea of using all three systems."

Therapeutic Drug Development Needs Work

Krainc said, "Identification of those mechanisms is crucial to developing drugs for clinical use in HD, to design a specific compound that will interfere with this mutant huntingtin. We are thinking of screening some molecules and peptides that potentially could reverse this abnormal finding that we see with mutant huntingtin," he said.

"I think that before thinking about drugs," he suggested, "it's important to know the details of molecular mechanisms that produce disease. For example, if we can identify the key molecular step that leads to neuronal toxicity in HD, then we can say this is the pathway. We are now asking: Can we design a specific drug, or screen a lot of drugs, against this pathway as a read-out, and see if any of the compounds will reverse the toxic effects in this disease setting?

"We are in very early stages of our drug development plans," Krainc pointed out. "We have some other collaborators - faculty members in our institute," he concluded, "who are interested in pursuing this drug discovery project with us."