DURHAM, N.C. -- As reported in the March issue of Nature Medicine, Duke
University Medical Center researchers have found a possible key to brain
disorders such as Huntington's disease, as well as discovering a new concept
of how mutant genes may produce disease.
The scientists discovered that these "triplet repeat" disease
genes produce proteins that errantly stick to an enzyme crucial to the production
of energy in brain cells. They believe that because this enzyme cannot,
therefore, do its job, brain tissue will malfunction and may, over years,
die. This would result in fatal physical and intellectual deterioration,
such as that seen in Huntington's disease patients.
Furthermore, their findings may answer why the number of genetic repeats
in these diseases determines how severe symptoms will be: More repeats produce
a protein that holds this "energy" enzyme more tightly, producing
widespread nerve cell failure in the brain.
The work offers biomedical researchers a new way to understand, and to potentially
treat these genetic diseases, the Duke scientists say.
"This is the first explanation of what these proteins produced by triplet
repeat genes may be doing in nerve cells," said neurologist and principal
investigator Dr. Warren Strittmatter. "It provides a new paradigm for
inherited neurologic disease, and that is the repeat region of these proteins
binds other proteins critical to glucose and oxygen metabolism in the brain.
And the larger the repeat region, the tighter they bind."
Authors of the study are from the same team that discovered the major gene
linked to Alzheimer's disease. In many ways, "this finding may be more
pivotal than our earlier work," said Dr. Allen Roses, scientific director
of the Deane Laboratory at Duke, where the experiments were conducted. "We
are beginning to cross the difficult area between gene discovery and abnormal
functions of the brain, and to open the door to target drugs," he said.
The primary researchers involved in the discovery are Duke neurologists
Strittmatter, James Burke and Jeffery Vance. Other collaborators include
Jan Enghild and Margaret Martin, both from Duke, and geneticists Yuh-Shan
Jou and Richard Myers from Stanford University School of Medicine.
Abnormal "triplet repeat" genes constitute the molecular equivalent
of stuttering. They repeat the same sequence of genetic units, called nucleotides,
over and over far more than do normal genes. While a normal gene might repeat
the three nucleotides cytosine, adenine and guanine (CAG) fewer than 30
times, genes that produce Huntington's disease and other such brain disorders
might repeat the sequence between 40 and 100 times. As the repetition grows,
the disease becomes more severe.
The Duke study explains why the number of nucleotide repeats in such diseases
relates to the severity of symptoms, the researchers say. The nucleotide
sequence CAG determines that the amino acid glutamine will be inserted into
the protein during its translation from genetic information to protein in
the brain cell. The greater the number of CAG repeats in the gene, the more
glutamines will be added, resulting in a structurally different protein
that binds strongly to a particular energy enzyme. As these crucial enzymes
become disabled, increased numbers of neurons malfunction and may die, destroying
brain tissue.
The researchers have found this effect in two neurological diseases -- Huntington's
and Haw River Syndrome. They believe the effect also works for at least
three other rare neurological disorders -- Kennedy's syndrome, Machado-Joseph
disease, and Spinocerebellar atrophy I -- that have the same expanded CAG
repeat.
"Traditional dogma is that mutant genes produce proteins that either
don't work at all or don't work well," said Burke, the first author.
"However, we found the proteins produced by these genes interact in
a different way, based on the number of repeated amino acids, producing
devastating consequences."
"The novel insight is that this extra domain produced by triple repeats
acts in the same way across all these diseases," Vance said. "Even
though we don't know what the normal function is of each of the disease
genes, the triple repeats are causing all the proteins to bind to this single
enzyme. Scientists have usually only thought about how a gene mutation produces
a change in that protein that results in a disease. This is an observation
that provides a mechanism for a group of diseases."
If drugs could block the protein interaction the researchers described,
then the discovery may offer treatment options, according to Roses.
The investigators became interested in the action of repeated gene sequences
in brain tissue two years ago, when they discovered Haw River Syndrome (also
known as DRPLA), an inherited neurological disorder that affects residents
of a rural North Carolina community.
The scientists found that the syndrome was like Huntington's disease in
that it was attributed to genes that acquire excess CAG nucleotide sequences.
Although the scientists do not know what the roles of the proteins produced
by Haw River or any of the triplet repeat diseases are, "Our hypothesis
is that these different proteins are all doing something similar, which
is binding to the same protein," Vance said. In other words, the abnormal
repeat in the DNA is adding a "docking area" domain in the protein,
which allowed substances that bind loosely to the normal protein to stick
more tightly to the enlarged disease protein.
To test that theory, the Duke team made artificial strings of glutamines,
either 20 glutamines, found in normal proteins, or 60 glutamines, found
in the mutant proteins. They then mixed these glutamine strings with brain
proteins to see what proteins bound. They found that one brain protein,
glyceraldehyde-3 phosphate dehydrogenase (GAPDH), bound to the long glutamine
string better than to the short glutamine string. GAPDH, found in all cells,
is known to produce energy, along with a number of other important functions.
In a second experiment, brain proteins were incubated with GAPDH. Huntingtin
<cq> protein and DRPLA protein bound to GAPDH, while other brain proteins
did not.
While he can hypothesize that binding of GAPDH may start the pathway of
errant protein interactions, Strittmatter said he can't yet define the cascade
of events that produces the disorders. "The brain is entirely dependent
on glucose metabolism, and this is a protein that is important in that pathway,"
he said. "But because GAPDH is expressed in every cell, not just in
brain cells, there has to be another level of complexity that we don't understand."
"This is at least an initial insight into the molecular genesis of
these diseases," Burke said. "It is a novel interaction that may
explain the increase in severity with increasing repeat size."
The researchers point out that their theory of triplet repeat diseases would
not apply to those that contain stuttering sequences in the "junk"
area of the genome, since those areas do not encode for protein production.
The genetic disease called myotonic dystrophy is an example of such a disorder.
The interdisciplinary research under Roses was a key to the discovery, Vance
said. "We are a group of clinicians, geneticists and molecular biologists.
Only by working across disciplines could we have come up with such an understanding,"
he said.
Additional funding or support for the study came from the Joseph and Kathleen
Bryan Alzheimer's Disease Research Center at Duke, the Duke Center for Human
Genetics, and the National Institutes of Health (specifically the National
Institute on Aging, and the National Institute of Neurological Disorders
and Stroke).