After linkage of the Huntington disease (HD) gene was found in 1983, it took ten years of work by an international group to identify the mutation in the gene interesting transcript 15 (IT15) that causes the disease.
HD is an autosomal dominant inherited neurodegenerative disease that becomes manifest in midlife and causes progressive motor, psychiatric, and cognitive dysfunction.
It is invariably terminal.
HD symptoms can begin as early as 2 years or as late as 90 years, although the average age of onset is in the late 30s and early 40s.
If a child inherits the gene from his or her father, a phenomenon called anticipation frequently occurs, whereby the child's age of onset is lower than the father's.
The IT15 gene is composed of 67 exons and encodes a protein of 3,144 amino acids, called huntingtin.
Exon 1 contains a CAG trinucleotide repeat that encodes the amino acid glutamine, followed by another repeat that encodes proline.
In unaffected individuals, there are 10-34 CAG repeats.
In those affected by HD, there are more than 40 repeats.
In those with 35-39 repeats, the disease is variably penetrant.
The age of onset of the disease varies inversely with the number of CAG repeats.
Individuals with juvenile onset usually have over 55 repeats, and they usually inherit the gene from their father.
Men occasionally have expanded repeats in their sperm.
The expansion is thought to occur via slippage during the DNA replication process.
Huntingtin contains a few domains that suggest particular functions, including WW domains and caspase cleavage sites, but the function of the protein remains unknown.
Huntingtin is expressed in the cytoplasm of most cells in the body.
In the brain, expression is found predominantly in neurons.
Within the cell, the protein is associated with the endoplasmic reticulum, the microtubules, and organelles such as the mitochondria and synaptic vesicles.
The association of huntingtin with these cellular components is loose, since during the process of differential centrifugation huntingtin can be separated from them.
The protein contains several caspase cleavage sites.
Huntingtin binds to a number of proteins, including Hap-1, α-adaptin, and several others.
Additional studies found that huntingtin binds to GAPDH.
Huntingtin was also recently shown to associate with PSD95, a protein found at the postsynaptic membrane and involved in anchoring of receptor proteins, particularly the N-methyl-D-aspartate (NMDA) receptor.
The huntingtin protein is necessary for normal development in mice, as knockouts do not survive beyond day 7-8 of embryogenesis.
Conditional knockouts, in which the gene is turned off during adulthood, develop a neurodegenerative disease.
Recently, cell models of huntingtin expression revealed that the wild type protein partially protects cells from noxious stimuli or from mutant huntingtin.
Huntingtin may also be important for cell survival through mechanisms such as growth factor stimulation, as BDNF rescued cells expressing mutant huntingtin.
Removal of the caspase sites in the protein is beneficial for cells, and wild-type huntingtin protects against mutant induced cell death.
Mutant huntingtin in transgenic mice
There are no antibodies that can distinguish mutant from wild-type protein, although there are antibodies that bind selectively to the polyglutamine repeat.
Presumed mutant huntingtin is found not only in the cytoplasm but also in the nucleus, where it forms aggregates (or neuronal intranuclear inclusions [NIIs]).
Aggregates also develop in neurites.
The aggregates are ubiquitinated, although antibodies against huntingtin appear to stain more aggregates than do antibodies against ubiquitin.
Analysis of HD brain tissue shows full-length huntingtin protein in the nuclear fraction as well as abundant immunopositive bands at lower molecular weight, suggesting proteolytic products in the nucleus.
In contrast, in control brains there was full-length protein in the total homogenate but no nuclear protein and few huntingtin fragments in any fraction.
Studies of HD brains show that there are more inclusions in the cortex than in the striatum and that cortical and striatal neurites contain numerous aggregates.
Postmortem studies of HD brains also show differential loss of projection neurons containing enkephalin, adenosine A2a, and dopamine D2 receptors compared with cells containing substance P, dynorphin, and dopamine D1 receptors.
In juvenile HD, both types of striatal projection neurons are equally affected.
In the cortex, neurons in the deeper layers (layers V and VI), which use the neurotransmitter glutamate, develop nuclear and neurite aggregates.
Aggregates in HD were first observed in an electro microscopy study of in vivo biopsies of HD brains.
This observation was not pursued at the time, but it was remembered in 1996 when studies of the first HD transgenic mouse (expressing the first exon of human huntingtin driven by the huntingtin promoter) were reported.
These mice develop normally until around 5 weeks of age, when they begin to lose weight and to perform less well on the Rotorod test.
Both brain weight and body weight diminish subsequently.
The animals develop diabetes and tremors and become less active.
They are finally moribund and die at around 13 weeks.
Extensive early studies of the brains of these animals showed no clear neurochemical abnormalities like those seen in postmortem HD brains.
Electro microscopy studies, however, showed intranuclear inclusions.
In the transgenic mice, it then became obvious from immunocytochemical studies that these NIIs were positive for the HD protein and for ubiquitin.
Furthermore, virtually all neurons in the brains of these so-called R6/2 mice contained NIIs.
These studies led scientists to revisit the examination of human brains in which NIIs were also found.
The frequency of NIIs in human HD brains was lower than in the transgenic mice, and the aggregates appeared as described above.
The formation of aggregates was subsequently thought to be the sine qua non of HD pathogenesis.
Two papers then appeared that suggested that the aggregates were an epiphenomenon, since cell death did not necessarily result from neuronal huntingtin aggregation, yet cell death did arise after the expression of mutant huntingtin.
It appeared that, to be toxic, the mutant protein had to get into the nucleus, since constructs with nuclear-export signals attenuated death resulting from exon 1 over expression.
Furthermore, inhibition of caspases rescued cells from death.
Examination of signaling proteins in the brains of R6 animals showed changes consistent with an alteration in gene transcription.
Genes encoding proteins, such as receptors, that had previously been found to change in HD were expressed at lower levels, in a pattern consistent with the human studies.
The analysis of the brains of R6 mice, interrogating over 6,500 mouse genes, revealed changes in expression levels mainly of genes involved in signaling pathways.
R6 mice have been treated with a variety of agents, including free radical scavengers, glutamate antagonists, creatine, and caspase inhibitors, and these are seen to prolong life by about 10-20%.
Indeed, even environmental enrichment prolongs life by 20-50%.
R6/2 mice have been crossed with dominant negative IL-1-converting enzyme (ICE) knockdowns, and offspring live longer than controls.
Intraventricular infusions of caspase inhibitors also prolong life, as does minocycline (an ICE inhibitor).
Electrophysiological studies of cells in brain slices revealed that, early in the disease, there are enhanced NMDA receptor responses, but eventually cells become resistant to NMDA agonists.
Conditional exon 1 transgenic mice show behavioral abnormalities, brain atrophy, NIIs, striatal gliosis, and reduction in dopamine D1 receptors when the transgene is expressed, and a reversal of these changes when the transgene is turned off in adulthood.
Conditional knockouts show a progressive neurodegenerative disorder when the wild-type mouse gene is turned off in adulthood.
Other whole-organism models
Worm, fruit fly, and yeast models of HD have been created primarily by transgene approaches using glutamine-encoding trinucleotide expansions.
These models have allowed the identification of suppressors and enhancers of expanded polyglutamine-induced pathology.
Studies of such compounds are now underway in several transgenic animals.
Cell culture models are also available.
In 1996, the Huntington Study Group was formed.
It is a multi-institutional organization of sites involved in collaborative studies of individuals with HD.
Clinical trials of a variety of putative neuroprotective agents have been carried out.
These include trials of the glutamate antagonists' lamotrigine, remacemide, and riluzole.
Riluzole and remacemide improved motor function, but lamotrigine had no effect.
Vitamin E, a free radical scavenger, was not an effective treatment; neither was idebenone, a booster of energy metabolism.
Recently, a two-by-two factorial study of coenzyme Q10 and remacemide showed a 13% slowing of progression with coenzyme Q10 and an improvement in motor function with remacemide, although the results were not statistically significant.
Studies of riluzole, creatine, and minocycline in HD patients are now underway.
As clinicians look toward treating individuals in the presymptomatic or early symptomatic phases of the disease, biomarkers or other characteristics of disease onset and progression need to be defined.
HD has received at great deal of attention in the field of neuroscience as a model of neurodegeneration.
Because it is relatively common and presymptomatic individuals can be identified, investigators have focused on the disease in the hope of finding therapies that can be given to gene carriers presymptomatically to prevent disease development.
The results to date suggest that huntingtin is necessary for developing and sustaining normal brain function.
In HD, the protein with the expanded polyQ may not function as effectively as the wild-type protein, and this may put neurons (especially in the striatum) under stress.
Striatal neurons then become vulnerable to the abundant glutamatergic input from the cerebral cortex.
Excitotoxicity, mitochondrial stress, and free radicals increase, and caspases within the cell are activated.
Mutant huntingtin is then cleaved, resulting in polyQ-containing fragments that are susceptible to aggregate formation.
The mutant fragments and aggregates recruit transcriptional factors vital to the normal function of neurons.
Cells survive in a dysfunctional state for some time, and it appears that turning off the mutant gene can result in reversal of neurodegeneration.
As many steps in the process of functional decline and cell death represent potential drug targets, scientists should eventually find a cure.