Ataxia Discoveries Open Window to Neurodegeneration

For the most part, modern medicine is no match for neurodegenerative diseases. But with advances in the study of genetics, the ability of scientists to get a molecular "handle" on such mysterious malfunctions promises to change all that. And perhaps the most useful such handle yet found is the phenomenon called trinucleotide repeat expansion. This occurs when any three of the four nucleotide subunits in DNA material begin to excessively repeat their adjacent appearance in a molecule. For example, in normal nucleic acid molecules, the adjacent occurrence of cytosine, adenosine, and guanine (CAG) may repeat itself up to 40 times before being interrupted by another combination. But in recent years, researchers have observed CAG tracts of up to several hundred repeats in DNA of the victims of certain diseases. Since the CAG tract codes for glutamine, such ailments have become known as polyglutamine disorders. At left, a slice from a cerebellum of an SCA7 patient who died of disease is shown on top. The lower section is from a comparable level of a cerebellum from an age-matched control who died from other causes. The SCA7 patient's cerebellum shows significant atrophy, characteristic of this disease. Above is an H&E (hematoxylin and eosin) stained picture of a slice from an SCA7 patient's cerebellum. The cellular granule cell layers (dark cellular portions) are outlined by the less cellular molecular layer. Large Purkinje cells normally populate the zone between these two layers but are almost completely absent in this patient's cerebellum. This reflects end-stage disease in a patient who is bedbound from severe late-stage SCA7. To date, eight neurodegenerative diseases have been shown to be caused by CAG expanded repeats located within specific genes. They include: Huntington's disease, dentatorrubral-pallidolysian atrophy, spinal-bulbar muscular atrophy, and five types of spinocerebellar ataxia (SCA) characterized by the inability to voluntarily control muscles. According to the Minneapolis-based, nonprofit National Ataxia Foundation, about one in 150,000 people in the United States suffers from ataxia that is hereditary or sporadic (not known to be familial). SCAs represent only one category of these diverse disorders, but they are subjects of particularly intense research nowadays. Dominantly inherited, with expansion transmitted more frequently through the father than the mother, they are much easier to map than more complex neurodegenerative disorders. Although their disease mechanisms are not yet understood, mapping of the SCA expanded repeats and positional cloning of specific mutations are accelerating as the Human Genome Project progresses. Along with growth in knowledge about SCAs comes the possibility of developing drugs to treat them and other disorders known or thought to be similarly caused, including dementias. The five SCAs that have been shown to result from CAG expansions are referred to as types 1, 2, 3, 6, and 7. At least three other types have been mapped, and researchers expect to discover still more SCAs, based on the indications of their clinical and laboratory work with families in which ataxia occurs. Efforts to pinpoint the genes responsible for SCAs 4, 5, and 10 are continuing, and researchers have not yet confirmed that those types are caused by triplet repeat expansions. The situation involving SCA9 is more mysterious. No paper has been published, and information about SCA9 is scant in the wider neurological community. CAG is the most common repeat expansion known to cause disease, but not the only one. The recessively inherited Friedreich's ataxia is caused by a GAA expansion. And CTG expansions (which include the fourth DNA nucleotide, thymine) are responsible for both myotonic dystrophy and SCA8. This month, the latter disease was shown to be the first dominantly inherited SCA arising from a repeat expansion other than CAG.1 Among the inherited ataxias (as opposed to those acquired through such nongenetic causes as alcoholism or tumors), SCAs are particularly difficult to classify clinically. Generally characterized by incoordination, the more specific signs in this heterogenous group, such as uncontrolled eye movements or speech difficulties, also often overlap from one disease to another. SCAs usually manifest in the third to fifth decades of life. There is a correlation between length of the repeat expansion, earlier age of onset, and increased severity of these diseases down through generations. Researchers studying families with histories of as-yet-unclassified inherited ataxias know that this correlation, called "anticipation," indicates that trinucleotide repeat expansion may be the culprit. Louis J. Ptacek Fundamental discoveries in this area were made in the 1980s and early 1990s by researchers studying fragile X syndrome, which is caused by a CGG expansion and is the most common form of inherited mental retardation.2,3 "It has blown open the field," declares Louis J. Ptacek, a neurology professor at the University of Utah. In 1996, Ptacek and colleagues mapped the SCA4 gene,4 and they now are "working very hard" to identify its exact position. "I'm not entirely convinced it has anticipation," he says, explaining that the clinical signs are not clear. His team therefore "won't put all our eggs in the trinucleotide repeat basket." Ptacek notes that it is still unknown why smaller, stable numbers of repeats cause no problems, while larger, unstable or varying numbers of repeats do. In most SCAs, the expanded repeats instruct the mutant gene to make a protein that is larger and heavier than normal. This gives it a neurotoxic "gain of function" that results in aggregations of polyglutamine in the neuron, called intranuclear inclusions. That condition has been considered a possible cause of such complex neurodegenerative diseases as Alzheimer's and Parkinson's. But a recent paper suggests that, in Huntington's disease, intranuclear inclusions may be a failed attempt by the cell to defend itself from death induced by the mutant gene.5 "Inclusions don't appear to be necessary for disease but they may be related to the rate of disease progression," Ptacek comments. Aside from their attempts to clone SCA4, Ptacek and colleagues currently are characterizing the biochemistry and cell biology of the SCA7 protein. In 1996, the team lost a race with a French group to publish the positional cloning of the SCA7 gene,6 a disease significantly different clinically from others in the group, in that it can cause blindness. "We think we have some important clues to the actual pathway of degeneration in SCA7," he reveals. Stefan-M. Pulst, neurology division director at Cedars-Sinai Medical Center and professor of medicine at the University of California, Los Angeles, and colleagues are "very actively" attempting to clone the SCA10 gene that they recently mapped.7 Pulst says because neurons of SCA10 victims are predisposed to fire abnormally, thus causing epileptic seizures, an increased understanding of this disease could reveal more about the thresholds of epileptic seizures, even though few forms of that disorder have yet been attributed to genetic defects. "I think a lot of research, even clinical research, is driven by these molecular studies," he comments. His team also mapped and cloned the SCA2 gene.8 They since have generated transgenic mice into which the human SCA2 gene has been inserted. These mice develop changes similar to human SCA2. Publication of those results is pending, and Pulst thinks that such SCA animal models eventually might lead to development of pharmaceutical interventions not only for ataxias but also for epilepsies, and perhaps for dementias. Already, introduction of the SCA6 gene has caused epileptic seizures in mice (see "Ataxia," Hot Papers). A collaborator in the identification of that gene, S.H. Subramony, vice chairman of neurology at the University of Mississippi Medical Center, notes, "Of all the CAG repeat diseases, [SCA6] is the one disorder that involved a protein that was already known. That is the calcium channel protein." Channel proteins control the movement of sodium, potassium, and calcium ions across membranes that enclose the proteins. "These ions are involved with excitability of the nervous system," Subramony explains. "With multiple examples of diseases that come from slightly different causes, we're likely to learn more about the mechanisms from each cause," observes Laura P. Ranum, assistant professor of neurology in the University of Minnesota's Institute of Human Genetics. Research groups at her university and at Baylor University collaborated in the positional cloning of the first SCA gene. 9,11 Ranum was lead author of a 1994 paper that announced the mapping of SCA5.10 In a dogged piece of detective work, she traced that disease to the grandparents of Abraham Lincoln. "Everyone knew their relationship to President Lincoln, but they didn't know their relationship to each other, or how much ataxia was in the family," she recalls. "So it was easier to work the family up, because of the Lincoln connection." Positional cloning of SCA5 has proved difficult, in part because of the large size of the region. The conventional method, of identifying candidate genes in a region that are known to code for proteins that could cause neurodegenerative disease, failed to produce the desired results. This prompted Ranum and colleagues to try something unconventional. National Ataxia Foundation 2600 Fernbrook Lane, Suite 119Minneapolis, MN 55447(612) 553-0020 Fax: (612) 553-0167 www.ataxia.org Taking DNA samples from patients with genetically undefined forms of dominantly inherited ataxias, they screened them for CAG repeats. A mother and daughter showed CAG expansions that analysis determined were not among those known to cause already-characterized SCAs. They then cloned a DNA fragment containing the expansion, and sequenced the genes in that clone, determining that the expansion was unlikely to be translated into a polyglutamine tract. Further screening of other families identified more ataxia patients with this expansion. Finally, analysis of cerebellar RNA from those patients detected an RNA transcribed through the repeat, but in the CTG orientation rather than CAG. They named the new disease SCA8. "We weren't looking for what we found," Ranum admits, "but I don't think we would have found it if we hadn't taken a different approach." Because the SCA8 repeat does not occur in protein-coding DNA, the pathology of disease is likely to be quite different from that of polyglutamine disorders. "Mechanistically, we don't know what the CTG expansion is doing," Ranum says, adding that molecularly, the disease resembles myotonic dystrophy, which also is caused by expansion of an untranslated CTG repeat. But clinically, SCA8 is "a run-of-the-mill ataxia." TEAM MINNESOTA: Standing are (L-R) Lawrence Schut, Kellie Benzow, and John Day. Seated are Laura Ranum, Melinda Mosely-Alldredge, and Michael Koob. Her colleague, University of Minnesota assistant professor of neurology Michael D. Koob, assembled evidence that SCA8 could be transcribed within a noncoding RNA. He raises the possibility that its mechanism of action is as an antisense gene, regulating transcription on the other strand. "It's possibly a decrease in the function of the sense gene, or of another gene right in the region," he suggests. "But even with the polyglutamine diseases, the mechanisms are not understood at all ... . From the research point of view, it's a wide open field." Steve Bunk is a contributing editor for The Scientist. M.D. Koob et al., "An untranslated CTG expansion causes a novel form of spinocerebellar ataxia (SCA8)," Nature Genetics, 21:379-84, April 1999. S.L. Sherman et al., "The marker (X) syndrome: a cytogenetic and genetic analysis," Annals of Human Genetics, 48[Pt 1]:21-37, 1984. Y.H. Fu et al., "Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox," Cell, 67:1047-58, 1991. K. Flanigan et al., "Autosomal dominant spinocerebellar ataxia with sensory axonal neuropathy (SCA4): clinical description and genetic localization to chromosome 16q22.1," American Journal of Human Genetics, 59:392-9, 1996. F. Saudou et al., "Huntingtin acts in the nucleus to induce apoptosis, but death does not correlate with the formation of intranuclear inclusions," Cell, 95:55-66, Oct. 2, 1998. G. David et al., "The gene for autosomal dominant cerebellar ataxia type II is located in a 5-cM region in 3p12-p13:genetic and physical mapping of the SCA7 locus," American Journal of Human Genetics, 59:1328-36, 1996. L. Zu et al., "Mapping of a new autosomal dominant spinocerebellar ataxia to chromosome 22," American Journal of Human Genetics, 64:594-9, February 1999. A. Nechiporuk et al., "Genetic mapping of the spinocerebellar ataxia type 2 gene on human chromosome 12," Neurology, 46:1731-5, 1996. H.T. Orr et al., "Expansion of an unstable trinucleotide CAG repeat in spinocerebellar ataxia type 1," Nature Genetics, 4:221-6, 1993. L.P. Ranum et al., "Spinocerebellar ataxia type 5 in a family descended from the grandparents of President Lincoln maps to chromosome 11," Nature Genetics, 8:280-4, 1994.

Ataxia Discoveries Open Window to Neurodegeneration

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