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Spinocerebellar Ataxias
Neurobiology of Disease 7, 523–527 (2000) doi:10.1006/nbdi.2000.0346, available online at http://www.idealibrary.com on
Spinocerebellar Ataxias Adapted from a Presentation by Huda Y. Zoghbi Howard Hughes Medical Institute, Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, BCM, Room T807, Houston, Texas 77030-3498
reach their seventies or eighties; the next generation to be affected will develop symptoms earlier, perhaps in their fifties; this pattern continues until children are developing the disease in their teens or even infancy, and dying within a few years. So far researchers have identified 10 distinct spinocerebellar ataxias, and have located the precise gene responsible for the disease in 7 of these disorders. Moreover, we know the nature of the mutation that causes trouble: instability in a triplet-repeat tract. The triplet repeat involved in the ataxias is a row of three nucleotide bases (cytosine, adenine, guanine) in the DNA that spell out CAG over and over and encode the amino acid glutamine. In normal genes, CAG may be repeated from 6 to 35 times, but mutant genes are expanded well beyond their normal length, encompassing 40 to more than 100 triplets. Because a number of neurodegenerative disorders, including Huntington’s disease, share this expansion of the polyglutamine tract, they are known collectively as polyglutamine or triplet repeat diseases. Interestingly, the longer the expansion, the more severe the disease, and the earlier its onset. The repeat is unstable and expands as it is passed from one generation to the next, particularly if the affected parent is the father—this explains the phenomenon of anticipation mentioned earlier. Figure 2 shows the strong inverse correlation between age of onset and number of repeats. So, what are the implications of finding the gene for several of these disorders and having the mutation in hand? The first ones are clinical. There is a simple and accurate diagnostic test to identify patients with these various disorders. Before 1993, many of these patients had to undergo extensive diagnostic workups, magnetic resonance imaging (MRI), computed axial tomography (CAT) scans, nerve conduction studies, and biopsies. Patients can now have a simple blood test to determine which of the ataxia subtypes they might have, and can be counseled accordingly. Greater un-
There are many questions yet to be answered about neurologic diseases. Why does one mutated copy of a gene cause disease despite the presence of a normal copy on the other chromosome? How can the disease process itself be quite slow and manifest itself clinically as a late-onset neurodegenerative disease? If the mutant gene is expressed in all neurons and other bodily tissues, why is it that only some neurons degenerate while others are spared? What can we do to spare the progression of these diseases and eventually prevent neurodegeneration? A 10- to 20-year delay of symptoms would dramatically improve daily life, even if there is never really a cure for these diseases. Once researchers thoroughly understand the molecular basis of these disorders, they will be able to specifically target processes that go awry for therapeutic intervention. The spinocerebellar ataxias are a group of diseases characterized by loss of balance and coordination. Although these are the features common to all the ataxias, the initial symptoms can vary greatly. An affected individual might first notice some slight difficulty maintaining balance. Others might note that their speech is becoming slurred or that their eyes suffer involuntary spasms. Some acquire a tremor; others may lose their sight or hearing. Then they develop the characteristic wide-based ataxic gait. Despite the varied onset, the end is the same: after 10 to 15 years of progressively worsening symptoms, patients die because they are no longer able to swallow or breathe. Studies of families with spinocerebellar ataxia type 1 illuminated two important facts about this group of diseases. First, the disease is autosomal dominant: each child of an affected parent has a 50% chance of inheriting the disease (Fig. 1). Second, each successive generation affected by spinocerebellar ataxia type 1 experiences anticipation; i.e., they will develop a more severe form of the disease at an earlier age. The first generation may display symptoms only when they 0969-9961/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
523
524
Huda Zoghbi
FIG. 1.
Diagram of multiple generations of a family with SCA1.
derstanding of the disease and how it is passed between generations offers patients and their families an opportunity to plan for their future. Most importantly, understanding of the basic principles allows researchers to study these diseases at their most fundamental level. If we can understand the pathophysiology of these diseases, we will be better equipped to develop effective therapies. Much progress has already been made by studying animal models of the disease. In the latter part of the 1990s, researchers have taken advantage of knowing the gene and the mutation and designed animal models to try to understand the pro-
FIG. 2. Inverse correlation between number of repeats and age of onset.
Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
gression of specific diseases. With an animal model, researchers are able to examine the disease process before symptoms appear. Through genetic studies of transgenic mice that carry copies of the mutated human spinocerebellar ataxia 1 (SCA1) gene, researchers have learned that the expanded glutamine sequence confers a novel and toxic function on the gene. Proteins are usually very complex molecules comprising a sequence of many amino acids. The sequence and nature of the amino acids determine the structure, properties, and function of the protein. As mentioned earlier, normal individuals have about 30 CAG repeats producing a normal protein with a normal sequence of the amino acid glutamine. Affected individuals synthesize a mutant protein with an expanded sequence of, say, 82 glutamines. This protein turns out to be quite toxic and causes juvenile onset disease in human patients. Mice that carry the normal human gene are perfectly normal and live 2 years without any abnormalities. But mice with the human mutation of 82 repeats develop problems with their gait, coordination, and balance. Furthermore, their Purkinje cells have the same alterations we see in human patients. Under the microscope, the mouse model reveals more about polyglutamine-induced toxicity. If we look at brain sections from the mouse with a normal human gene, the neurons look perfectly normal and the Purkinje cells look healthy. In mice with the SCA1 mutation, at 12 weeks of age, we notice that the Pur-
Spinocerebellar Ataxias
525
FIG. 3. Ubiquitin/proteasome-dependent degradation. Once a protein has accomplished its function, various enzymes (E1–E3) attach ubiquitins to the molecule to target it for degradation. The protein is then broken down by the proteasome, which has two main components (19S and 20S). The remaining cellular bits are recycled.
kinje cells start to look different, and vacuoles, or holes, appear in the cytoplasm. This vacuolation is often an indicator of damage. In addition, if the sections are stained to show the mutant SCAI protein, ataxin-1, we find a very intense, dark staining within the nucleus that does not appear in normal cells. It appears as though this protein is aggregating within the nucleus. The aggregated protein can occupy a considerable volume within the nucleus. Again, this is a sign of a dysfunctional cell.
In addition to the mouse models that can be developed to study the onset and progress of the disease, scientists have developed models in the fruit fly, Drosophila. Flies have one great advantage over mouse models: their life cycle is much shorter. Many generations can be studied more quickly. Neurons in the ventral cord of the fly nervous system are quite similar to neurons in the spinal cord of vertebrates; flies with the mutated ataxia genes have neurons and neuronal processes that degenerate very much like the mouse Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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Huda Zoghbi
FIG. 4. Brain stem neurons from SCA1 patient tissue. Left: Cells stained with ataxin-1 antibody show ataxin-1 aggregated into a single, insoluble clump in the nucleus. Right: Cells stained with ubiquitin antibody in the right panel show that these aggregates also contain ubiquitin, a molecule involved in the degradation of proteins.
model of the human disease. The proteins and the neurons can be observed under the microscope using fluorescent markers. We can insert normal and mutant human genes into the genetic material of flies, and see how this affects development of the neurons. Because flies develop quickly, we can generate and examine large numbers of flies with various mutations in different proteins. In addition, we can work on identifying proteins that might make their phenotype worse or better and therefore identify what we call modifier proteins or genes. This can open avenues for future molecular or gene therapy approaches to slow the onset of disease or treat the symptoms. What is it about the mutant protein that causes it to accumulate and aggregate? A cell can have too much protein for one of two reasons. Either the cell is making too much, or the protein is not being properly “degraded.” Normally proteins are synthesized within the cell, accomplish their function, and are then degraded, or broken down into their constituent amino acids by other proteins (proteases) and “recycled.” This happens through a process called ubiquitin/proteasome-dependent degradation (Fig. 3). Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Once the protein has performed its regular cellular function, multiple small proteins, called ubiquitins because they are found in all cells, attach to the protein. Once the ubiquitin chain is attached to the protein, it is fed through the proteasome, where it is degraded into reusable amino acids. This is a ubiquitin-dependent process. It cannot happen without ubiquitin attaching to the protein. The question is, therefore, can the mutant ataxin be accumulating because it is not degraded? Is that because ubiquitin cannot normally attach to these proteins or because there are other defects? We can stain the aggregates for this protein ubiquitin both in mice and in cells from patients. Figure 4 shows tissue from patients who died from spinocerebellar ataxia. Depicted are neurons from the brain stem that have large aggregates occupying a significant part of the nucleus. The cells are stained with the antibody for ubiquitin. The aggregates are ubiquitin-positive; i.e., ubiquitin is present in significant concentrations. This observation is consistent with the idea that there is abnormal recycling of the ataxin protein by ubiquitin. Looking at it slightly differently, perhaps the cellular apparatus recognizes
Spinocerebellar Ataxias
that the protein is abnormal, but the ubiquitin sent to degrade it is unable to, leading to an even greater aggregation of proteinaceous material. What is particularly interesting is that this discovery of protein aggregates in damaged cells has now been observed in many other progressive neurodegenerative diseases. Most, if not all, progressive, late-onset, neurodegenerative diseases are linked with mutant proteins that are probably structurally altered in such a way that they accumulate in the cells over time. This is a model being explored at least for the polyglutamine diseases, but it may apply to many other neurodegenerative diseases. Particular proteins have specific three-dimensional structures that, together with their chemical composition, determine their function within the cell. If, for any reason, this three-dimensional structure is significantly altered, the protein might not interact with proteins it was supposed to or it might find a new inappropriate partner within the cell. This may lead to dysfunction of a particular type of cell in a particular region of the brain and thereby help explain some of the distinct clinical phenotypes of these diseases. Proteins that do not have quite the right structure are thought to be “misfolded.” These are typically recognized and targeted for degradation. The ubiquitinated protein is sent through the proteasome. In SCA, the long glutamine tract makes it resistant to degradation; the long sequence of repeated amino acids within the mutant proteins either slows down or inhibits their degradation, resulting in a ubiquitinpositive nuclear aggregate.
527 There are proteins in the cell known as chaperones that are able to refold proteins and/or present them more aggressively to the proteasome for degradation. If this is indeed a misfolded protein, could these proteins be involved? Tissue from animals or humans with the mutated SCA1 gene show the presence of a particular chaperone within the nuclear aggregate. Several other chaperones, however, remain diffusely distributed throughout the nucleus. Could extra chaperones enhance the protein’s degradation? This possibility can first be tested in a cell culture assay in the laboratory by evaluating what happens to cells in the presence of mutant ataxin-1 with or without particular chaperones. These cultured cells can be engineered to express a lot of mutant ataxin-1, and the effect of adding chaperones to the culture can be evaluated. When a chaperone known to enable correct folding of proteins is genetically expressed in the cells, the percentage of cells with large aggregates decreases significantly. This suggests that the chaperone may either be helping to refold the protein so that it does not aggregate or it may be presenting the protein more efficiently for degradation. One or both of these processes may be taking place. This explanation and indicator of potential therapy is just one candidate worthy of further exploration. The various animal models and the cell culture system can be used to screen many candidate molecules and test different molecular interventions, as well as to help test more hypotheses as to how the disease develops.
Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Spinocerebellar Ataxias Adapted from a Presentation by Huda Y. Zoghbi Howard Hughes Medical Institute, Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, BCM, Room T807, Houston, Texas 77030-3498
reach their seventies or eighties; the next generation to be affected will develop symptoms earlier, perhaps in their fifties; this pattern continues until children are developing the disease in their teens or even infancy, and dying within a few years. So far researchers have identified 10 distinct spinocerebellar ataxias, and have located the precise gene responsible for the disease in 7 of these disorders. Moreover, we know the nature of the mutation that causes trouble: instability in a triplet-repeat tract. The triplet repeat involved in the ataxias is a row of three nucleotide bases (cytosine, adenine, guanine) in the DNA that spell out CAG over and over and encode the amino acid glutamine. In normal genes, CAG may be repeated from 6 to 35 times, but mutant genes are expanded well beyond their normal length, encompassing 40 to more than 100 triplets. Because a number of neurodegenerative disorders, including Huntington’s disease, share this expansion of the polyglutamine tract, they are known collectively as polyglutamine or triplet repeat diseases. Interestingly, the longer the expansion, the more severe the disease, and the earlier its onset. The repeat is unstable and expands as it is passed from one generation to the next, particularly if the affected parent is the father—this explains the phenomenon of anticipation mentioned earlier. Figure 2 shows the strong inverse correlation between age of onset and number of repeats. So, what are the implications of finding the gene for several of these disorders and having the mutation in hand? The first ones are clinical. There is a simple and accurate diagnostic test to identify patients with these various disorders. Before 1993, many of these patients had to undergo extensive diagnostic workups, magnetic resonance imaging (MRI), computed axial tomography (CAT) scans, nerve conduction studies, and biopsies. Patients can now have a simple blood test to determine which of the ataxia subtypes they might have, and can be counseled accordingly. Greater un-
There are many questions yet to be answered about neurologic diseases. Why does one mutated copy of a gene cause disease despite the presence of a normal copy on the other chromosome? How can the disease process itself be quite slow and manifest itself clinically as a late-onset neurodegenerative disease? If the mutant gene is expressed in all neurons and other bodily tissues, why is it that only some neurons degenerate while others are spared? What can we do to spare the progression of these diseases and eventually prevent neurodegeneration? A 10- to 20-year delay of symptoms would dramatically improve daily life, even if there is never really a cure for these diseases. Once researchers thoroughly understand the molecular basis of these disorders, they will be able to specifically target processes that go awry for therapeutic intervention. The spinocerebellar ataxias are a group of diseases characterized by loss of balance and coordination. Although these are the features common to all the ataxias, the initial symptoms can vary greatly. An affected individual might first notice some slight difficulty maintaining balance. Others might note that their speech is becoming slurred or that their eyes suffer involuntary spasms. Some acquire a tremor; others may lose their sight or hearing. Then they develop the characteristic wide-based ataxic gait. Despite the varied onset, the end is the same: after 10 to 15 years of progressively worsening symptoms, patients die because they are no longer able to swallow or breathe. Studies of families with spinocerebellar ataxia type 1 illuminated two important facts about this group of diseases. First, the disease is autosomal dominant: each child of an affected parent has a 50% chance of inheriting the disease (Fig. 1). Second, each successive generation affected by spinocerebellar ataxia type 1 experiences anticipation; i.e., they will develop a more severe form of the disease at an earlier age. The first generation may display symptoms only when they 0969-9961/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
523
524
Huda Zoghbi
FIG. 1.
Diagram of multiple generations of a family with SCA1.
derstanding of the disease and how it is passed between generations offers patients and their families an opportunity to plan for their future. Most importantly, understanding of the basic principles allows researchers to study these diseases at their most fundamental level. If we can understand the pathophysiology of these diseases, we will be better equipped to develop effective therapies. Much progress has already been made by studying animal models of the disease. In the latter part of the 1990s, researchers have taken advantage of knowing the gene and the mutation and designed animal models to try to understand the pro-
FIG. 2. Inverse correlation between number of repeats and age of onset.
Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
gression of specific diseases. With an animal model, researchers are able to examine the disease process before symptoms appear. Through genetic studies of transgenic mice that carry copies of the mutated human spinocerebellar ataxia 1 (SCA1) gene, researchers have learned that the expanded glutamine sequence confers a novel and toxic function on the gene. Proteins are usually very complex molecules comprising a sequence of many amino acids. The sequence and nature of the amino acids determine the structure, properties, and function of the protein. As mentioned earlier, normal individuals have about 30 CAG repeats producing a normal protein with a normal sequence of the amino acid glutamine. Affected individuals synthesize a mutant protein with an expanded sequence of, say, 82 glutamines. This protein turns out to be quite toxic and causes juvenile onset disease in human patients. Mice that carry the normal human gene are perfectly normal and live 2 years without any abnormalities. But mice with the human mutation of 82 repeats develop problems with their gait, coordination, and balance. Furthermore, their Purkinje cells have the same alterations we see in human patients. Under the microscope, the mouse model reveals more about polyglutamine-induced toxicity. If we look at brain sections from the mouse with a normal human gene, the neurons look perfectly normal and the Purkinje cells look healthy. In mice with the SCA1 mutation, at 12 weeks of age, we notice that the Pur-
Spinocerebellar Ataxias
525
FIG. 3. Ubiquitin/proteasome-dependent degradation. Once a protein has accomplished its function, various enzymes (E1–E3) attach ubiquitins to the molecule to target it for degradation. The protein is then broken down by the proteasome, which has two main components (19S and 20S). The remaining cellular bits are recycled.
kinje cells start to look different, and vacuoles, or holes, appear in the cytoplasm. This vacuolation is often an indicator of damage. In addition, if the sections are stained to show the mutant SCAI protein, ataxin-1, we find a very intense, dark staining within the nucleus that does not appear in normal cells. It appears as though this protein is aggregating within the nucleus. The aggregated protein can occupy a considerable volume within the nucleus. Again, this is a sign of a dysfunctional cell.
In addition to the mouse models that can be developed to study the onset and progress of the disease, scientists have developed models in the fruit fly, Drosophila. Flies have one great advantage over mouse models: their life cycle is much shorter. Many generations can be studied more quickly. Neurons in the ventral cord of the fly nervous system are quite similar to neurons in the spinal cord of vertebrates; flies with the mutated ataxia genes have neurons and neuronal processes that degenerate very much like the mouse Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
526
Huda Zoghbi
FIG. 4. Brain stem neurons from SCA1 patient tissue. Left: Cells stained with ataxin-1 antibody show ataxin-1 aggregated into a single, insoluble clump in the nucleus. Right: Cells stained with ubiquitin antibody in the right panel show that these aggregates also contain ubiquitin, a molecule involved in the degradation of proteins.
model of the human disease. The proteins and the neurons can be observed under the microscope using fluorescent markers. We can insert normal and mutant human genes into the genetic material of flies, and see how this affects development of the neurons. Because flies develop quickly, we can generate and examine large numbers of flies with various mutations in different proteins. In addition, we can work on identifying proteins that might make their phenotype worse or better and therefore identify what we call modifier proteins or genes. This can open avenues for future molecular or gene therapy approaches to slow the onset of disease or treat the symptoms. What is it about the mutant protein that causes it to accumulate and aggregate? A cell can have too much protein for one of two reasons. Either the cell is making too much, or the protein is not being properly “degraded.” Normally proteins are synthesized within the cell, accomplish their function, and are then degraded, or broken down into their constituent amino acids by other proteins (proteases) and “recycled.” This happens through a process called ubiquitin/proteasome-dependent degradation (Fig. 3). Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Once the protein has performed its regular cellular function, multiple small proteins, called ubiquitins because they are found in all cells, attach to the protein. Once the ubiquitin chain is attached to the protein, it is fed through the proteasome, where it is degraded into reusable amino acids. This is a ubiquitin-dependent process. It cannot happen without ubiquitin attaching to the protein. The question is, therefore, can the mutant ataxin be accumulating because it is not degraded? Is that because ubiquitin cannot normally attach to these proteins or because there are other defects? We can stain the aggregates for this protein ubiquitin both in mice and in cells from patients. Figure 4 shows tissue from patients who died from spinocerebellar ataxia. Depicted are neurons from the brain stem that have large aggregates occupying a significant part of the nucleus. The cells are stained with the antibody for ubiquitin. The aggregates are ubiquitin-positive; i.e., ubiquitin is present in significant concentrations. This observation is consistent with the idea that there is abnormal recycling of the ataxin protein by ubiquitin. Looking at it slightly differently, perhaps the cellular apparatus recognizes
Spinocerebellar Ataxias
that the protein is abnormal, but the ubiquitin sent to degrade it is unable to, leading to an even greater aggregation of proteinaceous material. What is particularly interesting is that this discovery of protein aggregates in damaged cells has now been observed in many other progressive neurodegenerative diseases. Most, if not all, progressive, late-onset, neurodegenerative diseases are linked with mutant proteins that are probably structurally altered in such a way that they accumulate in the cells over time. This is a model being explored at least for the polyglutamine diseases, but it may apply to many other neurodegenerative diseases. Particular proteins have specific three-dimensional structures that, together with their chemical composition, determine their function within the cell. If, for any reason, this three-dimensional structure is significantly altered, the protein might not interact with proteins it was supposed to or it might find a new inappropriate partner within the cell. This may lead to dysfunction of a particular type of cell in a particular region of the brain and thereby help explain some of the distinct clinical phenotypes of these diseases. Proteins that do not have quite the right structure are thought to be “misfolded.” These are typically recognized and targeted for degradation. The ubiquitinated protein is sent through the proteasome. In SCA, the long glutamine tract makes it resistant to degradation; the long sequence of repeated amino acids within the mutant proteins either slows down or inhibits their degradation, resulting in a ubiquitinpositive nuclear aggregate.
527 There are proteins in the cell known as chaperones that are able to refold proteins and/or present them more aggressively to the proteasome for degradation. If this is indeed a misfolded protein, could these proteins be involved? Tissue from animals or humans with the mutated SCA1 gene show the presence of a particular chaperone within the nuclear aggregate. Several other chaperones, however, remain diffusely distributed throughout the nucleus. Could extra chaperones enhance the protein’s degradation? This possibility can first be tested in a cell culture assay in the laboratory by evaluating what happens to cells in the presence of mutant ataxin-1 with or without particular chaperones. These cultured cells can be engineered to express a lot of mutant ataxin-1, and the effect of adding chaperones to the culture can be evaluated. When a chaperone known to enable correct folding of proteins is genetically expressed in the cells, the percentage of cells with large aggregates decreases significantly. This suggests that the chaperone may either be helping to refold the protein so that it does not aggregate or it may be presenting the protein more efficiently for degradation. One or both of these processes may be taking place. This explanation and indicator of potential therapy is just one candidate worthy of further exploration. The various animal models and the cell culture system can be used to screen many candidate molecules and test different molecular interventions, as well as to help test more hypotheses as to how the disease develops.
Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.