CAG Repeat Diseases Research Articles

Intra- and Intermolecular β-Pleated Sheet Formation in Glutamine-repeat Inserted Myoglobin as a Model for Polyglutamine Diseases

An aberrant structure of the expanded polyglutamine might be involved in the formation of aggregates in CAG repeat diseases. To elucidate structural properties of the expanded polyglutamine, we prepared sperm whale myoglobin (Mb) mutants, in which 12, 28, 35, and 50 repeats of glutamine were inserted at the corner between the C and D helices (Gln(12), Gln(28), Gln(35), and Gln(50), respectively). Circular dichroism and IR spectroscopies showed that the expanded polyglutamine, which was recognized by the monoclonal antibody 1C2 in Gln(28), Gln(35), and Gln(50) Mb forms an antiparallel beta-pleated sheet structure. Gln(50) Mb aggregates were found to comprise an intermolecular antiparallel beta-pleated sheet. Fluorescence together with (1)H NMR spectra revealed partial unfolding of the protein surface in Gln(35) and Gln(50) Mb, although the structural changes in the protein core were rather small. The present results indicate that the fluctuating beta-pleated sheet of the expanded polyglutamine exposed on the protein surface facilitates the formation of aggregates through intermolecular interactions. The present study has first established and characterized structural properties of a molecular model for polyglutamine diseases in which various lengths of polyglutamine including a pathologically expanded glutamine repeat were inserted into a structurally known protein.

Neuronal nuclear alterations in dentatorubral-pallidoluysian atrophy: ultrastructural and morphometric studies of the cerebellar granule cells

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disease caused by a CAG repeat expansion, resulting in ubiquitinated inclusions and diffuse accumulation of mutant atrophin-1 in the neuronal nuclei in many regions of the central nervous system. In the cerebellar cortex, such nuclear abnormalities occur in the granule cells. In the present study, we performed ultrastructural and morphometric analyses on the nuclei of the cerebellar granule cells from eight patients with DRPLA (four with juvenile-onset disease and four with adult-onset disease) in an attempt to obtain further insight into the neuronal nuclear alterations that occur in CAG-repeat diseases. Ultrastructurally, all patients had intranuclear filamentous inclusions (NIIs, neuronal intranuclear inclusions) and nuclear membrane indentations (NMIs) in some of their granule cells, and chromatin tended to be sparse in the nucleoplasm of the affected nuclei. No such changes were observed in the granule cells of four control subjects. In all patients there was an association between NIIs and NMIs, and nuclei with NIIs and/or NMIs were larger than those without such changes. However, the nuclear enlargement was not due solely to the NIIs — even nuclei without NIIs or NMIs were larger in the patients than in the controls. In the DRPLA patients, there was a significant inverse correlation between the cross-sectional area of the nuclei and the disease duration. These findings indicate that NIIs and NMIs are features in the disease and occur in association with each other, and that nuclear enlargement — the degree of which may decrease with time after onset of the illness — is a more prevalent abnormality than the formation of NIIs or NMIs.

Polyglutamine aggregation behavior in vitro supports a recruitment mechanism of cytotoxicity

In expanded CAG repeat diseases such as Huntington’s disease, proteins containing polyglutamine (poly(Gln)) sequences with repeat lengths of about 37 residues or more are associated with development of both disease symptoms and neuronal intranuclear inclusions (NIIs). Disease physiology in animal and cellular models does not always correlate with NII formation, however, and the mechanism by which aggregate formation might lead to cytotoxicity is unknown. To help evaluate various possible mechanisms, we determined the biophysical properties of a series of simple poly(Gln) peptides. The circular dichroism spectra of poly(Gln) peptides with repeat lengths of five, 15, 28 and 44 residues are all nearly identical and are consistent with a high degree of random coil structure, suggesting that the length-dependence of disease is not related to a conformational change in the monomeric states of expanded poly(Gln) sequences. In contrast, there is a dramatic increase in both the kinetics and the thermodynamic favorability of the spontaneous formation of ordered, amyloid-like aggregates for poly(Gln) peptides with repeat lengths of greater than 37 residues. At the same time, poly(Gln) peptides with repeat lengths in the 15–20 residue range, despite their poor abilities to support spontaneous, self-nucleated aggregation, are capable of efficiently adding to an already-formed aggregate. We also find that morphologically small, finely divided aggregates are much more efficient at recruiting poly(Gln) peptides than are large aggregates, suggesting a possible explanation for why disease pathology does not always correlate with the observable NII burden. Together, these data are consistent with a model for disease pathology in which critical cellular proteins possessing poly(Gln) sequences of modest length become inactivated when they are recruited into aggregates of an expanded poly(Gln) protein.

Meiotic instability of the CAG repeats in the SCA6/CACNA1A gene in two Japanese SCA6 families

Intergenerational stability of the CAG repeat number has been considered to be a specific molecular feature of SCA6 compared with other CAG repeat diseases. Nevertheless, we showed meiotic instability of the CAG repeats in the SCA6/CACNL1A gene in two Japanese SCA6 families, including de novo expansion. In one family, the CAG 20 allele expanded to the CAG 26 one during paternal transmission, and in the other family, the CAG 19 allele expanded to the CAG 20 one during maternal transmission. Although it is controversial as to whether the CAG 20 allele is pathological or not, this is the first case of haplotype analysis-proven de novo expansion in SCA6, confirming the derivation of an expanded allele from one normal allele. We should carefully follow up the individuals carrying the CAG 20 allele in our family who show normal neurological and radiological findings at present.

Solubilization and disaggregation of polyglutamine peptides.

A method is described for dissolving and disaggregating chemically synthesized polyglutamine peptides. Polyglutamine peptides longer than about Q20 have been reported to be insoluble in water, but dissolution in--and evaporation from--a mixture of trifluoroacetic acid and hexafluoroisopropanol converts polyglutamine peptides up to at least Q44 to a form readily soluble in aqueous buffers. This procedure also has a dramatic effect on peptides which appear to be completely soluble in water, by removing traces of aggregate that seed aggregation. The protocol makes possible solution studies-including in vitro aggregation experiments--on polyglutamine peptides with repeat lengths associated with increased risk of Huntington's Disease and other expanded CAG repeat diseases. It may also be useful in conducting reproducible, quantitative aggregation studies on other polypeptides.

Pathology of CAG repeat diseases.

Neuronal intranuclear inclusions have become the neuropathological signature of the CAG repeat diseases, although their cytotoxicity is a matter of controversy. It has been demonstrated that the inclusions in dentatorubral-pallidoluysian atrophy (DRPLA) and Machado-Joseph disease (MJD) were immunopositive for several transcription factors such as TATA-binding protein (TBP), TBP-associated factor (TAF(II)130), Sp1, cAMP-responsive element-binding protein (CREB) and CREB-binding protein, suggesting that neuronal degeneration in polyglutamine diseases may result from nuclear depletion of transcription factors containing the glutamine-rich domain. It was also revealed that, in the DRPLA brain, expanded polyglutamine stretches were diffusely accumulated in neuronal nucleoplasm. This nuclear pathology involved many neurons in various nervous system regions, such as the cerebral cortex, thalamus, substantia nigra, pontine nuclei, reticular formation and inferior olive, in addition to the previously recognized affected regions. The diffuse nuclear labeling was also detected in MJD, Huntington's disease, and spinal and bulbar muscular atrophy, suggesting that this nuclear pathology may be a characteristic feature and may exert certain influence on certain nuclear functions of many neurons in the CAG repeat diseases.

Pathology of CAG repeat diseases

Neuronal intranuclear inclusions have become the neuropathological signature of the CAG repeat diseases, although their cytotoxicity is a matter of controversy. It has been demonstrated that the inclusions in dentatorubral–pallidoluysian atrophy (DRPLA) and Machado–Joseph disease (MJD) were immunopositive for several transcription factors such as TATA‐binding protein (TBP), TBP‐associated factor (TAFII130), Sp1, cAMP‐responsive element‐binding protein (CREB) and CREB‐binding protein, suggesting that neuronal degeneration in polyglutamine diseases may result from nuclear depletion of transcription factors containing the glutamine‐rich domain. It was also revealed that, in the DRPLA brain, expanded polyglutamine stretches were diffusely accumulated in neuronal nucleoplasm. This nuclear pathology involved many neurons in various nervous system regions, such as the cerebral cortex, thalamus, substantia nigra, pontine nuclei, reticular formation and inferior olive, in addition to the previously recognized affected regions. The diffuse nuclear labeling was also detected in MJD, Huntington's disease, and spinal and bulbar muscular atrophy, suggesting that this nuclear pathology may be a characteristic feature and may exert certain influence on certain nuclear functions of many neurons in the CAG repeat diseases.

Neuronal intranuclear hyaline inclusion disease with polyglutamine-immunoreactive inclusions

Neuronal intranuclear hyaline inclusion disease (NIHID) is a group of neurodegenerative disorders characterized by the presence of intranuclear inclusions in neurons (NIs). We report here clinicopathological findings of a 25-year-old female patient who died after 13 years of a clinical course characterized by progressive gait disturbance and movement disorders. Histological examination revealed widespread NIs with neuronal loss in restricted regions; neuronal loss was severe in the subthalamic nucleus, internal pallidum, substantia nigra, Edinger-Westphal nucleus and Purkinje cell layer. Quantification of the NIs combined with a graded evaluation of neuronal loss revealed an overall tendency for more severe neuronal loss to be accompanied by a lower frequency of NIs. A morphological similarity to the nuclear inclusions recently identified in several CAG repeat diseases prompted us to examine the immunolocalization of ubiquitin and expanded polyglutamine stretches, which demonstrated the presence of ubiquitin at the periphery of most NIs. An expanded polyglutamine stretch was seen in the center of limited number of NIs. These findings indicate that abnormal fragments such as expanded polyglutamine regions are incorporated into the inclusion, aggregated in its center, and thereby metabolized by a ubiquitin-dependent proteolytic pathway. Although it remains to be elucidated how the formation of NIs is related to neuronal degeneration, our findings suggest that NIs are formed in the process of sequestering or degrading abnormal protein fragments and formation of NIs may not be immediately toxic to neurons.

Polyglutamine-induced ion channels: a possible mechanism for the neurotoxicity of Huntington and other CAG repeat diseases.

CAG repeats resulting in long polyglutamine tracts have been implicated in the pathogenesis of at least eight neurodegenerative diseases including Huntington. Expression of polyglutamine repeats is required for disease and increasing length of the repeats leads to earlier onset of illness (anticipation). Expression of polyglutamine repeats in cultured neurons leads to deposition of intracellular aggregates resembling those found in amyloid diseases, and to neurotoxicity. We report here that polyglutamine can induce large (19-220 pS), long-lived, (lifetime = 6 sec), non-selective (P(cation) = P(anion)) ion channels in planar phospholipid bilayer membranes, and that channel formation is enhanced by acidic pH. We propose that channel formation may be a mechanism of cellular toxicity in Huntington and other CAG repeat disease.

Tissue-Specific Somatic Mosaicism in Spinal and Bulbar Muscular Atrophy Is Dependent on CAG-Repeat Length and Androgen Receptor–Gene Expression Level

The factors influencing the tissue-specific pattern of somatic mosaicism in CAG-repeat diseases have not yet been fully resolved. We performed a detailed analysis of the degree of somatic mosaicism in various tissues from 20 patients with spinal and bulbar muscular atrophy (SBMA), including 4 who were deceased. The most outstanding feature was the prominent somatic mosaicism observed in the cardiac and skeletal muscles, composed predominantly of postmitotic cells, and in the skin, prostate, and testis. The CNS tissues, liver, and spleen showed the least mosaicism. The tissue distribution of somatic mosaicism in patients with SBMA was markedly different from that in patients with Huntington disease (HD) and from that in patients with dentatorubral-pallidoluysian atrophy (DRPLA). The degree of somatic mosaicism correlated with the CAG-repeat number but not with age at examination. Furthermore, tissues with a higher mosaicism level corresponded well to those with a higher expression level of androgen receptor protein. The tissue-specific pattern of somatic mosaicism related not only to cell composition with different cell turnover rates but to repeat size and gene expression levels, and postnatal cell division is unlikely to be a major cause of somatic mosaicism probably because of the relative stability of CAG repeat in SBMA.

Expression of Extended Polyglutamine Sequentially Activates Initiator and Effector Caspases

To date, eight neurodegenerative disorders, including Huntington's disease and dentatorubral-pallidoluysian atrophy, have been identified to be caused by expansion of a CAG repeat coding for a polyglutamine (polyQ) stretch. It is, however, unclear how polyQ expansion mediates neuronal cell death observed in these disorders. Here, we have established a tetracycline-regulated expression system producing 19 and 56 repeats of glutamine fused with green fluorescent protein. Induced expression of the 56 polyQ, but not of the 19 polyQ stretch caused marked nuclear aggregation and apoptotic morphological changes of the nucleus.In vitroenzyme assays and Western blotting showed that polyQ56expression sequentially activated initiator and effector caspases, such as caspase-8 or -9, and caspase-3, respectively. Furthermore, using cell-permeable fluorogenic substrate, the activation of caspase-3-like proteases was demonstrated in intact cells with aggregated polyQ. This is the first direct evidence that the expression of extended polyQ activates caspases and together with the previous findings that some of the products of genes responsible for CAG repeat diseases are substrates of caspase-3 indicates an important role of caspases in the pathogenesis of these diseases.

Single sperm analysis of the CAG repeats in the gene for dentatorubral-pallidoluysian atrophy (DRPLA): the instability of the CAG repeats in the DRPLA gene is prominent among the CAG repeat diseases.

Dentatorubral-pallidoluysian atrophy (DRPLA) is known to show the most prominent genetic anticipation among CAG repeat diseases. To investigate the mechanism underlying the meiotic instability of expanded CAG repeats in the gene for DRPLA, we determined the CAG repeat sizes of 427 single sperm from two individuals with DRPLA. The mean variance of the change in the CAG repeat size in sperm from the DRPLA patients (288.0) was larger than any variances of the CAG repeat size in sperm from patients with Machado-Joseph disease (38. 5), Huntington's disease (69.0) and spinal and bulbar muscular atrophy (16.3), which is consistent with the clinical observation that the genetic anticipation on the paternal transmission of DRPLA is the most prominent among CAG repeat diseases. The variance of the change in CAG repeat size was significantly different between the two DRPLA patients (F-test, P < 0.0001). However, the segregation ratio of single sperm with an expanded allele to ones with a normal allele is not statistically different ( P = 0.161) from the expected 1:1 segregation ratio, and thus segregation distortion of expanded alleles in meiosis in male patients with DRPLA was not demonstrated.

CAG repeats in various organisms studied by Southern blot analysis.

A 90-nucleotide (CAG)30, single-stranded DNA was used to probe Southern blots in order to indicate the quantity and distribution of long CAG repeats in selected genomes. Bovine and rat genomes were found to contain a particularly high content of CAG repeats, while the repeats were comparatively rare in the human genome. A particularly strong signal in the bovine genome was due to a CAG repeat associated with the 1.709 satellite. A similar element was found in goat and musk, but not in the other artiodactyls tested, suggesting that this particular CAG repeat developed some 10-20 million years ago within a 3.8-kb unit presently belonging to the satellite element and that this unit has later multiplied in the genome. Single-copy repeats could be discerned in yeast, but not in mammals. Thus the probe did not detect specific repeats in patients with CAG repeat diseases.

Nuclear inclusions in CAG repeat diseases: toxic or protective?

A variety of neurodegenerative disorders, including Huntington's disease (HD) and spinocerebellar ataxia type 1 (SCA1), are caused by the expansion of CAG repeats in the coding regions of the genes involved, giving rise to polyglutamine tracts in their mutant protein products (huntingtin and ataxin-1, respectively). The lack of homology (outside the CAG repeats) of the genes mutated in these disorders, the dominant nature of their inheritance, and the finding that mice with the relevant genes knocked out have no clinical symptoms have led to the conclusion that the expanded polyglutamine tract is a pathogenic gain-of-function mutation. The presence of nuclear inclusion bodies containing polyglutamine protein aggregates, both in patients with CAG repeat disorders and in mouse models of these diseases, suggested that these protein aggregates were toxic and therefore pathogenic. Two recent publications challenge this hypothesis.Klement and colleagues1xAtaxin-1 nuclear localization and aggregation: role in polyglutamine-induced disease in SCA1 transgenic mice. Klement, I.A. et al. Cell. 1998; 95: 41–53Abstract | Full Text | Full Text PDF | PubMed | Scopus (800)See all References have established transgenic mice that express a mutated form of ataxin-1 in which the self-association region of the protein is deleted. This region is essential for the formation of ataxin-1 aggregates, and the transgenic mice fail to form inclusion bodies. Surprisingly, the mice develop ataxia and Purkinje cell pathology similar to that of SCA1 mice whose ataxin-1 self-association region is intact, suggesting that nuclear aggregation of ataxin-1 is not required to initiate pathogenesis in transgenic mice. These authors also investigated the role of ataxin-1 subcellular localization in SCA1 pathogenesis. Normal ataxin-1 is nuclear, but the nuclear localization signal can be abolished by substituting Lys772 for Thr. In transgenic mice carrying this mutation, ataxin-1 is cytoplasmic; the mice show no Purkinje cell pathology and do not develop any clinical symptoms of the disease.Further evidence that localization to the nucleus of expanded repeat-containing proteins is required for neurodegeneration is provided by Saudou et al.2xHuntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions. 2 Saudou, F. et al. Cell. 1998; 95: 55–66Abstract | Full Text | Full Text PDF | PubMed | Scopus (1217)See all References They have found that in cultured striatal neurons, in which mutant huntingtin induces apoptosis, compounds that block nuclear localization of huntingtin suppress its ability to form intranuclear inclusions and to cause cell death. Experiments in the same culture system suggest that intranuclear inclusions are not required for neurodegeneration. In fact, suppressing inclusion formation increases death induced by mutant huntingtin in the cultured neurons, suggesting that inclusion formation might actually protect the cell from the toxic effects of expanded-repeat-containing proteins.These findings are another step in the elucidation of the mechanisms by which the polyglutamine expansions cause pathogenesis, and give us more tools to aid in the search for molecular targets that can be used for therapeutic strategies to treat these devastating conditions.

Neuronal Death in Cultured Murine Cortical Cells Is Induced by Inhibition of GAPDH and Triosephosphate Isomerase

Polyglutamine-containing proteins expressed in the CAG repeat diseases Huntington's disease and dentatorubralpallidoluyisian atrophy have recently been suggested to inhibit the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). To examine the consequences of GAPDH inhibition upon neuronal survival, we exposed murine neocortical cell cultures to the inhibitor of GAPDH and triosephosphate isomerase, α-monochlorohydrin. Cultures exposed to 6–15 mM α-monochlorohydrin for 48 h exhibited an increase in dihydroxyacetone phosphate and a decrease in neuronal ATP that was followed by progressive neuronal death; some glial death occurred at high drug concentrations. The neuronal death was characterized by cell body shrinkage and chromatin condensation and was sensitive to cycloheximide and to the caspase inhibitors Z-Val-Ala-Asp fluoromethylketone and tert-butoxycarbonyl-Asp fluoromethylketone. Neurons in striatal cell cultures were more vulnerable to death induced by exposure to α-monochlorohydrin, except that NADPH-diaphorase(+) neurons were selectively spared. Repeated addition of the glycolytic endpoint metabolite pyruvate to the bathing medium attenuated both the drop in neuronal ATP and the neuronal cell death.

CAG repeat diseases and intranuclear inclusions

CAG repeat diseases and intranuclear inclusions

CAG repeat expansion in autosomal dominant pure spastic paraplegia linked to chromosome 2p21-p24.

CAG repeat expansions have been identified as the disease-causing dynamic mutations in the coding regions of genes in several dominantly inherited neurodegenerative disorders, including spinobulbar muscular atrophy, Huntington's disease, dentatorubral-pallidoluysian atrophy, spinocerebellar ataxia type 1, 2 and 6 and Machado-Joseph disease. The CAG repeat expansions are translated to elongated polyglutamine tracts and an increased size of the polyglutamine tract correlates with anticipation, the cardinal feature, seen in all these diseases. Autosomal dominant pure spastic peraplegia (ADPSP) is a degenerative disorder of the central motor system clinically characterized by slowly progressive and unremitting spasticity of the legs, hyperreflexia and Babinski's sign. Like the established CAG repeat diseases ADPSP is characterized by both inter- and intrafamilial variation and anticipation. Using the Repeat Expansion Detection (RED) method, we have analyzed 21 affected individuals from six Danish families with the disease linked to chromosome 2p21-p24. We found that 20 of 21 affected individuals showed CAG repeat expansions versus two of 21 healthy spouses, demonstrating a strongly statistically significant association between the occurrence of the repeat expansion and the disease (Fisher's test, P < 10(-5)) suggesting that a CAG repeat expansion is involved presumably as a dynamic mutation in ADPSP linked to chromosome 2p21-p24. The size of the expansion is estimated to be > or = 60 CAG repeat copies in the affected individuals. The CAG repeat expansion is very likely translated and expressed as indicated by the detection of a polyglutamine-containing protein in an ADPSP patient.

Clinical aspects of CAG repeat diseases.

Seven neurodegenerative disorders are known to be caused by unstable expansions of the trinucleotide CAG within human genes, and more will be discovered in the coming years. These disorders share some clinical similarities, as well as some differences, which are summarized here. These diseases have unusual clinical genetic properties related to the dynamic nature of CAG repeat expansions, including instability of the repeat expansion in meiosis, particularly male meiosis; a strong correlation between onset age and size of the repeat expansion; anticipation (earlier disease onset in succeeding generations); new mutations arising from unstable, mutable alleles with a high-normal CAG repeat number; and reduced penetrance for alleles in the low-affected range. Much more remains to be learned about the molecular biology and clinical pathophysiology of this new class of genetic diseases.

The neuropathology of CAG repeat diseases: review and update of genetic and molecular features.

Classification of inherited neurodegenerative diseases is increasingly based on their genetic features, which supplement, clarify, and sometimes replace the older clinical and pathologic schemata. This change has been particularly rapid and impressive for the CAG repeat disorders. In Huntington's disease, X-linked spinobulbar muscular atrophy, dentatorubropallidoluysian atrophy, and a series of autosomal dominant cerebellar atrophies, genetic advances have resolved many nosologic issues, and opened new avenues for exploration of pathogenesis. In this review, we summarize classic and current concepts in neuropathology of these CAG repeat diseases.

CAG repeat number governs the development rate of pathology in Huntington's disease

We compared the number of CAG repeats, the age at death, and the severity of neuropathology in 89 Huntington's disease brains. We found a linear correlation between the CAG repeat number and the quotient of the degree of atrophy in the striatum (the brain region most severely affected in Huntington's disease) divided by age at death, with an intercept at 35.5 repeats. The largest CAG repeat length, therefore, at which no pathology is expected to develop is 35.5. These results imply that striatal damage in Huntington's disease is almost entirely a linear function of the length of the polyglutamine stretch beyond 35.5 glutamines multiplied by the age of the patient. Thus, it is predicted that the pathological process develops linearly from birth. Analysis of other measures of striatal function could test this hypothesis and might determine when treatment for CAG repeat diseases should start.