Polyglutamine pathogenesis.

An increasing number of neurodegenerative disorders have been found to be caused by expanding CAG triplet repeats that code for polyglutamine. Huntington's disease (HD) is the most common of these disorders and dentatorubral-pallidoluysian atrophy (DRPLA) is very similar to HD, but is caused by mutation in a different gene, making them good models to study. In this review, we will concentrate on the roles of protein aggregation, nuclear localization and proteolytic processing in disease pathogenesis. In cell model studies of HD, we have found that truncated N-terminal portions of huntingtin (the HD gene product) with expanded repeats form more aggregates than longer or full length huntingtin polypeptides. These shorter fragments are also more prone to aggregate in the nucleus and cause more cell toxicity. Further experiments with huntingtin constructs harbouring exogenous nuclear import and nuclear export signals have implicated the nucleus in direct cell toxicity. We have made mouse models of HD and DRPLA using an N-terminal truncation of huntingtin (N171) and full-length atrophin-1 (the DRPLA gene product), respectively. In both models, diffuse neuronal nuclear staining and nuclear inclusion bodies are observed in animals expressing the expanded glutamine repeat protein, further implicating the nucleus as a primary site of neuronal dysfunction. Neuritic pathology is also observed in the HD mice. In the DRPLA mouse model, we have found that truncated fragments of atrophin-1 containing the glutamine repeat accumulate in the nucleus, suggesting that proteolysis may be critical for disease progression. Taken together, these data lead towards a model whereby proteolytic processing, nuclear localization and protein aggregation all contribute to pathogenesis.     

Distribution of CAG repeat size in the dentatorubral and pallidoluysian atrophy (DRPLA) gene in a normal population in Taiwan

Dentatorubral and pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder with expansion of trinucleotide CAG repeats in the coding region of the gene. Expansion of the repeat tract beyond the normal range produces gene products with extended polyglutamine tracts. In this study, we analyzed the distribution of the CAG repeats in the DRPLA alleles in a normal Taiwanese population. We observed 15 different alleles and found that the range of the CAG repeat number was from 7-21. The most frequent allele contained 15 CAG repeats that represented 20% of the total analyzed alleles, followed by the 17 repeats (15.8%). The heterozygosity rate of this locus was 88%. Twelve parents-to-children transmissions of the DRPLA alleles in a Machado-Joseph disease family appeared to be normal without any alteration of the CAG repeat numbers. Phenotypes of DRPLA overlapped those of autosomal dominant cerebellar ataxia (ADCA). In order to identify DRPLA patients in Taiwan, we screened six autosomal dominant cerebellar ataxia patients without expansion in known spinocerebellar ataxia genes. All six patients had the repeat numbers within the normal range; thus, the possibility of DRPLA could be excluded.     

Somatic mosaicism of expanded CAG repeats in brains of patients with dentatorubral-pallidoluysian atrophy: cellular population-dependent dynamics of mitotic instability.

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disease caused by unstable expansion of a CAG repeat in the DRPLA gene. We performed detailed quantitative analysis of the size and the size distribution (range) of the expanded CAG repeats in various regions of the CNS of eight autopsied patients with DRPLA. Expanded alleles (AE) showed considerable variations in size, as well as in range, depending on the region of the CNS, whereas normal alleles did not show such variations, which indicates the occurrence of somatic mosaicism of AE in the CNS. The AE in the cerebellar cortex were consistently smaller by two to five repeat units than those in the cerebellar white matter. Moreover, the AE in the cerebral cortex were smaller by one to four repeat units than those in the cerebral white matter. These results suggest that the smaller AE in the cerebellar and cerebral cortices represent those of neuronal cells. The ranges of the AE in the cerebral cortex, cerebral white matter, and cerebellar white matter showed considerable variation ranging from 9 to 23 repeat units, whereas those in the cerebellar cortex showed little variance and were approximately 7 repeat units. The ranges of the AE in the cerebral cortex, cerebral white matter, and cerebellar white matter were much broader in patients with higher ages at death than they were in patients with lower ages at death, raising the possibility that the range of AE increases with time, as the result of mitotic instability of AE.     

Elongated CAG repeats of the B37 gene in a Danish family with dentato-rubro-pallido-luysian atrophy

Dentato-rubro-pallido-luysian atrophy (DRPLA) is considered to be rare in Europe. We describe a Danish family in which affected individuals in at least three generations have been diagnosed as suffering from Huntington's disease. Because analysis of the Huntingtin gene revealed normal alleles and various of the patients had seizures, we analysed the B37 gene and found significantly elongated CAG repeats as have been reported in DRPLA. Affected individuals with almost identical repeat lengths presented very different symptoms. Both expansion and contraction in paternal transmission was encountered.     

Southern analysis for detection of CAG repeat expansions associated with dentatorubral pallidoluysian atrophy

Dentatorubral pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder caused by expansion of an unstable, tandemly repeated trinucleotide sequence, (CAG)_n, in a novel gene on human chromosome 12p12-pter. Molecular diagnosis of DRPLA uses the polymerase chain reaction (PCR) to amplify and characterize the number of CAG repeats carried by individuals. The PCR analysis is fairly straightforward when two alleles are identified. However, when only a single allele is observed, it is difficult to know whether the sample is homozygous or whether there was failure to amplify the second allele. We describe a Southern analysis for detection of the DRPLA CAG repeat, providing an independent method for the assessment of expanded alleles. Received: 15 May 1996 / Revised: 23 September 1996     

Anticipation and Instability of IT-15 (CAG)N Repeats in Parent-Offspring Pairs with Huntington Disease

Huntington disease (HD) is an autosomal dominant degenerative disorder caused by an expanded and unstable trinucleotide repeat (CAG)n in a gene (IT-15) on chromosome 4. HD exhibits genetic anticipation—earlier onset in successive generations within a pedigree. From a population-based clinical sample, we ascertained parent-offspring pairs with expanded alleles, to examine the intergenerational behavior of the trinucleotide repeat and its relationship to anticipation. We find that the change in repeat length with paternal transmission is significantly correlated with the change in age at onset between the father and offspring. When expanded triplet repeats of affected parents are separated by median repeat length, we find that the longer paternal and maternal repeats are both more unstable on transmission. However, unlike in paternal transmission, in which longer expanded repeats display greater net expansion than do shorter expanded repeats, in maternal transmission there is no mean change in repeat length for either longer or shorter expanded repeats. We also confirmed the inverse relationship between repeat length and age at onset, the higher frequency of juvenile-onset cases arising from paternal transmission, anticipation as a phenomenon of paternal transmission, and greater expansion of the trinucleotide repeat with paternal transmission. Stepwise multiple regression indicates that, in addition to repeat length of offspring, age at onset of affected parent and sex of affected parent contribute significantly to the variance in age at onset of the offspring. Thus, in addition to triplet repeat length, other factors, which could act as environmental factors, genetic factors, or both, contribute to age at onset. Our data establish that further expansion of paternal repeats within the affected range provides a biological basis of anticipation in HD.     

Deregulation of BRCA1 Leads to Impaired Spatiotemporal Dynamics of γ-H2AX and DNA Damage Responses in Huntington’s Disease

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder of mid-life onset characterized by involuntary movements and progressive cognitive decline caused by a CAG repeat expansion in exon 1 of the Huntingtin (Htt) gene. Neuronal DNA damage is one of the major features of neurodegeneration in HD, but it is not known how it arises or relates to the triplet repeat expansion mutation in the Htt gene. Herein, we found that imbalanced levels of non-phosphorylated and phosphorylated BRCA1 contribute to the DNA damage response in HD. Notably, nuclear foci of γ-H2AX, the molecular component that recruits various DNA damage repair factors to damage sites including BRCA1, were deregulated when DNA was damaged in HD cell lines. BRCA1 specifically interacted with γ-H2AX via the BRCT domain, and this association was reduced in HD. BRCA1 overexpression restored γ-H2AX level in the nucleus of HD cells, while BRCA1 knockdown reduced the spatiotemporal propagation of γ-H2AX foci to the nucleoplasm. The deregulation of BRCA1 correlated with an abnormal nuclear distribution of γ-H2AX in striatal neurons of HD transgenic (R6/2) mice and BRCA1(+/-) mice. Our data indicate that BRCA1 is required for the efficient focal recruitment of γ-H2AX to the sites of neuronal DNA damage. Taken together, our results show that BRCA1 directly modulates the spatiotemporal dynamics of γ-H2AX upon genotoxic stress and serves as a molecular maker for neuronal DNA damage response in HD.     

Continuous and periodic expansion of CAG repeats in Huntington's disease R6/1 mice

Huntington's disease (HD) is one of several neurodegenerative disorders caused by expansion of CAG repeats in a coding gene. Somatic CAG expansion rates in HD vary between organs, and the greatest instability is observed in the brain, correlating with neuropathology. The fundamental mechanisms of somatic CAG repeat instability are poorly understood, but locally formed secondary DNA structures generated during replication and/or repair are believed to underlie triplet repeat expansion. Recent studies in HD mice have demonstrated that mismatch repair (MMR) and base excision repair (BER) proteins are expansion inducing components in brain tissues. This study was designed to simultaneously investigate the rates and modes of expansion in different tissues of HD R6/1 mice in order to further understand the expansion mechanisms in vivo. We demonstrate continuous small expansions in most somatic tissues (exemplified by tail), which bear the signature of many short, probably single-repeat expansions and contractions occurring over time. In contrast, striatum and cortex display a dramatic--and apparently irreversible--periodic expansion. Expansion profiles displaying this kind of periodicity in the expansion process have not previously been reported. These in vivo findings imply that mechanistically distinct expansion processes occur in different tissues.     

Abnormal dentatorubral-pallidoluysian atrophy (DRPLA) protein complex is pathologically ubiquitinated in DRPLA brains.

Dentatorubral-pallidoluysian atrophy (DRPLA) is caused by expansion of a glutamine repeat in DRPLA protein. DRPLA protein undergoes greater complex formation in DRPLA brain tissue, and expanded glutamine repeat enhances complex formation of DRPLA protein. Immunoblots with and without reduction show that the DRPLA protein complex is ubiquitinated only in DRPLA brain tissue. Moreover, immunoblots of regional DRPLA brain tissues reveal that pathological ubiquitination of DRPLA protein complex is found selectively in affected lesions. Double-labeling immunohistochemical studies with antibodies against DRPLA protein and ubiquitin demonstrate that the DRPLA protein is co-localized with ubiquitin in DRPLA neurons and show characteristic neuronal cytoplasmic inclusions with ubiquitinated DRPLA protein complex in the center. Our findings suggest that DRPLA protein undergoes abnormal complex formation with expanded glutamine repeat, and then the complex is pathologically ubiquitinated in DRPLA brain tissue. Pathological ubiquitination of abnormal DRPLA protein complex plays a role in DRPLA pathology.     

Molecular pathology of dentatorubral-pallidoluysian atrophy.

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant disorder characterized clinically by myoclonus, epilepsy, cerebellar ataxia, choreoathetosis and dementia. Cardinal pathological features of DRPLA are a combined degeneration of both the dentatorubral and the pallidoluysian systems. Although the early sporadic cases were reported by Western neuropathologists, a strong heritability and an age of onset-dependent variability of the clinical features were carefully deduced by Japanese clinicians. The disease is fairly common in Japan, but extremely rare in Caucasians. Since the gene was identified in 1994, DRPLA is known as one of the CAG repeat expansion diseases, in which the responsible gene is located on chromosome 12p and its product is called atrophin 1. DRPLA shows prominent 'anticipation', which is genetically clearly explained by a marked instability of the expanded CAG repeat length during spermatogenesis. Moreover, the instability of the CAG repeat length also seems to occur in the somatic cells, resulting in 'somatic mosaicism'. Possible mechanism(s) underlying the neuronal cell death in DRPLA are discussed in terms of molecular pathological points of view.     

Cell death in polyglutamine diseases

An increasing number of inherited neurodegenerative diseases are known to be caused by trinucleotide repeat expansions in the respective genes. At least nine disorders result from a CAG trinucleotide repeat expansion which is translated into a polyglutamine stretch in the respective proteins: Huntington's disease (HD), dentatorubral pallidolysian atrophy (DRPLA), spinal bulbar muscular atrophy (SBMA), and several of the spinocerebellar ataxias (SCA1, 2, 3, 6, 7 and 12). Although the molecular steps leading to the specific neuropathology of each disease are unknown and are still under intensive investigation, there is increasing evidence that some CAG repeat disorders involve the induction of apoptotic mechanisms. This review summarizes the clinical and genetic features of each CAG repeat disorder and focuses on the common mechanistic steps involved in the disease progression of these so-called polyglutamine diseases. Among the common molecular features the formation of intranuclear inclusions, the recruitment of interacting polyglutamine-containing proteins, the involvement of the proteasome and molecular chaperones, and the activation of caspases are discussed with regard to their potential implication for the induction of cell death.     

Cleavage of atrophin-1 at caspase site aspartic acid 109 modulates cytotoxicity

Dentatorubropallidoluysian atrophy (DRPLA) is one of eight autosomal dominant neurodegenerative disorders characterized by an abnormal CAG repeat expansion which results in the expression of a protein with a polyglutamine stretch of excessive length. We have reported recently that four of the gene products (huntingtin, atrophin-1 (DRPLA), ataxin-3, and androgen receptor) associated with these open reading frame triplet repeat expansions are substrates for the cysteine protease cell death executioners, the caspases. This led us to hypothesize that caspase cleavage of these proteins may represent a common step in the pathogenesis of each of these four neurodegenerative diseases. Here we present evidence that caspase cleavage of atrophin-1 modulates cytotoxicity and aggregate formation. Cleavage of atrophin-1 at Asp109 by caspases is critical for cytotoxicity because a mutant atrophin-1 that is resistant to caspase cleavage is associated with significantly decreased toxicity. Further, the altered cellular localization within the nucleus and aggregate formation associated with the expanded form of atrophin-1 are completely suppressed by mutation of the caspase cleavage site at Asp109. These results provide support for the toxic fragment hypothesis whereby cleavage of atrophin-1 by caspases may be an important step in the pathogenesis of DRPLA. Therefore, inhibiting caspase cleavage of the polyglutamine-containing proteins may be a feasible therapeutic strategy to prevent cell death.     

Dentatorubral-pallidoluysian atrophy (DRPLA): Close correlation of CAG repeat expansions with the wide spectrum of clinical presentations and prominent anticipation

Dentatorubral-pallidoluysian atrophy (DRPLA) is a rare autosomal dominant neurodegenerative disease characterized by various combinations of ataxia, choreoathetosis, myoclonus, epilepsy and dementia as well as various ages of onset. We have identified a specific unstable trinucleotide repeat expansion in a gene on the short arm of chromosome 12 as the pathogenic mutation for DRPLA. We investigated how the degree of the expansion of the CAG repeat affects the clinical manifestations of DRPLA. The sizes of the expanded alleles were well correlated with the ages of onset (r = −0.6955, P < 0.001). Patients with progressive myoclonus epilepsy (PME) phenotype had larger expansions (62–79 repeats) and earlier ages of onset (onset before age 20). Furthermore, most of the patients with PME phenotype inherited their expanded alleles from their affected fathers. On the other hand, patients with non-PME phenotype showed later ages of onset (onset after age 20) and smaller expansions (54–67 repeats). When ages of onset of each clinical symptom are compared with sizes of the CAG repeat, there is again a remarkably high correlation of the sizes of CAG repeat with each of the clinical symptoms. Thus the wide variation in clinical manifestations of DRPLA can now be clearly explained based on the degree of CAG repeat expansion, which strongly indicates that the expanded alleles are intimately involved in the neuronal degeneration in dentatofugal and pallidofugal systems.     

Population and family studies of CAG repeats in the IT-15 gene

A simple and effective method for typing of CAG repeats in the IT-15 gene has been suggested. This method was applied for examination of the CAG allele distribution in Huntington's disease (HD) patients in five different populations from the Commonwealth of Independent States. A total of 21 normal alleles with the sizes ranging from 9 to 32 triplet repeat units were revealed. Moreover, alleles with the size ranging from 16 to 20 repeats predominated constituting from 54.4 to 74.6% of all alleles in different populations. The number of repeats in one allele in HD patients exceeded 38 units (43 triplets on average). In two families an increase in the CAG repeat units number in the mutant allele upon its paternal transmission was recorded.     

High germinal instability of the (CTG)n at the SCA8 locus of both expanded and normal alleles

The autosomal dominant spinocerebellar ataxias (SCAs) are a group of late-onset, neurodegenerative disorders for which 10 loci have been mapped (SCA1, SCA2, SCA4-SCA8, SCA10, MJD, and DRPLA). The mutant proteins have shown an expanded polyglutamine tract in SCA1, SCA2, MJD/SCA3, SCA6, SCA7, and DRPLA; a glycine-to-arginine substitution was found in SCA6 as well. Recently, an untranslated (CTG)n expansion on chromosome 13q was described as being the cause of SCA8. We have now (1) assessed the repeat size in a group of patients with ataxia and a large number of controls, (2) examined the intergenerational transmission of the repeat, and (3) estimated the instability of repeat size in the sperm of one patient and two healthy controls. Normal SCA8 chromosomes showed an apparently trimodal distribution, with classes of small (15-21 CTGs), intermediate (22-37 CTGs), and large (40-91 CTGs) alleles; large alleles accounted for only0.7% of all normal-size alleles. No expanded alleles (>/=100 CTGs) were found in controls. Expansion of the CTG tract was found in five families with ataxia; expanded alleles (all paternally transmitted) were characterized mostly by repeat-size contraction. There was a high germinal instability of both expanded and normal alleles: in one patient, the expanded allele (152 CTGs) had mostly contraction in size (often into the normal range); in the sperm of two normal controls, contractions were also more frequent, but occasional expansions into the upper limit of the normal size range were also seen. In conclusion, our results show (1) no overlapping between control (15-91) and pathogenic (100-152) alleles and (2) a high instability in spermatogenesis (both for expanded and normal alleles), suggesting a high mutational rate at the SCA8 locus.     

Transglutaminase activity is related to CAG repeat length in patients with Huntington’s disease

Huntington’s disease (HD) is a neurodegenerative disorder associated with CAG repeat expansion. We measured transglutaminase (TGase) activity in lymphocytes from 35 HD patients and from healthy individuals to ascertain whether it was altered in this condition. TGase activity was above maximum control levels in 25% of HD patients; it was correlated with the age of the patient and inversely correlated with the CAG repeat length. These results suggest that: (1) HD could be biochemically heterogeneous, and (2) the length of the CAG repeat expansion/TGase ratio could be important in the manifestation of HD. Received: 25 March 1996 / Revised: 23 June 1996