Suppression of aggregate formation and apoptosis by transglutaminase inhibitors in cells expressing truncated DRPLA protein with an expanded polyglutamine stretch.

To elucidate the molecular mechanisms whereby expanded polyglutamine stretches elicit a gain of toxic function, we expressed full-length and truncated DRPLA (dentatorubral-pallidoluysian atrophy) cDNAs with or without expanded CAG repeats in COS-7 cells. We found that truncated DRPLA proteins containing an expanded polyglutamine stretch form filamentous peri- and intranuclear aggregates and undergo apoptosis. The apoptotic cell death was partially suppressed by the transglutaminase inhibitors cystamine and monodansyl cadaverine (but not putrescine), suggesting involvement of a transglutaminase reaction and providing a potential basis for the development of therapeutic measures for CAG-repeat expansion diseases.

Identification of the spinocerebellar ataxia type 2 gene using a direct identification of repeat expansion and cloning technique, DIRECT.

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant, neurodegenerative disorder that affects the cerebellum and other areas of the central nervous system. We have devised a novel strategy, the direct identification of repeat expansion and cloning technique (DIRECT), which allows selective detection of expanded CAG repeats and cloning of the genes involved. By applying DIRECT, we identified an expanded CAG repeat of the gene for SCA2. CAG repeats of normal alleles range in size from 15 to 24 repeat units, while those of SCA2 chromosomes are expanded to 35 to 59 repeat units. The SCA2 cDNA is predicted to code for 1,313 amino acids-with the CAG repeats coding for a polyglutamine tract. DIRECT is a robust strategy for identification of pathologically expanded trinucleotide repeats and will dramatically accelerate the search for causative genes of neuropsychiatric diseases caused by trinucleotide repeat expansions.

Expanded polyglutamine stretches interact with TAFII130, interfering with CREB-dependent transcription.

At least eight inherited neurodegenerative diseases are caused by expanded CAG repeats encoding polyglutamine (polyQ) stretches. Although cytotoxicities of expanded polyQ stretches are implicated, the molecular mechanisms of neurodegeneration remain unclear. We found that expanded polyQ stretches preferentially bind to TAFII130, a coactivator involved in cAMP-responsive element binding protein (CREB)-dependent transcriptional activation, and strongly suppress CREB-dependent transcriptional activation. The suppression of CREB-dependent transcription and the cell death induced by polyQ stretches were restored by the co-expression of TAFII130. Our results indicate that interference of transcription by the binding of TAFII130 with expanded polyQ stretches is involved in the pathogenetic mechanisms underlying neurodegeneration.

Neuropathology of sporadic amyotrophic lateral sclerosis of long duration.

We performed post-mortem examinations of three patients with progressive neurogenic amyotrophy of long duration. One patient had been clinically diagnosed as having sporadic amyotrophic lateral sclerosis (ALS) and two had been diagnosed with progressive spinal muscular atrophy (PSMA). The disease durations were 10, 17 and 20 years, respectively, and all of the patients died of respiratory failure with no artificial respiratory support. In all of the patients, both the upper and lower motor neuron systems were affected; degeneration of the former was definite, but was milder than that usually encountered in sporadic ALS patients, and the histopathology of the latter was identical to that of sporadic ALS. Light microscopy revealed Bunina bodies, which are characteristic of sporadic ALS, in the remaining anterior horn cells of each patient. In addition, ubiquitin-positive skein-like inclusions were also identified, immunohistochemically, in the remaining anterior horn cells of each patient. Neuron counts indicated that the number of neurons was preserved in Clarke's column in these patients, but was significantly reduced in the intermediolateral nucleus, compared with control subjects. Based on these findings, we think that these three patients, with long disease durations, were affected by essentially the same underlying disease process as that of sporadic, classical ALS. Moreover, we question the neuropathological occurrence of sporadic ALS without involvement of the upper motor neuron system, namely, pure PSMA or lower motor neuron disease.

[An acute axonal polyneuropathy affecting intrinsic hand muscles following Campylobacter infection--a case report].

A 24-year-old carpenter had the shakes and fever on March 13, 1990. He suffered from watery diarrhea on March 14 and 15. He left muscle weakness in his thumbs and fingers when he drove nails with a hammer on March 24. The weakness reached maximum by the 3rd day of illness. He was admitted to our hospital on day 4. Neurological examination revealed symmetrical weakness localized in the intrinsic hand muscles (MRC grade 2-4). The deep tendon reflexes were preserved. Sensation was intact except for mild disturbance of superficial sense on both plantar areas. Campylobacter jejuni was cultured from his stool. A complement fixation test indicated serologically preceding C. jejuni infection. Whereas maximum motor nerve conduction velocities were not reduced and distal latencies were not prolonged, compound muscle action potential recorded in the thenar and hypothenar muscles were remarkably reduced on day 5. Needle EMG showed neuropathic changes in four limbs. Sensory nerve conduction velocities and action potentials were normal. The weakness gradually improved in association with increased compound muscle action potentials in the thenar and hypothenar muscles. His muscle symptom fully resolved 2 months after the onset of his illness. Thin-layer chromatogram with immunostaining revealed that serum IgG from this patient reacted with GM1, GD1a, GD1b, but did not react with GM2 and GT1b. Enzyme-linked immunosorbent assay showed that anti-GM1, GD1a, GD1b antibodies titer (IgG) decreased concurrently with the clinical improvement.

[Home care of patients with advanced terminal cancer who had improved QOL after undergoing gastrostomy for gastrointestinal obstruction caused by peritonitis carcinomatosa].

Patients with gastrointestinal obstruction caused by peritonitis carcinomatosa suffer from chronic nausea, abdominal distension and inability to ingest food. Patients can receive home care only in rare cases. We performed a gastrostomy for the purpose of reducing gastrointestinal pressure in three patients with gastrointestinal obstruction caused by peritonitis carcinomatosa. The three patients had decreased mental and physical suffering and were able to be discharged from the hospital. We report the efficacy of gastrostomy because of the improvements in the three patients QOL at home.

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 symptoms 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.

Dentatorubral-pallidoluysian atrophy (DRPLA). Molecular basis for wide clinical features of DRPLA.

Dentatorubral-pallidoluysian atrophy (DRPLA) is a rare autosomal dominant neurodegenerative disorder characterized clinically by various combinations of myoclonus, epilepsy, cerebellar ataxia, choreoathetosis, dementia and psychiatric symptoms. Based on the phenomenon of anticipation, the gene for DRPLA was recently identified. DRPLA is caused by unstable expansion of a CAG repeat in the gene located on the short arm of chromosome 12. As have been observed in Huntington's disease and SCA1, there is a strong correlation between the age of onset and the size of CAG repeats. Furthermore, patients with larger repeats tend to show a PME (progressive myoclonus epilepsy) phenotype as well as earlier ages of onset. More prominent anticipation and larger intergenerational increase of CAG repeats in paternal transmission can be accounted for by the meiotic instability of CAG repeats in male gametogenesis. Comparison of size distributions of CAG repeats in Japanese, African-American and white populations revealed that 7.4% of the Japanese alleles had greater than 19 repeats, whereas none of the whites and 1% of the African-American alleles were of this size. The results may account for the ethnic predilection of DRPLA.

Sporadic motor neuron disease with severe sensory neuronopathy.

We report a 62-year-old man with sporadic motor neuron disease (MND) of 52 months' duration with progressive sensory disturbance and high cerebrospinal fluid protein content. Neuropathologically, both the upper and lower motor neuron systems were severely affected, and light and electron microscopy revealed Bunina bodies and skein-like inclusions, which are characteristic of amyotrophic lateral sclerosis, in the remaining anterior horn cells. Moreover, there was severe degeneration without inflammatory infiltrates in the spinal posterior columns, spinal ganglia, and peripheral sensory nerves. These findings suggest that this case may be an unusual variant of sporadic MND with severe somatic sensory system involvement.

Molecular cloning of a full-length cDNA for dentatorubral-pallidoluysian atrophy and regional expressions of the expanded alleles in the CNS.

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder characterized by genetic anticipation and variable combinations of symptoms including myoclonus, epilepsy, cerebellar ataxia, choreoathetosis, and dementia. Recently, we discovered that DRPLA is caused by unstable expansion of a CAG repeat of a gene on the short arm of chromosome 12. We determined the consensus DRPLA cDNA sequence containing the complete coding region for 1,185 amino acids. The CAG repeat, which is expanded in DRPLA, is located 1,462 bp downstream from the putative methionine initiation codon and encodes a poly-glutamine tract. Although poly-serine and proline tracts exist near the CAG repeats, these polyserine or proline tracts did not show any polymorphisms, which is in strong contrast to the high heterogeneity in the length of the CAG repeat. Northern blot analysis revealed a 4.7-kb transcript that is widely expressed in various tissues including heart, lung, kidney, placenta, skeletal muscle, and brain. Reverse transcription-PCR analysis revealed that the expanded alleles are transcribed to levels comparable to those of normal alleles. These results indicate that there is no difference in transcriptional efficiency between expanded and normal alleles. Furthermore, mRNA from cerebellar hemispheres of DRPLA patients showed smaller sizes of CAG repeats compared with other regions of the brain, which reflects somatic mosaicism of the expanded alleles of the DRPLA gene.

Non-Mendelian transmission in dentatorubral-pallidoluysian atrophy and Machado-Joseph disease: the mutant allele is preferentially transmitted in male meiosis.

Autosomal dominant dentatorubral-pallidoluysian atrophy (DRPLA) and Machado-Joseph disease (MJD) are neurodegenerative disorders caused by CAG trinucleotide repeat expansions. An inverse correlation of age at onset with the length of the expanded CAG trinucleotide repeats has been demonstrated, and the intergenerational instability of the length of the CAG trinucleotide repeats, which is more prominent in paternal than in maternal transmissions, has been shown to underlie the basic mechanisms of anticipation in DRPLA and MJD. Our previous observations on DRPLA and MJD pedigrees, as well as a review of the literature, have suggested that the numbers of affected offspring exceed those of unaffected offspring, which is difficult to explain by the Mendelian principle of random segregation of alleles. In the present study, we analyzed the segregation patterns in 211 transmissions in 24 DRPLA pedigrees and 80 transmissions in 7 MJD pedigrees, with the diagnoses confirmed by molecular testing. Significant distortions in favor of transmission of the mutant alleles were found in male meiosis, where the mutant alleles were transmitted to 62% of all offspring in DRPLA (chi2 = 7.69; P<.01) and 73% in MJD (chi2 = 6.82; P<.01). The results were consistent with meiotic drive in DRPLA and MJD. Since more prominent meiotic instability of the length of the CAG trinucleotide repeats is observed in male meiosis than in female meiosis and meiotic drive is observed only in male meiosis, these results raise the possibility that a common molecular mechanism underlies the meiotic drive and the meiotic instability in male meiosis.

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.

Single sperm analysis of the CAG repeats in the gene for Machado-Joseph disease (MJD1): evidence for non-Mendelian transmission of the MJD1 gene and for the effect of the intragenic CGG/GGG polymorphism on the intergenerational instability.

To investigate the mechanism of the meiotic instability of expanded CAG repeats in the gene for Machado-Joseph disease (MJD1), we analyzed the CAG repeat sizes of 1036 single sperm from six individuals with Machado-Joseph disease (MJD). The segregation ratio between single sperm with an expanded allele and those with a normal allele is significantly different (P <0.0001) from the expected 1:1 segregation ratio, which demonstrates segregation distortion of expanded alleles in male meiosis. In single sperm from individuals with the [expanded (CAG)n-CGG]/[normal (CAG)n-GGG] genotype, significantly greater instability of the CAG repeat was observed compared with single sperm from individuals with the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] genotype (F-test, P <0.001). These findings in single sperm confirm non-Mendelian transmission of the MJD1 gene and the effect of the intragenic CGG/GGG polymorphism on the intergenerational instability of the CAG repeats in the MJD1 gene, which have been observed in clinical and genetic studies. Our results indicate similarities and dissimilarities between MJD and Huntington's disease or myotonic dystrophy in terms of the inter-allelic interaction, segregation distortions and size distribution of trinucleotide repeats in mutant alleles. Further study is required to determine whether there is a common mechanism underlying the instability of the triplet repeats in 'triplet repeat diseases'.

Adenovirus-mediated expression of mutant DRPLA proteins with expanded polyglutamine stretches in neuronally differentiated PC12 cells. Preferential intranuclear aggregate formation and apoptosis.

To investigate the molecular mechanisms of neurodegeneration caused by expanded CAG repeats in dentatorubral-pallidoluysian atrophy (DRPLA), an autosomal dominant neuro degrees enerative disorder caused by unstable expansion of a CAG trinucleotide repeat in the DRPLA gene on 12p13.31, we established an efficient expression system for truncated and full-length DRPLA proteins with normal or expanded polyglutamine stretches in neuronally differentiated PC12 cells and fibroblasts using an adenovirus expression system. Although aggregate body formation was observed both in neuronally differentiated PC12 cells and in fibroblasts expressing truncated DRPLA proteins with Q82, >97% ( n = 3) of neuronally differentiated PC12 cells showed intra-nuclear inclusions, while only 31 21% ( n = 3) of fibro-blasts had intranuclear inclusions at 3 days after infection. The percentage of apoptotic cells was significantly higher in neuronally differentiated PC12 cells expressing the truncated DRPLA protein with Q82 than in fibroblasts, suggesting the possibility that intranuclear aggregate bodies are formed preferentially in neuronally differentiated PC12 cells and that these cells are more vulnerable than fibroblasts to the toxic effects of expanded polyglutamine stretches in the DRPLA protein. When the full-length DRPLA protein with Q82 was expressed, aggregate bodies were found exclusively in the nuclei of the neuronally differentiated PC12 cells, while they were found in the cytoplasm of fibroblasts. Despite the presence of aggregate bodies, apoptosis was not induced by expression of the full-length DRPLA protein with Q82 in either neuronally differentiated PC12 cells or fibroblasts, suggesting that the presence of intranuclear aggregate bodies is in itself not necessarily toxic to cells.

Japanese SCA families with an unusual phenotype linked to a locus overlapping with SCA15 locus.

The authors identified two Japanese spinocerebellar ataxia (SCA) families characterized by postural and action tremor and a very slow progression rate. A genome-wide linkage analysis revealed linkage to chromosome 3p26.1-25.3 with the highest multipoint lod score at D3S3728 (Zmax = 3.31 at theta = 0.00). The candidate region was 14.7 cM flanked by D3S1620 and D3S3691, which was partly overlapping with the locus of SCA15 characterized by pure cerebellar ataxia. Despite the difference in phenotypes, there remains a possibility that the causative gene for these Japanese SCA is allelic to SCA15.

Progressive atrophy of cerebellum and brainstem as a function of age and the size of the expanded CAG repeats in the MJD1 gene in Machado-Joseph disease.

Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disease characterized by cerebellar ataxia associated to varying degrees with pyramidal signs, extrapyramidal signs, or peripheral amyotrophy. It is caused by unstable expansion of the CAG repeat in the MJD1 gene on chromosome 14q32.1. To determine how the neurodegenerative process in the central nervous system of patients with MJD correlates with the size of expanded CAG repeats in the MJD1 gene and other factors, we performed detailed quantitative analyses of findings of magnetic resonance imaging of the central nervous system of 21 patients with MJD of various ages and with various sizes of expanded CAG repeats. We found that atrophy of the brainstem and cerebellar vermis in MJD patients is closely correlated not only with the size of expanded CAG repeat in the MJD1 gene but also with patient age, which suggests that the neurodegenerative process in MJD is regulated by the size of expanded CAG repeats as well as by the patient age.

Transgenic mice harboring a full-length human mutant DRPLA gene exhibit age-dependent intergenerational and somatic instabilities of CAG repeats comparable with those in DRPLA patients.

Dentatorubral-pallidoluysian atrophy (DRPLA) is one among an increasing number of hereditary neurodegenerative diseases determined as being caused by unstable expansion of CAG repeats coding for polyglutamine stretches. To investigate the molecular mechanisms underlying CAG repeat instability, we established three transgenic lines each harboring a single copy of a full-length human mutant DRPLA gene carrying a CAG repeat expansion. These transgenic mice exhibited an age-dependent increase (+0.31 per year) in male transmission and an age-dependent contraction (-1.21 per year) in female transmission. Similar tendencies in intergenerational instabilities were also observed in human DRPLA parent-offspring pairs. The intergenerational instabilities of the CAG repeats may be interpreted as being derived from the instability occurring during continuous cell division of spermatogonia in the male, and that occurring during the period of meiotic arrest in the female. The transgenic mice also exhibited an age-dependent increase in the degree of somatic mosaicism which occurred in a cell lineage-dependent manner, with the size range of CAG repeats being smaller in the cerebellum than in other tissues including the cerebrum, consistent with observations in autopsied tissues of DRPLA patients. Thus, the transgenic mice described in this study exhibited age-dependent intergenerational as well as somatic instabilities of expanded CAG repeats comparable with those observed in human DRPLA patients, and are therefore expected to serve as good models for investigating the molecular mechanisms of instabilities of CAG repeats.

Close associations between prevalences of dominantly inherited spinocerebellar ataxias with CAG-repeat expansions and frequencies of large normal CAG alleles in Japanese and Caucasian populations.

To test the hypothesis that the frequencies of normal alleles (ANs) with a relatively large number of CAG repeats (large ANs) are related to the prevalences of the dominant spinocerebellar ataxias (SCAs)-SCA types 1, 2, 3 (Machado-Joseph disease), 6, and dentatorubral-pallidoluysian atrophy (DRPLA)-we investigated the relative prevalences of these diseases in 202 Japanese and 177 Caucasian families and distributions of the number of CAG repeats of ANs at these disease loci in normal individuals in each population. The relative prevalences of SCA1 and SCA2 were significantly higher in Caucasian pedigrees (15% and 14%, respectively) than in Japanese pedigrees (3% and 5%, respectively), corresponding to the observation that the frequencies of large ANs of SCA1 (alleles >30 repeats) and of SCA2 (alleles >22 repeats) were significantly higher in Caucasians than in Japanese. The relative prevalences of MJD/SCA3, SCA6, and DRPLA were significantly higher in Japanese pedigrees (43%, 11%, and 20%, respectively) than in Caucasian pedigrees (30%, 5%, and 0%, respectively), corresponding to the observation that the frequencies of large ANs of MJD/SCA3 (>27 repeats), SCA6 (>13 repeats), and DRPLA (>17 repeats) were significantly higher in Japanese than in Caucasians. The close correlations of the relative prevalences of the dominant SCAs with the distributions of large ANs strongly support the assumption that large ANs contribute to generation of expanded alleles (AEs) and the relative prevalences of the dominant SCAs.

Intergenerational instability of the CAG repeat of the gene for Machado-Joseph disease (MJD1) is affected by the genotype of the normal chromosome: implications for the molecular mechanisms of the instability of the CAG repeat.

Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder caused by unstable expansion of a CAG repeat in the MJD1 gene at 14q32.1. To identify elements affecting the intergenerational instability of the CAG repeat, we investigated whether the CGG/GGG polymorphism at the 3' end of the CAG repeat affects intergenerational instability of the CAG repeat. The [expanded (CAG)n-CGG]/[normal (CAG)n-GGG] haplotypes were found to result in significantly greater instability of the CAG repeat compared to the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] or [expanded (CAG)nGGG]/[normal (CAG)n-GGG] haplotypes. Multiple stepwise logistic regression analysis revealed that the relative risk for a large intergenerational change in the number of CAG repeat units (< -2 or > 2) is 7.7-fold (95% CI: 2.5-23.9) higher in the case of paternal transmission than in that of maternal transmission and 7.4-fold (95% CI: 2.4-23.3) higher in the case of transmission from a parent with the [expanded (CAG)n-CGG]/[normal (CAG)n-GGG] haplotypes than in that of transmission from a parent with the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] or [expanded (CAG)n-GGG]/[normal (CAG)n-GGG] haplotypes. The combination of paternal transmission and the [expanded (CAG)n-CGG]/[normal (CAG)n-GGG] haplotypes resulted in a 75.2-fold (95% CI: 9.0-625.0) increase in the relative risk compared with that of maternal transmission and the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] or [expanded (CAG)n-GGG]/[normal (CAG)n-GGG] haplotypes. The results suggest that an inter-allelic interaction is involved in the intergenerational instability of the expanded CAG repeat.