Kiyoshi Iwabuchi

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 GAG 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.

Neuronal intranuclear inclusions in spinocerebellar ataxia type 2: triple-labeling immunofluorescent study

Spinocerebellar ataxia type 2 (SCA2) is associated with an expansion of CAG/polyglutamine-repeat of a gene of unknown function. We performed an immunohistochemical study to identify the immunolocalization of the disease protein ataxin-2 in normal and SCA2 patients. Although normal and expanded ataxin-2 were ubiquitously localized to the cytoplasm of neurons, ubiquitinated intranuclear inclusions were observed selectively in 1-2% of neurons of affected brain regions except the cerebellum. Triple-labeling immunofluorescence revealed that ataxin-2, expanded polyglutamine and ubiquitin were colocalized to these neuronal intranuclear inclusions (NIs), indicating that SCA2 shares morphological characteristics common to other neurological disorders associated with an expansion of CAG/polyglutamine-repeat. Lack of NIs in the cerebellar lesion, however, suggests the discrepancy between formation of NIs and neuronal degeneration in SCA2.

Linkage of autosomal recessive hereditary spastic paraplegia with mental impairment and thin corpus callosum to chromosome 15q13-15

To date, three loci for autosomal recessive hereditary spastic paraplegia (ARHSP) linked to chromosomes 8p12‐q13, 16qter, and 15q13–15 have been characterized. We have clinically characterized 13 Japanese ARHSP families and performed genetic linkage analyses. All 13 families were classified as having the “complicated” form, which manifests with mental impairment and thin corpus callosum. Linkage to the 8p12‐q13 and 16qter loci was excluded, although 10 of the 13 families showed marker data consistent with linkage to the 15q13–15 locus. The multipoint LOD score of the 10 families linked to chromosome 15 was above 9.00 in the 3‐centimorgan segment flanked by D15S994 and D15S659, with a maximum multipoint LOD score of 9.68 at a position 1.2 centimorgans telomeric from D15S994 to D15S659. We have shown that ARHSP with thin corpus callosum, a subtype of recessive spastic paraplegia, maps to chromosome 15q13–15. Ann Neurol 2000;48:108–112

Non-expanded polyglutamine proteins in intranuclear inclusions of hereditary ataxias--triple-labeling immunofluorescence study.

Abstract. Neuronal intranuclear inclusions (NIIs) found in CAG/polyglutamine-expansion disorders contain both expanded polyglutamine and the gene product without the CAG repeat. The gene product containing expanded polyglutamine has, therefore, been considered to be a major component of NIIs. In this immunohistochemical study, we showed recruitment of ataxin-2, ataxin-3 and TATA box binding protein (TBP) into NIIs of the pontine neurons of spinocerebellar ataxia type (SCA) 1, SCA2, SCA3 and dentatorubral-pallidoluysian atrophy brains. Triple-labeling immunofluorescence demonstrated colocalization of ataxin-2 and ataxin-3 in NIIs containing expanded polyglutamine, irrespective of the disease examined. These in vivo findings indicate that polyglutamine proteins recruited into NIIs are not restricted to their expanded form. Among these proteins, recruitment of ataxin-2 was least frequent in every case examined, suggesting that the rate of recruitment partly depends on the protein transported into NIIs. Because other proteins lacking polyglutamine motif were not detected in NIIs, it is suggested that the presence of polyglutamine is a prerequisite for these proteins to be recruited into nucleus and to form NIIs. Interaction between expanded and non-expanded polyglutamine may play roles during these processes.

PML nuclear bodies and neuronal intranuclear inclusion in polyglutamine diseases

In polyglutamine diseases, accumulation in the nucleus of mutant proteins induces the formation of neuronal intranuclear inclusions (NIIs). The nucleus is compartmentalized into structural and functional domains, which are involved in NII formation. Promyelocytic leukemia protein (PML), a major component of nuclear bodies, and mSin3A, a component of the transcription co-repressor complex, were used to investigate how the intranuclear domains/sites relate to NII formation in SCA2, SCA3, SCA7, SCA17 and DRPLA brains. We demonstrate that the size of PML-positive intranuclear structures was larger in pathological brains than in control ones and that these structures contained mutant proteins. PML colocalized only with small NIIs, which maintained the ring-like structure of normal nuclear bodies. Enlarged ring-like PML-positive structures, devoid of mutant proteins, were also found and might represent structures where mutant polyglutamine proteins have been successfully processed. These data suggest that NIIs originate from nuclear bodies, where mutant proteins accumulate for degradation.

Phenotype variation correlates with CAG repeat length in SCA2: A study of 28 Japanese patients

Spinocerebellar ataxia-2 (SCA2) is an autosomal dominant ataxia caused by an abnormal CAG repeat expansion in a novel gene on chromosome 12q24.1. The size of the mutant allele is unstable during transmission, and correlates inversely with age at onset. We studied eight Japanese SCA2 families, including 28 patients, to assess the effect of repeat length on the phenotype features of SCA2. Frequencies of slow eye movements (SEM), reflex activity, dementia, choreiform movements, and axial tremor correlated significantly with CAG repeat size. Parkinsonism was seen in a man homozygote for SCA2 mutation. The clinical variety of SCA2 is apparently influenced by the size of the mutant allele, as is the case in other CAG repeat disorders.

Asymmetrical temporal lobe atrophy with massive neuronal inclusions in multiple system atrophy

This report concerns a rare association of asymmetrical temporal lobe atrophy with multiple system atrophy (MSA). A 53-year-old Japanese woman developed cerebellar ataxia and parkinsonism and was diagnosed as olivopontocerebellar atrophy (OPCA). This patient showed forgetfulness and subsequent disorientation even in the early stage of the disease. She fell into a decorticate state at the age of 64, and died a year later. The autopsy showed MSA with asymmetrical atrophy of temporal lobes, intraneuronal globular inclusions mostly confined to the hippocampus, amygdaloid nucleus, and most abundant in the granule cells in the dentate fascia. These inclusions were intensely argyrophilic and expressed marked immunoreactivity to ubiquitin, but not to neurofilament (NF), tau and paired helical filaments (PHF). Ultrastructurally, they were composed of scattered short filamentous structures of 15 to 30 nm in diameter, ribosome-like granules, mitochondria and lipofuscin. The lack of immunoreactivity against tau, NF and PHF suggests that the inclusions are distinct from Pick bodies. To our knowledge, MSA in association with asymmetrical temporal lobe atrophy with the present neuronal inclusions has not been reported. This case is distinct from MSA combined with atypical Picks disease in the distribution and immunohistochemical properties of neuronal inclusions, and may present a new variant of MSA since the neuronal inclusions are similar, in many respects, to those of neuronal inclusions reported in MSA. Globular inclusions are also discussed in variants of Picks disease, amyotrophic lateral sclerosis and Alzheimers disease.

Attenuated nuclear shrinkage in neurons with nuclear aggregates: A morphometric study on pontine neurons of Machado-Joseph disease brains

Nuclear aggregates (NAs) and neurodegeneration in brains from patients with Machado-Joseph disease (MJD) are both triggered by pathological expansion of CAG/polyglutamine repeat in ataxin-3, but it remains to be clarified whether NA formation is associated with accelerated neurodegeneration. In an attempt to clarify a possible influence of NAs on neurons in human brains, we quantified the size and deformity of neuronal nuclei (those with or without NAs, separately) cross-sectioned on pontine preparations of autopsied brains from four patients with MJD and five controls. Nuclear shrinkage and deformity were more marked in MJD brains than in controls, and these changes were attenuated in neurons harboring NAs. NAs of MJD are presumably linked to a mechanism that attenuates rather than accelerates nuclear shrinkage and deformity. This finding leads us to consider that NAs are not necessarily toxic to neurons in diseased human brains.

Nasu-Hakola's disease (membranous lipodystrophy)

SummaryAn autopsy case of Nasu-Hakolas disease (membranous lipodystrophy) is reported. A 43-year-old Japanese man, whose parents were not consanguineous, had been suffering from frequent long bone fractures since the age of 10. Neuropsychiatric symptoms, which were characterized by euphoria, disturbance of attention and dementia, appeared at his thirties and generalized and/or localized seizures and apallial syndrome at the later stage. The neuropathology revealed diffuse leukoencephalopathy of the cerebrum. The peculiar aspects in this case were membranocystic changes in the lungs [Yagishita et al. Virchows Arch [A] 408:211–217 (1985)], diffuse degeneration of the cerebral cortex, chiefly in frontal and temporal lobes, and many axonal spheroids throughout the cerebral cortex. The ultrastructure of spheroids in the cerebral cortex demonstrated aggregations of mitochondria, dense bodies and minute concentric bodies and a small amount of neurofilaments.

JAPANESE CREUTZFELDT-JAKOB DISEASE PATIENTS EXHIBITING HIGH INCIDENCE OF THE E200K PRNP MUTATION AND LOCATED IN THE BASIN OF A RIVER

Seven cases with Creutzfeldt-Jakob disease (CJD) located in the basin of the Fuji river (Fuji area) in Japan were examined genetically and clinicopathologically. The onset of the disease was between 1989 and 1995. All cases were from different families, although 3 cases were family members of previously reported CJD patients. They had clinical and/or neuropathological features, corresponding to subacute spongiform encephalopathy. Five of the 7 cases, including the 3 familial cases, had the E200K mutation in the gene encoding prion protein (PRNP). It is suggested that there is a small cluster of CJD patients with a founder effect of the E200K mutation in the Fuji area, because the incidence of CJD with the E200K mutation appears to be much higher in this area than other areas in Japan. The disease penetrance of the 5 cases with the E200K mutation seems to be low, and they may have an age-related incidence in the Fuji area. These findings support the hypothesis that the phenotypes of CJD patients with the PRNP mutations are linked to the position of the mutation, but not related to ethnic or environmental factors.