Shigeki Kuzuhara

Lewy bodies are ubiquitinated

SummaryThe nature of Lewy bodies (LBs) in the brain stem and cerebral cortex in five cases of diffuse Lewy body disease and one case of Parkinsons disease with dementia were investigated immunocytochemically with various antibodies to cytoskeletal proteins, paired helical filaments (PHF) and ubiquitin. Antibodies to 200-kDa component of neurofilament, tau and PHF showed no significant reactions with most of LBs. Antibodies to high-molecular weight microtubule-associated proteins (HMWMAPs) moderately stained the periphery of a few of LBs. A monoclonal antibody to PHF (DF2) which recognizes ubiquitin, and polyclonal antibodies to ubiquitin immunostained virtually all of the typical and cortical LBs as intensely as Alzheimers neurofibrillary tangles and senile plaque neurites: the periphery of LBs was darkly stained, whereas the central core of typical LBs and central zone of cortical LBs were less intensely stained or remained unstained. Immunoelectron microscopy of the LBs with DF2 revealed that immune reaction products were located on the filaments exclusively in the periphery of LBs, but not on those in the center. These findings suggest that both types of LBs are immunocytochemically indistinguishable despite some structural differences, and that peripherally located filaments in LBs are tagged with ubiquitin, an element required for the ATP-dependent proteolysis system in the cell. Antibodies to ubiquitin are the most useful marker of LBs ever known.

Large-scale, multicenter study of cerebrospinal fluid tau protein phosphorylated at serine 199 for the antemortem diagnosis of Alzheimer's disease

We surveyed a total of 570 cerebrospinal fluid (CSF) samples from a variety of diseases, including Alzheimers disease (AD; n = 236), non‐AD‐demented and nondemented diseases (n = 239), and normal controls (n = 95) to quantitate levels of tau protein phosphorylated at serine 199 (CSF/phospho‐tau199) by a recently established sandwich ELISA. The CSF/phospho‐tau199 levels in the AD group were significantly elevated compared to those in all the other non‐AD groups. Receiver operating characteristics curves showed that the diagnostic sensitivity and specificity for the AD group versus all the other non‐AD groups using the CSF/phospho‐tau199 were 85.2% and 85.0%, respectively. Furthermore, there was a significant positive correlation between CSF/phospho‐tau199 and CSF/total‐tau levels in the AD group. Elevated CSF/phospho‐tau199 in the AD group was noted irrespective of age, gender, dementia severity, and number of apolipoprotein E4 alleles. Thus, we suggest that CSF/phospho‐tau199 may be a novel and logical biomarker in supporting antemortem diagnosis of AD.

CHCHD2 mutations in autosomal dominant late-onset Parkinson's disease: a genome-wide linkage and sequencing study

BACKGROUND Identification of causative genes in mendelian forms of Parkinsons disease is valuable for understanding the cause of the disease. We did genetic studies in a Japanese family with autosomal dominant Parkinsons disease to identify novel causative genes. METHODS We did a genome-wide linkage analysis on eight affected and five unaffected individuals from a family with autosomal dominant Parkinsons disease (family A). Subsequently, we did exome sequencing on three patients and whole-genome sequencing on one patient in family A. Variants were validated by Sanger sequencing in samples from patients with autosomal dominant Parkinsons disease, patients with sporadic Parkinsons disease, and controls. Participants were identified from the DNA bank of the Comprehensive Genetic Study on Parkinsons Disease and Related Disorders (Juntendo University School of Medicine, Tokyo, Japan) and were classified according to clinical information obtained by neurologists. Splicing abnormalities of CHCHD2 mutants were analysed in SH-SY5Y cells. We used the Fishers exact test to calculate the significance of allele frequencies between patients with sporadic Parkinsons disease and unaffected controls, and we calculated odds ratios and 95% CIs of minor alleles. FINDINGS We identified a missense mutation (CHCHD2, 182C>T, Thr61Ile) in family A by next-generation sequencing. We obtained samples from a further 340 index patients with autosomal dominant Parkinsons disease, 517 patients with sporadic Parkinsons disease, and 559 controls. Three CHCHD2 mutations in four of 341 index cases from independent families with autosomal dominant Parkinsons disease were detected by CHCHD2 mutation screening: 182C>T (Thr61Ile), 434G>A (Arg145Gln), and 300+5G>A. Two single nucleotide variants (-9T>G and 5C>T) in CHCHD2 were confirmed to have different frequencies between sporadic Parkinsons disease and controls, with odds ratios of 2·51 (95% CI 1·48-4·24; p=0·0004) and 4·69 (1·59-13·83, p=0·0025), respectively. One single nucleotide polymorphism (rs816411) was found in CHCHD2 from a previously reported genome-wide association study; however, there was no significant difference in its frequency between patients with Parkinsons disease and controls in a previously reported genome-wide association study (odds ratio 1·17, 95% CI 0·96-1·19; p=0·22). In SH-SY5Y cells, the 300+5G>A mutation but not the other two mutations caused exon 2 skipping. INTERPRETATION CHCHD2 mutations are associated with, and might be a cause of, autosomal dominant Parkinsons disease. Further genetic studies in other populations are needed to confirm the pathogenicity of CHCHD2 mutations in autosomal dominant Parkinsons disease and susceptibility for sporadic Parkinsons disease, and further functional studies are needed to understand how mutant CHCHD2 might play a part in the pathophysiology of Parkinsons disease. FUNDING Japan Society for the Promotion of Science; Japanese Ministry of Education, Culture, Sports, Science and Technology; Japanese Ministry of Health, Labour and Welfare; Takeda Scientific Foundation; Cell Science Research Foundation; and Nakajima Foundation.

Prospective 10-year surveillance of human prion diseases in Japan

We analysed the epidemiological data and clinical features of patients with prion diseases that had been registered by the Creutzfeldt-Jakob Disease Surveillance Committee, Japan, over the past 10 years, since 1999. We obtained information on 1685 Japanese patients suspected as having prion diseases and judged that 1222 patients had prion diseases, consisting of definite (n=180, 14.7%) and probable (n=1029, 84.2%) cases, except for dura mater graft-associated Creutzfeldt-Jakob disease which also included possible cases (n=13, 1.1%). They were classified into 922 (75.5%) with sporadic Creutzfeldt-Jakob disease, 216 (17.7%) with genetic prion diseases, 81 (6.6%) with acquired prion diseases, including 80 cases of dura mater graft-associated Creutzfeldt-Jakob disease and one case of variant Creutzfeldt-Jakob disease, and three cases of unclassified Creutzfeldt-Jakob disease (0.2%). The annual incidence rate of prion disease ranged from 0.65 in 1999 to 1.10 in 2006, with an average of 0.85, similar to European countries. Although methionine homozygosity at codon 129 polymorphism of the prion protein gene was reported to be very common (93%) in the general Japanese population, sporadic Creutzfeldt-Jakob disease in Japan was significantly associated with codon 129 homozygosity (97.5%), as reported in western countries. In sporadic Creutzfeldt-Jakob disease, MM1 type (Parchis classification) is the most common, as in western countries. Among atypical sporadic Creutzfeldt-Jakob disease cases, the MM2 type appeared most common, probably related to the very high proportion of methionine allele in the Japanese population. As for iatrogenic Creutzfeldt-Jakob disease, only dura mater graft-associated Creutzfeldt-Jakob disease cases were reported in Japan and, combined with the data from previous surveillance systems, the total number of dura mater graft-associated Creutzfeldt-Jakob disease was 138, comprising the majority of worldwide dura mater graft-associated Creutzfeldt-Jakob disease patients. Regarding genetic prion diseases, the most common mutation of prion protein gene was V180I (41.2%), followed by P102L (18.1%), E200K (17.1%) and M232R (15.3%), and this distribution was quite different from that in Europe. In particular, V180I and M232R were quite rare mutations worldwide. Patients with V180I or M232R mutations rarely had a family history of prion diseases, indicating that a genetic test for sporadic cases is necessary to distinguish these from sporadic Creutzfeldt-Jakob disease. In conclusion, our prospective 10-year surveillance revealed a frequent occurrence of dura mater graft-associated Creutzfeldt-Jakob disease, and unique phenotypes of sporadic Creutzfeldt-Jakob disease and genetic prion diseases related to the characteristic distribution of prion protein gene mutations and polymorphisms in Japan, compared with those in western countries.

Corticofugal projections to the motor nuclei of the brainstem and spinal cord in humans

We studied corticofugal projections to the motoneurons with Nauta-Gygaxs technique in a patient with cerebral infarction of both hemispheres. Motoneurons in the brainstem motor nuclei and spinal anterior horns seem to receive direct cortical projections, except for the oculomotor and abducens nuclei and Onuf s nucleus in the sacral cord.

Familial amyotrophic lateral sclerosis and parkinsonism-dementia complex of the Kii peninsula of Japan: Clinical and neuropathological study and tau analysis

We report the first case of neuropathologically verified parkinsonism‐dementia complex of the Kii peninsula, together with the patients brother, who had amyotrophic lateral sclerosis. The propositus woman developed parkinsonism and dementia at 63 years of age and died at 70 without displaying clinical features of amyotrophic lateral sclerosis. The brain exhibited marked atrophy of the frontal and temporal lobes. Microscopically, there were many neurofibrillary tangles in the central nervous system, most markedly in the mesial temporal lobe and deep nuclei, as well as changes of amyotrophic lateral sclerosis but no senile plaques or Lewy bodies. Neurofibrillary tangles exhibited twisted tubule structures on electon microscopic examination, and an analysis of insoluble tau protein extracted from the fresh brain revealed a 60‐, 64‐, 68‐kD triplet. The tau gene exhibited no mutations. Her brother developed progressive bulbar palsy–type amyotrophic lateral sclerosis at 45 years of age and died at 49 without presenting with dementia or parkinsonism. Neuropathological examination revealed not only pathologic features of amyotrophic lateral sclerosis but also a moderate number of neurofibrillary tangles in the temporal cortex and deep nuclei. The siblings were neuropathologically similar despite their different clinical manifestations. These findings suggest that amyotrophic lateral sclerosis and parkinsonism‐dementia complex of this family may be phenotypic variants of a tauopathy caused by genetic abnormalities. Ann Neurol 2001;49:501–511

Immunocytochemical and ultrastructural study of Lewy body-like hyaline inclusions in familial amyotrophic lateral sclerosis

SummaryLewy body-like hyaline inclusion (LI) in the neuronal soma and swollen cord-like processes is a characteristic feature in the anterior horn cells and neurons in thoracic nucleus (Clarke) of familial amyotrophic lateral sclerosis (ALS) with posterior column involvement. We have studied the LI in the case of two sisters with this disorders. Microscopically the LI consists of an eosinophilic “core” surrounded by a basophilic “halo”. Ultrastructurally the core consists of granule-associated filaments, while the halo consists of normal-looking neurofilament. Immunocytochemistry with anti-ubiquitin antibody shows that these granule-associated filaments in the core are highly ubiquitinated, while the normal-looking neurofilaments in the halo are not recognized by antiubiquitin antibody. Our study proves that LI consists of an aggregation of ubiquitinated filaments among a neurofilamentous accumulation, possibly representing a form of neuronal cytoskeletal disorganization in familial ALS.

C9ORF72 Repeat Expansion in Amyotrophic Lateral Sclerosis in the Kii Peninsula of Japan

BACKGROUND In the Kii peninsula of Japan, high prevalences of amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia complex have been reported. There are 2 major foci with a high prevalence, which include the southernmost region neighboring the Koza River (Kozagawa and Kushimoto towns in Wakayama prefecture) and the Hohara district (Mie prefecture). OBJECTIVE To delineate the molecular basis of ALS in the Kii peninsula of Japan, we analyzed hexanucleotide repeat expansion in the chromosome 9 open reading frame 72 (C9ORF72) gene, which has recently been identified as a frequent cause of ALS and frontotemporal dementia in the white population. DESIGN Case series. SETTING University hospitals. PATIENTS Twenty-one patients (1 familial patient and 20 sporadic patients) with ALS from Wakayama prefecture, and 16 patients with ALS and 16 patients with parkinsonism-dementia complex originating from Mie prefecture surveyed in 1994 through 2011 were enrolled in the study. In addition, 40 probands with familial ALS and 217 sporadic patients with ALS recruited from other areas of Japan were also enrolled in this study. MAIN OUTCOME MEASURES After screening by repeat-primed polymerase chain reaction, Southern blot hybridization analysis was performed to confirm the expanded alleles. RESULTS We identified 3 patients with ALS (20%) with the repeat expansion in 1 of the 2 disease foci. The proportion is significantly higher than those in other regions in Japan. Detailed haplotype analyses revealed an extended shared haplotype in the 3 patients with ALS, suggesting a founder effect. CONCLUSIONS Our findings indicate that the repeat expansion partly accounts for the high prevalence of ALS in the Kii peninsula.

Widespread expression of α-synuclein and τ immunoreactivity in Hallervorden-Spatz syndrome with protracted clinical course

Hallervorden-Spatz syndrome (HSS) is a rare autosomal recessive disorder clinically characterized by extrapyramidal signs and progressive dementia. In a typical case, the clinical symptoms become apparent during late childhood, and usually the course is protracted over a decade or more. We recently had an opportunity to study the brains of two cases of HSS with a clinical course of over 30 years. Case 1 was a 44-year-old female and case 2 was a 37-year-old male. Grossly, the brains showed severe fronto-temporal lobar atrophy with abundant spheroids and mild iron deposits in the globus pallidus, associated with features of motor neuron disease. In addition, there was diffuse sponginess in the atrophic cortex as well as widespread Alzheimers neurofibrillary tangles (NFTs) and Lewy bodies (LBs) in the cortical and subcortical regions, including the spinal cord. Ultrastructurally, NFTs were composed of paired helical filaments, and LBs of central dense cores with radiating fibrils. Discrete immunostaining was demonstrated in NFTs and neuropil threads with various antibodies against phosphorylated tau, and in LBs with antibody against alpha-synuclein. In addition, diffuse, overlapping immunoreactivity of alpha-synuclein and phosphorylated tau was seen within the cytoplasm of many neurons. However, when LBs and NFTs coexisted within the same neurons, they were clearly segregated. The findings of our present cases as well as those reported in the literature may indicate that simultaneous and extensive occurrence of abnormal phosphorylation of tau and accumulation of alpha-synuclein may constitute cardinal pathological features of HSS with protracted clinical course.

Activated brain regions in musicians during an ensemble: a PET study.

As in visual processing, we speculated that, in music processing, different brain regions would activate according to the mode of music listening. Using motets by a famous composer, we studied changes in regional cerebral blood flow (rCBF) with positron emission tomography associated with concentrating on the alto-part within the harmony (alto-part-listening condition) compared to listening to the harmony as a whole (harmony-listening condition). The alto-part-listening condition was associated with bilateral increases of rCBF in superior parietal lobules, precunei, premotor areas and orbital frontal cortices. Superior parietal lobules are likely to be responsible for auditory selective attention to the alto part within the harmony and the analysis of tone pitch on a mental score. The precuneus possibly participated in writing tones of the alto part on a mental score. Based on our findings, we propose that both auditory selective attention and analytic processing play an important role in concentrating on a certain vocal part within a harmony. During the harmony-listening condition, temporal poles, the anterior portion of the cingulate gyrus, occipital cortex and the medial surface of the cerebellum were bilaterally activated. Further studies are necessary to clarify the difference in music processing between musicians and nonmusicians.