Hidetoshi Date

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.

IFNβ-1b may severely exacerbate Japanese optic-spinal MS in neuromyelitis optica spectrum

Background: Interferon-β-1b (IFNβ-1b) has been used to prevent exacerbation of relapsing-remitting multiple sclerosis (RRMS) including optic-spinal multiple sclerosis (OSMS) in Japan. We encountered 2 patients with OSMS with unexpectedly severe exacerbation soon after the initiation of IFNβ-1b therapy. The experience urged us to retrospectively review the patients with RRMS who had been treated with IFNβ-1b to identify similar cases. Methods: At neurologic departments of 9 hospitals, the medical records of 56 patients with RRMS were reviewed to identify those who showed severe exacerbation soon after the initiation of IFNβ-1b therapy. Results: Of 56 patients with RRMS, we identified 7 who experienced severe exacerbation (exacerbation with increased scores of Expanded Disability Status Scale ≧7.0) within 90 days of the initiation of IFNβ-1b therapy. In all 7 patients, the exacerbations after the initiation of IFNβ-1b therapy were more severe than those experienced by the individual patients before the use of IFNβ-1b, and seemed to have occurred unexpectedly in a short time after the initiation of INFβ-1b therapy. A retrospective analysis revealed that all 7 patients had antibodies toward aquaporin 4, and the clinical features of all 7 patients after the exacerbation were consistent with those of neuromyelitis optica (NMO) spectrum. Conclusions: Our study suggests that IFNβ-1b may trigger severe exacerbation in patients with the NMO spectrum. In INFβ-1b therapy, cases in NMO spectrum should be carefully excluded.

Mutations in COQ2 in familial and sporadic multiple-system atrophy the multiple-system atrophy research collaboration

BACKGROUND Multiple-system atrophy is an intractable neurodegenerative disease characterized by autonomic failure in addition to various combinations of parkinsonism, cerebellar ataxia, and pyramidal dysfunction. Although multiple-system atrophy is widely considered to be a nongenetic disorder, we previously identified multiplex families with this disease, which indicates the involvement of genetic components. METHODS In combination with linkage analysis, we performed whole-genome sequencing of a sample obtained from a member of a multiplex family in whom multiple-system atrophy had been diagnosed on autopsy. We also performed mutational analysis of samples from members of five other multiplex families and from a Japanese series (363 patients and two sets of controls, one of 520 persons and one of 2383 persons), a European series (223 patients and 315 controls), and a North American series (172 patients and 294 controls). On the basis of these analyses, we used a yeast complementation assay and measured enzyme activity of parahydroxybenzoate-polyprenyl transferase. This enzyme is encoded by the gene COQ2 and is essential for the biosynthesis of coenzyme Q10. Levels of coenzyme Q10 in lymphoblastoid cells and brain tissue were measured on high-performance liquid chromatography. RESULTS We identified a homozygous mutation (M78V-V343A/M78V-V343A) and compound heterozygous mutations (R337X/V343A) in COQ2 in two multiplex families. Furthermore, we found that a common variant (V343A) and multiple rare variants in COQ2, all of which are functionally impaired, are associated with sporadic multiple-system atrophy. The V343A variant was exclusively observed in the Japanese population. CONCLUSIONS Functionally impaired variants of COQ2 were associated with an increased risk of multiple-system atrophy in multiplex families and patients with sporadic disease, providing evidence of a role of impaired COQ2 activities in the pathogenesis of this disease. (Funded by the Japan Society for the Promotion of Science and others.).

CpG Demethylation Enhances Alpha-Synuclein Expression and Affects the Pathogenesis of Parkinson's Disease

Background Alpha-synuclein (SNCA) gene expression is an important factor in the pathogenesis of Parkinsons disease (PD). Gene multiplication can cause inherited PD, and promoter polymorphisms that increase SNCA expression are associated with sporadic PD. CpG methylation in the promoter region may also influence SNCA expression. Methodology/Principal Findings By using cultured cells, we identified a region of the SNCA CpG island in which the methylation status altered along with increased SNCA expression. Postmortem brain analysis revealed regional non-specific methylation differences in this CpG region in the anterior cingulate and putamen among controls and PD; however, in the substantia nigra of PD, methylation was significantly decreased. Conclusions/Significance This CpG region may function as an intronic regulatory element for SNCA gene. Our findings suggest that a novel epigenetic regulatory mechanism controlling SNCA expression influences PD pathogenesis.

Mutations for Gaucher Disease Confer High Susceptibility to Parkinson Disease

BACKGROUND Increased frequency of pathogenic variants in GBA, the causative gene for Gaucher disease, has been suggested to be associated with Parkinson disease (PD). OBJECTIVES To conduct comprehensive resequencing of GBA to identify all sequence variants and to investigate the association of these variants with PD. DESIGN Case-control study. SETTING Multicenter university-based study. PARTICIPANTS Five hundred thirty-four patients with PD, 34 families in which multiple patients with PD are present, and 544 control subjects. MAIN OUTCOME MEASURES Disease status and GBA variations. RESULTS Comprehensive resequencing of GBA in 534 patients with PD and 544 controls revealed 27 sequence variants: 11 pathogenic variants associated with Gaucher disease, 11 nonsynonymous variants not associated with Gaucher disease, and 5 synonymous variants. Fifty patients with PD (9.4%) had 1 of the 11 pathogenic variants in the heterozygous state, whereas only 2 controls (0.37%) had such variants (odds ratio, 28.0). Among the pathogenic variants, R120W and L444P/RecNciI were highly prevalent, and each showed a significant association with PD. Furthermore, other rare pathogenic variants were found in 13 patients with PD but not in the controls, further confirming the role of these rare variants in the susceptibility to PD. Patients with PD carrying pathogenic variants were significantly younger than those not carrying them. In addition, concordance of PD states and pathogenic variants was observed in 8 multiplex families with PD. CONCLUSION Heterozygous pathogenic variants in GBA confer a high risk for sporadic PD, even for familial clustering, and are associated with significantly earlier age at onset of disease.

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.

Intranuclear Degradation of Polyglutamine Aggregates by the Ubiquitin-Proteasome System

Huntington disease and its related autosomal-dominant polyglutamine (pQ) neurodegenerative diseases are characterized by intraneuronal accumulation of protein aggregates. Studies on protein aggregates have revealed the importance of the ubiquitin-proteasome system as the front line of protein quality control (PQC) machinery against aberrant proteins. Recently, we have shown that the autophagy-lysosomal system is also involved in cytoplasmic aggregate degradation, but the nucleus lacked this activity. Consequently, the nucleus relies entirely on the ubiquitin-proteasome system for PQC. According to previous studies, nuclear aggregates possess a higher cellular toxicity than do their cytoplasmic counterparts, however degradation kinetics of nuclear aggregates have been poorly understood. Here we show that nuclear ubiquitin ligases San1p and UHRF-2 each enhance nuclear pQ aggregate degradation and rescued pQ-induced cytotoxicity in cultured cells and primary neurons. Moreover, UHRF-2 is associated with nuclear inclusion bodies in vitro and in vivo. Our data suggest that UHRF-2 is an essential molecule for nuclear pQ degradation as a component of nuclear PQC machinery in mammalian cells.

Aprataxin, the causative protein for EAOH is a nuclear protein with a potential role as a DNA repair protein

Early‐onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH) is an autosomal recessive neurodegenerative disorder characterized by early‐onset ataxia, ocular motor apraxia, and hypoalbuminemia. Recently, the causative gene for EAOH, APTX, has been identified. Of the two splicing variants of APTX mRNA, the short and the long forms, long‐form APTX mRNA was found to be the major isoform. Aprataxin is mainly located in the nucleus, and, furthermore, the first nuclear localization signal located near the amino terminus of the long‐form aprataxin is essential for its nuclear localization. We found, based on the yeast two‐hybrid and coimmunoprecipitation experiments, that the long‐form but not the short‐form aprataxin interacts with XRCC1 (x‐ray repair cross‐complementing group 1). Interestingly the amino terminus of the long‐form aprataxin is homologous with polynucleotidekinase‐3′‐phosphatase, which has been demonstrated to be involved in base excision repair, a subtype of single‐strand DNA break repair, through interaction with XRCC1, DNA polymerase β, and DNA ligase III. These results strongly support the possibility that aprataxin and XRCC1 constitute a multiprotein complex and are involved in single‐strand DNA break repair, and furthermore, that accumulation of unrepaired damaged DNA underlies the pathophysiological mechanisms of EAOH.

Aprataxin, causative gene product for EAOH/AOA1, repairs DNA single-strand breaks with damaged 3′-phosphate and 3′-phosphoglycolate ends

Aprataxin is the causative gene product for early-onset ataxia with ocular motor apraxia and hypoalbuminemia/ataxia with oculomotor apraxia type 1 (EAOH/AOA1), the clinical symptoms of which are predominantly neurological. Although aprataxin has been suggested to be related to DNA single-strand break repair (SSBR), the physiological function of aprataxin remains to be elucidated. DNA single-strand breaks (SSBs) continually produced by endogenous reactive oxygen species or exogenous genotoxic agents, typically possess damaged 3′-ends including 3′-phosphate, 3′-phosphoglycolate, or 3′-α, β-unsaturated aldehyde ends. These damaged 3′-ends should be restored to 3′-hydroxyl ends for subsequent repair processes. Here we demonstrate by in vitro assay that recombinant human aprataxin specifically removes 3′-phosphoglycolate and 3′-phosphate ends at DNA 3′-ends, but not 3′-α, β-unsaturated aldehyde ends, and can act with DNA polymerase β and DNA ligase III to repair SSBs with these damaged 3′-ends. Furthermore, disease-associated mutant forms of aprataxin lack this removal activity. The findings indicate that aprataxin has an important role in SSBR, that is, it removes blocking molecules from 3′-ends, and that the accumulation of unrepaired SSBs with damaged 3′-ends underlies the pathogenesis of EAOH/AOA1. The findings will provide new insight into the mechanism underlying degeneration and DNA repair in neurons.

ERBB4 Mutations that Disrupt the Neuregulin-ErbB4 Pathway Cause Amyotrophic Lateral Sclerosis Type 19

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disorder characterized by the degeneration of motor neurons and typically results in death within 3-5 years from onset. Familial ALS (FALS) comprises 5%-10% of ALS cases, and the identification of genes associated with FALS is indispensable to elucidating the molecular pathogenesis. We identified a Japanese family affected by late-onset, autosomal-dominant ALS in which mutations in genes known to be associated with FALS were excluded. A whole- genome sequencing and parametric linkage analysis under the assumption of an autosomal-dominant mode of inheritance with incomplete penetrance revealed the mutation c.2780G>A (p. Arg927Gln) in ERBB4. An extensive mutational analysis revealed the same mutation in a Canadian individual with familial ALS and a de novo mutation, c.3823C>T (p. Arg1275Trp), in a Japanese simplex case. These amino acid substitutions involve amino acids highly conserved among species, are predicted as probably damaging, and are located within a tyrosine kinase domain (p. Arg927Gln) or a C-terminal domain (p. Arg1275Trp), both of which mediate essential functions of ErbB4 as a receptor tyrosine kinase. Functional analysis revealed that these mutations led to a reduced autophosphorylation of ErbB4 upon neuregulin-1 (NRG-1) stimulation. Clinical presentations of the individuals with mutations were characterized by the involvement of both upper and lower motor neurons, a lack of obvious cognitive dysfunction, and relatively slow progression. This study indicates that disruption of the neuregulin-ErbB4 pathway is involved in the pathogenesis of ALS and potentially paves the way for the development of innovative therapeutic strategies such using NRGs or their agonists to upregulate ErbB4 functions.