Genome-wide association study identifies a new susceptibility locus in PLA2G4C for Multiple System Atrophy.

To elucidate the molecular basis of multiple system atrophy (MSA), a neurodegenerative disease, we conducted a genome-wide association study (GWAS) in a Japanese MSA case/control series followed by replication studies in Japanese, Korean, Chinese, European and North American samples. In the GWAS stage rs2303744 on chromosome 19 showed a suggestive association ( P = 6.5 × 10 -7 ) that was replicated in additional Japanese samples ( P = 2.9 × 10 -6 . OR = 1.58; 95% confidence interval, 1.30 to 1.91), and then confirmed as highly significant in a meta-analysis of East Asian population data ( P = 5.0 × 10 -15 . Odds ratio= 1.49; 95% CI 1.35 to 1.72). The association of rs2303744 with MSA remained significant in combined European/North American samples ( P =0.023. Odds ratio=1.14; 95% CI 1.02 to 1.28) despite allele frequencies being quite different between these populations. rs2303744 leads to an amino acid substitution in PLA2G4C that encodes the cPLA2γ lysophospholipase/transacylase. The cPLA2γ-Ile143 isoform encoded by the MSA risk allele has significantly decreased transacylase activity compared with the alternate cPLA2γ-Val143 isoform that may perturb membrane phospholipids and α-synuclein biology.

A Novel de novo KIF1A Mutation in a Patient with Ataxia, Intellectual Disability and Mild Foot Deformity.

Early-onset ataxias are often difficult to diagnose due to the genetic and phenotypic heterogeneity of patients. Whole exome sequencing (WES) is a powerful method for determining causative mutations of early-onset ataxias. We report a case in which a novel de novo KIF1A mutation was identified in a patient with ataxia, intellectual disability and mild foot deformity.A patient presented with sporadic forms of ataxia with mild foot deformity, intellectual disability, peripheral neuropathy, pyramidal signs, and orthostatic hypotension. WES was used to identify a novel de novo mutation in KIF1A, a known causative gene of neurodegeneration and spasticity with or without cerebellar atrophy or cortical visual impairment syndrome (NESCAVS).We report a novel phenotype of NESCAVS that is associated with a novel de novo missense mutation in KIF1A, which provides valuable information for the diagnosis of NESCAVS even in the era of WES. Early rehabilitation of patients with NESCAVS may prevent symptom worsening and improve the disease course.

Six years' accomplishment of the Initiative on Rare and Undiagnosed Diseases: nationwide project in Japan to discover causes, mechanisms, and cures.

The identification of causative genetic variants for hereditary diseases has revolutionized clinical medicine and an extensive collaborative framework with international cooperation has become a global trend to understand rare disorders. The Initiative on Rare and Undiagnosed Diseases (IRUD) was established in Japan to provide accurate diagnosis, discover causes, and ultimately provide cures for rare and undiagnosed diseases. The fundamental IRUD system consists of three pillars: IRUD diagnostic coordination, analysis centers (IRUD-ACs), and a data center (IRUD-DC). IRUD diagnostic coordination consists of clinical centers (IRUD-CLs) and clinical specialty subgroups (IRUD-CSSs). In addition, the IRUD coordinating center (IRUD-CC) manages the entire IRUD system and temporarily operates the IRUD resource center (IRUD-RC). By the end of March 2021, 6301 pedigrees consisting of 18,136 individuals were registered in the IRUD. The whole-exome sequencing method was completed in 5136 pedigrees, and a final diagnosis was established in 2247 pedigrees (43.8%). The total number of aberrated genes and pathogenic variants was 657 and 1718, among which 1113 (64.8%) were novel. In addition, 39 novel disease entities or phenotypes with 41 aberrated genes were identified. The 6-year endeavor of IRUD has been an overwhelming success, establishing an all-Japan comprehensive diagnostic and research system covering all geographic areas and clinical specialties/subspecialties. IRUD has accurately diagnosed diseases, identified novel aberrated genes or disease entities, discovered many candidate genes, and enriched phenotypic and pathogenic variant databases. Further promotion of the IRUD is essential for determining causes and developing cures for rare and undiagnosed diseases.

Muscle Transcriptomics Shows Overexpression of Cadherin 1 in Inclusion Body Myositis.

OBJECTIVE: This study aimed to elucidate the molecular features of inclusion body myositis (IBM). METHODS: We performed RNA sequencing analysis of muscle biopsy samples from 67 participants, consisting of 58 myositis patients with the pathological finding of CD8-positive T cells invading non-necrotic muscle fibers expressing major histocompatibility complex class I (43 IBM, 6 polymyositis, and 9 unclassifiable myositis), and 9 controls. RESULTS: Cluster analysis, principal component analysis, and pathway analysis showed that differentially expressed genes and pathways identified in IBM and polymyositis were mostly comparable. However, pathways related to cell adhesion molecules were upregulated in IBM as compared with polymyositis and controls (p < 0.01). Notably, CDH1, which encodes the epidermal cell junction protein cadherin 1, was overexpressed in the muscles of IBM, which was validated by another RNA sequencing dataset from previous publications. Western blotting confirmed the presence of mature cadherin 1 protein in the muscles of IBM. Immunohistochemical staining confirmed the positivity for anti-cadherin 1 antibody in the muscles of IBM, whereas there was no muscle fiber positive for anti-cadherin 1 antibody in immune-mediated necrotizing myopathy, antisynthetase syndrome, and controls. The fibers stained with anti-cadherin 1 antibody did not have rimmed vacuoles or abnormal protein accumulation. Experimental skeletal muscle regeneration and differentiation systems showed that CDH1 is expressed during skeletal muscle regeneration and differentiation. INTERPRETATION: CDH1 was detected as a differentially expressed gene, and immunohistochemistry showed that cadherin 1 exists in the muscles of IBM, whereas it was rarely seen in those of other idiopathic inflammatory myopathies. Cadherin 1 upregulation in muscle could provide a valuable clue to the pathological mechanisms of IBM. ANN NEUROL 2022;91:317-328.

Noncoding CGG repeat expansions in neuronal intranuclear inclusion disease, oculopharyngodistal myopathy and an overlapping disease.

Noncoding repeat expansions cause various neuromuscular diseases, including myotonic dystrophies, fragile X tremor/ataxia syndrome, some spinocerebellar ataxias, amyotrophic lateral sclerosis and benign adult familial myoclonic epilepsies. Inspired by the striking similarities in the clinical and neuroimaging findings between neuronal intranuclear inclusion disease (NIID) and fragile X tremor/ataxia syndrome caused by noncoding CGG repeat expansions in FMR1, we directly searched for repeat expansion mutations and identified noncoding CGG repeat expansions in NBPF19 (NOTCH2NLC) as the causative mutations for NIID. Further prompted by the similarities in the clinical and neuroimaging findings with NIID, we identified similar noncoding CGG repeat expansions in two other diseases: oculopharyngeal myopathy with leukoencephalopathy and oculopharyngodistal myopathy, in LOC642361/NUTM2B-AS1 and LRP12, respectively. These findings expand our knowledge of the clinical spectra of diseases caused by expansions of the same repeat motif, and further highlight how directly searching for expanded repeats can help identify mutations underlying diseases.

Expansions of intronic TTTCA and TTTTA repeats in benign adult familial myoclonic epilepsy.

Epilepsy is a common neurological disorder, and mutations in genes encoding ion channels or neurotransmitter receptors are frequent causes of monogenic forms of epilepsy. Here we show that abnormal expansions of TTTCA and TTTTA repeats in intron 4 of SAMD12 cause benign adult familial myoclonic epilepsy (BAFME). Single-molecule, real-time sequencing of BAC clones and nanopore sequencing of genomic DNA identified two repeat configurations in SAMD12. Intriguingly, in two families with a clinical diagnosis of BAFME in which no repeat expansions in SAMD12 were observed, we identified similar expansions of TTTCA and TTTTA repeats in introns of TNRC6A and RAPGEF2, indicating that expansions of the same repeat motifs are involved in the pathogenesis of BAFME regardless of the genes in which the expanded repeats are located. This discovery that expansions of noncoding repeats lead to neuronal dysfunction responsible for myoclonic tremor and epilepsy extends the understanding of diseases with such repeat expansion.

Molecular epidemiological study of familial amyotrophic lateral sclerosis in Japanese population by whole-exome sequencing and identification of novel HNRNPA1 mutation.

To elucidate the genetic epidemiology of familial amyotrophic lateral sclerosis (FALS) in the Japanese population, we conducted whole-exome sequencing analysis of 30 FALS families in whom causative mutations have not been identified in previous studies. Consequently, whole-exome sequencing analysis revealed novel mutations in HNRNPA1, TBK1, and VCP. Taken together with our previous results of mutational analyses by direct nucleotide sequencing analysis, a microarray-based resequencing method, or repeat-primed PCR analysis, causative mutations were identified in 41 of the 68 families (60.3%) with SOD1 being the most frequent cause of FALS (39.7%). Of the mutations identified in this study, a novel c.862/1018C>G (p.P288A/340A) mutation in HNRNPA1 located in the nuclear localization signal domain of hnRNPA1, enhances the recruitment of mutant hnRNPA1 into stress granules, indicating that an altered nuclear localization signal activity plays an essential role in amyotrophic lateral sclerosis pathogenesis.

Anti-TIF1-γ antibody and cancer-associated myositis: A clinicohistopathologic study.

OBJECTIVE: We aimed to analyze the clinical and histopathologic features of cancer-associated myositis (CAM) in relation to anti-transcriptional intermediary factor 1 γ antibody (anti-TIF1-γ-Ab), a marker of cancer association. METHODS: We retrospectively studied 349 patients with idiopathic inflammatory myopathies (IIMs), including 284 patients with pretreatment biopsy samples available. For the classification of IIMs, the European Neuromuscular Center criteria were applied. Patients with CAM with (anti-TIF1-γ-Ab[+] CAM) and without anti-TIF1-γ-Ab (anti-TIF1-γ-Ab[-] CAM) were compared with patients with IIM without cancers within and beyond 3 years of myositis diagnosis. RESULTS: Cancer was detected in 75 patients, of whom 36 (48%) were positive for anti-TIF1-γ-Ab. In anti-TIF1-γ-Ab(+) patients with CAM, cancers were detected within 1 year of myositis diagnosis in 35 (97%) and before 1 year of myositis diagnosis in 1. All the anti-TIF1-γ-Ab(+) patients with CAM satisfied the dermatomyositis (DM) criteria, including 2 possible DM sine dermatitis cases, and were characterized histologically by the presence of perifascicular atrophy, vacuolated fibers (VFs), and dense C5b-9 deposits on capillaries (dC5b-9). In contrast, 39 anti-TIF1-γ-Ab(-) patients with CAM were classified into various subgroups, and characterized by a higher frequency of necrotizing autoimmune myopathy (NAM). Notably, all 7 patients with CAM classified into the NAM subgroup were anti-TIF1-γ-Ab(-) and exhibited no dC5b-9 or VFs. CONCLUSIONS: CAM includes clinicohistopathologically heterogeneous disease entities. Among CAM entities, anti-TIF1-γ-Ab(+) CAM has characteristically shown a close temporal association with cancer detection and the histopathologic findings of dC5b-9 and VFs, and CAM with NAM is a subset of anti-TIF1-γ-Ab(-) CAM.

Variants associated with Gaucher disease in multiple system atrophy.

OBJECTIVE: Glucocerebrosidase gene (GBA) variants that cause Gaucher disease are associated with Parkinson disease (PD) and dementia with Lewy bodies (DLB). To investigate the role of GBA variants in multiple system atrophy (MSA), we analyzed GBA variants in a large case-control series. METHODS: We sequenced coding regions and flanking splice sites of GBA in 969 MSA patients (574 Japanese, 223 European, and 172 North American) and 1509 control subjects (900 Japanese, 315 European, and 294 North American). We focused solely on Gaucher-disease-causing GBA variants. RESULTS: In the Japanese series, we found nine carriers among the MSA patients (1.65%) and eight carriers among the control subjects (0.89%). In the European series, we found three carriers among the MSA patients (1.35%) and two carriers among the control subjects (0.63%). In the North American series, we found five carriers among the MSA patients (2.91%) and one carrier among the control subjects (0.34%). Subjecting each series to a Mantel-Haenszel analysis yielded a pooled odds ratio (OR) of 2.44 (95% confidence interval [CI], 1.14-5.21) and a P-value of 0.029 without evidence of significant heterogeneity. Logistic regression analysis yielded similar results, with an adjusted OR of 2.43 (95% CI 1.15-5.37) and a P-value of 0.022. Subtype analysis showed that Gaucher-disease-causing GBA variants are significantly associated with MSA cerebellar subtype (MSA-C) patients (P = 7.3 × 10(-3)). INTERPRETATION: The findings indicate that, as in PD and DLB, Gaucher-disease-causing GBA variants are associated with MSA.

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.

The TRK-fused gene is mutated in hereditary motor and sensory neuropathy with proximal dominant involvement.

Hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P) is an autosomal-dominant neurodegenerative disorder characterized by widespread fasciculations, proximal-predominant muscle weakness, and atrophy followed by distal sensory involvement. To date, large families affected by HMSN-P have been reported from two different regions in Japan. Linkage and haplotype analyses of two previously reported families and two new families with the use of high-density SNP arrays further defined the minimum candidate region of 3.3 Mb in chromosomal region 3q12. Exome sequencing showed an identical c.854C>T (p.Pro285Leu) mutation in the TRK-fused gene (TFG) in the four families. Detailed haplotype analysis suggested two independent origins of the mutation. Pathological studies of an autopsied patient revealed TFG- and ubiquitin-immunopositive cytoplasmic inclusions in the spinal and cortical motor neurons. Fragmentation of the Golgi apparatus, a frequent finding in amyotrophic lateral sclerosis, was also observed in the motor neurons with inclusion bodies. Moreover, TAR DNA-binding protein 43 kDa (TDP-43)-positive cytoplasmic inclusions were also demonstrated. In cultured cells expressing mutant TFG, cytoplasmic aggregation of TDP-43 was demonstrated. These findings indicate that formation of TFG-containing cytoplasmic inclusions and concomitant mislocalization of TDP-43 underlie motor neuron degeneration in HMSN-P. Pathological overlap of proteinopathies involving TFG and TDP-43 highlights a new pathway leading to motor neuron degeneration.

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.

Genotype-phenotype correlations in early onset ataxia with ocular motor apraxia and hypoalbuminaemia.

Early onset ataxia with ocular motor apraxia and hypoalbuminaemia/ataxia-oculomotor apraxia 1 is a recessively inherited ataxia caused by mutations in the aprataxin gene. We previously reported that patients with frameshift mutations exhibit a more severe phenotype than those with missense mutations. However, reports on genotype-phenotype correlation in early onset ataxia with ocular motor apraxia and hypoalbuminaemia are controversial. To clarify this issue, we studied 58 patients from 39 Japanese families, including 40 patients homozygous for c.689_690insT and nine patients homozygous or compound heterozygous for p.Pro206Leu or p.Val263Gly mutations who were compared with regard to clinical phenotype. We performed Kaplan-Meier analysis and log-rank tests for the ages of onset of gait disturbance and the inability to walk without assistance. The cumulative rate of gait disturbance was lower among patients with p.Pro206Leu or p.Val263Gly mutations than among those homozygous for the c.689_690insT mutation (P=0.001). The cumulative rate of inability to walk without assistance was higher in patients homozygous for the c.689_690insT mutation than in those with p.Pro206Leu or p.Val263Gly mutations (P=0.004). Using a Cox proportional hazards model, we found that the homozygous c.689_690insT mutation was associated with an increased risk for onset of gait disturbance (adjusted hazard ratio: 6.60) and for the inability to walk without assistance (adjusted hazard ratio: 2.99). All patients homozygous for the c.689_690insT mutation presented ocular motor apraxia at <15 years of age. Approximately half the patients homozygous for the c.689_690insT mutation developed cognitive impairment. In contrast, in the patients with p.Pro206Leu or p.Val263Gly mutations, only ∼50% of the patients exhibited ocular motor apraxia and they never developed cognitive impairment. The stepwise multivariate regression analysis using sex, age and the number of c.689_690insT alleles as independent variables revealed that the number of c.689_690insT alleles was independently and negatively correlated with median motor nerve conduction velocities, ulnar motor nerve conduction velocities and values of serum albumin. In the patient with c.[689_690insT]+[840delT], p.[Pro206Leu]+[Pro206Leu] and p.[Pro206Leu]+[Val263Gly] mutations, aprataxin proteins were not detected by an antibody to the N-terminus of aprataxin. Furthermore Pro206Leu and Val263Gly aprataxin proteins are unstable. However, the amount of the 689_690insT aprataxin messenger RNA was also decreased, resulting in more dramatic reduction in the amount of aprataxin protein from the c.689_690insT allele. In conclusion, patients with early onset ataxia with ocular motor apraxia and hypoalbuminaemia homozygous for the c.689_690insT mutation show a more severe phenotype than those with a p.Pro206Leu or p.Val263Gly mutation.

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 Parkinson's 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.

SNP HiTLink: a high-throughput linkage analysis system employing dense SNP data.

BACKGROUND: During this recent decade, microarray-based single nucleotide polymorphism (SNP) data are becoming more widely used as markers for linkage analysis in the identification of loci for disease-associated genes. Although microarray-based SNP analyses have markedly reduced genotyping time and cost compared with microsatellite-based analyses, applying these enormous data to linkage analysis programs is a time-consuming step, thus, necessitating a high-throughput platform. RESULTS: We have developed SNP HiTLink (SNP High Throughput Linkage analysis system). In this system, SNP chip data of the Affymetrix Mapping 100 k/500 k array set and Genome-Wide Human SNP array 5.0/6.0 can be directly imported and passed to parametric or model-free linkage analysis programs; MLINK, Superlink, Merlin and Allegro. Various marker-selecting functions are implemented to avoid the effect of typing-error data, markers in linkage equilibrium or to select informative data. CONCLUSION: The results using the 100 k SNP dataset were comparable or even superior to those obtained from analyses using microsatellite markers in terms of LOD scores obtained. General personal computers are sufficient to execute the process, as runtime for whole-genome analysis was less than a few hours. This system can be widely applied to linkage analysis using microarray-based SNP data and with which one can expect high-throughput and reliable linkage analysis.

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, 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'-alpha, beta-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'-alpha, beta-unsaturated aldehyde ends, and can act with DNA polymerase beta 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.

The FHA domain of aprataxin interacts with the C-terminal region of XRCC1.

Aprataxin (APTX) is the causative gene product for early-onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH/AOA1). In our previous study, we found that APTX interacts with X-ray repair cross-complementing group 1 (XRCC1), a scaffold protein with an essential role in single-strand DNA break repair (SSBR). To further characterize the functions of APTX, we determined the domains of APTX and XRCC1 required for the interaction. We demonstrated that the 20 N-terminal amino acids of the FHA domain of APTX are important for its interaction with the C-terminal region (residues 492-574) of XRCC1. Moreover, we found that poly (ADP-ribose) polymerase-1 (PARP-1) is also co-immunoprecipitated with APTX. These findings suggest that APTX, together with XRCC1 and PARP-1, plays an essential role in SSBR.