Kinya Ishikawa

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.).

Efficacy and safety of leuprorelin in patients with spinal and bulbar muscular atrophy (JASMITT study): a multicentre, randomised, double-blind, placebo-controlled trial

BACKGROUND Spinal and bulbar muscular atrophy is a hereditary motor neuron disease caused by the expansion of a polyglutamine tract in the androgen receptor. At present there are no treatments for spinal and bulbar muscular atrophy, although leuprorelin suppressed the accumulation of pathogenic androgen receptors in a phase 2 trial. We aimed to assess the efficacy and safety of leuprorelin for spinal and bulbar muscular atrophy. METHODS The Japan SBMA Interventional Trial for TAP-144-SR (JASMITT) was a 48-week, randomised, double-blind, placebo-controlled trial done at 14 hospitals between August, 2006, and March, 2008. Patients with spinal and bulbar muscular atrophy were randomly assigned (1:1) by minimisation to subcutaneous 11.25 mg leuprorelin or identical placebo every 12 weeks. Patients and investigators were masked to treatment allocation. The primary endpoint was pharyngeal barium residue, which indicates incomplete bolus clearance, measured at week 48 by videofluorography. All patients who were randomly assigned and who were assessed with videofluorography at least once were included in the analyses. This study is registered with the JMACCT clinical trials registry, number JMA-IIA00009, and the UMIN clinical trials registry, number UMIN000000465. FINDINGS 204 patients were randomly assigned and 199 started treatment: 100 with leuprorelin and 99 with placebo. At week 48, the pharyngeal barium residue after initial swallowing had changed by -5.1% (SD 21.0) in the leuprorelin group and by 0.2% (18.2) in the placebo group (difference between groups -5.3%; 95% CI -10.8 to 0.3; p=0.063). The mean difference in pharyngeal barium residue after piecemeal deglutition at week 48 was -3.2% (-6.4 to 0.0; p=0.049), but there was no significant difference between the groups after covariate adjustment for the baseline data (-4.1 to 1.6; p=0.392). In a predefined subgroup analysis, leuprorelin treatment was associated with a greater reduction in barium residue after initial swallowing than was placebo in patients with a disease duration less than 10 years (difference between groups -9.8, -17.1 to -2.5; p=0.009). There were no significant differences in the number of drug-related adverse events between groups (57 of 100 in the leuprorelin group and 54 of 99 in the placebo group; p=0.727). INTERPRETATION 48 weeks of treatment with leuprorelin did not show significant effects on swallowing function in patients with spinal and bulbar muscular atrophy, although it was well tolerated. Disease duration might influence the efficacy of leuprorelin and thus further clinical trials with sensitive outcome measures should be done in subpopulations of patients. FUNDING Large Scale Clinical Trial Network Project, Japan and Takeda Pharmaceuticals.

Japanese families with autosomal dominant pure cerebellar ataxia map to chromosome 19p13.1-p13.2 and are strongly associated with mild CAG expansions in the spinocerebellar ataxia type 6 gene in chromosome 19p13.1.

Autosomal dominant cerebellar ataxia is a group of clinically and genetically heterogeneous disorders. We carried out genomewide linkage analysis in 15 families with autosomal dominant pure cerebellar ataxia (ADPCA). Evidence for linkage to chromosome 19p markers was found in nine families, and combined multipoint analysis refined the candidate region to a 13.3-cM interval in 19p13.1-p13.2. The remaining six families were excluded for this region. Analysis of CAG-repeat expansion in the alpha1A-voltage-dependent calcium channel (CACNL1A4) gene lying in 19p13.1, recently identified among 8 small American kindreds with ADPCA (spinocerebellar ataxia type 6 [SCA6]), revealed that 8 of the 15 families studied had similar, very small expansion in this gene: all affected individuals had larger alleles (range of CAG repeats 21-25), compared with alleles observed in neurologically normal Japanese (range 5-20 repeats). Inverse correlation between the CAG-repeat number and the age at onset was found in affected individuals with expansion. The number of CAG repeats in expanded chromosomes was completely stable within each family, which was consistent with the fact that anticipation was not statistically proved in the SCA6 families that we studied. We conclude that more than half of Japanese cases of ADPCA map to 19p13.1-p13.2 and are strongly associated with the mild CAG expansion in the SCA6/CACNL1A4 gene.

Spinocerebellar ataxia type 6 knockin mice develop a progressive neuronal dysfunction with age-dependent accumulation of mutant CaV2.1 channels

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disorder caused by CAG repeat expansions within the voltage-gated calcium (CaV) 2.1 channel gene. It remains controversial whether the mutation exerts neurotoxicity by changing the function of CaV2.1 channel or through a gain-of-function mechanism associated with accumulation of the expanded polyglutamine protein. We generated three strains of knockin (KI) mice carrying normal, expanded, or hyperexpanded CAG repeat tracts in the Cacna1a locus. The mice expressing hyperexpanded polyglutamine (Sca684Q) developed progressive motor impairment and aggregation of mutant CaV2.1 channels. Electrophysiological analysis of cerebellar Purkinje cells revealed similar Ca2+ channel current density among the three KI models. Neither voltage sensitivity of activation nor inactivation was altered in the Sca684Q neurons, suggesting that expanded CAG repeat per se does not affect the intrinsic electrophysiological properties of the channels. The pathogenesis of SCA6 is apparently linked to an age-dependent process accompanied by accumulation of mutant CaV2.1 channels.

Ataxin-3 is translocated into the nucleus for the formation of intranuclear inclusions in normal and Machado-Joseph disease brains

Machado-Joseph disease (MJD)/spinocerebellar ataxia type 3 (SCA3) is one of the dominantly inherited cerebellar ataxias. The gene responsible for the disease, a novel gene of unknown function, encodes ataxin-3 containing a polyglutamine stretch. Although it has been known that ataxin-3 is incorporated into neuronal intranuclear inclusions (NIIs) in neurons of affected regions, the relationship between NII formation and neuronal degeneration still remains uncertain. In the present study we show two different conditions in which ataxin-3 is recruited into the nucleus and suggest a process to form nuclear inclusions. In normal brains, wild-type ataxin-3 localizes within the ubiquitin-positive nuclear inclusion, the Marinesco body, indicating that ataxin-3 is recruited into the nuclear inclusion even in the absence of pathologically expanded polyglutamine. In MJD/SCA3 brains, immunohistochemical analyses with anti-ataxin-3 antibody, anti-ubiquitin antibody, and monoclonal antibody 1C2 known to recognize expanded polyglutamine revealed differences in frequency and in diameter among NIIs recognized by each antibody. These results were confirmed in the same inclusions by double immunofluorescent staining, suggesting that expanded ataxin-3 forms a core, thereby recruiting wild-type ataxin-3 into the nucleus around the core portion, and then followed by activation of the ubiquitin/ATP-dependent pathway. Recruitment of ataxin-3 into the nucleus and formation of nuclear inclusion under two different conditions suggest that ataxin-3 may be translocated into the nucleus under certain conditions stressful on neuronal cells such as aging and polyglutamine neurotoxicity.

Cytoplasmic and nuclear polyglutamine aggregates in SCA6 Purkinje cells

Aggregations of the alpha1A-calcium channel protein have been previously demonstrated in spinocerebellar ataxia type 6 (SCA6). Here the authors show that small aggregates, labeled by a monoclonal antibody 1C2 that preferentially detects expanded polyglutamine larger than that in SCA6 mutation, are present mainly in the cytoplasm but also in the nucleus of Purkinje cells. Although the length of expansion is small in SCA6, the current finding might indicate that SCA6 conforms to the pathogenic mechanism(s) in other polyglutamine diseases.

Bax-inhibiting peptide protects cells from polyglutamine toxicity caused by Ku70 acetylation

Polyglutamine (polyQ) diseases, such as Huntingtons disease and Machado–Joseph disease (MJD), are caused by gain of toxic function of abnormally expanded polyQ tracts. Here, we show that expanded polyQ of ataxin-3 (Q79C), a gene that causes MJD, stimulates Ku70 acetylation, which in turn dissociates the proapoptotic protein Bax from Ku70, thereby promoting Bax activation and subsequent cell death. The Q79C-induced cell death was significantly blocked by Ku70 or Bax-inhibiting peptides (BIPs) designed from Ku70. Furthermore, expression of SIRT1 deacetylase and the addition of a SIRT1 agonist, resveratrol, reduced Q79C toxicity. In contrast, mimicking acetylation of Ku70 abolished the ability of Ku70 to suppress Q79C toxicity. These results indicate that Bax and Ku70 acetylation play important roles in Q79C-induced cell death, and that BIP may be useful in the development of therapeutics for polyQ diseases.

A gene on SCA4 locus causes dominantly inherited pure cerebellar ataxia

Background: Several different genes or their loci have been identified for autosomal dominant cerebellar ataxia (ADCA). However, other types of ataxia remain unassigned. Objective: To identify a new locus for ADCA. Methods: Six Japanese families with ADCA with pure cerebellar syndrome (ADCA type III) were examined. These families had been molecularly excluded for spinocerebellar ataxia (SCA) types 1 through 3, 5 through 8, and 10. Clinical examination was undertaken, and a genome-wide linkage search was performed on 250 microsatellite DNA markers. Results: Strong evidence for linkage was found with markers on human chromosome 16q, and haplotype and multipoint analyses further refined the gene locus in a 10.9-cM interval between D16S3089 and D16S515. Linkage disequilibrium was further found with the marker D16S3107 within the interval. The locus was exactly the candidate interval of SCA4, a rare form of ADCA clinically characterized by ataxia with sensory neuropathy and pyramidal tract signs. This would suggest that SCA4 and our ADCA type III are likely to be allelic disorders with different clinical features. Conclusion: The current study provides evidence that a gene on the SCA4 locus causes a pure cerebellar syndrome.

Morphological Purkinje cell changes in spinocerebellar ataxia type 6

Abstract Spinocerebellar ataxia type 6 (SCA6) was recently identified as a form of autosomal dominant spinocerebellar ataxia associated with a small CAG repeat expansion of the gene encoding an α 1 A-voltage-dependent calcium channel gene subunit on chromosome 19p13. In this study 50-μm-thick sections of cerebellar tissue from one patient with SCA6 were subjected to free-floating immunohistochemical staining with calbindin-D and parvalbumin antibodies. Severe loss of Purkinje cells was found, particularly in the vermis, and various morphological changes in Purkinje cells and their dendritic arborizations were demonstrated. Many of the remaining Purkinje cells were found to have heterotopic, irregularly shaped nuclei, an unclear cytoplasmic membrane outline, and somatic sprouts. Increased numbers of spine-like protrusions from swelling dendritic arborizations were found in the molecular layer. The axonal arrangement was disordered, and many torpedos were found in the granular layer and white matters. These morphological changes are completely different from those observed in paraneoplastic cerebellar degeneration (PCD) and multiple system atrophy (MSA) and are considered to be related to the genetic abnormality that causes abnormal development of Purkinje cells.

A clinical and genetic study in a large cohort of patients with spinocerebellar ataxia type 6

AbstractIn order to clarify the clinical and genetic features of SCA6, we retrospectively analyzed 140 patients. We observed an inverse correlation between the age of onset and the length of the expanded allele, and also between the age of onset and the sum of CAG repeats in the normal and the expanded alleles. The ages of onset of four homozygous patients correlated better with the sum of CAG repeats in both alleles rather than with the expanded allele calculated from heterozygous SCA6 subjects. Clinically, unsteadiness of gait was the main initial symptom, followed by vertigo and oscillopsia, and cerebellar signs were detected in nearly 100% of the patients. In contrast, extracerebellar signs were relatively mild and infrequent. The results of neuro-otological examination performed in 22 patients suggested the purely cerebellar abnormalities of ocular movements in nature. There was a close relationship between downbeat positioning nystagmus (DPN) and positioning vertigo, which became more common in the later stage. We conclude that total number of CAG repeat-units in both alleles is a good parameter for assessment of age of onset in SCA6 including homozygous patients. In addition, clinical and neuro-otological examinations suggested that SCA6 is a disease with predominantly cerebellar dysfunction.