Kazumasa Saigoh

Functional consequences of a CKIδ mutation causing familial advanced sleep phase syndrome

Familial advanced sleep phase syndrome (FASPS) is a human behavioural phenotype characterized by early sleep times and early-morning awakening. It was the first human, mendelian circadian rhythm variant to be well-characterized, and was shown to result from a mutation in a phosphorylation site within the casein kinase I (CKI)-binding domain of the human PER2 gene. To gain a deeper understanding of the mechanisms of circadian rhythm regulation in humans, we set out to identify mutations in human subjects leading to FASPS. We report here the identification of a missense mutation (T44A) in the human CKIδ gene, which results in FASPS. This mutant kinase has decreased enzymatic activity in vitro. Transgenic Drosophila carrying the human CKIδ-T44A gene showed a phenotype with lengthened circadian period. In contrast, transgenic mice carrying the same mutation have a shorter circadian period, a phenotype mimicking human FASPS. These results show that CKIδ is a central component in the mammalian clock, and suggest that mammalian and fly clocks might have different regulatory mechanisms despite the highly conserved nature of their individual components.

Mutations in the gene encoding p62 in Japanese patients with amyotrophic lateral sclerosis

Objective: The purpose of this study was to find mutations in the SQSTM1 gene encoding p62 in Japanese patients with amyotrophic lateral sclerosis (ALS), since this gene has been recently identified as a causative gene for familial and sporadic ALS in the United States. Methods: We sequenced this gene in 61 Japanese patients with sporadic and familial ALS. To our knowledge, we describe for the first time the clinical information of such mutation-positive patients. Results: We found novel mutations, p.Ala53Thr and p.Pro439Leu, in 2 patients with sporadic ALS. The clinical picture of the mutation-positive patients was that of typical ALS with varied upper motor neuron signs. Although this gene is causative for another disease, Paget disease of bone (PDB), none of our patients showed evidence of concomitant PDB. Conclusion: The presence of mutations in this racial population suggests worldwide, common involvement of the SQSTM1 gene in ALS.

An in-frame deletion in peripheral myelin protein-22 gene causes hypomyelination and cell death of the Schwann cells in the new Trembler mutant mice.

Cloning and sequencing of the peripheral myelin protein-22 cDNA and genomic DNA from newly found Trembler mice revealed an in-frame deletion including exon IV which codes for the second (TM2) and a part of third (TM3) transmembrane domain of peripheral myelin protein-22. This mutation was distinct from those in both other allelic Trembler and Trembler-J mice, which carry point mutations within the putative transmembrane spanning regions of peripheral myelin protein-22. Inheritance was autosomal dominant. The affected mice revealed an abnormal gait, which appeared at 15-20 days of age, followed by motor and sensory ataxia, which remained throughout life. Most of the affected mice could survive more than one year. One of the most notable pathological phenotypes was a giant vacuolar formation in the sciatic nerve of homozygotes. They vary in size within the cytoplasm of Schwann cells, which failed to assemble myelin at any ages studied. Heterozygotes showed normal myelination during the early postnatal stages, followed by a segmental demyelination at an advanced stage. Vacuolar formation was not so frequent as in the homozygotes. These results suggest that the missing of transmembrane spanning region (TM2 and TM3) of peripheral myelin protein-22 may disturb a dual biological function of peripheral myelin protein-22, leading to a dysmyelination of axons and to a vacuolar formation within the cytoplasm of the Schwann cells. The latter phenotype is discussed in conjunction with the disruption of an intracellular transport system and subsequent cell death.

Chondroitin beta-1,4- N -acetylgalactosaminyltransferase-1 missense mutations are associated with neuropathies

Chondroitin sulfate proteoglycans (CSPGs) in the peripheral nervous system likely participate as regulatory molecules in the process of axonal degeneration and regeneration. We investigated the chondroitin beta1,4-N-acetylgalactosaminyltransferase-1 (ChGn-1) gene in 114 patients affected with neuropathies including Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, hereditary motor and sensory neuropathy (HMSN) and unknown etiology. The controls were 196 patients with other neurological diseases. We found novel missense mutations in two patients with neuropathy (Bells palsy, unknown HMSN) in exons 5 (H234R) and 10 (M509R), respectively. None of the patients with other neurological diseases had either of these mutations. We then synthesized the two soluble forms of ChGn-1, containing each of the above mutations. Each of the soluble mutants was expressed in COS-1 cells and the mutant proteins were purified. The purified mutant proteins were used for western blotting analysis using an anti-ChGn-1 antibody and evaluated for glycosyltransferase activities. Although the expression of the ChGn-1 mutant proteins was confirmed by western blotting, they exhibited no N-acetylgalactosamineT-II activities. It is possible that these mutations are associated with the pathogenetic mechanisms of the peripheral neuropathies.

VCP gene analyses in Japanese patients with sporadic amyotrophic lateral sclerosis identify a new mutation

Accumulating evidence has proven that mutations in the VCP gene encoding valosin-containing protein (VCP) cause inclusion body myopathy with Paget disease of the bone and frontotemporal dementia. This gene was later found to be causative for amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease, occurring typically in elderly persons. We thus sequenced the VCP gene in 75 Japanese patients with sporadic ALS negative for mutations in other genes causative for ALS and found a novel mutation, p.Arg487His, in 1 patient. The newly identified mutant as well as known mutants rendered neuronal cells susceptible to oxidative stress. The presence of the mutation in the Japanese population extends the geographic region for involvement of the VCP gene in sporadic ALS to East Asia.

The stereo-specific effect of D-serine ethylester and the D-cycloserine in ataxic mutant mice

Spinocerebellar ataxia is one of the most common neurological disorders. However, few therapeutics are effective for the treatment of this disorder. In the present study, we investigated the efficacy of d-serine ethylester and a related substance, d-cycloserine, as therapeutic agents for ataxia in a murine model. Both compounds are known to stereospecific modulate N-methyl-d-aspartate type glutamate receptors, and impaired glutamate-mediated signaling has been implicated in spinocerebellar ataxia. Using a microdialysis method, we found that intraperitoneal administration of d-serine ethylester increases the extracellular content of endogenous d-serine in the mouse cerebellum for at least 3 h. Maximum elevation of the extracellular d-serine was observed at 40 min after injection. An open-field study was used to assay the effect of the d-serine derivatives on movement and ataxia. In mice exhibiting cytosine arabinoside-induced ataxia, d-serine ethylester reduced the falling index in a dose-dependent manner. The effect of d-serine ethylester was stereo-specific in that l-serine ethylester had no effect on the falling index at the maximum doses tested, and was partially inhibited by 5,7-dichlorokynurenate, an antagonist that binds to the glycine-binding site. Locomotor activity was not changed by the d-serine ethylester treatment. d-cycloserine also significantly reduced the falling index of the mice. Both d-serine ethylester and d-cycloserine had longer lasting effects than other potential therapeutic reagents for ataxia. Growing evidence suggests the essential involvement of endogenous d-serine in mammalian brain function, and our results suggest that d-serine derivatives may represent an effective new therapeutic for the treatment of spinocerebellar ataxia.

Oxidation and interaction of DJ-1 with 20S proteasome in the erythrocytes of early stage Parkinson’s disease patients

Parkinson’s disease (PD) is a progressive, age-related, neurodegenerative disorder, and oxidative stress is an important mediator in its pathogenesis. DJ-1, the product of the causative gene of a familial form of PD, plays a significant role in anti-oxidative defence to protect cells from oxidative stress. DJ-1 undergoes preferential oxidation at the cysteine residue at position 106 (Cys-106) under oxidative stress. Here, using specific antibodies against Cys-106-oxidized DJ-1 (oxDJ-1), it was found that the levels of oxDJ-1 in the erythrocytes of unmedicated PD patients (n = 88) were higher than in those of medicated PD patients (n = 62) and healthy control subjects (n = 33). Elevated oxDJ-1 levels were also observed in a non-human primate PD model. Biochemical analysis of oxDJ-1 in erythrocyte lysates showed that oxDJ-1 formed dimer and polymer forms, and that the latter interacts with 20S proteasome. These results clearly indicate a biochemical alteration in the blood of PD patients, which could be utilized as an early diagnosis marker for PD.

A novel mutation in the calcium channel gene in a family with hypokalemic periodic paralysis.

Hypokalemic periodic paralysis (HypoPP) type 1 is an autosomal dominant disease caused by mutations in the Ca(V)1.1 calcium channel encoded by the CACNA1S gene. Only seven mutations have been found since the discovery of the causative gene in 1994. We describe a patient with HypoPP who had a high serum potassium concentration after recovery from a recent paralysis, which complicated the correct diagnosis. This patient and other affected family members had a novel mutation, p.Arg900Gly, in the CACNA1S gene.

A chondroitin synthase-1 (ChSy-1) missense mutation in a patient with neuropathy impairs the elongation of chondroitin sulfate chains initiated by chondroitin N-acetylgalactosaminyltransferase-1.

BACKGROUND Previously, we identified two missense mutations in the chondroitin N-acetylgalactosaminyltransferase-1 gene in patients with neuropathy. These mutations are associated with a profound decrease in chondroitin N-acetylgalactosaminyltransferase-1 enzyme activity. Here, we describe a patient with neuropathy who is heterozygous for a chondroitin synthase-1 mutation. Chondroitin synthase-1 has two glycosyltransferase activities: it acts as a GlcUA and a GalNAc transferase and is responsible for adding repeated disaccharide units to growing chondroitin sulfate chains. METHODS Recombinant wild-type chondroitin synthase-1 enzyme and the F362S mutant were expressed. These enzymes and cells expressing them were then characterized. RESULTS The mutant chondroitin synthase-1 protein retained approximately 50% of each glycosyltransferase activity relative to the wild-type chondroitin synthase-1 protein. Furthermore, unlike chondroitin polymerase comprised of wild-type chondroitin synthase-1 protein, the non-reducing terminal 4-O-sulfation of GalNAc residues synthesized by chondroitin N-acetylgalactosaminyltransferase-1 did not facilitate the elongation of chondroitin sulfate chains when chondroitin polymerase that consists of the mutant chondroitin synthase-1 protein was used as the enzyme source. CONCLUSIONS The chondroitin synthase-1 F362S mutation in a patient with neuropathy resulted in a decrease in chondroitin polymerization activity and the mutant protein was defective in regulating the number of chondroitin sulfate chains via chondroitin N-acetylgalactosaminyltransferase-1. Thus, the progression of peripheral neuropathies may result from defects in these regulatory systems. GENERAL SIGNIFICANCE The elongation of chondroitin sulfate chains may be tightly regulated by the cooperative expression of chondroitin synthase-1 and chondroitin N-acetylgalactosaminyltransferase-1 in peripheral neurons and peripheral neuropathies may result from synthesis of abnormally truncated chondroitin sulfate chains.

Chondroitin sulfate β-1,4-N-acetylgalactosaminyltransferase-1 (ChGn-1) polymorphism: Association with progression of multiple sclerosis.

Chondroitin sulfate proteoglycans (CSPGs) are a constituent of the matrix of the central nervous system (CNS), likely participating as regulatory molecules in the process of demyelination, remyelination, axonal degeneration and regeneration in the CNS. ChGn-1 is a key enzyme for production of CSPGs and knock-out mice of this gene showed better recovery from spinal cord injury. We hypothesized that the clinical course of multiple sclerosis (MS) is influenced by the level of expression of ChGn-1 gene. We recruited 147 patients with MS and 181 healthy control subjects and analyzed single nucleotide polymorphisms (SNPs) of this gene. We found the coding SNP (cSNP: rs140161612) in approximately 10% of patients with MS as well as normal controls. The cSNP is changed from serine to leucine at position 126 (p.S126L). The expressed ChGn-1 mutant proteins exhibited no enzyme activities in COS-1 cells. In men, patients who had MS with S126L had a slower disease progression. This cSNP might be associated with the sex differences in clinical course of MS.