Satoshi Naruse

Expanded polyglutamine stretches interact with TAFII130, interfering with CREB-dependent transcription.

At least eight inherited neurodegenerative diseases are caused by expanded CAG repeats encoding polyglutamine (polyQ) stretches. Although cytotoxicities of expanded polyQ stretches are implicated, the molecular mechanisms of neurodegeneration remain unclear. We found that expanded polyQ stretches preferentially bind to TAFII130, a coactivator involved in cAMP-responsive element binding protein (CREB)-dependent transcriptional activation, and strongly suppress CREB-dependent transcriptional activation. The suppression of CREB-dependent transcription and the cell death induced by polyQ stretches were restored by the co-expression of TAFII130. Our results indicate that interference of transcription by the binding of TAFII130 with expanded polyQ stretches is involved in the pathogenetic mechanisms underlying neurodegeneration.

Long-term therapeutic efficacy and safety of low-dose tacrolimus (FK506) for myasthenia gravis

OBJECTIVE To elucidate the long-term therapeutic efficacy and safety of low-dose FK506 (tacrolimus) in patients with myasthenia gravis (MG). PATIENTS AND METHODS We treated nine patients with MG (all women: age range: 35-83 years (mean: 51.1 years); MGFA classification: 4 type IIa, 4 type IIb, and 1 type IVb patients) with FK506 for more than 24 months (observation period: 24-46 months). All the patients had undergone extended thymectomy before FK506 treatment; two patients (22.2%) had noninvasive thymoma and six (66.7%) had thymic hyperplasia. We evaluated total Quantitative MG (Q-MG) score, anti-acetylcholine receptor (AChR) antibody titer in the blood, interleukin 2 (IL-2) production in peripheral blood mononuclear cells (PBMCs), administration dosage of prednisolone (PSL), and adverse effects of FK506. RESULTS A reduction in steroid dosage of 50% without worsening of the symptoms was observed 1 year after FK506 administration in three out of six steroid-dependent MG patients (50.0%). The total Q-MG scores (range: 0-39 points) at 6 months and 1 year after FK506 administration improved by 3 points or more in six (66.7%) and seven (77.8%) out of nine patients, respectively. The efficacy of FK506 was maintained for more than 2 years. Although adverse effects were observed in three patients (33.3%), these were not serious. CONCLUSIONS Our study indicates that low-dose FK506 treatment may be efficacious not only in controlling intractable myasthenic symptoms, but also in reducing steroid dosage, and that FK506 is safe as an adjunctive drug to PSL for MG treatment for a maximum of 3 years.

Interference of CREB-dependent transcriptional activation by expanded polyglutamine stretches - augmentation of transcriptional activation as a potential therapeutic strategy for polyglutamine diseases

On the basis of the hypothesis that the interaction of mutant proteins with expanded polyglutamine stretches with transcriptional co‐activator, TAFII130, leads to transcriptional dysregulation, the transcriptional activation of c‐Fos and its suppression by expanded polyglutamine stretches was investigated. The phosphorylation of cAMP‐responsive element binding protein (CREB) and induction of c‐Fos in response to cAMP were strongly suppressed in Neuro2a cells expressing expanded polyglutamine. The suppression of CREB‐dependent transcriptional activation was reversibly rescued by increasing the concentration of cAMP. Expanded polyglutamine‐induced cytotoxicity was also substantially suppressed by augmenting CREB‐dependent transcriptional activation with a high concentration of cAMP. FR901228, a histone deacetylase inhibitor, was also demonstrated as rescuing the expanded polyglutamine‐induced suppression of CREB phosphorylation and c‐Fos expression. Furthermore, nuclear fragmentation was significantly suppressed by FR901228. The co‐expression of dominant‐negative CREB vectors considerably abrogated the suppressive effect of cAMP and FR901228 on the expanded polyglutamine‐induced nuclear fragmentation, suggesting that these compounds suppress polyglutamine‐induced cytotoxicity, largely, via the enhancement of CREB‐dependent transcriptional activation. These findings suggest that the interference of CREB‐dependent transcriptional activation by expanded polyglutamine stretches is involved in the pathogenetic mechanisms underlying neurodegeneration, and that the augmentation of CREB‐dependent transcriptional activation is a potential strategy in treating polyglutamine diseases.

New HSN2 mutation in Japanese patient with hereditary sensory and autonomic neuropathy type 2

The authors report a Japanese patient with hereditary sensory and autonomic neuropathy type 2 (HSAN2) who has a new mutation of the HSN2 gene. The pathologic findings of the patient matched those of Canadian patients. They identified a homozygous 1134-1135 ins T mutation, resulting in a frameshift, and the subsequent premature stop codon at residue 378. These observations support the hypothesis that HSN2 is a causative gene for HSAN2.

Novel GFAP mutation in patient with adult-onset Alexander disease presenting with spastic ataxia.

Adult-onset Alexander disease is rare and clinically characterized by slowly progressive signs of brainstem and spinal cord involvement. Missense mutations in the gene encoding the glial fibrillary acidic protein (GFAP) have been identified as a genetic basis for Alexander disease. We here report a Japanese patient with adult-onset Alexander disease with a novel GFAP mutation. A 36-year-old man of Japanese descent, a child of nonconsanguineous parents, with a 10-year history of slowly progressive gait disturbance, was referred to us. His early motor and intellectual development were normal. Neurological examination revealed rhythmic ocular nystagmoid movement, dysarthria, truncal and limb ataxia, increased muscle stretch reflex with bilateral Babinski sign, and spasticity in his lower extremities. Palatal myoclonus was not noted. He was ambulatory, but his gait was unsteady owing to ataxia and spasticity in the lower extremities. Brain MRI demonstrated a marked atrophy of the medulla oblongata and cervical spinal cord, and a mild atrophy of the cerebellar hemisphere (Fig. 1A). Fluid attenuation inversion recovery (FLAIR) images revealed abnormal hyperintensities in cerebellar dentate nucleus (Fig. 1B) and the periventricular white matter (Fig. 1C). Molecular genetic analysis of GFAP was performed using the patient’s genomic DNA after obtaining written informed consent. Sequence analysis revealed a heterozygous 302T > C substitution in exon 1 of GFAP, leading to an L101P substitution. The L101P substitution is located in the C-terminal end of the 1A rod domain of GFAP occurring in a highly conserved amino acid residue across species (Fig. 1D). The sequence change was confirmed by restriction fragment length polymorphism (RFLP) using enzyme digestion by BcgI in the patient and 100 normal control subjects (Fig. 1E). To obtain biochemical evidence of pathogenecity of the novel GFAP L101P mutant, we transfected wild-type and mutant GFAP, and examined the solubility of the GFAP protein. Samples were sequentially extracted with different stringent buffers and subjected to western blot analysis (see Supplementary methods). The well-characterized mutant R416W GFAP was largely recovered from the detergent-resistant S2 fraction because of the decreased solubility of mutant GFAP (Fig. 1F, lane 8) as previously reported. In this assay, wild-type GFAP was predominantly detected in the soluble S1 fraction (Fig. 1F, lane 2). In contrast, the mutant L101P GFAP was largely observed in the detergent-resistant S2 fraction (Fig. 1F, lane 7). Transfected cells were further analyzed for GFAP assembly by confocal microscopy (see Supplementary methods). Whereas wild-type GFAP displayed cytoplasmic distribution with a filamentous network, the L101P mutant yielded an irregular dot-like structure largely lacking the filamentous structure (Supp. Info. Fig.). In this study, we identified a novel GFAP mutation in a Japanese patient with adult-onset Alexander disease presenting with slowly progressive spastic ataxia. The parents of the patient are unaffected; hence, the mutation seems to arise de novo in the patient as in most cases of Alexander disease. Indeed, the mother did not carry the mutation. Unfortunately, DNA sample was unavailable from the father, who has recently died of heart disease. GFAP is a member of the intermediate filament family with a conserved central helical rod domain flanked by the head and tail domains (Fig. 1D). The L101P mutation detected in the patient is located in the coil 1A rod domain, which is considered to play an essential role in filament formation. Mutations of the a-helix regions in the rod domain are considered to alter the charge and hydrophobic interactions within coiled coils. Thus, mutations in the domain may affect the solubility of GFAP, which is supported by our biochemical experiments using cells expressing mutant GFAP. To date, more than 10 GFAP missense mutations associated with adult-onset Alexander disease have been found, with nearly all occurring in the rod domain. The genotype–phenotype correlation in Alexander disease has been poorly understood particularly in adult-onset cases, probably owing to the very small number of patients. Alexander disease in our patient is clinically characterized by slowly progressive spastic ataxia with bulbar signs without palatal myoclonus. In patients with adult-onset Alexander disease, bulbar symptoms, gait ataxia, and spasticity are common clinical features, whereas ocular motor abnormalities, autonomic dysfunctions, and palatal myoclonus have been reported with varying frequency. Our patient exhibited atrophy of the medulla and spinal cord, and abnormal hyperintensities of the periventricular white matter and cerebellar dentate nucleus on FLAIR images. The characteristic atrophy of the medulla and spinal cord is invariably present in adult-onset Alexander disease. In contrast, leukoencephalopathy and abnormal signal intensities of the cerebellum are not always observed in adultonset cases. The question of why missense mutations in the same critical domain of GFAP result in such different clinical phenotypes and MRI findings in Alexander disease is intriguing and deserves further attention and elucidation.

Mutational analysis of the amyloid precursor protein gene in Japanese familial Alzheimer's disease kindreds.

We sequenced the entire coding region of the amyloid precursor protein (APP) genes of 11 unrelated patients with Japanese familial Alzheimers disease (FAD) in order to determine the exact frequency of known APP gene mutations and to search for novel mutations responsible for FAD. Three out of 11 (27.3%) FAD patients showed the known Val to Ile mis-sense mutation at codon 717, but no other mutations were detected in the entire coding region. Analysis of exons 16 and 17 in 30 Japanese with sporadic AD revealed no mutations. Moreover, there were no significant differences in the allele frequencies of the DNA polymorphism in intron 9 among the 11 FAD, 39 sporadic AD, and 110 control subjects.

Absence of linkage disequilibrium at amyloid precursor protein gene locus in Japanese familia Alzheimer's disease with 717Val→Ile mutation

To date, eleven independent FAD pedigrees with the 717Val-->Ile mutation have been identified. Interestingly, five pedigrees were of Japanese origin and four were of British origin. The apparent ethnic prediction of this mutation raises the possibility that there is a founder effect in these two island nations. We did not observe any significant linkage disequilibrium in any locus of APP and GT12 loci in the five Japanese FAD pedigrees with the 717Val-->Ile mutation. A founder effect would probably not be present in Japanese FAD pedgrees with the 717Val-->Ile mutation.