Kimiaki Utsugisawa

Reduction with age in methylcytosine in the promoter region -224∼ - 101 of the amyloid precursor protein gene in autopsy human cortex

Methylation status of cytosines and its changes with age in the promoter region (−226∼−101) of the amyloid precursor protein (APP) was analyzed using bisulfite genomic sequencing in the cerebral cortex of human autopsy brain. Cytosines at 13 locations were methylated in at least one of the cases studied. Methylcytosines at these locations was more frequent in cases ≤70 years old (26%) than in cases >70 years old (8%) (p 70 years old (5%) (p<0.01). These cytosines constituted one of the 9-bp-long GC-rich elements (GGGCGC G/A GG) or an 11-bp inverted repeat (GGCCGT CGGCC). The present findings indicate that some cytosines, particularly those at −207∼−182, in the promoter region of the APP gene are frequently methylated and suggest that their demethylation with age may have some significance in the development of Aβ deposition in the aged brain. The relative importance of these elements in the total promoter activity of the APP gene remains to be definitively established.

Effects of age on messenger RNA expression of glucocorticoid, thyroid hormone, androgen, and estrogen receptors in postmortem human hippocampus

We studied messenger RNA (mRNA) expressions of receptors for glucocorticoid (GR), thyroid hormone (TR), androgen (AR), and estrogen (ER) and their changes with age in the hippocampal subregions in postmortem human brain. In situ hybridization was done with biotin-labeled antisense synthetic oligonucleotide probes. About 80% or more of the pyramidal neurons in the hippocampal subregions expressed mRNAs for individual receptors in the brains of subjects younger than 65. The ratio of mRNA-containing neuron density to total neuron density significantly decreased with age for GR in CA1 and CA3, and for AR in CA1. Non-significant trends in the reduction with age in the ratio of ER mRNA-containing neurons in CA1 and the ratio of GR mRNA-containing neurons in the hilus also were found. Age-related reductions in nuclear receptor protein mRNA expression in neurons in the hippocampal subfields may be important in the impairments of cognition, emotion, and responses to acute stress in the aged.

Autoimmune Targets of Heart and Skeletal Muscles in Myasthenia Gravis

OBJECTIVE To investigate the clinical, histological, and immunological features of patients with myasthenia gravis (MG) who also developed myocarditis and/or myositis. DESIGN Observational and retrospective case series. SETTING Keio University, Hanamaki General Hospital, Kanazawa University, Nagasaki University, and Juntendo University. PATIENTS A cohort of 8 patients with MG with clinically defined inflammatory myopathies. INTERVENTIONS Clinical and histological features were described. Serological analyses included MG-related antistriational autoantibodies (those to titin, ryanodine receptor, muscular voltage-gated potassium channel Kv1.4) and myositis-specific autoantibodies. RESULTS Of 924 patients with MG, 8 (0.9%) had inflammatory myopathies. The mean (SD) onset age of MG was 55.3 (10.3) years. All patients showed severe symptoms with bulbar involvement; 5 patients had myasthenic crisis and 4 had invasive thymoma. Myocarditis was found in 3 patients and myositis in 6. Myocarditis, developing 13 to 211 months after the MG onset, was characterized by heart failure and arrhythmias. Myositis, developing before or at the same time as MG, affected limb and paraspinal muscles. Histological findings of skeletal muscles showed CD8(+) lymphocyte infiltration. Seven patients had 1 of these antistriational autoantibodies but not myositis-specific autoantibodies. Immunomodulatory therapy was required for all patients and was effective for both MG and inflammatory myopathies, although 1 patient died. CONCLUSIONS Heart and skeletal muscles are autoimmune targets in some patients with MG. This autoimmunity has a broad clinical spectrum with antistriational autoantibodies.

Efficacy of low-dose FK506 in the treatment of Myasthenia gravis--a randomized pilot study.

To determine the efficacy of low-dose FK506 in the treatment of myasthenia gravis (MG), untreated de novo patients were randomly selected to receive treatment with (n = 18) or without (n = 16) FK506, and were evaluated for 1 year after treatment with limitation of daily dose of prednisolone. Low-dose FK506 reduced the duration of early-phase therapy in hospital (p < 0.05) and the need for combined therapy with plasmapheresis and high-dose intravenous methylprednisolone or high-dose intravenous methylprednisolone alone (p < 0.05). It also reduced the daily dose of prednisolone (p < 0.05) required to maintain minimal manifestations of MGFA postintervention status. None of the patients exhibited significant side effects up to 1 year after treatment. These findings suggest that low-dose FK506 is safe and efficacious for the treatment of de novo MG patients.

Agraphia and acalculia after a left prefrontal (F1, F2) infarction.

A patient presented with agraphia and acalculia associated with a left frontal (F1, F2) infarction. He made mainly phonological but also lexical errors in writing (syllabograms), but his ability to write kanji (morphograms) was relatively preserved. Although he could add and subtract numbers, he could neither multiply nor divide them because of a difficulty in retrieving the multiplication tables and calculation procedures. Positron emission tomography showed decreased cerebral blood flow and metabolism limited to the infarct site. These findings suggest that agraphia and acalculia may occur associated with a left prefrontal lesion, and that the retrieval of arithmetic processes is modality specific.

The methylation status of cytosines in a τ gene promoter region alters with age to downregulate transcriptional activity in human cerebral cortex

Changes with age in the methylation status of cytosines in a promoter region of the tau gene were investigated in autopsy human cerebral cortex, using the bisulfite method, polymerase chain reaction (PCR) and direct sequencing of PCR products. While the total number of methylcytosines decreased with age, the changes in methylation status differed among transcription factor binding sites. Cytosines in the AP2-binding sites were never methylated in any of the cases studied at any age. Methylcytosines in the binding sites for Sp1, a transcriptional activator, significantly increased with age, whereas those in the binding sites for GCF, a repressor of GC-rich promoters, significantly decreased with age. These findings suggest that the methylation status of cytosines in the promoter region of the tau gene alters with age to decrease its transcriptional activity in the human cerebral cortex.

The MG-QOL15 Japanese version: validation and associations with clinical factors.

Our study aim was to produce a Japanese translation of the 15‐item Myasthenia Gravis Quality‐of‐Life Scale (MG‐QOL15), assess its reliability and validity, and examine clinical factors affecting self‐perceived QOL in MG.

Protective effect of nicotine through nicotinic acetylcholine receptor α7 on hypoxia-induced membrane disintegration and DNA fragmentation of cultured PC12 cells

To investigate the effect of nicotine on hypoxic neuronal damage, cultured PC12 cells were exposed to hypoxia for 9 h and then reoxygenated for 72 h. The cells were stained by propidium iodide (PI), a marker of cell membrane disintegration and the TUNEL method, which indicates DNA fragmentation. In control cultures, the ratio of PI-positive cells to total cells progressively increased during and after exposure to hypoxia, constituting 39% of total cells at 72 h posthypoxia. This increase in PI-positive cells was completely inhibited by nicotine until 12 h posthypoxia, and was partially and dose-dependently inhibited thereafter. The ratio of TUNEL-positive cells to total cells started to increase at 24 h posthypoxia and reached 36% at 72 h in control cultures. This ratio was also dose-dependently inhibited by nicotine. These inhibitory effects of nicotine on the increase in PI-positive and TUNEL-positive cells were abolished by the addition to the medium of alpha-bungarotoxin, an antagonistic ligand for nicotinic acetylcholine receptor (AChR) alpha7. These findings suggest that nicotine inhibits, through AChR alpha7, hypoxia-induced cell membrane disintegration and DNA fragmentation of cultured PC12 cells exposed to hypoxia.

Age-related changes in nicotinic acetylcholine receptor subunits α4 and β2 messenger RNA expression in postmortem human frontal cortex and hippocampus

Age-related changes in nicotinic acetylcholine receptor (nAChR) subunit α4 and β2 messenger RNA (mRNA) expression in the postmortem human frontal cortex and hippocampus was investigated using the reverse transcription-polymerase chain reaction (RT-PCR). In the frontal cortex, both α4 and β2 subunit mRNA expression decreased with age. In the hippocampus, α4 subunit mRNA expression was unaltered, while β2 subunit mRNA expression significantly decreased with age. These findings suggest that nAChR transcription decreases during aging with differing vulnerability between subunits and brain regions, which could in part contribute to the reduction in cognitive functions seen in the elderly.

Overexpression of α7 nicotinic acetylcholine receptor prevents G1‐arrest and DNA fragmentation in PC12 cells after hypoxia

We investigated the neuroprotective function of α7 nicotinic acetylcholine receptor (α7nAChR) after transient hypoxia (12 h) and reoxygenation (0–72 h), comparing rat pheochromocytoma (PC12) cells overexpressing FLAG‐tagged α7nAChR (α7pCMV cells) and control PC12 cells (non‐transfected or transfected with vector only) in medium with and without nicotine. Plasma membrane degradation in the early phase after hypoxia was inhibited in PC12 cells with nicotine, and more profoundly in α7pCMV cells with nicotine. Inhibition of DNA fragmentation in the late phase after hypoxia was most remarkable in α7pCMV cells with nicotine, but, surprisingly, it was more remarkable in α7pCMV cells without nicotine than in PC12 cells with nicotine. G1‐arrest of the cell cycle, observed in control PC12 cells at 12 h after hypoxia, preceding DNA fragmentation, was not evident in α7pCMV cells, with or without nicotine. Furthermore, in α7pCMV cells with and without nicotine, the basal expression levels of total Akt were approximately 1.5‐fold higher, and the up‐regulation of Akt phosphorylated at Ser473 after hypoxia was strikingly enhanced, compared with control PC12 cells. These findings suggest that α7nAChR functions constitutively in PC12 cells, that its overexpression raises tolerance against G1‐arrest and DNA fragmentation after hypoxia, and that it can be considered a candidate target for treatment against hypoxia‐induced acute membrane degradation and delayed DNA fragmentation in neurons.