Kazuo Miyanaga

In vitro processing of amyloid precursor protein by cathepsin D

The formation of beta A4 amyloid in the brains of individuals with Alzheimers disease requires the proteolytic cleavage of amyloid precursor protein. Several lines of evidence suggest that cathepsin D, the major lysosomal/endosomal aspartic protease, may be involved in this process. In this work, we used a sensitive in vitro method of detection to investigate the role of cathepsin D in the proteolytic processing of a 100-amino acid C-terminal fragment (C100) inclusive of beta A4 and cytoplasmic domain of APP. Digestion of C100 with cathepsin D resulted in cleavage at the amyloidogenic gamma-cleavage sites. This occurred preferentially at Thr43-Val44 and at Ala42-Thr43, generating full length beta A4 43 and beta A4 42 amyloid peptides, respectively. Cathepsin D was also found to cleave the substrate at the following nonamyloidogenic sites; Leu34-Met35, Thr48-Leu49 and Leu49-Val50. A high concentration of cathepsin D resulted in cleavage also occurring at Phe19-Phe20, Phe20-Ala21 and Phe93-Phe94 of the C100, suggesting that these sites are somewhat less sensitive to the action of cathepsin D. Digestion of C100 using different solublizing agents indicated that the cleavage of C100 by cathepsin D is greatly influenced by the structural integrity of the substrate. However, our results suggest that cathepsin D could generate the pathogenic beta A4 amyloid peptides from its precursor in vitro, which may indicate a role in the amyloidogenesis of Alzheimers disease.

Two autopsy cases with Pelizaeus-Merzbacher disease phenotype of adult onset, without mutation of proteolipid protein gene.

Abstract We report the autopsy cases of two brothers which are pathologically compatible with Pelizaeus-Merzbacher disease (PMD). Both patients had a late onset (at the ages of 29 and 42 years) and chronic neurological symptoms including tremor, ataxia and dementia. The T2-weighted magnetic resonance imaging of the younger brother demonstrated increased signal areas with sparing of small areas in the cerebral white matter. The postmortem examinations, obtained at the ages of 45 and 61 years, showed similar neuropathological findings. Histologically, a cardinal finding was a lack of myelin in large parts of white matter with the preservation of islands of intact myelin, resulting in a “tigroid” appearance. Only small amounts of sudanophilic material were present. The axons were relatively well preserved, but oligodendrocytes were numerically reduced. Ultrastructurally, myelin sheaths in the white matter were markedly thin. Immunohistochemistry showed that proteolipid protein (PLP) was reduced in the affected white matter. However, genetic studies did not reveal exonic mutations or duplications of the PLP gene. We conclude that the two cases are a rare type of dysmyelinating disorder with PMD phenotype of adult onset and could be caused by previously unrecognized abnormalities of the PLP gene or other genes.

Declining of memory functions of normal elderly persons

Two studies examined the declining of memory functions in normal elderly persons using the Yokota memory test (YMT), which includes 15 items concerning verbal and non‐verbal memory functions. In the first study, 552 subjects over 40 years of age in five age groups were examined. Factor analysis revealed that YMT consisted of two factors pertaining to short‐term/working memory, and two factors pertaining to long‐term memory. It is suggested that the former was more affected than the latter, with aging. In the second study, YMT was examined in relation to the revised version of Hasegawa dementia scale (HDS‐R), which was the most popular intelligence scale for the elderly in Japan. As a result, memory functions differentially declined with the decreasing score of HDS‐R, which suggests that memory functions differentially declined with progressive risk of dementia.

Plasma α1,3-Fucosyltransferase Deficiency in Schizophrenia

Levels of plasma α1,3-fucosyltransferase (α1,3FT) were assayed in 44 patients with schizophrenia and in 50 healthy controls. Significantly reduced enzyme activities were observed in patients (p < 0.05) and 4 unrelated patients were found, for the first time in Japan, to be deficient in the enzyme activity. Two point mutations in the coding region of the FUT6 gene encoding plasma α1,3FT that were responsible for the inactivation of the enzyme activity were detected in those patients. Genotyping of the Le gene (FUT3) in these patients demonstrated that 2 of them were also FUT3 deficient and were grouped as Lewis– individuals whereas the rest were Lewis+.

Molecular Analysis of Plasma α1,3-Fucosyltransferase Deficiency and Development of the Methods for Its Genotyping

Four patients with mental illness were found to be deficient in plasma α1,3-fucosyltransferase for the first time in Japan [Exp Clin Immunogenet 1999;16:125–130]. Complete sequencing of FUT6 genes in these individuals revealed the presence of two point mutations, i.e., G739 to A (Glu→247 to Lys) and C945 to A (Tyr→315 to stop). In addition to two reported alleles having G739 to A (pf1) and G739 to A and C945 to A (pf3), a new mutated allele having C945 to A (pf2) was found to be present and all the individuals who lack α1,3-fucosyltransferase activity in plasma were found to possess pf genes homozygously (pf/pf). In order to detect such lethal mutations in FUT6 genes easily, PCR-RFLP methods have also been developed and applied for the screening of FUT6 deficiency in a large number of samples which resulted in the demonstration of three additional FUT6-deficient individuals. The absence of α1,3-fucosylated molecules on α1-acid glycoprotein in plasma from all the 7 individuals was confirmed to result from the plasma α1,3-fucosyltransferase deficiency.

Persistent Synchronous Periodic Discharges Caused by Anoxic Encephalopathy Due to Cardiopulmonary Arrest

We report a patient with vegetative state induced by anoxic encephalopathy due to cardiopulmonary arrest who showed synchronous periodic discharges for a prolonged period. The patient was a 47-year-old man admitted to our hospital for depression, who suddenly developed cardiopulmonary arrest of unknown etiology, and entered chronic vegetative state as a result of anoxic encephalopathy. Electroencephalography (EEG) revealed that synchronous periodic discharges were present for up to 5 months. The wave pattern, periodicity and duration of appearance of synchronous periodic discharges were similar to those of synchronous periodic discharges in Creutzfeldt-Jakob disease (CJD). The periodicity of synchronous periodic discharges was gradually prolonged, with the course again similar to the discharges in CJD. The mechanism of occurrence is considered to be similar to that of synchronous periodic discharges in CJD.

Multicenter population-based study on the prevalence of early onset dementia in Japan: Vascular dementia as its prominent cause

In Japan, the government and media have become aware of the issues of early onset dementia (EOD), but policies for EOD have not yet been established and support systems are inadequate. To provide practical data about EOD, a two‐step postal survey was performed.

Prevalence and illnesses causing early-onset dementia in Japan: A multi-center study

aim of this study is to evaluate the frequency of cognitive deficit and depressive symptoms in patients stratified according to the risk of developing cardiovascular events, as well as to compare the cognitive performance between low and high risk groups. Methods: The Framingham Score was used to stratify 110 patients in low, medium or high risk for cardiovascular disease. The Mini Mental State Examination (MMSE) and the Geriatric Depression Scale (GDS-15) were used to evaluate the cognitive performance and the presence of depressive symptoms. The cutoff for the presence of depressive symptoms was considered 6 on GDS. Results: The high risk group showed significant lower scores on MMSE (p<0.001) regardless of education when compared to the other groups. The GDS-15 results did not differ significantly among the groups of cardiovascular risk (p1⁄40.161) however, they showed that the prevalence of depressive symptoms was higher (37%) in the group with cardiovascular risk than in the general population (5-17%). Conclusions: The present findings corroborate the differential cognitive performance of patients with high cardiovascular risk. It was also noted the high prevalence of depressive symptoms in patients with cardiovascular risk, although it was not significantly different among the groups, it was higher than in the general population.

Screening with Monoclonal Anti Beta-Protein Antibody for Immuno-Reactivity in the Sera of Patients with Alzheimer’s Disease

A monoclonal antibody(aSP01) and a polyclonal antibody was raised against a synthetic peptide corresponding to residues(N:1-10, SP01) of the beta-protein. Using the aSP01 and sandwitch ELISA technique immunoreactivities in sera were determined for four groups, three affected(Alzheimer’s disease, senile dementia of Alzheimer’s type(AD/ SDAT), multi-infarct dementia(MID), dementia of not otherwise satisfied (NOS)), and a non-affected group. Whereas a high value in the cross-reactivity was observed on AD(35.11±7.91), relatively low values were obtained for the latter groups(MID; 16.76±2.58, N0S;13.28±2.20, non-demented; 14.41 ±.1. 94) • No difference in the mean value of the CRM was shown between male and female groups(male;16.04±3.51, female; 14.99±2.47). Practically no correration was observed between the age and CRM of the two affected groups(AD;r=-0.0585, MID;r=0.0353)and non-affected group(r=0.2758).

Preparation of a Monoclonal Antibody Against a Synthetic Brain Amyloid Beta Peptide(N1–10) and Distribution of an Immunoreactivity in Serum

Alzheimer’s disease(AD) is a progressive neurodegenerative disorder of the human central nervous system characterized by formation of neurofibrillary tangles(NFT), senile(neuritic) plaques, and cerebrovascular amyloid-osis1,2,3. The mature senile plaques(SP) consist of degenerating neurites surrounding a core(SPC) of proteinaceous filaments which have structural properties of amyloid. Similar filaments are also found in the walls of meningeal and intracortical blood vessels(CVA). The initial isolation and partial sequencing of a 4.2kD polypeptide(called β protein or A4 peptide) reported for amyloid isolated from extraparenchymal meningeal vessels in AD aswell as in Down’s syndrome(DS)4 and, subsequently, from SPC. These findings suggesting a common origin for both types of amyloid, may also indicate that a common or a very similar mechanism may involve in the formation of amyloid in AD and DS. Recently, the base sequence has been determined by several laboratories of a cDNA for a precursor amyloid β protein6,7 Subsequently, the gene has been mapped in the vicinity of theSOD locus on chromosome 21. The putative precursor of amyloid β protein consists of 695 amino acid residues and has a structure characteristic of a cell surface receptor.