Seigo Tanaka

Vascular Factors and Risk of Dementia: Design of the Three-City Study and Baseline Characteristics of the Study Population

Objective: To describe the baseline characteristics of the participants in the Three-City (3C) Study, a study aiming to evaluate the risk of dementia and cognitive impairment attributable to vascular factors. Methods: Between 1999 and 2001, 9,693 persons aged 65 years and over, noninstitutionalized, were recruited from the electoral rolls of three French cities, i.e. Bordeaux, Dijon and Montpellier. Health-related data were collected during face-to-face interviews using standardized questionnaires. The baseline examination included cognitive testing and diagnosis of dementia, and assessment of vascular risk factors, including blood pressure measurements, ultrasound examination of the carotid arteries, and measurement of biological parameters (glycemia, total, high-density lipoprotein and low-density lipoprotein cholesterol, triglycerides, creatinemia); 3,442 magnetic resonance imaging (MRI) examinations were performed in subjects aged 65–79. Measurements of ultrasound, blood, and MRI parameters were centralized. Two follow-up examinations (at 2 and 4 years) were planned. Results: After exclusion of the participants who had subsequently refused the medical interview, the 3C Study sample consisted of 3,649 men (39.3%) and 5,645 women, mean age 74.4 years, with a relatively high level of education and income. Forty-two percent of the participants reported to be followed up for hypertension, about one third for hypercholesterolemia, and 8% for diabetes; 65% had elevated blood pressure measures (systolic blood pressure ≧140 or diastolic blood pressure ≧90). The proportion of Mini-Mental State Examination scores below 24 was 7% and dementia was diagnosed in 2.2% of the participants. Conclusion: Distribution of baseline characteristics of the 3C Study participants suggests that this study will provide a unique opportunity to estimate the risk of dementia attributable to vascular factors.

Caspase dependent DNA fragmentation might be associated with excitotoxicity in Alzheimer disease

Recent studies have shown that deficient functioning of glutamate transporters (GTs) in Alzheimer disease (AD) might lead to neurodegeneration via excitotoxicity; however, the characteristics of cell death and pathways involved are not yet clear. The main objective of the present study was to determine if deficient GT functioning in AD could be associated with cell damage and caspase activation. For this purpose, we analyzed the levels of caspase-1 and 3 immunoreactivity in AD and control brains and correlated this data with the numbers of cells displaying DNA fragmentation, GT activity, and amyloid precursor protein (APP) mRNA expression. Compared to controls, AD cases showed extensive positive labeling of neurons and glial cells with an assay for DNA fragmentation suggestive of cell damage, as well as increased neuronal caspase-3 and Bcl-2 immunoreactivity. Linear regression analysis showed a strong negative correlation between GT activity and apoptosis, and between deficient GT functioning and caspase-3 immunoreactivity. Neurons displaying DNA fragmentation presented more intense caspase-3 immunoreactivity than intact neurons. In addition, the altered ratio between the spliced forms of APP correlated with DNA fragmentation and caspase-3 immunolabeling. Taken together, these results support the possibility that excitotoxic injury associated with deficient GT functioning and an imbalance in ratio of spliced APP forms might lead to cell death via caspase-3 activation.

Tissue-specific expression of three types of β-protein precursor mRNA: Enhancement of protease inhibitor-harboring types in Alzheimer's disease brain

Abstract Expression of three types of mRNA encoding amyloid β-protein precursor (APP) in various tissues was analysed, using a ribonuclease protection assay, with special reference to Alzheimers disease (AD). The total content and the proportion of APP mRNAs were specific to each tissue. Among eight tissues examined, the brain was distinct in that the expression level was highest and APP695 mRNA was expressed in abundance. The ratio of APP770/APP751/APP695 mRNAs was approximately 1:10:20 in the cerebral cortex of control brain. The proportions of APP770 mRNA and APP770-plus-APP751 mRNAs increased up to 2.6- and 1.4-fold, respectively, in various regions of AD brain compared with control. The enhanced expression of protease inhibitor-haboring types (APP770 and APP751) may disturb the balance between biosynthesis and degradation of APPs and ultimately lead to accumulation of β-protein as amyloid.

Behavioral characteristics of the SAM-P/8 strain in Sidman active avoidance task

The behavior of the senescence-accelerated mouse (SAM-P/8) at the age of 1, 2, 4 and 10-11 months in Sidman active avoidance learning was analyzed, and compared to findings in the controls (SAM-R/1). At the age of 1 and 2 months, learning was comparable in these two strains. At the age of 4 and 10-11 months, SAM-P/8 but not SAM-R/1 learned active avoidance. We propose that SAM-P/8 can serve as a valid model of deficits in learning and memory.

Apolipoprotein E polymorphism in Japanese patients with Alzheimer's disease or vascular dementia.

Apolipoprotein E (ApoE) plays a key part in lipid metabolism both in the liver, and in the CNS. To clarify the association of ApoE polymorphism with Alzheimers disease and vascular dementia in Japan, 13 patients with early onset (age > or = 65) sporadic Alzheimers disease, 40 patients with late onset (age < or = 65) sporadic Alzheimers disease, 19 patients with vascular dementia, and 49 non-demented control subjects were analysed. The results showed a significantly increased frequency of the epsilon 4 allele in the patients with late onset sporadic Alzheimers disease (0.25), but not in the patients with early onset sporadic Alzheimers disease (0.04) or in the patients with vascular dementia (0.13) compared with controls (0.09). The raised frequency of the epsilon 4 allele in the patients with late onset sporadic Alzheimers disease was of a lower magnitude than that in United States and Canadian studies. This may in part be due to a lower epsilon 4 frequency in the normal Japanese population and reflect the lower morbidity from Alzheimers disease in Japan.

Transferrin receptor-dependent cytotoxicity of artemisinin–transferrin conjugates on prostate cancer cells and induction of apoptosis

Artemisinin, a natural product isolated from Artemisia annua, contains an endoperoxide group that can be activated by intracellular iron to generate toxic radical species. Cancer cells over-express transferrin receptors (TfR) for iron uptake while most normal cells express nearly undetectable levels of TfR. We prepared a series of artemisinin-tagged transferrins (ART-Tf) where different numbers of artemisinin units are attached to the N-glycoside chains of transferrin (Tf). The Tf bearing approximately 16 artemisinins retains the functionality of both Tf and artemisinin. Reduction of TfRs by TfR siRNA transfection significantly impaired the ability of ART-Tf, but not dihydroartemisinin, to kill cells. We also demonstrate that the ART-Tf conjugate kills the prostate carcinoma cell line DU 145 by the mitochondrial pathway of apoptosis.

The commonality of protein interaction networks determined in neurodegenerative disorders (NDDs)

MOTIVATION Neurodegenerative disorders (NDDs) are progressive and fatal disorders, which are commonly characterized by the intracellular or extracellular presence of abnormal protein aggregates. The identification and verification of proteins interacting with causative gene products are effective ways to understand their physiological and pathological functions. The objective of this research is to better understand common molecular pathogenic mechanisms in NDDs by employing protein-protein interaction networks, the domain characteristics commonly identified in NDDs and correlation among NDDs based on domain information. RESULTS By reviewing published literatures in PubMed, we created pathway maps in Kyoto Encyclopedia of Genes and Genomes (KEGG) for the protein-protein interactions in six NDDs: Alzheimers disease (AD), Parkinsons disease (PD), amyotrophic lateral sclerosis (ALS), Huntingtons disease (HD), dentatorubral-pallidoluysian atrophy (DRPLA) and prion disease (PRION). We also collected data on 201 interacting proteins and 13 compounds with 282 interactions from the literature. We found 19 proteins common to these six NDDs. These common proteins were mainly involved in the apoptosis and MAPK signaling pathways. We expanded the interaction network by adding protein interaction data from the Human Protein Reference Database and gene expression data from the Human Gene Expression Index Database. We then carried out domain analysis on the extended network and found the characteristic domains, such as 14-3-3 protein, phosphotyrosine interaction domain and caspase domain, for the common proteins. Moreover, we found a relatively high correlation between AD, PD, HD and PRION, but not ALS or DRPLA, in terms of the protein domain distributions. AVAILABILITY http://www.genome.jp/kegg/pathway/hsa/hsa01510.html (KEGG pathway maps for NDDs).

Determination of amyloid β protein precursors harboring active form of proteinase inhibitor domains in cerebrospinal fluid of Alzheimer's disease patients by trypsin-antibody sandwich ELISA

beta-Amyloid protein precursors (APP) having proteinase inhibitor domains (APPI) were quantified by a new sandwich enzyme linked immunosorbent assay for detection of active (free) form of proteinase inhibitors by using trypsin in place of the first antibody and by denaturation of APPI-trypsin complex in the microtiterplate. The concentration of APPs having APPI in cerebrospinal fluid of Alzheimers disease patients was found, by this method, to be significantly elevated compared with those of multi-infarct dementia.

Age-related changes in the proportion of amyloid precursor protein mRNAs in Alzheimer's disease and other neurological disorders.

In the human brain, alternative splicing of amyloid precursor protein (APP) gene transcript generates at least three types of mRNA coding for APP770, APP751 and APP695. The former two types harbor, but the latter one lacks a domain of Kunitz-type serine protease inhibitor (KPI). We studied, by using the RNase protection technique, the expression of APP mRNAs in brains of Alzheimers disease (AD) and other neurological disorders with special reference to aging. We found that the ratio of (APP770 mRNA+APP751 mRNA)/APP695 mRNA in the frontal cortex increased approximately 1.5-fold in AD compared with other neurodegenerative or cerebrovascular disorders. The ratio in other neurological disorders did not change significantly from control even in their affected brain regions. On the other hand, we found a positive correlation between the ratio and age; the ratio (y) increased gradually with the advance of age (x) as expressed by y = 0.005x + 0.014 (r = 0.372) for the AD group, and y = 0.004x -0.037 (r = 0.486) for the non-AD group. These correlations indicate that the AD brain reached the same ratio of KPI-harboring to lacking APP mRNAs a few decades earlier than the non-AD brain in senescence. This finding of AD-specific and age-related change led us to the idea that a relative increase in KPI-harboring APPs over a KPI-lacking APP may perturb normal degradation of APPs, thereby leading to deposition of beta A4 protein as amyloid.

Mitochondrial impairment induced by poly(ADP-ribose) polymerase-1 activation in cortical neurons after oxygen and glucose deprivation

Neuronal cells injured by ischemia and reperfusion to a certain extent are committed to death in necrotic or apoptotic form. Necrosis is induced by gross ATP depletion or ‘energy crisis’ of the cell, whereas apoptosis is induced by a mechanism still to be defined in detail. Here, we investigated this mechanism by focusing on a DNA damage‐sensor, poly(ADP‐ribose) polymerase‐1 (PARP‐1). A 2‐h oxygen and glucose deprivation (OGD) followed by reoxygenation (Reox) induced apoptosis, rather than necrosis, in rat cortical neurons. During the Reox, PARP‐1 was much activated and autopoly(ADP‐ribosyl)ated, consuming the substrate, NAD+. Induction of apoptosis by OGD/Reox was suppressed by overexpression of Bcl‐2, indicating mitochondrial impairment in this induction process. Mitochondrial permeability transition (MPT), or membrane depolarization, and a release of proapoptotic proteins, i.e. cytochrome c, apoptosis‐inducing factor and endonuclease G, from mitochondria were observed during the Reox. These apoptotic changes of mitochondria and the nucleus were attenuated by PARP‐1 inhibitors, 1,5‐dihydroxyisoquinoline and benzamide, and also by small interfering RNA specific for PARP‐1. These results indicated that PARP‐1 plays a principal role in inducing mitochondrial impairment that ultimately leads to apoptosis of neurons after cerebral ischemia.