Nobuya Kitaguchi

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.

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.

Enzyme specificity of proteinase inhibitor region in amyloid precursor protein of Alzheimer's disease: different properties compared with protease nexin I

Senile plaques, often surrounded by abnormally grown neurites, are characteristic of Alzheimers diseased brain. The core of the plaque is mainly composed of amyloid beta protein (beta-AP), two of whose three precursors (APP) have serine proteinase inhibitor regions (APPI). APPI derivatives containing 60, 72 or 88 amino-acid fragments (APPI-60, APPI-72 and APPI-88, respectively) of the longest APP were produced in COS-1 cell culture medium, with the APPI cDNA ligated to the signal sequence of tissue plasminogen activator. The secreted APPIs were purified by sequential acetone precipitation followed by affinity chromatography using immobilized trypsin. These three APPIs and O-glycosylation-site-mutated APPI showed similar inhibitory activity against trypsin, chymotrypsin and plasmin. The purified APPI-72 was found to inhibit trypsin (Ki = 1.1 x 10(-10) M) and chymotrypsin (Ki = 5.8 x 10(-9) M) most strongly, and to inhibit leukocyte elastase (Ki = 7.9 x 10(-7) M) and several blood coagulation proteinases (Ki = 0.46-12 x 10(-7) M), but not urokinase or thrombin. The observed inhibition pattern was quite different from that of protease nexin I, one of serine proteinase inhibitors possessing neurite outgrowth activity. This suggests that the physiological roles of APPI are different from those of protease nexin I, and that APPI could not cause aberrant growth of neurite into the plaque. The presence of APPI having strong inhibitory activity in the brain might lead to the formation of amyloid deposits by preventing complete degradation of APPs.

Cell display library for gene cloning of variable regions of human antibodies to hepatitis B surface antigen

A novel cell display system was developed for cloning the variable region (V) genes of antigen-specific human antibodies. The system is based on an antibody library displayed on the surface of COS cells, using a plasmid vector designed to direct expression of membrane-bound antibodies. COS cells expressing antigen-specific antibodies were separated using a flow cytometer for their binding to a fluorescent dye-labeled antigen. To test the performance of this system. We cloned V genes of 4 antibodies directed against hepatitis B surface antigen (HBsAg) from a library prepared from peripheral blood lymphocytes of a vaccinated donor. These membrane-bound anti-HBsAg antibodies were easily converted to soluble forms, all of which showed a size similar to human serum IgG in SDS-PAGE and the same specific binding to HBsAg as membrane-bound forms in ELISA. All VH and VK gene segments of the 4 clones isolated in this study belonged to VHIII and VKI subgroups, respectively. These findings demonstrate the potential and selection capabilities of our cell display system for cloning the V genes of antigen-specific human antibodies.

Reduction of Alzheimer's disease amyloid-β in plasma by hemodialysis and its relation to cognitive functions.

Background/Aims: Rapid removal of plasma amyloid-β (Aβ) by blood purification may serve as a peripheral Aβ sink from the brain for Alzheimer’s disease therapy. We investigated the reduction of plasma Aβ during hemodialysis and cognitive states. Methods: Aβ concentrations and Mini-Mental State Examinations (MMSE) were investigated in 37 hemodialysis patients (68.9 ± 4.1 years). Results: The dialyzers effectively removed Aβ1–40 and Aβ1–42, 63.9 ± 14.4 and 51.6 ± 17.0% at 4 h dialysis, resulting in the reduction of Aβs in whole-body circulation by 51.1 ± 8.9 and 32.7 ± 12.0%, respectively. Although the plasma Aβs before dialysis (750.8 ± 171.3 pg/ml for Aβ1–40) were higher than those reported for Alzheimer’s disease patients, the cognitive states of hemodialysis patients were relatively normal, especially of longer dialysis vintages. Conclusions: Dialyzers effectively reduced Aβs in whole-body circulation. Repeated rapid decrease of plasma Aβs might maintain cognitive state.

Occurrence of acetylcholinesterase activity closely associated with amyloid β/A4 protein is not correlated with acetylcholinesterase-positive fiber density in amygdala of Alzheimer's disease

SummaryTo investigate the possible relationship between acetylcholinesterase (AChE)-containing fiber density and senile plaque density and between AChE-positive plaques and β/A4 protein deposition, AChE histochemistry, the modified Bielschowskys method and β/A4 protein immunohistochemistry were performed on the amygdala of Alzheimers disease (AD) and aged control cases. Abundant AChE-positive senile plaques were found in the amygdala and related structures in AD. These AChE-positive plaques were mainly of the primitive or diffuse type. In addition to senile plaques of typical morphologies a variety of AChE-positive structures were observed in the amygdala and related regions in AD. A comparison of serial sections stained alternatively with AChE histochemistry and β/A4 protein immunohistochemistry has revealed that these AChE-positive structures with variable morphological appearances displayed β/A4 protein immunoreactivity, indicating that AChE is localized in a variety of β/A4 protein deposition including the diffuse plaque. Thus, it is suggested that AChE is present in some senile plaques at the earliest stage. However, there was no apparent correlation between the numerical density of AChE-positive fibers and senile plaque density. These findings suggest that the degeneration of cholinergic neurons is not attributed to the occurrence of AChE activity in β/A4 protein.

Patients that have Undergone Hemodialysis Exhibit Lower Amyloid Deposition in the Brain: Evidence Supporting a Therapeutic Strategy for Alzheimer’s Disease by Removal of Blood Amyloid

As a proof of concept that removal of blood amyloid-β (Aβ) can reduce Aβ deposition in the brains of patients with Alzheimers disease, cortices of patients who had undergone hemodialysis (HD), which removes Aβ from the blood, were histochemically analyzed; postmortem brain sections were stained with anti-Aβ antibodies. Brains from patients who had undergone HD had significantly fewer senile plaques than those of patient who had not undergone HD. This significant difference was also confirmed by silver staining. Our findings suggest that removal of blood Aβ by hemodialysis results in lower accumulation of Aβ in the brain.

Immunohistochemical localization of the proteinase inhibitor region of amyloid precursor proteins in the neocortex of Alzheimer's disease and aged controls

SummaryThe immunohistochemical localization of the proteinase inhibitor region of amyloid protein precursors (APPI) in the postmortem human neocortex was studied using a polyclonal antibody raised against a purified recombinant human APPI derivative produced by COS-1 cells. APPI-like immunoreactivity (APPI-LI) was found diffusely in the human neocortex. APPI-LI appeared as irregularly shaped granular structures. The size of the APPI-LI structures was 1–4 μm in diameter. APPI-LI usually formed a cluster of 10- to 20-μm diameter in the cortical gray matter and 20- to 40-μm diameter in the subcortical white matter. Double staining for APPI and glial fibrillary acidic protein indicated that APPI-LI in the white matter and molecular layer was localized exclusively in the fibrillary astrocytes. In contrast, APPI-LI was found in neurons as well as in the fibrillary astrocytes in layers II through to VI. Under fluorescence microscopy, APPI-LI in both neurons and fibrillary astrocytes were found in close association with lipofuscin. The present observations indicate that APPI is localized in neurons and astrocytes in the human neocortex and that APPI may be associated with lipofuscin or lysosome in the human neocortex.

Removal of Blood Amyloid As a Therapeutic Strategy for Alzheimer’s Disease: The Influence of Smoking and Nicotine

Accumulation of amyloid β protein (Aβ) in the brain causes cognitive impairment in Alzheimer’s disease (AD). The nature of the relationship between smoking and AD or dementia has been controversial. However, a recent meta-analysis revealed that smoking is a risk factor for AD. With regard to nicotinic acetylcholinergic receptors (nAChRs), both AD and control patients that smoke have been reported to show an increase in 3H-cytisine (an α4β4 nAChR agonist) binding in the temporal cortex. The α7 nAChR is also a key factor in AD pathology, particularly in relation to internalization of Aβs. Furthermore, there are many reports showing the neuroprotective effects of nicotine. The internalization of Aβ may lead to Aβ clearance in the brain.

REMOVAL OF Aβ OLIGOMERS FROM THE BLOOD USING HOLLOW FIBERS: A POTENTIAL THERAPEUTIC SYSTEM FOR ALZHEIMER’S DISEASE

antibodies generated, we selected a molecule showing a KD value of 6 nM for isoAsp7-Ab and displaying w 500fold selectivity over non-modified Ab. Immunohistochemical staining of tissue from human post mortem brain showed exclusive labelling of amyloid plaques. This pattern was also observed in brain samples from transgenic mice, where we observed a characteristic increase of isoAsp detection beginning with onset of plaque deposition and increasing with age and amyloid pathology. Based on Ab extraction and ELISA quantification, we conclude that the isoAsp content is low in micewith onset of pathology (4%) and increases with ageing of 5xFAD mice. A 12weeks treatment of transgenic mice led to a reduction of isoAsp-modified and total Ab load, suggesting that selective opsonization of modified amyloid is eliciting phagocytosis of non-modified peptides. In a second trial, we observed an amelioration of a behavioral phenotype in elevated plus maze upon treatment with the antibody. Conclusions:In addition to previous studies applying antibodies directed against pGlu-modified Ab, we show here that also isoAsp-modified Ab represents a vital target for immunotherapy. The results support the general concept that targeting modified Ab peptides, which might be underrepresented compared to full-length Ab, are attractive anchor points for a more directed approach of immunotherapy in order to reduce common side effects.