Yasuo Tokushima

Trophic effect of β-amyloid precursor protein on cerebral cortical neurons in culture

We investigated the effect of human beta-amyloid precursor protein (APP) on rat primary cerebral cortical neurons cultured in a serum-free medium. Two secretory APP species (APP667 and APP592) with and without the protease inhibitor domain were produced by COS-1 cells transfected with APP cDNAs, which encode the N-terminal portions of APP770 and APP695. Both highly purified APP species, when added to the medium, enhanced neuronal survival and neurite extension in a dose-dependent manner with a maximum effect at approximately 100 nM. These results suggest that secreted forms of APP have trophic activity for cerebral cortical neurons.

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.

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.

Amyloid β Protein Precursors having Proteinase Inhibitor Regions are Highly Expressed in Alzheimer’s Disease Brains

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder of the aged and is characterized by cerebral deposits of amyloid β -protein (β -AP) comprising about 40 amino acids as senile plaque core and vascular amyloid.1,2 Since there is a correlation between the number of plaques and the degree of dementia, 3 it has been suggested that the formation of senile plaque is one of the pathogenetic features of AD. A complementary DNA (cDNA) clone of a β-AP precursor (APP) has been proved to encode a 695-amino acid precursor (APP695) having structural features characteristic of cell surface glycoproteins.4