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

BACKGROUND/AIMS: Rapid removal of plasma amyloid-ß (Aß) by blood purification may serve as a peripheral Aß sink from the brain for Alzheimer's disease therapy. We investigated the reduction of plasma Aß during hemodialysis and cognitive states. METHODS: Aß concentrations and Mini-Mental State Examinations (MMSE) were investigated in 37 hemodialysis patients (68.9 ± 4.1 years). RESULTS: The dialyzers effectively removed Aß(1-40) and Aß(1-42), 63.9 ± 14.4 and 51.6 ± 17.0% at 4 h dialysis, resulting in the reduction of Aßs in whole-body circulation by 51.1 ± 8.9 and 32.7 ± 12.0%, respectively. Although the plasma Aßs before dialysis (750.8 ± 171.3 pg/ml for Aß(1-40)) 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 Aßs in whole-body circulation. Repeated rapid decrease of plasma Aßs might maintain cognitive state.

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 Alzheimer's 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.

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 Alzheimer's 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.

Structure prediction of protease inhibitor region in amyloid precursor protein of Alzheimer's disease.

Recent findings of the protease inhibitor domain in amyloid precursor protein of Alzheimer's disease (APPI) raised a novel hypothesis on the mechanism of amyloid deposition in the brain. APPI has significant amino acid sequence homology with Kunitz-type basic trypsin inhibitor super-family proteins, and the gene expression product showed real inhibitory activity. Since the three-dimensional model of APPI would help in understanding biological phenomena in molecular detail, we constructed an atomic model of APPI based on the structure of bovine pancreatic trypsin inhibitor (BPTI). The substitution of BPTI side chains by best-fitting corresponding amino acid structures was followed by the removal of van der Waals overlappings by molecular mechanics energy minimization with the AMBER force field, to give the feasible model of APPI. We also built serine protease models based on the structure of trypsin and investigated the target enzyme specificity of the inhibitory activity by the active-site mapping method. The models can explain the relative enzyme spectra of APPI and BPTI.

Novel precursor of Alzheimer's disease amyloid protein shows protease inhibitory activity.

Alzheimer's disease is characterized by cerebral deposits of amyloid beta-protein (AP) as senile plaque core and vascular amyloid, and a complementary DNA encoding a precursor of this protein (APP) has been cloned from human brain. From a cDNA library of a human glioblastoma cell line, we have isolated a cDNA identical to that previously reported, together with a new cDNA which contains a 225-nucleotide insert. The sequence of the 56 amino acids at the N-terminal of the protein deduced from this insert is highly homologous to the basic trypsin inhibitor family, and the lysate from COS-1 cells transfected with the longer APP cDNA showed an increased inhibition of trypsin activity. Partial sequencing of the genomic DNA encoding APP showed that the 225 nucleotides are located in two exons. At least three messenger RNA species, apparently transcribed from a single APP gene by alternative splicing, were found in human brain. We suggest that protease inhibition by the longer APP(s) could be related to aberrant APP catabolism.

A new compound (AZ36041) promotes the survival of the neurons and reduces neurotoxicity of Alzheimer's beta-amyloid protein.

Alzheimer's beta-amyloid protein (A beta) is the main component of senile plaques, which are characteristic hallmarks of the Alzheimer's disease brain. Recently, there have been several reports that A beta has toxic effects on both cultured neurons and in the brain. We confirmed the neurotoxicity of A beta in vitro and found a new compound, called AZ36041 (4-chloro-N-(5-nitro-2-tiazoyl)benzenesulfone amide), which dramatically reduced A beta neurotoxicity. This compound was also found to have a neuroprotective effect against toxicity of glutamate and enhanced neuronal survival in the absence of neurotoxic compounds. AZ36041 may be a useful tool for investigating the mechanism of A beta neurotoxicity in vitro and in vivo.

Amyloid beta protein precursor with Kunitz-type protease inhibitor domains (APPI) in cerebrospinal fluid and APPI mRNAs in cultured skin fibroblasts of patients with Alzheimer's disease.

We studied amyloid beta protein precursor with Kunitz-type protease inhibitor domains (APPI) concentration in cerebrospinal fluid (CSF) and APPI mRNA in skin fibroblast in Alzheimer's disease (AD). The subjects consisted of AD, cerebrovascular dementia (VD) and age-matched controls. We measured the APPI concentration in the CSF by the trypsin antibody sandwich ELISA. APPI mRNA was examined by the RT-PCR technique. The CSF APPI concentration in AD was significantly higher than that of VD and controls. The CSF APPI concentration in AD was high in the initial stage of dementia and decreased during the course. The expression of APPI mRNA in skin fibroblasts in AD was also significantly higher than that of VD and controls. These results suggest that measurements of APPI levels in CSF and APPI mRNA in skin fibroblasts may be useful for early diagnosis of AD.

Determination of amyloid beta 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 Alzheimer's disease patients was found, by this method, to be significantly elevated compared with those of multi-infarct dementia.

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

The 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 microns in diameter. APPI-LI usually formed a cluster of 10- to 20-microns diameter in the cortical gray matter and 20- to 40-microns 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.

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

To investigate the possible relationship between acetylcholinesterase (AChE)-containing fiber density and senile plaque density and between AChE-positive plaques and beta/A4 protein deposition, AChE histochemistry, the modified Bielschowsky's method and beta/A4 protein immunohistochemistry were performed on the amygdala of Alzheimer's 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 beta/A4 protein immunohistochemistry has revealed that these AChE-positive structures with variable morphological appearances displayed beta/A4 protein immunoreactivity, indicating that AChE is localized in a variety of beta/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 beta/A4 protein.

Amyloid beta protein precursors with kunitz-type inhibitor domains and acetylcholinesterase in cerebrospinal fluid from patients with dementia of the Alzheimer type.

We used the ELISA to measure the concentration of amyloid protein precursor with Kunitz type trypsin inhibitor domains (APPI) in CSF of dementia of the Alzheimer type (DAT) and examined the correlation of APPI with acetylcholinesterase (AChE) and somatostatin (SRIF). We found the APPI concentration in CSF of DAT to be significantly elevated compared with that of multi-infarct dementia and controls. We could significantly correlate APPI with AChE, but not correlate APPI with SRIF. The present results suggest that measurement of CSF APPI levels may be useful for diagnosis of DAT and the change of APPI may closely be associated with abnormality of acetylcholine system in DAT that has been reported.

Trophic effect of beta-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.

Three types of amyloid protein precursor mRNA in human brain: their differential expression in Alzheimer's disease.

Three types of amyloid protein precursor (APP) mRNA, produced by alternative splicing, were detected by Northern blotting in human brains, both control and Alzheimer's disease. These mRNAs encode APP695 consisting of 695 amino acids, APP751 harboring a 56 amino acid insert homologous to a Kunitz-type trypsin inhibitor inside APP695, and APP770 containing an additional 19 amino acid insert. Another possible APP mRNA which encodes "APP714" containing a 19 amino acid insert was not found in brain samples tested. Quantitative analysis revealed that, although the relative expression levels of the three mRNAs were variable among individuals, there was no remarkable change in expression of APP695 and APP751 mRNAs in Alzheimer's disease compared with control, but that APP770 mRNA level was elevated significantly in Alzheimer's disease.

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

Expression of three types of mRNA encoding amyloid beta-protein precursor (APP) in various tissues was analysed, using a ribonuclease protection assay, with special reference to Alzheimer's 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-harboring types (APP770 and APP751) may disturb the balance between biosynthesis and degradation of APPs and ultimately lead to accumulation of beta-protein as amyloid.