Amino-terminally elongated Aß peptides are generated by the secreted metalloprotease ADAMTS4 and deposit in a subset of Alzheimer's disease brains.

AIMS: The aggregation and deposition of amyloid-ß (Aß) peptides in the brain is thought to be the initial driver in the pathogenesis of Alzheimer's disease (AD). Aside from full-length Aß peptides starting with an aspartate residue in position 1, both N-terminally truncated and elongated Aß peptides are produced by various proteases from the amyloid precursor protein (APP) and have been detected in brain tissues and body fluids. Recently, we demonstrated that the particularly abundant N-terminally truncated Aß4-x peptides are generated by ADAMTS4, a secreted metalloprotease that is exclusively expressed in the oligodendrocyte cell population. In this study, we investigated whether ADAMTS4 might also be involved in the generation of N-terminally elongated Aß peptides. METHODS: We used cell-free and cell-based assays in combination with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) and electrochemiluminescence sandwich immunoassays to identify and quantify N-terminally elongated Aß peptide variants. Antibodies against these Aß variants were characterised by peptide microarrays and employed for the immunohistochemical analyses of human brain samples. RESULTS: In this study, we discovered additional ADAMTS4 cleavage sites in APP. These were located N-terminal to Asp-(1) in the Aß peptide sequence between residues Glu-(-7) and Ile-(-6) as well as Glu-(-4) and Val-(-3), resulting in the release of N-terminally elongated Aß-6-x and Aß-3-x peptides, of which the latter serve as a component in a promising Aß-based plasma biomarker. Aß-6/-3-40 peptides were detected in supernatants of various cell lines and in the cerebrospinal fluid (CSF), and ADAMTS4 enzyme activity promoted the release of Aß-6/-3-x peptides. Furthermore, by immunohistochemistry, a subset of AD cases displayed evidence of extracellular and vascular localization of N-terminally elongated Aß-6/-3-x peptides. DISCUSSION: The current findings implicate ADAMTS4 in both the pathological process of Aß peptide aggregation and in the early detection of amyloid pathology in AD.

Assessment of immunoprecipitation with subsequent immunoassays for the blood-based diagnosis of Alzheimer's disease.

The Aß42/40 ratio and the concentration of phosphorylated Tau181 in blood plasma represent attractive biomarkers for Alzheimer's disease. As a means for reducing potential matrix effects, which may interfere with plasma immunoassays, we have previously developed a pre-analytical sample workup by semi-automated immunoprecipitation. Here we test the compatibility of pre-analytical immunoprecipitations with automated Aß1-40, Aß1-42 and phosphorylated Tau181 immunoassays on the Lumipulse platform and compare the diagnostic performance of the respective immunoprecipitation immunoassay approaches with direct plasma measurements. 71 participants were dichotomized according to their Aß42/40 ratios in cerebrospinal fluid into the diagnostic groups amyloid-positive (n = 32) and amyloid-negative (n = 39). The plasma Aß1-42/1-40 ratio and phosphorylated Tau181 levels were determined on the Lumipulse G600II platform (Fujirebio) by direct measurements in EDTA-plasma or after Aß- or Tau-immunoprecipitation, respectively. Pre-analytical immunoprecipitation of Aß turned out to be compatible with the Lumipulse Aß assays and resulted in a numerical, yet statistically not significant increase in the area under the ROC curve for plasma Aß1-42/1-40. Additionally, we observed a significant increase in the standardised effect size (Cohen's D). Pre-analytical immunoprecipitation of Tau resulted in increased differences between the diagnostic groups in terms of median and mean phosphorylated Tau 181 levels. Furthermore, we observed a greater Cohen's d (p < 0.001) and a larger area under the ROC curve (p = 0.038) after Tau-IP. Our preliminary findings in a small, preselected sample indicate that pre-analytical immunoprecipitation may have the potential to improve the diagnostic performance of plasma biomarker immunoassays for Aß1-42/1-40 and phosphorylated Tau181 to predict brain amyloid deposition.

A Combination of Caffeine Supplementation and Enriched Environment in an Alzheimer's Disease Mouse Model.

A variety of factors has been associated with healthy brain aging, and epidemiological studies suggest that physical activity and nutritional supplements such as caffeine may reduce the risk of developing dementia and, in particular, Alzheimer's disease (AD) in later life. Caffeine is known to act as a cognitive enhancer but has been also shown to positively affect exercise performance in endurance activities. We have previously observed that chronic oral caffeine supplementation and a treatment paradigm encompassing physical and cognitive stimulation by enriched environment (EE) housing can improve learning and memory performance and ameliorate hippocampal neuron loss in the Tg4-42 mouse model of AD. Here, we investigated whether these effects were synergistic. To that end, previous findings on individual treatments were complemented with unpublished, additional data and analyzed in depth by ANOVA followed by Bonferroni multiple comparison post tests. We further evaluated whether plasma neurofilament light chain levels reflect neuropathological and behavioral changes observed in the experimental groups. While a treatment combining physical activity and caffeine supplementation significantly improved learning and memory function compared to standard-housed vehicle-treated Tg4-42 in tasks such as the Morris water maze, no major additive effect outperforming the effects of the single interventions was observed.

Detection and quantification of Aß-3-40 (APP669-711) in cerebrospinal fluid.

Neurochemical biomarkers can support the diagnosis of Alzheimer's disease and may facilitate clinical trials. In blood plasma, the ratio of the amyloid-ß (Aß) peptides Aß-3-40/Aß1-42 can predict cerebral amyloid-ß pathology with high accuracy (Nakamura et al., 2018). Whether or not Aß-3-40 (aka. amyloid precursor protein (APP) 669-711) is also present in cerebrospinal fluid (CSF) is not clear. Here, we investigated whether Aß-3-40 can be detected in CSF and to what extent the CSF Aß-3-40/Aß42 ratio is able to differentiate between individuals with or without amyloid-ß positron emission tomography (PET) evidence of brain amyloid. The occurrence of Aß-3-40 in human CSF was assessed by immunoprecipitation followed by mass spectrometry. For quantifying the CSF concentrations of Aß-3-40 in 23 amyloid PET-negative and 17 amyloid PET-positive subjects, we applied a sandwich-type immunoassay. Our findings provide clear evidence of the presence of Aß-3-40 and Aß-3-38 in human CSF. While there was no statistically significant difference in the CSF concentration of Aß-3-40 between the two diagnostic groups, the CSF Aß-3-40/Aß42 ratio was increased in the amyloid PET-positive individuals. We conclude that Aß-3-40 appears to be a regular constituent of CSF and may potentially serve to accentuate the selective decrease in CSF Aß42 in Alzheimer's disease.

Solid-Phase Synthesis and Characterization of N-Terminally Elongated Aß-3-x -Peptides.

In addition to the prototypic amyloid-ß (Aß) peptides Aß1-40 and Aß1-42 , several Aß variants differing in their amino and carboxy termini have been described. Synthetic availability of an Aß variant is often the key to study its role under physiological or pathological conditions. Herein, we report a protocol for the efficient solid-phase peptide synthesis of the N-terminally elongated Aß-peptides Aß-3-38 , Aß-3-40 , and Aß-3-42 . Biophysical characterization by NMR spectroscopy, CD spectroscopy, an aggregation assay, and electron microscopy revealed that all three peptides were prone to aggregation into amyloid fibrils. Immunoprecipitation, followed by mass spectrometry, indicated that Aß-3-38 and Aß-3-40 are generated by transfected cells even in the presence of a tripartite ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor. The elongated Aß peptides starting at Val(-3) can be separated from N-terminally-truncated Aß forms by high-resolution isoelectric-focusing techniques, despite virtually identical isoelectric points. The synthetic Aß variants and the methods presented here are providing tools to advance our understanding of the potential roles of N-terminally elongated Aß variants in Alzheimer's disease.

Astrocytes and microglia but not neurons preferentially generate N-terminally truncated Aß peptides.

The neuropathological hallmarks of Alzheimer's disease include extracellular neuritic plaques and neurofibrillary tangles. The neuritic plaques contain ß-amyloid peptides (Aß peptides) as the major proteinaceous constituent and are surrounded by activated microglia and astrocytes as well as dystrophic neurites. N-terminally truncated forms of Aß peptides are highly prevalent in neuritic plaques, including Aß 3-x beginning at Glu eventually modified to pyroglutamate (Aß N3pE-x), Aß 2-x, Aß 4-x, and Aß 5-x. The precise origin of the different N-terminally modified Aß peptides currently remains unknown. To assess the contribution of specific cell types to the formation of different N-terminally truncated Aß peptides, supernatants from serum-free primary cell cultures of chicken neurons, astrocytes, and microglia, as well as human astrocytes, were analyzed by Aß-ELISA and one- and two-dimensional SDS-urea polyacrylamide gel electrophoresis followed by immunoblot analysis. To evaluate the contribution of ß- and γ-secretase to the generation of N-terminally modified Aß, cultured astrocytes were treated with membrane-anchored "tripartite ß-secretase (BACE1) inhibitors" and the γ-secretase inhibitor DAPT. Neurons, astrocytes, and microglia each exhibited cell type-specific patterns of secreted Aß peptides. Neurons predominantly secreted Aß peptides that begin at Asp1, whereas those released from astrocytes and microglia included high proportions of N-terminally modified Aß peptides, presumably including Aß 2/3-x and 4/5-x. The inhibition of BACE1 reduced the amount of Aß 1-x in cell culture supernatants but not the amount of Aß 2-x.

High-fat diet induced isoform changes of the Parkinson's disease protein DJ-1.

Genetic and environmental factors mediate via different physiological and molecular processes a shifted energy balance leading to overweight and obesity. To get insights into the underlying processes involved in energy intake and weight gain, we compared hypothalamic tissue of mice kept on a high-fat or control diet for 10 days by a proteomic approach. Using two-dimensional difference gel electrophoresis in combination with LC-MS/MS, we observed significant abundance changes in 15 protein spots. One isoform of the protein DJ-1 was elevated in the high-fat diet group in three different mouse strains SWR/J, C57BL/6N, and AKR/J analyzed. Large-scale validation of DJ-1 isoforms in individual samples and tissues confirmed a shift in the pattern of DJ-1 isoforms toward more acidic isoforms in several brain and peripheral tissues after feeding a high-fat diet for 10 days. The identification of oxidation of cysteine 106 as well as 2-succinyl modification of the same residue by mass spectrometry not only explains the isoelectric shift of DJ-1 but also links our results to similar shifts of DJ-1 observed in neurodegenerative disease states under oxidative stress. We hypothesize that DJ-1 is a common physiological sensor involved in both nutrition-induced effects and neurodegenerative disease states.

Matrix Development for the Detection of Phosphorylated Amyloid-ß Peptides by MALDI-TOF-MS.

Amyloid-ß (Aß) peptides, including post-translationally modified variants thereof, are believed to play a key role in the onset and progression of Alzheimer's disease. Suggested modified Aß species with potential disease relevance include Aß peptides phosphorylated at serine in position eight (pSer8-Aß) or 26 (pSer26-Aß). However, the published studies on those Aß peptides essentially relied on antibody-based approaches. Thus, complementary analyses by mass spectrometry, as shown for other modified Aß variants, will be necessary not only to unambiguously verify the existence of phosphorylated Aß species in brain samples but also to reveal their exact identity as to phosphorylation sites and potential terminal truncations. With the aim of providing a novel tool for addressing this still-unresolved issue, we developed a customized matrix formulation, referred to as TOPAC, that allows for improved detection of synthetic phosphorylated Aß species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. When TOPAC was compared with standard matrices, we observed higher signal intensities but minimal methionine oxidation and phosphate loss for intact pSer8-Aß(1-40) and pSer26-Aß(1-40). Similarly, TOPAC also improved the mass spectrometric detection and sequencing of the proteolytic cleavage products pSer8-Aß(1-16) and pSer26-Aß(17-28). We expect that TOPAC will facilitate future efforts to detect and characterize endogenous phosphorylated Aß species in biological samples and that it may also find its use in phospho-proteomic approaches apart from applications in the Aß field.

Brain Region-Specific Differences in Amyloid-ß Plaque Composition in 5XFAD Mice.

Senile plaques consisting of amyloid-beta (Aß) peptides are a major pathological hallmark of Alzheimer's disease (AD). Aß peptides are heterogeneous regarding the exact length of their amino- and carboxy-termini. Aß1-40 and Aß1-42 are often considered to represent canonical "full-length" Aß species. Using immunohistochemistry, we analyzed the distribution of Aß1-x, Aßx-42 and Aß4-x species in amyloid deposits in the subiculum, hippocampus and cortex in 5XFAD mice during aging. Overall plaque load increased in all three brain regions, with the subiculum being the area with the strongest relative plaque coverage. In the subiculum, but not in the other brain regions, the Aß1-x load peaked at an age of five months and decreased thereafter. In contrast, the density of plaques positive for N-terminally truncated Aß4-x species increased continuously over time. We hypothesize that ongoing plaque remodeling takes place, leading to a conversion of deposited Aß1-x peptides into Aß4-x peptides in brain regions with a high Aß plaque burden.

Optimisation of BACE1 inhibition of tripartite structures by modification of membrane anchors, spacers and pharmacophores - development of potential agents for the treatment of Alzheimer's disease.

Systematic variation of membrane anchor, spacer and pharmacophore building blocks leads to an optimisation of the inhibitory effect of tripartite structures towards BACE1-induced cleavage of the amyloid precursor protein (APP).

ERK1/2 protein and mRNA levels in human blood are linked to smoking behavior.

From studies in cultured cells and animal models, nicotine and alcohol are known to regulate extracellular signal-regulated kinase 1 and 2 (ERK1/2). Alterations of ERK1/2 are thought to contribute to the drugs' rewarding effects. Accumulating evidence supports the importance of ERK1/2 in the molecular pathophysiology of depression and affective regulation in the hippocampus. We recently showed that the expression and phosphorylation of cyclic adenosine monophosphate response element (CRE)-binding protein (CREB) in human buffy coat were associated with smoking behavior. Because ERK1/2 is known to effect phosphorylation of CREB, the aim of the present study was to further elucidate whether cigarette smoking leads to alterations in terms of ERK1/2 in human buffy coat as well. In a comparison of 53 smokers with 146 non-smoking controls, we found significantly higher levels of ERK1/2 protein (P=0.004). In contrast, phospho-ERK1/2, phospho-/total-ERK1/2 ratio, mRNA-ERK1 and mRNA-ERK2 were not significantly different. Multiple regression analysis revealed a significant relation among the number of cigarettes smoked daily (R(2)=0.266, P=0.003), the Fagerström Test for Nicotine Dependence score (R(2)=0.149, P=0.032) and the mRNA expression of ERK1. Moreover, our analysis suggests that the mRNA expression of ERK2 might be linked to mood (model summary: R(2)=0.087, P=0.019; mRNA-ERK2: P=0.026). Given that the ERK1/2 signaling pathway plays an important role in the physiology and pathophysiology of affective and addictive behavior, our findings provide a rationale basis for additional mechanistic studies that may lead to the development of novel signaling pathway selective therapeutics in humans.

Is plasma amyloid-ß 1-42/1-40 a better biomarker for Alzheimer's disease than AßX-42/X-40?

BACKGROUND: A reduced amyloid-ß (Aß)42/40 peptide ratio in blood plasma represents a peripheral biomarker of the cerebral amyloid pathology observed in Alzheimer's disease brains. The magnitude of the measurable effect in plasma is smaller than in cerebrospinal fluid, presumably due to dilution by Aß peptides originating from peripheral sources. We hypothesized that the observable effect in plasma can be accentuated to some extent by specifically measuring Aß1-42 and Aß1-40 instead of AßX-42 and AßX-40. METHODS: We assessed the plasma AßX-42/X-40 and Aß1-42/1-40 ratios in an idealized clinical sample by semi-automated Aß immunoprecipitation followed by closely related sandwich immunoassays. The amyloid-positive and amyloid-negative groups (dichotomized according to Aß42/40 in cerebrospinal fluid) were compared regarding the median difference, mean difference, standardized effect size (Cohen's d) and receiver operating characteristic curves. For statistical evaluation, we applied bootstrapping. RESULTS: The median Aß1-42/1-40 ratio was 20.86% lower in amyloid-positive subjects than in the amyloid-negative group, while the median AßX-42/X-40 ratio was only 15.56% lower. The relative mean difference between amyloid-positive and amyloid-negative subjects was -18.34% for plasma Aß1-42/1-40 compared to -15.50% for AßX-42/X-40. Cohen's d was 1.73 for Aß1-42/1-40 and 1.48 for plasma AßX-42/X-40. Unadjusted p-values < 0.05 were obtained after .632 bootstrapping for all three parameters. Receiver operating characteristic analysis indicated very similar areas under the curves for plasma Aß1-42/1-40 and AßX-42/X-40. CONCLUSIONS: Our findings support the hypothesis that the relatively small difference in the plasma Aß42/40 ratio between subjects with and without evidence of brain amyloidosis can be accentuated by specifically measuring Aß1-42/1-40 instead of AßX-42/X-40. A simplified theoretical model explaining this observation is presented.

Evaluation of cerebrospinal fluid glycoprotein NMB (GPNMB) as a potential biomarker for Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder associated with extracellular amyloid-ß peptide deposition and progressive neuron loss. Strong evidence supports that neuroinflammatory changes such as the activation of astrocytes and microglia cells are important in the disease process. Glycoprotein nonmetastatic melanoma protein B (GPNMB) is a transmembrane glycoprotein that has recently been associated with an emerging role in neuroinflammation, which has been reported to be increased in post-mortem brain samples from AD and Parkinson's disease patients. METHODS: The present study describes the partial "fit for purpose" validation of a commercially available immunoassay for the determination of GPNMB levels in the cerebrospinal fluid (CSF). We further assessed the applicability of GPNMB as a potential biomarker for AD in two different cohorts that were defined by biomarker-supported clinical diagnosis or by neuroimaging with amyloid positron emission tomography, respectively. RESULTS: The results indicated that CSF GPNMB levels could not distinguish between AD or controls with other neurological diseases but correlated with other parameters such as aging and CSF pTau levels. CONCLUSIONS: The findings of this study do not support GPNMB in CSF as a valuable neurochemical diagnostic biomarker of AD but warrant further studies employing healthy control individuals.

Phagocytosis and LPS alter the maturation state of ß-amyloid precursor protein and induce different Aß peptide release signatures in human mononuclear phagocytes.

BACKGROUND: The classic neuritic ß-amyloid plaque of Alzheimer's disease (AD) is typically associated with activated microglia and neuroinflammation. Similarly, cerebrovascular ß-amyloid (Aß) deposits are surrounded by perivascular macrophages. Both observations indicate a contribution of the mononuclear phagocyte system to the development of ß-amyloid. METHODS: Human CD14-positive mononuclear phagocytes were isolated from EDTA-anticoagulated blood by magnetic activated cell sorting. After a cultivation period of 72 hours in serum-free medium we assessed the protein levels of amyloid precursor protein (APP) as well as the patterns and the amounts of released Aß peptides by ELISA or one-dimensional and two-dimensional urea-based SDS-PAGE followed by western immunoblotting. RESULTS: We observed strong and significant increases in Aß peptide release upon phagocytosis of acetylated low density lipoprotein (acLDL) or polystyrene beads and also after activation of the CD14/TLR4 pathway by stimulation with LPS. The proportion of released N-terminally truncated Aß variants was increased after stimulation with polystyrene beads and acLDL but not after stimulation with LPS. Furthermore, strong shifts in the proportions of single Aß1-40 and Aß2-40 variants were detected resulting in a stimulus-specific Aß signature. The increased release of Aß peptides was accompanied by elevated levels of full length APP in the cells. The maturation state of APP was correlated with the release of N-terminally truncated Aß peptides. CONCLUSIONS: These findings indicate that mononuclear phagocytes potentially contribute to the various N-truncated Aß variants found in AD ß-amyloid plaques, especially under neuroinflammatory conditions.

Smoking behaviour is associated with expression and phosphorylation of CREB in human buffy coat.

Nicotine induces various acute und chronic pharmacological effects which can be long lasting and might lead to nicotine dependence. Nicotinic acetylcholine receptors (nAChRs) are involved in nicotine-induced phosphorylation of CREB (cyclic AMP response element-binding protein) in PC12h cells. Several studies, mainly done in animal models, report that CREB plays a role in anxiety, memory and substance abuse as well as in affective disorders. Information regarding nicotine effects on gene expression in humans in vivo is rare. The aim of our study was to determine whether or not there are differences between smokers and non-smoking controls in terms of CREB expression and phosphorylation in human buffy coat. Comparing 32 smokers with 76 non-smoking controls we found significantly elevated relative (p=0.043) and absolute (p=0.040) CREB phosphorylation in the blood of smokers who had smoked two cigarettes in the past 6 h. In contrast, the score of the State and Trait Anxiety Inventory, total-CREB and mRNA-CREB were not significantly different. Multiple regression analysis revealed a significant relation between the number of cigarettes smoked daily (R2=0.143, p=0.023), the Fagerström Test for Nicotine Dependence score (R2=0.145, p=0.022) and the expression of CREB. Moreover, in accord with previously published data our analysis suggests gender and age as factors that significantly influence expression and phosphorylation of CREB. It appears that human buffy coat is suitable for studying pharmacological effects of substances such as nicotine on selected signal transduction pathways in humans in vivo. This kind of study may be helpful for translating findings from animal models and cell cultures.

Structural design, solid-phase synthesis and activity of membrane-anchored ß-secretase inhibitors on Aß generation from wild-type and Swedish-mutant APP.

Covalent coupling of ß-secretase inhibitors to a raftophilic lipid anchor via a suitable spacer by using solid-phase peptide synthesis leads to tripartite structures displaying substantially improved inhibition of cellular secretion of the ß-amyloid peptide (Aß). Herein, we describe a series of novel tripartite structures, their full characterization by NMR spectroscopy and mass spectrometry, and the analysis of their biological activity in cell-based assays. The tripartite structure concept is applicable to different pharmacophores, and the potency in terms of ß-secretase inhibition can be optimized by adjusting the spacer length to achieve an optimal distance of the inhibitor from the lipid bilayer. A tripartite structure containing a transition-state mimic inhibitor was found to be less potent on Aß generation from Swedish-mutant amyloid precursor protein (APP) than from the wild-type protein. Moreover, our observations suggest that specific variants of Aß are generated from wild-type APP but not from Swedish-mutant APP and are resistant to ß-secretase inhibition. Efficient inhibition of Aß secretion by tripartite structures in the absence of appreciable neurotoxicity was confirmed in a primary neuronal cell culture, thus further supporting the concept.

The Role of Cathepsin B in the Degradation of Aß and in the Production of Aß Peptides Starting With Ala2 in Cultured Astrocytes.

Astrocytes may not only be involved in the clearance of Amyloid beta peptides (Aß) in Alzheimer's disease (AD), but appear to produce N-terminally truncated Aß (Aßn-x) independently of BACE1, which generates the N-Terminus of Aß starting with Asp1 (Aß1-x). A candidate protease for the generation of Aßn-x is cathepsin B (CatB), especially since CatB has also been reported to degrade Aß, which could explain the opposite roles of astrocytes in AD. In this study, we investigated the influence of CatB inhibitors and the deletion of the gene encoding CatB (CTSB) using CRISPR/Cas9 technology on Aß2-x and Aß1-x levels in cell culture supernatants by one- and two-dimensional Urea-SDS-PAGE followed by immunoblot. While the cell-permeant inhibitors E64d and CA-074 Me did not significantly affect the Aß1-x levels in supernatants of cultured chicken and human astrocytes, they did reduce the Aß2-x levels. In the glioma-derived cell line H4, the Aß2-x levels were likewise decreased in supernatants by treatment with the more specific, but cell-impermeant CatB-inhibitor CA-074, by CA-074 Me treatment, and by CTSB gene deletion. Additionally, a more than 2-fold increase in secreted Aß1-x was observed under the latter two conditions. The CA-074 Me-mediated increase of Aß1-x, but not the decrease of Aß2-x, was influenced by concomitant treatment with the vacuolar H+-ATPase inhibitor Bafilomycin A1. This indicated that non-lysosomal CatB mediated the production of Aß2-x in astrocytes, while the degradation of Aß1-x seemed to be dependent on lysosomal CatB in H4 cells, but not in primary astrocytes. These findings highlight the importance of considering organelle targeting in drug development to promote Aß degradation.

Distinct fractional Abeta release patterns in human mononuclear phagocytes.

The presence of cells of the mononuclear phagocyte lineage surrounding neuritic and vascular beta-amyloid (Abeta) plaques is a hallmark of Alzheimer's disease (AD) neuropathology. The fractional Abeta peptide patterns released by human mononuclear phagocyte (MNP), lymphocyte and PBMC cultures were assessed by Abeta-SDS-PAGE/immunoblot and compared with the Abeta patterns in neuronal supernatants, in cerebrospinal fluid (CSF), and plasma. MNP released substantial amounts of Abeta peptides and the Abeta pattern contrasted with that in neuronal supernatants, CSF and plasma by reduced Abeta(1-42) and increased proportions of N-truncated Abeta species. MNP derived from early AD patients released significantly more total Abeta peptides than age-matched controls. The proportion of Abeta(1-42) was not altered.

Adherence-dependent shifts in the patterns of beta-amyloid peptides secreted by human mononuclear phagocytes.

Cells of the mononuclear phagocyte system are closely associated with vascular and neuritic beta-amyloid deposits in Alzheimer's disease. Using one-dimensional and newly developed two-dimensional Abeta-SDS-PAGE Western immunoblot techniques (1D/2D-Abeta-WIB) we investigated the patterns of Abeta peptides released by primary non-adherent and adherence-activated human mononuclear phagocytes in vitro. An overall increase of total released Abeta peptides (Abeta(total)) was observed in adherence-activated mononuclear phagocyte cultures. 2D-Abeta-WIB revealed that the proportion of Abeta(1-40) decreased significantly to 50.2+/-5.4% (n=10) of Abeta(total) compared to 65.9+/-5.6% (n=7) in non-adherent cultures (p<0.0001, t=5.82). Abeta(1-42) accounted for only 3.0+/-2.1% of Abeta(total) and its proportion did not change significantly upon adherence (2.8+/-0.5% of Abeta(total)). In adherence-activated cultures we detected pronounced shifts in the fractional pattern of released Abeta peptides in favour of N-truncated species. The second most prominent Abeta peptide accounted for as much as 12.7+/-3.0% of Abeta(total) (2.0+/-1.2% in non-adherent cultures; p<0.0001, t=9.00) and was identified as Abeta(2-40) by comigration with a synthetic peptide and by N-terminal-specific antibodies. A strong increase of a further Abeta immunoreactive spot migrating at pI 5.45 was observed. It accounted for 9.2+/-1.7% of Abeta(total) as compared to 1.0+/-0.9% in non-adherent cultures (p<0.0001, t=11.61) and presumably represented a variant of Abeta(2-40) as determined by C-terminal Abeta(40)-specific immunoprecipitation and N-terminal-specific immunodetection. Thus, mononuclear phagocytes might be one source of the N-truncated Abeta peptides regularly found in human plasma and are less likely to contribute substantially to plasma Abeta(1-42).