Validation of soluble amyloid-ß precursor protein assays as diagnostic CSF biomarkers for neurodegenerative diseases.

Analytical validation of a biomarker assay is essential before implementation in clinical practice can occur. In this study, we analytically validated the performance of assays detecting soluble amyloid-ß precursor protein (sAPP) α and ß in CSF in two laboratories according to previously standard operating procedures serving this goal. sAPPα and sAPPß ELISA assays from two vendors (IBL-international, Meso Scale Diagnostics) were validated. The performance parameters included precision, sensitivity, dilutional linearity, recovery, and parallelism. Inter-laboratory variation, biomarker comparison (sAPPα vs. sAPPß) and clinical performance was determined in three laboratories using 60 samples of patients with subjective memory complaints, Alzheimer's disease, or frontotemporal dementia. All performance parameters of the assays were similar between labs and within predefined acceptance criteria. The only exceptions were minor out-of-range results for recovery at low concentrations and, despite being within predefined acceptance criteria, non-comparability of the results for evaluation of the dilutional linearity and hook-effect. Based on the inter-laboratory correlation between Lab #1 and Lab #2, the IBL-international assays were more robust (sAPPα: r(2) = 0.92, sAPPß: r(2) = 0.94) than the Meso Scale Diagnostics (MSD) assay (sAPPα: r(2) = 0.70, sAPPß: r(2) = 0.80). Specificity of assays was confirmed using assay-specific peptide competitors. Clinical validation showed consistent results across the clinical groups in the different laboratories for all assays. The validated sAPP assays appear to be of sufficient technical quality and perform well. Moreover, the study shows that the newly developed standard operating procedures provide highly useful tools for the validation of new biomarker assays. A recommendation was made for renewed instructions to evaluate the dilutional linearity and hook-effect. We analytically validated the performance of assays detecting soluble amyloid-ß precursor protein (sAPP) α and ß in CSF according to SOPs in agreement with ISO15189 guidelines. The validated sAPP assays appear to be of sufficient technical quality and perform well. Moreover, this study proofs that the newly developed SOPs, with a minor modification, provide highly useful tools for the validation of new biomarker assays.

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.

Analysis of amino-terminal variants of amyloid-ß peptides by capillary isoelectric focusing immunoassay.

Here we present a novel assay for the separation and detection of amino-terminal amyloid-ß (Aß) peptide variants by capillary isoelectric focusing (CIEF) immunoassay. Specific amino-terminally truncated Aß peptides appear to be generated by ß-secretase (BACE1)-independent mechanisms and have previously been observed in cerebrospinal fluid (CSF) after BACE1 inhibitor treatment in an animal model. CIEF immunoassay sensitivity is sufficient to detect total Aß in CSF without preconcentration. To analyze low-abundance amino-terminally truncated Aß peptides from cell culture supernatants, we developed a CIEF-compatible immunoprecipitation protocol, allowing for selective elution of Aß peptides with very low background. CIEF immunoassay and immunoprecipitation mass spectrometry analysis identified peptides starting at residue Arg(5) as the main amino-terminal Aß variants produced in the presence of tripartite BACE1 inhibitor in our cell culture model. The CIEF immunoassay allows for robust relative quantification of Aß peptide patterns in biological samples. To assess the future possibility of absolute quantification, we have prepared the Aß peptides Aß(x-10), Aß(x-16), and Aß(5-38(D23S)) by using solid phase peptide synthesis as internal standards for the CIEF immunoassay.

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).

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.

Pharmacological Inhibition of Amyloidogenic APP Processing and Knock-Down of APP in Primary Human Macrophages Impairs the Secretion of Cytokines.

It has been previously shown that the amyloid precursor protein (APP) support the innate immune defense as an immune receptor. Amyloid ß (Aß) peptides seem to have properties of an antimicrobial peptide and can act as opsonines. In APP-deficient mouse models, a reduced secretion of cytokines has been observed. Still, it is unclear whether this can be attributed to the lack of APP or to the missing secretion of Aß peptides. We inhibited the secretion of Aß peptides in primary human monocyte derived macrophages with the γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine-t-butyl-ester (DAPT) or the ß-secretase inhibitor GL-189. Alternatively, we knocked down APP by transfection with siRNA. We measured tumor necrosis factor α (TNFα), interleukin 6 (IL-6) and interleukin (IL-10) by enzyme linked immunosorbent assay (ELISA) and evaluated the phagocytotic activity by flow cytometry. We observed reduced concentrations of TNFα and IL-6 in the media of APPk/d macrophages and after inhibition of the ß-, or γ-secretase, especially after additional immunological activation with lipopolysaccharide (LPS). Secretion of IL-10 was increased after pharmacological inhibition of APP processing when the macrophages were not immunologically activated but was decreased during LPS-induced inflammation in APPk/d macrophages. No changes of the phagocytotic activity were observed. We conclude that macrophage APP and Aß peptides support the initiation of an immune response and are involved in the regulation of TNFα, IL-6, and IL-10 secretion by human monocyte-derived macrophages.