Identification of the Aß37/42 peptide ratio in CSF as an improved Aß biomarker for Alzheimer's disease.

INTRODUCTION: Identifying CSF-based biomarkers for the ß-amyloidosis that initiates Alzheimer's disease (AD) could provide inexpensive and dynamic tests to distinguish AD from normal aging and predict future cognitive decline. METHODS: We developed immunoassays specifically detecting all C-terminal variants of secreted amyloid ß-protein and identified a novel biomarker, the Aß 37/42 ratio, that outperforms the canonical Aß42/40 ratio as a means to evaluate the γ-secretase activity and brain Aß accumulation. RESULTS: We show that Aß 37/42 can distinguish physiological and pathological status in (1) presenilin-1 mutant vs wild-type cultured cells, (2) AD vs control brain tissue, and (3) AD versus cognitively normal (CN) subjects in CSF, where 37/42 (AUC 0.9622) outperformed 42/40 (AUC 0.8651) in distinguishing CN from AD. DISCUSSION: We conclude that the Aß 37/42 ratio sensitively detects presenilin/γ-secretase dysfunction and better distinguishes CN from AD than Aß42/40 in CSF. Measuring this novel ratio alongside promising phospho-tau analytes may provide highly discriminatory fluid biomarkers for AD.

Generation and application of semi-synthetic p-Tau181 calibrator for immunoassay calibration.

Evidence suggests that plasma levels of tau protein phosphorylated at specific residues such as p-T181, p-T217, and p-T231 can be used as biomarkers for Alzheimer's disease (AD) diagnosis and prognosis. Accurate tools to calibrate immunoassays (calibrators) to precisely detect phosphorylated residues on tau protein will provide important gains in reliability and specificity. This study sought to establish a method to generate those accurate calibrators. We generated a semi-synthetic (chimeric) p-Tau181 calibrator by coupling a recombinant tau fragment (residues 1-174) with a synthetic peptide containing a single phosphorylated residue (p-T181) via thioester bond formation. The generation of a semi-synthetic protein containing both the N-terminal region of tau and the pT181 epitope was demonstrated by mobility shift assays using CBB staining and immunoblotting with N-terminal and pT181-specific antibodies. p-Tau 181 assays performed with the novel calibrator on multiple platforms revealed LLoQs as low as 0.14 pg/ml. Our facile and inexpensive method generates a semi-synthetic tau pT181 calibrator suitable for different immunoassay platforms. The same method can easily be adapted to other AD-relevant phospho-epitopes such as pT217 and pT231.

Hydrophilic loop 1 of Presenilin-1 and the APP GxxxG transmembrane motif regulate γ-secretase function in generating Alzheimer-causing Aß peptides.

γ-Secretase is responsible for the proteolysis of amyloid precursor protein (APP) into amyloid-beta (Aß) peptides, which are centrally implicated in the pathogenesis of Alzheimer's disease (AD). The biochemical mechanism of how processing by γ-secretase is regulated, especially as regards the interaction between enzyme and substrate, remains largely unknown. Here, mutagenesis reveals that the hydrophilic loop-1 (HL-1) of presenilin-1 (PS1) is critical for both γ-secretase step-wise cleavages (processivity) and its allosteric modulation by heterocyclic γ-modulatory compounds. Systematic mutagenesis of HL-1, including all of its familial AD mutations and additional engineered variants, and quantification of the resultant Aß products show that HL-1 is necessary for proper sequential γ-secretase processivity. We identify Y106, L113, and Y115 in HL-1 as key targets for heterocyclic γ-secretase modulators (GSMs) to stimulate processing of pathogenic Aß peptides. Further, we confirm that the GxxxG domain in the APP transmembrane region functions as a critical substrate motif for γ-secretase processivity: a G29A substitution in APP-C99 mimics the beneficial effects of GSMs. Together, these findings provide a molecular basis for the structural regulation of γ-processivity by enzyme and substrate, facilitating the rational design of new GSMs that lower AD-initiating amyloidogenic Aß peptides.

Multiple BACE1 inhibitors abnormally increase the BACE1 protein level in neurons by prolonging its half-life.

INTRODUCTION: There is keen interest in elucidating the biological mechanisms underlying recent failures of ß-site amyloid precursor protein-cleaving enzyme-1 (BACE1) inhibitors in Alzheimer's disease trials. METHODS: We developed a highly sensitive and specific immunoassay for BACE1 in cell lines and iPSC-derived human neurons to systematically analyze the effects of eight clinically relevant BACE1 inhibitors. RESULTS: Seven of 8 inhibitors elevated BACE1 protein levels. Among protease inhibitors tested, the elevation was specific to BACE1 inhibitors. The inhibitors did not increase BACE1 transcription but extended the protein's half-life. BACE1 became elevated at concentrations below the IC50 for amyloid ß (Aß). DISCUSSION: Elevation of BACE1 by 7 of 8 BACE1 inhibitors raises new concerns about advancing such ß-secretase inhibitors for AD. Chronic elevation could lead to intermittently uninhibited BACE1 when orally dosed inhibitors reach trough levels, abnormally increasing substrate processing. Compounds such as roburic acid that lower Aß by dissociating ß/γ secretase complexes are better candidates because they neither inhibit ß- and γ-secretase nor increase BACE1 levels.