Paper-based origami assisted and enhanced electroanalytical detection of β-Amyloid peptide in plasma samples

BackgroundNeurogranin (Ng) is a cerebrospinal fluid (CSF) biomarker for synaptic dysfunction at early stage of Alzheimer’s disease (AD). It has been reported that a variety of truncated peptides from Ng is increased in AD brain tissue. However, the clinical significance of Ng peptides in plasma has remained unclear. We have developed a simultaneous detection method of a plurality of amyloid βpeptides in plasma by using immunoprecipitation combined with matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (IP‐MALDI‐MS) (Kaneko et al., Proc Jpn Acad Ser B Phys Biol Sci. 2014). In this study, we applied the IP‐MALDI‐MS technique to Ng measurement.MethodNg peptides and proteins were immunoprecipitated from human plasma or standard sample spiked by stable isotope‐labeled (SIL) Ng50‐78 using magnetic beads coupled with anti‐Ng monoclonal antibody. After the IP was performed two times consecutively, Ng proteins and peptides eluted from antibody beads were applied on the MALDI plate. SIL‐Ng50‐78 was used as an internal standard for normalizing an intensity of each Ng peptide.ResultWe optimized the condition such as a kind of detergent and an amount of antibody beads in IP for enhancing Ng signal intensity in MS. In the examination of detergents, UDM showed the highest sensitivity among six tested detergents. Using IP‐MALDI‐MS, we tried to detect Ng peptides and proteins in human plasma. As a result, some peaks corresponding to a variety of Ng peptides (m/z m/z > 5000.0) were detected in a plasma sample. To evaluate our semi‐quantitative assay using the internal standard, we selected some Ng peptides with low and high mass (m/z 2331.5–4084.7) to examine a linearity of the normalized intensity of Ng peptides. The accuracy in linear regression model showed within 80‐120% over a range of 5–160 pM in Ng48‐75 and 10‐160 pM in Ng50–78 and Ng33–75. Intra assay precision of Ng peptides in plasma was less than 20%CV.ConclusionIP‐MALDI‐MS enable the highly sensitive and simultaneous semi‐quantitative measurement of multiple Ng peptides levels. It could be helpful in the analysis of plasma Ng peptides for AD biomarker.

BACE1 (beta-site amyloid precursor protein-cleaving enzyme-1) is a membrane-bound aspartic protease that cleaves amyloid precursor protein to produce a neurotoxic peptide, amyloid beta-peptide, and has been implicated in triggering the pathogenesis of Alzheimer disease. We showed previously that BACE1 cleaves beta-galactoside alpha2,6-sialyltransferase I (ST6Gal I) to initiate its secretion, but it remained unclear how BACE1 affects the cellular level of alpha2,6-sialylation. Here, we found that BACE1 overexpression in Hep3B cells increased the sialylation of soluble secreted glycoproteins, but did not affect cell-surface sialylation. The sialylation of soluble glycoproteins was not increased by ST6Gal I overexpression alone, but was increased by co-overexpression of ST6Gal I and BACE1 or by expression of the soluble form of ST6Gal I, suggesting that soluble ST6Gal I produced by BACE1 plays, at least in part, a role in the sialylation of soluble glycoproteins. We also found that plasma glycoproteins from BACE1-deficient mice exhibited reduced levels of alpha2,6-sialylation compared with those from wild-type mice. We propose a novel regulatory mechanism in which cleavage and secretion of ST6Gal I enhance the sialylation of soluble glycoprotein substrates.

Crosslinked zwitterionic polymeric ionic liquid-functionalized nitinol wires for fiber-in-tube solid-phase microextraction and UHPLC-MS/MS as an amyloid beta peptide binding protein assay in biological fluids

Alzheimer's disease (AD), a neurological disorder, is a major public health concern and the most common form of dementia. Its typical symptoms include memory loss, confusion, changes in personality, and cognitive impairment, which result in patients gradually losing independence. Over the last decades, some studies have focused on searching for effective biomarkers as early diagnostic indicators of AD. Amyloid-β (Aβ) peptides have been consolidated as reliable AD biomarkers and have been incorporated into modern diagnostic research criteria. However, quantitative analysis of Aβ peptides in biological samples remains a challenge because both the sample and the physical-chemical properties of these peptides are complex. During clinical routine, Aβ peptides are measured in the cerebrospinal fluid by immunoassays, but the availability of a specific antibody is critical-in some cases, an antibody may not exist, or its specificity may be inadequate, leading to low sensitivity and false results. HPLC-MS/MS has been reported as a sensitive and selective method for determining different fragments of Aβ peptides in biological samples simultaneously. Developments in sample preparation techniques (preconcentration platforms) such as immunoprecipitation, 96-well plate SPME,onlineSPME, andfiber-in-tubeSPME have enabled not only effective enrichment of Aβ peptides present at trace levels in biological samples, but also efficient exclusion of interferents from the sample matrix (sample cleanup). This high extraction efficiency has provided MS platforms with higher sensitivity. Recently, methods affording LLOQ values as low as 5pgmL-1 have been reported. Such low LLOQ values are adequate for quantifying Aβ peptides in complex matrixes including cerebrospinal fluid (CSF) and plasma samples. This review summarizes the advances in mass spectrometry (MS)-based methods for quantifying Aβ peptides and covers the period 1992-2022. Important considerations regarding the development of the HPLC-MS/MS method such as the sample preparation step, optimization of the HPLC-MS/MS parameters, and matrix effects are described. Clinical applications, difficulties related to analysis of plasma samples, and future trends of these MS/MS-based methods are also discussed.

BackgroundPlasma amyloid-β (Aβ) peptides and tau proteins are promising biomarkers of Alzheimer’s disease (AD), not only for predicting Aβ and tau pathology but also for differentiating AD from other neurodegenerative diseases. However, reference intervals for plasma biomarkers of AD in healthy elderly Chinese individuals have not yet been established.MethodsBiomarkers of AD were measured using single-molecule array (Simoa) assays in plasma samples from 193 healthy, cognitively unimpaired Chinese individuals aged 50–89 years. The 95% reference intervals for plasma Aβ42, Aβ40, t-tau, p-tau181, and derived ratios were calculated by using log-transformed parametric methods.ResultsPlasma Aβ42, Aβ40, and p-tau181 levels were positively correlated with age, while the Aβ42/Aβ40 ratio was negatively correlated with age. The 95% reference intervals for plasma Aβ42 and Aβ40 were 2.72–11.09 pg/mL and 61.4–303.9 pg/mL, respectively, and the 95% reference intervals for plasma t-tau and p-tau181 were 0.20–3.12 pg/mL and 0.49–3.29 pg/mL, respectively. The 95% reference intervals for the Aβ42/Aβ40 ratio, p-tau181/t-tau ratio, and p-tau181/Aβ42 ratio were 0.022–0.064, 0.38–6.34, and 0.05–0.55, respectively.ConclusionReference intervals for plasma biomarkers of AD may assist clinicians in making accurate clinical decisions.

Alzheimer's disease (AD) molecular hallmarks are the accumulation of β-amyloid (Aβ) peptides and phosphorylated tau proteins in the patients' cerebral cortex. For this reason, the measurement of cerebrospinal fluid (CSF) levels of phosphorylated tau at threonine 181 (pTAU181), Aβ40, and Aβ42 is a routine clinical evaluation that, together with other diagnostic tools, helps providing a correct diagnosis. However, CSF collection is an invasive procedure. Hence, this study aims to compare these biomarkers concentration in CSF and plasma, assess their diagnostic accuracy within AD clinical continuum and determine the clinical variant and profile effect on their concentrations. Patients were assigned to a specific diagnosis, variant (typical vs atypical AD) and clinical profile (single or multidomain) following the NIA-AA criteria. Plasma samples were collected from a total of 85 healthy control (HC), 17 mild-cognitive impairment (MCI non-AD), 14 MCI presenting positive AD markers (MCI-AD) and 51 AD patients. CSF was obtained from 10 MCI non-AD, 12 MCI-AD and 42 AD dementia cases. APOE genotype was assessed using TaqMan™ SNP Genotyping Assay (Applied Biosystems, USA). Both plasma and CSF biomarkers measurements were performed using the LUMIPULSE® G600II instrument (Fujirebio, Japan) with relative kits. Plasma concentrations of the three biomarkers and relative ratios followed a trend confirmed both by CSF measurements and by literature. Furthermore, both biofluid biomarker levels appeared to change coherently with the change in patients' MMSE score, further supporting that cognitive function impairment is mirrored by a variation in CSF and most importantly blood biomarker levels. Considering the clinical variant, atypical patients displayed higher pTAU181 and lower Aβ42 concentrations, reflecting their lower MMSE score. As for the clinical profile, the Posterior Cortical Atrophy (PCA) one appeared to deviate from the others, showing higher pTAU181 and lower Aβ42 levels. Lastly, pTAU181 and Aβ42 were observed to be the best AD predictors. pTAU181, Aβ42, and the Aβ42/Aβ40 ratio were confirmed to be good indicators of AD pathology. Although further analyses are needed to accurately define the role of these biomarkers in distinguishing between the clinical variants, data hints at a possible difference between AD typical and atypical form.

Metal nanoparticles based electrochemical biosensing of neutrophil gelatinase-associated lipocalin biomarker for monitoring acute kidney injury

ApoE and Aβ in Alzheimer’s Disease: Accidental Encounters or Partners?

Alzheimer's disease (AD) is a neurodegenerative disorder characterized clinically by the progressive decline of memory and cognition. Histopathologically, two main hallmarks have been identified in AD: amyloid-β peptide extracellular neuritic plaques and neurofibrillary tangles formed by posttranslational modified tau protein. A definitive diagnosis can only be achieved after the post mortem verification of the histological mentioned alterations. Therefore, the development of biomarkers that allow an early diagnosis and/or predict disease progression is imperative. The prospect of a blood-based biomarker is possible with the finding of circulating microRNAs (miRNAs), a class of small non-coding RNAs of 22–25 nucleotides length that regulate mRNA translation rate. miRNAs travel through blood and recent studies performed in potential AD cases suggest the possibility of finding pathology-associated differences in circulating miRNA levels that may serve to assist in early diagnosis of the disease. However, these studies analyzed samples at a single time-point, limiting the use of miRNAs as biomarkers in AD progression. In this study we evaluated miRNA levels in plasma samples at different time-points of the evolution of an AD-like pathology in a transgenic mouse model of the disease (3xTg-AD). We performed multiplex qRT-PCR and compared the plasmatic levels of 84 miRNAs previously associated to central nervous system development and disease. No significant differences were detected between WT and transgenic young mice. However, age-related significant changes in miRNA abundance were observed for both WT and transgenic mice, and some of these were specific for the 3xTg-AD. In agreement, variations in the levels of particular miRNAs were identified between WT and transgenic old mice thus suggesting that the age-dependent evolution of the AD-like pathology, rather than the presence and expression of the transgenes, modifies the circulating miRNA levels in the 3xTg-AD mice.

BackgroundInducing brain ATP-binding cassette 1 (ABCA1) activity in Alzheimer’s disease (AD) mouse models is associated with improvement in AD pathology. The purpose of this study was to investigate the effects of the ABCA1 agonist peptide CS-6253 on amyloid-β peptides (Aβ) and lipoproteins in plasma and cerebrospinal fluid (CSF) of cynomolgus monkeys, a species with amyloid and lipoprotein metabolism similar to humans.MethodsCS-6253 peptide was injected intravenously into cynomolgus monkeys at various doses in three different studies. Plasma and CSF samples were collected at several time points before and after treatment. Levels of cholesterol, triglyceride (TG), lipoprotein particles, apolipoproteins, and Aβ were measured using ELISA, ion-mobility analysis, and asymmetric-flow field-flow fractionation (AF4). The relationship between the change in levels of these biomarkers was analyzed using multiple linear regression models and linear mixed-effects models.ResultsFollowing CS-6253 intravenous injection, within minutes, small plasma high-density lipoprotein (HDL) particles were increased. In two independent experiments, plasma TG, apolipoprotein E (apoE), and Aβ42/40 ratio were transiently increased following CS-6253 intravenous injection. This change was associated with a non-significant decrease in CSF Aβ42. Both plasma total cholesterol and HDL-cholesterol levels were reduced following treatment. AF4 fractionation revealed that CS-6253 treatment displaced apoE from HDL to intermediate-density- and low density-lipoprotein (IDL/LDL)-sized particles in plasma. In contrast to plasma, CS-6253 had no effect on the assessed CSF apolipoproteins or lipids.ConclusionsTreatment with the ABCA1 agonist CS-6253 appears to favor Aβ clearance from the brain.

ADAM10 is the main α-secretase that participates in the non-amyloidogenic cleavage of amyloid precursor protein (APP) in neurons, inhibiting the production of β-amyloid peptide (Aβ) in Alzheimer’s disease (AD). Strong recent evidence indicates the importance of the localization of ADAM10 for its activity as a protease. In this study, we investigated ADAM10 activity in plasma and CSF samples of patients with amnestic mild cognitive impairment (aMCI) and mild AD compared with cognitively healthy controls. Our results indicated that plasma levels of soluble ADAM10 were significantly increased in the mild AD group, and that in these samples the protease was inactive, as determined by activity assays. The same results were observed in CSF samples, indicating that the increased plasma ADAM10 levels reflect the levels found in the central nervous system. In SH-SY5Y neuroblastoma cells, ADAM10 achieves its major protease activity in the fraction obtained from plasma membrane lysis, where the mature form of the enzyme is detected, confirming the importance of ADAM10 localization for its activity. Taken together, our results demonstrate the potential of plasma ADAM10 to act as a biomarker for AD, highlighting its advantages as a less invasive, easier, faster, and lower-cost processing procedure, compared to existing biomarkers.

Amyloid‐β peptide (Aβ 1‐42 ) plays a pathogenic role in Alzheimer´s disease. Its physiological functions are, however, still debated. Aβ 1‐42 can induce the secretion of the pro‐inflammatory cytokine intereukin‐1β (IL‐1β) by immune cells. Known interaction partners of Aβ 1‐42 are α7 nicotinic acetylcholine receptors (nAChRs). Recently, we identified a cholinergic mechanism that inhibits the ATP‐associated release of IL‐1β by human monocytes via nAChRs containing α7, α9 and/or α10 subunits. Moreover, we discovered novel nAChR agonists that efficiently inhibit monocytic IL‐1β release via a metabotropic mechanism. The objective of this study was to test if Aβ 1‐42 can modulate this cholinergic mechanism. Human monocytic U937 cells and freshly isolated human peripheral blood mononuclear cells (approved by the ethics committee of the medical faculty Giessen, Germany, and performed in accordance with the Helsinki Declaration) were primed with lipopolysaccharide and ATP‐induced IL‐1β release in the presence or absence nAChR agonists and Aβ 1‐42 was measured by enzyme‐linked immunosorbent assay. We found that Aβ 1‐42 dose‐dependently (IC 50 = 2.5 µM) antagonized the inhibitory effect of canonical and novel non‐canonical nicotinic agonists like phosphocholine (100 µM), C‐reactive protein (5 µg/mL) and glycerophosphocholine (10 µM), indicating a novel pro‐inflammatory Aβ 1‐42 function that enables monocytic IL‐1β release in the presence of nAChR agonists. In whole‐cell patch‐clamp and calcium imaging experiments on human P2X7 receptor overexpressing HEK293 cells, no direct effect of Aβ 1‐42 on P2X7 receptor activity was observed. In blood plasma samples from major surgery patients, IL‐1β was mostly detected together with increased Aβ 1‐42 levels, which is in line with our hypothesis. Taken together, our results suggest that Aβ 1‐42 can antagonize the cholinergic control of ATP‐mediated monocytic IL‐1β‐release and, thus, provide evidence for a novel function of Aβ 1‐42 that also might be considered in situations of systemic inflammation.

An optimized UPLC-MS/MS method for human plasma amyloid-β 42 and 40 measurement and application in Alzheimer's disease diagnosis

Electroanalytical overview: The electroanalytical detection of theophylline

Glutaminyl cyclases (QC) catalyze the formation of neurotoxic pGlu-modified amyloid-β peptides found in the brains of people with Alzheimer's disease (AD). Reports of several-fold increases in soluble QC (sQC) expression in the brain and peripheral circulation of AD individuals has prompted the development of QC inhibitors as potential AD therapeutics. There is, however, a lack of standardized quantitative data on QC expression in human tissues, precluding inter-laboratory comparison and validation. We tested the hypothesis that QC is elevated in AD tissues by quantifying levels of sQC protein and activity in post-mortem brain tissues from AD and age-matched control individuals. We found a modest but statistically significant increase in sQC protein, which paralleled a similar increase in enzyme activity. In plasma samples sourced from the Australian Imaging, Biomarker and Lifestyle study we determined that QC activity was not different between the AD and control group, though a modest increase was observed in female AD individuals compared to controls. Plasma QC activity was further correlated with levels of circulating monocytes in AD individuals. These data provide quantitative evidence that alterations in QC expression are associated with AD pathology.