Amyloidogenic Nanoplaques in Blood Serum of Patients with Alzheimer’s Disease Revealed by Time-Resolved Thioflavin T Fluorescence Intensity Fluctuation Analysis

Abstract

Background: Biomarkers are central to current research on molecular mechanisms underlying Alzheimer’s disease (AD). Their further development is of paramount importance for understanding pathophysiological processes that eventually lead to disease onset. Biomarkers are also crucial for early disease detection, before clinical manifestation, and for development of new disease modifying therapies. Objective: The overall aim of this work is to develop a minimally invasive method for fast, ultra-sensitive and cost-effective detection of structurally modified peptide/protein self-assemblies in the peripheral blood and in other biological fluids. Specifically, we focus here on using this method to detect structured amyloidogenic oligomeric aggregates in the blood serum of apparently healthy individuals and patients in early AD stage, and measure their concentration and size. Methods: Time-resolved detection of Thioflavin T (ThT) fluorescence intensity fluctuations in a sub-femtoliter observation volume element was used to identify in blood serum ThT-active structured amyloidogenic oligomeric aggregates, hereafter called nanoplaques, and measure with single-particle sensitivity their concentration and size. Results: The concentration and size of structured amyloidogenic nanoplaques are significantly higher in the blood serum of individuals diagnosed with AD than in control subjects. Conclusion: A new method with the ultimate, single-particle sensitivity was successfully developed. The proposed approach neither relies on the use of immune-based probes, nor on the use of radiotracers, signal-amplification or protein separation techniques, and provides a minimally invasive test for fast and cost-effective early determination of structurally modified peptides/proteins in the peripheral blood, as shown here, but also in other biological fluids.

Single-molecule studies of amyloid proteins: from biophysical properties to diagnostic perspectives

“…Thus, ultrasensitive analytical methods are needed to improve the accuracy of blood-based biomarkers for early-stage AD prediction. Recently, Tiiman et al (2019) identified the Aβ aggregates in blood serum from AD patients and found a higher concentration and larger size of ThT-reactive amyloidogenic nanoplaque compared to healthy controls through the SM FCS-ThT method. This result demonstrated the feasibility of detecting the actual amyloid states in the blood that corresponds to the fibrils in the brain.…”

A Bird’s-Eye View of the Multiple Biochemical Mechanisms that Propel Pathology of Alzheimer’s Disease: Recent Advances and Mechanistic Perspectives on How to Halt the Disease Progression Targeting Multiple Pathways

“…A possible diagnosis tool for AD has been the use of positron emission tomography (PET) with radiotracers to detect the presence of fibrillar deposits of amyloid in the brain that are greater than 1 mm in size. PET scans are quite expensive and there are issues in correlating deposits of amyloid with progression of AD, as its been found that plaques also deposit in the brains of healthy individual [107]. Detecting A␤ via antibodies in serum and cerebrospinal fluid (CSF) have also shown to be promising biomarkers but the predicative power of these biomarkers has not been particularly encouraging [108,109].…”

“…Detecting A␤ via antibodies in serum and cerebrospinal fluid (CSF) have also shown to be promising biomarkers but the predicative power of these biomarkers has not been particularly encouraging [108,109]. Furthermore, attempts to correlate serum/CSF with PET are not very convincing as of yet [107]. A potential minimally invasive variant to measuring amyloid without the use of expensive radiotracers or immunebased probes was developed by Tiiman et al [107].…”

“…Furthermore, attempts to correlate serum/CSF with PET are not very convincing as of yet [107]. A potential minimally invasive variant to measuring amyloid without the use of expensive radiotracers or immunebased probes was developed by Tiiman et al [107]. This technique measures amyloidogenic oligomeric aggregates (called nanoplaques) in blood via a specialized ThT (Thioflavin T) fluorescence assay [107].…”

“…A potential minimally invasive variant to measuring amyloid without the use of expensive radiotracers or immunebased probes was developed by Tiiman et al [107]. This technique measures amyloidogenic oligomeric aggregates (called nanoplaques) in blood via a specialized ThT (Thioflavin T) fluorescence assay [107]. Tiiman et al was able to show that there was a significantly higher amount of larger nanoplaques in the blood of AD patients compared to control subjects.…”