Different soluble aggregates of Aβ42 can give rise to cellular toxicity through different mechanisms.

Protein aggregation is a complex process resulting in the formation of heterogeneous mixtures of aggregate populations that are closely linked to neurodegenerative conditions, such as Alzheimer's disease. Here, we find that soluble aggregates formed at different stages of the aggregation process of amyloid beta (Aβ42) induce the disruption of lipid bilayers and an inflammatory response to different extents. Further, by using gradient ultracentrifugation assay, we show that the smaller aggregates are those most potent at inducing membrane permeability and most effectively inhibited by antibodies binding to the C-terminal region of Aβ42. By contrast, we find that the larger soluble aggregates are those most effective at causing an inflammatory response in microglia cells and more effectively inhibited by antibodies targeting the N-terminal region of Aβ42. These findings suggest that different toxic mechanisms driven by different soluble aggregated species of Aβ42 may contribute to the onset and progression of Alzheimer's disease.This study is supported by the Marie-Curie Individual Fellowship programme (S.D.), EPSRC Studentship (D.C.W.), Boehringer Ingelheim Fonds (P.F.), Studienstiftung des deutschen Volkes (P.F.), Senior Research Fellowship from the Alzheimer's Society, Grant Number 317, AS-SF-16-003, UK (F.A.A), Swiss National Fondation for Science and Darwin College grant number P2ELP2_162116 and P300P2_171219 (F.S.R.), Borysiewicz Biomedical Fellowship from the University of Cambridge(P.S), the UK Biotechnology and Biochemical Sciences Research Council (C.M.D.); the Wellcome Trust (C.M.D) the Cambridge Centre for Misfolding Diseases (P.F., F.A.A., P.S., C.M.D., and M.V.) and the European Research Council Grant Number 669237 (D.K.) and the Royal Society (D.K.)

Soluble amyloid beta-containing aggregates are present throughout the brain at early stages of Alzheimer's disease.

Protein aggregation likely plays a key role in the initiation and spreading of Alzheimer's disease pathology through the brain. Soluble aggregates of amyloid beta are believed to play a key role in this process. However, the aggregates present in humans are still poorly characterized due to a lack of suitable methods required for characterizing the low concentration of heterogeneous aggregates present. We have used a variety of biophysical methods to characterize the aggregates present in human Alzheimer's disease brains at Braak stage III. We find soluble amyloid beta-containing aggregates in all regions of the brain up to 200 nm in length, capable of causing an inflammatory response. Rather than aggregates spreading through the brain as disease progresses, it appears that aggregation occurs all over the brain and that different brain regions are at earlier or later stages of the same process, with the later stages causing increased inflammation

The Development and Application of Ultra-sensitive Protein Aggregate Detection Techniques

There are more than 55 million patients suffering from dementia worldwide, yet there is no definitive diagnostic tool or effective treatment for the disease. The thesis focuses on the development and application of ultra-sensitive techniques for detecting protein aggregates. It begins by studying the role of apolipoprotein E (apoE) in amyloid- β (Aβ) pathology in Alzheimer’s disease (AD). The isoform-specific and lipidation-specific behaviours of apoE and the co-aggregates formed with A β are explored. The discussion proposes potential therapeutic strategies involving the promotion of apoE lipidation. Then, the focus shifts to the development of ultra-sensitive techniques for quantifying protein aggregates in human biofluid. The resulting single molecule array (SiMoA)-based assays exhibit high sensitivity in the lower picomolar range with satisfactory precision. The aggregate quantification assay developed was then used in biomarker discovery for AD diagnosis. The multifaceted approach, combining individual assay measurements and ratios, exhibited a more reliable blood-based prediction for the disease than individual measurements. The SiMoA assays were also employed to evaluate the effectiveness of MEDI1341 in a clinical trial, providing insights into antibody interference and the drug’s impact on physiological protein aggregate concentration. In addition, the thesis introduces a fluorescence image analysis suite, ACT, which proves to be a valuable tool in protein aggregate studies. ACT was rigorously tested and found to be particularly user-friendly. It demonstrated great performance in identifying and characterising aggregate in fluorescence microscopy image.The work was sponsored by an AstraZeneca studentship

Mineralization and ore genesis of the Qiaoxiahala Fe-Cu-(Au) deposit in the northern margin of East Junggar terrane, Central Asian Orogenic Belt: Constraints from fluid inclusions and stable isotopes

The Qiaoxiahala Fe-Cu-(Au) deposit (1.44 Mt ore @ 43-53% Fe, 0.55-2.21% Cu and 0.13-2.4 g/t Au) is located in the northern margin of East Junggar terrane, NW China. The deposit is hosted in the upper part of the Middle Devonian Beitashan Formation mafic and felsic volcanic rocks and sedimentary interbeds, and is spatially closely related to local fractures. Six hypogene hydrothermal stages have been identified, including (Stage I) early skarn, (Stage II) late skarn, (Stage III) magnetite mineralization, (Stage IV) magnetite-pyrite mineralization, (Stage V) chalcopyrite mineralization and (Stage VI) late veins. These six stages include three major mineralization events, as represented by the (Stage III) magnetite-epi dote-K-feldspar-quartz-calcite, (Stage IV) magnetite-pyrite-garnet-quartz-calcite and (Stage V) chalcopyrite-chlorite assemblages. The Stage III magnetite mineralization fluids were likely of high temperature (354-386 degrees C), low-medium salinity (9.9-14.7 wt% NaCl equiv.), Mg/Fe-rich and organic-rich with lithostatic pressure of 1.3-1.5 kbar and corresponding depth of 4.3-4.8 km. Oxygen and hydrogen isotopes showed that the O-18-rich (9.7-11.2 parts per thousand) and D-rich (-26.3 parts per thousand) fluids were possibly modified from the Early Carboniferous seawater interacting with the Beitashan Formation organic-rich limestone or tuff. In comparison, the Stage IV magnetite-pyrite mineralization fluids were likely of medium-high temperature (272-453 degrees C), low-medium salinity (2.9-19.5 wt% NaCl equiv.), Mg/Fe-rich, with pressure of 0.8-1.2 kbar and corresponding depth of 2.6-4.0 km. The Stage V chalcopyrite mineralization fluids were likely of low-medium temperature (186-270 degrees C), low-medium salinity (1.4-21.8 wt% NaCl equiv.), Ca- or Na-rich, with pressure of 0.4-0.6 kbar and corresponding depth of 1-2 km. Hydrogen and oxygen isotopic compositions (Stage IV: delta D-water = -141 to -92 parts per thousand; delta O-18(water) = 8.9-11.1 parts per thousand Stage V: delta D-water = -104 parts per thousand; delta O-18(water) = 7.2 parts per thousand) suggest that the ore fluids were of connate marine origin. Furthermore, Stage IV delta C-13(water) values (-0.6 to 0 parts per thousand) also indicate interactions between seawater and sedimentary wall-rocks. The negative sulfur isotopic compositions of chalcopyrite and pyrite (-4.2 to -0.2 parts per thousand) may demonstrate a biogenic sulfur source. Compared with the arc-related IOCG deposits in the central Andean orogen, the Qiaoxiahala Fe-Cu-(Au) deposit shares many common features in geology, mineralization, alteration and various sources of fluids, indicating a possible IOCG-like mineralization-style and potential to discover other Late Paleozoic arc-related IOCG deposits in East Junggar. (C) 2017 Elsevier B.V. All rights reserved

An economic, square-shaped flat-field illumination module for TIRF-based super-resolution microscopy.

Super-resolution microscopy allows complex biological assemblies to be observed with remarkable resolution. However, the presence of uneven Gaussian-shaped illumination hinders its use in quantitative imaging or high-throughput assays. Methods developed to circumvent this problem are often expensive, hard to implement, or not applicable to total internal reflection fluorescence imaging. We herein demonstrate a cost-effective method to overcome these challenges using a small square-core multimodal optical fiber as the coupler. We characterize our method with synthetic, recombinant, and cellular systems imaged under total internal reflection fluorescence and highly inclined and laminated optical sheet illuminations to demonstrate its ability to produce highly uniform images under all conditions

A computational suite for the structural and functional characterization of amyloid aggregates

We developed the Aggregate Characterisation Toolkit (ACT); a fully automated computational suite based on existing and widely used core algorithms to measure the number, size and permeabilizing activity of recombinant and human-derived aggregates imaged with diffraction-limited and super-resolution microscopy methods at high throughput. We have validated ACT on simulated ground-truth images of aggregates mimicking those from diffraction-limited and super-resolution microscopies and showcased its use in characterising protein aggregates from Alzheimer’s disease. ACT is developed for high-throughput, batch processing of images collected from multiple samples and is available as an open-source code. Given its accuracy, speed and accessibility, ACT is expected to be a fundamental tool in studying human and non-human amyloid intermediates, developing early disease stage diagnostics and screening for antibodies that bind toxic and heterogeneous human amyloid aggregates

Imaging protein aggregates in the serum and cerebrospinal fluid in Parkinson's disease.

Aggregation of α-synuclein plays a key role in the development of Parkinson's disease. Soluble aggregates are present not only within human brain but also the CSF and blood. Characterizing the aggregates present in these biofluids may provide insights into disease mechanisms and also have potential for aiding diagnosis. We used two optical single-molecule imaging methods called aptamer DNA-PAINT and single-aggregate confocal fluorescence, together with high-resolution atomic force microscopy for specific detection and characterization of individual aggregates with intermolecular β-sheet structure, present in the CSF and serum of 15 early stage Parkinson's disease patients compared to 10 healthy age-matched controls. We found aggregates ranging in size from 20 nm to 200 nm, in both CSF and serum. There was a difference in aggregate size distribution between Parkinson's disease and control groups with a significantly increased number of larger aggregates (longer than 150 nm) in the serum of patients with Parkinson's disease. To determine the chemical composition of the aggregates, we performed aptamer DNA-PAINT on serum following α-synuclein and amyloid-β immunodepletion in an independent cohort of 11 patients with early stage Parkinson's disease and 10 control subjects. β-Sheet aggregates in the serum of Parkinson's disease patients were found to consist of, on average, 50% α-synuclein and 50% amyloid-β in contrast to 30% α-synuclein and 70% amyloid-β in control serum [the differences in the proportion of these aggregates were statistically significant between diseased and control groups (P = 1.7 × 10-5 for each species)]. The ratio of the number of β-sheet α-synuclein aggregates to β-sheet amyloid-β aggregates in serum extracted using our super-resolution method discriminated Parkinson's disease cases from controls with an accuracy of 98.2% (AUC = 98.2%, P = 4.3 × 10-5). Our data suggest that studying the protein aggregates present in serum can provide information about the disruption of protein homeostasis occurring in Parkinson's disease and warrants further investigation as a potential biomarker of disease

Small soluble α-synuclein aggregates are the toxic species in Parkinson's disease.

Funder: Royal SocietySoluble α-synuclein aggregates varying in size, structure, and morphology have been closely linked to neuronal death in Parkinson's disease. However, the heterogeneity of different co-existing aggregate species makes it hard to isolate and study their individual toxic properties. Here, we show a reliable non-perturbative method to separate a heterogeneous mixture of protein aggregates by size. We find that aggregates of wild-type α-synuclein smaller than 200 nm in length, formed during an in vitro aggregation reaction, cause inflammation and permeabilization of single-liposome membranes and that larger aggregates are less toxic. Studying soluble aggregates extracted from post-mortem human brains also reveals that these aggregates are similar in size and structure to the smaller aggregates formed in aggregation reactions in the test tube. Furthermore, we find that the soluble aggregates present in Parkinson's disease brains are smaller, largely less than 100 nm, and more inflammatory compared to the larger aggregates present in control brains. This study suggests that the small non-fibrillar α-synuclein aggregates are the critical species driving neuroinflammation and disease progression.This work was supported by Parkinson’s UK (G-1901), UK Dementia Research Institute, which receives its funding from DRI Ltd. funded by the UK Medical Research Council and by the European Research Council Grant Number 669237 and the Royal Society. Y.Z .was supported by the UK Engineering and Physical Sciences Research Council (EP/R005397/1) and the NIHR Cambridge Biomedical Research Centre Dementia and Neurodegeneration Theme (146281)