Co-aggregation with Apolipoprotein E modulates the function of Amyloid-ß in Alzheimer's disease.

Which isoforms of apolipoprotein E (apoE) we inherit determine our risk of developing late-onset Alzheimer's Disease (AD), but the mechanism underlying this link is poorly understood. In particular, the relevance of direct interactions between apoE and amyloid-ß (Aß) remains controversial. Here, single-molecule imaging shows that all isoforms of apoE associate with Aß in the early stages of aggregation and then fall away as fibrillation happens. ApoE-Aß co-aggregates account for ~50% of the mass of diffusible Aß aggregates detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. We show how dynamic interactions between apoE and Aß tune disease-related functions of Aß aggregates throughout the course of aggregation. Our results connect inherited APOE genotype with the risk of developing AD by demonstrating how, in an isoform- and lipidation-specific way, apoE modulates the aggregation, clearance and toxicity of Aß. Selectively removing non-lipidated apoE4-Aß co-aggregates enhances clearance of toxic Aß by glial cells, and reduces secretion of inflammatory markers and membrane damage, demonstrating a clear path to AD therapeutics.

Single-Molecule Characterization and Super-Resolution Imaging of Alzheimer's Disease-Relevant Tau Aggregates in Human Samples.

Hyperphosphorylation and aggregation of the protein tau play key roles in the development of Alzheimer's disease (AD). While the molecular structure of the filamentous tau aggregates has been determined to atomic resolution, there is far less information available about the smaller, soluble aggregates, which are believed to be more toxic. Traditional techniques are limited to bulk measures and struggle to identify individual aggregates in complex biological samples. To address this, we developed a novel single-molecule pull-down-based assay (MAPTau) to detect and characterize individual tau aggregates in AD and control post-mortem brain and biofluids. Using MAPTau, we report the quantity, as well as the size and circularity of tau aggregates measured using super-resolution microscopy, revealing AD-specific differences in tau aggregate morphology. By adapting MAPTau to detect multiple phosphorylation markers in individual aggregates using two-color coincidence detection, we derived compositional profiles of the individual aggregates. We find an AD-specific phosphorylation profile of tau aggregates with more than 80 % containing multiple phosphorylations, compared to 5 % in age-matched non-AD controls. Our results show that MAPTau is able to identify disease-specific subpopulations of tau aggregates phosphorylated at different sites, that are invisible to other methods and enable the study of disease mechanisms and diagnosis.

Cerebral organoids with chromosome 21 trisomy secrete Alzheimer's disease-related soluble aggregates detectable by single-molecule-fluorescence and super-resolution microscopy.

Understanding the role of small, soluble aggregates of beta-amyloid (Aß) and tau in Alzheimer's disease (AD) is of great importance for the rational design of preventative therapies. Here we report a set of methods for the detection, quantification, and characterisation of soluble aggregates in conditioned media of cerebral organoids derived from human iPSCs with trisomy 21, thus containing an extra copy of the amyloid precursor protein (APP) gene. We detected soluble beta-amyloid (Aß) and tau aggregates secreted by cerebral organoids from both control and the isogenic trisomy 21 (T21) genotype. We developed a novel method to normalise measurements to the number of live neurons within organoid-conditioned media based on glucose consumption. Thus normalised, T21 organoids produced 2.5-fold more Aß aggregates with a higher proportion of larger (300-2000 nm2) and more fibrillary-shaped aggregates than controls, along with 1.3-fold more soluble phosphorylated tau (pTau) aggregates, increased inflammasome ASC-specks, and a higher level of oxidative stress inducing thioredoxin-interacting protein (TXNIP). Importantly, all this was detectable prior to the appearance of histological amyloid plaques or intraneuronal tau-pathology in organoid slices, demonstrating the feasibility to model the initial pathogenic mechanisms for AD in-vitro using cells from live genetically pre-disposed donors before the onset of clinical disease. Then, using different iPSC clones generated from the same donor at different times in two independent experiments, we tested the reproducibility of findings in organoids. While there were differences in rates of disease progression between the experiments, the disease mechanisms were conserved. Overall, our results show that it is possible to non-invasively follow the development of pathology in organoid models of AD over time, by monitoring changes in the aggregates and proteins in the conditioned media, and open possibilities to study the time-course of the key pathogenic processes taking place.

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

We developed the aggregate characterization 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 characterizing 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.

Real-Time Growth Kinetics Analysis of Macromolecular Assemblies in Cells with Single Molecule Resolution.

Single molecule fluorescence microscopy has the unique advantage to provide real-time information on the spatiotemporal assembly of individual protein complexes in cellular membranes. This includes the assembly of proteins into oligomer species of numerous copy numbers. However, there is a need for improved tracing analysis of the real-time growth kinetics of these assemblies in cells with single molecule resolution. Here, we present an automated analysis software to accurately measure the real-time kinetics of assembly of individual high-order oligomer complexes. Our software comes with a simple Graphical User Interface (GUI), is available as a source code and an executable, and can analyze a full data set of several hundred to thousand molecules in less than 2 minutes. Importantly, this software is suitable for the analysis of intracellular protein oligomers, whose stoichiometry is usually more difficult to quantify due to variability in signal detection in the different areas of the cell. We validated our method with simulated ground-truth data and time-lapse images of diffraction-limited oligomeric assemblies of BAX and BAK proteins on mitochondria of cells undergoing apoptosis. Our approach provides the broad community of biologists with a fast, user-friendly tool to trace the compositional evolution of macromolecular assemblies, and potentially model their growth for a deeper understanding of the structural and biophysical mechanisms underlying their functions.

Constructing a cost-efficient, high-throughput and high-quality single-molecule localization microscope for super-resolution imaging.

Single-molecule localization microscopy (SMLM) leverages the power of modern optics to unleash ultra-precise structural nanoscopy of complex biological machines in their native environments as well as ultra-sensitive and high-throughput medical diagnostics with the sensitivity of a single molecule. To achieve this remarkable speed and resolution, SMLM setups are either built by research laboratories with strong expertise in optical engineering or commercially sold at a hefty price tag. The inaccessibility of SMLM to life scientists for technical or financial reasons is detrimental to the progress of biological and biomedical discoveries reliant on super-resolution imaging. In this work, we present the NanoPro, an economic, high-throughput, high-quality and easy-to-assemble SMLM for super-resolution imaging. We show that our instrument performs similarly to the most expensive, best-in-class commercial microscopes and rivals existing open-source microscopes at a lower price and construction complexity. To facilitate its wide adoption, we compiled a step-by-step protocol, accompanied by extensive illustrations, to aid inexperienced researchers in constructing the NanoPro as well as assessing its performance by imaging ground-truth samples as small as 20 nm. The detailed visual instructions make it possible for students with little expertise in microscopy engineering to construct, validate and use the NanoPro in <1 week, provided that all components are available.

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.

The interplay between BAX and BAK tunes apoptotic pore growth to control mitochondrial-DNA-mediated inflammation.

BAX and BAK are key apoptosis regulators that mediate the decisive step of mitochondrial outer membrane permeabilization. However, the mechanism by which they assemble the apoptotic pore remains obscure. Here, we report that BAX and BAK present distinct oligomerization properties, with BAK organizing into smaller structures with faster kinetics than BAX. BAK recruits and accelerates BAX assembly into oligomers that continue to grow during apoptosis. As a result, BAX and BAK regulate each other as they co-assemble into the same apoptotic pores, which we visualize. The relative availability of BAX and BAK molecules thereby determines the growth rate of the apoptotic pore and the relative kinetics by which mitochondrial contents, most notably mtDNA, are released. This feature of BAX and BAK results in distinct activation kinetics of the cGAS/STING pathway with implications for mtDNA-mediated paracrine inflammatory signaling.

Systematic Assessment of the Accuracy of Subunit Counting in Biomolecular Complexes Using Automated Single-Molecule Brightness Analysis.

Analysis of single-molecule brightness allows subunit counting of high-order oligomeric biomolecular complexes. Although the theory behind the method has been extensively assessed, systematic analysis of the experimental conditions required to accurately quantify the stoichiometry of biological complexes remains challenging. In this work, we develop a high-throughput, automated computational pipeline for single-molecule brightness analysis that requires minimal human input. We use this strategy to systematically quantify the accuracy of counting under a wide range of experimental conditions in simulated ground-truth data and then validate its use on experimentally obtained data. Our approach defines a set of conditions under which subunit counting by brightness analysis is designed to work optimally and helps in establishing the experimental limits in quantifying the number of subunits in a complex of interest. Finally, we combine these features into a powerful, yet simple, software that can be easily used for the analysis of the stoichiometry of such complexes.

Quantum Emitter Localization in Layer-Engineered Hexagonal Boron Nitride.

Hexagonal boron nitride (hBN) is a promising host material for room-temperature, tunable solid-state quantum emitters. A key technological challenge is deterministic and scalable spatial emitter localization, both laterally and vertically, while maintaining the full advantages of the 2D nature of the material. Here, we demonstrate emitter localization in hBN in all three dimensions via a monolayer (ML) engineering approach. We establish pretreatment processes for hBN MLs to either fully suppress or activate emission, thereby enabling such differently treated MLs to be used as select building blocks to achieve vertical (z) emitter localization at the atomic layer level. We show that emitter bleaching of ML hBN can be suppressed by sandwiching between two protecting hBN MLs, and that such thin stacks retain opportunities for external control of emission. We exploit this to achieve lateral (x-y) emitter localization via the addition of a patterned graphene mask that quenches fluorescence. Such complete emitter site localization is highly versatile, compatible with planar, scalable processing, allowing tailored approaches to addressable emitter array designs for advanced characterization, monolithic device integration, and photonic circuits.

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.

DeepSinse: deep learning-based detection of single molecules.

MOTIVATION: Imaging single molecules has emerged as a powerful characterization tool in the biological sciences. The detection of these under various noise conditions requires the use of algorithms that are dependent on the end-user inputting several parameters, the choice of which can be challenging and subjective. RESULTS: In this work, we propose DeepSinse, an easily trainable and useable deep neural network that can detect single molecules with little human input and across a wide range of signal-to-noise ratios. We validate the neural network on the detection of single bursts in simulated and experimental data and compare its performance with the best-in-class, domain-specific algorithms. AVAILABILITYAND IMPLEMENTATION: Ground truth ROI simulating code, neural network training, validation code, classification code, ROI picker, GUI for simulating, training and validating DeepSinse as well as pre-trained networks are all released under the MIT License on www.github.com/jdanial/DeepSinse. The dSTORM dataset processing code is released under the MIT License on www.github.com/jdanial/StormProcessor. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Single molecule imaging of protein aggregation in Dementia: Methods, insights and prospects.

The aggregation of misfolded proteins is a fundamental pathology in neurodegeneration which remains poorly understood due to its exceptional complexity and lack of appropriate characterization tools that can probe the role of the low concentrations of heterogeneous protein aggregates formed during the progression of the disease. In this review, we explain the principles underlying the operation of single molecule microscopy, an imaging method that can resolve molecules one-by-one, its application to imaging and characterizing individual protein aggregates in human samples and in vitro as well as the important questions in neurobiology this has answered and can answer.

Computed Histological Quantification of Atherosclerotic Plaque Microcalcifications.

Inflammation has a central role in atherosclerotic plaque formation and rupture. Intense macrophage inflammatory activity results in microcalcifications which are strongly associated with plaque vulnerability. Microcalcifications with specific critical size between 5 and 65 µ, located in the fibrous cap producing local mechanical stress on the plaque surface and may directly contribute to plaque rupture. Hence, accurate assessment of microcalcifications size and dimension has significant clinical importance. Current invasive and noninvasive plaque imaging has limited spatial resolution which limits accurate definition of microcalcifications in the atherosclerotic plaques. We describe a new imaging technique with high spatial resolution, based on confocal microscopic analysis, using a dedicated software which allows automatic characterization of microcalcifications and quantitative assessment of their extent and localization.

Coronary Artery Microcalcification: Imaging and Clinical Implications.

Strategies to prevent acute coronary and cerebrovascular events are based on accurate identification of patients at increased cardiovascular (CV) risk who may benefit from intensive preventive measures. The majority of acute CV events are precipitated by the rupture of the thin cap overlying the necrotic core of an atherosclerotic plaque. Hence, identification of vulnerable coronary lesions is essential for CV prevention. Atherosclerosis is a highly dynamic process involving cell migration, apoptosis, inflammation, osteogenesis, and intimal calcification, progressing from early lesions to advanced plaques. Coronary artery calcification (CAC) is a marker of coronary atherosclerosis, correlates with clinically significant coronary artery disease (CAD), predicts future CV events and improves the risk prediction of conventional risk factors. The relative importance of coronary calcification, whether it has a protective effect as a stabilizing force of high-risk atherosclerotic plaque has been debated until recently. The extent of calcium in coronary arteries has different clinical implications. Extensive plaque calcification is often a feature of advanced and stable atherosclerosis, which only rarely results in rupture. These macroscopic vascular calcifications can be detected by computed tomography (CT). The resulting CAC scoring, although a good marker of overall coronary plaque burden, is not useful to identify vulnerable lesions prone to rupture. Unlike macrocalcifications, spotty microcalcifications assessed by intravascular ultrasound or optical coherence tomography strongly correlate with plaque instability. However, they are below the resolution of CT due to limited spatial resolution. Microcalcifications develop in the earliest stages of coronary intimal calcification and directly contribute to plaque rupture producing local mechanical stress on the plaque surface. They result from a healing response to intense local macrophage inflammatory activity. Most of them show a progressive calcification transforming the early stage high-risk microcalcification into the stable end-stage macroscopic calcification. In recent years, new developments in noninvasive cardiovascular imaging technology have shifted the study of vulnerable plaques from morphology to the assessment of disease activity of the atherosclerotic lesions. Increased disease activity, detected by positron emission tomography (PET) and magnetic resonance (MR), has been shown to be associated with more microcalcification, larger necrotic core and greater rates of events. In this context, the paradox of increased coronary artery calcification observed in statin trials, despite reduced CV events, can be explained by the reduction of coronary inflammation induced by statin which results in more stable macrocalcification.

Quantitative analysis of super-resolved structures using ASAP.

Super-resolution microscopy allows imaging of cellular structures with high throughput and detail. However, the efficient and quantitative analysis of images generated is challenging with existing tools. Here, we develop ASAP (automated structures analysis program) to enable rapid and automated detection, classification and quantification of super-resolved structures. We validate ASAP on ground truth data and demonstrate its broad applicability by analyzing images of nucleoporins, TORC1 complexes, endocytic vesicles and Bax pores.

Correction: On demand modulation of lipid composition in an individual bilayer.

Correction for 'On demand modulation of lipid composition in an individual bilayer' by John S. H. Danial et al., Soft Matter, 2017, 13, 1788-1793.

On demand modulation of lipid composition in an individual bilayer.

Changes in local lipid composition are thought to play a key role in regulating many complex cellular processes. By studying lipid organization in artificial lipid bilayers the physical principles underlying these process can be studied in detail. However, such in vitro measurements are often hindered by heterogeneities in the lipid composition of individual bilayers prepared by current bulk methods. Here, the lipid composition of an individual droplet interface bilayer is varied by lipid titration into the bilayer from the oil phase in a microfluidic device. Control of lipid composition allows the reversible switching between single- and two-phase regions and sampling of specific lipid compositions in an individual bilayer. This method enables controlled modulation of composition-sensitive processes in a single lipid membrane.

Advanced fluorescence microscopy techniques for the life sciences.

The development of super-resolved fluorescence microscopy, for which the Nobel Prize was awarded in 2014, has been a topic of interest to physicists and biologists alike. It is inevitable that numerous questions in biomedical research cannot be answered by means other than direct observation. In this review, advances to fluorescence microscopy are covered in a widely accessible fashion to facilitate its use in decisions related to its acquisition and utilization in biomedical research.