Super-resolution imaging of alpha-synuclein polymorphisms and their potential role in neurodegeneration

The conversion of soluble, functional proteins into amyloid fibrils has been linked to the development of neurodegenerative disorders, including Parkinson's and Alzheimer's disease. In the brains of patients with these disorders, the increasing presence of amyloid-containing plaques corresponds to neuronal cell death and the worsening of symptoms. However, protein amyloids are not merely confined to dying cells. Rather, some show a propensity to be transmitted to, and enter adjacent cells and induce the polymerization of the native monomer population. Whether this process is directly associated with toxicity or not is still highly debated. In this mini review, we will discuss structural polymorphisms of α-synuclein, as determined by super-resolution imaging techniques, and how these may be related to neuronal toxicity.This work was funded by grants from the UK Medical Research Council (MR/K015850/1 and MR/K02292X/1), Alzheimer’s Research UK (ARUK-EG2012A-1), the UK Engineering and Physical Sciences Research Council (EP/H018301/1), and the Wellcome Trust (089703/ Z/09/Z)

Intrinsically aggregation-prone proteins form amyloid-like aggregates and contribute to tissue aging in C. elegans

Reduced protein homeostasis leading to increased protein instability is a common molecular feature of aging, but it remains unclear whether this is a cause or consequence of the aging process. In neurodegenerative diseases and other amyloidoses, specific proteins self-assemble into amyloid fibrils and accumulate as pathological aggregates in different tissues. More recently, widespread protein aggregation has been described during normal aging. Until now, an extensive characterization of the nature of age-dependent protein aggregation has been lacking. Here, we show that age-dependent aggregates are rapidly formed by newly synthesized proteins and have an amyloid-like structure resembling that of protein aggregates observed in disease. We then demonstrate that age-dependent protein aggregation accelerates the functional decline of different tissues in C. elegans. Together, these findings imply that amyloid-like aggregates contribute to the aging process and therefore could be important targets for strategies designed to maintain physiological functions in the late stages of life

Transmission electron microscopy characterization of fluorescently labelled amyloid β 1-40 and α-synuclein aggregates

<p>Abstract</p> <p>Background</p> <p>Fluorescent tags, including small organic molecules and fluorescent proteins, enable the localization of protein molecules in biomedical research experiments. However, the use of these labels may interfere with the formation of larger-scale protein structures such as amyloid aggregates. Therefore, we investigate the effects of some commonly used fluorescent tags on the morphologies of fibrils grown from the Alzheimer's disease-associated peptide Amyloid β 1-40 (Aβ40) and the Parkinson's disease-associated protein α-synuclein (αS).</p> <p>Results</p> <p>Using transmission electron microscopy (TEM), we verify that N-terminal labeling of Aβ40 with AMCA, TAMRA, and Hilyte-Fluor 488 tags does not prevent the formation of protofibrils and amyloid fibrils of various widths. We also measure the two-photon action cross-section of Aβ40 labelled with Hilyte Fluor 488 and demonstrate that this tag is suitable for use with two-photon fluorescence techniques. Similarly, we find that Alexa Fluor 488 labelling of αS variant proteins near either the N or C terminus (position 9 or 130) does not interfere with the formation of amyloid and other types of αS fibrils. We also present TEM images of fibrils grown from αS C-terminally labelled with enhanced green fluorescent protein (EGFP). Near neutral pH, two types of αS-EGFP fibrils are observed via TEM, while denaturation of the EGFP tag leads to the formation of additional species.</p> <p>Conclusions</p> <p>We demonstrate that several small extrinsic fluorescent tags are compatible with studies of amyloid protein aggregation. However, although fibrils can be grown from αS labelled with EGFP, the conformation of the fluorescent protein tag affects the observed aggregate morphologies. Thus, our results should assist researchers with label selection and optimization of solution conditions for aggregation studies involving fluorescence techniques.</p

A new era for understanding amyloid structures and disease

The aggregation of proteins into amyloid fibrils and their deposition into plaques and intracellular inclusions is the hallmark of amyloid disease. The accumulation and deposition of amyloid fibrils, collectively known as amyloidosis, is associated with many pathological conditions that can be associated with ageing, such as Alzheimer disease, Parkinson disease, type II diabetes and dialysis-related amyloidosis. However, elucidation of the atomic structure of amyloid fibrils formed from their intact protein precursors and how fibril formation relates to disease has remained elusive. Recent advances in structural biology techniques, including cryo-electron microscopy and solid-state NMR spectroscopy, have finally broken this impasse. The first near-atomic-resolution structures of amyloid fibrils formed in vitro, seeded from plaque material and analysed directly ex vivo are now available. The results reveal cross-β structures that are far more intricate than anticipated. Here, we describe these structures, highlighting their similarities and differences, and the basis for their toxicity. We discuss how amyloid structure may affect the ability of fibrils to spread to different sites in the cell and between organisms in a prion-like manner, along with their roles in disease. These molecular insights will aid in understanding the development and spread of amyloid diseases and are inspiring new strategies for therapeutic intervention

Imaging Aβ(1–42) fibril elongation reveals strongly polarised growth and growth incompetent states

The major hallmark of Alzheimer’s disease is the deposition of plaques of amyloid fibrils formed from amyloid-beta (Ab) peptides. Kinetic studies have contributed significantly towards a mechanistic understanding of amyloid fibril self-assembly, however dynamic features of the aggregation process cannot be captured using ensemble methods. Here we present an assay for imaging Ab42 aggregation dynamics at the single fibril level, allowing for the quantitative extraction of concentration and temperature dependent kinetic parameters. From direct observation of elongation using TIRF and superresolution optical microscopy, we find that Ab42 fibril growth is strongly polarized, with fast and slow growing ends arising from different elongation rates, but also from a growth incompetent state, which dominates the process at the slow growing end. Our findings reveal the surprising complexity of the Ab42 fibril elongation reaction at the microscopic level

α-Synuclein – Regulator of Exocytosis, Endocytosis, or Both?

α-Synuclein is known as a presynaptic protein that binds to small synaptic vesicles. Recent studies suggest that α-synuclein is not only attracted to these tiny and therewith highly curved membranes, but that in fact the sensing and regulation of membrane curvature is part of its physiological function. Moreover, recent studies have suggested that α-synuclein plays a role in the endocytosis of synaptic vesicles, and have provided support for a function of α-synuclein during exo- and endocytosis in which curvature sensing and membrane stabilization are crucial steps. This review aims to highlight recent research in the field and adds a new picture on the function of α-synuclein in maintaining synaptic homeostasis upon intense and repetitive neuronal activity.J.L. was supported by a research fellowship from the Deutsche Forschungsgemeinschaft (DFG; award LA 3609/2-1 ). C.F.K. acknowledges funding from the UK Engineering and Physical Sciences Research Council ( EPSRC ). G.S.K. and C.F.K. acknowledge funding from the Wellcome Trust, the UK Medical Research Council (MRC), and Alzheimer Research UK (ARUK)

Graphene for Biosensing Applications in Point-of-Care Testing

Graphene and graphene-related materials (GRMs) exhibit a unique combination of electronic, optical, and electrochemical properties, which make them ideally suitable for ultrasensitive and selective point-of-care testing (POCT) devices. POCT device-based applications in diagnostics require test results to be readily accessible anywhere to produce results within a short analysis timeframe. This review article provides a summary of methods and latest developments in the field of graphene and GRM-based biosensing in POCT and an overview of the main applications of the latter in nucleic acids and enzymatic biosensing, cell detection, and immunosensing. For each application, we discuss scientific and technological advances along with the remaining challenges, outlining future directions for widespread use of this technology in biomedical applications

Microelectrode Arrays for Simultaneous Electrophysiology and Advanced Optical Microscopy

Advanced optical imaging techniques address important biological questions in neuroscience, where structures such as synapses are below the resolution limit of a conventional microscope. At the same time, microelectrode arrays (MEAs) are indispensable in understanding the language of neurons. Here, the authors show transparent MEAs capable of recording action potentials from neurons and compatible with advanced microscopy. The electrodes are made of the conducting polymer poly(3,4-ethylenedioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS) and are patterned by optical lithography, ensuring scalable fabrication with good control over device parameters. A thickness of 380 nm ensures low enough impedance and >75% transparency throughout the visible part of the spectrum making them suitable for artefact-free recording in the presence of laser illumination. Using primary neuronal cells, the arrays record single units from multiple nearby sources with a signal-to-noise ratio of 7.7 (17.7 dB). Additionally, it is possible to perform calcium (Ca2+) imaging, a measure of neuronal activity, using the novel transparent electrodes. Different biomarkers are imaged through the electrodes using conventional and super-resolution microscopy (SRM), showing no qualitative differences compared to glass substrates. These transparent MEAs pave the way for harnessing the synergy between the superior temporal resolution of electrophysiology and the selectivity and high spatial resolution of optical imaging

Multishank Thin-Film Neural Probes and Implantation System for High-Resolution Neural Recording Applications

Silicon probes have played a key role in studying the brain. However, the stark mechanical mismatch between these probes and the brain leads to chronic damage in the surrounding neural tissue, limiting their application in research and clinical translation. Mechanically flexible probes made of thin plastic shanks offer an attractive tissue-compatible alternative but are difficult to implant into the brain and struggle to achieve the electrode density and layout necessary for the high-resolution applications their silicon counterparts excel at. Here, we present a multi-shank high-density flexible neural probe design which emulates the functionality of stiff silicon arrays for neural population recordings across multiple sites within a given region. The flexible probe is accompanied by a detachable 3D printed implanter which delivers the probe by means of hydrophobic-coated shuttles. The shuttles can then be retracted with minimal movement, and the implanter houses the electronics necessary for in vivo recording applications. Validation of the probes through extracellular recordings from multiple brain regions and histological evidence of minimal foreign body response opens the path to long-term chronic monitoring of neural ensemble