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Ball, S. R.

Publications and source records attributed to Ball, S. R..

4 recordsLinked to original sources

Molecular mechanism of α-synuclein aggregation on lipid membranes revealed.

The central hallmark of Parkinsons disease pathology is the aggregation of the -synuclein protein, which, in its healthy form, is associated with lipid membranes. Purified monomeric -synuclein is relatively stable in vitro, but its aggregation can be triggered by the presence of lipid vesicles. Despite this central importance of lipids in the context of -synuclein aggregation, their mechanistic role in this process has not been established to date. Here, we use chemical kinetics to develop a detailed mechanistic model that is able to globally describe the aggregation behaviour of -synuclein in the presence of DMPS lipid vesicles, across a range of lipid and protein concentrations. Through the application of our kinetic model to experimental data, we find that the reaction is a co-aggregation process involving both protein and lipids and that lipids promote aggregation predominantly by enabling the elongation process. Moreover, we find that the initial formation of aggregates, via primary nucleation, takes place not on the surface of lipid vesicles but at the interfaces present in vitro. Our model will enable mechanistic insights, also in other lipid-protein co-aggregation systems, which will be crucial in the rational design of drugs that inhibit aggregate formation and act at the key points in the -synuclein aggregation cascade.

biophysics↗

Two-color coincidence single-molecule pull-down for the specific detection of disease-associated protein aggregates

The misfolding and aggregation of protein is a characteristic of many neurodegenerative disorders, including Alzheimers and Parkinsons disease. The wide range of sizes and structures of oligomers and fibrils generated have previously been studied using single-molecule and super-resolution microscopy. These methods, however, tend to rely on the use of either directly labeled protein, or on the addition of non-specific amyloid stains, such as thioflavin-T. This has prevented the characterization of protein aggregate composition in complex biological samples. Here, we have developed a single-molecule two-color aggregate pull-down (STAPull) assay to overcome this challenge by probing immobilized proteins using orthogonally labeled antibodies targeting the same epitope. By looking at colocalized signals, we can eliminate monomeric protein, and specifically quantify aggregated proteins. Using the aggregation-prone alpha-synuclein protein as a model, we demonstrate that this approach can specifically detect aggregates with a limit of detection of 5 pM. Furthermore, we show that STAPull can be used in a range of samples, including in human biofluids. STAPull is generally applicable to protein aggregates from a variety of disorders, and will aid in the identification of biomarkers that are crucial in the effort to diagnose these diseases.

neuroscience↗

iPSC-Derived PSEN2 (N141I) Astrocytes and Microglia Exhibit a Primed Inflammatory Phenotype

BackgroundWidescale evidence points to the involvement of glia and immune pathways in the progression of Alzheimers disease (AD). AD-associated iPSC-derived glial cells show a diverse range of AD-related phenotypic states encompassing cytokine/chemokine release, phagocytosis and morphological profiles, but to date studies are limited to cells derived from PSEN1, APOE and APP mutations or sporadic patients. The aim of the current study was to successfully differentiate iPSC-derived microglia and astrocytes from patients harbouring an AD-causative PSEN2 (N141I) mutation and characterise the inflammatory and morphological profile of these cells. MethodsiPSCs from three healthy control individuals and three familial AD patients harbouring a heterozygous PSEN2 (N141I) mutation were used to derive astrocytes and microglia-like cells and cell identity and morphology were characterised through immunofluorescent microscopy. Cellular characterisation involved the stimulation of these cells by LPS and A{beta}42 and analysis of cytokine/chemokine release was conducted through ELISAs and multi-cytokine arrays. The phagocytic capacity of these cells was then indexed by the uptake of fluorescently labelled fibrillar A{beta}42. ResultsAD-derived astrocytes and microglia-like cells exhibited an atrophied and less complex morphological appearance than healthy controls. AD-derived astrocytes showed increased basal expression of GFAP, S100{beta} and increased secretion and phagocytosis of A{beta}42 while AD-derived microglia-like cells showed decreased IL-8 secretion compared to healthy controls. Upon immunological challenge AD-derived astrocytes and microglia-like cells show exaggerated secretion of the pro-inflammatory IL-6, CXCL1, ICAM-1 and IL-8 from astrocytes and IL-18 and MIF from microglia. ConclusionOur study showed, for the first time, the differentiation and characterisation of iPSC-derived astrocytes and microglia-like cells harbouring a PSEN2 (N141I) mutation. PSEN2 (N141I)-mutant astrocytes and microglia-like cells presented with a primed phenotype characterised by reduced morphological complexity, exaggerated pro-inflammatory cytokine secretion and altered A{beta}42 production and phagocytosis.

neuroscience↗

Perphenazine-macrocycle conjugates divert Aβ42 towards non-toxic aggregates by monomer sequestration.

Alzheimers disease is imposing a growing social and economic burden worldwide and effective therapies are required. Strategies aimed at the removal of fibrillar plaques formed by the amyloid-{beta} peptide have not proved therapeutic and the focus has shifted to approaches that target the cytotoxic oligomeric amyloid-{beta} species that are populated before fibrils are deposited. We have designed and synthesized perphenazine-cyclam conjugates that specifically and rapidly bind to the monomeric form of A{beta}42, reducing the production of both cytotoxic oligomers and amyloid fibrils. We have applied detailed kinetic analysis and NMR spectroscopy to show that the perphenazine-cyclam conjugates divert the A{beta}42 monomer into amorphous aggregates that are not toxic to differentiated SH-SY5Y cells in vitro. Unlike most other amyloid inhibitors studied to date, these conjugates inhibit oligomer and fibril assembly even in the presence of pre-formed fibrillar seeds, demonstrating that they act through a monomer sequestration mechanism. These modular, three-dimensional conjugates therefore effectively prevent monomer-dependent secondary nucleation, the autocatalytic process that generates the majority of toxic oligomers.

biophysics↗