6 research outputs found
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SingleâMolecule Characterization and SuperâResolution Imaging of Alzheimer's DiseaseâRelevant Tau Aggregates in Human Samples
Hyperphosphorylation and aggregation of the protein tau play key roles in the development of Alzheimerâs 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 singleâmolecule pullâdownâbased assay (MAPTau) to detect and characterize individual tau aggregates in AD and control postâmortem brain and biofluids. Using MAPTau, we report the quantity, as well as the size and circularity of tau aggregates measured using superâresolution microscopy, revealing ADâspecific differences in tau aggregate morphology. By adapting MAPTau to detect multiple phosphorylation markers in individual aggregates using twoâcolor 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 ageâmatched nonâAD controls. Our results show that MAPTau is able to identify diseaseâspecific subpopulations of tau aggregates phosphorylated at different sites, that are invisible to other methods and enable the study of disease mechanisms and diagnosis.This work was supported by the UK Dementia Research Institute, which receives its funding from DRI Ltd., funded by the UK Medical Research Council, Alzheimerâs Society, and Alzheimerâs Research. D.C. was supported by the Lady Edith Wolfson Junior Non-Clinical Research Fellowship awarded by the MND Association UK (Cox 971-799). J.Y.L.L. is supported by the Croucher Foundation Limited (Hong Kong). W.A.M and T.K. received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement 116060 (IMPRiND). This Joint Undertaking receives support from the European Unionâs Horizon 2020 Research and Innovation Program and EFPIA. This work is supported by the Swiss State Secretariat for Education, Research, and Innovation (SERI) under contract 17.00038. JBR is supported by the Wellcome Trust (103838; 220258), the Medical Research Council (MC_UU_00030/14); JBR and the Cambridge Brain Bank are supported by the NIHR Cambridge Biomedical Research Centre (NIHR203312)
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Single-Molecule Characterization and Super-Resolution Imaging of Alzheimer's Disease-Relevant Tau Aggregates in Human Samples.
Publication status: PublishedFunder: UK Dementia Research Institute; doi: http://dx.doi.org/10.13039/501100017510Funder: Croucher Foundation Limited (Hong Kong)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
Orthogonal fluorescent chemogenetic reporters for multicolor imaging
International audienceSpectrally separated fluorophores allow the observation of multiple targets simultaneously inside living cells, leading to a deeper understanding of the molecular interplay that regulates cell function and fate. Chemogenetic systems combining a tag and a synthetic fluorophore provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. Here, we present the engineering of a set of spectrally orthogonal fluorogen-activating tags based on the fluorescence-activating and absorption shifting tag (FAST) that are compatible with two-color, live-cell imaging. The resulting tags, greenFAST and redFAST, demonstrate orthogonality not only in their fluorogen recognition capabilities, but also in their one- and two-photon absorption profiles. This pair of orthogonal tags allowed the creation of a two-color cell cycle sensor capable of detecting very short, early cell cycles in zebrafish development and the development of split complementation systems capable of detecting multiple proteinâprotein interactions by live-cell fluorescence microscopy
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SingleâMolecule Characterization and SuperâResolution Imaging of Alzheimer's DiseaseâRelevant Tau Aggregates in Human Samples
Publication status: PublishedFunder: UK Dementia Research Institute; doi: http://dx.doi.org/10.13039/501100017510Funder: Croucher Foundation Limited (Hong Kong)AbstractHyperphosphorylation 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 singleâmolecule pullâdownâbased assay (MAPTau) to detect and characterize individual tau aggregates in AD and control postâmortem brain and biofluids. Using MAPTau, we report the quantity, as well as the size and circularity of tau aggregates measured using superâresolution microscopy, revealing ADâspecific differences in tau aggregate morphology. By adapting MAPTau to detect multiple phosphorylation markers in individual aggregates using twoâcolor coincidence detection, we derived compositional profiles of the individual aggregates. We find an ADâspecific phosphorylation profile of tau aggregates with more than 80â% containing multiple phosphorylations, compared to 5â% in ageâmatched nonâAD controls. Our results show that MAPTau is able to identify diseaseâspecific subpopulations of tau aggregates phosphorylated at different sites, that are invisible to other methods and enable the study of disease mechanisms and diagnosis.This work was supported by the UK Dementia Research Institute, which receives its funding from DRI Ltd., funded by the UK Medical Research Council, Alzheimerâs Society, and Alzheimerâs Research. D.C. was supported by the Lady Edith Wolfson Junior Non-Clinical Research Fellowship awarded by the MND Association UK (Cox 971-799). J.Y.L.L. is supported by the Croucher Foundation Limited (Hong Kong). W.A.M and T.K. received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement 116060 (IMPRiND). This Joint Undertaking receives support from the European Unionâs Horizon 2020 Research and Innovation Program and EFPIA. This work is supported by the Swiss State Secretariat for Education, Research, and Innovation (SERI) under contract 17.00038. JBR is supported by the Wellcome Trust (103838; 220258), the Medical Research Council (MC_UU_00030/14); JBR and the Cambridge Brain Bank are supported by the NIHR Cambridge Biomedical Research Centre (NIHR203312)
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Cerebral organoids with chromosome 21 trisomy secrete Alzheimer's disease-related soluble aggregates detectable by single-molecule-fluorescence and super-resolution microscopy.
Acknowledgements: DK is funded by UK Dementia Research Institute, which receives its funding from DRI Ltd. funded by the UK Medical Research Council and the Royal Society. DN is funded by The Wellcome Trust Collaborative Award in Science 217199/Z/19/Z. AM was awarded a William Harvey Academy Fellowship, co-funded by the People Programme (Marie Curie Actions) under REA no. 608765 and also a JĂ©rĂŽme Lejeune Foundation Post-doctoral Fellowship. IA was awarded an Adris Foundation grant. JYLL is supported by the Croucher Foundation Limited (Hong Kong).Funder: Royal Society; doi: https://doi.org/10.13039/501100000288Funder: D.K. is funded by UK Dementia Research Institute, which receives its funding from DRI Ltd. funded by the UK Medical Research Council.Funder: A.M. was awarded a William Harvey Academy Fellowship, co-funded by the People Programme (Marie Curie Actions) under REA no. 608765 and also a JĂ©rĂŽme Lejeune Foundation Post-doctoral Fellowship.Funder: J.Y.L.L. is supported by the Croucher Foundation Limited (Hong Kong).Funder: I.A. was awarded an Adris Foundation grant.Understanding the role of small, soluble aggregates of beta-amyloid (AÎČ) 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 (AÎČ) 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 AÎČ aggregates with a higher proportion of larger (300-2000ânm2) 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.D.K. is funded by UK Dementia Research Institute, which receives its funding from DRI Ltd. funded by the UK Medical Research Council and the Royal Society. D.N. is funded by The Wellcome Trust Collaborative Award in Science 217199/Z/19/Z. A.M. was awarded a William Harvey Academy Fellowship, co-funded by the People Programme (Marie Curie Actions) under REA no. 608765 and also a JĂ©rĂŽme Lejeune Foundation Post-doctoral Fellowship. I.A. was awarded an Adris Foundation grant. J.Y.L.L. is supported by the Croucher Foundation Limited (Hong Kong)