11 research outputs found
The fluorescent pentameric oligothiophene pFTAA identifies filamentous tau in live neurons cultured from adult P301S tau mice.
Identification of fluorescent dyes that label the filamentous protein aggregates characteristic of neurodegenerative disease, such as β-amyloid and tau in Alzheimer's disease, in a live cell culture system has previously been a major hurdle. Here we show that pentameric formyl thiophene acetic acid (pFTAA) fulfills this function in living neurons cultured from adult P301S tau transgenic mice. Injection of pFTAA into 5-month-old P301S tau mice detected cortical and DRG neurons immunoreactive for AT100, an antibody that identifies solely filamentous tau, or MC1, an antibody that identifies a conformational change in tau that is commensurate with neurofibrillary tangle formation in Alzheimer's disease brains. In fixed cultures of dorsal root ganglion (DRG) neurons, pFTAA binding, which also identified AT100 or MC1+ve neurons, followed a single, saturable binding curve with a half saturation constant of 0.14 μM, the first reported measurement of a binding affinity of a beta-sheet reactive dye to primary neurons harboring filamentous tau. Treatment with formic acid, which solubilizes filamentous tau, extracted pFTAA, and prevented the re-binding of pFTAA and MC1 without perturbing expression of soluble tau, detected using an anti-human tau (HT7) antibody. In live cultures, pFTAA only identified DRG neurons that, after fixation, were AT100/MC1+ve, confirming that these forms of tau pre-exist in live neurons. The utility of pFTAA to discriminate between living neurons containing filamentous tau from other neurons is demonstrated by showing that more pFTAA+ve neurons die than pFTAA-ve neurons over 25 days. Since pFTAA identifies fibrillar tau and other misfolded proteins in living neurons in culture and in animal models of several neurodegenerative diseases, as well as in human brains, it will have considerable application in sorting out disease mechanisms and in identifying disease-modifying drugs that will ultimately help establish the mechanisms of neurodegeneration in human neurodegenerative diseases.This work was funded by grant number NC/L000741/1 from the National Council of the 3Rs (NC3Rs) and Alzheimer's Research UK (ARUK). KPR Nilsson is funded by an ERC Starting Independent Researcher Grant (Project: MUMID, number 260604).This is the final published version of the article. It was originally published in Frontiers in Neuroscience (Brelstaff J, Ossola B, Neher JJ, Klingstedt T, Nilsson KPR, Goedert M, Spillantini MG, Tolkovsky AM, Frontiers in Neuroscience, 2015, 9:184, doi:10.3389/fnins.2015.00184). The final version is available at http://dx.doi.org/10.3389/fnins.2015.0018
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Sensory Neurons from Tau Transgenic Mice and Their Utility in Drug Screening.
Tau misfolding is a major cause of neurodegeneration, tauopathies being a growing group of diseases in which tau forms insoluble aggregates, best known in Alzheimer disease as neurofibrillary tangles (NFTs). Many transgenic mouse models of tauopathies have been generated, but it has been difficult to demonstrate disease in primary brain neurons from these mice because neurons need to be harvested within a few days of birth and tau fails to produce NFTs. Transgenic mice have been generated that express the 0N4R isoform of human tau mutated at amino acid 301 (P301S mice) under the Thy1.2 promoter. These mice, which model an inherited form of frontotemporal dementia, develop NFTs around 5Â months of age. Taking advantage of the fact that Thy1.2 is expressed in the peripheral nervous system, we found that dorsal root ganglion (DRG) neurons express P301S tau and develop tau pathology along a similar time course to that found in central nervous system neurons in mice. Thus, NFTs are well-developed around 5Â months of age. Because DRG neurons can be cultured from adult mice for months, they have proven to be an excellent model for studying how tau pathology develops and for screening compounds that may ameliorate tau pathology. Here we present a detailed protocol for the preparation of long-term DRG neuron cultures and describe how to study whether activation of autophagy ameliorates tau pathology
pFTAA - a high affinity oligothiophene probe that detects filamentous tau in vivo and in cultured neurons
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The delayed kinetics of Myddosome formation explains why amyloid-beta aggregates trigger Toll-like receptor 4 less efficiently than lipopolysaccharide.
Peer reviewed: TrueAcknowledgements: The electron microscopy work was performed using the facilities at CAIC (Cambridge Advanced Imaging Centre). This work was funded by the Alzheimer’s research UK, EPSRC (EP/W015005/1), BBSRC (BB/V000276/1), the Royal Society, and the UK Dementia Research Institute which receives its funding from UK DRI Ltd, funded by the UK Medical Research Council, Alzheimer’s Society, and Alzheimer’s Research UK.The Myddosome is a key innate immune signalling platform. It forms at the cell surface and contains MyD88 and IRAK proteins which ultimately coordinate the production of pro-inflammatory cytokines. Toll-like receptor 4 (TLR4) signals via the Myddosome when triggered by lipopolysaccharide (LPS) or amyloid-beta (Aβ) aggregates but the magnitude and time duration of the response are very different for reasons that are unclear. Here, we followed the formation of Myddosomes in live macrophages using local delivery of TLR4 agonist to the cell surface and visualisation with 3D rapid light sheet imaging. This was complemented by super-resolution imaging of Myddosomes in fixed macrophages to determine the size of the signalling complex at different times after triggering. Myddosomes formed more rapidly after LPS than in response to sonicated Aβ 1-42 fibrils (80 vs 372 s). The mean lifetimes of the Myddosomes were also shorter when triggered by LPS compared to sonicated Aβ fibrils (170 and 220 s), respectively. In both cases, a range of Myddosome of different sizes (50-500 nm) were formed. In particular, small round Myddosomes around 100 nm in size formed at early time points, then reduced in proportion over time. Collectively, our data suggest that compared to LPS the multivalency of Aβ fibrils leads to the formation of larger Myddosomes which form more slowly and, due to their size, take longer to disassemble. This explains why sonicated Aβ fibrils results in less efficient triggering of TLR4 signalling and may be a general property of protein aggregates