Extracellular Tau Aggregates Research Articles

Measuring Antibody-Mediated Tau Fibril Uptake in Microglia by Flow Cytometry.

Microglia are brain-resident phagocytic cells, considered to be the innate immune cells of the central nervous system. Microglia respond to both infectious pathogens in the brain and sterile cellular debris, including the proteinaceous aggregates that accumulate in the brains of patients with Alzheimer's disease (AD). Microtubule-associated protein Tau is an intracellular protein that self-aggregates into neurofibrillary tangles in Alzheimer's disease and many other neurodegenerative diseases. Ongoing clinical trials are testing whether therapeutic antibodies specific to Tau protein aggregates can reduce pathological protein deposition and improve the course of disease. Data suggest that Tau-specific antibodies act on extracellular Tau aggregates by promoting uptake into microglia cells and thus preventing its prion-like spread to unaffected neurons. Here we describe a protocol to test the effect of Tau-specific antibodies on Tau uptake into microglia by flow cytometry. Recombinant Tau protein is fibrillized in vitro and tagged with a fluorescent label. Then, fibrillized Tau is incubated with the antibody of interest and applied to microglial cells in culture. Uptake of Tau into microglia is then assessed by measuring fluorescence intensity by flow cytometry. With slight modifications, this assay can be used to test effects of many antibodies, various Tau protein compositions, and different microglial sources in a high-throughput format.

PI3K-AKT activation resculpts integrin signaling to drive filamentous tau-induced proinflammatory astrogliosis

BackgroundMicrotubule-binding protein tau is a misfolding-prone protein associated with tauopathies. As tau undergoes cell-to-cell transmission, extracellular tau aggregates convert astrocytes into a pro-inflammatory state via integrin activation, causing them to release unknown neurotoxic factors.ResultsHere, we combine transcriptomics with isotope labeling-based quantitative mass spectrometry analysis of mouse primary astrocyte secretome to establish PI3K-AKT as a critical differentiator between pathogenic and physiological integrin activation; simultaneous activation of PI3K-AKT and focal adhesion kinase (FAK) in tau fibril-treated astrocytes changes the output of integrin signaling, causing pro-inflammatory gene upregulation, trans-Golgi network restructuring, and altered secretory flow. Furthermore, NCAM1, as a proximal signaling component in tau-stimulated integrin and PI3K-AKT activation, facilitates the secretion of complement C3 as a main neurotoxic factor. Significantly, tau fibrils-associated astrogliosis and C3 secretion can be mitigated by FAK or PI3K inhibitors.ConclusionsThese findings reveal an unexpected function for PI3K-AKT in tauopathy-associated reactive astrogliosis, which may be a promising target for anti-inflammation-based Alzheimer’s therapy.

TAU-INDUCED ASTROCYTE SENESCENCE: A NOVEL MECHANISM FOR NEURONAL DYSFUNCTION IN ALZHEIMER’S DISEASE

Chronic sterile inflammation is a pathological feature of Alzheimer’s disease (AD) and other neurodegenerative diseases. The mechanisms that drive neuroinflammation and its impact on AD progression are still incompletely understood. The accumulation of molecular damage in somatic cells can trigger cellular senescence, an irreversible state of cell cycle arrest accompanied by the expression of proinflammatory mediators known collectively as the “senescence-associated secretory phenotype” (SASP). Misfolded tau forms pathogenic soluble aggregates that are released extracellularly and are transmitted trans-neuronally, promoting native tau phosphorylation and aggregation in target cells. We recently showed that, in addition to neurons, pathogenic soluble extracellular tau aggregates propagate to brain microvascular endothelial cells, where microtubule destabilization triggers senescence/SASP. Because astrocytes have a critical role in the regulation of both synaptic function and cerebral blood flow and are directly exposed to tau at its site of release at the tripartite synapse, we conducted studies to define whether soluble aggregated tau propagates to astrocytes, inducing astrocyte senescence/SASP and neuronal dysfunction/damage. Our studies indicate that tau can be propagated transcellularly to astrocytes, triggering cellular senescence/SASP. Our studies suggest that astrocyte senescence is detrimental to dendritic and synaptic structure and density, suggesting that pathogenic soluble tau-induced astrocyte senescence may contribute to synaptic dysfunction and loss in AD. Drugs that eliminate senescent cells are FDA-approved and antibody-based approaches to remove tau from brain are already in clinical trials. Our studies suggest that these interventions could be effective in the treatment of AD and other tauopathies.

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia.

Alzheimer's disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing neuronal dysfunction which eventually leads to cognitive decline. Hyperphosphorylated tau aggregates in the neuron are believed to cause most of the pathology associated with AD. These aggregates are assumed to be released into the extracellular compartment and taken up by adjacent healthy neurons where they induce further tau aggregation. This "prion-like" spreading can be interrupted by antibodies capable of binding and "neutralizing" extracellular tau aggregates as shown in preclinical mouse models of AD. One of the proposed mechanisms by which therapeutic antibodies reduce pathology is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia. Here, we describe a quantitative cell-based assay to assess tau uptake by microglia. This assay uses the mouse microglial cell line BV-2, allows for high specificity, low variability and medium throughput. Data generated with this assay can contribute to a better characterization of anti-tau antibody effector functions.

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

Alzheimer's disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing neuronal dysfunction which eventually leads to cognitive decline. Hyperphosphorylated tau aggregates in the neuron are believed to cause most of the pathology associated with AD. These aggregates are assumed to be released into the extracellular compartment and taken up by adjacent healthy neurons where they induce further tau aggregation. This "prion-like" spreading can be interrupted by antibodies capable of binding and "neutralizing" extracellular tau aggregates as shown in preclinical mouse models of AD. One of the proposed mechanisms by which therapeutic antibodies reduce pathology is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia. Here, we describe a quantitative cell-based assay to assess tau uptake by microglia. This assay uses the mouse microglial cell line BV-2, allows for high specificity, low variability and medium throughput. Data generated with this assay can contribute to a better characterization of anti-tau antibody effector functions.

Anti-Tau Monoclonal Antibodies Derived from Soluble and Filamentous Tau Show Diverse Functional Properties in vitro and in vivo.

The tau spreading hypothesis provides rationale for passive immunization with an anti-tau monoclonal antibody to block seeding by extracellular tau aggregates as a disease-modifying strategy for the treatment of Alzheimer's disease (AD) and potentially other tauopathies. As the biochemical and biophysical properties of the tau species responsible for the spatio-temporal sequences of seeding events are poorly defined, it is not yet clear which epitope is preferred for obtaining optimal therapeutic efficacy. Our internal tau antibody collection has been generated by immunizations with different tau species: aggregated- and non-aggregated tau and human postmortem AD brain-derived tau fibrils. In this communication, we describe and characterize a set of these anti-tau antibodies for their biochemical and biophysical properties, including binding, tissue staining by immunohistochemistry, and epitope. The antibodies bound to different domains of the tau protein and some were demonstrated to be isoform-selective (PT18 and hTau56) or phospho-selective (PT84). Evaluation of the antibodies in cellular- and in vivo seeding assays revealed clear differences in maximal efficacy. Limited proteolysis experiments support the hypothesis that some epitopes are more exposed than others in the tau seeds. Moreover, antibody efficacy seems to depend on the structural properties of fibrils purified from tau Tg mice- and postmortem human AD brain.

Internalization of the Extracellular Full-Length Tau Inside Neuro2A and Cortical Cells Is Enhanced by Phosphorylation.

Tau protein is mainly intracellular. However, several studies have demonstrated that full-length Tau can be released into the interstitial fluid of the brain. The physiological or pathological function of this extracellular Tau remains unknown. Moreover, as evidence suggests, extracellular Tau aggregates can be internalized by neurons, seeding Tau aggregation. However, much less is known about small species of Tau. In this study, we hypothesized that the status of phosphorylation could alter the internalization of recombinant Tau in Neuro2A and cortical cells. Our preliminary results revealed that the highly phosphorylated form of Tau entered the cells ten times more easily than a low phosphorylated one. This suggests that hyperphosphorylated Tau protein could spread between neurons in pathological conditions such as Alzheimer’s disease.

Anti-Tau Antibodies that Block Tau Aggregate Seeding In Vitro Markedly Decrease Pathology and Improve Cognition In Vivo

Tau aggregation occurs in neurodegenerative diseases including Alzheimer's disease and many other disorders collectively termed tauopathies. trans-cellular propagation of tau pathology, mediated by extracellular tau aggregates, may underlie pathogenesis of these conditions. P301S tau transgenic mice express mutant human tau protein and develop progressive tau pathology. Using a cell-based biosensor assay, we screened anti-tau monoclonal antibodies for their ability to block seeding activity present in P301S brain lysates. We infused three effective antibodies or controls into the lateral ventricle of P301S mice for 3 months. The antibodies markedly reduced hyperphosphorylated, aggregated, and insoluble tau. They also blocked development of tau seeding activity detected in brain lysates using the biosensor assay, reduced microglial activation, and improved cognitive deficits. These data imply a central role for extracellular tau aggregates in the development of pathology. They also suggest that immunotherapy specifically designed to block trans-cellular aggregate propagation will be a productive treatment strategy.

In Vivo Spreading of Tau Pathology

In Vivo Spreading of Tau Pathology

In VivoMicrodialysis Reveals Age-Dependent Decrease of Brain Interstitial Fluid Tau Levels in P301S Human Tau Transgenic Mice

Although tau is a cytoplasmic protein, it is also found in brain extracellular fluids, e.g., CSF. Recent findings suggest that aggregated tau can be transferred between cells and extracellular tau aggregates might mediate spread of tau pathology. Despite these data, details of whether tau is normally released into the brain interstitial fluid (ISF), its concentration in ISF in relation to CSF, and whether ISF tau is influenced by its aggregation are unknown. To address these issues, we developed a microdialysis technique to analyze monomeric ISF tau levels within the hippocampus of awake, freely moving mice. We detected tau in ISF of wild-type mice, suggesting that tau is released in the absence of neurodegeneration. ISF tau was significantly higher than CSF tau and their concentrations were not significantly correlated. Using P301S human tau transgenic mice (P301S tg mice), we found that ISF tau is fivefold higher than endogenous murine tau, consistent with its elevated levels of expression. However, following the onset of tau aggregation, monomeric ISF tau decreased markedly. Biochemical analysis demonstrated that soluble tau in brain homogenates decreased along with the deposition of insoluble tau. Tau fibrils injected into the hippocampus decreased ISF tau, suggesting that extracellular tau is in equilibrium with extracellular or intracellular tau aggregates. This technique should facilitate further studies of tau secretion, spread of tau pathology, the effects of different disease states on ISF tau, and the efficacy of experimental treatments.

Propagation of Tau Misfolding from the Outside to the Inside of a Cell

Tauopathies are neurodegenerative diseases characterized by aggregation of the microtubule-associated protein Tau in neurons and glia. Although Tau is normally considered an intracellular protein, Tau aggregates are observed in the extracellular space, and Tau peptide is readily detected in the cerebrospinal fluid of patients. Tau aggregation occurs in many diseases, including Alzheimer disease and frontotemporal dementia. Tau pathology begins in discrete, disease-specific regions but eventually involves much larger areas of the brain. It is unknown how this propagation of Tau misfolding occurs. We hypothesize that extracellular Tau aggregates can transmit a misfolded state from the outside to the inside of a cell, similar to prions. Here we show that extracellular Tau aggregates, but not monomer, are taken up by cultured cells. Internalized Tau aggregates displace tubulin, co-localize with dextran, a marker of fluid-phase endocytosis, and induce fibrillization of intracellular full-length Tau. These intracellular fibrils are competent to seed fibril formation of recombinant Tau monomer in vitro. Finally, we observed that newly aggregated intracellular Tau transfers between co-cultured cells. Our data indicate that Tau aggregates can propagate a fibrillar, misfolded state from the outside to the inside of a cell. This may have important implications for understanding how protein misfolding spreads through the brains of tauopathy patients, and it is potentially relevant to myriad neurodegenerative diseases associated with protein misfolding.