Preparation of stable tau oligomers for cellular and biochemical studies

Karikari, Thomas K.; Nagel, David A.; Grainger, Alastair I.; Clarke-Bland, Charlotte; Hill, Eric J.; Moffat, K.

doi:10.1016/j.ab.2018.10.013

Cited by 29 publications

(41 citation statements)

References 41 publications

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“…We therefore, sought to take advantage of the conformational and aggregation differences of the tau K18 variants to investigate their possible influences on the cellular internalization of exogenously applied oligomers. To achieve this, Alexa Fluor-488-C5-maleimide-labeled oligomers were first prepared as detailed in section “Materials and Methods” and elsewhere (Karikari et al, 2019) before being used in in vitro cell culture assays. These stable oligomers were granular and non-fibrillar (Supplementary Figure S4).…”

Section: Resultsmentioning

confidence: 99%

Distinct Conformations, Aggregation and Cellular Internalization of Different Tau Strains

Karikari

Nagel

Grainger

et al. 2019

Front. Cell. Neurosci.

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The inter-cellular propagation of tau aggregates in several neurodegenerative diseases involves, in part, recurring cycles of extracellular tau uptake, initiation of endogenous tau aggregation, and extracellular release of at least part of this protein complex. However, human brain tau extracts from diverse tauopathies exhibit variant or “strain” specificity in inducing inter-cellular propagation in both cell and animal models. It is unclear if these distinctive properties are affected by disease-specific differences in aggregated tau conformation and structure. We have used a combined structural and cell biological approach to study if two frontotemporal dementia (FTD)-associated pathologic mutations, V337M and N279K, affect the aggregation, conformation and cellular internalization of the tau four-repeat domain (K18) fragment. In both heparin-induced and native-state aggregation experiments, each FTD variant formed soluble and fibrillar aggregates with remarkable morphological and immunological distinctions from the wild type (WT) aggregates. Exogenously applied oligomers of the FTD tau-K18 variants (V337M and N279K) were significantly more efficiently taken up by SH-SY5Y neuroblastoma cells than WT tau-K18, suggesting mutation-induced changes in cellular internalization. However, shared internalization mechanisms were observed: endocytosed oligomers were distributed in the cytoplasm and nucleus of SH-SY5Y cells and the neurites and soma of human induced pluripotent stem cell-derived neurons, where they co-localized with endogenous tau and the nuclear protein nucleolin. Altogether, evidence of conformational and aggregation differences between WT and disease-mutated tau K18 is demonstrated, which may explain their distinct cellular internalization potencies. These findings may account for critical aspects of the molecular pathogenesis of tauopathies involving WT and mutated tau.

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Section: Resultsmentioning

confidence: 99%

Distinct Conformations, Aggregation and Cellular Internalization of Different Tau Strains

Karikari

Nagel

Grainger

et al. 2019

Front. Cell. Neurosci.

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“…Briefly, Escherichia coli BL21 (DE3) carrying pProEX plasmids (Promega) coding for wild-type full-length tau-441 (Uniprot ID: P10636-8) with N-terminal 6xHis and FLAG tags and cysteine modifications (C291A/C322A/I260C), (Figure 1) were inoculated into Luria broth (15 ml) containing ampicillin (100 μg/ml) and chloramphenicol (35 μg/ml) and incubated at 37°C at 180 rpm overnight. The purpose of the cysteine modifications was to have a single cysteine residue located outside the microtubule-binding region that can be specifically labeled by a fluorophore without potentially interfering with the protein’s functions; this approach has been widely used and shown to have no detrimental effects (Kumar et al, 2014; Michel et al, 2014; Shammas et al, 2015; Karikari et al, 2019). The starter cultures were added to 750-ml fresh LB broth with ampicillin (100 μg ml −1 ) and returned to the shaking incubator for 90 min.…”

Section: Methodsmentioning

confidence: 99%

Introduction of Tau Oligomers into Cortical Neurons Alters Action Potential Dynamics and Disrupts Synaptic Transmission and Plasticity

Hill

Karikari

Moffat

et al. 2019

eNeuro

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Significance Statement The protein tau is highly expressed in neurons and is involved in maintaining neuronal structure. In diseases such as Alzheimer's disease (AD), tau can form oligomers, which consist of tau molecules joined together. There is growing evidence that these tau oligomers are toxic to neurons, although their precise actions are still being characterized. We have taken the approach of introducing structurally-defined tau-441 oligomers into neurons via the recording electrode (a method previously published by Kaufmann et al., 2016). This method allowed us to provide detailed characterization of the concentration and time-dependent actions of tau oligomers on neuronal properties. We have found that tau interferes with the action potential wave form, modifies synaptic transmission and can block events that probably underlie memory storage.

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“…As the truncated Tau [31] and repeat Tau domains [51] have been already reported to form globular oligomeric structures [55], we were interested in identifying the nanostructures of hTau40 WT oligomers. TEM (Fig.…”

Section: Preparation and Stabilization Of Htau40 Wt Oligomersmentioning

confidence: 99%

Phagocytosis of full-length Tau oligomers by Actin-remodeling of activated microglia

Das

Balmik

Chinnathambi

2020

J Neuroinflammation

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Background: Alzheimer's disease is associated with the accumulation of intracellular Tau tangles within neurons and extracellular amyloid-β plaques in the brain parenchyma, which altogether results in synaptic loss and neurodegeneration. Extracellular concentrations of oligomers and aggregated proteins initiate microglial activation and convert their state of synaptic surveillance into a destructive inflammatory state. Although Tau oligomers have fleeting nature, they were shown to mediate neurotoxicity and microglial pro-inflammation. Due to the instability of oligomers, in vitro experiments become challenging, and hence, the stability of the full-length Tau oligomers is a major concern. Methods: In this study, we have prepared and stabilized hTau40 WT oligomers, which were purified by size-exclusion chromatography. The formation of the oligomers was confirmed by western blot, thioflavin-S, 8-anilinonaphthaalene-1sulfonic acid fluorescence, and circular dichroism spectroscopy, which determine the intermolecular cross-β sheet structure and hydrophobicity. The efficiency of N9 microglial cells to phagocytose hTau40 WT oligomer and subsequent microglial activation was studied by immunofluorescence microscopy with apotome. The one-way ANOVA was performed for the statistical analysis of fluorometric assay and microscopic analysis. Results: Full-length Tau oligomers were detected in heterogeneous globular structures ranging from 5 to 50 nm as observed by high-resolution transmission electron microscopy, which was further characterized by oligomer-specific A11 antibody. Immunocytochemistry studies for oligomer treatment were evidenced with A11 + Iba1 high microglia, suggesting that the phagocytosis of extracellular Tau oligomers leads to microglial activation. Also, the microglia were observed with remodeled filopodia-like actin structures upon the exposure of oligomers and aggregated Tau. Conclusion: The peri-membrane polymerization of actin filament and co-localization of Iba1 relate to the microglial movements for phagocytosis. Here, these findings suggest that microglia modified actin cytoskeleton for phagocytosis and rapid clearance of Tau oligomers in Alzheimer's disease condition.

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Preparation of stable tau oligomers for cellular and biochemical studies

Cited by 29 publications

References 41 publications

Distinct Conformations, Aggregation and Cellular Internalization of Different Tau Strains

Distinct Conformations, Aggregation and Cellular Internalization of Different Tau Strains

Introduction of Tau Oligomers into Cortical Neurons Alters Action Potential Dynamics and Disrupts Synaptic Transmission and Plasticity

Phagocytosis of full-length Tau oligomers by Actin-remodeling of activated microglia

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