Tau K18 Research Articles

Irisin inhibits microglial senescence via TFAM-mediated mitochondrial metabolism in a mouse model of tauopathy

BackgroundThe accumulation of senescent microglia has been highlighted as a critical contributor to the progression of tauopathies. Irisin, a muscle-derived hormone produced by the proteolytic cleavage of Fibronectin-domain III containing 5 (FNDC5), mediates the pleiotropic effects of exercise on the physical body. Herein, we investigate the potential role of irisin in microglial senescence in tauopathies.MethodsTo model tauopathies both in vivo and in vitro, we utilized P301S tau transgenic mice and tau K18 fibril-treated microglia BV2 cells, respectively. We first examined the expression of the irisin expression and senescence phenotypes of microglia in tauopathies. Subsequently, we investigated the impact of irisin on microglial senescence and its underlying molecular mechanisms.ResultWe observed a reduction in irisin levels and an onset of premature microglial senescence both in vivo and in vitro. Irisin administration was found to counteract microglial senescence and ameliorate cognitive decline in P301S mice. Mechanistically, irisin effectively inhibited microglial senescence by stimulating the expression of mitochondrial transcription factor A (TFAM), a master regulator of mitochondrial respiratory chain biogenesis, thereby enhancing mitochondrial oxidative phosphorylation (OXPHOS). Silencing TFAM eliminated the inhibitory effect of irisin on microglial senescence as well as the restorative effect of irisin on mitochondrial OXPHOS. Furthermore, the SIRT1/PGC1α signaling pathway appeared to be implicated in irisin-mediated upregulation of TFAM.ConclusionTaken together, our study revealed that irisin mitigated microglial senescence via TFAM-driven mitochondrial biogenesis, suggesting a promising new avenue for therapeutic strategies targeting tauopathies.

Therapeutic Potential of Traditional Oriental Medicines in Targeting Tau Pathology: Insights from Cell-free and Cell-based Screening.

Traditional Oriental Medicines (TOMs) formulated using a variety of medicinal plants have a low risk of side effects. In previous studies, five TOMs, namely Dangguijakyaksan, Hwanglyeonhaedoktang, Ukgansan, Palmijihwanghwan, and Jowiseungchungtang have been commonly used to treat patients with Alzheimer's disease. However, only a few studies have investigated the effects of these five TOMs on tau pathology. This study aimed to examine the effect of five TOMs on various tau pathologies, including post-translational modifications, aggregation and deposition, tau-induced neurotoxicity, and tau-induced neuroinflammation. Immunocytochemistry was used to investigate the hyperphosphorylation of tau induced by okadaic acid. In addition, the thioflavin T assay was used to assess the effects of the TOMs on the inhibition of tau K18 aggregation and the dissociation of tau K18 aggregates. Moreover, a water-soluble tetrazolium-1 assay and a quantitative reverse transcription polymerase chain reaction were used to evaluate the effects of the TOMs on tau-induced neurotoxicity and inflammatory cytokines in HT22 and BV2 cells, respectively. The five TOMs investigated in this study significantly reduced okadaic acid-induced tau hyperphosphorylation. Hwanglyeonhaedoktang inhibited the aggregation of tau and promoted the dissociation of tau aggregates. Dangguijakyaksan and Hwanglyeonhaedoktang attenuated tau-induced neurotoxicity in HT22 cells. In addition, Dangguijakyaksan, Hwanglyeonhaedoktang, Ukgansan, and Palmijihwanghwan reduced tauinduced pro-inflammatory cytokine levels in BV2 cells. Our results suggest that five TOMs are potential therapeutic candidates for tau pathology. In particular, Hwanglyeonhaedoktang showed the greatest efficacy among the five TOMs in cell-free and cell-based screening approaches. These findings suggest that Hwanglyeonhaedoktang is suitable for treating AD patients with tau pathology.

Korean red ginseng polysaccharide as a potential therapeutic agent targeting tau pathology in Alzheimer's disease

Tau is a microtubule-associated protein that plays a critical role in the stabilization and modulation of neuronal axons. Tau pathology is stronger associated with cognitive decline in patients with Alzheimer's disease (AD) than amyloid beta (Aβ) pathology. Hence, tau targeting is a promising approach for the treatment of AD. Previous studies have demonstrated that the non-saponin fraction with rich polysaccharide (NFP) from Korean red ginseng (KRG) can modulate tau aggregation and exert a therapeutic effect on AD. Therefore, we investigated the efficacy of NFP isolated from KRG on tau pathology in experimental models of AD. Our results showed that NFP from KRG ameliorated deposition and hyperphosphorylation of tau in the brain of 3xTg mice. Moreover, NFP from KRG modulated the aggregation and dissociation of tau K18 in vitro. We demonstrated the alleviatory effects of NFP from KRG on hyperphosphorylated tau and tau kinase in okadaic acid-treated HT22 cells. Furthermore, NFP from KRG mitigated Aβ deposition, neurodegeneration, and neuroinflammation in 3xTg mice. We revealed the neuroprotective effects of NFP from KRG on tau-induced neuronal loss in HT22 cells. Our results indicate that NFP extracted from KRG is a novel therapeutic agent for the treatment of AD associated with tau pathology.

Genipin and pyrogallol: Two natural small molecules targeting the modulation of disordered proteins in Alzheimer's disease

Alzheimer's disease (AD) is characterized by the aggregation of disordered proteins, such as amyloid beta (Aβ) and tau, leading to neurotoxicity and disease progression. Despite numerous efforts, effective inhibitors of Aβ and tau aggregates have not been developed. Thus, we aimed to screen natural small molecules from crude extracts that target various pathologies and are prescribed for patients with neurological diseases. In this study, we screened 162 natural small molecules prescribed for neurological diseases and identified genipin and pyrogallol as hit compounds capable of simultaneously regulating the aggregation of Aβ and tau K18. Moreover, we confirmed the dual modulatory effects of these compounds on the reduction of amyloid-mediated neurotoxicity in vitro and the disassembly of preformed Aβ42 and tau K18 fibrils. Furthermore, we observed the alleviatory effects of genipin and pyrogallol against AD-related pathologies in triple transgenic AD mice. Molecular dynamics and docking simulations revealed the molecular interaction dynamics of genipin and pyrogallol with Aβ42 and tau K18, providing insights into their suppression of aggregation. Our findings suggest the therapeutic potential of genipin and pyrogallol as dual modulators for the treatment of AD by inhibiting aggregation or promoting dissociation of Aβ and tau.

Different tau fibril types reduce prion level in chronically and de novo infected cells

Neurodegenerative diseases are often characterized by the codeposition of different amyloidogenic proteins, normally defining distinct proteinopathies. An example is represented by prion diseases, where the classical deposition of the aberrant conformational isoform of the prion protein (PrPSc) can be associated with tau insoluble species, which are usually involved in another class of diseases called tauopathies. How this copresence of amyloidogenic proteins can influence the progression of prion diseases is still a matter of debate. Recently, the cellular form of the prion protein, PrPC, has been investigated as a possible receptor of amyloidogenic proteins, since its binding activity with Aβ, tau, and α-synuclein has been reported, and it has been linked to several neurotoxic behaviors exerted by these proteins. We have previously shown that the treatment of chronically prion-infected cells with tau K18 fibrils reduced PrPSc levels. In this work, we further explored this mechanism by using another tau construct that includes the sequence that forms the core of Alzheimer's disease tau filaments invivo to obtain a distinct fibril type. Despite a difference of six amino acids, these two constructs form fibrils characterized by distinct biochemical and biological features. However, their effects on PrPSc reduction were comparable and probably based on the binding to PrPC at the plasma membrane, inhibiting the pathological conversion event. Our results suggest PrPC as receptor for different types of tau fibrils and point out a role of tau amyloid fibrils in preventing the pathological PrPC to PrPSc conformational change.

Dual modulators of aggregation and dissociation of amyloid beta and tau: In vitro, in vivo, and in silico studies of Uncaria rhynchophylla and its bioactive components

A prominent characteristic of Alzheimer's disease (AD) is the deposition of both amyloid-β (Aβ) peptide and tau protein in the brain. Aβ and tau not only induce toxicity through self-aggregation but also induce more potent toxicity through the synergistic action of Aβ and tau. In particular, neurotoxic aggregates of Aβ and tau directly affect several AD pathologies including neuroinflammation and cognitive decline. Therefore, there is increasing interest in strategies to modulate the aggregation and dissociation of Aβ and tau for treatment of AD. Our recent study found that Uncaria rhynchophylla (UR) has a therapeutic effect on AD via the inhibition of Aβ aggregation and attenuating Aβ-mediated pathogenesis of AD. However, no studies have investigated whether UR has anti- and disaggregation effects on both Aβ and tau. In this study, we showed the significant effects of UR on aggregation and dissociation of Aβ42 and tau K18 using a thioflavin T (ThT) assay. In addition, histological study revealed an inhibitory effect of UR on the accumulation of Aβ and tau and AD-related pathologies in 3xTg mice with both Aβ and tau pathology. Furthermore, we found that rhynchophylline and corynoxeine, bioactive components of UR, could modulate the aggregation and dissociation of both Aβ and tau using molecular docking simulation, isothermal titration calorimetry, and ThT assays. In conclusion, our results demonstrate that UR can inhibit the aggregation of Aβ and tau and promote the degradation of their aggregates in AD.

Tetramerization of the S100B Chaperone Spawns a Ca2+ Independent Regulatory Surface that Enhances Anti-aggregation Activity and Client Specificity

Alzheimer’s disease (AD) hallmarks include the aggregation of amyloid-β (Aβ), tau and neuroinflammation promoted by several alarmins. Among these is S100B, a small astrocytic homodimeric protein, upregulated in AD, whose multiple biological activities depend on localization, concentration, and assembly state. S100B was reported to inhibit the aggregation and toxicity of Aβ42 and tau similarly to a holdase-type chaperone. This activity is dependent of Ca2+-binding, which triggers the exposure of a regulatory binding cleft at the S100B dimer interface with which amyloidogenic clients dynamically interact. Although the dimer prevails, a significant portion of secreted S100B in the human brain occurs as higher order multimers, whose protective functions remain uncharacterized and which we here investigate. Resorting to ThT-monitored aggregation kinetics, we determined that unlike the dimer, tetrameric S100B inhibits Aβ42 aggregation at sub/equimolar ratios, an effect that persists in the absence of Ca2+ binding. Structural analysis revealed that S100B tetramerization spawns a novel extended cleft accommodating an aggregation-prone surface that mediates interactions with monomeric Aβ client via hydrophobic interactions, as corroborated by Bis-ANS fluorescence and docking analysis. Correspondingly, at high ionic strength that reduces solvation and favours hydrophobic contacts, the inhibition of Aβ42 aggregation by tetrameric S100B is 3-fold increased. Interestingly, this extended Ca2+-independent surface favours Aβ42 as substrate, as tau K18 aggregation is not inhibited by the apo tetramer. Overall, results illustrate a mechanism through which oligomerization of the S100B chaperone fine-tunes anti-aggregation activity and client specificity, highlighting the potential functional relevance of S100B multimers in the regulation of AD proteotoxicity.

Global Structure of the Intrinsically Disordered Protein Tau Emerges from Its Local Structure.

The paradigmatic disordered protein tau plays an important role in neuronal function and neurodegenerative diseases. To disentangle the factors controlling the balance between functional and disease-associated conformational states, we build a structural ensemble of the tau K18 fragment containing the four pseudorepeat domains involved in both microtubule binding and amyloid fibril formation. We assemble 129-residue-long tau K18 chains with atomic detail from an extensive fragment library constructed with molecular dynamics simulations. We introduce a reweighted hierarchical chain growth (RHCG) algorithm that integrates experimental data reporting on the local structure into the assembly process in a systematic manner. By combining Bayesian ensemble refinement with importance sampling, we obtain well-defined ensembles and overcome the problem of exponentially varying weights in the integrative modeling of long-chain polymeric molecules. The resulting tau K18 ensembles capture nuclear magnetic resonance (NMR) chemical shift and J-coupling measurements. Without further fitting, we achieve very good agreement with measurements of NMR residual dipolar couplings. The good agreement with experimental measures of global structure such as single-molecule Förster resonance energy transfer (FRET) efficiencies is improved further by ensemble refinement. By comparing wild-type and mutant ensembles, we show that pathogenic single-point P301L, P301S, and P301T mutations shift the population from the turn-like conformations of the functional microtubule-bound state to the extended conformations of disease-associated tau fibrils. RHCG thus provides us with an atomically detailed view of the population equilibrium between functional and aggregation-prone states of tau K18, and demonstrates that global structural characteristics of this intrinsically disordered protein emerge from its local structure.

Homogalacturonan from squash: Characterization and tau-binding pattern of a sulfated derivative

A pectic polysaccharide (WAP) was isolated from squash and identified as a homogalacturonan with a molecular mass of 83.2 kDa by GPC, monosaccharide composition analysis, FT-IR and NMR spectra. Sulfation modification of WAP was carried out and a sulfated derivative (SWAP) was obtained with a substitution degree of 1.81. The NMR spectrum indicated that the sulfation modification mainly occurred at the C-2 and C-3 positions of galacturonan residues. The binding pattern of SWAP to tau K18 protein was observed in 2D 1H15N HSQC spectra of tau, which resembled the tau-heparin interaction, with R2 domain as the major binding region. These results suggest that SWAP has the potential to act as a heparin mimic to inhibit the transcellular spread of tau; thus natural polysaccharide from squash may be developed into therapies for AD and related tauopathies.

Fisetin inhibits tau aggregation by interacting with the protein and preventing the formation of β-strands

Alzheimer's disease is a neurodegenerative disease which severely impacts the health of the elderly. Current treatments are only able to alleviate symptoms, but not prevent or cure the disease. The neurofibrillary tangles formed by tau protein aggregation are one of the defining characteristics of Alzheimer's disease, so tau protein has become a key target for the drug design. In this study, we show that fisetin, a plant-derived polyphenol compound, can inhibit aggregation of the tau fragment, K18, and can disaggregate tau K18 filaments in vitro. Meanwhile it is able to prevent the formation of tau aggregates in cells. Both experimental and computational studies indicate that fisetin could directly interact with tau K18 protein. The binding is mainly created by hydrogen bond and van der Waal force, prevents the formation of β-strands at the two hexapeptide motifs, and does not perturb the secondary structure or the tubulin binding ability of tau protein. In summary, fisetin might be a candidate for further development as a potential preventive or therapeutic drug for Alzheimer's disease.

Exploring IDP-Ligand Interactions: tau K18 as A Test Case.

Over the past decade intrinsically disordered proteins (IDPs) have emerged as a biologically important class of proteins, many of which are of therapeutic relevance. Here, we investigated the interactions between a model IDP system, tau K18, and nine literature compounds that have been reported as having an effect on tau in order to identify a robust IDP–ligand system for the optimization of a range of biophysical methods. We used NMR, surface plasmon resonance (SPR) and microscale thermophoresis (MST) methods to investigate the binding of these compounds to tau K18; only one showed unambiguous interaction with tau K18. Several near neighbors of this compound were synthesized and their interactions with tau K18 characterized using additional NMR methods, including 1D ligand-observed NMR, diffusion-ordered spectroscopy (DOSY) and 19F NMR. This study demonstrates that it is possible to detect and characterize IDP–ligand interactions using biophysical methods. However, care must be taken to account for possible artefacts, particularly the impact of compound solubility and where the protein has to be immobilized.

The protein-surfactant stoichiometry governs the conformational switching and amyloid nucleation kinetics of tau K18.

Amyloids are pathological hallmarks of a number of debilitating neurodegenerative diseases. Understanding the molecular mechanism of protein amyloid assembly with an emphasis on structural characterization of early, key prefibrillar species is important for targeted drug design and clinical interventions. Tau is an intrinsically disordered, microtubule-binding protein which is also implicated in various neurodegenerative disorders such as frontotemporal dementia, Down's syndrome, Alzheimer's disease, etc. Earlier reports have demonstrated that tau aggregation in vitro is triggered by anionic inducers, presumably due to charge compensation which facilitates intermolecular association between the tau polypeptide chains. However, the molecular mechanism of tau amyloid aggregation, involving the structural characterization of amyloidogenic intermediates formed especially during early key steps, remains elusive. In this work, we have employed a spectroscopic toolbox to elucidate the mechanism of anionic surfactant-induced disorder-to-order amyloid transition of a tau segment. This study revealed that the amyloid assembly is mediated via binding-induced conformational switching into an early partially helical amyloid-competent intermediate. Additionally, protein and inducer concentration-dependent studies indicated that at the higher protein and/or inducer concentrations, competing off-pathway intermediates dampen the amyloid assembly which implies that the stoichiometry of protein and inducer plays a key regulatory role in the amyloid nucleation and fibril elongation kinetics.

Red Ginseng Inhibits Tau Aggregation and Promotes Tau Dissociation In Vitro.

Tau, a microtubule-associated protein expressed in mature neurons, interacts with tubulin to promote the assembly and stabilization of microtubules. However, abnormally hyperphosphorylated tau dissociates from microtubules and self-aggregates. Tau aggregates, including paired helical filaments and neurofibrillary tangles, promote neuronal dysfunction and death and are the defining neuropathological feature of tauopathies. Therefore, suppressing tau aggregation or stimulating the dissociation of tau aggregates has been proposed as an effective strategy for treating neurodegenerative diseases associated with tau pathology such as Alzheimer's disease (AD) and frontotemporal dementia. Interestingly, ginsenosides extracted from Panax ginseng reduced the hippocampal and cortical expression of phosphorylated tau in a rat model of AD. However, no studies have been conducted into the effect of red ginseng (RG) and its components on tau pathology. Here, we evaluated the effect of Korean red ginseng extract (KRGE) and its components on the aggregation and disassociation of tau. Using the thioflavin T assay, we monitored the change in fluorescence produced by the aggregation or disassociation of tau K18, an aggregation-prone fragment of tau441 containing the microtubule-binding domain. Our analysis revealed that KRGE not only inhibited tau aggregation but also promoted the dissociation of tau aggregates. In addition, the KRGE fractions, such as saponin, nonsaponin, and nonsaponin fraction with rich polysaccharide, also inhibited tau aggregation and promoted the dissociation of tau aggregates. Our observations suggest that RG could be a potential therapeutic agent for the treatment of neurodegenerative diseases associated with tauopathy.

Fuzzy Interactions Between 14‐3‐3ζ And Tau Regulate Tau Aggregation

The microtubule associated protein tau is vital for elongation and stability of microtubules in the central nervous system. However, aberrant accumulation of tau amyloid fibrils in neurons is a character of a neurodegenerative pathology called Alzheimer’s disease. Extensive studies have revealed that several factors promote aggregation of tau, including mutations, polyanions, phosphorylation, and interactions with other proteins. Previous studies have shown that the conserved scaffolding protein family 14‐3‐3 proteins interact with tau in a phosphorylation dependent manner and 14‐3‐3 proteins promote tau aggregation. Crystal structure of 14‐3‐3/tau complex revealed that phosphorylated tau segments bind to the ligand binding grooves of 14‐3‐3 dimer. Consequently, it is promising that 14‐3‐3 specific binding ligands will reduce/inhibit tau aggregation. Indeed, rational designed inhibitors of 14‐3‐3/tau interaction have been found to reduce binding of PKA‐phosphorylated tau protein to 14‐3‐3ζ. Although thermodynamics characterization showed that non‐phosphorylated tau segments bind to 14‐3‐3 much weaker than phosphorylated tau segments, 14‐3‐3ζ also promotes non‐phosphorylated tau aggregation. In this work, we investigated the interactions between tau K18 construct and 14‐3‐3ζ. We found that non‐phosphorylated and PKA‐phosphorylated tau proteins showed similar aggregating propensity in the presence of 14‐3‐3ζ. Replacing Ser324 with Ala had little effect of tau aggregation mediated by 14‐3‐3ζ protein. We further saturated the 14‐3‐3ζ ligand binding grooves by a high affinity peptide R18 and found that the presence of R18 could not reduce tau aggregation. Molecular dynamics simulations showed that tau K18 interact with 14‐3‐3ζ via extensive fuzzy interactions. Our results suggest that besides binding to the canonical ligand binding groove, tau also interacts with 14‐3‐3ζ via nonspecific interactions which are responsible for tau aggregation and cannot be inhibited by 14‐3‐3ζ specific inhibitors.Support or Funding InformationThis work was supported by Hubei University of Technology

Distinct Conformations, Aggregation and Cellular Internalization of Different Tau Strains.

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.

ATOMIC STRUCTURES OF A SINGLE CHAIN ANTIBODY THAT BINDS AND INHIBITS SEEDING BY TAU OLIGOMERS

Alzheimer disease (AD) is the only disease among the 10 leading causes of death that cannot be cured, prevented, or even slowed. Amyloid plaques and tau neurofibrillary tangles are the pathological hallmarks of AD, and levels of tau aggregation are tightly linked to cognitive decline. Several lines of evidence suggest that toxic tau oligomers play a pivotal role in the spread of tau pathology, and passive immunization using antibodies that neutralize tau oligomers offers promise in the battle to delay, and potentially even prevent onset of AD. We engineered scFv-M204 antibody, and cloned into pMES4 vector using Gibson assembly method. Next, we evaluated the ability of scFv-M204 antibody to bind tau oligomers, and inhibit seeding by brain-derived tau from tauopathy donors with AD and CTE in tau biosensor cells. Here we report a monoclonal antibody (M204) that binds tau oligomers of the full-length tau (Tau 40) and also the four-repeat domain of tau (Tau k18). M204 binds the aggregation-prone segments VQIINK and SVQIV in tau, and inhibits cell-to-cell seeding by tau oligomers, confirming the powerful role of theses sequences in promoting tau oligomerization. To minimize the size of the antibody, we sought to produce M204 as a single-chain variable-fragment (scFv) in Escherichia coli. The scFv-M204 was found to purify in three different conformations: monomer, dimer and trimer when expressed in the bacterial periplasm. The dimeric and trimeric conformations bind tau oligomers better than the monomeric form of scFv-M204, and importantly also inhibit tau aggregation in vitro and in HEK293 biosensor cells seeded by brain homogenates from human patients with tauopathies including AD and Chronic Traumatic Encephalopathy (CTE). The crystal structures of the monomer, dimer and trimer were determined revealing differences that could contribute to the increased binding and inhibition that we observed by higher order structures of scFv-M204. We speculate that by binding toxic oligomers, which are hypothesized to be early seeding-competent species that are formed by tau, our scFv-M204 antibody may have potential as an early-stage diagnostic for AD and other tauopathies, in addition to holding promise as a prospective therapeutic.

Nonspecific Electrostatic Interactions between Tau and 14‐3‐3ζ Promote Tau Aggregating

Aberrant accumulation of microtubule associated protein tau and formation of amyloid fibrils are pathological hallmarks of neurodegenerative tauopathies. Previous studies have suggested that the scaffolding protein 14‐3‐3ζ promotes tau aggregating by interacting specifically with the pSer324 14‐3‐3ζ‐binding motif within the third microtubule binding repeat. Therefore, the tau/14‐3‐3ζ interaction is a promising drug target for Alzheimer's disease treatment. Indeed, inhibitors of 14‐3‐3/tau interaction have been found to reduce binding of PKA‐phosphorylated tau protein to 14‐3‐3ζ. However, 14‐3‐3ζ has also been shown to promote aggregation of unphosphorylated tau protein, suggesting that nonspecific interactions between tau and 14‐3‐3ζ may be also involved in tau aggregating. In this work, the interactions between tau K18 construct and 14‐3‐3ζ were investigated. We compared the aggregating propensity for three tau species (i.e. WT, pS324, and S324A) in the presence or absence of 14‐3‐3ζ. Although phosphorylation at Ser324 increased the binding affinity of tau K18 to 14‐3‐3ζ, phosphorylation only showed marginal effect on 14‐3‐3ζ mediated tau aggregating. Furthermore, WT and S324A tau proteins showed similar aggregating propensity in the presence of 14‐3‐3ζ. Although 14‐3‐3ζ specific inhibitors reduced the binding affinity of pS324 tau with 14‐3‐3ζ, NMR spectroscopy showed that nonspecific interactions between pS324 tau and 14‐3‐3ζ remained when 14‐3‐3ζ specific inhibitors were present. Our results suggest that nonspecific interactions between tau and 14‐3‐3ζ play an important role in the 14‐3‐3ζ mediated tau aggregating process.Support or Funding InformationThis work was supported by National Natural Science Foundation of China (Grant number: 21603121) and Hubei University of Technology.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Understanding the Kinetic Roles of 14-3-3ζ during Tau Filament Formation

The microtubule associated protein tau and 14-3-3ζ protein are two proteins highly expressed in the central nervous system. Tau protein promotes microtubule assembly and aberrant accumulation of tau and formation of amyloid fibrils are pathological hallmarks of neurodegenerative tauopathies including Alzheimer's disease. Extensive studies have suggested that polyanionic inducers, such as heparin, interact with tau directly and induce specific conformational changes within tau, accelerating the amyloidogenesis process. 14-3-3ζ proteins form homodimers and bind their targets through specific pSer/pThr motifs. It has been found that tau protein contains two 14-3-3ζ-binding motifs, i.e., pSer214 and pSer324, and tau protein interacts with 14-3-3ζ protein directly. Furthermore, 14-3-3ζ is able to induce tau aggregation, although it's still controversial whether this process depends on tau phosphorylation. Here, we investigated the kinetic roles of 14-3-3ζ during tau filament formation. We used the K18 constructs of tau that consists of the four microtubule repeats and the pSer324 14-3-3ζ-binding motifs. Our results showed that 14-3-3ζ induced tau K18 aggregation at a rate much larger than that from full-length tau. Kinetics analyses showed that the lag time and rate of aggregation were dependent on the stoichiometry of tau to 14-3-3ζ. Although phosphorylation at Ser324 increased the binding affinity of tau K18 to 14-3-3ζ, phosphorylation only showed marginal effect on 14-3-3ζ mediated tau aggregation. Our results suggest that the formation of the 2:1 complex of tau monomer and 14-3-3ζ dimer plays an important role in the 14-3-3ζ mediated tau aggregation process.

Preparation of stable tau oligomers for cellular and biochemical studies

Increasing evidence suggests that small oligomers are the principal neurotoxic species of tau in Alzheimer's disease and other tauopathies. However, mechanisms of tau oligomer-mediated neurodegeneration are poorly understood. The transience of oligomers due to aggregation can compromise the stability of oligomers prepared in vitro. Consequently, we sought to develop an efficient method which maintains the stability and globular conformation of preformed oligomers. This study demonstrates that labeling a single-cysteine form of the pro-aggregant tau four-repeat region (K18) with either Alexa Fluor 488-C5-maleimide or N-ethylmaleimide in reducing conditions stabilizes oligomers by impeding their further aggregation. Furthermore, the use of this approach to study the propagation of labeled extracellular tau K18 oligomers into human neuroblastoma cells and human stem cell-derived neurons is described. This method is potentially applicable for preparing stabilized oligomers of tau for diagnostic and biomarker tests, as well as for in vitro structure-activity relationship assays.

Synergistic Amyloid Switch Triggered by Early Heterotypic Oligomerization of Intrinsically Disordered α-Synuclein and Tau

Amyloidogenic intrinsically disordered proteins, α-synuclein and tau are linked to Parkinson's disease and Alzheimer's disease, respectively. A body of evidence suggests that α-synuclein and tau, both present in the presynaptic nerve terminals, co-aggregate in many neurological ailments. The molecular mechanism of α-synuclein-tau hetero-assembly is poorly understood. Here we show that amyloid formation is synergistically facilitated by heterotypic association mediated by binding-induced misfolding of both α-synuclein and tau K18. We demonstrate that the intermolecular association is largely driven by the electrostatic interaction between the negatively charged C-terminal segment of α-synuclein and the positively charged tau K18 fragment. This heterotypic association results in rapid formation of oligomers that readily mature into hetero-fibrils with a much shorter lag phase compared to the individual proteins. These findings suggested that the critical intermolecular interaction between α-synuclein and tau can promote facile amyloid formation that can potentially lead to efficient sequestration of otherwise long-lived lethal oligomeric intermediates into innocuous fibrils. We next show that a well-known familial Parkinson's disease mutant (A30P) that is known to aggregate slowly via accumulation of highly toxic oligomeric species during the long lag phase converts into amyloid fibrils significantly faster in the presence of tau K18. The early intermolecular interaction profoundly accelerates the fibrillation rate of A30P α-synuclein and impels the disease mutant to behave similar to wild-type α-synuclein in the presence of tau. Our findings suggest a mechanistic underpinning of bypassing toxicity and suggest a general strategy by which detrimental amyloidogenic precursors are efficiently sequestered into more benign amyloid fibrils.