Recombinant Tau Research Articles

Effect of dietary n-3 polyunsaturated fatty acid rich fish oil on the endometrial prostaglandin production in the doe (Capra hircus)

Recently, we showed that dietary supplementation of n-3 PUFA rich fish oil (FO) decreased the metabolites of serum prostaglandin (PG) F2α and E2 during the window of pregnancy recognition in the doe. In this study, we investigated its effect on the changes on endometrial PG production in vitro. Cycling does (n = 12) of Rohilkhand region were divided into two equal groups and fed a concentrate diet supplemented with either FO containing 26% n-3 PUFA (TRT; n = 6) or palm oil (CON; n = 6) @ 0.6 mL/kg body weight for 57 days. Estrus was synchronized by two injections of PGF2α analogue viz, on day 25 and 36 of supplementation and laparo-hysterotomy was performed to obtain endometrial tissue on day 16 of the synchronized estrus. Endometrial explant culture was done using a defined medium.The basal PG production was assayed at 6 and 12 h. Endometrial explant was stimulated with oxytocin (OXT) and/or recombinant ovine interferon tau (roIFN-τ) and PGs were assayed at 3 and 12 h post-treatment. The relative expression of genes related to PG metabolism in the endometrium was done by Quantitative Real Time PCR technique (qRT-PCR). There was a significant (P < 0.05) decline in the basal production of PGF2α and PGE2 in the TRT as compared to the CON group. The cultured endometrial tissue produced PGF2α in a time- dependent fashion in both the groups (P < 0.05). Neither OXT nor roIFN-τ had a significant (P > 0.05) effect on the PGF2α and PGE2 production in the TRT group. Similarly, the PG production in the OXT and roIFN-τ was comparable with the control in TRT. Expression of mRNA for cyclooxygenase-2 (COX-2), cytosolic phospholipase A2 (cPLA2) and PGF synthase (PGFS) was lower (P < 0.05) whereas, PGE synthase and peroxisome proliferator-activated receptors such as PPAR-γ and δ was increased (P < 0.05) in n-3 PUFA fed doe. In conclusion, dietary supplementation of FO decreased the endometrial production of PGF2α and PGE2 by downregulating the COX-2, cPLA2 and PGFS transcripts in the doe. The findings suggest that n-3 PUFA influence embryo survival by modulating the endometrial PG.

α-Synuclein Oligomers Induce a Unique Toxic Tau Strain

BackgroundThe coexistence of α-synuclein and tau aggregates in several neurodegenerative disorders, including Parkinson’s disease and Alzheimer’s disease, raises the possibility that a seeding mechanism is involved in disease progression. MethodsTo further investigate the role of α-synuclein in the tau aggregation pathway, we performed a set of experiments using both recombinant and brain-derived tau and α-synuclein oligomers to seed monomeric tau aggregation in vitro and in vivo. Brain-derived tau oligomers were isolated from well-characterized cases of progressive supranuclear palsy (n = 4) and complexes of brain-derived α-synuclein/tau oligomers isolated from patients with Parkinson’s disease (n = 4). The isolated structures were purified and characterized by standard biochemical methods, then injected into Htau mice (n = 24) to assess their toxicity and role in tau aggregation. ResultsWe found that α-synuclein induced a distinct toxic tau oligomeric strain that avoids fibril formation. In vivo, Parkinson’s disease brain–derived α-synuclein/tau oligomers administered into Htau mouse brains accelerated endogenous tau oligomer formation concurrent with increasing cell loss. ConclusionsOur findings provide evidence, for the first time, that α-synuclein enhances the harmful effects of tau, thus contributing to disease progression.

JAK3 and PI3K mediate bovine Interferon-tau stimulated gene expression in the blood neutrophils.

Interferon tau, a 23 kDa trophoblast derived protein diffuses out from the uterus into the circulation and leads to the expression of IFNτ stimulated genes viz. ISG15 and OAS1 in blood neutrophils. The IFNτ pathway is species as well as tissue specific. To unsnarl the IFNτ downstream signaling pathway, the blood neutrophils were incubated simultaneously with 10 ng/ml of recombinant bovine interferon tau and the inhibitors of JAK2 (AG490), JAK3 (CP690550), p38 (SB202190), PI3K/Akt (LY294002), and MAPK/Erk (U0126) at specific doses for 4-hr duration. The IFNτ pathway was determined through real-time gene expression of ISG15 and OAS1; immunocytochemistry of ISG15; and Western blotting of ISG15, OAS1, pJAK3 and PI3K. The ISG15 and OAS1 expression decreased significantly (p < 0.001) in the presence of pJAK3 and PI3K inhibitors as compared to a positive control where only interferon tau was used. Immunocytochemistry revealed an attenuated ISG15 response while stimulating blood neutrophils with pJAK3 inhibitor (CP690550) and PI3K inhibitor (LY294002). Similarly, Western blot analysis of neutrophil protein fraction showed weak signals of ISG15, OAS1, pJAK3 and PI3K in the presence of pJAK3 and PI3K inhibitors. The expression profile, immunocytochemistry and western blot analysis revealed a JAK3 and PI3K mediated interferon-tau stimulated gene expression in blood neutrophils.

Purification of Soluble Recombinant Human Tau Protein from Bacteria Using Double-tag Affinity Purification.

Dysfunction of the microtubule-associated protein Tau (encoded by the MAPT gene) has been implicated in more than twenty neurodegenerative diseases, including Alzheimer's. As such, the physiological and disease-relevant functions of Tau have garnered great interest in the research community. One barrier hampering investigations into the functions of Tau and the generation of pharmacological agents targeting Tau has been the difficulty of obtaining soluble Tau protein in purified form. Here, we describe a protocol that uses dual affinity tag purification to selectively purify soluble recombinant Tau protein from bacteria that is functionally active for downstream applications including immunization, microtubule binding assays, and protein-protein interaction studies.

Purification and Fibrillation of Recombinant Human Amyloid-β, Prion Protein, and Tau Under Native Conditions.

Protein misfolding, aggregation, and amyloid formation is involved in a large number of diseases. Recombinantly expressed proteins to study the amyloid fibril formation process are important for mechanistic studies. We here report protocols for production, purification, and fibrillation of three different proteins commonly found in cerebral amyloid; Aβ and Tau found in Alzheimer's disease, Chronic traumatic brain injury, Corticobasal degeneration, and Progressive Supranuclear Palsy and human prion protein found in Creutzfeldt-Jakob's disease. The three protocols have in common that the protein is in a pH-neutral phosphate saline buffer during fibrillation to mimic their endogenous near physiological environment.

Preparation of Tau Oligomers After the Protein Extraction from Bacteria and Brain Cortices.

Oligomerization of soluble tau protein is attracting the attention of an increasingly larger number of scientists involved in research on Alzheimer's disease and other tauopathies. A variety of methods have been developed for the purification of proteins from biological tissues and bacterial cells. Various types of high performance liquid chromatography (HPLC) and affinity tags represent the most common techniques for isolating proteins. Here, we describe a procedure for extracting recombinant tau protein from bacterial cells, utilizing a 6×His affinity tag, or endogenous tau from brain cortices using acid extraction followed by fast protein liquid chromatography (FPLC). Additionally, we introduce a method for oligomerization based on reduction and oxidation of cysteine residues. Our preparation assures high yield of tau protein, while preserving its physiological function.

Kinase-loaded magnetic beads for sequential in vitro phosphorylation of peptides and proteins.

Post-translational modifications, including phosphorylation, greatly impact the physiological function of proteins, especially those that are natively unfolded and implicated in many neurodegenerative diseases. However, structural and functional studies of such proteins require fully defined phosphorylation, including those that are not physiological. Thus, the kinases ERK2 and GSK-3β were immobilized to various superparamagnetic beads with carboxylic, aldehyde, Ni2+, or Co3+ functional groups, with a view to efficiently phosphorylate peptides and proteins in vitro. Full phosphorylation of specific synthetic peptides confirmed that beads were successfully loaded with kinases. Remarkably, enzymes covalently immobilized on carboxylated SeraMag beads remained active upon reuse, with residual activity after 10 uses 99.5 ± 0.34% for GSK-3β and 36.2 ± 2.01% for ERK2. The beads were also used to sequentially phosphorylate recombinant tau, which in vivo is a biomarker of Alzheimer's disease. Thus, a system consisting of two fully active kinases immobilized to magnetic beads is demonstrated for the first time. In comparison to soluble enzymes, the beads are easier to handle, reusable, and thus low-cost. Importantly, these beads are also convenient to remove from reactions to minimize contamination of phosphorylated products or to exchange with other kinases.

Can any “non-specific charge modification within microtubule binding domains of Tau” be a prerequisite of the protein amyloid aggregation? An in vitro study on the 1N4R isoform

The aggregation of Tau into amyloid fibrils is a hallmark of neurodegenerative diseases such as Alzheimer’s disease (AD). Compared to the Aβ peptide, tau pathology more closely tracks changes in brain function that are responsible for the onset of early symptoms in AD. Tau belongs to the class of intrinsically disordered protein and folds into an ordered β-structure during aggregation, a process that appears in many cases to be preceded by hyperphosphorylation of Tau monomers. Although Tau fibrils can be formed by heparin-induced aggregation of un-phosphorylated recombinant Tau, it is important to understanding the paradox of Tau’s random-like conformations and aggregation propensity. In this study, to look into the effect of charge neutralization on Tau aggregation propensity, solvent accessible lysine residues were chemically acetylated/pseudo-phosphorylated. All Tau variants did not aggregate in the absence of the polyanionic factor; however, in contrast to the wild-type protein, acetylated and pseudo-phosphorylated variants were not able to aggregate even in the presence of the polyanionic cofactor. These aggregation incompetent Tau variants may be good analogs for the phosphorylated Tau, to explore more about the exact role of the phosphorylated Tau monomers in AD progress.

The Molecular Chaperone Artemin Efficiently Blocks Fibrillization of TAU Protein In Vitro.

Objective Aggregation of the TAU proteins in the form of neurofibrillary tangles (NFTs) in the brain is a common risk factor in tauopathies including Alzheimer’s disease (AD). Several strategies have been implemented to target NFTs, among which chaperones, which facilitate the proper folding of proteins, appear to hold great promise in effectively inhibiting TAU polymerization. The aim of this study was to analyze the impact of the chaperone Artemin on TAU aggregation in vitro. Materials and Methods In this experimental study, recombinant TAU- or Artemin proteins were expressed in E.coli bacteria, and purified using ion-exchange and affinity chromatography. Sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) was used to run the extracted proteins and check their purity. Heparin was used as an aggregation inducer. The interaction kinetics of TAU aggregation and disassembly was performed using thioflavin T (ThT) fluorescence analysis and circular dichroism (CD) spectroscopy. Results Ion-exchange and affinity chromatography yielded highly pure TAU and Artemin proteins for subsequent analyses. In addition, we found that heparin efficiently induced TAU fibrillization 48 hours post-incubation, as evidenced by ThT assay. Importantly, Artemin was observed to effectively block the aggregation of both physiologic- and supra- physiologic TAU concentrations in a dose-dependent manner, as judged by ThT and CD spectroscopy analyses. ConclusionOur collective results show, for the first time, that the chaperone Artemin could significantly inhibit aggregation of the TAU proteins in a dose-dependent manner, and support Artemin as a potential potent blocker of TAU aggregation in people with AD.

Characterization of isolated tau-reactive antibodies from the IVIG product, plasma of patients with Alzheimer's disease and cognitively normal individuals

The presence of natural tau-reactive antibodies was reported in human blood. In this study, we isolated and characterized natural tau-reactive antibodies occurring in IVIG product Flebogamma, plasma of patients with Alzheimer's disease (AD) and older cognitively normal persons (controls). Using blotting immunoassays and ELISA, we showed reactivity of antibodies obtained from IVIG and controls against a recombinant fragment of tau (155–421aa) and aggregates present in brains of AD patients. In contrast, antibodies isolated from plasma of AD patients reacted mainly with the recombinant full-length tau form and tau monomeric forms in brain tissue.

Dual Mechanism of Toxicity for Extracellular Injection of Tau Oligomers versus Monomers in Human Tau Mice.

The mechanism of tau toxicity is still unclear. Here we report that recombinant tau oligomers and monomers, intraventricularly injected in mice with a pure human tau background, foster tau pathology through different mechanisms. Oligomeric forms of tau alter the conformation of tau in a paired helical filament-like manner. This effect occurs without tau hyperphosphorylation as well as activation of specific kinases, suggesting that oligomers of tau induce tau assembly through a nucleation effect. Monomers, in turn, induce neurodegeneration through a calpain-mediated tau cleavage that leads to accumulation of a 17 kDa neurotoxic peptide and induction of apoptotic cell death.

PPID CONTROLS TOXIC AMYLOID FORMATION THROUGH PROLINE ISOMERIZATION

Controlling the toxicity of pathogenic amyloidogenic proteins, like tau in Alzheimer's disease, is key to the prevention of many neurodegenerative diseases. PPID, a peptidyl-prolyl isomerase (PPIase), which we have shown to interact with tau, suppresses aggregation across several biological models. Recombinant PPID and P301L tau were combined and aggregation observed by Thioflavin T (ThT) fluorescence for 72 hours. Human HEK cells overexpressing P301L tau were transfected with PPID and sarkosyl insoluble tau levels quantified by Western blot. rTg4510 mice (n=8), overexpressing human P301L tau, were injected with AAV9-PPID or AAV9-GFP and tau species levels were measured by Gallyas silver-staining, and Western blots. Neuronal count was measured by unbiased stereology after tissue was stained with NeuN. PPID significantly reduced the aggregation of P301L tau in in vitro. Insoluble tau levels were significantly reduced in HEK cells after PPID transfection. Injection of AAV9-PPID into mouse hippocampi significantly decreased the production of insoluble tau and was neuroprotective compared to control. PPID can significantly lower insoluble tau levels across several models including mice overexpressing human tau. This provides the novel PPIase as a promising therapeutic target.

Novel human neuronal tau model exhibiting neurofibrillary tangles and transcellular propagation

Tauopathies are a class of neurodegenerative diseases, including Alzheimer's disease, frontotemporal dementia and progressive supranuclear palsy, which are associated with the pathological aggregation of tau protein into neurofibrillary tangles (NFT). Studies have characterized tau as a “prion-like” protein given its ability to form distinct, stable amyloid conformations capable of transcellular and multigenerational propagation in clonal fashion. It has been proposed that progression of tauopathy could be due to the prion-like propagation of tau, suggesting the possibility that end-stage pathologies, like NFT formation, may require an instigating event such as tau seeding. To investigate this, we applied a novel human induced pluripotent stem cell (hiPSC) system we have developed to serve as a human neuronal model. We introduced the tau repeat domain (tau-RD) with P301L and V337M (tau-RD-LM) mutations into hiPSC-derived neurons and observed expression of tau-RD at levels similar to total tau in postmortem AD brains. Tau aggregation occurred without the addition of recombinant tau fibrils. The conditioned media from tau-RD cultures contained tau-RD seeds, which were capable of inducing aggregate formation in homotypic mode in non-transduced recipient neuronal cultures. The resultant NFTs were thioflavin-positive, silver stain-positive, and assumed fibrillary appearance on transmission electron microscopy (TEM) with immunogold, which revealed paired helical filament 1 (PHF1)-positive NFTs, representing possible recruitment of endogenous tau in the aggregates. Functionally, expression of tau-RD caused neurotoxicity that manifested as axon retraction, synaptic density reduction, and enlargement of lysosomes. The results of our hiPSC study were reinforced by the observation that Tau-RD-LM is excreted in exosomes, which mediated the transfer of human tau to wild-type mouse neurons in vivo. Our hiPSC human neuronal system provides a model for further studies of tau aggregation and pathology as well as a means to study transcellular propagation and related neurodegenerative mechanisms.

Generation of recombinant bovine interferon tau in the human embryonic kidney cell line and its biological activity

The objective of this study was to generate recombinant bovine interferon tau (rbIFNT) in mammalian hosts. The complementary DNA encoding bovine IFNT2 was cloned for the construction of pRcRSV-bIFNT2 expression vector. The expression vector was transfected to 293 cells. Transfected cells harboring expression vector were selected with G418. Highly expressing clonal line was adapted to serum-free suspension culture in a spinner flask. The recombinant protein had 24 kDa apparent molecular mass, suggesting being expressed as a glycoprotein, and was purified from serum-free conditioned medium by the combination of Diethylaminoethanol Sepharose ion exchange and Sephacryl S-200 HR gel filtration. A total of 7.3 mg rbIFNT was obtained from 13.5L conditioned medium. Generated rbIFNT was biologically active in terms of antiviral activity measured by the plaque inhibition assay with Madin-Darby bovine kidney cells and the vesicular stomatitis virus. The recombinant protein was also utilized for immunization to raise antibodies in the rabbit. The generated antibody was capable of use in both Western blotting and the binding assay. The results in the present study suggest that a certain amount of rbIFNT is raised in mammalian hosts by using conventional plasmid vector and its antibody provides useful tools for studies in the biology of bovine IFNT.

Production of recombinant tau oligomers in vitro.

The pathological aggregation of the tau protein is a common characteristic of many neurodegenerative diseases. There is strong interest in characterizing the potentially toxic nature of tau oligomers. These nonfibrillar, soluble multimers appear to be more toxic than neurofibrillary tangles made up of filamentous tau. However, reliable production, purification, and verification of tau oligomers can provide certain challenges. Here, we provide a series of methods that address these issues. First, recombinant tau is produced using Escherichia coli, purified through affinity, size-exclusion, and anion-exchange chromatography steps and quantified using an SDS Lowry protein quantitation assay. Aggregation of tau is induced using arachidonic acid, and oligomers are purified by centrifugation over a sucrose step gradient. Finally, we describe a sandwich enzyme-linked immunosorbent assay that utilizes the tau oligomer-specific TOC1 antibody to confirm the presence of oligomeric tau. Together, these steps provide a very simple and reliable method for producing tau oligomers that can be used in downstream applications.

Expression and isolation of recombinant tau.

In this chapter, we describe methods for the purification of both untagged and polyhistidine-tagged tau protein. These protocols utilize a bacterial expression system to produce the tau isoform of interest, followed by heat treatment and column chromatography to separate tau from impurities. These techniques yield a biochemically pure protein with which to pursue any number of questions regarding the mechanisms of tau action.

Passive immunization targeting the N-terminal region of phosphorylated tau (residues 68–71) improves spatial memory in okadaic acid induced tauopathy model rats

Alzheimer's disease (AD) is the leading cause of dementia, characterized by progressive loss of memory and other cognitive functions. The cognitive impairment in patients with AD is closely associated with loss of synapses and the formation of neurofibrillary tangles (NFT) containing hyperphosphorylated tau in the hippocampus. Effective treatment for AD is still not available. In this study, the sequence comprising of residues 50–71 in the N-terminal region of tau, containing theoretically predicted B- and T-cell epitopes in close proximity to pathologically relevant phospho-serine (residue 68) and phospho-threonine (residues 69, 71) was selected as a potential immunotherapeutic peptide. This 22-residue long phospho-peptide (50TPTEDGSEEPGSETSDAKpSpTPpT71) was custom synthesized and its therapeutic potential was tested in experimental rats. For this purpose, adult Sprague-Dawley rats were intranasally treated with okadaic acid (OA), a selective inhibitor of protein phosphatase PP2A. Within a day of OA administration, these rats showed marked impairment in cognitive functions with a significant increase in p-tau/t-tau ratio in the hippocampal homogenates. Passive immunization studies conducted in these OA treated rats with polyclonal anti-phospho-peptide antibodies resulted in a significant improvement in learning and memory functions in Barne's maze task. Further, p-tau levels in the hippocampal homogenates were reduced. In addition, these antibodies effectively prevented the aggregation of recombinant tau in vitro. These results demonstrate that targeting N-terminal region of tau harbouring the phospho-residue cluster 68–71 would be beneficial and may present an effective therapeutic opportunity for AD and other tauopathies.

Production of O-GlcNAc Modified Recombinant Tau in E. coli and Detection of Ser400 O-GlcNAc Tau In Vivo.

Assembly of the microtubule-associated protein tau (tau) into paired helical filaments that ultimately give rise to neurofibrillary tangles (NFTs) makes up one half of the two hallmark pathologies of Alzheimer's disease (AD). Tau has been shown to be modified with O-linked N-acetylglucosamine residues (O-GlcNAc), which is the modification of serine and threonine residues of nucleocytoplasmic proteins with N-acetyl-D-glucosamine (GlcNAc) moieties. Increasing O-GlcNAc in mouse models of tauopathy has been shown to hinder the progression of symptoms in these mice and impair the aggregation of tau into NFTs. In order to study how O-GlcNAc on tau may contribute to the protective effects observed in tauopathy mouse models, it is beneficial to study O-GlcNAc modified tau in vitro. Here we describe a method for producing, purifying and enriching recombinant tau that is O-GlcNAc modified. These methods have enabled the identification of O-GlcNAc modification sites on tau including Ser400. We further describe the detection of Ser400 O-GlcNAc on tau from brain lysates.

Assays for the Screening and Characterization of Tau Aggregation Inhibitors.

Aggregation of tau protein is a pathological hallmark of Alzheimer's disease and other neurodegenerative tauopathies. Inhibition of tau aggregation could provide a method for the treatment of these disorders. Methods to identify tau aggregation inhibitors (TAIs) in vitro are useful and here we describe assays for TAIs using purified recombinant tau protein fragments in a cell-free immunoassay format and a stably transfected cell model to create a more physiological environment.

Formation, release, and internalization of stable tau oligomers in cells.

Tau is a neuronal microtubule-binding protein that, in Alzheimer's disease and other neurodegenerative diseases, can form oligomeric and large fibrillar aggregates, which deposit in neurofibrillary tangles. Tau's physiological state of multimerization appears to vary across conditions, and a stable dimeric form of soluble tau has been suggested from experiments using recombinant tau invitro. We tested if tau dimerization or oligomerization, also occurs in cells, and if soluble tau oligomers are relevant for the release and internalization of tau. We developed a sensitive tau split-luciferase assay to show the rapid intracellular formation of stable tau dimers that are released and taken up by cells. Our data further suggest that tau dimerization can be accelerated slightly by aggregation catalysts. We conclude that tau oligomers are a stable physiological form of tau, and that tau oligomerization does not necessarily lead to tau aggregation.