Structural and Functional Characterization of Neurotoxic Oligomers of Pro-aggregant Tau Repeat Domain : A model for Tauopathy disease

Tau protein is a microtubule associated protein present abundantly in the neurons of the central nervous system where it stabilizes the axonal microtubules thereby providing structural architecture for the axons of neurons. Aggregation of Tau occurs in many neurodegenerative diseases collectively termed tauopathies including Alzheimer disease (AD) and frontotemporal dementia (FTD). The mutation ΔK280 of Tau was originally discovered in cases of FTD (Rizzu et al., 1999). In vitro it leads to a pronounced propensity of the protein to aggregate (Barghorn et al., 2000). The repeat domain of Tau protein with this pro-aggregant mutation (TauRDΔ) induces toxicity in transgenic mice and organotypic hippocampal slice culture models (Sydow et al., 2011, Messing et al., 2013). One current concept of Tau-mediated toxicity in Alzheimer disease and related tau dependent pathologies is that it is based on low-n oligomeric species, rather than higher aggregated forms (fibers and neurofibrillary tangles). To test this we characterized oligomers from TauRDΔ protein assembled and purified in vitro. Since Tau oligomers are in dynamic equilibrium during aggregation, we tried to capture and stabilize only the oligomeric forms of Tau using EGCG (Epigallocatechin gallate). EGCG reduces the formation of fibrils and increases the SDS stable oligomers. However, the oligomers are not separable by gel filtration chromatography. Therefore we stabilized the tau oligomers using a low concentration of glutaraldehyde as a cross-linking reagent. This yielded SDS stable low-n oligomers predominantly in the form of dimers, trimers, tetramers with very low amounts of higher order species. The cross-linked TauRDΔ oligomers can be purified by hydrophobic interaction chromatography with ~95% purity. They exhibit enhanced fluorescence with the dye ANS, arguing for an altered conformation (compared with monomers) and possibly exposed hydrophobic surface patches. However, they do not contain substantial ß-sheet structure, as analyzed by thioflavin S fluorescence and circular dichroism. Atomic force microscopy (AFM) of TauRDΔ oligomers reveals that the particles are roughly globular in shape, with diameters in the range 1.6-5.4 nm (AFM height values). The hydrodynamic radius of TauRDΔ oligomers (~5.2 nm) is dominated by that of tetramers, as measured by dynamic light scattering. The size of TauRDΔ oligomers reveals that they contain up to 4-5 molecules of Tau, consistent with the SDS gel analysis. The TauRDΔ oligomers do not exhibit global toxicity towards rat primary neurons when applied to the extracellular medium, as judged by MTT and LDH assays. However, functional impairment can be deduced from a pronounced (up to 50%) decrease of dendritic spines and a shift from mushroom-shaped to stubby spines. Consistent with this, the expression of cytoskeletal proteins which are necessary to maintain the mushroom spines is reduced. The neurons also show an increase in reactive oxygen species and influx of calcium. In summary, low-n oligomers of TauRDΔ do not cause gross changes in viability, but induce subtle functional defects, leading to an increase in Ca++ and ROS, and consequently to loss of spines and associated shape changes. Since Tau is an intracellular protein and the formation of oligomers occurs inside the cells, we introduced low-n Tau oligomers by protein transfection into SH-SY5Y cells and primary rat hippocampal neurons and analyzed them by flow cytometry and western blot analysis. This showed that only the cells transfected with TauRDΔ oligomers (but not monomers) induce the intracellular aggregation of Tau and recruitment of endogenous Tau into the aggregates. This is accompanied by the hyperphosphorylation of aggregated Tau. Although TauRDΔ oligomer transfected cells do not undergo cell death within 15 h of transfection, we found the presence of annexin V positive cells. When compared to monomers and fibrils, the oligomer transfected cells show a 5 fold increase in annexin V positive cells suggesting enhanced apoptosis. We conclude that TauRDΔ oligomers applied extracellularly cause degeneration of spines without affecting cell viability, whereas introducing oligomers intracellularly leads to Tau aggregation and apoptosis

A novel D-amino acid peptide with therapeutic potential (ISAD1) inhibits aggregation of neurotoxic disease-relevant mutant Tau and prevents Tau toxicity in vitro

Background: Alzheimer's disease (AD), the most common form of dementia, is a progressive neurodegenerative disorder that mainly affects older adults. One of the pathological hallmarks of AD is abnormally aggregated Tau protein that forms fibrillar deposits in the brain. In AD, Tau pathology correlates strongly with clinical symptoms, cognitive dysfunction, and neuronal death. Methods: We aimed to develop novel therapeutic D-amino acid peptides as Tau fibrillization inhibitors. It has been previously demonstrated that D-amino acid peptides are protease stable and less immunogenic than L-peptides, and these characteristics may render them suitable for in vivo applications. Using a phage display procedure against wild type full-length Tau (TauFL), we selected a novel Tau binding L-peptide and synthesized its D-amino acid version ISAD1 and its retro inversed form, ISAD1rev, respectively. Results: While ISAD1rev inhibited Tau aggregation only moderately, ISAD1 bound to Tau in the aggregation-prone PHF6 region and inhibited fibrillization of TauFL, disease-associated mutant full-length Tau (TauFLΔK, TauFL-A152T, TauFL-P301L), and pro-aggregant repeat domain Tau mutant (TauRDΔK). ISAD1 and ISAD1rev induced the formation of large high molecular weight TauFL and TauRDΔK oligomers that lack proper Thioflavin-positive β-sheet conformation even at lower concentrations. In silico modeling of ISAD1 Tau interaction at the PHF6 site revealed a binding mode similar to those known for other PHF6 binding peptides. Cell culture experiments demonstrated that ISAD1 and its inverse form are taken up by N2a-TauRDΔK cells efficiently and prevent cytotoxicity of externally added Tau fibrils as well as of internally expressed TauRDΔK. Conclusions: ISAD1 and related peptides may be suitable for therapy development of AD by promoting off-pathway assembly of Tau, thus preventing its toxicity. Keywords: Alzheimer’s disease; D-amino acid peptides; Phage display; Tau aggregation inhibitors; Therap

Potent Tau Aggregation Inhibitor D-Peptides Selected against Tau-Repeat 2 Using Mirror Image Phage Display

Alzheimer's disease and other Tauopathies are associated with neurofibrillary tangles composed of Tau protein, as well as toxic Tau oligomers. Therefore, inhibitors of pathological Tau aggregation are potentially useful candidates for future therapies targeting Tauopathies. Two hexapeptides within Tau, designated PHF6* (275-VQIINK-280) and PHF6 (306-VQIVYK-311), are known to promote Tau aggregation. Recently, the PHF6* segment has been described as the more potent driver of Tau aggregation. We therefore employed mirror-image phage display with a large peptide library to identify PHF6* fibril binding peptides consisting of D-enantiomeric amino acids. The suitability of D-enantiomeric peptides for in vivo applications, which are protease stable and less immunogenic than L-peptides, has already been demonstrated. The identified D-enantiomeric peptide MMD3 and its retro-inverso form, designated MMD3rev, inhibited in vitro fibrillization of the PHF6* peptide, the repeat domain of Tau as well as full-length Tau. Dynamic light scattering, pelleting assays and atomic force microscopy demonstrated that MMD3 prevents the formation of tau β-sheet-rich fibrils by diverting Tau into large amorphous aggregates. NMR data suggest that the D-enantiomeric peptides bound to Tau monomers with rather low affinity, but ELISA (enzyme-linked immunosorbent assay) data demonstrated binding to PHF6* and full length Tau fibrils. In addition, molecular insight into the binding mode of MMD3 to PHF6* fibrils were gained by in silico modelling. The identified PHF6*-targeting peptides were able to penetrate cells. The study establishes PHF6* fibril binding peptides consisting of D-enantiomeric amino acids as potential molecules for therapeutic and diagnostic applications in AD research

The release and trans-synaptic transmission of Tau via exosomes

BACKGROUND Tau pathology in AD spreads in a hierarchical pattern, whereby it first appears in the entorhinal cortex, then spreads to the hippocampus and later to the surrounding areas. Based on this sequential appearance, AD can be classified into six stages ("Braak stages"). The mechanisms and agents underlying the progression of Tau pathology are a matter of debate. Emerging evidence indicates that the propagation of Tau pathology may be due to the transmission of Tau protein, but the underlying pathways and Tau species are not well understood. In this study we investigated the question of Tau spreading via small extracellular vesicles called exosomes. METHODS Exosomes from different sources were analyzed by biochemical methods and electron microscopy (EM) and cryo-EM. Microfluidic devices that allow the culture of cell populations in different compartments were used to investigate the spreading of Tau. RESULTS We show that Tau protein is released by cultured primary neurons or by N2a cells overexpressing different Tau constructs via exosomes. Neuron-derived exosomal Tau is hypo-phosphorylated, compared with cytosolic Tau. Depolarization of neurons promotes release of Tau-containing exosomes, highlighting the importance of neuronal activity. Using microfluidic devices we show that exosomes mediate trans-neuronal transfer of Tau depending on synaptic connectivity. Tau spreading is achieved by direct transmission of exosomes between neurons. In organotypic hippocampal slices, Tau-containing exosomes in conditioned medium are taken up by neurons and microglia, not astrocytes. In N2a cells, Tau assemblies are released via exosomes. They can induce inclusions of other Tau molecules in N2a cells expressing mutant human Tau. We also studied exosomes from cerebrospinal fluid in AD and control subjects containing monomeric and oligomeric Tau. Split-luciferase complementation reveals that exosomes from CSF can promote Tau aggregation in cultured cells. CONCLUSION Our study demonstrates that exosomes contribute to trans-synaptic Tau transmission, and thus offer new approches to control the spreading of pathology in AD and other tauopathies

The Plasma Factor XIII Heterotetrameric Complex Structure: Unexpected Unequal Pairing within a Symmetric Complex

International audienceFactor XIII (FXIII) is a predominant determinant of clot stability, strength, and composition. Plasma FXIII circulates as a pro-transglutaminase with two catalytic A subunits and two carrier-protective B subunits in a heterotetramer (FXIII-A2B2). FXIII-A2 and -B2 subunits are synthesized separately and then assembled in plasma. Following proteolytic activation by thrombin and calcium-mediated dissociation of the B subunits, activated FXIII (FXIIIa) covalently cross links fibrin, promoting clot stability. The zymogen and active states of the FXIII-A subunits have been structurally characterized; however, the structure of FXIII-B subunits and the FXIII-A2B2 complex have remained elusive. Using integrative hybrid approaches including atomic force microscopy, cross-linking mass spectrometry, and computational approaches, we have constructed the first all-atom model of the FXIII-A2B2 complex. We also used molecular dynamics simulations in combination with isothermal titration calorimetry to characterize FXIII-A2B2 assembly, activation, and dissociation. Our data reveal unequal pairing of individual subunit monomers in an otherwise symmetric complex, and suggest this unusual structure is critical for both assembly and activation of this complex. Our findings enhance understanding of mechanisms associating FXIII-A2B2 mutations with disease and have important implications for the rational design of molecules to alter FXIII assembly or activity to reduce bleeding and thrombotic complications

Reversal of Tau-Dependent Cognitive Decay by Blocking Adenosine A1 Receptors: Comparison of Transgenic Mouse Models with Different Levels of Tauopathy

The accumulation of tau is a hallmark of several neurodegenerative diseases and is associatedwith neuronal hypoactivity and presynaptic dysfunction. Oral administration of the adenosineA1 receptor antagonist rolofylline (KW-3902) has previously been shown to reverse spatial memorydeficits and to normalize the basic synaptic transmission in a mouse line expressing full-lengthpro-aggregant tau (TauDK) at low levels, with late onset of disease. However, the efficacy of treatmentremained to be explored for cases of more aggressive tauopathy. Using a combination of behavioralassays, imaging with several PET-tracers, and analysis of brain tissue, we compared the curativereversal of tau pathology by blocking adenosine A1 receptors in three mouse models expressingdifferent types and levels of tau and tau mutants. We show through positron emission tomographyusing the tracer [18F]CPFPX (a selective A1 receptor ligand) that intravenous injection of rolofyllineeffectively blocks A1 receptors in the brain. Moreover, when administered to TauDK mice, rolofyllinecan reverse tau pathology and synaptic decay. The beneficial effects are also observed in a line withmore aggressive tau pathology, expressing the amyloidogenic repeat domain of tau (TauRDDK) withhigher aggregation propensity. Both models develop a progressive tau pathology with missorting,phosphorylation, accumulation of tau, loss of synapses, and cognitive decline. TauRDDK causespronounced neurofibrillary tangle assembly concomitant with neuronal death, whereas TauDK accumulatesonly to tau pretangles without overt neuronal loss. A third model tested, the rTg4510line, has a high expression of mutant TauP301L and hence a very aggressive phenotype starting at~3 months of age. This line failed to reverse pathology upon rolofylline treatment, consistent with ahigher accumulation of tau-specific PET tracers and inflammation. In conclusion, blocking adenosineA1 receptors by rolofylline can reverse pathology if the pathological potential of tau remains below athreshold value that depends on concentration and aggregation propensity

Disruption of Structural Disulfides of Coagulation FXIII-B Subunit; Functional Implications for a Rare Bleeding Disorder

Congenital FXIII deficiency is a rare bleeding disorder in which mutations are detected in F13A1 and F13B genes that express the two subunits of coagulation FXIII, the catalytic FXIII-A, and protective FXIII-B. Mutations in FXIII-B subunit are considerably rarer compared to FXIII-A. Three mutations in the F13B gene have been reported on its structural disulfide bonds. In the present study, we investigate the structural and functional importance of all 20 structural disulfide bonds in FXIII-B subunit. All disulfide bonds were ablated by individually mutating one of its contributory cysteine’s, and these variants were transiently expressed in HEK293t cell lines. The expression products were studied for stability, secretion, the effect on oligomeric state, and on FXIII-A activation. The structural flexibility of these disulfide bonds was studied using classical MD simulation performed on a FXIII-B subunit monomer model. All 20 FXIII-B were found to be important for the secretion and stability of the protein since ablation of any of these led to a secretion deficit. However, the degree of effect that the disruption of disulfide bond had on the protein differed between individual disulfide bonds reflecting a functional hierarchy/diversity within these disulfide bonds

Additional file 1: Figure S1. of The release and trans-synaptic transmission of Tau via exosomes

Tau in exosomes from cortical neurons is not phosphorylated at PHF1 and AT8 sites. Tau in neuronal lysates is phosphorylated at PHF1 and AT8 sites (lanes 1, 4), whereas Tau in dephosphorylated neuronal lysates and exosomal Tau are not detectably phosphorylated at these sites (lane 2, 3 and 5, 6). (PNG 30 kb

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