Site-Specific Hyperphosphorylation Inhibits, Rather than Promotes, Tau Fibrillization, Seeding Capacity, and Its Microtubule Binding.

The consistent observation of phosphorylated tau in the pathology of Alzheimer's disease has contributed to the emergence of a model where hyperphosphorylation triggers both tau disassociation from microtubules and its subsequent aggregation. Herein, we applied a total chemical synthetic approach to site-specifically phosphorylate the microtubule binding repeat domain of tau (K18) at single (pS356) or multiple (pS356/pS262 and pS356/pS262/pS258) residues. We show that hyperphosphorylation of K18 inhibits 1) its aggregation in vitro, 2) its seeding activity in cells, 3) its binding to microtubules, and 4) its ability to promote microtubule polymerization. The inhibition increased with increasing the number of phosphorylated sites, with phosphorylation at S262 having the strongest effect. Our results argue against the hyperphosphorylation hypothesis and underscore the importance of revisiting the role of site-specific hyperphosphorylation in regulating tau functions in health and disease.

Protein Semisynthesis Provides Access to Tau Disease-Associated Post-translational Modifications (PTMs) and Paves the Way to Deciphering the Tau PTM Code in Health and Diseased States.

The microtubule-associated protein Tau plays a central role in neurodegeneration and is a leading therapeutic target for the treatment of Alzheimer's disease (AD). Several lines of evidence suggest that post-translational modifications (PTMs) regulate the function(s) of Tau, including its subcellular localization, clearance, aggregation, toxicity, and pathological spreading. However, the lack of tools and methodologies that allow site-specific introduction of PTMs in Tau have limited our ability to dissect the role of PTMs in regulating Tau functions in health and disease. To facilitate deciphering the Tau PTM code, we have developed, for the first time, semisynthetic strategies that allow for the site-specific introduction of single or multiple physiological or disease-associated PTMs that occur within residues 246-441 of Tau, which includes the microtubule-binding domain (MTBD). As a proof of concept, we produced unmodified Tau and three Tau variants with single or multiple disease-associated PTMs that were not previously accessible as homogeneously modified proteins, AcK280, pY310, and pS396/pS404. We then focused on investigating the effect of acetylation at lysine 280 (AcK280) on the structure, aggregation, and microtubule binding properties of Tau. Our results show that site-specific acetylation at K280 significantly enhances the aggregation rate of Tau and impairs microtubule assembly. Surprisingly, compared with unmodified Tau, which forms long and flexible filaments, AcK280 Tau forms predominantly globular oligomers and short fibrils (<200 nm) that exhibit a reduced propensity to assemble into long filaments. These findings are consistent with the increased aggregation propensity and pathogenicity of this mutant in animal models of AD and suggest that acetylation at this residue might enhance the seeding capacity or formation of toxic Tau species in vivo. Beyond acetylation and phosphorylation, the development of this semisynthetic strategy provides new opportunities to investigate other types of Tau PTMs and to study the cross-talk between PTMs that occurs within residues 246-441, which were previously inaccessible, thereby paving the way to deciphering the Tau PTM code in health and disease.

The Heidenhain variant of Creutzfeldt-Jakob disease and concomitant tau pathology: A case report.

The Heidenhain form of Creutzfeldt-Jakob disease (CJD) is a rare CJD variant with predominantly visual symptoms in the early stages. Clinical manifestations of metamorphopsia, hemianopia and Balint's syndrome correlate with the involvement of the posterior cortical regions. A 71-year old healthy and very active man was admitted because of impaired visual acuity, hemianopia, and gait disturbance progressing over one week. MRI found typical cortical hyperintensities in the occipital regions while rhythm slowing and sharp waves were seen in the occipital regions on EEG. Protein 14-3-3 was detected in the cerebrospinal fluid. Postmortem neuropathology revealed typical histopathological changes consistent with CJD. Moreover, we found deposits of phosphorylated tau protein in the limbic regions that met the criteria for primary age-related tauopathy (PART); representing an additional and interesting finding in our case.

Tag-Free Semi-Synthesis of the Tau Protein.

Expressed protein ligation (EPL) is a valuable tool to study site-specific functionalities on proteins such as posttranslational modifications. The purification of such ligation products from EPL mixtures can be cumbersome due to a small size difference between the expressed protein portion and the desired ligated protein. Therefore, affinity tags are often required, which remain on the protein after purification. Herein, we present an efficient protocol to install a photocleavable biotin building block on synthetic C-terminal tau[390-441] and describe its use for purification of full-length semi-synthetic tau[1-441].

Semi-synthesis of a tag-free O-GlcNAcylated tau protein by sequential chemoselective ligation.

In this paper, the first semi-synthesis of the Alzheimer-relevant tau protein carrying an O-GlcNAcylation is demonstrated by using sequential chemoselective ligation. The 52-amino acid C-terminus of tau was obtained by native chemical ligation between two synthetic peptide fragments, one carrying the O-GlcNAc moiety on Ser400, which has recently been demonstrated to inhibit tau phosphorylation and to hinder tau oligomerization, and the other equipped with a photocleavable biotin handle. After desulfurization to deliver a native alanine at the ligation junction, the N-terminal cysteine was unmasked, and the peptide was further used for expressed protein ligation to generate the full-length tau protein, which was purified by a photocleavable biotin tag. We thus provide a synthetic route to obtain a homogenous tag-free O-GlcNAcylated tau protein that can further help to elucidate the significance of posttranslational modification on the tau protein and pave the way for evaluating possible drug targets in Alzheimer's disease. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Native chemical ligation between asparagine and valine: application and limitations for the synthesis of tri-phosphorylated C-terminal tau.

We present the successful native chemical ligation (NCL) at an Asn-Val site employing ß-mercaptovaline and subsequent desulfurization in the synthesis of native phosphorylated C-terminal tau, relevant for Alzheimer's disease related research. Despite recent progress in the field of NCL we illustrate limitations of this ligation site that stem from thioester hydrolysis and predominantly aspartimide formation. We systematically investigated the influence of pH, temperature, peptide concentration and thiol additives on the outcome of this ligation and identified conditions under which the ligation can be driven toward complete conversion, which required the deployment of a high surplus of thioester. Application of the optimized conditions allowed us to gain access to challenging tri-phosphorylated C-terminal tau peptide in practical yields.

Structural characterization by NMR of a double phosphorylated chimeric peptide vaccine for treatment of Alzheimer's disease.

Rational design of peptide vaccines becomes important for the treatment of some diseases such as Alzheimer's disease (AD) and related disorders. In this study, as part of a larger effort to explore correlations of structure and activity, we attempt to characterize the doubly phosphorylated chimeric peptide vaccine targeting a hyperphosphorylated epitope of the Tau protein. The 28-mer linear chimeric peptide consists of the double phosphorylated B cell epitope Tau229₋237[pThr231/pSer235] and the immunomodulatory T cell epitope Ag85B241₋255 originating from the well-known antigen Ag85B of the Mycobacterium tuberculosis, linked by a four amino acid sequence -GPSL-. NMR chemical shift analysis of our construct demonstrated that the synthesized peptide is essentially unfolded with a tendency to form a ß-turn due to the linker. In conclusion, the -GPSL- unit presumably connects the two parts of the vaccine without transferring any structural information from one part to the other. Therefore, the double phosphorylated epitope of the Tau peptide is flexible and accessible.

Binding to the minor groove of the double-strand, tau protein prevents DNA from damage by peroxidation.

Tau, an important microtubule associated protein, has been found to bind to DNA, and to be localized in the nuclei of both neurons and some non-neuronal cells. Here, using electrophoretic mobility shifting assay (EMSA) in the presence of DNA with different chain-lengths, we observed that tau protein favored binding to a 13 bp or a longer polynucleotide. The results from atomic force microscopy also showed that tau protein preferred a 13 bp polynucleotide to a 12 bp or shorter polynucleotide. In a competitive assay, a minor groove binder distamycin A was able to replace the bound tau from the DNA double helix, indicating that tau protein binds to the minor groove. Tau protein was able to protect the double-strand from digestion in the presence of DNase I that was bound to the minor groove. On the other hand, a major groove binder methyl green as a negative competitor exhibited little effect on the retardation of tau-DNA complex in EMSA. This further indicates the DNA minor groove as the binding site for tau protein. EMSA with truncated tau proteins showed that both the proline-rich domain (PRD) and the microtubule-binding domain (MTBD) contributed to the interaction with DNA; that is to say, both PRD and MTBD bound to the minor groove of DNA and bent the double-strand, as observed by electron microscopy. To investigate whether tau protein is able to prevent DNA from the impairment by hydroxyl free radical, the chemiluminescence emitted by the phen-Cu/H(2)O(2)/ascorbate was measured. The emission intensity of the luminescence was markedly decreased when tau protein was present, suggesting a significant protection of DNA from the damage in the presence of hydroxyl free radical.

[Clinically atypical CJD: diagnostic relevance of cerebrospinal fluid markers and molecular genetic analysis?]. / Die klinisch atypische CJK - Diagnostische Bedeutung von Liquormarkern und molekulargenetischen Untersuchungen? -

HISTORY AND CLINICAL FINDINGS: A 66-year-old woman with known remitting depressive episodes was admitted to a psychiatric hospital where a severe major depression was diagnosed. The patient failed to respond to SSRI medication. In addition, she developed a pronounced speech disturbance and gait ataxia. Therefore she was referred to the department of neurology 5 months after disease onset. She presented with a predominant cerebellar syndrome. Because of a severe dysarthria testing of cognitive functions were impossible. INVESTIGATIONS: EEG revealed a general slowing of the basic activity. Periodic sharp wave complexes were not seen during the entire disease course. Cerebrospinal fluid analysis (CSF) was normal with regard to cell count and blood-csf-barrier function. 14- 3-3 proteins were detected in CSF, and CSF levels of tau-protein and neuron-specific enolase were strongly elevated. A point mutation was detected at codon 196 (E196K) of the prion proteine gene. In addition, codon 129 was found homozygous for valine. TREATMENT AND COURSE: During the following 10 weeks the patient developed pyramidal and extrapyramidal signs, myoclonus and akinetic mutism. Complications were decubital ulcers and infections of the urinary tract. The myoclonus slightly regressed following treatment with benzodiazepines. The patient died 9 months after disease onset. CONCLUSION: A rapid disease course and typical changes of surrogate markers in CSF may support the in-vivo diagnosis of CJD despite incomplete clinical criteria (e. g. lack of dementia). In such cases, detection of mutations by molecular genetic analysis is of importance to further support the diagnosis of CJD.

Tyrosine- versus serine-phosphorylation leads to conformational changes in a synthetic tau peptide.

One of the major immunodominant epitopes of the paired helical filaments (PHF) of Alzheimer's disease is the peptide sequence GAEIVYKSPVVSGD (T3), comprising amino acids 389-402 of the microtubule-associated protein, tau, when it is phosphorylated at the first serine residue. While the corresponding anti-PHF monoclonal antibody recognizes the peptide phosphorylated at either serine, it does not recognize the tyrosine-phosphorylated peptide. Here we describe the effect of serine- versus tyrosine-phosphorylation on the conformation of a synthetic tau peptide. While adding a phosphate to the serine residue has practically no impact on the structure of the non-phosphorylated peptide, phosphorylation of the tyrosine results in considerable conformational changes.

Spectroscopic evidence that monoclonal antibodies recognize the dominant conformation of medium-sized synthetic peptides.

Spectroscopic methods have amply documented that small- and medium-sized peptides tend to assume unordered conformations in water. The conformational tendencies, however, manifest in halogenated alcohols, and the preferred secondary structures are apparent from the circular dichroism (CD) spectra. Here we report the results of immobilizing peptide and protein antigens from various mixtures of trifluoroethanol and water during enzyme-linked immunosorbent assays. The increased recognition by the appropriate monoclonal antibodies (mAbs) is correlated with the increase of the alpha helical, beta turn, or beta pleated sheet content of the peptides presented in the different solvent mixtures. Remarkably, the antibody binding can be detected at considerably lower antigen levels if the antigen is immobilized from trifluoroethanol. The antigens we used corresponded to fragments of normal human neurofilaments and tau protein found in the paired helical filaments of Alzheimer's disease, and the nucleoprotein of rabies virus. The conformation of myoglobin is as stable in water as in trifluoroethanol, and therefore acted as a negative control. Indeed, the recognition of myoglobin did not increase upon increasing the trifluoroethanol concentration in the solvent used to apply the antigen to the plate. The possibility of imperfect binding to the plastic carrier or nonspecific binding to irrelevant antibodies is excluded by using control experiments. We offer the first direct evidence that the mAbs recognize the secondary structure of epitopes, and that it is possible to correlate the binding conformation of the epitopes with CD measurements made in trifluoroethanol-water mixtures.

Chemically synthesized 182-235 segment of tau protein and analogue peptides are efficient in vitro microtubule assembly inducers of low apparent sequence specificity.

A 54-amino acid peptide reproducing the first and second repeats and intervening spacer sequence of the tubulin binding motif (residues 182-235) of murine tau protein, and several congeners representing different degrees of sequence scrambling have been prepared by solid phase methods and fully characterized chemically. These double-repeat peptides have been shown to induce microtubule formation at concentrations about one order of magnitude lower than single-repeat controls, under conditions close to the critical concentration needed for tubulin self-assembly. On the other hand, partial loss of microtubule-inducing capacity was observed for peptides with primary structures increasingly disorganized with respect to the canonical peptide. These results call into question the assumption that a high degree of primary structure specificity is involved in the tau-tubulin interaction leading to in vitro microtubule formation.