Post-translational modifications within tau paired helical filament nucleating motifs perturb microtubule interactions and oligomer formation

Post-translationally modified tau is the primary component of tau neurofibrillary tangles, a pathological hallmark of Alzheimer''s disease and other tauopathies. Post-translational modifications (PTMs) within the tau microtubule (MT)-binding domain (MBD), which encompasses two hexapeptide motifs that act as critical nucleating regions for tau aggregation, can potentially modulate tau aggregation as well as interactions with MTs and membranes. Here, we characterize the effects of a recently discovered tau PTM, lysine succinylation, on tau–tubulin interactions and compare these to the effects of two previously reported MBD modifications, lysine acetylation and tyrosine phosphorylation. As generation of site-specific PTMs in proteins is challenging, we used short synthetic peptides to quantify the effects on tubulin binding of three site-specific PTMs located within the PHF6^∗ (paired helical filament [PHF] residues 275–280) and PHF6 (residues 306–311) hexapeptide motifs: K280 acetylation, Y310 phosphorylation, and K311 succinylation. We compared these effects to those observed for MBD PTM-mimetic point mutations K280Q, Y310E, and K311E. Finally, we evaluated the effects of these PTM-mimetic mutations on MBD membrane binding and membrane-induced fibril and oligomer formation. We found that all three PTMs perturb tau MT binding, with Y310 phosphorylation exerting the strongest effect. PTM-mimetic mutations partially recapitulated the effects of the PTMs on MT binding and also disrupted tau membrane binding and membrane-induced oligomer and fibril formation. These results imply that these PTMs, including the novel and Alzheimer''s disease–specific succinylation of tau K311, may influence both the physiological and pathological interactions of tau and thus represent targets for therapeutic intervention.     

Structural transitions in tau k18 on micelle binding suggest a hierarchy in the efficacy of individual microtubule‐binding repeats in filament nucleation

The protein tau is found in an aggregated filamentous state in the intraneuronal paired helical filament deposits characteristic of Alzheimer's disease and other related dementias and mutations in tau protein and mRNA cause frontotemproal dementia. Tau isoforms include a microtubule‐binding domain containing either three or four imperfect tandem microtubule binding repeats that also form the core of tau filaments and contain hexapaptide motifs that are critical for tau aggregation. The tau microtubule‐binding domain can also engage in direct interactions with detergents, fatty acids, or membranes, which can greatly facilitate tau aggregation and may also mediate some tau functions. Here, we show that the alternatively spliced second microtubule‐binding repeat exhibits significantly different structural characteristics compared with the other three repeats in the context of the intact repeat domain. Most notably, the PHF6* hexapeptide motif located at the N‐terminus of repeat 2 has a lower propensity to form strand‐like structure than the corresponding PHF6 motif in repeat 3, and unlike PHF6 converts to partially helical structure in the micelle‐bound state. Interestingly, the behavior of the Module‐B motif, located at the beginning of repeat 4, resembles that of PHF6* rather than PHF6. Our observations, combined with previous results showing that PHF6* and Module‐B are both less effective than PHF6 in nucleating tau aggregation, suggest a hierarchy in the efficacy of these motifs in nucleating tau aggregation that originates in differences in their intrinsic propensities for extended strand‐like structure and the resistance of these propensities to changes in tau's environment.     

Integrating in vitro and in silico approaches to evaluate the “dual functionality” of palmatine chloride in inhibiting and disassembling Tau-derived VQIVYK peptide fibrils

Background

Alzheimer's disease (AD) is the most common neurodegenerative disorder which is characterized by the deposits of intra-cellular tau protein and extra-cellular amyloid-β (Aβ) peptides in the human brain. Understanding the mechanism of protein aggregation and finding compounds that are capable of inhibiting its aggregation is considered to be highly important for disease therapy.

Intrinsically Disordered Proteins in the Neurodegenerative Processes: Formation of Tau Protein Paired Helical Filaments and Their Analysis

1. Several intrinsically disordered proteins (IDPs) play principal role in the neurodegenerative processes of various types. Among them, α-synuclein is involved in Parkinson's disease, prion protein in transmissible spongiform encephalopathies, and tau protein in Alzheimer's disease (AD) and related tauopathies. Neuronal damage in AD is accompanied by the presence of tau protein fibrils composed of paired helical filaments (PHF).2. Tau protein represents a typical IDP. IDPs do not exhibit any stable secondary structure in the free form, but they are able to fold after binding to targets and contain regions with large propensity to adopt a defined type of secondary structure. Binding–folding event at tau protein leading to PHF generation is believed to happen in the course of tauopathies.3. Detailed molecular topology of PHF formation is unknown. There are evidences about the cross-beta structure in PHF core; however the precise arrangement of the tau polypeptide chain is unclear. In this review we summarize current attempts at in vitro PHF reconstruction and the development of methods for PHF structure determination. The emphasis is put on the monoclonal antibodies used as structural molecular probes for research on the role of IDPs in pathogenesis of neurodegenerative diseases.Dedicated to the late Peter Kontsek.     

Charge-pairing mechanism of phosphorylation effect upon amyloid fibrillation of human tau core peptide

Phosphorylation of a fibrillogenic protein, human tau, is believed to play crucial roles in the pathogenesis of Alzheimer's disease. For elucidating molecular mechanisms of the phosphorylation effect on tau fibrillation, we synthesized a peptide, VQIVY 310K (PHF6) and its phosphorylated derivative (PHF6pY). PHF6 is a partial peptide surrounding a plausible in vivo phosphorylation site Tyr310 and forms amyloid-type fibrils similar to those generated by full-length tau. Fibrillation of PHF6 and PHF6pY were studied by spectroscopic and microscopic methods, and the critical concentration of the fibrillation was determined for comparing the fibril stability. The results showed that the phosphorylation strongly influenced the fibrillation propensity of PHF6 by changing its dependency on pH and ionic strength. On the basis of the observations, we suggested that charged sites on the phosphate group and its electrostatic pairing with the neighboring charged residues were physical origins of the phosphorylation effect. To verify this charge-pairing mechanism, we conducted experiments using a series of PHF6 derivatives with non-native charge distributions. The electrostatic interaction in an intermolecular mode was also demonstrated by the system composed of two different peptide species, which found that fibrillation of nonphosphorylated PHF6 was drastically enhanced when a trace amount of phosphorylated PHF6 molecules coexisted. A simulation analysis utilizing crystal coordinates of the PHF6 fibril was also performed for interpreting the experimental results in a molecular level. The present study using the model peptide system gave us a microscopically insightful view on the roles of tau phosphorylation in amyloid-related diseases.     

Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local beta-structure

The microtubule-associated protein tau is a natively unfolded protein in solution, yet it is able to polymerize into the ordered paired helical filaments (PHF) of Alzheimer's disease. In the splice isoforms lacking exon 10, this process is facilitated by the formation of beta-structure around the hexapeptide motif PHF6 ((306)VQIVYK(311)) encoded by exon 11. We have investigated the structural requirements for PHF polymerization in the context of adult tau isoforms containing four repeats (including exon 10). In addition to the PHF6 motif there exists a related PHF6* motif ((275)VQIINK(280)) in the repeat encoded by the alternatively spliced exon 10. We show that this PHF6* motif also promotes aggregation by the formation of beta-structure and that there is a cross-talk between the two hexapeptide motifs during PHF aggregation. We also show that two of the tau mutations found in hereditary frontotemporal dementias, DeltaK280 and P301L, have a much stronger tendency for PHF aggregation which correlates with their high propensity for beta-structure around the hexapeptide motifs.     

Affinity of Tau antibodies for solubilized pathological Tau species but not their immunogen or insoluble Tau aggregates predicts in vivo and ex vivo efficacy

Background

A few tau immunotherapies are now in clinical trials with several more likely to be initiated in the near future. A priori, it can be anticipated that an antibody which broadly recognizes various pathological tau aggregates with high affinity would have the ideal therapeutic properties. Tau antibodies 4E6 and 6B2, raised against the same epitope region but of varying specificity and affinity, were tested for acutely improving cognition and reducing tau pathology in transgenic tauopathy mice and neuronal cultures.

Results

Surprisingly, we here show that one antibody, 4E6, which has low affinity for most forms of tau acutely improved cognition and reduced soluble phospho-tau, whereas another antibody, 6B2, which has high affinity for various tau species was ineffective. Concurrently, we confirmed and clarified these efficacy differences in an ex vivo model of tauopathy. Alzheimer’s paired helical filaments (PHF) were toxic to the neurons and increased tau levels in remaining neurons. Both toxicity and tau seeding were prevented by 4E6 but not by 6B2. Furthermore, 4E6 reduced PHF spreading between neurons. Interestingly, 4E6’s efficacy relates to its high affinity binding to solubilized PHF, whereas the ineffective 6B2 binds mainly to aggregated PHF. Blocking 4E6's uptake into neurons prevented its protective effects if the antibody was administered after PHF had been internalized. When 4E6 and PHF were administered at the same time, the antibody was protective extracellularly.

Conclusions

Overall, these findings indicate that high antibody affinity for solubilized PHF predicts efficacy, and that acute antibody-mediated improvement in cognition relates to clearance of soluble phospho-tau. Importantly, both intra- and extracellular clearance pathways are in play. Together, these results have major implications for understanding the pathogenesis of tauopathies and for development of immunotherapies.

     

Understanding molecular features of aggregation-resistant tau conformer using oxidized monomer

BackgroundAggregation of tau into paired helical filament (PHF) is a hallmark of Alzheimer's disease (AD), and Cys-mediated disulfide bond formation plays a vital role in tau fibrillation. While intermolecular disulfide bond between Cys residues in microtubule-binding repeat (MTBR) region facilitates tau aggregation, intramolecular disulfide bond attenuates the same, though the molecular basis for such phenomenon remains obscure. Thus intramolecular disulfide-bonded tau monomer might be an excellent model to understand the unique features of aggregation-resistant tau conformer.MethodsWe synthesized the Cys cross-linked tau40 monomer by oxidation and characterized the altered conformational dynamics in the molecule by Hydrogen-deuterium exchange, limited proteolysis and fluorescence quenching.ResultsDeuterium exchange study showed that rigidity was imparted in the core PHF region of oxidized tau40 in MTBR segment, consisting of the fundamental PHF6 motif. Conformational rigidity was prominent in C-terminal tail region also. Limited proteolysis supported reduced accessibility of MTBR region in the molecule.ConclusionsPHF formation of oxidized tau40 might be attenuated either by induction of intramolecular H-bonding between the regions of high β-structure propensity in second and third MTBR (R₂, R₃), thus preventing intermolecular interaction between them, or by imparted rigidity in R₂–R₃, preventing the formation of extended β-structure preceding fibrillation. Data indicated plausible effect of conformational adaptation on the nucleation process of oxidized tau40 assembly.General significanceOur findings unravel the essential molecular features of aggregation-resistant tau conformer. Therapeutics stabilizing such conformers in vivo might be of high benefit in arresting tau assembly during AD and other tauopathies.     

Using intramolecular disulfide bonds in tau protein to deduce structural features of aggregation-resistant conformations

Because tau aggregation likely plays a role in a number of neurodegenerative diseases, understanding the processes that affect tau aggregation is of considerable importance. One factor that has been shown to influence the aggregation propensity is the oxidation state of the protein itself. Tau protein, which contains two naturally occurring cysteine residues, can form both intermolecular disulfide bonds and intramolecular disulfide bonds. Several studies suggest that intermolecular disulfide bonds can promote tau aggregation in vitro. By contrast, although there are data to suggest that intramolecular disulfide bond formation retards tau aggregation in vitro, the precise mechanism underlying this observation remains unclear. While it has been hypothesized that a single intramolecular disulfide bond in tau leads to compact conformations that cannot form extended structure consistent with tau fibrils, there are few data to support this conjecture. In the present study we generate oxidized forms of the truncation mutant, K18, which contains all four microtubule binding repeats, and isolate the monomeric fraction, which corresponds to K18 monomers that have a single intramolecular disulfide bond. We study the aggregation propensity of the oxidized monomeric fraction and relate these data to an atomistic model of the K18 unfolded ensemble. Our results argue that the main effect of intramolecular disulfide bond formation is to preferentially stabilize conformers within the unfolded ensemble that place the aggregation-prone tau subsequences, PHF6* and PHF6, in conformations that are inconsistent with the formation of cross-β-structure. These data further our understanding of the precise structural features that retard tau aggregation.     

Single particle analysis of tau oligomer formation induced by metal ions and organic solvents

Pathological aggregates of tau protein are found in several neurodegenerative diseases termed ‘tauopathies’. Increasing evidence indicates that tau oligomer species rather than the large amyloid cytoplasmic inclusions relevant for histopathological diagnosis might be crucial for cellular damage and neurodegeneration. Trivalent metal ions and polyanionic structures like heparin or arachidonic acid have been shown to induce tau aggregation. However, little is known about early processes of tau aggregation. In this study, we applied fluorescence correlation spectroscopy (FCS) and scanning for intensely fluorescent targets (SIFT) to investigate oligomer formation of tau protein at nanomolar protein concentrations at the single-particle level. Our results indicate that the formation of distinct tau oligomers is induced by the trivalent metal ions Fe³⁺ and Al³⁺ and by organic solvents like DMSO, respectively. In contrast, bivalent metal ions (Cu²⁺, Zn²⁺, Mn²⁺, Ca²⁺, Mg²⁺) had no effect. While DMSO-induced small tau oligomers are relatively stable in solution, dynamic remodeling can be initiated by non-ionic detergents. Moreover Al³⁺ induces rapid formation of a different oligomer species of larger size. Our results provide further insights into early tau oligomerization and aggregation dynamics.     

Yeast surface display of full-length human microtubule-associated protein tau

Microtubule-associated protein tau is an intrinsically disordered, highly soluble protein found primarily in neurons. Under normal conditions, tau regulates the stability of axonal microtubules and intracellular vesicle transport. However, in patients of neurodegeneration such as Alzheimer's disease (AD), tau forms neurofibrillary deposits, which correlates well with the disease progression. Identifying molecular signatures in tau, such as posttranslational modification, truncation, and conformational change has great potential to detect earliest signs of neurodegeneration and develop therapeutic strategies. Here, we show that full-length human tau, including the longest isoform found in the adult brain, can be robustly displayed on the surface of yeast Saccharomyces cerevisiae. Yeast-displayed tau binds to anti-tau antibodies that cover epitopes ranging from the N-terminus to the 4R repeat region. Unlike tau expressed in the yeast cytosol, surface-displayed tau was not phosphorylated at sites found in AD patients (probed by antibodies AT8, AT270, AT180, and PHF-1). However, yeast-displayed tau showed clear binding to paired helical filament (PHF) tau conformation-specific antibodies Alz-50, MC-1, and Tau-2. Although the tau possessed a conformation found in PHFs, oligomerization or aggregation into larger filaments was undetected. Taken together, yeast-displayed tau enables robust measurement of protein interactions and is of particular interest for characterizing conformational change.     

Anthraquinones inhibit tau aggregation and dissolve Alzheimer's paired helical filaments in vitro and in cells

The abnormal aggregation of tau protein into paired helical filaments (PHFs) is one of the hallmarks of Alzheimer's disease. Aggregation takes place in the cytoplasm and could therefore be cytotoxic for neurons. To find inhibitors of PHF aggregation we screened a library of 200,000 compounds. The hits found in the PHF inhibition assay were also tested for their ability to dissolve preformed PHFs. The results were obtained using a thioflavin S fluorescence assay for the detection and quantification of tau aggregation in solution, a tryptophan fluorescence assay using tryptophan-containing mutants of tau, and confirmed by a pelleting assay and electron microscopy of the products. Here we demonstrate the feasibility of the approach with several compounds from the family of anthraquinones, including emodin, daunorubicin, adriamycin, and others. They were able to inhibit PHF formation with IC50 values of 1-5 microm and to disassemble preformed PHFs at DC50 values of 2-4 microm. The compounds had a similar activity for PHFs made from different tau isoforms and constructs. The compounds did not interfere with the stabilization of microtubules by tau. Tau-inducible neuroblastoma cells showed the formation of tau aggregates and concomitant cytotoxicity, which could be prevented by inhibitors. Thus, small molecule inhibitors could provide a basis for the development of tools for the treatment of tau pathology in AD and other tauopathies.     

Epitope mapping and structural basis for the recognition of phosphorylated tau by the anti‐tau antibody AT8

Microtubule‐associated protein tau becomes abnormally phosphorylated in Alzheimer's disease and other tauopathies and forms aggregates of paired helical filaments (PHF‐tau). AT8 is a PHF‐tau‐specific monoclonal antibody that is a commonly used marker of neuropathology because of its recognition of abnormally phosphorylated tau. Previous reports described the AT8 epitope to include pS202/pT205. Our studies support and extend previous findings by also identifying pS208 as part of the binding epitope. We characterized the phosphoepitope of AT8 through both peptide binding studies and costructures with phosphopeptides. From the cocrystal structure of AT8 Fab with the diphosphorylated (pS202/pT205) peptide, it appeared that an additional phosphorylation at S208 would also be accommodated by AT8. Phosphopeptide binding studies showed that AT8 bound to the triply phosphorylated tau peptide (pS202/pT205/pS208) 30‐fold stronger than to the pS202/pT205 peptide, supporting the role of pS208 in AT8 recognition. We also show that the binding kinetics of the triply phosphorylated peptide pS202/pT205/pS208 was remarkably similar to that of PHF‐tau. The costructure of AT8 Fab with a pS202/pT205/pS208 peptide shows that the interaction interface involves all six CDRs and tau residues 202–209. All three phosphorylation sites are recognized by AT8, with pT205 acting as the anchor. Crystallization of the Fab/peptide complex under acidic conditions shows that CDR‐L2 is prone to unfolding and precludes peptide binding, and may suggest a general instability in the antibody. Proteins 2016; 84:427–434. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Passive immunization targeting pathological phospho-tau protein in a mouse model reduces functional decline and clears tau aggregates from the brain

Targeting hyperphosphorylated tau by immunotherapy is emerging as a promising approach to treat tauopathies such as Alzheimer's disease and frontotemporal dementia. We have previously reported that active tau immunization clears tau aggregates from the brain and attenuates or prevents functional impairments in two different tangle mouse models. Here, we assessed the efficacy of passive immunization with the PHF1 antibody, which targets a phospho-epitope within one of our active immunogens. Homozygous female tangle mice (JNPL3, 2-3 months) were injected intraperitoneally once per week with PHF1 or pooled mouse IgG (250 μg/125 μL; n = 10 per group) for a total of 13 injections. Their behavior was assessed at 5-6 months of age and brain tissue was subsequently harvested for analyses of treatment efficacy. The treated mice performed better than controls on the traverse beam task (p < 0.03), and had 58% less tau pathology in the dentate gyrus of the hippocampus (p = 0.02). As assessed by western blots, the antibody therapy reduced the levels of insoluble pathological tau by 14-27% (PHF1, p < 0.05; PHF1/total tau, p < 0.0001) and 34-45% (CP13 or CP13/total tau, p < 0.05). Levels of soluble tau and sarkosyl soluble tau were unchanged, compared with controls, as well as total tau levels in all the fractions. Plasma levels of PHF1 correlated inversely with tau pathology in the brainstem (p < 0.01), with a strong trend in the motor cortex (p < 0.06) as well as with insoluble total tau levels (p < 0.02), indicating that higher dose of antibodies may have a greater therapeutic effect. Significant correlation was also observed between performance on the traverse beam task and PHF1 immunoreactivity in the dentate gyrus (p < 0.05) as well as with insoluble PHF1/total tau ratio on western blots (p < 0.04). These results show that passive immunization with tau antibodies can decrease tau pathology and functional impairments in the JNPL3 model. Future studies will determine the feasibility of this approach with other monoclonals and in different tangle models in which thorough cognitive assessment can be performed.     

Characterization of two VQIXXK motifs for tau fibrillization in vitro

Tau proteins are building blocks of the filaments that form neurofibrillary tangles of Alzheimer's disease (AD) and related neurodegenerative tauopathies. It was recently reported that two VQIXXK motifs in the microtubule (MT) binding region, named PHF6 and PHF6*, are responsible for tau fibrillization. However, the exact role each of these motifs plays in this process has not been analyzed in detail. Using a recombinant human tau fragment containing only the four MT-binding repeats (K18), we show that deletion of either PHF6 or PHF6* affected tau assembly but only PHF6 is essential for filament formation, suggesting a critical role of this motif. To determine the amino acid residues within PHF6 that are required for tau fibrillization, a series of deletion and mutation constructs targeting this motif were generated. Deletion of VQI in either PHF6 or PHF6* lessened but did not eliminate K18 fibrillization. However, removal of the single K311 residue from PHF6 completely abrogated the fibril formation of K18. K311D mutation of K18 inhibited tau filament formation, while K311A and K311R mutations had no effect. These data imply that charge change at position 311 is important in tau fibril formation. A similar requirement of nonnegative charge at this position for fibrillization was observed with the full-length human tau isoform (T40), and data from these studies indicate that the formation of fibrils by T40K311D and T40K311P mutants is repressed at the nucleation phase. These findings provide important insights into the mechanisms of tau fibrillization and suggest targets for AD drug discovery to ameliorate neurodegeneration mediated by filamentous tau pathologies.     

Effects of different anti-tau antibodies on tau fibrillogenesis: RTA-1 and RTA-2 counteract tau aggregation

Tau is the major antigenic component of neurofibrillary pathology in tauopathy, including Alzheimer's disease. Although conversion of soluble tau to an insoluble polymerized fibrillar form is a key factor in the pathogenesis of tauopathy, the mechanism of the change is unclear and no inhibitors of fibril formation are available. Monoclonal antibodies against the 1st or 2nd repeat of the microtubule binding domain, but not the C-terminal 16 residues, completely inhibited tau aggregation into PHF. Furthermore, they did not inhibit tau-induced tubulin assembly. Thus, they are useful to investigate tau protein conversion and will be useful therapeutic lead materials.     

Passive Immunization with Phospho-Tau Antibodies Reduces Tau Pathology and Functional Deficits in Two Distinct Mouse Tauopathy Models

In Alzheimer’s disease (AD), an extensive accumulation of extracellular amyloid plaques and intraneuronal tau tangles, along with neuronal loss, is evident in distinct brain regions. Staging of tau pathology by postmortem analysis of AD subjects suggests a sequence of initiation and subsequent spread of neurofibrillary tau tangles along defined brain anatomical pathways. Further, the severity of cognitive deficits correlates with the degree and extent of tau pathology. In this study, we demonstrate that phospho-tau (p-tau) antibodies, PHF6 and PHF13, can prevent the induction of tau pathology in primary neuron cultures. The impact of passive immunotherapy on the formation and spread of tau pathology, as well as functional deficits, was subsequently evaluated with these antibodies in two distinct transgenic mouse tauopathy models. The rTg4510 transgenic mouse is characterized by inducible over-expression of P301L mutant tau, and exhibits robust age-dependent brain tau pathology. Systemic treatment with PHF6 and PHF13 from 3 to 6 months of age led to a significant decline in brain and CSF p-tau levels. In a second model, injection of preformed tau fibrils (PFFs) comprised of recombinant tau protein encompassing the microtubule-repeat domains into the cortex and hippocampus of young P301S mutant tau over-expressing mice (PS19) led to robust tau pathology on the ipsilateral side with evidence of spread to distant sites, including the contralateral hippocampus and bilateral entorhinal cortex 4 weeks post-injection. Systemic treatment with PHF13 led to a significant decline in the spread of tau pathology in this model. The reduction in tau species after p-tau antibody treatment was associated with an improvement in novel-object recognition memory test in both models. These studies provide evidence supporting the use of tau immunotherapy as a potential treatment option for AD and other tauopathies.     

Oxidative stress induces transient O‐GlcNAc elevation and tau dephosphorylation in SH‐SY5Y cells

O‐linked β‐N‐acetlyglucosamine or O‐GlcNAc modification is a dynamic post‐translational modification occurring on the Ser/Thr residues of many intracellular proteins. The chronic imbalance between phosphorylation and O‐GlcNAc on tau protein is considered as one of the main hallmarks of Alzheimer's disease. In recent years, many studies also showed that O‐GlcNAc levels can elevate upon acute stress and suggested that this might facilitate cell survival. However, many consider chronic stress, including oxidative damage as a major risk factor in the development of the disease. In this study, using the neuronal cell line SH‐SY5Y we investigated the dynamic nature of O‐GlcNAc after treatment with 0.5 mM H₂O₂ for 30 min. to induce oxidative stress. We found that overall O‐GlcNAc quickly increased and reached peak level at around 2 hrs post‐stress, then returned to baseline levels after about 24 hrs. Interestingly, we also found that tau protein phosphorylation at site S262 showed parallel, whereas at S199 and PHF1 sites showed inverse dynamic to O‐Glycosylation. In conclusion, our results show that temporary elevation in O‐GlcNAc modification after H₂O₂‐induced oxidative stress is detectable in cells of neuronal origin. Furthermore, oxidative stress changes the dynamic balance between O‐GlcNAc and phosphorylation on tau proteins.     

A comparison of the neuronal dysfunction caused by <Emphasis Type="Italic">Drosophila</Emphasis> tau and human tau in a <Emphasis Type="Italic">Drosophila</Emphasis> model of tauopathies

Hyperphosphorylation and aggregation of tau into tangles is a feature of disorders such as Alzheimer’s disease and other Tauopathies. To model these disorders in Drosophila melanogaster, human tau has been over-expressed and a variety of phenotypes have been observed including neurotoxicity, disrupted neuronal and synaptic function and locomotor impairments. Neuronal dysfunction has been seen prior to neuronal death and in the absence of tangle formation. The Drosophila tau protein shares a large degree of homology with human tau but differs in the crucial microtubule binding domains. Although like human tau Drosophila tau can induce neurotoxicity, little is known about its ability to disrupt neuronal function. In this study we demonstrate that like human tau, over-expression of Drosophila tau results in disrupted axonal transport, altered neuromuscular junction morphology and locomotor impairments. This indicates that like human tau, over-expression of Drosophila tau compromises neuronal function despite significant differences in microtubule binding regions.