Microtubule-binding Region Of Tau Research Articles

E2814: an anti‐tau therapy engages its CNS target and affects the downstream tangle‐specific biomarker MTBR‐tau243 in Dominantly Inherited Alzheimer’s Disease

AbstractBackgroundE2814 is a human IgG1 antibody that binds to microtubule binding region of tau (MTBR‐tau), a critical domain for the seeding and spreading of tau pathology in Alzheimer’s Disease (AD). It is being developed for the treatment of AD by stopping tau propagation and slowing the associated cognitive decline. E2814 has been evaluated in healthy and Dominantly Inherited Alzheimer’s Disease (DIAD) participants for safety, pharmacokinetics (PK), and cerebrospinal fluid (CSF) target engagement.MethodSeventy‐two healthy participants were enrolled in single and multiple ascending dose cohorts of Study 001 (NCT04231513) and received randomized, double‐blind, placebo‐controlled treatment of E2814. Intravenous infusion of single and multiple doses (once every 4 weeks for three times) were administered. In Study 103 (NCT04971733), 7 DIAD participants with confirmed mutations and mild to moderate cognitive impairment were enrolled and received open‐label treatment of E2814. Four sequential doses were evaluated as intravenous infusion every 4 weeks. Serum and CSF PK, and CSF target engagement (by analyzing both bound and unbound MTBR‐tau299 and MTBR‐tau354 [containing the epitopes of E2814]) were measured in both healthy and DIAD participants, as well as safety assessments. CSF MTBR‐tau243 recapitulating AD tau pathology was also measured in DIAD participants using methods previously described (Horie et al., J Prev Alz Dis 2022).ResultE2814 was safe and well‐tolerated over a wide dose range. No infusion‐related reactions or other clinically significant safety signals were observed with single or repeated dose administration for up to 15 months. E2814 showed a dose‐related increase in serum and CSF exposure with CSF‐to‐serum ratio of ∼0.2%, comparable between healthy and DIAD participants. CSF target engagement was demonstrated by concentration‐related and sustained MTBR‐tau299 and MTBR‐tau354 binding in healthy and DIAD participants. More importantly, E2814 elicited a rapid and robust reduction on MTBR‐tau243, a neurofibrillary tangle‐specific biomarker, in DIAD participants.ConclusionE2814 demonstrated promising safety, PK and CSF target engagement profile in DIAD participants, and showed evidence of downstream effects on tangle‐specific biomarker MTBR‐tau243. Data supports further evaluation in the ongoing Ph2/3 DIAN‐TU Tau NexGen Platform Study in DIAD (NCT05269394) and future exploration in sporadic AD.

Change in Cerebrospinal Fluid Tau Microtubule Binding Region Detects Symptom Onset, Cognitive Decline, Tangles, and Atrophy in Dominantly Inherited Alzheimer's Disease.

Identifying cerebrospinal fluid measures of the microtubule binding region of tau (MTBR-tau) species that reflect tau aggregation could provide fluid biomarkers that track Alzheimer's disease related neurofibrillary tau pathological changes. We examined the cerebrospinal fluid (CSF) MTBR-tau species in dominantly inherited Alzheimer's disease (DIAD) mutation carriers to assess the association with Alzheimer's disease (AD) biomarkers and clinical symptoms. Cross-sectional and longitudinal CSF from 229 DIAD mutation carriers and 130 mutation non-carriers had sequential characterization of N-terminal/mid-domain phosphorylated tau (p-tau) followed by MTBR-tau species and tau positron emission tomography (tau PET), other soluble tau and amyloid biomarkers, comprehensive clinical and cognitive assessments, and brain magnetic resonance imaging of atrophy. CSF MTBR-tau species located within the putative "border" region and one species corresponding to the "core" region of aggregates in neurofibrillary tangles (NFTs) increased during the presymptomatic stage and decreased during the symptomatic stage. The "border" MTBR-tau species were associated with amyloid pathology and CSF p-tau; whereas the "core" MTBR-tau species were associated stronger with tau PET and CSF measures of neurodegeneration. The ratio of the border to the core species provided a continuous measure of increasing amounts that tracked clinical progression and NFTs. Changes in CSF soluble MTBR-tau species preceded the onset of dementia, tau tangle increase, and atrophy in DIAD. The ratio of 4R-specific MTBR-tau (border) to the NFT (core) MTBR-tau species corresponds to the pathology of NFTs in DIAD and change with disease progression. The dynamics between different MTBR-tau species in the CSF may serve as a marker of tau-related disease progression and target engagement of anti-tau therapeutics. ANN NEUROL 2023;93:1158-1172.

Comprehensive CSF Tau Profiling Identifies Soluble Tau Pathophysiological Stages in Dominantly Inherited Alzheimer Network (DIAN): Implications for the DIAN‐TU Tau Next Generation Platform

AbstractBackgroundBrain tau aggregation is a pathological hallmark of Alzheimer’s disease (AD) and the tau microtubule binding region (MTBR) is the core of AD tau tangles. Recently, we identified the enrichment of some specific MTBR species in sporadic AD brain tangles. Corresponding soluble MTBR species in the CSF increased together with clinical progression and tau pathology measured by tau‐PET. Because extracellular MTBR‐tau was discovered in human AD, MTBR‐tau has become a high priority target for antibodies binding this region. In this study, we sought to determine when these MTBR‐tau changes occurred and the relationship to clinical, cognitive and biomarker changes in the DIAN.MethodWe designed a mass spectrometry‐based method to quantify more than a dozen tau species in CSF. To profile MTBR‐tau, we conducted the immunoprecipitation using the anti‐MTBR antibody, E2814 which is bi‐epitopic to R2 and R4 regions and a tau drug in clinical trial. We analyzed CSF from 227 mutation carriers (MCs) (152 asymptomatic, 77 symptomatic) and 141 non‐carriers enrolled in the DIAN‐observational cohort.ResultCSF MTBR‐tau specifically increased in MCs and changed at different times over the disease course. The change of MTBR‐tau299 (residue 299‐317) in R2‐3 occurred at an estimated years to symptom onset (EYO) of −22, close to the first detection of change in p‐tau217 occupancy (EYO −21). The increase of MTBR‐tau354 (residue 354‐369) in R4 saturated in late clinical stages, potentially due to the deposition into brain tangles. Notably, the ratio of MTBR‐tau299/354 which expected to recapitulate tau pathophysiology highly correlated with p‐tau217 occupancy, an early amyloid marker, in especially asymptomatic MCs (r = 0.82). This indicates MTBR‐tau and p‐tau217 could be useful as biomarkers to assess anti‐MTBR therapies in tau‐PET negative populations.ConclusionWe identified a soluble measure of tau tangles, CSF MTBR species, with changes many years before symptom onset and continuing through symptomatic stages, suggesting early pre‐symptomatic prevention trials can be implemented with anti‐MTBR‐tau antibodies, such as E2814. These results have been utilized to design the tau next generation platform in the DIAN Trials Unit (DIAN‐TU) E2814 drug arm to target soluble MTBR‐tau in independent cohorts of asymptomatic and symptomatic mutation carriers.

Molecular crowding and RNA synergize to promote phase separation, microtubule interaction, and seeding of Tau condensates.

Biomolecular condensation of the neuronal microtubule‐associated protein Tau (MAPT) can be induced by coacervation with polyanions like RNA, or by molecular crowding. Tau condensates have been linked to both functional microtubule binding and pathological aggregation in neurodegenerative diseases. We find that molecular crowding and coacervation with RNA, two conditions likely coexisting in the cytosol, synergize to enable Tau condensation at physiological buffer conditions and to produce condensates with a strong affinity to charged surfaces. During condensate‐mediated microtubule polymerization, their synergy enhances bundling and spatial arrangement of microtubules. We further show that different Tau condensates efficiently induce pathological Tau aggregates in cells, including accumulations at the nuclear envelope that correlate with nucleocytoplasmic transport deficits. Fluorescent lifetime imaging reveals different molecular packing densities of Tau in cellular accumulations and a condensate‐like density for nuclear‐envelope Tau. These findings suggest that a complex interplay between interaction partners, post‐translational modifications, and molecular crowding regulates the formation and function of Tau condensates. Conditions leading to prolonged existence of Tau condensates may induce the formation of seeding‐competent Tau and lead to distinct cellular Tau accumulations.

CSF and blood plasma mass spectrometry measures of Aβ, tau, and NfL species and longitudinal relationship to preclinical and clinical staging of amyloid and tau aggregation and clinical stage of Alzheimer’s disease

AbstractBackgroundRecent advances in the development of novel Alzheimer’s disease (AD) measures of amyloid, tau, and neurodegeneration in cerebrospinal fluid (CSF) and blood have enabled a better understanding of the links between amyloid‐beta (Aβ), tau species and neurodegeneration in the brain, CSF, and blood. The discoveries of novel tau species in CSF and blood, including specific phospho‐tau (p‐tau) and truncated species containing the microtubule binding region of tau (MTBR‐tau) which aggregates in tau tangles, have greatly expanded our understanding of tau biology and tau target development. The longitudinal Aβ, tau, and neurofilament light chain (NfL) changes previously measured in CSF are now being measured accurately in blood, enabling interpretation and understanding of AD progression in much larger and more robust populations.MethodWe analyzed longitudinal CSF and/or blood plasma mass spectrometry measures of Aβ42/Aβ40, multiple p‐tau species (including p‐tau217, 231, 181, 205, etc.), total tau, MTBR‐tau (e.g., MTBR‐tau243, MTBR‐tau299, and MTBR‐tau354), and NfL in sporadic AD and dominantly inherited AD cohorts. Mass spectrometry results were compared to amyloid and tau aggregation measures by Positron Emission Tomography (PET) scans and clinical and cognitive measures in local and international clinical cohorts including ADNI, AIBL, BioFINDER, Mayo Clinic, the Knight ADRC, and the DIAN.ResultThe longitudinal results indicate that a pathophysiological cascade of events begin with altered CSF and blood plasma Aβ42/Aβ40 ratio, followed by increases in amyloid plaques as measured by amyloid PET, associated with increased phosphorylation of specific CSF tau species (e.g., p‐tau217, p‐tau181, etc.), before increases in p‐tau205, NfL, total tau concentrations, hypometabolism, and atrophy. Finally, some species of MTBR‐tau increase before or with tau aggregation by tau PET and onset of clinical symptoms and correlate with longitudinal tau aggregation and clinical progression.ConclusionThese findings indicate that CSF and blood plasma Aβ, p‐tau, MTBR‐tau and NfL measures are highly precise biomarkers of brain amyloidosis, tauopathy, and neurodegeneration and can accurately identify stages of preclinical and clinical AD.

The design of Dominantly Inherited Alzheimer Disease Trial of the anti‐tau antibody, E‐2814, on the DIAN‐TU Tau Next Generation Platform

AbstractBackgroundTau pathology is closely associated with the onset and progression of Alzheimer’s disease. Recent advances in drugs that target tau suggest the potential to prevent or attenuate the pathophysiology and pathology of tau. Novel CSF and blood tau species including specific phospho‐tau (p‐tau) and truncated species containing the microtubule binding region of tau (MTBR‐tau) which aggregates in tau tangles, increases 10 years or more before tau tangles are detected by tau‐PET. Two stages of AD are identified and studied using soluble p‐tau and MTBR‐tau forms in the asymptomatic stage and Tau PET in the post‐tangle symptomatic stage. E2814, an anti‐tau monoclonal antibody targets MTBR‐tau and decreases tau pathology in animal models.MethodWe designed an anti‐tau trial to target soluble tau species in two cohorts including pre‐tangle asymptomatic and post‐tangle symptomatic participants in dominantly inherited AD (DIAD). We analyzed longitudinal clinical, cognitive, tau PET and CSF measures of tau species including, multiple p‐tau species (e.g., p‐tau217, 231, 181, 205), total tau, MTBR‐tau (e.g., MTBR‐tau243, MTBR‐tau299, and MTBR‐tau354), and NfL in DIAD cohorts from DIAN and DIAN‐TU. Novel trial design and statistical approaches have been developed to address primary and secondary outcomes, magnitude of effect by stage of disease (pre‐tangle vs. post‐tangle), and potential for prevention.ResultWe designed a trial of two cohorts, each powered, with different biomarker outcomes: in the pre‐tangle, tau‐PET negative cohort, CSF ptau217/tau217 is the endpoint; for the post‐tangle tau‐PET positive cohort, tau‐PET is the endpoint. Further, each cohort will measure target engagement by CSF MTBR‐tau and downstream effects on soluble total tau, p‐tau and MTBR tau with common measures of neurodegeneration by CSF and blood NfL, atrophy by MRI, hypometabolism by FDG‐PET, effects on amyloid by abeta42/40 and amyloid‐PET, and cognitive and clinical measures. This design is facilitated by the predictable pattern of biomarker changes observed in DIAD allowing appropriate powering of the study even in this rare condition.ConclusionThe Tau Next Generation Platform will address disease progression, before and after tangle formation begins, assessing when an anti‐MTBR tau monoclonal antibody will have the largest effects. It is the first anti‐tau trial for DIAD.

Facile Installation of Post-translational Modifications on the Tau Protein via Chemical Mutagenesis.

Post-translational modifications of proteins are ubiquitous in living organisms, as they enable an accurate control of the interactions of these macromolecules. For mechanistic studies, it would be highly advantageous to be able to produce in vitro post-translationally modified proteins with site-specificity. Here, we demonstrate one facile way to achieve this goal through the use of post-translational chemical mutagenesis. We illustrate this approach by performing site-specific phosphorylation and methylation of tau, a protein that stabilizes microtubules and whose aggregation is closely linked with Alzheimer's disease. We then verify the effects of the post-translational modifications on the ability of tau to control microtubule polymerization, revealing in particular an unexpected role for phosphorylation at S199, which is outside the microtubule-binding region of tau. These results show how the chemical mutagenesis approach that we present enables the systematic analysis of site-specific post-translational modifications of a key protein involved in the pathogenesis of Alzheimer's disease.

Tau microtubule binding region in cerebrospinal fluid as a biomarker to differentiate tau pathology in Alzheimer’s disease

AbstractBackgroundTau deposition in brain is a pathological hallmark of some neurodegenerative disorders including Alzheimer’s disease (AD). Braak staging of post‐mortem tissue suggests pathological tau spreads through the brain via synaptically connected pathways, and specific tau isoforms containing the microtubule binding region (MTBR) are thought to be essential to form extracellular seeds that propagate pathology. Tau is cleaved at the mid‐domain and the N‐ter to mid‐domain species are actively secreted extracellularly, whereas MTBR‐tau show faster turn‐over and deposition in brain. Recently, we identified the MTBR regions (residues 299‐317 (HVPG) and 354‐369 (IGSL)) and the nearby region (residue 243‐254 (LQTA)) were specifically enriched in AD brain aggregates. However, if and how these species change in extracellular spaces under pathophysiogical conditions were not known. This work describes the behavior of the pathological species in extracellular spaces by analyzing human cerebrospinal fluid (CSF) with changes of disease progression.MethodWe established a method to quantify MTBR‐tau in CSF, consisting of chemical extraction followed by tryptic digestion and mass spectrometry analysis. This method was applied to the CSF analysis of AD participants (n=50 amyloid negative with CDR=0‐1, n=50 amyloid positive with CDR=0‐2). A subset of these participants was followed longitudinally (2‐9 years).ResultOur method revealed that (1) MTBR‐tau were specifically increased in amyloid positive group and HVPG was the most significantly increased even in clinically asymptomatic stages, (2) the increases of HVPG and IGSL were saturated after symptomatic onset, whereas LQTA continued to increase even after the clinical onset, and (3) the concentration of LQTA showed the highest correlation with tau pathology as measured by positron emission tomography (PET) for tau (Pearson r=0.84, n=35) as well as cognitive testing measures.ConclusionMTBR‐tau enriched in AD brain aggregates are increased in AD CSF. These findings suggest HVPG and LQTA in CSF could be promising biomarkers to recapitulate amyloid status and tau pathology in AD, respectively. Notably, only LQTA showed the continuous increase along to disease progression in terms of tau‐PET as well as amyloid status and cognitive decline, which suggests the region is key to differentiate tau pathology in AD.

Pre-clinical characterisation of E2814, a high-affinity antibody targeting the microtubule-binding repeat domain of tau for passive immunotherapy in Alzheimer\u2019s disease

Tau deposition in the brain is a pathological hallmark of many neurodegenerative disorders, including Alzheimer’s disease (AD). During the course of these tauopathies, tau spreads throughout the brain via synaptically-connected pathways. Such propagation of pathology is thought to be mediated by tau species (“seeds”) containing the microtubule binding region (MTBR) composed of either three repeat (3R) or four repeat (4R) isoforms. The tau MTBR also forms the core of the neuropathological filaments identified in AD brain and other tauopathies. Multiple approaches are being taken to limit tau pathology, including immunotherapy with anti-tau antibodies. Given its key structural role within fibrils, specifically targetting the MTBR with a therapeutic antibody to inhibit tau seeding and aggregation may be a promising strategy to provide disease-modifying treatment for AD and other tauopathies. Therefore, a monoclonal antibody generating campaign was initiated with focus on the MTBR. Herein we describe the pre-clinical generation and characterisation of E2814, a humanised, high affinity, IgG1 antibody recognising the tau MTBR. E2814 and its murine precursor, 7G6, as revealed by epitope mapping, are antibodies bi-epitopic for 4R and mono-epitopic for 3R tau isoforms because they bind to sequence motif HVPGG. Functionally, both antibodies inhibited tau aggregation in vitro. They also immunodepleted a variety of MTBR-containing tau protein species. In an in vivo model of tau seeding and transmission, attenuation of deposition of sarkosyl-insoluble tau in brain could also be observed in response to antibody treatment. In AD brain, E2814 bound different types of tau filaments as shown by immunogold labelling and recognised pathological tau structures by immunohistochemical staining. Tau fragments containing HVPGG epitopes were also found to be elevated in AD brain compared to PSP or control. Taken together, the data reported here have led to E2814 being proposed for clinical development.

Independent tubulin binding and polymerization by the proline-rich region of Tau is regulated by Tau's N-terminal domain

Tau is an intrinsically disordered, microtubule-associated protein that has a role in regulating microtubule dynamics. Despite intensive research, the molecular mechanisms of Tau-mediated microtubule polymerization are poorly understood. Here we used single-molecule fluorescence to investigate the role of Tau's N-terminal domain (NTD) and proline-rich region (PRR) in regulating interactions of Tau with soluble tubulin. We assayed both full-length Tau isoforms and truncated variants for their ability to bind soluble tubulin and stimulate microtubule polymerization. We found that Tau's PRR is an independent tubulin-binding domain that has tubulin polymerization capacity. In contrast to the relatively weak interactions with tubulin mediated by sites distributed throughout Tau's microtubule-binding region (MTBR), resulting in heterogeneous Tau: tubulin complexes, the PRR bound tubulin tightly and stoichiometrically. Moreover, we demonstrate that interactions between the PRR and MTBR are reduced by the NTD through a conserved conformational ensemble. On the basis of these results, we propose that Tau's PRR can serve as a core tubulin-binding domain, whereas the MTBR enhances polymerization capacity by increasing the local tubulin concentration. Moreover, the NTD appears to negatively regulate tubulin-binding interactions of both of these domains. The findings of our study draw attention to a central role of the PRR in Tau function and provide mechanistic insight into Tau-mediated polymerization of tubulin.

Tau's Proline Rich Region Dominates Tubulin Binding

Tau is an intrinsically disordered, microtubule associated protein best known for its role neurodegenerative tauopathies such as Alzheimer's disease. Tau's physiological roles include stabilizing microtubules and regulating microtubule dynamic instability. Considerable attention has been given to tau's microtubule binding region (MTBR), and more recently to its MTBR-flanking R’ region, and their role in tau binding to both microtubules and soluble tubulin. N-terminally flanking the MTBR is the proline rich region (PRR), which is widely accepted as critical for tau-mediated polymerization of microtubules and also cited as demonstrating weak microtubule binding. Here, we investigate the role of the PRR in tau binding to soluble tubulin. We find that the PRR dominates binding of tau to soluble tubulin. The PRR has two equivalent binding sites for soluble tubulin dimers, and surprisingly, competes with the MTBR-R’ for binding to soluble tubulin suggesting at least partial overlap of the binding sites in these separate regions. In the absence of the MTBR, PRR is capable of binding microtubules and polymerizing tubulin, albeit weakly. Collectively, our data suggests the PRR region is an important binding motif for the soluble tubulin pool. Interestingly, this domain is not visible in the recent cryo-EM structure of microtubule-bound tau. Future work will focus on describing the relationship between PRR and MTBR-R’ on the microtubule.

Native Top-Down Mass Spectrometry and Ion Mobility Spectrometry of the Interaction of Tau Protein with a Molecular Tweezer Assembly Modulator.

Native top-down mass spectrometry (MS) and ion mobility spectrometry (IMS)were applied to characterize the interaction of a molecular tweezer assembly modulator, CLR01, with tau, a protein believed to be involved in a number of neurodegenerative disorders, including Alzheimer's disease. The tweezer CLR01 has been shown to inhibit aggregation of amyloidogenic polypeptides without toxic side effects. ESI-MS spectra for different forms of tau protein (full-length, fragments, phosphorylated, etc.) in the presence of CLR01 indicate a primary binding stoichiometry of 1:1. The relatively high charging of the protein measured from non-denaturing solutions is typical of intrinsically disordered proteins, such as tau. Top-down mass spectrometry using electron capture dissociation (ECD) is a tool usedto determine not only the sites of post-translational modifications but also the binding site(s) of non-covalent interacting ligands to biomolecules. The intact protein and the protein-modulator complex were subjected to ECD-MS to obtain sequence information, map phosphorylation sites, and pinpoint the sites of inhibitor binding. TheESI-MSstudy of intact tau proteins indicates that top-down MS is amenable to the study of various tau isoforms and their post-translational modifications (PTMs). The ECD-MS data point to a CLR01 binding site in the microtubule-binding region of tau, spanning residues K294-K331, which includes a six-residue nucleating segment PHF6 (VQIVYK) implicated in aggregation. Furthermore, ion mobility experiments on the tau fragmentin the presence of CLR01 and phosphorylated taureveal a shift towards a more compact structure. The mass spectrometry study suggests a picture for the molecular mechanism ofthe modulation of protein-protein interactions in tau by CLR01. Graphical Abstract ᅟ.

InP Quantum Dots: Probing the Active Domain of Tau Peptide Using Energy Transfer

Aggregation of Tau, a natively unfolded protein, is responsible for tauopathies, a class of neurodegenerative disorders. An active peptide sequence containing 20 amino acids is selected from the Tau microtubule binding region, which includes the essential V306-K311 residue, to monitor the structural change that initiates aggregation at very low concentrations. The synthesis of a peptide sequence is accomplished by employing solid-phase protocols. The active domain of Tau possesses an amino functionality on lysine and free thiol on cysteine. The former end is selectively labeled to rhodamine 101 which is further bound to the InP/ZnS quantum dot surface through the thiol linkage. Efficient resonance energy transfer is observed in its unfolded conformation which is confirmed using various steady state fluorescence techniques. The average distance between the quantum dot core and the chromophore is probed by Förster resonance energy transfer (FRET) as 24.5 ± 0.8 Å. Heparin, a negatively charged glycosaminoglycan, is used for inducing aggregation of the active domain of the Tau peptide. Structural changes in the peptide monomer, on addition of heparin, could be monitored at nanomolar concentrations through the inhibition of energy transfer from quantum dots to rhodamine dye.

AADvac1, an Active Immunotherapy for Alzheimer's Disease and Non Alzheimer Tauopathies: An Overview of Preclinical and Clinical Development.

Neurofibrillary tau protein pathology is closely associated with the progression and phenotype of cognitive decline in Alzheimer's disease and other tauopathies, and a high-priority target for disease-modifying therapies. Herein, we provide an overview of the development of AADvac1, an active immunotherapy against tau pathology, and tau epitopes that are potential targets for immunotherapy. The vaccine leads to the production of antibodies that target conformational epitopes in the microtubule-binding region of tau, with the aim to prevent tau aggregation and spreading of pathology, and promote tau clearance. The therapeutic potential of the vaccine was evaluated in transgenic rats and mice expressing truncated, non mutant tau protein, which faithfully replicate of human tau pathology. Treatment with AADvac1 resulted in reduction of neurofibrillary pathology and insoluble tau in their brains, and amelioration of their deleterious phenotype. The vaccine was highly immunogenic in humans, inducing production of IgG antibodies against the tau peptide in 29/30 treated elderly patients with mild-to-moderate Alzheimer's. These antibodies were able to recognise insoluble tau proteins in Alzheimer patients' brains. Treatment with AADvac1 proved to be remarkably safe, with injection site reactions being the only adverse event tied to treatment. AADvac1 is currently being investigated in a phase 2 study in Alzheimer's disease, and a phase 1 study in non-fluent primary progressive aphasia, a neurodegenerative disorder with a high tau pathology component.

Curcumin Inhibits Tau Aggregation and Disintegrates Preformed Tau Filaments in vitro.

The pathological aggregation of tau is a common feature of most of the neuronal disorders including frontotemporal dementia, Parkinson's disease, and Alzheimer's disease. The inhibition of tau aggregation is considered to be one of the important strategies for treating these neurodegenerative diseases. Curcumin, a natural polyphenolic molecule, has been reported to have neuroprotective ability. In this work, curcumin was found to bind to adult tau and fetal tau with a dissociation constant of 3.3±0.4 and 8±1 μM, respectively. Molecular docking studies indicated a putative binding site of curcumin in the microtubule-binding region of tau. Using several complementary techniques, including dynamic light scattering, thioflavin S fluorescence, 90° light scattering, electron microscopy, and atomic force microscopy, curcumin was found to inhibit the aggregation of tau. The dynamic light scattering analysis and atomic force microscopic images revealed that curcumin inhibits the oligomerization of tau. Curcumin also disintegrated preformed tau oligomers. Using Far-UV circular dichroism, curcumin was found to inhibit the β-sheets formation in tau indicating that curcumin inhibits an initial step of tau aggregation. In addition, curcumin inhibited tau fibril formation. Furthermore, the effect of curcumin on the preformed tau filaments was analyzed by atomic force microscopy, transmission electron microscopy, and 90° light scattering. Curcumin treatment disintegrated preformed tau filaments. The results indicated that curcumin inhibited the oligomerization of tau and could disaggregate tau filaments.

A 31-residue peptide induces aggregation of tau's microtubule-binding region in cells.

The self-propagation of misfolded conformations of tau underlies neurodegenerative diseases, including Alzheimer's. There is considerable interest in discovering the minimal sequence and active conformational nucleus that defines this self-propagating event. The microtubule-binding region, spanning residues 244-372, reproduces much of the aggregation behaviour of tau in cells and animal models. Further dissection of the amyloid-forming region to a hexapeptide from the third microtubule-binding repeat resulted in a peptide that rapidly forms fibrils in vitro. We show that this peptide lacks the ability to seed aggregation of tau244-372 in cells. However, as the hexapeptide is gradually extended to 31 residues, the peptides aggregate more slowly and gain potent activity to induce aggregation of tau244-372 in cells. X-ray fibre diffraction, hydrogen-deuterium exchange and solid-state NMR studies map the beta-forming region to a 25-residue sequence. Thus, the nucleus for self-propagating aggregation of tau244-372 in cells is packaged in a remarkably small peptide.

Exploring the Functional and Structural Impact of Disease-Associated Mutants of Tau

Tau is an intrinsically disordered microtubule associated protein, whose pathological self-association is linked to several tauopathies, including Alzheimer's disease. A number of lines of evidence support tau aggregation as well as loss of its native interactions with microtubules (MTs) as contributing to pathology. Prior work from our lab showed that both disease-relevant as well as designed point mutations to tau's microtubule binding region (MTBR) enhance binding of tau to soluble tubulin. This observation indicates the mechanism of MT destabilization in disease may arise, in part, from an altered distribution of tubulin- and microtubule-bound tau. Here we determine whether the increase in affinity observed previously generally holds for disease-relevant tau mutants. Specifically, we characterize variants within all four repeats in the MTBR and at both N- (R5L and R5H) and C-termini (R406W). To investigate this, we used fluorescence correlation spectroscopy (FCS) to quantify the binding of tau variants to soluble tubulin. Changes in the affinity and function were correlated with the topological features of each tau mutant in solution or upon interaction with soluble tubulin by using single-molecule FRET. Together, our work allows to identify domain/region specific conformational changes in tau relevant to tauopathies and to explore the effect on its intrinsically disordered features. This provides a structural basis for understanding the impact of tau disease mutations in the tau-tubulin complex.

Rational Methods to Pharmacologically Target IDPs: Developing Modulators of Tau Aggregation

Despite a large amount of time and effort invested, the development of drugs targeting intrinsically disordered proteins (IDPs) is a major open problem in pharmacology. Classic drug development strategies are impeded by the highly dynamic nature of IDPs. We propose a strategy to identify inhibitors of the pathological aggregation of the IDP tau, implicated in Alzheimer's disease and other neurodegenerative disorders. We approach this problem by utilizing computational approaches and in vitro experiments to develop new modulators of tau aggregation as discussed below.Initially, computational simulations were used to generate conformational ensembles of tau244-372, the aggregation-prone microtubule binding region of tau. For this purpose, conventional molecular dynamics (MD) simulations were compared against enhanced sampling methods, including a recently developed novel approach based on repeated simulated annealing (ReSA).Preferentially sampled conformations within these dynamic ensembles were identified based on local and global structural similarity. To do this, the structures were grouped together based on root mean standard deviations (RMSD) between conformations and representative structures then generated from these groups. Subsequent computational screening identified selective ligands of these preferentially sampled structures by creating receptor force-fields from the representative structures and scoring potential ligand affinity based on steric, electrostatic and hydrophobic properties.Finally, computational hits were validated using a variety of biophysical methods including fibril-formation assays and single-molecule fluorescence approaches such as fluorescence correlation spectroscopy.This strategy has the potential to enhance our ability to rationally target IDPs for pharmacological intervention and may prove valuable in tackling other IDP-related pathologies such as Parkinson's disease, Diabetes, and Huntington's disease.

Immunotherapy targeting Alzheimer's phospho-tau epitope within the microtubule binding region of tau clears pathological tau and prevents functional decline in a mouse model of tauopathy

Immunotherapy targeting Alzheimer's phospho-tau epitope within the microtubule binding region of tau clears pathological tau and prevents functional decline in a mouse model of tauopathy

Structural insights into Alzheimer filament assembly pathways based on site‐directed mutagenesis and S‐glutathionylation of three‐repeat neuronal Tau protein

Although Tau and MAP2 readily assemble into straight filaments (SFs), Tau's unique ability to form paired-helical filaments (PHFs) may offer clues as to why Tau's microtubule-binding region (MTBR) is the exclusive building block of the neurofibrillary tangles that accumulate during Alzheimer's disease. To learn more about the factors permitting Tau to form both SFs and PHFs, we investigated the microtubule binding, thiol oxidation, and polymerization reactions of the monomer and dimer forms of Tau and MAP2 MTBRs. This review focuses on electron microscopic evidence (1) that facilitated the identification of amino acid residues within 3-repeat Tau that promote PHF formation; and (2) provided experimental evidence for the polymerization of S-glutathionylated three-repeat Tau, a reaction that unambiguously demonstrates that disulfide-linked Tau-S-S-Tau dimer formation is not a compulsory step in filament assembly. We also consider these findings within the context of current views on the genetic and biochemical basis of Tau fibrillogenesis.