Repeat Domain Of Tau Research Articles

Astaxanthin inhibits apoptosis in a cell model of tauopathy by attenuating endoplasmic reticulum stress and unfolded protein response

The accumulation of misfolded proteins is a common pathological characteristic shared by many neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease. The disruption of proteostasis triggers endoplasmic reticulum (ER) stress, during which the unfolded protein response (UPR) is initiated by the activation of protein kinase R-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6). These three branches of UPR signals act in concert to reduce the levels of abnormal proteins and restore ER homeostasis. However, the overactivation of UPR impairs cell function and induces apoptosis, which has been implicated in neurodegeneration. Astaxanthin is a xanthophyll carotenoid which has been shown to have neuroprotective effects in both cell and animal models; however, its effects on ER stress and UPR induced by disrupted proteostasis remain unclear. In this study, the effects of astaxanthin on ER stress and cytotoxicity were investigated in N2a cells stably expressing the pro-aggregant tau repeat domain carrying FTDP-17 mutation ΔK280 (Tau4RDΔK280). The results demonstrated that astaxanthin significantly inhibited Tau4RDΔK280-induced loss of cell viability and apoptosis, attenuating Tau4RDΔK280-induced caspase-3 activation and decrease of Bcl-2. Further studies revealed that astaxanthin treatment alleviated Tau4RDΔK280-induced ER stress and suppressed the activation of PERK, IRE1 and ATF6 signaling pathways. These findings suggested that astaxanthin might inhibit Tau4RDΔK280-induced cytotoxicity by attenuating UPR and ER stress. In addition, astaxanthin treatment resulted in a great reduction in the production of intracellular reactive oxygen species and a significant decrease in calcium influx induced by Tau4RDΔK280, which also contributed to the protective effects of astaxanthin against Tau4RDΔK280-induced cytotoxicity.

Development of an anti-tauopathy mucosal vaccine specifically targeting pathologic conformers

Alzheimer’s disease (AD) and related tauopathies are associated with pathological tau protein aggregation, which plays an important role in neurofibrillary degeneration and dementia. Targeted immunotherapy to eliminate pathological tau aggregates is known to improve cognitive deficits in AD animal models. The tau repeat domain (TauRD) plays a pivotal role in tau-microtubule interactions and is critically involved in the aggregation of hyperphosphorylated tau proteins. Because TauRD forms the structural core of tau aggregates, the development of immunotherapies that selectively target TauRD-induced pathological aggregates holds great promise for the modulation of tauopathies. In this study, we generated recombinant TauRD polypeptide that form neurofibrillary tangle-like structures and evaluated TauRD-specific immune responses following intranasal immunization in combination with the mucosal adjuvant FlaB. In BALB/C mice, repeated immunizations at one-week intervals induced robust TauRD-specific antibody responses in a TLR5-dependent manner. Notably, the resulting antiserum recognized only the aggregated form of TauRD, while ignoring monomeric TauRD. The antiserum effectively inhibited TauRD filament formation and promoted the phagocytic degradation of TauRD aggregate fragments by microglia. The antiserum also specifically recognized pathological tau conformers in the human AD brain. Based on these results, we engineered a built-in flagellin-adjuvanted TauRD (FlaB-TauRD) vaccine and tested its efficacy in a P301S transgenic mouse model. Mucosal immunization with FlaB-TauRD improved quality of life, as indicated by the amelioration of memory deficits, and alleviated tauopathy progression. Notably, the survival of the vaccinated mice was dramatically extended. In conclusion, we developed a mucosal vaccine that exclusively targets pathological tau conformers and prevents disease progression.

Stable ubiquitin conjugation for biological interrogation of ubiquitinated tau repeat domain

Protein semisynthesis approaches are key for gaining insights into the effects of post-translational modifications (PTMs) on the structure and function of modified proteins. Among PTMs, ubiquitination involves the conjugation of a small protein modifier to a substrate amino acid residue and is unique in controlling a variety of cellular processes. Interest has grown in understanding the role of ubiquitination in neurodegenerative conditions, including tauopathies. The latter are characterized by the accumulation of the intrinsically disordered protein tau in the form of neurofibrillary tangles in the brains of patients. The presence of ubiquitinated tau in the pathological aggregates suggests that ubiquitination might play a role in the formation of abnormal protein deposits. In this study, we developed a new strategy, based on dehydroalanine chemistry, to install wild type ubiquitin on a tau repeat domain construct with site-specificity. We optimized a three-step reaction which yielded a good amount of highly pure tau repeat domain ubiquitinated in position 353. The structural features of the conjugate were examined by circular dichroism and NMR spectroscopy. The ubiquitinated tau was challenged in a number of assays: fibrils formation under aggregating conditions in vitro, chemical stability upon exposure to a variety of biological media including cell extracts, and internalization into astrocytes. The results demonstrated the wide applicability of the new semisynthetic strategy for the investigation of ubiquitinated substrates in vitro or in cell, and in particular for studying if ubiquitination has a role in the molecular mechanisms that underlie the aberrant transition of tau into pathological aggregates.

VCP regulates early tau seed amplification via specific cofactors.

Neurodegenerative tauopathies may progress based on seeding by pathological tau assemblies, whereby an aggregate is released from one cell, gains entry to an adjacent or connected cell, and serves as a specific template for its own replication in the cytoplasm. In vitro seeding reactions typically take days, yet seeding into the complex cytoplasmic milieu happens within hours, implicating a machinery with unknown players that controls this process in the acute phase. We used proximity labeling to identify factors that control seed amplification within 5h of seed exposure. We fused split-APEX2 to the C-terminus of tau repeat domain (RD) to reconstitute peroxidase activity 5h after seeded intracellular tau aggregation. Valosin containing protein (VCP/p97) was the top hit. VCP harbors dominant mutations that underlie two neurodegenerative diseases, multisystem proteinopathy and vacuolar tauopathy, but its mechanistic role is unclear. We used immortalized cells and human neurons to study the effects of VCP on tau seeding. We exposed cells to fibrils or brain homogenates in cell culture media and measured effects on uptake and induction of intracellular tau aggregation following various genetic and chemical manipulations of VCP. VCP knockdown reduced tau seeding. Chemical inhibitors had opposing effects on aggregation in HEK293T tau biosensor cells and human neurons alike: ML-240 increased seeding efficiency, whereas NMS-873 decreased it. The inhibitors were effective only when administered within 8h of seed exposure, indicating a role for VCP early in seed processing. We screened 30 VCP co-factors in HEK293T biosensor cells by genetic knockout or knockdown. Reduction of ATXN3, NSFL1C, UBE4B, NGLY1, and OTUB1 decreased tau seeding, as did NPLOC4, which also uniquely increased soluble tau levels. By contrast, reduction of FAF2 increased tau seeding. Divergent effects on tau seeding of chemical inhibitors and cofactor reduction indicate that VCP regulates this process. This is consistent with a dedicated cytoplasmic processing complex based on VCP that directs seeds acutely towards degradation vs. amplification.

Hairpin trimer transition state of amyloid fibril

Protein fibril self-assembly is a universal transition implicated in neurodegenerative diseases. Although fibril structure/growth are well characterized, fibril nucleation is poorly understood. Here, we use a computational-experimental approach to resolve fibril nucleation. We show that monomer hairpin content quantified from molecular dynamics simulations is predictive of experimental fibril formation kinetics across a tau motif mutant library. Hairpin trimers are predicted to be fibril transition states; one hairpin spontaneously converts into the cross-beta conformation, templating subsequent fibril growth. We designed a disulfide-linked dimer mimicking the transition state that catalyzes fibril formation, measured by ThT fluorescence and TEM, of wild-type motif - which does not normally fibrillize. A dimer compatible with extended conformations but not the transition-state fails to nucleate fibril at any concentration. Tau repeat domain simulations show how long-range interactions sequester this motif in a mutation-dependent manner. This work implies that different fibril morphologies could arise from disease-dependent hairpin seeding from different loci.

Interaction of Tau with G-Protein-Coupled Purinergic P2Y12 Receptor by Molecular Docking and Molecular Dynamic Simulation.

Alzheimer's disease, a progressive neurological disorder, is characterized by the accumulation of neurofibrillary tangles and senile plaques by Tau and amyloid-β, respectively, in the brain microenvironment. The misfolded protein aggregates interact with several components of neuronal and glial cells such as membrane lipids, receptors, transporters, enzymes, cytoskeletal proteins, etc. Under pathological conditions, Tau interacts with several G-protein-coupled receptors (GPCRs), which undergoes either receptor signaling or desensitization followed by internalization of the protein complex. The purinergic GPCR, P2Y12 which is expressed in microglial cells, plays a key role in its activation and migration. Microglial cells sense and migrate to the site of injury aided by P2Y12 receptor that interacts with ADP released from damaged cells. P2Y12 receptor also interacts with misfolded Tau accumulated at the extracellular space and promotes receptor-mediated internalization. Immunocolocalization and co-immunoprecipitation studies demonstrated the interaction of Tau species with the P2Y12 receptor. Later, in-silico analyses were carried out with the repeat domain of Tau (TauRD), which has been identified as the interacting partner of P2Y12 receptor by in-vitro studies. Molecular docking and molecular dynamics simulation studies show the stability and the type of interaction in TauRD-receptor complex. Tau interaction with P2Y12 receptor plays a significant role in maintaining the active state of microglia which could lead to neuroinflammation and neuronal damage in AD brain. Hence, blocking P2Y12-Tau interaction and P2Y12-mediated Tau internalization in microglial cells could be possible therapeutic strategies in downregulating the severity of neuroinflammation in AD.

Atomic insights into the inhibition of R3 domain of tau protein by epigallocatechin gallate, quercetin and gallic acid

Inhibiting tau protein aggregation has become a prospective avenue for the therapeutic development of tauopathies. The third microtubule-binding repeat (R3) domain of tau is confirmed as the most aggregation-favorable fragment of the whole protein. As dimerization is the first step of the aggregation of tau into amyloid fibrils, impeding the dimerization of the R3 domain is critical to prevent the full-length tau aggregation. Natural polyphenol small molecules epigallocatechin gallate (EGCG), quercetin (QE) and gallic acid (GA) are proven to inhibit the aggregation of the full-length recombinant tau (For EGCG and QE) or the R3 domain (For GA) of tau in vitro. However, the underlying molecular mechanisms of the inhibitive effects on the R3 domain of tau remain largely unknown. In this study, we conducted numerous all-atom molecular dynamics simulations on R3 dimers with and without EGCG, QE or GA, respectively. The results reveal that all three molecules can effectively decrease the β structure composition of the R3 dimer, induce the dimer to adopt loosely-packed conformations, and weaken interchain interactions, thus impeding the dimerization of the R3 peptide chains. The specific preferentially binding sites for the three molecules exhibit similarities and differences. Hydrophobic, π-π stacking and hydrogen-bonding interactions collectively drive EGCG, QE and GA respectively binding on the R3 dimer, while QE also binds with the dimer through cation-π interaction. Given the incurable nature of tauopathies hitherto, our research provides helpful knowledge for the development of drugs to treat tauopathies.

Frontotemporal Dementia P301L Mutation Potentiates but Is Not Sufficient to Cause the Formation of Cytotoxic Fibrils of Tau.

The P301L mutation in tau protein is a prevalent pathogenic mutation associated with neurodegenerative frontotemporal dementia, FTD. The mechanism by which P301L triggers or facilitates neurodegeneration at the molecular level remains unclear. In this work, we examined the effect of the P301L mutation on the biochemical and biological characteristics of pathologically relevant hyperphosphorylated tau. Hyperphosphorylated P301L tau forms cytotoxic aggregates more efficiently than hyperphosphorylated wildtype tau or unphosphorylated P301L tau in vitro. Mechanistic studies establish that hyperphosphorylated P301L tau exacerbates endoplasmic reticulum (ER) stress-associated gene upregulation in a neuroblastoma cell line when compared to wildtype hyperphosphorylated tau treatment. Furthermore, the microtubule cytoskeleton is severely disrupted following hyperphosphorylated P301L tau treatment. A hyperphosphorylated tau aggregation inhibitor, apomorphine, also inhibits the harmful effects caused by P301L hyperphosphorylated tau. In short, the P301L single mutation within the core repeat domain of tau renders the underlying hyperphosphorylated tau more potent in eliciting ER stress and cytoskeleton damage. However, the P301L mutation alone, without hyperphosphorylation, is not sufficient to cause these phenotypes. Understanding the conditions and mechanisms whereby selective mutations aggravate the pathogenic activities of tau can provide pivotal clues on novel strategies for drug development for frontotemporal dementia and other related neurodegenerative tauopathies, including Alzheimer's disease.

Hematoxylin modulates tau-RD protein fibrillization and ameliorates Alzheimer's disease-like symptoms in a yeast model

Alzheimer's disease (AD) is one of the most serious neurodegenerative diseases with no effective treatment options available. The formation of insoluble amyloid fibrils of the hyperphosphorylated tau protein is intimately associated with AD, hence the tau protein has been a key target for AD drug development. In this work, hematoxylin was discovered as a dual functional compound, that is, acting in the inhibition of repeat domain of tau (tau-RD) protein fibrillogenesis and remodeling of pre-formed tau-RD fibrils in vitro. Meanwhile, hematoxylin was able to reduce the accumulation of tau-RD aggregates in Saccharomyces cerevisiae. Experimental and computational studies indicated that hematoxylin directly interacts with tau-RD protein through hydrophobic forces, hydrogen bonds, π-cation interactions, and π-π stackings. In addition, cellular viability assays showed that hematoxylin greatly reduced cytotoxicity induced by tau-RD aggregates. In summary, hematoxylin might be a promising candidate for further development as a potential therapeutic drug for AD patients.

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 associated with neuronal hypoactivity and presynaptic dysfunction. Oral administration of the adenosine A1 receptor antagonist rolofylline (KW-3902) has previously been shown to reverse spatial memory deficits and to normalize the basic synaptic transmission in a mouse line expressing full-length pro-aggregant tau (TauΔK) at low levels, with late onset of disease. However, the efficacy of treatment remained to be explored for cases of more aggressive tauopathy. Using a combination of behavioral assays, imaging with several PET-tracers, and analysis of brain tissue, we compared the curative reversal of tau pathology by blocking adenosine A1 receptors in three mouse models expressing different types and levels of tau and tau mutants. We show through positron emission tomography using the tracer [18F]CPFPX (a selective A1 receptor ligand) that intravenous injection of rolofylline effectively blocks A1 receptors in the brain. Moreover, when administered to TauΔK mice, rolofylline can reverse tau pathology and synaptic decay. The beneficial effects are also observed in a line with more aggressive tau pathology, expressing the amyloidogenic repeat domain of tau (TauRDΔK) with higher aggregation propensity. Both models develop a progressive tau pathology with missorting, phosphorylation, accumulation of tau, loss of synapses, and cognitive decline. TauRDΔK causes pronounced neurofibrillary tangle assembly concomitant with neuronal death, whereas TauΔK accumulates only to tau pretangles without overt neuronal loss. A third model tested, the rTg4510 line, 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 a higher accumulation of tau-specific PET tracers and inflammation. In conclusion, blocking adenosine A1 receptors by rolofylline can reverse pathology if the pathological potential of tau remains below a threshold value that depends on concentration and aggregation propensity.

Expanded Conformations of Monomeric Tau Initiate Its Amyloidogenesis.

Understanding early amyloidogenesis is key to rationally develop therapeutic strategies. Tau protein forms well-characterized pathological deposits but its aggregation mechanism is still poorly understood. Using single-molecule force spectroscopy based on a mechanical protection strategy, we studied the conformational landscape of the monomeric tau repeat domain (tau-RD244-368 ). We found two sets of conformational states, whose frequency is influenced by mutations and the chemical context. While pathological mutations Δ280K and P301L and a pro-amyloidogenic milieu favored expanded conformations and destabilized local structures, an anti-amyloidogenic environment promoted a compact ensemble, including a conformer whose topology might mask two amyloidogenic segments. Our results reveal that to initiate aggregation, monomeric tau-RD244-368 decreases its polymorphism adopting expanded conformations. This could account for the distinct structures found in vitro and across tauopathies.

Expanded Conformations of Monomeric Tau Initiate Its Amyloidogenesis**

AbstractUnderstanding early amyloidogenesis is key to rationally develop therapeutic strategies. Tau protein forms well‐characterized pathological deposits but its aggregation mechanism is still poorly understood. Using single‐molecule force spectroscopy based on a mechanical protection strategy, we studied the conformational landscape of the monomeric tau repeat domain (tau‐RD244‐368). We found two sets of conformational states, whose frequency is influenced by mutations and the chemical context. While pathological mutations Δ280K and P301L and a pro‐amyloidogenic milieu favored expanded conformations and destabilized local structures, an anti‐amyloidogenic environment promoted a compact ensemble, including a conformer whose topology might mask two amyloidogenic segments. Our results reveal that to initiate aggregation, monomeric tau‐RD244‐368 decreases its polymorphism adopting expanded conformations. This could account for the distinct structures found in vitro and across tauopathies.

Site-directed double monoubiquitination of the repeat domain of the amyloid-forming protein tau impairs self-assembly and coacervation

In Alzheimer’s disease and related disorders called tauopathies, the microtubule-associated protein tau accumulates in the brain in the form of amyloid-like supramolecular filaments. As an intrinsically disordered protein, tau undergoes many post-translational modifications, including ubiquitination. Alterations to the levels of ubiquitination of tau have been observed at various stages of neurodegenerative conditions.We focus on proteoform-specific interrogations to obtain mechanistic insight into the effects of ubiquitination on disease-related conformational transitions of tau. Single and double ubiquitination of tau at residues Lys311 and Lys317 is strongly associated with pathological conditions. In this study, we leveraged disulfide-directed chemistry to install ubiquitin at one or both of those positions in the isolated microtubule-binding repeat domain of tau. We obtained homogeneously modified tau proteins and observed that they retained disordered character in solution.We found that ubiquitination in position 317 (with or without ubiquitination in position 311) impaired the formation of ordered fibrillar structures via oligomeric intermediates. Since the transition to fibrillar species may proceed via an alternative condensation pathway involving liquid droplet intermediates, we further tested the ability of the ubiquitinated proteoforms to phase separate. Single monoubiquitinated tau species were able to coacervate, however no liquid droplets were observed for the double ubiquitinated form.Taken together, the data indicate that double ubiquitination in the third repeat of tau disfavors the formation of amyloid aggregates by distinct mechanisms, suggesting that the presence of ubiquitinated residues 311 and 317 in insoluble tau may result from modifications in advanced stages of aggregation. These findings contribute to our understanding of the influence of site-specific ubiquitination on the pathological conformational transitions of a prototypical intrinsically disordered protein.

Conformation‐specific anti‐tau antibodies distinguish human tauopathies

AbstractBackgroundNeurodegenerative tauopathies are defined by progressive brain accumulation of tau aggregates, and include Alzheimer disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Pick disease (PiD). Tau is an intrinsically disordered protein, yet it adopts diverse, highly ordered amyloid assemblies in disease states. These may amplify within cells by acting as “seeds,” or templates for their own replication. Evidence suggests that tau assemblies transfer between cells of the brain, which has inspired the development of immunotherapies that are in clinical trials. Analyses by cryo‐EM of insoluble tau filaments from tauopathies have revealed distinct fibril core structures, but these are not readily distinguished by immunohistochemistry. The tau repeat domain (RD) consists of three or four repeated segments and contains the amino acids comprising each tau filament core. The RD is required for tau assembly and has a different conformation in seed‐competent tau than in inert tau. Antibodies specific for distinct RD conformations have promise as diagnostic and therapeutic agents.MethodWe generated novel anti‐tau monoclonal antibodies against structurally defined synthetic linear peptide epitopes predicted to be exposed in seed‐competent tau. We employed a screening protocol using biosensor cell‐based seeding assays to select antibodies for sensitivity and specificity in binding soluble tau seeds from AD and non‐AD tauopathies.ResultTwo antibodies, MD2.2 and MD3.1, preferentially bind soluble tau seeds versus inert tau monomer, and distinguish seeds in AD and PSP from those of CBD and PiD. They stain tauopathy brain tissue with a pattern distinct from that of AT8.ConclusionWe have generated novel anti‐tauRD antibodies with specificity for seed‐competent conformations and which distinguish tauopathies. These may be valuable for biochemical and neuropathological investigation of tauopathies and may also improve development of immunotherapy for AD and PSP.

Proanthocyanidins prevent tau protein aggregation and disintegrate tau filaments

Occurrence of neurofibrillary tangles of the tau protein is a hallmark of tau-related neurodegenerative diseases, i.e. Alzheimer's disease (AD) and frontotemporal dementia. The pathological mechanism underlying AD remains poorly understood, and effective treatments are still unavailable to mitigate the disease. Inhibiting of tau aggregation and disrupting the existing fibrils are key targets in drug discovery towards preventing or curing AD. In this study, grape seed proanthocyanidins (GSPs) was found to effectively inhibit the repeat domain of tau (tau-RD) aggregation and disaggregate tau-RD fibrils in a concentration-dependent manner by inhibiting β-sheet formation of tau-RD. In cells, GSPs relieved cytotoxicity induced by tau-RD aggregates. Molecular dynamics simulations indicated that strong hydrogen bonding, hydrophobic interaction and π-π stacking between GSPs and tau-RD protein were major reasons why GSPs had high inhibitory activity on tau-RD fibrillogenesis. These results provide preliminary data to develop GSPs into medicines, foodstuffs or nutritional supplements for AD patients, suggesting that GSPs could be a candidate molecule in the drug design for AD therapeutics.

Virtual Screening and Testing of GSK-3 Inhibitors Using Human SH-SY5Y Cells Expressing Tau Folding Reporter and Mouse Hippocampal Primary Culture under Tau Cytotoxicity.

Glycogen synthase kinase-3β (GSK-3β) is an important serine/threonine kinase that implicates in multiple cellular processes and links with the neurodegenerative diseases including Alzheimer's disease (AD). In this study, structure-based virtual screening was performed to search database for compounds targeting GSK-3β from Enamine's screening collection. Of the top-ranked compounds, 7 primary hits underwent a luminescent kinase assay and a cell assay using human neuroblastoma SH-SY5Y cells expressing Tau repeat domain (TauRD) with pro-aggregant mutation ΔK280. In the kinase assay for these 7 compounds, residual GSK-3β activities ranged from 36.1% to 90.0% were detected at the IC50 of SB-216763. In the cell assay, only compounds VB-030 and VB-037 reduced Tau aggregation in SH-SY5Y cells expressing ΔK280 TauRD-DsRed folding reporter. In SH-SY5Y cells expressing ΔK280 TauRD, neither VB-030 nor VB-037 increased expression of GSK-3α Ser21 or GSK-3β Ser9. Among extracellular signal-regulated kinase (ERK), AKT serine/threonine kinase 1 (AKT), mitogen-activated protein kinase 14 (P38) and mitogen-activated protein kinase 8 (JNK) which modulate Tau phosphorylation, VB-037 attenuated active phosphorylation of P38 Thr180/Tyr182, whereas VB-030 had no effect on the phosphorylation status of ERK, AKT, P38 or JNK. However, both VB-030 and VB-037 reduced endogenous Tau phosphorylation at Ser202, Thr231, Ser396 and Ser404 in neuronally differentiated SH-SY5Y expressing ΔK280 TauRD. In addition, VB-030 and VB-037 further improved neuronal survival and/or neurite length and branch in mouse hippocampal primary culture under Tau cytotoxicity. Overall, through inhibiting GSK-3β kinase activity and/or p-P38 (Thr180/Tyr182), both compounds may serve as promising candidates to reduce Tau aggregation/cytotoxicity for AD treatment.

Tau R2 and R3 are essential regions for tau aggregation, seeding and propagation

Tauopathies are characterised by intracellular deposits of fibrillar tau tangles. However, the interneuronal spread of pathological tau species precedes the development of major tau burdens. Two amyloid motifs, VQIINK in repeat 2 and VQIVYK in repeat 3, of tau repeat domain, assemble into β-sheet-rich fibrils on their own but alone do not form seed-competent fibrils. In contrast, the entire R3 region self-aggregates and forms seed-competent fibrils. Our study aimed to identify the minimal regions in the tau repeat domain that define seeding and its impact on intracellular tau phosphorylation and aggregation. Using peptides of individual repeats, we show that R2, like R3, forms seed-competent fibrils when assembled in the presence of heparin. However, R3, but not R2, forms seed-competent fibrils when assembled without heparin, even though both R2 and R3 have identical N-terminal hexapeptide and cysteine residue sequences. Moreover, cysteine to alanine substitution in R3 abrogates its self-aggregation and seeding potency. Tau RD P301S biosensor cells and Tau P301L (0N4R)-expressing HEK293 cells seeded with R2 and R3 fibrils show the induction of pathological phosphorylation of tau at Ser262/Ser396/Ser404 positions and oligomerisation of native tau. Protein fractions of biosensor cells seeded with R2 and R3 fibrils reseed endogenous tau aggregation when introduced into a fresh set of biosensor cells. Our findings suggest that R3 may be the minimal region for pathological seed generation under physiological conditions, whereas R2 might need polyanionic cofactors to generate pathogenic seeds. Lastly, R2 and R3 fibrils induce template-induced misfolding and pathological hyperphosphorylation of intracellular tau, making intracellular tau seed-competent.

Tau seeding in chronic traumatic encephalopathy parallels disease severity.

Chronic traumatic encephalopathy (CTE), a neurodegenerative tauopathy, is associated with behavioral, mood and cognitive impairment, including dementia. Tauopathies are neurodegenerative diseases whose neuropathological phenotypes are characterized by distinct histopathologic features of tau pathology, which progressively deposit throughout the brain. In certain tauopathies, especially Alzheimer's disease (AD), tau deposition appears to follow brain network connections. Experimental evidence suggests that the progression of tau pathology in humans, mouse and cell models could be explained by tau seeds that adopt distinct conformations and serve as templates for their own amplification to mediate transcellular propagation of pathology. Tau seeds are efficiently detected by the induction of aggregation in cell-based "biosensors" that express tau repeat domain (RD) with a disease-associated mutation (P301S) fused to complementary fluorescent protein tags (cyan and yellow fluorescent protein). Biosensors enable quantification of tau seeding in fixed and fresh-frozen brain tissue. Phospho-tau deposition in CTE follows progressive stages (I-IV), but the relationship of seeding to this deposition is unclear. We have used an established biosensor assay to independently quantify tau seeding as compared to AT8 phospho-tau histopathology in thin sections of fixed tissues of 11 brain regions from 27 patients with CTE, 5 with other tauopathies, and 5 negative controls. In contrast to prior studies of AD, we detected tau seeding late in the course of CTE (predominantly stages III and IV). It was less anatomically prevalent than AT8-positive inclusions, which were relatively widespread. We especially observed seeding in the limbic system (amygdala, thalamus, basal ganglia), which may explain the dominant cognitive and behavior impairments that characterize CTE.

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.

Continuous Monitoring of Tau-Induced Neurotoxicity in Patient-Derived iPSC-Neurons.

Tau aggregation within neurons is a critical feature of Alzheimer's disease (AD) and related tauopathies. It is believed that soluble pathologic tau species seed the formation of tau aggregates in a prion-like manner and propagate through connected neurons during the progression of disease. Both soluble and aggregated forms of tau are thought to have neurotoxic properties. In addition, different strains of misfolded tau may cause differential neurotoxicity. In this work, we present an accelerated human neuronal model of tau-induced neurotoxicity that incorporates both soluble tau species and tau aggregation. Using patient-derived induced pluripotent stem cell (iPSC) neurons expressing a tau aggregation biosensor, we develop a cell culture system that allows continuous assessment of both induced tau aggregation and neuronal viability at single-cell resolution for periods of >1 week. We show that exogenous tau “seed” uptake, as measured by tau repeat domain (TauRD) reporter aggregation, increases the risk for subsequent neuronal death in vitro. These results are the first to directly visualize neuronal TauRD aggregation and subsequent cell death in single human iPSC neurons. Specific morphologic strains or patterns of TauRD aggregation are then identified and associated with differing neurotoxicity. Furthermore, we demonstrate that familial AD iPSC neurons expressing the PSEN1 L435F mutation exhibit accelerated TauRD aggregation kinetics and a tau strain propagation bias when compared with control iPSC neurons.SIGNIFICANCE STATEMENT Neuronal intracellular aggregation of the microtubule binding protein tau occurs in Alzheimer's disease and related neurodegenerative tauopathies. Tau aggregates are believed to spread from neuron to neuron via prion-like misfolded tau seeds. Our work develops a human neuronal live-imaging system to visualize seeded tau aggregation and tau-induced neurotoxicity within single neurons. Using an aggregation-sensing tau reporter, we find that neuronal uptake and propagation of tau seeds reduces subsequent survival. In addition, human induced pluripotent stem cell (iPSC) neurons carrying an Alzheimer's disease-causing mutation in presenilin-1 undergo tau seeding more rapidly than control iPSC neurons. However, they do not show subsequent differences in neuronal survival. Finally, specific morphologies of tau aggregates are associated with increased neurotoxicity.