4-repeat Tau Research Articles

Structure challenges in the multivalency of Tau-microtubule interactions.

Structural studies aiming to visualize the interaction of Tau with microtubules (MTs) face several challenges, the main concerning the fact that Tau has multiple MT-interacting regions. In particular, the four (or three) pseudo-repeats of Tau bind to identical elements along the MT lattice but do it through non-identical residues. In addition, any given Tau molecule can use all its repeats or just one for its engagement with MTs. Finally, the binding of one Tau is not necessarily in register with respect to the next one. The mismatch in the MT and Tau repeats, therefore, challenges conventional modes of image analysis when visualizing these samples using cryo-electron microscopy. This commentary is dedicated to those challenges and ways to circumvent them while aiming for an atomic description of the Tau-tubulin interaction.

Progressive Supranuclear Palsy: A Review

Progressive Supranuclear Palsy (PSP) is a rare neurodegenerative disease which is caused by 4 repeat tau proteins. PSP has a low diagnosis rate because it is underdiagnosed or misdiagnosed as other common diseases such as Parkinson’s Disease (PD). A systematic and comprehensive literature review has been conducted with evidence from various sources in the databases of PubMed, Google Scholar, and JSTOR. A synthesis was conducted based on “Progressive Supranuclear Palsy” in etiology and differential diagnosis was conducted based on “‘Parkinson’s disease’ on the way to progressive supranuclear palsy: a review on PSP-parkinsonism.” From the results we can see that there is a lack of research overall for PSP and it is greatly understudied. One area that needs to be focused on is the treatment for disease-modifying symptoms of PSP as mostly only PD medications are used even though both the diseases are vastly different. A systematic literature review has been done on the topic of PSP regarding the current advances. In general, the diagnosis of PSP is lacking as there is misdiagnosis between one of the PSP subtypes, PSP-Parkinsonism (PSP-P), and PD while there is also underdiagnosis as they symptoms of PSP-Richardson’s Syndrome is seen as natural aging. The lack of early diagnosis and validated biomarkers makes it difficult to find a treatment for PSP. With the lack of information about PSP it is imperative that systematic research needs to be done so that patients have a favorable course and better standard of living.

Analysis of inflammatory markers and tau deposits in an autopsy series of nine patients with anti-IgLON5 disease

Anti-IgLON5 disease is a rare neurological, probably autoimmune, disorder associated in many cases with a specific tauopathy. Only a few post-mortem neuropathological studies have been reported so far. Little is known about the pathogenic mechanisms that result in neurodegeneration. We investigated the neuropathology of anti-IgLON5 disease and characterized cellular and humoral inflammation. We included nine cases (six of them previously published). Median age of patients was 71 years (53–82 years), the median disease duration was 6 years (0.5–13 years), and the female to male ratio was 5:4. Six cases with a median disease duration of 9 years presented a prominent tauopathy. Five of them had a classical anti-IgLON5-related brainstem tauopathy and another presented a prominent neuronal and glial 4-repeat tauopathy, consistent with progressive supranuclear palsy (PSP). Three cases with short disease duration (median 1.25 years) only showed a primary age-related neurofibrillary pathology. Inflammatory infiltrates of T and B cells were mild to moderate and did not significantly differ between anti-IgLON5 disease cases with or without tauopathy. In contrast, we found an extensive neuropil deposition of IgG4 in the tegmentum of the brainstem, olivary nucleus, and cerebellar cortex that was most prominent in two patients with short disease duration without the typical IgLON5-related tauopathy. The IgG4 deposits were particularly prominent in the cerebellar cortex and in these regions accompanied by mild IgG1 deposits. Activated complement deposition (C9neo) was absent. Our study indicates that IgLON5-related tau pathology occurs in later disease stages and may also present a PSP-phenotype with exclusively 4-repeat neuronal and glial tau pathology. The prominent deposition of anti-IgLON5 IgG4 at predilection sites for tau pathology suggests that anti-IgLON5 antibodies precede the tau pathology. Early start of immunotherapy might prevent irreversible neuronal damage and progression of the disease, at least in a subgroup of patients.

A heterozygous splicing variant IVS9-7A > T in intron 9 of the MAPT gene in a patient with right-temporal variant frontotemporal dementia with atypical 4 repeat tauopathy

Right temporal variant frontotemporal dementia, also called right-predominant semantic dementia, often has an unclear position within the framework of the updated diagnostic criteria for behavioral variant frontotemporal dementia or primary progressive aphasia. Recent studies have suggested that this population may be clinically, neuropathologically, and genetically distinct from those with behavioral variant frontotemporal dementia or left-predominant typical semantic variant primary progressive aphasia. Here we describe a Japanese case of right temporal variant frontotemporal dementia with novel heterozygous MAPT mutation Adenine to Thymidine in intervening sequence (IVS) 9 at position -7 from 3ʹ splicing site of intron 9/exon 10 boundary (MAPT IVS9-7A > T). Postmortem neuropathological analysis revealed a predominant accumulation of 4 repeat tau, especially in the temporal lobe, amygdala, and substantia nigra, but lacked astrocytic plaques or tufted astrocytes. Immunoelectron microscopy of the tau filaments extracted from the brain revealed a ribbon-like structure. Moreover, a cellular MAPT splicing assay confirmed that this novel variant promoted the inclusion of exon 10, resulting in the predominant production of 4 repeat tau. These data strongly suggest that the MAPT IVS9-7 A > T variant found in our case is a novel mutation that stimulates the inclusion of exon 10 through alternative splicing of MAPT transcript and causes predominant 4 repeat tauopathy which clinically presents as right temporal variant frontotemporal dementia.

NMR Relaxation Experiments Probe Monomer-Fibril Interaction and Identify Critical Interacting Residues Responsible for Distinct Tau Fibril Morphologies.

Tau aggregation is governed by secondary processes, a major pathological pathway for tau protein fibril propagation, yet its molecular mechanism remains unknown. This work uses saturation transfer and lifetime line-broadening experiments to identify the critical residues involved in these secondary processes. Distinct residue-specific NMR relaxation parameters were obtained for the truncated three repeat tau construct (K19) in equilibrium with structurally different, self-aggregated (saK19) or heparin-induced (hK19) fibrils. The interacting residues are restricted to R3 repeat for hK19 and to R3, R4, and R' repeats for saK19 fibrils. Furthermore, the relaxation profiles of tau monomers in equilibrium with the structurally comparable, in vitro pathological fibrils (tauAD and tauCTE) were similar but distinct from hK19 or saK19 fibrils. Thus, residue-specific relaxation identifies the important residues involved in the binding of monomers to the fibrils. The relaxation profile of the monomers in equilibrium with the NMR invisible fibril seeds potentially distinguishes the distinct structures of tau fibrils.

Photo-Excited Toluidine Blue Disaggregates the Repeat Tau and Modulates End-Binding Protein EB1, Cytoskeletal Structure in Neuronal Cells

Alzheimer's disease is a progressive neurological disorder characterized by the intracellular accumulation of Tau protein aggregates. In the present work, we studied the effect of Toluidine Blue and photo-excited Toluidine Blue on the aggregation of repeat Tau using in vitro assays. The in vitro experiments were carried out on recombinant repeat Tau which was purified by cation exchange chromatography. The ThS fluorescence analysis was used to study the aggregation kinetics of Tau. CD spectroscopy and electron microscopy were used to study the secondary structure and morphology of Tau respectively. The actin cytoskeleton modulation was studied in Neuro2a cells with help of immunofluorescent microscopy. Results showed that Toluidine Blue efficiently inhibited the formation of higher-order aggregates, which was evidenced by Thioflavin S fluorescence assay, SDS-PAGE, and TEM. Immunofluorescence studies on the cytoskeleton of Neuro2a cells showed that Toluidine Blue and photo-excited Toluidine Blue treatment at a non-toxic concentration of 0.5 µM stimulated the formation of actin-rich lamellipodia and filopodia structures. Tubulin networks were also differentially modulated after the treatment of Toluidine Blue and photo-excited Toluidine Blue. End-binding protein 1 (EB1) levels were observed to increase after Toluidine Blue and photo-excited Toluidine Blue treatment indicating accelerated microtubule polymerization. The overall study suggested that Toluidine Blue inhibited the aggregation of soluble Tau and photo-excited Toluidine Blue disaggregated the pre-formed Tau filaments. In our study, TB and PE-TB were observed to be potent against Tau aggregation. We observed a distinctive modulation of actin, tubulin networks, and EB1 levels after TB and PE-TB treatment, which suggested that TB and PE-TB have potency against cytoskeleton deformities.

Differential Binding and Conformational Dynamics of Tau Microtubule-Binding Repeats with a Preformed Amyloid-β Fibril Seed.

Both senile plaques formed by amyloid-β (Aβ) and neurofibrillary tangles (NFTs) comprised of tau are pathological hallmarks of Alzheimer's disease (AD). The accumulation of NFTs better correlates with the loss of cognitive function than senile plaques, but NFTs are rarely observed without the presence of senile plaques. Hence, cross-seeding of tau by preformed Aβ amyloid fibril seeds has been proposed to drive the aggregation of tau and exacerbate AD progression, but the molecular mechanism remains unknown. Here, we first identified cross-interaction hotspots between Aβ and tau using atomistic discrete molecular dynamics simulations (DMD) and confirmed the critical role of the four microtubule-binding repeats of tau (R1-R4) in the cross-interaction with Aβ. We further investigated the binding structure and dynamics of each tau repeat with a preformed Aβ fibril seed. Specifically, R1 and R3 preferred to bind the Aβ fibril lateral surface instead of the elongation end. In contrast, R2 and R4 had higher binding propensities to the fibril elongation end than the lateral surface, enhancing β-sheet content by forming hydrogen bonds with the exposed hydrogen bond donors and acceptors. Together, our results suggest that the four repeats play distinct roles in driving the binding of tau to different surfaces of an Aβ fibril seed. Binding of tau to the lateral surface of Aβ fibril can increase the local concentration, while the binding to the elongation surface promotes β-sheet formation, both of which reduce the free energy barrier for tau aggregation nucleation and subsequent fibrillization.

Aquaporin 4 is differentially increased and dislocated in association with tau and amyloid-beta

AimsNeurovascular-glymphatic dysfunction plays an important role in Alzheimer's disease and has been analysed mainly in relation to amyloid-beta (Aβ) pathology. Here, we aim to investigate the neurovascular alterations and mapping of aquaporin 4 (AQP4) distribution and dislocation associated with tau and Aβ. Materials and methodsPerfusion, susceptibility weighted imaging and structural magnetic resonance imaging (MRI) were performed in the pR5 mouse model of 4-repeat tau and the arcAβ mouse model of amyloidosis. Immunofluorescence staining was performed using antibodies against AQP4, vessel, astroglia, microglia, phospho-tau and Aβ in brain tissue slices from pR5, arcAβ and non-transgenic mice. Key findingspR5 mice showed regional atrophy, preserved cerebral blood flow, and reduced cerebral vessel density compared to non-transgenic mice, while arcAβ mice showed cerebral microbleeds and reduced cerebral vessel density. AQP4 dislocation and peri-tau enrichment in the hippocampus and increased AQP4 levels in the cortex and hippocampus were detected in pR5 mice compared to non-transgenic mice. In comparison, cortical AQP4 dislocation and cortical/hippocampal peri-plaque increases were observed in arcAβ mice. Increased expression of reactive astrocytes were detected around the tau inclusions in pR5 mice and Aβ plaques in arcAβ mice. SignificanceWe demonstrated the neurovascular alterations, microgliosis, astrogliosis and increased AQP4 regional expression in pR5 tau and arcAβ mice. We observed a divergent region-specific AQP4 dislocation and association with phospho-tau and Aβ pathologies.

Phosphorylation at Ser289 Enhances the Oligomerization of Tau Repeat R2.

In tauopathies such as Alzheimer's disease (AD), aberrant phosphorylation causes the dissociation of tau proteins from microtubules. The dissociated tau then aggregates into sequent forms from soluble oligomers to paired helical filaments and insoluble neurofibrillary tangles (NFTs). NFTs is a hallmark of AD, while oligomers are found to be the most toxic form of the tau aggregates. Therefore, understanding tau oligomerization with regard to abnormal phosphorylation is important for the therapeutic development of AD. In this study, we investigated the impact of phosphorylated Ser289, one of the 40 aberrant phosphorylation sites of full-length tau proteins, on monomeric and dimeric structures of tau repeat R2 peptides. We carried out intensive replica exchange molecular dynamics simulation with a total simulation time of up to 0.1 ms. Our result showed that the phosphorylation significantly affected the structures of both the monomer and the dimer. For the monomer, the phosphorylation enhanced ordered-disordered structural transition and intramolecular interaction, leading to more compactness of the phosphorylated R2 compared to the wild-type one. As to the dimer, the phosphorylation increased intermolecular interaction and β-sheet formation, which can accelerate the oligomerization of R2 peptides. This result suggests that the phosphorylation at Ser289 is likely to promote tau aggregation. We also observed a phosphorylated Ser289-Na+-phosphorylated Ser289 bridge in the phosphorylated R2 dimer, suggesting an important role of cation ions in tau aggregation. Our findings suggest that phosphorylation at Ser289 should be taken into account in the inhibitor screening of tau oligomerization.

Differential binding dynamics of four microtubule-binding repeats of tau with an amyloid-β fibril seed.

Both senile plaques formed by amyloid-beta (Aβ) and neurofibrillary tangles comprised of tau are hallmarks of Alzheimer's disease (AD). The accumulation of neurofibrillary tangles better correlates with the loss of cognitive function than senile plaques, but neurofibrillary tangles are rarely observed without the presence of senile plaques. Hence, cross-seeding of tau by preformed Aβ amyloid fibril seeds has been proposed to drive the aggregation of tau and exacerbate AD progression, but the molecular mechanism remains poorly understood. Herein, we identified cross-interaction hotspots between Aβ and tau using atomistic discrete molecular dynamics simulations (DMD) and confirmed the critical role of the four microtubule-binding repeats of tau (R1-R4) in the cross-interaction with Aβ. We further investigated the binding structure and dynamics of each tau repeat with a preformed Aβ fibril seed. Specifically, R1 and R3 preferred to bind the lateral instead of elongation surfaces of the fibril, preserving similar secondary structures as in the solution. In contrast, R2 and R4 had higher binding propensities to the elongation than lateral surfaces, adopting β-sheet structures by forming hydrogen bonds with the exposed hydrogen bond donors and acceptors. Together, our results suggest that four MTBD repeats play distinct roles in driving the binding of tau to two different surfaces of an Aβ fibril seed. The binding of tau to the lateral surface of Aβ fibril can increase the local concentration, while the binding in the elongation surface promotes β-sheet formation, both of which reduce the free energy barrier for tau aggregation nucleation and subsequent fibrillization. Our computational study offers molecular insight into the experimentally observed cross-seeding of Tau aggregation by pre-formed Aβ fibril seeds and helps better understand the pathological synergy of Aβ and tau aggregation in the etiology of AD.

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.

Phosphorylation of Tau R2 Repeat Destabilizes Its Binding to Microtubules: A Molecular Dynamics Simulation Study.

Phosphorylation, the most popular post-translational modification of tau protein, plays an important role in regulating tau physiological functions. However, aberrant phosphorylation attenuates the binding affinity of tau to a microtubule (MT), resulting in MT destabilization followed by accumulation of neurofibrillary tangles in the brain. There are in total 85 potential phosphorylation sites in a full-length tau protein, and about half of them are abnormal as they occur in tau of Alzheimer's disease (AD) brain only. In this work, we investigated the impact of abnormal Ser289, Ser293, and Ser289/Ser293 phosphorylation on tau R2-MT binding and the conformation of tau R2 using molecular dynamics simulation. We found that the phosphorylation significantly affected R2-MT interaction and reduced the binding affinity of tau R2 peptides to MTs. Free energy decomposition analysis suggested that the post-translational modified residues themselves made a significant contribution to destabilize tau repeat R2-MT binding. Therefore, the phosphorylation may attenuate the binding affinity of tau to MTs. Additionally, the phosphorylation also enhanced helix-coil transition of monomeric R2 peptides, which may result in the acceleration of tau aggregation. Since these phosphorylated sites have not been examined in previous experimental studies, our finding through all-atom molecular dynamics simulations and free energy analysis can inspire experimental scientists to investigate the impact of the phosphorylation on MT binding and aggregation of full-length tau and the pathological roles of the phosphorylation at those sites in AD development through in vitro/in vivo assays.

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.

Symptomatology in 4-repeat tauopathies is associated with data-driven topology of [18F]-PI-2620 tau-PET signal

In recent years in vivo visualization of tau deposits has become possible with various PET radiotracers. The tau tracer [18F]PI-2620 proved high affinity both to 3-repeat/4-repeat tau in Alzheimer’s disease as well as to 4-repeat tau in progressive supranuclear palsy (PSP) and corticobasal syndrome (CBS). However, to be clinically relevant, biomarkers should not only correlate with pathological changes but also with disease stage and progression. Therefore, we aimed to investigate the correlation between topology of [18F]PI-2620 uptake and symptomatology in 4-repeat tauopathies.72 patients with possible or probable 4-repeat tauopathy, i.e. 31 patients with PSP-Richardson’s syndrome (PSP-RS), 30 with amyloid-negative CBS and 11 with PSP-non-RS/CBS, underwent [18F]PI-2620-PET. Principal component analysis was performed to identify groups of similar brain regions based on 20–40 min p.i. regional standardized uptake value ratio z-scores. Correlations between component scores and the items of the PSP Rating Scale were explored.Motor signs like gait, arising from chair and postural instability showed a positive correlation with tracer uptake in mesial frontoparietal lobes and the medial superior frontal gyrus and adjacent anterior cingulate cortex. While the signs disorientation and bradyphrenia showed a positive correlation with tracer uptake in the parietooccipital junction, the signs disorientation and arising from chair were negatively correlated with tau-PET signal in the caudate nucleus and thalamus. Total PSP Rating Scale Score showed a trend towards a positive correlation with mesial frontoparietal lobes and a negative correlation with caudate nucleus and thalamus. While in CBS patients, the main finding was a negative correlation of tracer binding in the caudate nucleus and thalamus and a positive correlation of tracer binding in medial frontal cortex with gait and motor signs, in PSP-RS patients various correlations of clinical signs with tracer binding in specific cerebral regions could be detected.Our data reveal [18F]PI-2620 tau-PET topology to correlate with symptomatology in 4-repeat tauopathies. Longitudinal studies will be needed to address whether a deterioration of signs and symptoms over time can be monitored by [18F]PI-2620 in 4-repeat tauopathies and whether [18F]PI-2620 may serve as a marker of disease progression in future therapeutic trials. The detected negative correlation of tracer binding in the caudate nucleus and thalamus with the signs disorientation and arising from chair may be due to an increasing atrophy in these regions leading to partial volume effects and a relative decrease of tracer uptake in the disease course. As cerebral regions correlating with symptomatology differ depending on the clinical phenotype, a precise knowledge of clinical signs and symptoms is necessary when interpreting [18F]PI-2620 PET results.

Sequence-Dependent Conformational Properties of PGGG Motif in Tau Repeats: Insights from Molecular Dynamics Simulations of Narrow Pick Filament.

Tauopathies are a class of neurodegenerative diseases correlated with the presence of pathological Tau fibrils as a diagnostic marker. The microtubule-binding repeat region of Tau protein, which includes R1, R2, R3, and R4 repeats, constitutes the core of these fibrils. Each repeat consists of a semiconserved C-terminal hexapeptide flanked by KxGS and PGGG motifs. Previous studies have shown the influence of these peptides on protein aggregation, yet their repeat-specific properties are less explored. Using molecular dynamics, we probed the sequence-specific influence of the C-terminal hexapeptide (264ENLKHQ269) in determining the compact local conformation of the R1 repeat of the narrow Pick filament (NPF) with a homologous E264G mutation. In addition to that, we also studied the influence of 262S phosphorylation on this conformation as the phosphorylation is proposed to alleviate the pathogenesis of Pick's disease. Interestingly, we determined that E264G mutation induces a conformational shift of 270PGGG273 from a turn to a random coil. This conformational dependence is experimentally verified with the R1R3-E264G mutant construct, which displayed accelerated aggregation compared with the R1R3 wild-type construct. A significant delay in aggregation of the R1R3-G326E mutant further demonstrates the importance of 326G in determining the conformation of the R3 repeat. Thus, we conclude that the conformational properties of the PGGG motif in Tau repeats are strongly dependent on the repeat-specific sequence of the C-terminal hexapeptide.

Tuberous sclerosis complex is associated with a novel human tauopathy.

Tuberous sclerosis complex (TSC) is a neurogenetic disorder leading to epilepsy, developmental delay, and neurobehavioral dysfunction. The syndrome is caused by pathogenic variants in TSC1 (coding for hamartin) or TSC2 (coding for tuberin). Recently, we reported a progressive frontotemporal dementia-like clinical syndrome in a patient with a mutation in TSC1, but the neuropathological changes seen in adults with TSC with or without dementia have yet to be systematically explored. Here, we examined neuropathological findings in adults with TSC (n = 11) aged 30-58years and compared them to age-matched patients with epilepsy unrelated to TSC (n = 9) and non-neurological controls (n = 10). In 3 of 11 subjects with TSC, we observed a neurofibrillary tangle-predominant "TSC tauopathy" not seen in epilepsy or non-neurological controls. This tauopathy was observed in the absence of pathological amyloid beta, TDP-43, or alpha-synuclein deposition. The neurofibrillary tangles in TSC tauopathy showed a unique pattern of post-translational modifications, with apparent differences between TSC1 and TSC2 mutation carriers. Tau acetylation (K274, K343) was prominent in both TSC1 and TSC2, whereas tau phosphorylation at a common phospho-epitope (S202) was observed only in TSC2. TSC tauopathy was observed in selected neocortical, limbic, subcortical, and brainstem sites and showed a 3-repeat greater than 4-repeat tau isoform pattern in both TSC1 and TSC2 mutation carriers, but no tangles were immunolabeled with MC1 or p62 antibodies. The findings suggest that individuals with TSC are at risk for a unique tauopathy in mid-life and that tauopathy pathogenesis may involve TSC1, TSC2, and related molecular pathways.

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.

Neuropathology of classic myotonic dystrophy type 1 is characterized by both early initiation of primary age-related tauopathy of the hippocampus and unique 3-repeat tauopathy of the brainstem.

Myotonic dystrophy type 1 (DM1) is an inherited autosomal-dominant condition that induces altered splicing of transcripts, including MAPT, leading to a distinctive abnormal deposition of tau protein in the CNS. We characterized the tau isoforms of abnormal depositions in the brains of 4 patients with classic DM1 by immunohistochemistry using isoform-specific antibodies. All patients, including those of presenile age, showed numerous neurofibrillary tangles (NFTs) of both 3-repeat and 4-repeat tau in the limbic area and mild involvement in the cerebral cortex. Amyloid-β deposition was only seen in 1 senile case while cortical tauopathy in all other cases was consistent with primary age-related tauopathy (PART). In the putamen and globus pallidus, only a few tau deposits were observed. Tau deposits in the brainstem frequently showed a DM1-specific pattern with 3-repeat tau dominant NFTs. Additionally, tau-positive astrocytes morphologically similar to tufted astrocytes and astrocytic plaques were occasionally observed in the brainstem; however, they were predominantly composed of 3-repeat tau. Thus, the classic DM1 showed both early onset of PART-like pathology in the limbic areas as a progeroid syndrome of DM1 and an abnormal splicing event in the brainstem leading to 3-repeat tau dominant accumulation with both neuronal and astrocytic involvement.

Characterization of microtubule-associated protein tau isoforms and Alzheimer’s disease-like pathology in normal sheep (Ovis aries): relevance to their potential as a model of Alzheimer’s disease

Alzheimer’s disease is a chronic neurodegenerative disease that accounts for up to 80% of all dementias. Characterised by deteriorations of memory and cognitive function, the key neuropathological features are accumulations of β-amyloid and hyperphosphorylated tau, as ‘plaques’ and ‘tangles’, respectively. Despite extensive study, however, the exact mechanism underlying aggregate formation in Alzheimer’s disease remains elusive, as does the contribution of these aggregates to disease progression. Importantly, a recent evaluation of current Alzheimer’s disease animal models suggested that rodent models are not able to fully recapitulate the pathological intricacies of the disease as it occurs in humans. Therefore, increasing attention is being paid to species that might make good alternatives to rodents for studying the molecular pathology of Alzheimer’s disease. The sheep (Ovis aries) is one such species, although to date, there have been few molecular studies relating to Alzheimer’s disease in sheep. Here, we investigated the Alzheimer’s disease relevant histopathological characteristics of 22 sheep, using anti-β-amyloid (Abcam 12267 and mOC64) and phosphorylation specific anti-tau (AT8 and S396) antibodies. We identified numerous intraneuronal aggregates of both β-amyloid and tau that are consistent with early Alzheimer’s disease-like pathology. We confirmed the expression of two 3-repeat (1N3R, 2N3R) and two 4-repeat (1N4R, 2N4R) tau isoforms in the ovine brain, which result from the alternative splicing of two tau exons. Finally, we investigated the phosphorylation status of the serine396 residue in 30 sheep, and report that the phosphorylation of this residue begins in sheep aged as young as 2 years. Together, these data show that sheep exhibit naturally occurring β-amyloid and tau pathologies, that reflect those that occur in the early stages of Alzheimer’s disease. This is an important step towards the validation of the sheep as a feasible large animal species in which to model Alzheimer’s disease.