Tau forms oligomeric complexes on microtubules that are distinct from tau aggregates

Alquezar C., Arya S., Kao A.W.

Post-translational modifications (PTMs) on tau have long been recognized as affecting protein function and contributing to neurodegeneration. The explosion of information on potential and observed PTMs on tau provides an opportunity to better understand these modifications in the context of tau homeostasis, which becomes perturbed with aging and disease. Prevailing views regard tau as a protein that undergoes abnormal phosphorylation prior to its accumulation into the toxic aggregates implicated in Alzheimer's disease (AD) and other tauopathies. However, the phosphorylation of tau may, in fact, represent part of the normal but interrupted function and catabolism of the protein. In addition to phosphorylation, tau undergoes another forms of post-translational modification including (but not limited to), acetylation, ubiquitination, glycation, glycosylation, SUMOylation, methylation, oxidation, and nitration. A holistic appreciation of how these PTMs regulate tau during health and are potentially hijacked in disease remains elusive. Recent studies have reinforced the idea that PTMs play a critical role in tau localization, protein-protein interactions, maintenance of levels, and modifying aggregate structure. These studies also provide tantalizing clues into the possibility that neurons actively choose how tau is post-translationally modified, in potentially competitive and combinatorial ways, to achieve broad, cellular programs commensurate with the distinctive environmental conditions found during development, aging, stress, and disease. Here, we review tau PTMs and describe what is currently known about their functional impacts. In addition, we classify these PTMs from the perspectives of protein localization, electrostatics, and stability, which all contribute to normal tau function and homeostasis. Finally, we assess the potential impact of tau PTMs on tau solubility and aggregation. Tau occupies an undoubtedly important position in the biology of neurodegenerative diseases. This review aims to provide an integrated perspective of how post-translational modifications actively, purposefully, and dynamically remodel tau function, clearance, and aggregation. In doing so, we hope to enable a more comprehensive understanding of tau PTMs that will positively impact future studies.

Arakhamia T., Lee C.E., Carlomagno Y., Kumar M., Duong D.M., Wesseling H., Kundinger S.R., Wang K., Williams D., DeTure M., Dickson D.W., Cook C.N., Seyfried N.T., Petrucelli L., Steen J.A., et. al.

Tau aggregation into insoluble filaments is the defining pathological hallmark of tauopathies. However, it is not known what controls the formation and templated seeding of strain-specific structures associated with individual tauopathies. Here, we use cryo-electron microscopy (cryo-EM) to determine the structures of tau filaments from corticobasal degeneration (CBD) human brain tissue. Cryo-EM and mass spectrometry of tau filaments from CBD reveal that this conformer is heavily decorated with posttranslational modifications (PTMs), enabling us to map PTMs directly onto the structures. By comparing the structures and PTMs of tau filaments from CBD and Alzheimer's disease, it is found that ubiquitination of tau can mediate inter-protofilament interfaces. We propose a structure-based model in which cross-talk between PTMs influences tau filament structure, contributing to the structural diversity of tauopathy strains. Our approach establishes a framework for further elucidating the relationship between the structures of polymorphic fibrils, including their PTMs, and neurodegenerative disease.

Hill E., Karikari T.K., Moffat K.G., Richardson M.J., Wall M.J.

Tau is a highly soluble microtubule-associated protein that acts within neurons to modify microtubule stability. However, abnormally phosphorylated tau dissociates from microtubules to form oligomers and fibrils which associate in the somatodendritic compartment. Although tau can form neurofibrillary tangles (NFTs), it is the soluble oligomers that appear to be the toxic species. There is, however, relatively little quantitative information on the concentration-dependent and time-dependent actions of soluble tau oligomers (oTau) on the electrophysiological and synaptic properties of neurons. Here, whole-cell patch clamp recording was used to introduce known concentrations of oligomeric full-length tau-441 into mouse hippocampal CA1 pyramidal and neocortical Layer V thick-tufted pyramidal cells. oTau increased input resistance, reduced action potential amplitude and slowed action potential rise and decay kinetics. oTau injected into presynaptic neurons induced the run-down of unitary EPSPs which was associated with increased short-term depression. In contrast, introduction of oTau into postsynaptic neurons had no effect on basal synaptic transmission, but markedly impaired the induction of long-term potentiation (LTP). Consistent with its effects on synaptic transmission and plasticity, oTau puncta could be observed in the soma, axon and in the distal dendrites of injected neurons.

Bohrer C.H., Yang X., Weng X., Tenner B., Thakur S., McQuillen R., Ross B., Wooten M., Chen X., Lakadamyali M., Zhang J., Roberts E., Xiao J.

AbstractIn single-molecule localization based super-resolution microscopy (SMLM), a fluorophore stochastically switches between fluorescent- and dark-states, leading to intermittent emission of fluorescence, a phenomenon known as blinking. Intermittent emissions create multiple localizations belonging to the same molecule, resulting in blinking-artifacts within SMLM images. These artifacts are often interpreted as true biological assemblies, confounding quantitative analyses and interpretations. Multiple methods have been developed to eliminate these artifacts, but they either require additional experiments, arbitrary thresholds, or specific photo-kinetic models. Here we present a method, termed Distance Distribution Correction (DDC), to eliminate blinking-caused repeat localizations without any additional calibrations. The approach relies on the finding that the true pairwise distance distribution of different fluorophores in an SMLM image can be naturally obtained from the imaging sequence by using distances between localizations separated by a time much longer than the average fluorescence survival time. We show that using the true pairwise distribution we can define and then maximize the likelihood of obtaining a particular set of localizations void of blinking-artifacts, generating an accurate reconstruction of the underlying cellular structure. Using both simulated and experimental data, we show that DDC surpasses all previous existing blinking-artifact correction methodologies, resulting in drastic improvements in obtaining the closest estimate of the true spatial organization and number of fluorescent emitters in a wide range of applications. The simplicity and robustness of DDC will allow it to become the field standard in SMLM imaging, enabling the most accurate reconstruction and quantification of SMLM images to date.

Li D., Cho Y.K.

Antibodies raised against defined phosphorylation sites of the microtubule-associated protein tau are widely used in scientific research and being applied in clinical assays. However, recent studies have revealed an alarming degree of non-specific binding found in these antibodies. In order to quantify and compare the specificity phospho-tau antibodies and other post-translational modification site-specific antibodies in general, a measure of specificity is urgently needed. Here, we report a robust flow cytometry assay using human embryonic kidney cells that enables the determination of a specificity parameter termed Φ, which measures the fraction of non-specific signal in antibody binding. We validate our assay using anti-tau antibodies with known specificity profiles, and apply it to measure the specificity of seven widely used phospho-tau antibodies (AT270, AT8, AT100, AT180, PHF-6, TG-3, and PHF-1) among others. We successfully determined the Φ values for all antibodies except AT100, which did not show detectable binding in our assay. Our results show that antibodies AT8, AT180, PHF-6, TG-3, and PHF-1 have Φ values near 1, which indicates no detectable non-specific binding. AT270 showed Φ value around 0.8, meaning that approximately 20% of the binding signal originates from non-specific binding. Further analyses using immunocytochemistry and western blotting confirmed the presence of non-specific binding of AT270 to non-tau proteins found in human embryonic kidney cells and the mouse hippocampus. We anticipate that the quantitative approach and parameter introduced here will be widely adopted as a standard for reporting the specificity for phospho-tau antibodies, and potentially for post-translational modification targeting antibodies in general. Cover Image for this issue: doi: 10.1111/jnc.14727.

Siahaan V., Krattenmacher J., Hyman A.A., Diez S., Hernández-Vega A., Lansky Z., Braun M.

Tau is an intrinsically disordered protein, which diffuses on microtubules1. In neurodegenerative diseases, collectively termed tauopathies, malfunction of tau and its detachment from axonal microtubules are correlated with axonal degeneration2. Tau can protect microtubules from microtubule-degrading enzymes such as katanin3. However, how tau carries out this regulatory function is still unclear. Here, using in vitro reconstitution, we show that tau molecules on microtubules cooperatively form cohesive islands that are kinetically distinct from tau molecules that individually diffuse on microtubules. Dependent on the tau concentration in solution, the islands reversibly grow or shrink by addition or release of tau molecules at their boundaries. Shielding microtubules from kinesin-1 motors and katanin, the islands exhibit regulatory qualities distinct from a comparably dense layer of diffusible tau. Superprocessive kinesin-8 motors penetrate the islands and cause their disassembly. Our results reveal a microtubule-dependent phase of tau that constitutes an adaptable protective layer on the microtubule surface. We anticipate that other intrinsically disordered axonal proteins display a similar cooperative behaviour and potentially compete with tau in regulating access to the microtubule surface. Tan et al. and Siahaan et al. present distinct but complementary data showing that microtubule regulates dynamic condensation of tau molecules, and this in turn affects microtubule biology and function.

Tan R., Lam A.J., Tan T., Han J., Nowakowski D.W., Vershinin M., Simó S., Ori-McKenney K.M., McKenney R.J.

Tau is an abundant microtubule-associated protein in neurons. Tau aggregation into insoluble fibrils is a hallmark of Alzheimer’s disease and other types of dementia1, yet the physiological state of tau molecules within cells remains unclear. Using single-molecule imaging, we directly observe that the microtubule lattice regulates reversible tau self-association, leading to localized, dynamic condensation of tau molecules on the microtubule surface. Tau condensates form selectively permissible barriers, spatially regulating the activity of microtubule-severing enzymes and the movement of molecular motors through their boundaries. We propose that reversible self-association of tau molecules, gated by the microtubule lattice, is an important mechanism of the biological functions of tau, and that oligomerization of tau is a common property shared between the physiological and disease-associated forms of the molecule. Tan et al. and Siahaan et al. present distinct but complementary data showing that microtubule regulates dynamic condensation of tau molecules, and this in turn affects microtubule biology and function.

Falcon B., Zivanov J., Zhang W., Murzin A.G., Garringer H.J., Vidal R., Crowther R.A., Newell K.L., Ghetti B., Goedert M., Scheres S.H.

Chronic traumatic encephalopathy (CTE) is a neurodegenerative tauopathy that is associated with repetitive head impacts or exposure to blast waves. First described as punch-drunk syndrome and dementia pugilistica in retired boxers1–3, CTE has since been identified in former participants of other contact sports, ex-military personnel and after physical abuse4–7. No disease-modifying therapies currently exist, and diagnosis requires an autopsy. CTE is defined by an abundance of hyperphosphorylated tau protein in neurons, astrocytes and cell processes around blood vessels8,9. This, together with the accumulation of tau inclusions in cortical layers II and III, distinguishes CTE from Alzheimer’s disease and other tauopathies10,11. However, the morphologies of tau filaments in CTE and the mechanisms by which brain trauma can lead to their formation are unknown. Here we determine the structures of tau filaments from the brains of three individuals with CTE at resolutions down to 2.3 Å, using cryo-electron microscopy. We show that filament structures are identical in the three cases but are distinct from those of Alzheimer’s and Pick’s diseases, and from those formed in vitro12–15. Similar to Alzheimer’s disease12,14,16–18, all six brain tau isoforms assemble into filaments in CTE, and residues K274–R379 of three-repeat tau and S305–R379 of four-repeat tau form the ordered core of two identical C-shaped protofilaments. However, a different conformation of the β-helix region creates a hydrophobic cavity that is absent in tau filaments from the brains of patients with Alzheimer’s disease. This cavity encloses an additional density that is not connected to tau, which suggests that the incorporation of cofactors may have a role in tau aggregation in CTE. Moreover, filaments in CTE have distinct protofilament interfaces to those of Alzheimer’s disease. Our structures provide a unifying neuropathological criterion for CTE, and support the hypothesis that the formation and propagation of distinct conformers of assembled tau underlie different neurodegenerative diseases. Cryo-electron microscopy structures of tau filaments from the brains of three individuals with chronic traumatic encephalopathy reveal distinct assembled tau conformers, with a novel protofilament fold enclosing hydrophobic molecules.

Zhang W., Falcon B., Murzin A.G., Fan J., Crowther R.A., Goedert M., Scheres S.H.

Assembly of microtubule-associated protein tau into filamentous inclusions underlies a range of neurodegenerative diseases. Tau filaments adopt different conformations in Alzheimer’s and Pick’s diseases. Here, we used cryo- and immuno- electron microscopy to characterise filaments that were assembled from recombinant full-length human tau with four (2N4R) or three (2N3R) microtubule-binding repeats in the presence of heparin. 2N4R tau assembles into multiple types of filaments, and the structures of three types reveal similar ‘kinked hairpin’ folds, in which the second and third repeats pack against each other. 2N3R tau filaments are structurally homogeneous, and adopt a dimeric core, where the third repeats of two tau molecules pack in a parallel manner. The heparin-induced tau filaments differ from those of Alzheimer’s or Pick’s disease, which have larger cores with different repeat compositions. Our results illustrate the structural versatility of amyloid filaments, and raise questions about the relevance of in vitro assembly.

Merezhko M., Brunello C.A., Yan X., Vihinen H., Jokitalo E., Uronen R., Huttunen H.J.

Tauopathies are characterized by cerebral accumulation of Tau protein aggregates that appear to spread throughout the brain via a cell-to-cell transmission process that includes secretion and uptake of pathological Tau, followed by templated misfolding of normal Tau in recipient cells. Here, we show that phosphorylated, oligomeric Tau clusters at the plasma membrane in N2A cells and is secreted in vesicle-free form in an unconventional process sensitive to changes in membrane properties, particularly cholesterol and sphingomyelin content. Cell surface heparan sulfate proteoglycans support Tau secretion, possibly by facilitating its release after membrane penetration. Notably, secretion of endogenous Tau from primary cortical neurons is mediated, at least partially, by a similar mechanism. We suggest that Tau is released from cells by an unconventional secretory mechanism that involves its phosphorylation and oligomerization and that membrane interaction may help Tau to acquire properties that allow its escape from cells directly through the plasma membrane.

Ondrejcak T., Klyubin I., Corbett G.T., Fraser G., Hong W., Mably A.J., Gardener M., Hammersley J., Perkinton M.S., Billinton A., Walsh D.M., Rowan M.J.

Intracellular neurofibrillary tangles (NFTs) composed of tau protein are a neuropathological hallmark of several neurodegenerative diseases, the most common of which is Alzheimer's disease (AD). For some time NFTs were considered the primary cause of synaptic dysfunction and neuronal death, however, more recent evidence suggests that soluble aggregates of tau are key drivers of disease. Here we investigated the effect of different tau species on synaptic plasticity in the male rat hippocampusin vivo. Intracerebroventricular injection of soluble aggregates formed from either wild-type or P301S human recombinant tau potently inhibited hippocampal long-term potentiation (LTP) at CA3-to-CA1 synapses. In contrast, tau monomers and fibrils appeared inactive. Neither baseline synaptic transmission, paired-pulse facilitation nor burst response during high-frequency conditioning stimulation was affected by the soluble tau aggregates. Similarly, certain AD brain soluble extracts inhibited LTP in a tau-dependent manner that was abrogated by either immunodepletion with, or coinjection of, a mid-region anti-tau monoclonal antibody (mAb), Tau5. Importantly, this tau-mediated block of LTP was prevented by administration of mAbs selective for the prion protein (PrP). Specifically, mAbs to both the mid-region (6D11) and N-terminus (MI-0131) of PrP prevented inhibition of LTP by both recombinant and brain-derived tau. These findings indicate that PrP is a mediator of tau-induced synaptic dysfunction.SIGNIFICANCE STATEMENTHere we report that certain soluble forms of tau selectively disrupt synaptic plasticity in the live rat hippocampus. Further, we show that monoclonal antibodies to cellular prion protein abrogate the impairment of long-term potentiation caused both by recombinant and Alzheimer's disease brain-derived soluble tau. These findings support a critical role for cellular prion protein in the deleterious synaptic actions of extracellular soluble tau in tauopathies, including Alzheimer's disease. Thus, approaches targeting cellular prion protein, or downstream pathways, might provide an effective strategy for developing therapeutics.

Falcon B., Zhang W., Murzin A.G., Murshudov G., Garringer H.J., Vidal R., Crowther R.A., Ghetti B., Scheres S.H., Goedert M.

The ordered assembly of tau protein into abnormal filamentous inclusions underlies many human neurodegenerative diseases1. Tau assemblies seem to spread through specific neural networks in each disease2, with short filaments having the greatest seeding activity3. The abundance of tau inclusions strongly correlates with disease symptoms4. Six tau isoforms are expressed in the normal adult human brain—three isoforms with four microtubule-binding repeats each (4R tau) and three isoforms that lack the second repeat (3R tau)1. In various diseases, tau filaments can be composed of either 3R or 4R tau, or of both. Tau filaments have distinct cellular and neuroanatomical distributions5, with morphological and biochemical differences suggesting that they may be able to adopt disease-specific molecular conformations6,7. Such conformers may give rise to different neuropathological phenotypes8,9, reminiscent of prion strains10. However, the underlying structures are not known. Using electron cryo-microscopy, we recently reported the structures of tau filaments from patients with Alzheimer’s disease, which contain both 3R and 4R tau11. Here we determine the structures of tau filaments from patients with Pick’s disease, a neurodegenerative disorder characterized by frontotemporal dementia. The filaments consist of residues Lys254–Phe378 of 3R tau, which are folded differently from the tau filaments in Alzheimer’s disease, establishing the existence of conformers of assembled tau. The observed tau fold in the filaments of patients with Pick’s disease explains the selective incorporation of 3R tau in Pick bodies, and the differences in phosphorylation relative to the tau filaments of Alzheimer’s disease. Our findings show how tau can adopt distinct folds in the human brain in different diseases, an essential step for understanding the formation and propagation of molecular conformers. The structures of tau filaments from patients with the neurodegenerative disorder Pick’s disease show that the filament fold is different from that of the tau filaments found in Alzheimer’s disease.

Qiang L., Sun X., Austin T.O., Muralidharan H., Jean D.C., Liu M., Yu W., Baas P.W.

It is widely believed that tau stabilizes microtubules in the axon [1-3] and, hence, that disease-induced loss of tau from axonal microtubules leads to their destabilization [3-5]. An individual microtubule in the axon has a stable domain and a labile domain [6-8]. We found that tau is more abundant on the labile domain, which is inconsistent with tau's proposed role as a microtubule stabilizer. When tau is experimentally depleted from cultured rat neurons, the labile microtubule mass of the axon drops considerably, the remaining labile microtubule mass becomes less labile, and the stable microtubule mass increases. MAP6 (also called stable tubule-only polypeptide), which is normally enriched on the stable domain [9], acquires a broader distribution across the microtubule when tau is depleted, providing a potential explanation for the increase in stable microtubule mass. When MAP6 is depleted, the labile microtubule mass becomes even more labile, indicating that, unlike tau, MAP6 is a genuine stabilizer of axonal microtubules. We conclude that tau is not a stabilizer of axonal microtubules but is enriched on the labile domain of the microtubule to promote its assembly while limiting the binding to it of genuine stabilizers, such as MAP6. This enables the labile domain to achieve great lengths without being stabilized. These conclusions are contrary to tau dogma.

Kellogg E.H., Hejab N.M., Poepsel S., Downing K.H., DiMaio F., Nogales E.

Tackling microtubule-tau interactions Alzheimer's disease is a major cause of death in the elderly. Disease progression is associated with the accumulation of neurofibrillary tangles composed of tau, a protein important for neuronal development and function. Tangle formation is preceded by phosphorylation events that cause tau to dissociate from its native binding partner, microtubules. Microtubule-tau interactions have been mysterious. Kellogg et al. used cryo–electron microscopy and molecular modeling to show how tau interacts with the outer surface of the microtubule, stapling together tubulin subunits and thus stabilizing the polymer. A key tau amino acid within the tightly bound segment between tubulin subunits corresponds to a clinically relevant site of tau phosphorylation, explaining the competition between microtubule interaction and tau aggregation. Science , this issue p. 1242

Spiess M., Hernandez-Varas P., Oddone A., Olofsson H., Blom H., Waithe D., Lock J.G., Lakadamyali M., Strömblad S.

Integrins are the core constituents of cell–matrix adhesion complexes such as focal adhesions (FAs) and play key roles in physiology and disease. Integrins fluctuate between active and inactive conformations, yet whether the activity state influences the spatial organization of integrins within FAs has remained unclear. In this study, we address this question and also ask whether integrin activity may be regulated either independently for each integrin molecule or through locally coordinated mechanisms. We used two distinct superresolution microscopy techniques, stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion microscopy (STED), to visualize active versus inactive β1 integrins. We first reveal a spatial hierarchy of integrin organization with integrin molecules arranged in nanoclusters, which align to form linear substructures that in turn build FAs. Remarkably, within FAs, active and inactive β1 integrins segregate into distinct nanoclusters, with active integrin nanoclusters being more organized. This unexpected segregation indicates synchronization of integrin activities within nanoclusters, implying the existence of a coordinate mechanism of integrin activity regulation.

Rahman M.W., Sharma P., Chattopadhyay T., Saroja S.R.

Tsoi P.S., Lucas L., Rhoades D., Ferreon J.C., Ferreon A.C.

Biomolecular condensates (BMCs) are membrane-less protein compartments with physiological and pathological relevance. The formation of BMCs is driven by a process known as liquid–liquid phase separation (LLPS), a field that has largely focused on the study of micron-sized condensates. However, there have been recent studies showing that proteins that undergo LLPS also form nanometer-sized condensates. These nanometer-sized condensates, or nanocondensates, are distinct from microcondensates and potentially exhibit more relevance in cell biology. The field of nanocondensate research is in its infancy, with limited biophysical studies of these structures. Here, we studied condensate formation and dissolution of wild-type and disease-linked (hyperphosphorylated and missense mutated) Tau. We investigated the effects of solution condition modulation on nanocondensate formation and dissolution, and observed that Tau condensation is strongly regulated by electrostatic forces and less affected by hydrophobic disruption. We observed that all three Tau variants studied shared condensate formation properties when in solution conditions with the same ionic strength. However, hyperphosphorylated and missense-mutated Tau exhibited higher resistance to dissolution compared to wild-type Tau. This study uncovers additional distinctions between different types of condensates, which provides further insight into the distinctions between physiological and pathological condensates.

Lucas L., Tsoi P.S., Quan M.D., Choi K., Ferreon J.C., Ferreon A.C.

Proteins phase-separate to form condensates that partition and concentrate biomolecules into membraneless compartments. These condensates can exhibit dichotomous behaviors in biology by supporting cellular physiology or instigating pathological protein aggregation1–3. Tau and α- synuclein (αSyn) are neuronal proteins that form heterotypic (Tau:αSyn) condensates associated with both physiological and pathological processes. Tau and αSyn functionally regulate microtubules8–12, but are also known to misfold and co-deposit in aggregates linked to various neurodegenerative diseases4,5,6,7, which highlights the paradoxically ambivalent effect of Tau:αSyn condensation in health and disease. Here, we show that tubulin modulates Tau:αSyn condensates by promoting microtubule interactions, competitively inhibiting the formation of homotypic and heterotypic pathological oligomers. In the absence of tubulin, Tau-driven protein condensation accelerates the formation of toxic Tau:αSyn heterodimers and amyloid fibrils. However, tubulin partitioning into Tau:αSyn condensates modulates protein interactions, promotes microtubule polymerization, and prevents Tau and αSyn oligomerization and aggregation. We distinguished distinct Tau and αSyn structural states adopted in tubulin-absent (pathological) and tubulin-rich (physiological) condensates, correlating compact conformations with aggregation and extended conformations with function. Furthermore, using various neuronal cell models, we showed that loss of stable microtubules, which occurs in Alzheimer’s disease and Parkinsons disease patients13,14, results in pathological oligomer formation and loss of neurites, and that functional condensation using an inducible optogenetic Tau construct resulted in microtubule stablization. Our results identify that tubulin is a critical modulator in switching Tau:αSyn pathological condensates to physiological, mechanistically relating the loss of stable microtubules with disease progression. Tubulin restoration strategies and Tau-mediated microtubule stabilization can be potential therapies targeting both Tau-specific and Tau/αSyn mixed pathologies.

Lucas L., Tsoi P.S., Ferreon J.C., Ferreon A.C.

Tau is a microtubule-associated protein that undergoes liquid–liquid phase separation (LLPS) to form condensates under physiological conditions, facilitating microtubule stabilization and intracellular transport. LLPS has also been implicated in pathological Tau aggregation, which contributes to tauopathies such as Alzheimer’s disease. While LLPS is known to promote Tau aggregation, the relationship between Tau’s structural states and its phase separation behavior remains poorly defined. Here, we examine how oligomerization modulates Tau LLPS and uncover key distinctions between monomeric, oligomeric, and amyloidogenic Tau species. Using dynamic light scattering and fluorescence microscopy, we monitored oligomer formation over time and assessed oligomeric Tau’s ability to undergo LLPS. We found that Tau monomers readily phase separate and form condensates. As oligomerization progresses, Tau’s propensity to undergo LLPS diminishes, with oligomers still being able to phase separate, albeit with reduced efficiency. Interestingly, oligomeric Tau is recruited into condensates formed with 0-day-aged Tau, with this recruitment depending on the oligomer state of maturation. Early-stage, Thioflavin T (ThT)-negative oligomers co-localize with 0-day-aged Tau condensates, whereas ThT-positive oligomers resist condensate recruitment entirely. This study highlights a dynamic interplay between Tau LLPS and aggregation, providing insight into how Tau’s structural and oligomeric states influence its pathological and functional roles. These findings underscore the need to further explore LLPS as a likely modulator of Tau pathogenesis and distinct pathogenic oligomers as viable therapeutic targets in tauopathies.

Chinnathambi S., Rangappa N., Chandrashekar M.

Fan X., Okada K., Lin H., Ori-McKenney K.M., McKenney R.J.

AbstractPhosphorylation plays a crucial role in both normal and disease processes involving the microtubule-associated protein tau. Physiologically, phosphorylation regulates tau’s subcellular localization within neurons and is involved in fetal development and animal hibernation. However, abnormal phosphorylation of tau is linked to the formation of neurofibrillary tangles (NFTs) in various human tauopathies. Interestingly, the patterns of tau phosphorylation are similar in both normal and abnormal processes, leaving unclear whether phosphorylated tau retains its functional role in normal processes. The relationship between tau phosphorylation and NFT assembly in tauopathies is also still debated. To address these questions, we investigated the effects of tau phosphorylation on microtubule binding, cooperative protein envelope formation, and NFT filament assembly relevant to tauopathies. Consistent with previous results, our findings show that tau phosphorylation decreases tau’s overall affinity for microtubules, but we reveal that phosphorylation more dramatically impacts the cooperativity between tau molecules during tau envelope formation along microtubules. Additionally, we observed that the specific pattern of phosphorylation, rather than overall phosphorylation level, strongly impacts the assembly of tau filamentsin vitro. Our results reveal new insights into how tau phosphorylation impacts tau’s physiological roles on microtubules and its pathoconversion into NFTs.

Jamerlan A.M., Shim K.H., Sharma N., An S.S.

Depositions of protein aggregates are typical pathological hallmarks of various neurodegenerative diseases (NDs). For example, amyloid-beta (Aβ) and tau aggregates are present in the brain and plasma of patients with Alzheimer’s disease (AD); α-synuclein in Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA); mutant huntingtin protein (Htt) in Huntington’s disease (HD); and DNA-binding protein 43 kD (TDP-43) in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and limbic-predominant age-related TDP-43 encephalopathy (LATE). The same misfolded proteins can be present in multiple diseases in the form of mixed proteinopathies. Since there is no cure for all these diseases, understanding the mechanisms of protein aggregation becomes imperative in modern medicine, especially for developing diagnostics and therapeutics. A Multimer Detection System (MDS) was designed to distinguish and quantify the multimeric/oligomeric forms from the monomeric form of aggregated proteins. As the unique epitope of the monomer is already occupied by capturing or detecting antibodies, the aggregated proteins with multiple epitopes would be accessible to both capturing and detecting antibodies simultaneously, and signals will be generated from the oligomers rather than the monomers. Hence, MDS could present a simple solution for measuring various conformations of aggregated proteins with high sensitivity and specificity, which may help to explore diagnostic and treatment strategies for developing anti-aggregation therapeutics.

Cehlar O., Njemoga S., Horvath M., Cizmazia E., Bednarikova Z., Barrera E.E.

In this review, we focus on the biophysical and structural aspects of the oligomeric states of physiologically intrinsically disordered proteins and peptides tau, amyloid-β and α-synuclein and partly disordered prion protein and their isolations from animal models and human brains. These protein states may be the most toxic agents in the pathogenesis of Alzheimer’s and Parkinson’s disease. It was shown that oligomers are important players in the aggregation cascade of these proteins. The structural information about these structural states has been provided by methods such as solution and solid-state NMR, cryo-EM, crosslinking mass spectrometry, AFM, TEM, etc., as well as from hybrid structural biology approaches combining experiments with computational modelling and simulations. The reliable structural models of these protein states may provide valuable information for future drug design and therapies.

Khodja L.A., Campanacci V., Lippens G., Gigant B.

Abstract Tau is a protein involved in the regulation of axonal microtubules in neurons. In pathological conditions, it forms filamentous aggregates which are molecular markers of neurodegenerative diseases known as tauopathies. Structures of Tau in fibrils or bound to the microtubule have been reported. We present here a structure of a Tau construct comprising the PHF6 motif, an oligopeptide involved in Tau aggregation, as a complex with tubulin. This Tau fragment binds as a dimer to a new site which, when transposed to the microtubule, would correspond to a pore between protofilaments. These results raise new hypotheses on Tau-induced microtubule assembly and stabilization and on Tau oligomerization.

Wang N., Cui J., Sun Z., Chen F., He X.

Soeda Y., Yoshimura H., Bannai H., Koike R., Shiiba I., Takashima A.

Intracellular tau aggregation requires a local protein concentration increase, referred to as "droplets". However, the cellular mechanism for droplet formation is poorly understood. Here, we expressed OptoTau, a P301L mutant tau fused with CRY2olig, a light-sensitive protein that can form homo-oligomers. Under blue light exposure, OptoTau increased tau phosphorylation and was sequestered in aggresomes. Suppressing aggresome formation by nocodazole formed tau granular clusters in the cytoplasm. The granular clusters disappeared by discontinuing blue light exposure or 1,6-hexanediol treatment suggesting that intracellular tau droplet formation requires microtubule collapse. Expressing OptoTau-ΔN, a species of N-terminal cleaved tau observed in the Alzheimer's disease brain, formed 1,6-hexanediol and detergent-resistant tau clusters in the cytoplasm with blue light stimulation. These intracellular stable tau clusters acted as a seed for tau fibrils in vitro. These results suggest that tau droplet formation and N-terminal cleavage are necessary for neurofibrillary tangles formation in neurodegenerative diseases.

Gregory J.A., Hickey C.M., Chavez J., Cacace A.M.

This minireview explores the burgeoning field of targeted protein degradation (TPD) and its promising applications in neuroscience and clinical development. TPD offers innovative strategies for modulating protein levels, presenting a paradigm shift in small-molecule drug discovery and therapeutic interventions. Importantly, small-molecule protein degraders specifically target and remove pathogenic proteins from central nervous system cells without the drug delivery challenges of genomic and antibody-based modalities. Here, we review recent advancements in TPD technologies, highlight proteolysis targeting chimera (PROTAC) protein degrader molecules with proximity-induced degradation event-driven and iterative pharmacology, provide applications in neuroscience research, and discuss the high potential for translation of TPD into clinical settings.

Hugelier S., Tang Q., Kim H.H., Gyparaki M.T., Bond C., Santiago-Ruiz A.N., Porta S., Lakadamyali M.

Single-molecule localization microscopy (SMLM) has gained widespread use for visualizing the morphology of subcellular organelles and structures with nanoscale spatial resolution. However, analysis tools for automatically quantifying and classifying SMLM images have lagged behind. Here we introduce Enhanced Classification of Localized Point clouds by Shape Extraction (ECLiPSE), an automated machine learning analysis pipeline specifically designed to classify cellular structures captured through two-dimensional or three-dimensional SMLM. ECLiPSE leverages a comprehensive set of shape descriptors, the majority of which are directly extracted from the localizations to minimize bias during the characterization of individual structures. ECLiPSE has been validated using both unsupervised and supervised classification on datasets, including various cellular structures, achieving near-perfect accuracy. We apply two-dimensional ECLiPSE to classify morphologically distinct protein aggregates relevant for neurodegenerative diseases. Additionally, we employ three-dimensional ECLiPSE to identify relevant biological differences between healthy and depolarized mitochondria. ECLiPSE will enhance the way we study cellular structures across various biological contexts. Enhanced Classification of Localized Point clouds by Shape Extraction (ECLiPSE) is a robust feature extraction and classification pipeline for diverse and heterogeneous structures in both 2D and 3D single-molecule localization microscopy data.

Hossen F., Sun G.Y., Lee J.C.

Despite of yet unknown mechanism, microvascular deposition of oligomeric Tau (oTau) has been implicated in alteration of the Blood-Brain Barrier (BBB) function in Alzheimer's disease (AD) brains. In this study, we employed an in vitro BBB model using primary mouse cerebral endothelial cells (CECs) to investigate the mechanism underlying the effects of oTau on BBB function. We found that exposing CECs to oTau induced oxidative stress through NADPH oxidase, increased oxidative damage to proteins, decreased proteasome activity, and expressions of tight junction (TJ) proteins including occludin, zonula occludens-1 (ZO-1) and claudin-5. These effects were suppressed by the pretreatment with Fasudil, a RhoA/ROCK signaling inhibitor. Consistent with the biochemical alterations, we found that exposing the basolateral side of CECs to oTau in the BBB model disrupted the integrity of the BBB, as indicated by an increase in FITC-dextran transport across the model, and a decrease in trans endothelial electrical resistance (TEER). oTau also increased the transmigration of peripheral blood mononuclear cells (PBMCs) in the BBB model. These functional alterations in the BBB induced by oTau were also suppressed by Fasudil. Taken together, our findings suggest that targeting the RhoA/ROCK pathway can be a potential therapeutic strategy to maintain BBB function in AD.

Yi L.X., Zeng L., Wang Q., Tan E.K., Zhou Z.D.

Alzheimer's disease (AD) is the most common neurodegenerative disorder that affects the cerebral cortex and hippocampus, and is characterised by progressive cognitive decline and memory loss. A recent report of a patient carrying a novel gain-of-function variant of RELN (H3447R, termed RELN-COLBOS) who developed resilience against presenilin-linked autosomal-dominant AD (ADAD) has generated enormous interest. The RELN-COLBOS variant enhances interactions with the apolipoprotein E receptor 2 (ApoER2) and very-low-density lipoprotein receptor (VLDLR), which are associated with delayed AD onset and progression. These findings were validated in a transgenic mouse model. Reelin is involved in neurodevelopment, neurogenesis, and neuronal plasticity. The evidence accumulated thus far has demonstrated that the Reelin pathway links apolipoprotein E4 (ApoE4), amyloid-β (Aβ), and tubulin-associated unit (Tau), which are key proteins that have been implicated in AD pathogenesis. Reelin and key components of the Reelin pathway have been highlighted as potential therapeutic targets and biomarkers for AD.