Toxic Tau Oligomers Research Articles

RNA‐Binding Proteins Musashi and Tau Aggresome in Alzheimer’s disease

AbstractBackgroundTauopathies, such as Alzheimer’s disease (AD), are characterized by the accumulation of toxic tau oligomers in the brain and accumulation of proteinaceous inclusions stored in cellular structures called aggresomes. Recent studies suggest that AD also exhibits pathology related to RNA binding proteins (RBPs), including Musashi proteins. Our lab has previously discovered that Musashi 1 (MSI1) and Musashi 2 (MSI2) aggregate in human AD brains and we have also visualized co‐localization with tau oligomers, suggesting toxic direct crosstalk between tau and MSI proteins. With this discovery and the growing evidence indicating MSI proteins as contributors to AD pathology, through their loss of functions it is crucial to elucidate how the interactions between misfolded tau and MSI influence tau aggregation and perhaps formation of cellular aggresomes.MethodIn this study, we characterize which tau oligomeric species, including phosphorylated and ubiquitinated ones, interact with MSI within the human AD brain (Braak Stage V‐VI). We investigate the deposition and cellular location of MSI and tau aggregates in AD and age‐matched control human cortical brain tissues by biochemical, immunohistochemistry and co‐immunofluorescence assays. Using high‐resolution microscopy and imaging analysis we measured the differences in size and morphology of MSI/tau co‐aggregates.ResultInvestigation of these co‐aggregates showed specific accumulation in neurons as well as in glial cells (astrocytes and microglia). We performed biochemical assays using commercial and in‐house antibodies to qualitatively investigate MSI1, MSI2, and tau across in AD brains. Co‐IF was performed to investigate the spatial and quantitative distribution of MSI1, MSI2, and tau aggregates. MSI/tau aggregate morphological characterization was conducted to profile their size and shape in AD cortices.ConclusionFor the first time, we show that the presence and quantity of tau oligomeric species and MSI co‐aggregates vary across the AD brains, showing inter‐subjects’ variability in size and deposition density. We also identify that tau conformers with MSI1 and MSI2 form different co‐aggregates in size and morphology quantifying their colocalization in high‐resolution 3D imaging. Further investigations are warranted to determine the temporal and regional distribution of these tau species in association with MSI proteins as well as the mechanisms underlying their aggregation.

De Novo Design of a β-Helix Tau Protein Scaffold: An Oligomer-Selective Vaccine Immunogen Candidate for Alzheimer's Disease.

Tau pathology is associated with many neurodegenerative disorders, including Alzheimer's disease (AD), where the spatio-temporal pattern of tau neurofibrillary tangles strongly correlates with disease progression, which motivates therapeutics selective for misfolded tau. Here, we introduce a new avidity-enhanced, multi-epitope approach for protein-misfolding immunogen design, which is predicted to mimic the conformational state of an exposed epitope in toxic tau oligomers. A predicted oligomer-selective tau epitope 343KLDFK347 was scaffolded by designing a β-helix structure that incorporated multiple instances of the 16-residue tau fragment 339VKSEKLDFKDRVQSKI354. Large-scale conformational ensemble analyses involving Jensen-Shannon Divergence and the embedding depth showed that the multi-epitope scaffolding approach, employed in designing the β-helix scaffold, was predicted to better discriminate toxic tau oligomers than other "monovalent" strategies utilizing a single instance of an epitope for vaccine immunogen design. Using Rosetta, 10,000 sequences were designed and screened for the linker portions of the β-helix scaffold, along with a C-terminal stabilizing α-helix that interacts with the linkers, to optimize the folded structure and stability of the scaffold. Structures were ranked by energy, and the lowest 1% (82 unique sequences) were verified using AlphaFold. Several selection criteria involving AlphaFold are implemented to obtain a lead-designed sequence. The structure was further predicted to have free energetic stability by using Hamiltonian replica exchange molecular dynamics (MD) simulations. The synthesized β-helix scaffold showed direct binding in surface plasmon resonance (SPR) experiments to several antibodies that were raised to the structured epitope using a designed cyclic peptide. Moreover, the strength of binding of these antibodies to in vitro tau oligomers correlated with the strength of binding to the β-helix construct, suggesting that the construct presents an oligomer-like conformation and may thus constitute an effective oligomer-selective immunogen.

Targeted Musashi1 knockdown leads to reduce tau pathology and improves cognitive function in aged P301L mouse model

AbstractBackgroundThe buildup of toxic tau species in the brain is thought to lead to the development of Alzheimer’s disease. While our current understanding of the mechanisms underlying this aggregation is limited, growing evidence has pointed to RNA‐binding proteins (RBPs) playing a role. The dysregulation of RBPs is associated with various neurodegenerative diseases. For example, during amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTLD), dysregulated TDP‐43 accumulates in the brain. Similarly, our recent studies showed that the RBP, Musashi‐1 (MSI1), may co‐aggregate with toxic tau oligomers in Alzheimer’s disease. Clarifying the connection between these two proteins may provide novel insights into the mechanisms behind toxic tau buildup in Alzheimer’s, allowing for new therapeutic targets in treatment development.MethodIn this study, we investigated the role of MSI1 on the aggregation and deposition of tau in aged (18‐months old) P301L mouse models, which express the P301L mutant form of tau. MSI1 expression was knocked down (KD) by performing intracerebroventricular injections of MSI1 antisense oligonucleotides into the hippocampus. The effects of MSI1 KD on tau accumulation and pathology were studied in the hippocampus, cortex, and cerebellum.ResultWestern blot and immunofluorescence (IF) analysis revealed a significant reduction in MSI1 and tau proteins levels and aggregation in the P301L mouse brain, mainly in the hippocampus. Furthermore, brain Co‐IF microscopy and Dot Blot analysis of the PBS‐ and Sarkosyl‐soluble fractions showed that there was a significant reduction of MSI1 and tau oligomeric species in the hippocampus, cortex, and cerebellum in MSI1 KD mice. In addition, phosphorylated tau forms (pSer422, pSer202/Thr205, pThr212/Ser214 and pThr231/Ser235) were significantly decreased in the cortex, cerebellum, and most prominently, in the hippocampus. Increase in synaptic marker Synaptophysin suggesting an increase of synaptogenesis. Behavioral tests, Novel Object Recognition (NOR) and Y‐Maze spontaneous alternations tests, MSI1‐KD mice showed significant improvements.ConclusionThese results suggest that MSI1 plays a major role in tau toxicity, revealing it as a possible target for treating Alzheimer’s disease.

Heterogeneous Tau Oligomers as Molecular Targets for Alzheimer’s Disease and Related Tauopathies

Tauopathies, including Alzheimer’s disease (AD), are a group of neurodegenerative disorders characterized by pathological aggregation of microtubule binding protein tau. The presence of tau neurofibrillary tangles, which are insoluble β-sheet fibrils, in the brain has been the histopathological hallmark of these diseases as their level correlates with the degree of cognitive impairment. However, recent studies suggest that tau oligomers, which are soluble proteins that are formed prior to insoluble fibrils, are the principal toxic species impairing neurons and inducing neurodegeneration. Targeting toxic tau oligomers is challenging, as they are mostly unstructured and adopting multiple conformations. The heterogeneity of tau oligomers is further illustrated by the different oligomeric species formed by various methods. The current models and technologies to study tau oligomerization represent important resources and avenues to push the forefront of elucidating the true toxic tau species. In this review, we will summarize the distinct tau oligomers generated using different strategies and discuss their conformational characteristics, neurotoxicity, relevance to pathological phenotypes, as well as their applications in drug discovery. This information will provide insights to understanding heterogeneous tau oligomers and their role as molecular targets for AD and related tauopathies.

Single cell transcriptomic profiling of a neuron-astrocyte assembloid tauopathy model

The use of iPSC derived brain organoid models to study neurodegenerative disease has been hampered by a lack of systems that accurately and expeditiously recapitulate pathogenesis in the context of neuron-glial interactions. Here we report development of a system, termed AstTau, which propagates toxic human tau oligomers in iPSC derived neuron-astrocyte assembloids. The AstTau system develops much of the neuronal and astrocytic pathology observed in tauopathies including misfolded, phosphorylated, oligomeric, and fibrillar tau, strong neurodegeneration, and reactive astrogliosis. Single cell transcriptomic profiling combined with immunochemistry characterizes a model system that can more closely recapitulate late-stage changes in adult neurodegeneration. The transcriptomic studies demonstrate striking changes in neuroinflammatory and heat shock protein (HSP) chaperone systems in the disease process. Treatment with the HSP90 inhibitor PU-H71 is used to address the putative dysfunctional HSP chaperone system and produces a strong reduction of pathology and neurodegeneration, highlighting the potential of AstTau as a rapid and reproducible tool for drug discovery.

Aging-associated REGγ proteasome decline predisposes to tauopathy

The REGγ-20S proteasome is an ubiquitin- and ATP-independent degradation system, targeting selective substrates, possibly helping to regulate aging. The studies we report here demonstrate that aging-associated REGγ decline predisposes to decreasing tau turnover, as in a tauopathy. The REGγ proteasome promotes degradation of human and mouse tau, notably phosphorylated tau and toxic tau oligomers that shuttle between the cytoplasm and nuclei. REGγ-mediated proteasomal degradation of tau was validated in 3- to 12-month-old REGγ KO mice, REGγ KO;PS19 mice, and PS19 mice with forebrain conditional neuron-specific overexpression of REGγ (REGγ OE) and behavioral abnormalities. Coupled with tau accumulation, we found with REGγ-deficiency, neuron loss, dendrite reduction, tau filament accumulation, and microglial activation are much more prominent in the REGγ KO;PS19 than the PS19 model. Moreover, we observed that the degenerative neuronal lesions and aberrant behaviors were alleviated in REGγ OE;PS19 mice. Memory and other behavior analysis substantiate the role of REGγ in prevention of tauopathy-like symptoms. In addition, we investigated the potential mechanism underlying aging-related REGγ decline. This study provides valuable insights into the novel regulatory mechanisms and potential therapeutic targets for tau-related neurodegenerative diseases.

Pathological tau signatures and nuclear alterations in neurons, astrocytes and microglia in Alzheimer's disease, progressive supranuclear palsy, and dementia with Lewy bodies.

Accumulation of pathological tau aggregates is a prominent feature in tauopathies that leads during the course of the diseases to neuronal dysfunction before and cell death after. Microglia and astrocytes have been described as playing important roles in synaptic spreading of toxic tau in several neurodegenerative diseases (NDs). Here, we have investigated the immunological and biochemical properties of aggregated tau species in different brain cell types in tau-induced neurodegenerative diseases such as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and dementia with Lewy bodies (DLB). Additionally, we examined nuclear size, nuclear density, and chromatin compaction in neuronal and glial cells from diseased brain tissues. Microscopic-histological examination was performed using in-house mouse monoclonal antibodies for toxic tau conformers (TTC-M1 and TTC-M2) and tau oligomers (TOMA1-4). By immunohistochemistry and co-immunofluorescence assays using TOMA/TTC-Ms and cell-type specific markers for neurons, astrocytes, and microglia, we observed that TOMA/TTC-Ms were immunoreactive to diverse tau species in different cell types. Analysis of colocalization coefficients indicated an increased pathological tau deposition mainly in the neurons. Western blot analysis of brain homogenates using TOMA/TTC-Ms revealed distinct patterns of tau aggregation in each disease, suggesting that TOMA/TTC-Ms can distinguish between different tau aggregates present in different tauopathies. Additionally, using DAPI staining, we observed that neuronal and astrocytic nuclei had significantly greater nuclear area and increased chromatin compaction in AD cortices compared to non-demented controls. In contrast, reduction in nuclear density/area and more relaxed chromatin was noticed in DLB neurons, astrocytes and microglia and PSP astrocytes and microglia. Cell-type specific tropism of toxic tau species in tauopathies will provide a greater understanding of the involvement of different brain cell types in tau pathology. In this study, we observed that each disease presented cell-type specific nuclear phenotype and tau deposition pattern.

In vivo validation of therapeutic efficacy of a small molecule lead targeting tau self‐association in JNPL3 mice

AbstractBackgroundSmall molecules were screened and optimized for inhibiting tau self‐association, the initial step in the formation of soluble toxic tau oligomers and larger insoluble aggregates. This approach is differentiated from other tau aggregation inhibitor programs that have largely focused on inhibiting formation and/or dissociating large and relatively inert fibrils which could generate toxic tau oligomer seeds. Small molecules inhibitors are agnostic to specific post‐translational modifications, conformational changes or strains of tau aggregates, and have superior access to their target in the CNS relative to immunotherapeutic approaches. We have previously shown in vivo efficacy of a lead compound in preventive studies in htau (Davidowitz et al., 2020) and human tau P301L JNPL3 mouse models of tauopathy. The tau P301L mutation is the most common FTDP‐17 mutation and causes tau aggregation and motor deficits with age (Lewis et al., 2000).MethodBlinded studies of homozygous female tau 4R/0N P301L JNPL3 mice were performed at an independent laboratory where mice were treated for five months by administration of our lead compound in feed from 7 to 12 months‐of‐age. The four groups of mice in the study were baseline (7 months; n=20), vehicle, and two treatment groups (40 and 80 mg/kg doses; n=25 each). To quantify levels of Sarkosyl‐insoluble tau, self‐associated tau, and phosphorylated tau in the treated and control groups of mice ELISAs were performed (Acker et al., 2012; Forest et al., 2019). Rotarod and open field studies were performed to evaluate motor behavior.ResultEfficacy was shown in the cortex of treated mice that had reduced levels of self‐associated tau below baseline, baseline levels of insoluble tau aggregates, and reduced levels of aberrantly phosphorylated tau with statistical significance. In the hindbrain there was reduction of insoluble tau aggregates in the 40 mg/kg group compared to vehicle. Treatment groups showed improved Rotarod performance compared to baseline and vehicle groups.ConclusionTreatment with the lead compound reduced tau aggregation and improved motor behavior in aged JNPL3 mice and support preclinical development of the lead for clinical studies.

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.

Rutin prevents tau pathology and neuroinflammation in a mouse model of Alzheimer\u2019s disease

BackgroundTau pathology is a hallmark of Alzheimer’s disease (AD) and other tauopathies. During disease progression, abnormally phosphorylated forms of tau aggregate and accumulate into neurofibrillary tangles, leading to synapse loss, neuroinflammation, and neurodegeneration. Thus, targeting of tau pathology is expected to be a promising strategy for AD treatment.MethodsThe effect of rutin on tau aggregation was detected by thioflavin T fluorescence and transmission electron microscope imaging. The effect of rutin on tau oligomer-induced cytotoxicity was assessed by MTT assay. The effect of rutin on tau oligomer-mediated the production of IL-1β and TNF-α in vitro was measured by ELISA. The uptake of extracellular tau by microglia was determined by immunocytochemistry. Six-month-old male Tau-P301S mice were treated with rutin or vehicle by oral administration daily for 30 days. The cognitive performance was determined using the Morris water maze test, Y-maze test, and novel object recognition test. The levels of pathological tau, gliosis, NF-kB activation, proinflammatory cytokines such as IL-1β and TNF-α, and synaptic proteins including synaptophysin and PSD95 in the brains of the mice were evaluated by immunolabeling, immunoblotting, or ELISA.ResultsWe showed that rutin, a natural flavonoid glycoside, inhibited tau aggregation and tau oligomer-induced cytotoxicity, lowered the production of proinflammatory cytokines, protected neuronal morphology from toxic tau oligomers, and promoted microglial uptake of extracellular tau oligomers in vitro. When applied to Tau-P301S mouse model of tauopathy, rutin reduced pathological tau levels, regulated tau hyperphosphorylation by increasing PP2A level, suppressed gliosis and neuroinflammation by downregulating NF-kB pathway, prevented microglial synapse engulfment, and rescued synapse loss in mouse brains, resulting in a significant improvement of cognition.ConclusionIn combination with the previously reported therapeutic effects of rutin on Aβ pathology, rutin is a promising drug candidate for AD treatment based its combinatorial targeting of tau and Aβ.

C1 Inhibits Liquid-Liquid Phase Separation and Oligomerization of Tau and Protects Neuroblastoma Cells against Toxic Tau Oligomers.

The pathological aggregation of tau is one of the major contributing factors for several neurodegenerative tauopathies, including Alzheimer's disease. Here, we report that C1, a synthetic derivative of curcumin, strongly inhibited both the aggregation and filament formation of purified tau and protected neuroblastoma cells from the deleterious effects of the tau oligomers. Using confocal microscopy, C1 was found to reduce both the size and number of the tau droplets and increased the critical concentration of tau required for the droplet formation in vitro indicating that C1 suppressed the liquid-liquid phase separation of tau. C1 inhibited the aggregation of tau with a half-maximal inhibitory concentration of 1.5 ± 0.1 μM. An analysis of the aggregation kinetics data indicated that C1 strongly reduced the initial rate of the aggregation of tau. A dot blot analysis using tau-oligomer-specific antibody indicated that C1 inhibited the oligomerization of tau. Furthermore, dynamic light scattering experiments suggested that C1 strongly reduced the mean diameter of the tau oligomers. Atomic force microscopy experiments showed that C1 treatment reduced both the size and number of tau oligomers, suppressed the transition of tau oligomers into filaments, and also disintegrated preformed tau filaments. Also, the binding interaction of C1 with tau was monitored using absorbance and fluorescence spectroscopy. C1 bound to Y310W-tau with a dissociation constant of 2.0 ± 0.5 μM. The findings suggested that C1 is a potent inhibitor of tau aggregation and provided insights into the inhibitory mechanism of C1 on the oligomerization and fibril formation of tau.

TIA1 potentiates tau phase separation and promotes generation of toxic oligomeric tau

Tau protein plays an important role in the biology of stress granules and in the stress response of neurons, but the nature of these biochemical interactions is not known. Here we show that the interaction of tau with RNA and the RNA binding protein TIA1 is sufficient to drive phase separation of tau at physiological concentrations, without the requirement for artificial crowding agents such as polyethylene glycol (PEG). We further show that phase separation of tau in the presence of RNA and TIA1 generates abundant tau oligomers. Prior studies indicate that recombinant tau readily forms oligomers and fibrils in vitro in the presence of polyanionic agents, including RNA, but the resulting tau aggregates are not particularly toxic. We discover that tau oligomers generated during copartitioning with TIA1 are significantly more toxic than tau aggregates generated by incubation with RNA alone or phase-separated tau complexes generated by incubation with artificial crowding agents. This pathway identifies a potentially important source for generation of toxic tau oligomers in tau-related neurodegenerative diseases. Our results also reveal a general principle that phase-separated RBP droplets provide a vehicle for coassortment of selected proteins. Tau selectively copartitions with TIA1 under physiological conditions, emphasizing the importance of TIA1 for tau biology. Other RBPs, such as G3BP1, are able to copartition with tau, but this happens only in the presence of crowding agents. This type of selective mixing might provide a basis through which membraneless organelles bring together functionally relevant proteins to promote particular biological activities.

The role of wild-type tau in Alzheimer's disease and related tauopathies.

Tau oligomers have recently emerged as the principal toxic species in Alzheimer's disease (AD) and tauopathies. Tau oligomers are spontaneously self-assembled soluble tau proteins that are formed prior to fibrils, and they have been shown to play a central role in neuronal cell death and in the induction of neurodegeneration in animal models. As the therapeutic paradigm shifts to targeting toxic tau oligomers, this suggests the focus to study tau oligomerization in species that are less susceptible to fibrillization. While truncated and mutation containing tau as well as the isolated repeat domains are particularly prone to fibrillization, the wild-type (WT) tau proteins have been shown to be resistant to fibril formation in the absence of aggregation inducers. In this review, we will summarize and discuss the toxicity of WT tau both in vitro and in vivo, as well as its involvement in tau oligomerization and cell-to-cell propagation of pathology. Understanding the role of WT tau will enable more effective biomarker development and therapeutic discovery for treatment of AD and tauopathies.

Reduction of the RNA Binding Protein TIA1 Exacerbates Neuroinflammation in Tauopathy.

Neuroinflammatory processes play an integral role in the exacerbation and progression of pathology in tauopathies, a class of neurodegenerative disease characterized by aggregation of hyperphosphorylated tau protein. The RNA binding protein (RBP) T-cell Intracellular Antigen 1 (TIA1) is an important regulator of the innate immune response in the periphery, dampening cytotoxic inflammation and apoptosis during cellular stress, however, its role in neuroinflammation is unknown. We have recently shown that TIA1 regulates tau pathophysiology and toxicity in part through the binding of phospho-tau oligomers into pathological stress granules, and that haploinsufficiency of TIA1 in the P301S mouse model of tauopathy results in reduced accumulation of toxic tau oligomers, pathologic stress granules, and the development of downstream pathological features of tauopathy. The putative role of TIA1 as a regulator of the peripheral immune response led us to investigate the effects of TIA1 on neuroinflammation in the context of tauopathy, a chronic stressor in the neural environment. Here, we evaluated indicators of neuroinflammation including; reactive microgliosis and phagocytosis, pro-inflammatory cytokine release, and oxidative stress in hippocampal neurons and glia of wildtype and P301S transgenic mice expressing TIA1+/+, TIA1+/–, and TIA1–/– in both early (5 month) and advanced (9 month) disease states through biochemical, ultrastructural, and histological analyses. Our data show that both TIA1 haploinsufficiency and TIA1 knockout exacerbate neuroinflammatory processes in advanced stages of tauopathy, suggesting that TIA1 dampens the immune response in the central nervous system during chronic stress.

A Complex Containing HNRNPA2B1 and N <sup>6</sup>-Methyladenosine Modified Transcripts Mediates Actions of Toxic Tau Oligomers

The microtubule associated protein tau oligomerizes in response to stress and disease, but the function of oligomeric tau (oTau) and the ultimate mechanisms of toxicity are unknown. To gain insights, we have now used Cry2-based optogenetics to induce tau oligomers (oTau-c) in neuronal cultures. oTau-c can seed tau aggregation and biochemical fractionatesin a manner similar to oTau. Optical induction of oTau elicits a translational stress response that includes cytoplasmic translocation of the TIA1, abundant stress granules(SGs) and reduced protein synthesis. Proteomic analysis identifies HNRNPA2B1 as a principle target of oTau. Imaging and immunoprecipitation verify the HNRNPA2B1 association with endogenous oTau in neurons, animal models and human Alzheimer brain tissue. Mechanistic studies demonstrate that HNRNPA2B1 functions as a linker, connecting oTau with N6-methyladenosine modified transcripts (m6A). Knockdown of HNRNPA2B1 prevents oTau from associating with m6A, prevents oTau-induced reductions in protein synthesis and reduces oTau-induced toxicity. Finally, we show striking increases in m6A-oTau and -HNRNPA2B1 complexes in brains of Alzheimer subjects and P301S tau mice. These results reveal a novel complex containing oTau, HNRNPA2B1 and m6A that contributes to the integrated stress response of oTau.

Toxic Tau Oligomers Modulated by Novel Curcumin Derivatives

The pathological aggregation and accumulation of tau, a microtubule-associated protein, is a common feature amongst more than 18 different neurodegenerative diseases that are collectively known as tauopathies. Recently, it has been demonstrated that the soluble and hydrophobic tau oligomers are highly toxic in vitro due to their capacity towards seeding tau misfolding, thereby propagating the tau pathology seen across different neurodegenerative diseases. Modulating the aggregation state of tau oligomers through the use of small molecules could be a useful therapeutic strategy to target their toxicity, regardless of other factors involved in their formation. In this study, we screened and tested a small library of newly synthesized curcumin derivatives against preformed recombinant tau oligomers. Our results show that the curcumin derivatives affect and modulate the tau oligomer aggregation pathways, converting to a more aggregated non-toxic state as assessed in the human neuroblastoma SH-SY5Y cell line and primary cortical neuron cultures. These results provide insight into tau aggregation and may become a basis for the discovery of new therapeutic agents, as well as advance the diagnostic field for the detection of toxic tau oligomers.

Targeting the ensemble of heterogeneous tau oligomers in cells: A novel small molecule screening platform for tauopathies

ObjectiveUnderstanding the heterogeneous pathology in Alzheimer's disease and related tauopathies is one of the most urgent and fundamental challenges facing the discovery of novel disease-modifying therapies. Through monitoring ensembles of toxic and nontoxic tau oligomers spontaneously formed in cells, our biosensor technology can identify tool compounds that modulate tau oligomer structure and toxicity, providing much needed insight into the nature and properties of toxic tau oligomers. BackgroundTauopathies are a group of neurodegenerative disorders characterized by pathologic aggregation of the microtubule binding protein tau. Recent studies suggest that tau oligomers are the primary toxic species in tauopathies. New/Updated HypothesisWe hypothesize that tau biosensors capable of monitoring tau oligomer conformation are able to identify tool compounds that modulate the structure and conformation of these tau assemblies, providing key insight into the unique structural fingerprints of toxic tau oligomers. These fingerprints will provide gravely needed biomarker profiles to improve staging of early tauopathy pathology and generate lead compounds for potential new therapeutics. Our time-resolved fluorescence resonance energy transfer biosensors provide us an exquisitely sensitive technique to monitor minute structural changes in monomer and oligomer conformation. In this proof-of-concept study, we identified a novel tool compound, MK-886, which directly binds tau, perturbs the conformation of toxic tau oligomers, and rescues tau-induced cytotoxicity. Furthermore, we show that MK-886 alters the conformation of tau monomer at the proline-rich and microtubule binding regions, stabilizing an on-pathway oligomer. Major Challenges for the HypothesisOur approach monitors changes in the ensemble of assemblies that are spontaneously formed in cells but does not specifically isolate or enrich unique toxic tau species. However, time-resolved fluorescence resonance energy transfer does not provide high-resolution, atomic scale information, requiring additional experimental techniques to resolve the structural features stabilized by different tool compounds. Linkage to Other Major TheoriesOur biosensor technology is broadly applicable to other areas of tauopathy therapeutic development. These biosensors can be readily modified for different isoforms of tau, specific post-translational modifications, and familial Alzheimer's disease–associated mutations. We are eager to explore tau interactions with chaperone proteins, monitor cross-reactivity with other intrinsically disordered proteins, and target seeded oligomer pathology.

Intraneuronal Tau Misfolding Induced by Extracellular Amyloid-β Oligomers.

Abnormal folding and aggregation of the microtubule-associated protein, tau, is a hallmark of several neurodegenerative disorders, including Alzheimer's disease (AD). Although normal tau is an intrinsically disordered protein, it does exhibit tertiary structure whereby the N- and C-termini are often in close proximity to each other and to the contiguous microtubule-binding repeat domains that extend C-terminally from the middle of the protein. Unfolding of this paperclip-like conformation might precede formation of toxic tau oligomers and filaments, like those found in AD brain. While there are many ways to monitor tau aggregation, methods to monitor changes in tau folding are not well established. Using full length human 2N4R tau doubly labeled with the Förster resonance energy transfer (FRET) compatible fluorescent proteins, Venus and Teal, on the N- and C-termini, respectively (Venus-Tau-Teal), intensity and lifetime FRET measurements were able to distinguish folded from unfolded tau in living cells independently of tau-tau intermolecular interactions. When expression was restricted to low levels in which tau-tau aggregation was minimized, Venus-Tau-Teal was sensitive to microtubule binding, phosphorylation, and pathogenic oligomers. Of particular interest is our finding that amyloid-β oligomers (AβOs) trigger Venus-Tau-Teal unfolding in cultured mouse neurons. We thus provide direct experimental evidence that AβOs convert normally folded tau into a conformation thought to predominate in toxic tau aggregates. This finding provides further evidence for a mechanistic connection between Aβ and tau at seminal stages of AD pathogenesis.

Tau oligomers mediate aggregation of RNA-binding proteins Musashi1 and Musashi2 inducing Lamin alteration.

The exact mechanisms leading to neurodegeneration in Alzheimer's disease (AD) and other tauopathies are not yet entirely understood. However, it is known that several RNA‐binding proteins (RBPs) form toxic aggregates and also interact with tau in such granules in tauopathies, including AD. The Musashi (MSI) family of RBPs, consisting of two homologues: Musashi1 and Musashi2, have not been extensively investigated in neurodegenerative diseases. Here, using a tau inducible HEK (iHEK) model we investigate whether MSI proteins contribute to the aggregation of toxic tau oligomers (TauO). Wild‐type and mutant P301L tau iHEK cells are used to study the effect of different tau variants on the cellular localization of MSI proteins. Interestingly, we observe that tau co‐localizes with MSI in the cytoplasm and nuclei, altering the nuclear transport of MSI. Furthermore, incremental changes in the size and density of nuclear MSI/tau foci are observed. We also report here that TauO interact with MSI to cause the formation of distinct nuclear aggregates. Moreover, tau/MSI aggregates induce structural changes to LaminB1, leading to nuclear instability. These results illustrate a possible mechanism of neurodegeneration mediated by the aggregation of MSI proteins and TauO, suggesting that MSI plays a critical role in cellular dysfunction.

EFhd2 Affects Tau Liquid-Liquid Phase Separation.

The transition of tau proteins from its soluble physiological conformation to the pathological aggregate forms found in Alzheimer’s disease and related dementias, is poorly understood. Therefore, understanding the process that modulates the formation of toxic tau oligomers and their conversion to putative neuroprotective neurofibrillary tangles will lead to better therapeutic strategies. We previously identified that EFhd2 is associated with aggregated tau species in AD brains and the coiled-coil domain in EFhd2 mediates the interaction with tau. To further characterize the association between EFhd2 and tau, we examined whether EFhd2 could affect the liquid–liquid phase separation properties of tau under molecular crowding conditions. We demonstrate that EFhd2 alters tau liquid phase behavior in a calcium and coiled-coil domain dependent manner. Co-incubation of EFhd2 and tau in the absence of calcium leads to the formation of solid-like structures containing both proteins, while in the presence of calcium these two proteins phase separate together into liquid droplets. EFhd2’s coiled-coil domain is necessary to alter tau’s liquid phase separation, indicating that protein–protein interaction is required. The results demonstrate that EFhd2 affects the liquid–liquid phase separation of tau proteins in vitro, suggesting that EFhd2 modulates the structural dynamics of tau proteins.