Liquid-liquid Phase Separation Of Tau Research Articles

Pathogenic Mutation ΔK280 Promotes Hydrophobic Interactions Involving Microtubule-Binding Domain and Enhances Liquid-Liquid Phase Separation of Tau.

Liquid-liquid phase separation (LLPS) of tau protein can initiate its aggregation which is associated with Alzheimer's disease. The pathogenic mutation ΔK280 can enhance the aggregation of K18, a truncated tau variant comprising the microtubule-binding domain. However, the impact of ΔK280 on K18 LLPS and underlying mechanisms are largely unexplored. Herein, the conformational ensemble and LLPS of ΔK280 K18 through multiscale molecular simulations and microscopy experiments are investigated. All-atom molecular dynamic simulations reveal that ΔK280 significantly enhances the collapse degree and β-sheet content of the K18 monomer, indicating that ΔK280 mutation may promote K18 LLPS, validated by coarse-grained phase-coexistence simulations and microscopy experiments. Importantly, ΔK280 mutation promotes β-sheet formation of six motifs (especially PHF6), increases the hydrophobic solvent exposure of PHF6* and PHF6, and enhances hydrophobic, hydrogen bonding, and cation-π interactions involving most of the motifs, thus facilitating the phase separation of K18. Notably, ΔK280 alters the interaction network among the six motifs, inducing the formation of K18 conformations with high β-sheet contents and collapse degree. Coarse-grained simulations on full-length tau reveal that ΔK280 promotes tau LLPS by enhancing the hydrophobic interactions involving the microtubule-binding domain. These findings offer detailed mechanistic insights into ΔK280-induced tau pathogenesis, providing potential targets for therapeutic intervention.

Chemical regulation of Tau oligomers in phase separation in Alzheimer’s disease

A huge number of patients suffering from certain neurodegenerative disorder, which are collectively called as tauopathies, may exhibit pathological tau protein aggregates in their brains. This category of diseases includes Alzheimer's disease (AD). In AD, post translational modifications such as phos phorylations, glycosylations, truncations, and subsequent aggregation into oligomers, paired helical filaments (PHFs), and neurofibrillary tangles (NFTs) are closely associated with cognitive decline and neurodegeneration. As a result, tau oligomers have emerged as the primary toxic species in AD and tauo pathies. Tau oligomers are soluble, self-assembled tau proteins that are formed prior to fibrils and have been demonstrated to play a pivotal role in neuronal cell death and the induction of neurodegeneration in animal models. In this succinct review, we collate and summarize literature pertaining to tau oligomer formation and its role in Alzheimer's disease. Secondly, we explore the crucial role of zinc ions (Zn²⁺) in tau aggregation, as studies suggest that zinc induces reversible tau oligomerization and can lead to tau hyper phosphorylation. The concentration of zinc is critical, as excessive levels can promote harmful tau aggregation, while normal levels are essential for physiological functions. We also examine natural and chemical compounds that can modulate tau aggregation, and lastly, we discuss how Tau protein can undergo liquid-liquid phase separation (LLPS) in neurons, forming droplets that can later develop into toxic oligomers, which are the primary hallmark of AD. We mention some molecules, such as proteins, nucleic acids, and metal ions, that influence tau LLPS and aggregation.

Tannic acid as a biphasic modulator of tau protein liquid–liquid phase separation

Tannic acid (TA) is a natural polyphenol that shows great potential in the field of biomedicine due to its anti-inflammatory, anti-oxidant, anti-bacterial, anti-tumor, anti-virus, and neuroprotective activities. Recent studies have revealed that liquid−liquid phase separation (LLPS) is closely associated with protein aggregation. Therefore, modulating LLPS offers new insights into the treatment of neurodegenerative diseases. In this study, we investigated the influence of TA on the LLPS of the Alzheimer's-related protein tau and the underlying mechanism. Our findings indicate that TA affects the LLPS of tau in a biphasic manner, with initial promotion and subsequent suppression as the TA to tau molar ratio increases. TA modulates tau phase separation through a combination of hydrophobic interactions and hydrogen bonds. The balance between TA-tau and tau-tau interactions is found to be relevant to the material properties of TA-induced tau condensates. We further illustrate that the modulatory activity of TA in phase separation is highly dependent on the target proteins. These findings enhance our understanding of the forces driving tau LLPS under different conditions, and may facilitate the identification and optimization of compounds that can rationally modulate protein phase transition in the future.

Hybrid molecules synergistically mitigate ferroptosis and amyloid-associated toxicities in Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the build-up of extracellular amyloid β (Aβ) plaques and intracellular neurofibrillary tangles (NFTs). Ferroptosis, an iron (Fe)-dependent form of cell death plays a significant role in the multifaceted AD pathogenesis through generation of reactive oxygen species (ROS), mitochondrial damage, lipid peroxidation, and reduction in glutathione peroxidase 4 (GPX4) enzyme activity and levels. Aberrant liquid-liquid phase separation (LLPS) of tau drives the growth and maturation of NFTs contributing to AD pathogenesis. In this study, we strategically combined the structural and functional properties of gallic acid (GA) and cyclic dipeptides (CDPs) to synthesize hybrid molecules that effectively target both ferroptosis and amyloid toxicity in AD. This innovative approach marks a paradigm shift from conventional therapeutic strategies. This is the first report of a synthetic small molecule (GCTR) that effectively combats ferroptosis, simultaneously restoring enzymatic activity and enhancing cellular levels of its master regulator, GPX4. Further, GCTR disrupts Fe3+-induced LLPS of tau, and aids in attenuation of abnormal tau fibrillization. The synergistic action of GCTR in combating both ferroptosis and amyloid toxicity, bolstered by GPX4 enhancement and modulation of Fe3+-induced tau LLPS, holds promise for the development of small molecule-based novel therapeutics for AD.

EB1 Increases the Dynamics of Tau Droplets and Inhibits Tau Aggregation: Implications in Tauopathies.

EB1, a microtubule plus end-tracking protein (+TIP), regulates microtubule dynamics. Recent evidence indicates cross-talk between EB proteins and tau, a microtubule-associated neuronal protein that is important for the growth and stability of microtubules. We investigated the interaction between tau and EB1 and the effect of binding of EB1 on tau function and aggregation. EB1 colocalized with tau in SH-SY5Y cells and coimmunoprecipitated with tau. Further, purified EB1 impaired the ability of adult tau to induce tubulin polymerization in vitro. EB1 bound to tau with a dissociation constant of 2.5 ± 0.7 μM. EB1 reduced heparin-induced tau aggregation with a half-maximal inhibitory concentration of 4.3 ± 0.2 μM, and increased the dynamics of tau in phase-separated droplets. The fluorescence recovery rate in tau droplets increased from 0.02 ± 0.01 to 0.07 ± 0.03 s-1, while the half-time of recovery decreased from 44.5 ± 14 to 13.5 ± 6 s in the presence of 8 μM EB1, suggesting a delay in the transition of tau from the soluble to aggregated form in tau liquid-liquid phase separation. EB1 decreased the rate of aggregation and increased the critical concentration of tau aggregation. Dynamic light scattering, atomic force microscopy, dot blot assays, and SDS-PAGE analysis showed that EB1 inhibited the formation of oligomers and higher-order aggregates of tau. The data suggest a novel role for EB1 as a regulator of tau function and aggregation, and the findings indicated the role of the EB family proteins in neuronal function and neurodegeneration.

Polycatechols inhibit ferroptosis and modulate tau liquid-liquid phase separation to mitigate Alzheimer's disease.

Alzheimer's disease (AD) is a complex neurodegenerative disorder that affects learning, memory, and cognition. Current treatments targeting amyloid-β (Aβ) and tau have shown limited effectiveness, necessitating further research on the aggregation and toxicity mechanisms. One of these mechanisms involves the liquid-liquid phase separation (LLPS) of tau, contributing to the formation of pathogenic tau aggregates, although their conformational details remain elusive. Another mechanism is ferroptosis, a type of iron-dependent lipid peroxidation-mediated cell death, which has been implicated in AD. There is a lack of therapeutic strategies that simultaneously target amyloid toxicity and ferroptosis. This study aims to explore the potential of polycatechols, PDP and PLDP, consisting of dopamine and L-Dopa, respectively, as multifunctional agents to modulate the pathological nexus between ferroptosis and AD. Polycatechols were found to sequester the labile iron pool (LIP), inhibit Aβ and tau aggregation, scavenge free radicals, protect mitochondria, and prevent ferroptosis, thereby rescuing neuronal cell death. Interestingly, PLDP promotes tau LLPS, and modulates their intermolecular interactions to inhibit the formation of toxic tau aggregates, offering a conceptually innovative approach to tackle tauopathies. This is a first-of-its-kind polymer-based integrative approach that inhibits ferroptosis, counteracts amyloid toxicity, and modulates tau LLPS to mitigate the multifaceted toxicity of AD.

Biphasic modulation of tau liquid-liquid phase separation by polyphenols.

Molecular tools that modulate tau liquid-liquid phase separation (LLPS) promise to treat tauopathies. We screened a set of polyphenols and demonstrated concentration-dependent biphasic modulation of tau LLPS by gallic acid (GA), showcasing its ability to expedite the liquid-to-gel transition in tau condensates and effectively impede the formation of deleterious fibrillar aggregates.

Heterotypic liquid-liquid phase separation of tau and α-synuclein: Implications for overlapping neuropathologies

Tauopathies and synucleinopathies are characterized by the aggregation of Tau and α-synuclein (AS) into amyloid structures, respectively. Individuals with these neuropathies have an elevated risk of developing subsequent neurodegenerative or comorbid disorders. Intriguingly, post-mortem brain examinations have revealed co-localization of Tau and AS aggregates, suggesting a synergistic pathological relationship with an adverse prognosis. The role of liquid-liquid phase separation (LLPS) in the development of neurodegenerative diseases is currently receiving significant attention, as it can contribute to the aggregation and co-deposition of amyloidogenic proteins. In this study, we investigated the phase separation behavior of Tau and AS under various insults, some of which are implicated in disease progression. Our findings demonstrate the formation of heterotypic droplets composed of Tau and AS at physiologically relevant mole ratios that mimic neurons' soma and terminal buttons. Importantly, these heterotypic droplets exhibit increased resistance to electrostatic screening compared to homotypic condensates. Moreover, we observed that biologically relevant biomolecules, known to be dysregulated in disease, exert different effects on these droplets. Additionally, we provide evidence that phase separation itself influences the amyloid aggregation of Tau and AS, underscoring the significance of this process in the development of aggregopathies.

Domain-specific modulatory effects of phosphomimetic substitutions on liquid-liquid phase separation of tau protein

Aggregation of tau is one of the major pathogenic events in Alzheimer's disease and several other neurodegenerative disorders. Recent reports demonstrated that tau can condense into liquid droplets that undergo time-dependent transition to a solid-like state, suggesting that liquid condensates may be on the pathway to pathological aggregation of tau. While hyperphosphorylation is a key feature of tau isolated from brains of patients with Alzheimer's disease and other tauopathies, the mechanistic role of phosphorylation in tau liquid-liquid phase separation (LLPS) remains largely unexplored. In an attempt to bridge this gap, here we performed systematic studies by introducing phosphomimetic substitutions of Ser/Thr residues with negatively charged Asp/Glu residues in different regions of the protein. Our data indicate that the phosphorylation patterns that increase the polarization of charge distribution in full-length tau (tau441) promote protein LLPS, whereas those that decrease charge polarization have an opposite effect. Overall, this study further supports the notion that tau LLPS is driven by attractive intermolecular electrostatic interactions between the oppositely charged domains. We also show that the phosphomimetic tau variants with low intrinsic propensity for LLPS can be efficiently recruited to droplets formed by the variants with high LLPS propensity. Furthermore, the present data demonstrate that phosphomimetic substitutions have a major effect on time-dependent material properties of tau droplets, generally slowing down their aging. The latter effect is most dramatic for the tau variant with substitutions within the repeat domain, which correlates with the decreased fibrillation rate of this variant.

YTHDF1 phase separation triggers the fate transition of spermatogonial stem cells by activating the IκB-NF-κB-CCND1 axis

N6-methyladenosine (m6A) modification controls cell fate determination. Here, we show that liquid-liquid phase separation (LLPS) of YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), a pivotal m6A "reader" protein, promotes the transdifferentiation of spermatogonial stem cells (SSCs) into neural stem cell-like cells by activating the IκB-nuclear factor κB (NF-κB)-CCND1 axis. The inhibition of IκBα/β mRNA translation mediated by YTHDF1 LLPS is the key to the activation of the IκB-NF-κB-CCND1 axis. Disrupting either YTHDF1 LLPS or NF-κB activation inhibits transdifferentiation efficiency. Moreover, overexpression of the YTH domain of YTHDF1 inhibits the activation of the IκB-NF-κB-CCND1 axis by promoting IκBα/β mRNA translation. Overexpression of the tau-YTH fusion protein reactivates IκB-NF-κB-CCND1 axis by inhibiting the translation of IκBα/β mRNAs, and tau LLPS is observed, which can restore transdifferentiation efficiency. Our findings demonstrate that the protein-RNA LLPS plays essential roles in cell fate transition and provide insights into translational medicine and the therapy of neurological diseases.

14-3-3ζ Participates in the Phase Separation of Phosphorylated and Glycated Tau and Modulates the Physiological and Pathological Functions of Tau.

Tau plays a major role in Alzheimer's disease (AD) and several other neurodegenerative diseases. Tau undergoing liquid-liquid phase separation (LLPS) performs specific physiological functions, induces pathological processes, and contributes to neurodegeneration. Regulating Tau phase separation helps maintain physiological functions of Tau and inhibits pathological aggregation. Here, we show that the 14-3-3 zeta isoform (14-3-3ζ) participates in Tau LLPS. 14-3-3ζ can undergo co-phase separation with WT Tau, participate in and stabilize Tau droplets, and inhibit Tau droplet-driven tubulin assembly. On the other hand, 14-3-3ζ disrupts the LLPS of phosphorylated and glycated Tau, thereby inhibiting the amyloid aggregation initiated by LLPS.

Investigations into the phase separation and nuclear role of tau.

Tau is a microtubule-associated protein and pathological hallmark of Alzheimer's disease (AD), most infamous for becoming hyperphosphorylated and fibrilizing into neurofibrillary tangles (NFTs). Beyond this role, mounting evidence suggests that tau localizes into the nucleus and plays unknown roles in DNA protection and heterochromatin regulation. Intriguingly, frontotemporal dementia mutants (P301L) of tau show loss of genetically silent heterochromatin clusters. This has been associated with aberrant expression of heterochromatic genes and in other studies, of transposable element activation in AD patient brain tissue. Similar effects of heterochromatin relaxation and gene dysregulation have been observed in tau knockouts, suggesting that loss of tau is pathologically relevant. Recent literature showed that tau undergoes liquid-liquid phase separation (LLPS). Studies have not fully described the functional role of nuclear tau in gene regulation, and whether this involves LLPS. My work demonstrates that tau has an intrinsic ability to phase separate with, compact, and oligomerize chromatin, likely through its DNA-binding domain, and this is regulated by phosphorylation. In addition, tau phase separates with heterochromatin protein 1α (HP1α), an essential heterochromatin constituent, which may promote its partitioning into heterochromatin despite its lack of DNA-sequence specificity. These data imply a potential role for tau LLPS in the maintenance of chromatin compaction with aging.

Tau liquid-liquid phase separation is modulated by the Ca2+ -switched chaperone activity of the S100B protein.

Aggregation of the microtubule-associated protein tau is implicated in several neurodegenerative tauopathies including Alzheimer's disease (AD). Recent studies evidenced tau liquid-liquid phase separation (LLPS) into droplets as an early event in tau pathogenesis with the potential to enhance aggregation. Tauopathies like AD are accompanied by sustained neuroinflammation and the release of alarmins at early stages of inflammatory responses encompass protective functions. The Ca2+ -binding S100B protein is an alarmin augmented in AD that was recently implicated as a proteostasis regulator acting as a chaperone-type protein, inhibiting aggregation and toxicity through interactions of amyloidogenic clients with a regulatory surface exposed upon Ca2+ -binding. Here we expand the regulatory functions of S100B over protein condensation phenomena by reporting its Ca2+ -dependent activity as a modulator of tau LLPS induced by crowding agents (PEG) and metal ions (Zn2+ ). We observe that apo S100B has a negligible effect on PEG-induced tau demixing but that Ca2+ -bound S100B prevents demixing, resulting in a shift of the phase diagram boundary to higher crowding concentrations. Also, while incubation with apo S100B does not compromise tau LLPS, addition of Ca2+ results in a sharp decrease in turbidity, indicating that interactions with S100B-Ca2+ promote transition of tau to the mixed phase. Further, electrophoretic analysis and FLIM-FRET studies revealed that S100B incorporates into tau liquid droplets, suggesting an important stabilizing and chaperoning role contributing to minimize toxic tau aggregates. Resorting to Alexa488-labeled tau we observed that S100B-Ca2+ reduces the formation of tau fluorescent droplets, without compromising liquid-like behavior and droplet fusion events. The Zn2+ -binding properties of S100B also contribute to regulate Zn2+ -promoted tau LLPS as droplets are decreased by Zn2+ buffering by S100B, in addition to the Ca2+ -triggered interactions with tau. Altogether this work uncovers the versatility of S100B as a proteostasis regulator acting on protein condensation phenomena of relevance across the neurodegeneration continuum.

(-)-Epigallocatechin-3-gallate, a Polyphenol from Green Tea, Regulates the Liquid-Liquid Phase Separation of Alzheimer's-Related Protein Tau.

The microtubule-associated protein tau is involved in Alzheimer's disease and other tauopathies. Recently, tau has been shown to undergo liquid-liquid phase separation (LLPS), which is implicated in the physiological function and pathological aggregation of tau. In this report, we demonstrate that the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) promotes the formation of liquid tau droplets at neutral pH by creating a network of hydrophobic interactions and hydrogen bonds, mainly with the proline-rich domain of tau. We further show that EGCG oxidation, tau phosphorylation, and the chemical structure of the polyphenol influence the efficacy of EGCG in facilitating tau LLPS. Complementary to the inhibitory activity of EGCG in tau fibrillization, our findings provide novel insights into the biological activity of EGCG and offer new clues for future studies on the molecular mechanism by which EGCG alleviates neurodegenerative diseases.

Cyclic dipeptide-based small molecules modulate zinc-mediated liquid-liquid phase separation of tau.

Liquid-liquid phase separation (LLPS) is a complex physicochemical phenomenon mediated by multivalent transient weak interactions among macromolecules like polymers, proteins, and nucleic acids. It has implications in cellular physiology and disease conditions like cancer and neurodegenerative disorders. Many proteins associated with neurodegenerative disorders like RNA binding protein FUS (FUsed in Sarcoma), alpha-synuclein (α-Syn), TAR DNA binding protein 43 (TDP-43), and tau are shown to undergo LLPS. Recently, the tau protein responsible for Alzheimer's disease (AD) and other tauopathies is shown to phase separate into condensates in vitro and in vivo. The diverse noncovalent interactions among the biomolecules dictate the complex LLPS phenomenon. There are limited chemical tools to modulate protein LLPS which has therapeutic potential for neurodegenerative disorders. We have rationally designed cyclic dipeptide (CDP)-based small-molecule modulators (SMMs) by integrating multiple chemical groups that offer diverse chemical interactions to modulate tau LLPS. Among them, compound 1c effectively inhibits and dissolves Zn-mediated tau LLPS condensates. The SMM also inhibits tau condensate-to-fibril transition (tau aggregation through LLPS). This approach of designing SMMs of LLPS establishes a novel platform that has potential implication for the development of therapeutics for neurodegenerative disorders.

Binding of two zinc ions promotes liquid-liquid phase separation of Tau

Tau is a naturally disordered microtubule associated protein which forms intraneuronal aggregates in several neurodegenerative diseases including Alzheimer's disease (AD). It was reported that zinc interaction with tau protein can trigger its aggregation. Recently we identified three zinc binding sites located in the N-terminal part, repeat region and the C-terminal part of tau. Here we characterized zinc binding to each of the three sites using isothermal titration calorimetry (ITC) and determined the impact of each site on aggregation using dynamic light scattering (DLS) assays. First, we confirmed the presence of three zinc binding sites on tau and determined the thermodynamic parameters of binding of zinc to these sites. We found a high-affinity zinc binding site located in the repeat region of tau and two N- and C-terminus binding sites with a lower binding constant for zinc. Second, we showed that tau aggregation necessitates zinc binding to the high affinity site in the R2R3 region, while LLPS necessitates zinc binding to any two binding sites. With regard to the role of zinc ions in the aggregation of proteins in neurodegenerative diseases, these findings bring new insights to the understanding of the aggregation mechanism of tau protein induced by zinc.

Light Microscopy and Dynamic Light Scattering to Study Liquid-Liquid Phase Separation of Tau Proteins In Vitro.

Tau is an intrinsically disordered protein that binds and stabilizes axonal microtubules (MTs) in neurons of the central nervous system. The binding of Tau to MTs is mediated by its repeat domain and flanking proline-rich domains. The positively charged (basic) C-terminal half of Tau also mediates the assembly Tau into fibrillar aggregates in Alzheimer's disease (AD) and tauopathy brains. In recent years, another assembly form of Tau has been identified: Tau can undergo liquid-liquid phase separation (LLPS), which leads to its condensation into liquid-dense phases, either by complex coacervation with polyanions like heparin or RNA or through "self-coacervation" at high Tau concentrations. Condensation of Tau in the absence of polyanions can be enhanced by the presence of molecular crowding agents in a dilute Tau solution. In vitro experiments using recombinant purified Tau are helpful to study the physicochemical determinants of Tau LLPS, which can then be extrapolated into the cellular context. Tau condensation is a new aspect of Tau biology that may play a role for the initiation of Tau aggregation, but also for its physiological function(s), for example, the binding to microtubules. Here we describe how to study the condensation of Tau in vitro using light microscopy, including fluorescence recovery after photobleaching (FRAP), to assess the shape and molecular diffusion in the condensates, a proxy for the degree of condensate percolation. We also describe turbidity measurements of condensate-containing solutions to assess the overall amount of LLPS and time-resolved dynamic light scattering (trDLS) to study the formation and size of Tau condensates.

Tau liquid-liquid phase separation: At the crossroads of tau physiology and tauopathy.

Abnormal deposition of tau in neurons is a hallmark of Alzheimer's disease and several other neurodegenerative disorders. In the past decades, extensive efforts have been made to explore the mechanistic pathways underlying the development of tauopathies. Recently, the discovery of tau droplet formation by liquid-liquid phase separation (LLPS) has received a great deal of attention. It has been reported that tau condensates have a biological role in promoting and stabilizing microtubule (MT) assembly. Furthermore, it has been hypothesized that the transition of phase-separated tau droplets to a gel-like state and then to fibrils is associated with the pathology of neurodegenerative diseases. In this review, we outline LLPS, the structural disorder that facilitates tau droplet formation, the effects of posttranslational modification of tau on condensate formation, the physiological function of tau droplets, the pathways from droplet to toxic fibrils, and the therapeutic strategies for tauopathies that might evolve from toxic droplets. We expect a deeper understanding of tau LLPS will provide additional insights into tau physiology and tauopathies.

Tau liquid-liquid phase separation in neurodegenerative diseases.

Aggregation of the microtubule-associated protein tau plays a major role in Alzheimer's disease and several other neurodegenerative disorders. An exciting recent development is the finding that, akin to some other proteins associated with neurodegenerative disease, tau has a high propensity to condensate via the mechanism of liquid-liquid phase separation (LLPS). Here, we discuss the evidence for tau LLPS in vitro, the molecular mechanisms of this reaction, and the role of post-translational modifications and pathogenic mutations in tau phase separation. We also discuss recent studies on tau LLPS in cells and the insights these studies provide regarding the link between LLPS and neurodegeneration in tauopathies.

Zinc enhances liquid-liquid phase separation of Tau protein and aggravates mitochondrial damages in cells

Intraneuronal neurofibrillary tangles composed of Tau aggregates have been widely accepted as an important pathological hallmark of Alzheimer's disease. Liquid-liquid phase separation (LLPS) of Tau can lead to its aggregation, and Tau aggregation can then be enhanced by zinc. However, it is unclear whether zinc modulates the formation of Tau stress granules in cells. We herein report that zinc promotes the formation of stress granules containing a pathological mutant ΔK280 of full-length human Tau. Furthermore, zinc promotes LLPS of ΔK280 of full-length Tau, shifting the equilibrium phase boundary to a lower protein concentration, and modulates the liquid nature of droplets formed by this pathological mutation. Zinc also promotes pathological phosphorylation of ΔK280 in neuronal cells, and aggravates mitochondrial damage and elevates reactive oxygen species production induced by Tau aggregation. Importantly, we show that treatment of cells with zinc increases the interaction between full-length Tau and G3BP1 inside stress granules to promote the formation of Tau filaments and increase Tau toxicity in neuronal cells. Collectively, these results demonstrate how Tau condensation and mitochondrial damages induced by Tau aggregation are enhanced by zinc to deteriorate the pathogenesis of Alzheimer's disease, bridging the gap between Tau LLPS and aggregation in neuronal cells.