Do Tau Fibrils Form Via Liquid Droplets?

How do tau fibrils develop? In the July 19 Structure online, scientists led by Yoshiyuki Soeda and Akihiko Takashima at Gakushuin University, Tokyo, proposed that the protein first condenses into droplets, then folds into insoluble β sheets. When the authors forced mutant human tau in a mouse cell line to come together, it underwent liquid-liquid phase separation (LLPS), forming spherical globs in the cytoplasm.

  • Mutant human tau jammed together in cells forms liquid droplets.
  • Snipping tau’s N-terminus promotes formation of β sheets within droplets.
  • Truncated tau is common in aged brain, hinting at biological relevance.

This tau was highly phosphorylated and oligomeric, but also dissolvable by alcohol or detergent. Once the scientists deleted the N-terminal region, however, tau became prone to aggregate. Insoluble β sheets capable of seeding fibrils formed inside droplets. N-terminal cleavage and LLPS may be intermediate steps on the way to neurofibrillary tangles, the authors suggested.

Ben Wolozin at Boston University praised the study’s excellent use of common cell biology tools to gain new insights into tau aggregation. “[The paper] conveys an important difference between potentially functional tau oligomerization—which proceeds through LLPS—and pathological tau oligomerization and aggregation, which correlates with formation of insoluble, fibrillar tau,” he wrote (comment below).

Previous studies had linked liquid droplets of tau to the production of toxic forms, but haven’t answered the question of how this happens even in cell culture, much less the aging human brain (Aug 2017 news; Wegmann et al., 2018; Kanaan et al., 2020). Moreover, some scientists have found that tau can aggregate via other routes, without first forming droplets (Aug 2017 conference news; Apr 2024 news).

Stages of Fibrillization? When mutant human tau (purple) fused to a light-responsive protein (OptoTau, left) is induced to congregate via blue light, it accumulates in aggresomes of neuroblastoma cells (middle left). If aggresome transport is blocked by microtubule (green) disassembly, tau droplets appear in the cytoplasm (middle right). When this condensed tau lacks its N-terminus, it folds into β sheets (far right). Nucleus is blue. [Courtesy of Soeda et al., Structure.]

To gain mechanistic insight, Takashima’s group turned to a highly artificial system. First author Soeda expressed human P301L tau, which aggregates readily, in a mouse neuroblastoma cell line. Tau was fused to a second protein, the plant photoreceptor arabidopsis cryptochrome 2 (CRY2). When the authors shone blue light onto the cells, CRY2 oligomerized, bringing tau monomers close to each other. This tool is commonly used to promote protein aggregation, and it accelerated tau deposition, i.e., clumps of hyperphosphorylated, oligomeric tau accumulating near the nucleus.

The authors believed these clumps might represent aggresomes, protein snarls that build up when a cell’s degradation machinery is overwhelmed. In such cases, cells transport indigestible protein bits along their microtubules and sequester them near the nucleus. Supporting this idea, when the authors added the microtubule buster nocodazole to the cell media, tau no longer accumulated near the nucleus. Instead, it appeared in round droplets throughout the cytoplasm.

The droplets gradually disappeared when the authors turned off the blue light, meaning the tau remained soluble. In addition, adding the alcohol 1,6-hexanediol to the cells dispersed the droplets, as it does for other protein droplets. Likewise, when the authors treated cell lysates with the detergent Sarkozy, tau clumps broke up, again showing it contained no insoluble β sheets.

To spark fibrillization, the authors snipped off tau’s N-terminus. Insoluble tau deposits tend to lack this tail, suggesting its presence may hinder β-sheet formation (Dec 2020 news). Sure enough, the truncated tau/CRY2 chimera made eight times as many oligomers after exposure to blue light as did full-length tau/CRY2. Moreover, the tau clumps now contained β sheets. Droplets were no longer spherical, and they bound the fibril marker thioflavin S. They also stayed stable after the blue light was switched off, and resisted degradation by 1,6-hexanediol and sarkosyl.

These tau aggregates were not long enough to be considered fibrils, the authors found. They did, however, seed aggregation in solutions of monomeric tau, confirming the presence of β sheets.

The authors believe the findings are relevant to what happens in the brain. N-terminally truncated tau increases with age in tau transgenic mice, suggesting this form could spur aggregation (Matsumoto et al., 2015). “Our findings support a model whereby P301L tau clustering is accumulated in aggresomes initially, and disruption of the aggresomal process leads to tau aggregation through LLPS, ultimately forming a tau seed,” they wrote.—Madolyn Bowman Rogers

Comments

  1. This article from the Takashima laboratory makes excellent use of the tau::Cry2 system to explore mechanisms of tau aggregation. The CRY2 system was first used to optically induce liquid-liquid phase separation (LLPS) of RNA-binding proteins by the Brangwynne laboratory (Shin et al., 2017). The Kosik/Han labs, as well as my lab, then applied this to study tau aggregation using light-induced oligomerization (Zhang et al., 2020; Jiang et al., 2021). We did our studies in primary cultures of cortical neurons, where we observed rapid tau oligomerization, occurring within 8 min of light exposure. A similar result can be achieved in cell lines with longer exposure to blue light (24 hours) (Zhang et al., 2020).

    The Takashima lab used the cell line/long exposure approach to investigate mechanisms of tau aggregation in mouse N2a cells. This powerful system allows one to modulate tau structure directly, which avoids the pleiotropic effects observed when one induces tau aggregation by activating stress kinases or inhibiting proteostasis. The Takashima group shows that oligomerization of tau is sufficient to drive pathological (proline directed) phosphorylation, and that optically induced oligomerization of N-terminally truncated opto-Tau containing mainly the microtubule repeat-binding domain (MTBR) rapidly forms fibrils without requiring or associating with microtubules. These results confirm observations from the prior studies (Zhang et al., 2020; Jiang et al., 2021). 

    The studies in this manuscript then provide several additional new insights into mechanisms of tau aggregation. This manuscript nicely shows that optically induced tau oligomerization forms membraneless granules through a process of LLPS. This is through studies demonstrating dispersion of the tau granules with 1, 6 hexanediol and by demonstrating circularity of the phase-separated tau granules.

    The manuscript brings out another important point, in showing that abrogating tau interactions with microtubules leads to tau fibrillization. The group shows that cell treatment with the tau depolymerizing agent nicodozole induces tau fibrillization. These fibrils no longer associate with aggresomes, and instead are shunted to lysosomes. Using phospho-mimetic mutants, they demonstrate that pathological phosphorylation of tau, i.e., proline-directed phosphorylation, also causes formation of tau fibrils.

    The distinction between tau LLPS and tau fibrillization is important because it conveys a difference between potentially functional tau oligomerization—which proceeds through LLPS—and pathological tau oligomerization and aggregation— which correlates with formation of insoluble, fibrillar tau and the type of structures seen in CryoEM studies of tau fibrils (Shi et al., 2021; Fitzpatrick et al., 2017; Lovestam et al., 2023; Zhang et al., 2020). 

    Takashima and colleagues conclude that formation of fibrils proceeds through LLPS. Such a mechanism is possible if one imagines that oligomeric tau first associates with microtubules, then upon pathological phosphorylation dissociates from microtubules and forms fibrils. An alternate pathway might also occur in parallel. This alternate pathway would proceed through pathological tau phosphorylation, which is known to prevent tau from interacting with microtubules. In this alternate pathway, tau might fibrillize without proceeding through LLPS (since in the Takashima mechanism, tau LLPS requires microtubule association).

    In interpreting this manuscript, it is important to be cognizant of a key nuance. The optically induced oligomerization of tau in cell lines, such as N2a, requires up to 24 hours of treatment, which is slow. In contrast, the LLPS process in primary neurons is much quicker, requiring only 5 to 10 minutes of mild light exposure (blue light from an LED is sufficient) (Jiang et al., 2021). The rapid kinetics of tau LLPS in primary neurons allows the tau oligomerization pathway to be much more active and achieve much more physiological activity before proline-directed phosphorylation leads to fibrillization. Our work shows that this physiological activity regulates RNA metabolism. In contrast, in cell lines, the LLPS process is very slow and microtubule association appears to play a much greater role. My hunch is that the greater role of microtubules in this process directs tau oligomers/granules more toward aggresomes than would occur in primary neuronal cultures, which is certainly an important point to examine in future studies.

    Another classic worry about tau::CRY2 is whether the large CRY2 protein changes tau aggregates. We examined this question extensively, and found that proteins pulled down with the tau::CRY2 oligomeric construct could also be pulled down by IP of native tau oligomers. However, it is possible that some interactions of native tau oligomers might not occur with the tau::CRY2 chimera because of steric hindrance. All groups studying the tau::CRY2 construct observe that this construct readily forms granules and forms fibrils in a manner similar to that observed with unlabeled tau; this might be the case because the CRY2 hangs off of the tau protein in a manner that is distant and physically distinct from the microtubule binding domain.

    References:

    Shin Y, Berry J, Pannucci N, Haataja MP, Toettcher JE, Brangwynne CP. Spatiotemporal Control of Intracellular Phase Transitions Using Light-Activated optoDroplets. Cell. 2017 Jan 12;168(1-2):159-171.e14. Epub 2016 Dec 29 PubMed.

    Zhang X, Vigers M, McCarty J, Rauch JN, Fredrickson GH, Wilson MZ, Shea JE, Han S, Kosik KS. The proline-rich domain promotes Tau liquid-liquid phase separation in cells. J Cell Biol. 2020 Nov 2;219(11) PubMed.

    Jiang L, Lin W, Zhang C, Ash PE, Verma M, Kwan J, van Vliet E, Yang Z, Cruz AL, Boudeau S, Maziuk BF, Lei S, Song J, Alvarez VE, Hovde S, Abisambra JF, Kuo MH, Kanaan N, Murray ME, Crary JF, Zhao J, Cheng JX, Petrucelli L, Li H, Emili A, Wolozin B. Interaction of tau with HNRNPA2B1 and N6-methyladenosine RNA mediates the progression of tauopathy. Mol Cell. 2021 Oct 21;81(20):4209-4227.e12. Epub 2021 Aug 27 PubMed.

    Shi Y, Zhang W, Yang Y, Murzin AG, Falcon B, Kotecha A, van Beers M, Tarutani A, Kametani F, Garringer HJ, Vidal R, Hallinan GI, Lashley T, Saito Y, Murayama S, Yoshida M, Tanaka H, Kakita A, Ikeuchi T, Robinson AC, Mann DM, Kovacs GG, Revesz T, Ghetti B, Hasegawa M, Goedert M, Scheres SH. Structure-based classification of tauopathies. Nature. 2021 Oct;598(7880):359-363. Epub 2021 Sep 29 PubMed.

    Fitzpatrick AW, Falcon B, He S, Murzin AG, Murshudov G, Garringer HJ, Crowther RA, Ghetti B, Goedert M, Scheres SH. Cryo-EM structures of tau filaments from Alzheimer's disease. Nature. 2017 Jul 13;547(7662):185-190. Epub 2017 Jul 5 PubMed.

    Lövestam S, Li D, Wagstaff JL, Kotecha A, Kimanius D, McLaughlin SH, Murzin AG, Freund SM, Goedert M, Scheres SH. Disease-specific tau filaments assemble via polymorphic intermediates. Nature. 2024 Jan;625(7993):119-125. Epub 2023 Nov 29 PubMed.

    Zhang W, Tarutani A, Newell KL, Murzin AG, Matsubara T, Falcon B, Vidal R, Garringer HJ, Shi Y, Ikeuchi T, Murayama S, Ghetti B, Hasegawa M, Goedert M, Scheres SH. Novel tau filament fold in corticobasal degeneration. Nature. 2020 Apr;580(7802):283-287. Epub 2020 Feb 12 PubMed.

    View all comments by Benjamin Wolozin

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References

News Citations

  1. More Droplets of Tau
  2. Monomeric Seeds and Oligomeric Clouds—Proteopathy News from AAIC
  3. Tau Toggling Peptides: One Seeds Fibrils; the Other Dismantles Them
  4. Mounting Modifications Move Tau Toward Aggregation in Alzheimer’s Brain

Paper Citations

  1. Wegmann S, Eftekharzadeh B, Tepper K, Zoltowska KM, Bennett RE, Dujardin S, Laskowski PR, MacKenzie D, Kamath T, Commins C, Vanderburg C, Roe AD, Fan Z, Molliex AM, Hernandez-Vega A, Muller D, Hyman AA, Mandelkow E, Taylor JP, Hyman BT. Tau protein liquid-liquid phase separation can initiate tau aggregation. EMBO J. 2018 Apr 3;37(7) Epub 2018 Feb 22 PubMed.
  2. Kanaan NM, Hamel C, Grabinski T, Combs B. Liquid-liquid phase separation induces pathogenic tau conformations in vitro. Nat Commun. 2020 Jun 4;11(1):2809. PubMed.
  3. Matsumoto SE, Motoi Y, Ishiguro K, Tabira T, Kametani F, Hasegawa M, Hattori N. The twenty-four KDa C-terminal tau fragment increases with aging in tauopathy mice: implications of prion-like properties. Hum Mol Genet. 2015 Nov 15;24(22):6403-16. Epub 2015 Sep 15 PubMed.

Further Reading

Primary Papers

  1. Soeda Y, Yoshimura H, Bannai H, Koike R, Takashima A. Intracellular Tau Fragment Droplets Serve as Seeds for Tau Fibrils. 2023 Sep 11 10.1101/2023.09.10.557018 (version 1) bioRxiv.

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