Fowler SL, Behr TS, Turkes E, Cauhy PM, Foiani MS, Schaler A, Crowley G, Bez S, Ficulle E, Tsefou E, O'Brien DP, Fischer R, Geary B, Gaur P, Miller C, D'Acunzo P, Levy E, Duff KE, Ryskeldi-Falcon B. Tau filaments are tethered within brain extracellular vesicles in Alzheimer's disease. bioRxiv. 2023 Apr 30; PubMed.
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The University of Queensland
The work by Fowler and colleagues is beautiful, and the images visualizing tau filaments within extracellular vesicles by cryo-electron microscopy are simply stunning. I had always asked myself what would confer seeding capability to extracellular vesicles including exosomes. Is it a particular phosphorylation pattern of tau (or any other form of post-translational modification), a particular conformation or association with other proteins, or a particular aggregation state? Interestingly, this new work shows all of this.
It reveals filaments (although the discussion whether filaments or oligomers are the major toxic species is still open), there is tethering to the limiting membrane, and the enrichment with endo-lysosomal proteins hints at particular interactions and also subcellular origins. This foundational work is indeed inspiring for all of us working on tau spreading and seeding.
View all comments by Jürgen GötzEmory University
Extracellular vesicles (EVs) have emerged in recent years as important vehicles of intercellular communication in many domains of biology. In the brain, EVs are produced by neurons and non-neuronal cells; their intercellular transfer can support homeostasis, but they also are capable of disseminating pathogenic protein seeds that drive neurodegenerative diseases. The once-murky details of this remarkable mechanism are now yielding to increasingly sophisticated analyses, and the characterization of polymeric tau in Alzheimer brain-derived vesicles by Fowler and colleagues is a welcome contribution.
In a series of striking ultrastructural images, they show that some vesicles contain short segments of paired helical and straight filaments of tau. These intravesicular mini-fibrils can be abundant, and their molecular structure is similar to that of the longer tau fibrils that course through the neuronal cytosol as neurofibrillary tangles. The analysis further demonstrates that fibril-containing vesicles are enriched in proteins associated with the endosomal/lysosomal system.
Which cell type(s) give rise to vesicles bearing fibrillar tau is not certain; microglia and neurons are two obvious (and not mutually exclusive) candidates. Microglia have long been known to be intimately involved in the pathobiology of AD, including Aβ plaques (e.g., Dansokho and Heneka, 2018; Walker, 2020) and tauopathy (e.g. Hansen et al., 2018; Hopp et al., 2018; Maphis et al., 2015). Previous studies have found that inhibiting microglia or the release of EVs blocks the spread of tauopathy (Asai et al., 2015).
Another potential source of the fibril-bearing vesicles is neurons themselves. In humans, the abnormal neuronal processes surrounding Aβ plaques can contain tau fibrils along with a rich variety of vesicles (“autophagic vacuoles”) that constitute an intermediate stage in lysosomal degradation (Nixon, 2007). In this regard, it is worth considering the possibility that at least some of the tau fibril-bearing vesicles derived from tissue homogenates are intracellular entities that have been released by the preparation of the samples. An electron-microscopic search for tau mini-fibrils in intracellular vesicles, especially in dystrophic neurites, might help inform this question. Leaky or ruptured dystrophic neurites still could be a source of EVs in vivo, and indeed they might account for the strong correlation between phospho-tau in the CSF and cerebral Aβ load (Therriault et al., 2023).
The researchers note that mini-fibrils appear to be end-tethered to the vesicular membrane, suggesting that they are selectively packaged, but how is it that the fibrils are conveniently sized to fit inside the vesicles? Are they truncated segments of the much longer fibrils that typify neurofibrillary tangles, suggestive of microglial processing? Or are they growing fibrils that happen to be captured before they reach a limiting length, suggestive of neuronal origin?
Finally, seeding-competent oligomeric Aβ has been found in EVs (Sardar Sinha et al., 2018). It would be interesting to know if both aberrant Aβ and tau are present in the same EVs, and if so, whether they interact in any way. This investigation provides a model for addressing some of the many questions surrounding the pathogenicity of aberrant protein assemblies in the brain.
References:
Asai H, Ikezu S, Tsunoda S, Medalla M, Luebke J, Haydar T, Wolozin B, Butovsky O, Kügler S, Ikezu T. Depletion of microglia and inhibition of exosome synthesis halt tau propagation. Nat Neurosci. 2015 Nov;18(11):1584-93. Epub 2015 Oct 5 PubMed.
Dansokho C, Heneka MT. Neuroinflammatory responses in Alzheimer's disease. J Neural Transm (Vienna). 2018 May;125(5):771-779. Epub 2017 Dec 22 PubMed.
Hansen DV, Hanson JE, Sheng M. Microglia in Alzheimer's disease. J Cell Biol. 2018 Feb 5;217(2):459-472. Epub 2017 Dec 1 PubMed.
Hopp SC, Lin Y, Oakley D, Roe AD, DeVos SL, Hanlon D, Hyman BT. The role of microglia in processing and spreading of bioactive tau seeds in Alzheimer's disease. J Neuroinflammation. 2018 Sep 18;15(1):269. PubMed.
Maphis N, Xu G, Kokiko-Cochran ON, Jiang S, Cardona A, Ransohoff RM, Lamb BT, Bhaskar K. Reactive microglia drive tau pathology and contribute to the spreading of pathological tau in the brain. Brain. 2015 Jun;138(Pt 6):1738-55. Epub 2015 Mar 31 PubMed.
Nixon RA. Autophagy, amyloidogenesis and Alzheimer disease. J Cell Sci. 2007 Dec 1;120(Pt 23):4081-91. PubMed.
Sardar Sinha M, Ansell-Schultz A, Civitelli L, Hildesjö C, Larsson M, Lannfelt L, Ingelsson M, Hallbeck M. Alzheimer's disease pathology propagation by exosomes containing toxic amyloid-beta oligomers. Acta Neuropathol. 2018 Jul;136(1):41-56. Epub 2018 Jun 13 PubMed.
Therriault J, Vermeiren M, Servaes S, Tissot C, Ashton NJ, Benedet AL, Karikari TK, Lantero-Rodriguez J, Brum WS, Lussier FZ, Bezgin G, Stevenson J, Rahmouni N, Kunach P, Wang YT, Fernandez-Arias J, Socualaya KQ, Macedo AC, Ferrari-Souza JP, Ferreira PC, Bellaver B, Leffa DT, Zimmer ER, Vitali P, Soucy JP, Triana-Baltzer G, Kolb HC, Pascoal TA, Saha-Chaudhuri P, Gauthier S, Zetterberg H, Blennow K, Rosa-Neto P. Association of Phosphorylated Tau Biomarkers With Amyloid Positron Emission Tomography vs Tau Positron Emission Tomography. JAMA Neurol. 2023 Feb 1;80(2):188-199. PubMed.
Walker LC. Aβ Plaques. Free Neuropathol. 2020;1 Epub 2020 Oct 30 PubMed.
View all comments by Lary WalkerMayo Clinic Florida
This study demonstrates for the first time the presence of PHF or SF of tau, which are the truncated form encompassing residues 305-379 and detected as 12 or 24 kD by TauC. The authors beautifully show the structure of PHF by cryo-electron tomography and single-particle cryo-EM, which also revealed the structure of PHF as two C-shaped protofilaments and interesting anionic molecules between residue R349 and K375.
The study also demonstrates that AD brain-derived EVs have tau seeding activity both in vitro and in vivo. This is a very interesting finding and provides evidence that brain-derived EVs carry PHF tau seeds capable of tau propagation.
We detected tau protein by Tau13 antibody, which may be attributed to the difference in the isolation method of EVs, sensitivity of the WB technique, or loading volume of the EV samples. We also detected globular tau aggregates in brain EVs (Ruan et al., 2021), which may also be present in Fowler et al.'s preparation.
The authors also show tethering of PHF to the lumen of EVs at the tail end of PHF, suggesting the presence of a unique tau-interacting molecule for the insertion of PHF tau to the EVs. Tau interactome studies will be useful for the identification of the tau interaction molecule for the molecular understanding of the sorting system.
References:
Ruan Z, Pathak D, Venkatesan Kalavai S, Yoshii-Kitahara A, Muraoka S, Bhatt N, Takamatsu-Yukawa K, Hu J, Wang Y, Hersh S, Ericsson M, Gorantla S, Gendelman HE, Kayed R, Ikezu S, Luebke JI, Ikezu T. Alzheimer's disease brain-derived extracellular vesicles spread tau pathology in interneurons. Brain. 2021 Feb 12;144(1):288-309. PubMed. Correction.
View all comments by Tsuneya IkezuUCLA
Scientists have long postulated that pathological tau fibrils spread throughout the brain in a prion-like fashion using intercellular transfer mediated by extracellular vesicles (EVs). Benjamin Falcon’s lab in the MRC Laboratory of Molecular Biology now reports isolation of EVs from AD patients and shows intercellular tau transport in action.
In this preprint, the authors isolated EV fractions from postmortem AD patient brain tissues that contain microvesicles, exosomes, and the newly discovered mitovesicles. After confirming the presence of sarkosyl insoluble tau in the EVs, the authors performed electron cryotomography (cryo-ET) on these freshly extracted vesicles. Cryo-ET combines images of the specimen as it is rotated in the electron beam, producing a picture of the tau fibers inside intact vesicles. Averaging images (“subtomogram averaging”) of fibers inside EVs gave low-resolution densities resembling tau paired helical filaments (PHFs) and straight filaments (SFs).
Subsequent single-particle cryo-EM performed on extracted fibers confirmed the identity of tau PHFs inside EVs. The only difference between the EV-extracted tau PHFs and other previously solved PHF structures from tau extracted from total brain homogenates is the presence of an additional density between the positively charged side chains of Arginine 349 and Lysine 375, giving this PHF an ever-so-slightly-more-compact C-shaped fold.
While studying the tomograms of tau containing EVs, the authors detected an elongated density tethering tau filament ends to the surrounding membrane. It would appear that not all filaments are tethered. Rather, most filaments make lateral contacts with the tethered filament and each other, resulting in bundles of tau filaments enveloped by the EV. Tau was found in the membrane fraction following EV fractionation, further demonstrating direct membrane interaction between tau and the EV membrane.
Whereas these findings may suggest hypotheses about tau being actively sorted into vesicles, and may point to new therapeutic targets, the identity and contact points of the postulated tethers are not yet established. Overall, this study elevates ex vivo tau studies by placing tau directly in a cellular context. It also shows the promise of in situ cryo-ET to study neurodegeneration in a native biological environment.
—Xinyi Cheng is co-author of this comment.
View all comments by David EisenbergMake a Comment
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