Tau tangles accumulate within neurons, but they are also thought to travel from cell to cell via extracellular vesicles. A study posted April 30 on bioRXiv provides startling support for this idea. For the first time, scientists used cryo-electron tomography to spy on the spatial organization of proteins within vesicles isolated from the brains of people with Alzheimer's disease. Why, hello there: They spotted filaments of tau nestled inside.
- Cryo-electron tomography spots tau filaments inside extracellular vesicles from AD brain.
- Filaments are tethered to each other, and to the membrane.
- Mystery molecules forge these links.
The filaments did not appear to have been sloppily shoved into these vesicles. Rather, an unidentified protein neatly tethered them to the vesicle inner membrane. Under the gaze of the cryo-electron microscope, the core of the filaments revealed a back-to-back C-shaped fold, akin to its counterpart extracted from AD brain lysates, albeit with some differences. Vesicle filaments were shorter, and decorated by unknown molecules that the scientists speculate may have ushered the tau inside.
Led by Karen Duff at University College London and Benjamin Ryskeldi-Falcon of the Medical Research Council Laboratory of Molecular Biology, Cambridge, U.K., the scientists also reported that these tau filaments seeded propagation of tau pathology within cell lines and in transgenic mouse brain. In all, the study casts extracellular vesicles (EVs) as “good contenders” for tau transport vehicles, and illuminates “a whole new biology for tau,” Duff told Alzforum.
“The work by Fowler and colleagues is beautiful, and the images visualizing tau filaments within extracellular vesicles by cryo-electron microscopy are simply stunning," commented Jürgen Götz at the University of Queensland, Brisbane, Australia. Lary Walker of Emory University, Atlanta, noted that while the intercellular transfer of EVs within the brain can support homeostasis, it can also disseminate pathogenic protein seeds that drive neurodegenerative disease. “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 ... is a welcome contribution,” he wrote to Alzforum (comments below).
From free-floating tau to tunneling nanotubes to EVs, different modes of transport have been proposed to explain the spread of tau pathology in tauopathies. EVs are an attractive option, because they could explain how tau appears to travel via synaptic circuitry and other routes.
EVs come in different flavors, distinguished by their cellular origins. Best known are exosomes, which derive from multivesicular bodies that fuse with the plasma membrane, releasing their brood of intraluminal vesicles into the extracellular space. Neurons can secrete exosomes, but microglia crank them out with gusto when activated. Indeed, microglia have been implicated as prime disseminators of pathological tau in mouse models, where the cells gobble up tau from neurons and then spew it out, either in free-floating form, or packaged within exosomes (Oct 2015 news; Clayton et al., 2021; Oct 2021 news; Apr 2023 conference news).
But do tau filaments travel this way in the human brain? To find out, co-first authors Stephanie Fowler and Tiana Behr studied tau within EVs derived from human brain samples. Using density gradient centrifugation, they fractionated EVs of various heft from frontal, temporal, and hippocampal AD brain extracts, and used mass spectrometry to probe the origins of EVs in the fractions. They identified three kinds: exosomes, which were loaded with endolysosomal proteins; microvesicles, which fuse directly with the plasma membrane and contain a glut of membrane-derived proteins; and mitovesicles, which come from mitochondria.
The scientists also detected fragments of tau, predominantly within exosomes. This tau was truncated at both the N- and C-termini and contained the third and fourth of the four microtubule binding domains. It resisted dissolution by sarkosyl and bound to antibodies for paired helical filaments, suggesting it was fibrillar.
Cryo-electron tomography revealed how tau was arranged within the vesicles. Though lower resolution than cryo-EM, cryo-ET visualizes the ultrastructure and organization of proteins in situ. This is the first time the technique has been applied to human brain tissue, said Ryskeldi-Falcon. The tomographs revealed both paired helical filaments and straight filaments of tau within the lumen of EVs. Individual vesicles contained three to 50 filaments each. Compared to the micrometer-long filaments that form neurofibrillary tangles, the vesicle filaments were short, measuring 75 to 100 nanometers in length.
Strikingly, in each vesicle at least one of these stubby tau filaments was tethered to the inner membrane, while the remaining fibrils seemed to cling to the tethered filament, and/or to each other. In this way, it appears that all tau filaments may be attached—either directly or indirectly—to the inner membrane. To confirm that all the tau filaments were indeed attached in this way, the scientists extracted the membrane fraction of the vesicles. They found tau filaments still attached, while no filaments were detected among the intraluminal proteins.
Tau fibrils latched on to the membrane exclusively by their filament ends. There, flexible, elongated densities, likely representing a mysterious tethering protein, wedged in between the filament tip and the membrane (image below).
Cryo-ET also revealed smaller vesicles within the EVs. Tau filaments occasionally made contact with those (first image below). Duff does not know what these are, but said that tau filaments were never found within them, nor did tau appear tied specifically to their surface. Finally, globular entities decorated the sides of the filaments. Some of these hangers-on followed the helical symmetry of the fibril, suggesting they bound specific sequences.
Tethered Tau. Tomographs of EV cross-sections show tau filaments (pink asterisks) hitched to the luminal side of the EV membrane (yellow arrows) via unknown densities (orange arrows). [Courtesy of Fowler et al., bioRXiv, 2023.]
To Ryskeldi-Falcon, this intricate arrangement of tau filaments means that their presence is no accident. “It suggests there is something selective that’s packaging tau filaments into these vesicles,” he said.
To zero in on the fibrils' atomic structure, the scientists resolved individual paired helical filaments with cryo-EM. The protofilament structure that emerged—back-to-back C-shaped protofilaments packaged with helical symmetry—was nearly identical to the one previously identified from AD brain homogenates by Michel Goedert and Sjors Scheres's groups at the MRC in Cambridge (Jul 2017 news). One notable distinction: an unknown, negatively charged molecule bridged positively charged tau residues on each tip of the C. This interloper essentially closed up the C, resulting in a more compact protofilament core (see image below).
Closing the Loop. Positioned on each end of the C-shaped protofilament near positively charged residues, negatively charged molecules (gray blob, arrow) pull the ends of the C shape closer together. [Courtesy of Fowler et al., bioRxiv, 2023.]
These EV-specific structural tweaks left tau’s ability to seed aggregates intact. The scientists found that tau filaments from EVs potently seeded tau aggregation in biosensor cell lines, and within the brains of P301S-tau transgenic mice.
Marc Diamond of UT Southwestern Medical Center in Houston called the findings “fundamentally interesting.” He cautioned that implicating EV tau in seeding or propagation should involve ruling out involvement of extravesicular tau, and testing the relative seeding efficiency of tau encased in different vesicle fractions. He thinks further context on how common these EVs with tau filaments are compared to the total population of EVs will help the field understand their biological significance.
To the authors, the findings suggest that tau filaments are actively packaged into vesicles of endolysosomal origin, which are then secreted as exosomes and, if taken up by recipient cells, may seed tauopathy. One open question is which cell types do this. Duff said this is under active investigation. She noted that neurons might be goaded into producing exosomes in response to hyperactivity brought on by exposure to Aβ oligomers.
Another suspect: viruses. The cellular response they provoke is increasingly implicated in AD, most recently with reports that viral infection ramps up EV production by neuron and that ancient viral response pathways might stoke inflammation (Apr 2023 news; Apr 2023 conference news).
Microglia may crank up EV production in response to inflammation, Duff added. Fowler said that defects in the endolysosomal system also promote the release of EVs, particularly EVs of endolysosomal origin—the type found to contain tau seeds (Cataldo et al., 2000; Hessvik et al., 2016; Abdulrahman et al., 2018).
Tsuneya Ikezu, at the Mayo Clinic in Jacksonville, Florida, was intrigued by the tethering of tau filaments to the lumen of EVs. Tau interactome studies in EVs could identify the molecules involved, and explain how the sorting works, he said. Ikezu noted that the exosomes housing the tau filaments were quite large, as exosomes go, and that tau filaments, even short ones, may be unable to squeeze inside of smaller vesicles.
Walker wondered why the fibrils happen to be conveniently sized to fit inside these 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?” Another question is whether tau filaments cross paths with oligomeric Aβ inside exosomes, since these aggregates have also been spotted within EVs (Sardar Sinha et al., 2018). “This investigation provides a model for addressing some of the many questions surrounding the pathogenicity of aberrant protein assemblies in the brain,” Walker wrote.—Jessica Shugart
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