09 Apr 2023

For almost 10 years, scientists around the world have been using cell-based fluorescent sensors to study the protofibrils that spawn neurofibrillary tangles in tauopathies. Some have claimed the tau chimeras seeded in these cells cannot twist into the same type of fibrils found in the brain. Not so, said Sarah Shahmoradian, University of Texas Southwestern Medical Center, Dallas, at the International Conference on Alzheimer's and Parkinson's Diseases 2023, held March 28 to April 1 in Gothenburg, Sweden. Her high-resolution two-dimensional and three-dimensional cryo-electron microscopy and tomography images showed that these chimeras fold and stack into a typical amyloid structure, with their fluorescent peptide moieties evenly spaced along the outside of the fibrils. Her findings support the contention that these cells make good models for studying tau fibril formation in the brain.

Indeed, in a separate AD/PD presentation, Ulrich Hartl, Max Planck Institute of Biochemistry, Martinsried, Germany, reported that the fibrils in these same cells are pulled apart by valosin-containing protein. This chaperone then tosses the tau fragments into the proteasome for degradation. While that sounds like a blessing, VCP can also release new seeds that might fuel the spread of tangles in tauopathies, Hartl discovered.

Together, these studies elucidate the dynamic nature of tau fibrillization in cells, perhaps with broad consequences for tau pathology in the brain. Still, whether the structures formed in these cell lines are identical to those found in the tauopathies remains to be seen.

Marc Diamond, now at UT Southwestern, had developed the biosensor cell lines in question when he was at Washington University, St. Louis. The HEK293 cells express two tau chimeras. Each comprises an aggregation-prone repeat domain of tau coupled to cyan (CFP) or yellow fluorescent protein (YFP). When the chimeras are close together—as in a fibril—fluorescence from the CFP excites YFP in a process called fluorescence resonance energy transfer. FRET is visible under the microscope, and indeed, Diamond and colleagues used it to identify morphologically distinct polymorphs of fluorescent tau in sensor cells seeded by extracts taken from different tauopathies (May 2014 news). The cells even detected tau seeds in brains that had no overt signs of tau pathology (Oct 2014 news).

Scientists in other labs began using the cells. Then, three years ago, Eckhard Mandelkow’s lab at the German Center for Neurodegenerative Diseases, Bonn, threw cold water on them, claiming that the bulky fluorescent proteins prevented templated misfolding and fibrilization of tau as it happens in the human brain (May 2020 news).

Shahmoradian, a biophysicist, deployed some high-resolution cryo-electron microscopy techniques to take a closer look. In Gothenburg, she showed cryo-EM and cryo-electron tomography images of fibrils isolated from HEK sensor cells that had been seeded with brain extract from the tauopathy corticobasal syndrome. After placing fibrils from the sensor cells on an EM grid, Shahmoradian used their fluorescence to pinpoint them for cryo-EM, then tilted the grid through multiple angles to obtain three-dimensional cryo-ET images. This vastly increases the amount of information one can get from one fibril, she said.

From individual fibrils, Shahmoradian found hints of teeny blobs dotting their periphery. By summing analysis of 1.3 million of these fibril particles, she found that these moieties densely decorated the tau fibrils and that they had the same dimensions as—YFP. In short, her lab’s data indicated that brain extracts had seeded fibrils in the sensor cells, and that those fibrils were formed by the tau-YFP chimeras (image below).

But did these in vitro fibrils resemble those found in human brain? Fast Fourier transform analysis was able to resolve repeated features in the fibrils. It predicted that protein chains were stacked perpendicularly to the axis of the fibril at 4.7Å apart, and that they formed cross-β sheets in the fibril core. These are typical features and dimensions of amyloid fibrils, including the paired-helical fragments (PHFs) of tau that aggregate into neurofibrillary tangles.

Mandelkow’s group had calculated that tau-YFP chimeras couldn’t form PHFs, because YFP would not fit into the 4.7Å gap. “We cannot explain why the Mandelkow group could not produce recombinant tau-YFP fibrils,” Shahmoradian wrote to Alzforum. “Their interpretation that their negative data indicates biosensor cells cannot create tau fibrils, however, is incorrect,” she added. She concluded that steric hindrance does not keep these chimeras from templating fibrils.

Shahmoradian found similar fibrils within iPSC-derived human neurons. When she seeded such neurons expressing the FRET twins P301L-tau clover and P301L-tau ruby, fibrils formed that were unlike any natural filaments, such as microtubules or neurofilaments. These were detected by plunge-freezing the neurons, homing in on the FRET fluorescence, and using cryo-focus ion beam milling—a cellular equivalent of sand blasting—to thin the material sufficiently for cryo-TEM. The findings hint that, in human neurons, the fluorescent reporters form the same type of amyloid Shahmoradian had found in the sensor cells. She has no high-resolution cryo-EM tomography on those fibrils yet.

Breaking Up Tau Fibrils
Hartl collaborates with Diamond and uses the same sensory lines. He also believes that the fibrils in these cells are amyloids. Evidence came from a collaboration with Mark Hipp and Wolfgang Baumeister, also at Max Planck in Martinsried. Qiang Guo and Rubén Fernández-Busnadiego in Baumeister’s lab noticed that the fibrils looked quite different than microtubules and other cytoskeletal polymers, such as actin filaments, and that they bound the amyloid dye Amylo-Glo.

Cryo-ET suggested that the tau fibrils associate with organelles such as mitochondria, the Golgi network, and the endoplasmic reticulum. The latter is famous for its ability to disassemble protein complexes as part of the ER-associated proteasome pathway for degradation. Could that system dismantle these tau fibrils, too?

To test if the fibrils come apart, Itika Saha and colleagues in Hartl’s lab stopped tau-YFP production in sensor lines that had been seeded, then watched to see what happened to tau. The number of inclusions in the cells dropped 10-fold within a day and they shrank to half their size.

For tau fibrils to vanish so quickly, there would have to be some sort of machinery in the cells tearing them asunder. To find out what that might be, Saha compared the proteomes of cells with and without tau inclusions. The former had upregulated components of the proteasome—and VCP.

The chaperone piqued Hartl’s interest, because mutations in its gene have been associated with TDP-43 proteopathies, and even cause a rare form of frontotemporal dementia (Neuman et al., 2007; Oct 2020 news).

The hexameric VCP complex forms a ring structure with a central pore (image at right). Powered by two ATPase subunits, it grabs the ends of proteins and pulls polypeptide chains through the pore, unravelling the whole thing in the process. Dubbed a protein extractor, it is highly conserved and essential for proteostasis from yeast to people.

Is VCP the dis-aggregase that pulls tau fibrils apart? Saha added an inhibitor of VCP's ATPases to tau sensor cells after they had formed tau inclusions. This time when she shut down tau production, the inclusions hung around (see image below). When she added the inhibitor to cells that were making fibrils, inclusions grew larger. “We see a net reaction between formation of aggregates and constant disaggregation,” said Hartl. Blocking the proteasome also prevented disassembly, suggesting a coupling between VCP's and the proteasome's actions.

Disassembly of tau inclusions is good, right? Not always. Patricia Yuste-Checa in Hartl’s lab wondered if blocking VCP would affect the spread of toxic forms of tau from cell to cell. She added it to sensor cells as they were actively fibrillizing tau, extracted their aggregates, then used the aggregates to seed a new set of sensor cells. Surprisingly, the VCP inhibitor halved the seeds' potency, suggesting that the chaperone helped these toxic protofibrils to form. Blocking the chaperone Hsp70, or the proteasome, had no effect.

Hartl proposes that when VCP unravels the end of a fibril, it might release monomers of tau that get degraded by the proteasome, but if VCP starts pulling fibrils apart in the middle, this might release fragments that then act as seeds for the growth of more fibrils (see model below). These seeds might be involved in transcellular seeding of tau fibrils in the brain. If so, VCP would increase the danger of this phenomenon. The work appeared on February 2 in Nature Communications.

Dicey Disassembly. In pulling apart tau fibrils, VCP might release toxic seeds that accelerate aggregation of normal tau. [Courtesy of Saha et al., Nature Communications, 2023.]

Does this dynamic regulation happen in the human brain? “Although I would be surprised if VCP did not function in disaggregation in the brain, this remains to be addressed,” Hartl wrote to Alzforum. One hint that it might play out that way came from mouse primary neurons expressing fluorescent reporter tau chimeras. Blocking VCP dramatically increased the number of tau inclusions.

All that said, a major question remains. Are the amyloid fibrils formed in these sensor lines, and in induced human neurons, truly the same as those that form in the human brain? “At this point, we cannot be sure that the fibrils of tau that are disaggregated in our various cellular models are the same as those in patient brain,” Hartl wrote.

In Gothenburg, Sjors Scheres, MRC Laboratory of Molecular Biology, Cambridge, England, cautioned that the structures that form in seeding experiments may be different than those in the seed. His and Michel Goedert's labs had previously shown that α-synuclein fibrils formed in vitro bore little resemblance to the structure of the α-synuclein used to seed them (Lövestam et al., 2021). In the case of tau, more than 70 different fibril structures formed in response to varying fibrillization conditions, and only two were identical to tau structures found in disease (Lövestam et al., 2022). 

Still, Scheres believes that experimental systems, be they in vitro, in cells, or in animals, will be crucial for studying how these protein fibrils form in the brain. “Are the structures that are formed the same as those in disease? We should answer this question. If so, then one could hope that some of the molecular mechanisms in the models are relevant for disease,” he said at AD/PD.

Shahmoradian is on it. “Our lab is now working on solving the core structure of these cell-extracted fibrils so that we can compare them to human brain tissue-extracted fibrils,” she wrote to Alzforum.—Tom Fagan

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References

News Citations

1. Like Prions, Tau Strains Are True to Form 22 May 2014
2. Cellular Biosensor Detects Tau Seeds Long Before They Sprout Pathology 9 Oct 2014
3. Widely Used Tau Seeding Assay Challenged 22 May 2020
4. VCP Coding Mutation Causes a Tauopathy With Vacuoles 2 Oct 2020

Paper Citations

1. Neumann M, Mackenzie IR, Cairns NJ, Boyer PJ, Markesbery WR, Smith CD, Taylor JP, Kretzschmar HA, Kimonis VE, Forman MS. TDP-43 in the ubiquitin pathology of frontotemporal dementia with VCP gene mutations. J Neuropathol Exp Neurol. 2007 Feb;66(2):152-7. PubMed.
2. Lövestam S, Schweighauser M, Matsubara T, Murayama S, Tomita T, Ando T, Hasegawa K, Yoshida M, Tarutani A, Hasegawa M, Goedert M, Scheres SH. Seeded assembly in vitro does not replicate the structures of α-synuclein filaments from multiple system atrophy. FEBS Open Bio. 2021 Apr;11(4):999-1013. Epub 2021 Feb 24 PubMed.
3. Lövestam S, Koh FA, van Knippenberg B, Kotecha A, Murzin AG, Goedert M, Scheres SH. Assembly of recombinant tau into filaments identical to those of Alzheimer's disease and chronic traumatic encephalopathy. Elife. 2022 Mar 4;11 PubMed.

Further Reading

News

1. Flock of New Folds Fills in Tauopathy Family Tree 5 Oct 2021
2. Double Spiral Sets TDP-43 Apart from Other Amyloids 10 Dec 2021
3. Cryo-EM Unveils Distinct Aβ42 Fibril Structures for Sporadic, Familial AD 14 Jan 2022
4. Surprise! TMEM106b Fibrils Found in Neurodegenerative Diseases 12 Apr 2022
5. In Parkinson’s, Synuclein Fibrils Fold Differently Than in MSA 21 Jul 2022