Many research groups study tau misfolding and propagation using in vitro models, but interpreting findings from artificial systems can be dicey. In a preprint on bioRxiv, researchers led by Eckhard Mandelkow at the German Center for Neurodegenerative Diseases, Bonn, question whether a widely used assay of tau propagation truly detects transmission of a toxic conformation.
- A fluorescent tag prevents tau fragments from forming paired helical filaments.
- This implies tau inclusions in a common propagation assay may not be PHFs.
- The finding casts doubt on whether the assay detects prion-like propagation.
In this assay, researchers add extracts from Alzheimer’s brain to cultured cells that contain fluorescently labeled tau fragments. When seeded, these fragments aggregate and light up via fluorescence resonance energy transfer (FRET), suggesting a prion-like spread of misfolded tau from the outside of the cell to its inside. However, Mandelkow and colleagues found that the bulky fluorescent label on these tau fragments creates a steric hindrance that prevents them from forming paired helical filaments (PHFs), the building blocks of tangles. Instead, the labeled fragments coalesce into amorphous clumps.
The findings suggest that this assay cannot detect prion-like propagation. This would throw open the possibility that something else in the brain extract might be responsible for inducing tau aggregation, the authors argue. “Factors other than tau should be considered as the [extracellular] agents propagating tau pathology,” Mandelkow wrote to Alzforum.
Packing Interference. Unlabeled aggregation-prone tau fragments form long fibrils (left). Those with an N-terminal fluorescent tag do not (middle), and those with a C-terminal tag form short, aberrant fibrils (right). [Courtesy of Kaniyappan et al., bioRxiv.]
Other scientists disagree with this conclusion. They accept the finding that these fluorescently labeled tau fragments cannot form PHFs, but still believe the cellular assay flags the presence of pathological tau in the brain extract. “I believe these types of cellular aggregation assays are useful and powerful tools to detect seeding-competent assemblies from human or animal brain,” Wouter Peelaerts at the Van Andel Research Institute in Grand Rapids, Michigan, wrote to Alzforum. To Brad Hyman at Massachusetts General Hospital in Charlestown, the data highlight the need to pay attention to the limitations of cellular assays. “There was never any question that the conformational structure of the FRET-based bioreporter would be the same as tau aggregates in the brain … Like most models, its utility depends heavily on understanding its strengths and weaknesses,” he wrote (full comment below). Hyman uses the assay in his own work.
The FRET assay was originally developed by Marc Diamond, now at the University of Texas Southwestern Medical Center in Dallas. He engineered HEK293 cells to express aggregation-prone fragments of tau that are tagged with either a donor (cyan) or acceptor (yellow) fluorescent molecule. When researchers add tau fibrils to the culture medium, these labeled tau pieces come together and fluoresce (Oct 2014 news). The assay has since been adopted by many other labs as a way to measure tau’s pathological activity.
To take a closer look at the structure of these fluorescent tau aggregates, joint first authors Senthilvelrajan Kaniyappan and Katharina Tepper in Mandelkow’s group created tagged tau constructs similar to those used in the FRET assay. They took the short repeat domain of tau containing the pro-aggregant deletion mutation ΔK280, and fused it with a GFP tag at either the N- or C-terminus. Then they incubated these constructs with the nucleating agent heparin in cell-free solution and analyzed the results by UV light scattering. Unlabeled tau fragments aggregated readily, while C-terminally tagged tau formed only a third as many aggregates, and N-terminally tagged tau formed none. Electron microscopy revealed that C-terminally labeled tau aggregated into short fibrils, about twice as thick as those generated by unlabeled tau, with a distinct appearance from PHFs. Meanwhile, N-labeled tau clumped up randomly (see image above).
Additional structural studies added evidence that labeled tau assembles differently than unlabeled. Scanning transmission electron microscopy showed that unlabeled tau fragments formed filaments with a mass-per-length ratio of 4.4 molecules per nanometer, in good agreement with the expected value of 4.3 for PHFs. Labeled tau fragments, on the other hand, formed aggregates with a mass-per-length value around 2. “These values are clearly incompatible with a PHF-like packing of molecules,” the authors wrote. Mandelkow noted that GFP has a diameter of about 3 nm, while the rungs of PHFs are about half a nanometer apart, so there is no room to fit a GFP tag on every rung. The size of the GFP tag prevents close packing of the tau protein, Mandelkow concluded.
In a comment on bioRxiv, Diamond and colleagues question whether the findings from these cell-free studies apply to their cellular assay. Diamond noted several technical differences, chief among them his group’s use of a longer linker sequence, 21 amino acids instead of 13, to attach the fluorescent tag. That would allow GFP to float farther from the fibril core and give it more room to pack. The FRET assay uses a different tau mutation, P301S, and different fluorescent tags, CFP and YFP. Diamond also noted that PHFs are highly polymorphic, with diameters that can vary by more than 2.6-fold, and pointed to a prior study that found evidence for tau aggregates assuming a β-sheet structure in this cellular assay (Barghorn and Mandelkow, 2002; Wegmann et al., 2010; Sanders et al., 2014).
Mandelkow disputes the idea that the GFP linker would make a significant difference, noting that because amino acid strands coil up in solution, the difference in length would be only about one nanometer, not enough to allow the GFP molecules to pack together.
Lary Walker at Emory University, Atlanta, said the Mandelkow group’s conclusions were reasonable within the context of their experimental conditions, and noted that the cellular environment complicates things. “To settle the issue, it would be useful to run controlled comparisons of technical differences such as the linker length in both paradigms,” Walker wrote to Alzforum (full comment below).
Peelaerts cautioned that perhaps none of these in vitro systems reflect the behavior of tau in the brain. “PHFs are just one part of a bigger puzzle. Aggregated tau exists in many conformations, which are dynamic and driven by the equilibrium between the cellular environment and the protein itself. Comparing in vitro assembled seeds with more physiological conditions is therefore always a difficult exercise,” he wrote.
Beyond the structural issue, the scientists also disagreed on the broader interpretation of a positive FRET signal in this assay, and whether that indicates the presence of misfolded tau in the brain extract. Ben Wolozin at Boston University concurred with Hyman and Peelaerts that the assay responds to misfolded tau. “Multiple published studies show that the FRET-sensor lines reliably detect the presence of aggregation-competent tau in brain tissues,” Wolozin wrote. He noted that his company, Aquinnah Pharmaceuticals, has found good concordance between a positive signal in this assay and detection of tau aggregates in the same brain extract using biochemistry or immunohistochemistry. Aquinnah searches for ways to eliminate stress granules, which are associated with Alzheimer’s disease and amyotrophic lateral sclerosis.
For his part, Mandelkow believes the intracellular tau deposits seen in the FRET assay may represent a response to cellular stress or inflammatory stimuli, rather than to aggregated tau in the extract. He noted that tau in primary mouse neurons can be induced to aggregate simply by exposure to activated microglia, or treatment with the proinflammatory cytokine TNFα (Gorlovoy et al., 2009). In addition, a recent study implicated activated microglia in promoting tangles in a tau mouse model (Nov 2019 news). Perhaps inflammation, rather than prion-like propagation, stokes the spread of misfolded tau through brain, Mandelkow suggested.—Madolyn Bowman Rogers
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- Antibodies Stop Toxic Tau in Its Extracellular Tracks
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- Human Tau Strains Propagate Faithfully in Wild-Type Mice
- Tau Filaments from the Alzheimer’s Brain Revealed at Atomic Resolution
- Does Tau’s Third Repeat Propagate Misfolding in Vivo?
- Kaniyappan S, Tepper K, Biernat J, Chandupatla RR, Hübschmann S, Irsen S, Bicher S, Klatt C, Mandelkow EM, Mandelkow E. FRET-based tau seeding assay does not represent prion-like templated assembly of tau fibers. bioRxiv March 25, 2020. BioRxiv.
- Kaniyappan S, Tepper K, Biernat J, Chandupatla RR, Hübschmann S, Irsen S, Bicher S, Klatt C, Mandelkow EM, Mandelkow E. FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau filaments. Mol Neurodegener. 2020 Jul 16;15(1):39. PubMed.