Far from being inert intracellular pebbles, inclusions formed by misfolded tau are quite dynamic, according to a paper in the January 26 Acta Neuropathologica. Todd Golde and colleagues at University of Florida, Gainesville, reported seeing turnover of tau inclusions in cultured slices of brain taken from young mice. Inclusions formed within 96 hours of seeding with tau fibrils, or infection with adenoviruses expressing mutant tau. The tau wandered in and out of the inclusions, with a half-life of about one week. As the aggregates aged, however, turnover slowed and half-life tripled. Knowing that inclusions are dynamic, researchers might find new ways to clear them, the authors suggested.
- Tau’s half-life in inclusions is about one week, longer in older inclusions.
- Inclusions seeded by mutant or wild-type tau turned over at same rate.
- Brain-slice model could be useful for studying inclusion-busting drugs.
“This article elegantly shows rapid turnover of soluble tau, while increasingly fibrillar tau shows increasingly slower turnover,” Benjamin Wolozin, Boston University, wrote to Alzforum (full comment below).
Previously, first author Cara Croft had collaborated with Benoit Giasson and colleagues, also at UF, to create a cell-culture model that did not require addition of tau fibrils to seed tangles (Strang et al., 2018). They infected cells with a recombinant adeno-associated virus (rAAV) containing human tau with two mutations, P301L and S320F, that cause frontotemporal dementia and parkinsonism linked to chromosome 17 (Rosso et al., 2002; Hutton et al., 1998). The scientists then used the same rAAV construct to infect organotypic mouse brain slices (Feb 2019 news). After 28 days, tau inclusions formed within neurons.
Now, Croft has tracked how those inclusions behave over time. She infected the brain slices with rAAVs containing human wild-type or P301L/S320F tau tagged with a Dendra2 motif. This fluorescent peptide is photoswitchable, meaning it changes irreversibly from emitting green photons to red after brief exposure to short-wavelength light. After this conversion, newly made tau can be distinguished by its green fluorescence, allowing the researchers to track new and older tau in real time.
In the slices, P301L/S320F tau formed inclusions within 96 hours and as quickly as 24 hours. After 10 days, about 87 percent of cells had inclusions of fibrillar tau, which lit up when stained with the β-sheet dye thiazine red. The Dendra-2-tagged tau clumped similarly to the unadulterated tau, indicating that the fluorescent motif was neither impeding nor aiding aggregation.
To track tau dynamics over time, Croft and colleagues cultured the transfected brain slices for 10 days, then shone light onto the cells to convert all the green-fluorescent tau to red. Then they repeatedly imaged the cells over 21 days, measuring how much tau was old versus new. Neurons produced wild-type and mutant tau at about the same rate. Wild-type formed no inclusions and had a half-life of about 2.7 days.
Tau in inclusions lasted longer, but not by much. New tau replaced old, gradually transitioning red clumps to green without affecting the size of the inclusion (see image below). Inclusion tau had a half-life of about eight days. Golde was surprised by how quickly tau turned over.
Tau Turnover. In mouse brain slices cultured for 10 days, green-fluorescent Dendra2-tau was zapped with light, turning the protein red (top row). Over the next 21 days, old tau turned over (left columns) as new tau formed (middle columns). Right columns show the mix. Mutant tau (right three columns) in inclusions lingered longer than did wild-type tau (left three columns) in the cytosol. [Courtesy of Croft et al., Acta Neuropathologica, 2021.]
Because tauopathies do not usually begin until midlife and inclusions are known to stick around for years, the authors wondered if tau dynamics changed as the tissue aged. The researchers cultured the brain slices for 30 or 60 days before converting the green-labeled tau to red, then measured turnover in the inclusions, again for at least 21 more days. The older the tissue culture, the longer tau’s half-life. It grew from eight days when photoswitching was done in 10-day-old slices to 16 days and 23 days when 30- and 60-day-old tissue was used, respectively. Even wild-type tau languished slightly longer as it aged, with a half-life of about four days in both 30- and 60-day-old slices, up from the 2.7 in 10-day-old tissue.
What about inclusions caused by fibrils of wild-type tau, such as those found in Alzheimer’s disease? Croft took fibrils made in Escherichia coli expressing the microtubule-binding domain of four-repeat (4R) tau and added them to 14-day-old slice cultures. These did not seed inclusions in wild-type tissue, but did seed in slices expressing P301L tau. P301L slices on their own did not form inclusions.
Inclusions formed within 96 hours of seeding. After 10 days, Croft shone light on the slices and measured inclusion turnover for another 10 days. Tau half-life was about eight days, or just the same as the P301L/S320F tau. In 30- and 60-day-old slices post-seeding, it was 18 and 24 days, respectively, again similar to half-lives cause by the double mutant.
Why tau turnover slows over time remains a mystery. Croft thinks that microglia and astrocytes may change over time, impeding tau clearance. Golde agreed, noting that tau turnover must be facilitated. “An isolated tangle does not spontaneously dissolve, so I do not see how it could be a passive process,” he told Alzforum.
Tara Spires-Jones, University of Edinburgh, was intrigued by the longer half-life in older cultures. “It begs the question of what the turnover is like in the aged human brain,” she wrote to Alzforum (full comment below). “Is this difference a function of the tissue or of the age of the inclusion?” Golde wondered the same thing. “There is evidence that cross-linking and other post-translational modifications occur in tau inclusions,” he said. “We can imagine that something cross-linked would be harder to remove.”
Marc Diamond, University of Texas Southwestern, Dallas, was not surprised by these findings. “We have known for a few decades that inclusions are not ‘rocks’ but can be disassembled,” he wrote to Alzforum. “Presumably the aggregates get more consolidated, as other proteins pile on, and it is a bigger mess to disassemble.”
Previously, researchers in David Holtzman’s lab at Washington University School of Medicine, St. Louis, examined tau turnover in the brains of transgenic mice (Yamada et al., 2015). Soluble tau turned over faster than insoluble tau, with half-lives of about 10 and 34 days, respectively. Phosphorylated soluble tau cleared even faster, with half-lives between five to 10 days, depending on which amino acid was modified. The authors noted that insoluble tau likely represented oligomers, fibrils, and other aggregates, but they did not look at inclusions specifically.
“Overall, our findings are reasonably consistent with Croft’s despite using very different methods,” Holtzman told Alzforum. “We both show that tau aggregates are in equilibrium with soluble tau.”
Also at Wash U, Randy Bateman, Celeste Karch, and colleagues studied tau turnover in human induced pluripotent stem cell (iPSC)-derived neurons (March 2018 news). Using stable isotope labeling, the researchers tracked how much tau was present over time via mass spectrometry. In cultured neurons, tau’s half-life was seven days, with phosphorylated and 4R tau turning over quicker than the three-repeat variety.
What about other tau mutations or other tauopathies besides FTD and PD? Michel Goedert, MRC Laboratory of Molecular Biology, Cambridge, England, U.K., noted that inclusion turnover rates likely depend on filament structures and modifications of their components, which differ between diseases. “For example, tau filament folds differ among AD, chronic traumatic encephalopathy, FTDP-17, and corticobasal degeneration,” he wrote to Alzforum (full comment below).
Holtzman proposed that because of this ebb and flow of tau, one could possibly get rid of the aggregates by removing the surrounding soluble tau. Claire Durrant, University of Edinburgh, warned against potentially unwanted effects of this, such as releasing toxic, synapse-disrupting oligomers. “This model could be useful for testing this principle, by correlating increased or decreased turnover with measures of neuronal health and synaptic function,” she wrote to Alzforum (full comment below). Diamond agreed. “This is a strong system to study the biological basis of aggregate turnover, and could have important implications for identifying factors involved in this process.”—Chelsea Weidman Burke
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