Opposites attract, sometimes so much so that they retreat into their own little bubble. This seems to be the case with tau, a positively charged protein, and RNA, a negatively charged nucleic acid. According to a study published July 6 in PLOS Biology, the pair tightly coupled together in neurons, and coalesced into droplets in a dish. While tau appeared unfettered by the close quarters at first, the protein started showing early signs of fibrillization after prolonged residency in the droplets. The researchers, led by Kenneth Kosik and Songi Han at the University of California, Santa Barbara, proposed that these droplets could play a role in tau aggregation within neurons.
The findings add tau to the growing list of neurodegenerative disease-associated proteins known to undergo liquid-liquid phase separation (LLPS), a phenomenon increasingly implicated in all manner of cellular functions. LLPS occurs when proteins and/or nucleic acids associate closely, changing the viscosity of their local environment (see Li et al., 2012). In so doing, they can form transient “membrane-less organelles,” like pop-up shops, to accomplish specific cellular functions. Stress granules, the nucleolus, and even lipid rafts of signaling receptors are examples. RNA binding proteins, and other sticky proteins that contain so-called low-complexity domains, are common inhabitants of these liquid organelles. Neurodegenerative bad boys TDP-43, FUS, C9ORF72 dipeptide repeats, hnRNPA1, and hnRNPA2B1 are among those spotted within the droplets, where they are proposed to derail the machinery within them (Oct 2015 webinar; Oct 2016 news; May 2017 conference news).
The idea that tau, too, could dabble in droplets stems from observations that it associates with RNA. Though tau is not a bona fide RNA-binding protein or a bearer of low-complexity domains, the protein’s positive charge and intrinsic disordered state make it a prime candidate for an electrostatic liaison with RNA. Previous studies reported that tau’s association with RNA coaxed the protein into fibrils, though with less vigor than the polyanionic aggregation inducer, heparin (see Kamper et al., 1996; Wang et al., 2006).
In the present study, first author Xuemei Zhang and colleagues sought to investigate the nature of tau’s association with RNA. Using a cross-linking technique called PAR-iCLIP, the researchers found that both wild-type and mutant forms of tau associated with RNA in human embryonic kidney (HEK) 293T cells and in neurons derived from induced pluripotent stem cells (iPSCs). A closer inspection of the RNA bound to tau revealed that they were predominantly tiny species.
To the researchers’ surprise, transfer RNA (tRNAs) made up the overwhelming majority of tau’s RNA partners. While tau comingled with many tRNAs, it gave preferential treatment to some. Of the 231 tRNAs tau buddied up with iPSC-derived neurons, it preferred tRNAArg most. The researchers found that at low tau-to-RNA ratios, tau associated with RNA species as a dimer, while ratcheting up the tau concentration generated much larger tau-to-RNA complexes.
Mixing tau and RNA together outside of cells created a turbid solution. Under the brightfield microscope, the researchers spied droplets full of tau and RNA. The droplets formed when using full-length tau or Δtau187, and when using tRNA, poly(A)RNA, or poly(U)RNA. Regardless of the type of RNA molecule, droplets existed in a 1:1 charge ratio with their tau associates. They were highly dynamic, readily merged, and the researchers found they could toggle droplet formation up or down by adjusting protein/RNA concentrations, salt concentration, pH, or temperature. Importantly, droplets formed under physiological conditions mimicking those in neurons.
How did the droplet-bound life change tau? Not much at first, the researchers reported. Spin-labeling experiments of tau/RNA droplets revealed that tau did not change its conformation despite its high concentration inside droplets. In contrast, heparin induced dramatic changes corresponding to β-sheet formation. However, Thioflavin T (ThT) fluorescence, an indicator of β-sheets, did gradually rise over 15 hours after the droplets formed. Like other groups studying liquid-liquid phase transition of neurodegenerative disease proteins, Kosik and colleagues suggested that, given enough time and under certain conditions, the droplets could be a prime venue for tau aggregation. In support of this idea, they saw an increase in sarkosyl-insoluble tau when they transfected iPSC-derived neurons with an overload of tRNA.
Kosik told Alzforum that the negatively charged RNA in the droplet likely beckons tau to squeeze more closely together than it would without the attractive charge, and to do so without changing its conformation. A major caveat of the study is that it provides no proof that the tau/RNA droplets exist within cells, Kosik acknowledged. However, he said that if the droplets do form in cells, their integrity could be modulated by cellular stress, a state known to change parameters such as pH and salt concentration. “We think the in vitro tuning conditions we observed for the droplets may have some correlates to stress condition in a living cell,” he said.
Benjamin Wolozin of Boston University praised the study for its rigor, and agreed that cellular stress would influence such droplets inside a cell. For his part, Wolozin has reported that tau facilitates the formation of stress granules, a type of membrane-less organelle that sequesters nonessential RNA transcripts from translation (May 2016 news). He also reported that in response to stress, tau moves from axons into the somatodendritic compartment, where it is likelier to encounter RNA. Perhaps when stress goes on too long, this relationship promotes tau aggregation, he said. This would align with the inklings of aggregation that Kosik and colleagues observed hours after tau/RNA droplets formed.
Wolozin was intrigued by tau’s preference for tRNA, noting that if this association is confirmed, it could explain previous observations that tau inhibits translation (Meier et al., 2016). Perhaps tRNAs deliver tau to the translation machinery, where it gums up the works, he said.
Though still unpublished, findings from other researchers are converging on the idea that tau forms droplets. At a recent meeting in Leuven, Belgium, Susanne Wegmann of Massachusetts General Hospital in Charlestown reported as much. In collaboration with Anthony Hyman at the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden, Germany, Wegmann found that the phosphorylation of tau facilitated droplet formation, and that the droplets could form in neurons (May 2017 conference news). Wegmann speculated that the droplets could provide cells with a ready supply of tau for stabilization of microtubules. This idea is supported by findings the researchers recently posted on bioRχiv (Hernandez-Vega et al., 2017).
Markus Zweckstetter of the Max Planck Institute for Biophysical Chemistry in Gottingen, Germany, will also soon report on liquid phase separation of tau, including differences between tau splice variants in their tendency to form droplets. Zweckstetter told Alzforum that contents of the tau droplets he observed differ from those described in Kosik’s work. Stay tuned for details when Alzforum covers this upcoming paper.—Jessica Shugart
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