18 Aug 2017

When it rains, sometimes it pours. In the second paper describing taus ability to undergo liquid-liquid phase separation in the past month, researchers led by Markus Zweckstetter of the German Center for Neurodegenerative Diseases in Gttingen report that phosphorylation of tau dramatically enhances droplet formation, and that taus repeat domains play a key role in the process. In their August 17 paper in Nature Communications, the researchers made the case that droplets are an essential precursor in the formation of toxic tau tangles, though experiments were all conducted in cell-freeconditions.

Tau is one of many proteins involved in neurodegenerative disease that have been spotted mingling in liquid droplets (Oct 2015 webinar, Oct 2016 news). Inside the cell, the process of liquid-liquid phase separation (LLPS) leads to membrane-less organelles, including stress granules and the nucleolus. Interactions between proteinsespecially those donning low complexity domainsand nucleic acids trigger the process, and researchers have proposed the close quarters in the droplets could breed toxic aggregates, or derail essential cellular functions (May 2016 news). A recent study led by Kenneth Kosik and Songi Han of the University of California, Santa Barbara reported that tau coalesced into liquid droplets in a dish, and that interactions between positively charged tau and negatively charged RNA made the magic happen (Jul 2017 news). Researchers led by Anthony Hyman of Germanys Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, along with Bradley Hyman and Suzanne Wegmann of Massachusetts General Hospital in Charlestown, have also reported that tau forms droplets in vitro and in neurons (May 2017 news).

Fluorescently labeled tau (green) forms droplets at body temperature (bottom), but not at 5C (top). [Image courtesy of Ambadipudi et al., Nature Communications,2017]

In the current study, first author Susmitha Ambadipudi and colleagues investigated if, and under what conditions, tau undergoes LLPS, and how that relates to its propensity to aggregate into fibrils. They started by analyzing various regions of the tau protein with catGranule, a program that predicts propensity of a given protein region to undergo phase separation. While much of taus N-terminus scored low, its repeat domains scored high. The researchers therefore conducted most of their experiments using the K18 fragment of tau, which contains only the four repeatdomains.

They reported that under reducing conditions similar to those inside a cell, K18 formed a turbid solution. Bright field and confocal microscopy revealed K18 formed droplets under Goldilocks conditionsnot too cold (5C), not too hot (above 65C), but just right (37C). Under these conditions, K18 did not appear to form outright fibrils, although CD and NMR spectroscopy suggested the protein started exhibiting signs of b-sheet structure and that droplet-resident tau proteins formed tight molecular interactions, akin to amesh.

Could this mesh facilitate fibril formation under certain conditions? To get closer to answering this question, the researchers toggled multiple parameters, including temperature and pH, and added the polyanion heparin into the mix. They found that heparin triggered fibril formation most efficiently and under the very same conditions that facilitate LLPS, suggesting the two processes are linked. Polyanions have long been used to promote formation of tau fibrils (Goedert et al., 1996).

In the cell, tau occurs in six isoforms due to alterative splicing, and can be further processed by proteolytic fragmentation and a variety of post-translation modifications. How might these permutations affect LLPS? They found that droplet formation correlated with the number of repeats, and did not occur at all in an N-terminal fragment that lacked repeats. They also found that phosphorylation of repeat domains by the MARK2 kinase promoted LLPS. Notably, phosphorylated tau underwent LLPS at just 2 mM, a concentration similar to that inside of neurons. Interestingly, another recent study found that phosphorylation had the opposite effect on phase separation of the FUS protein, which plays a role in amyotrophic lateral sclerosis (Aug 2017 news).

In this proposed model of tangle formation, phosphorylation of tau promotes the formation of liquid droplets, which crowds tau, recruits polyanions, and triggers aggregation. [Courtesy of Ambadipudi et al., Nature Communications,2017]

The in vitro findings mesh with Kosik and Hans recent study, which found that tau formed droplets in the presence of RNA. Though Zweckstetter used heparin as a polyanion instead of RNA, both studies point to the importance of electrostatic interactions between tau and negatively charged molecules in promoting LLPS and aggregation. As a post-translational modification bearing a negative charge, phosphorylation may play a similar role, Zweckstetter pointed out. Zweckstetter added that phase separation of full-length, unphosphorylated tau did not occur in their hands. Mostly likely phosphorylation or interactions with RNA would be needed to facilitate that, he toldAlzforum.

The latter finding contradicts the findings of Wegmann and colleagues who did detect phase separation of full-length tau, and even of N-terminal tau completely devoid of repeat domains, she told Alzforum (May 2017 conference news). Wegmann was fascinated by this difference, adding that it underscores the complex process of phase separation, pointing to varying contributions of different regions of the tau protein in the process. For her part, Wegmann is working on pinning down the presence of liquid droplets of tau in cultured neurons, and in AD braintissue.

In a joint commentary to Alzforum, Kosik and Han agreed that understanding the physiological significance of these droplets was crucial: The in vitro studies lay the groundwork for the next big step: how might these phenomena operate in vivo where life is not only many-fold more complicated but rife with emergent properties.JessicaShugart

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More Droplets of Tau - Alzforum