How Does a Neuron Avoid Aggregation of Liquid Protein Droplets?

The liquid liquid phase transition. [Courtesy of March et al., 2016, Brain Research under a CC BY 4.0 license.]

Researchers believe that many proteins linked to neurodegenerative diseases function when condensed into liquid droplets. Unfortunately, for reasons that elude scientists, those droplets can harden into toxic protein aggregates. Two recent papers offer some insight into how a cell might regulate condensation and aggregation of proteins that undergo liquid-liquid phase transitions. Researchers led by Nicolas Fawzi at Brown University in Providence, Rhode Island, and Jeetain Mittal at Lehigh University, Bethlehem, Pennsylvania, drilled down to the atomic level to investigate. In the January 17 Molecular Cell, they reported that pathogenic mutations in the RNA-binding protein hnRNPA2 enhanced its propensity to form β-sheets, spurring fibrillization of droplets. On the other hand, methylation of hnRNPA2 disrupted protein contacts and slowed condensation.

Meanwhile, researchers led by Jared Sterneckert at Technische Universität Dresden, Germany, found that revving up autophagy, a cellular disposal system, helped shrink RNA stress granules by degrading FUS, another protein that forms liquid droplets. Enhanced autophagy improved survival in cells and flies carrying mutant FUS, they wrote in the January 17 issue of Stem Cell Reports. Both hnRNPA2 and FUS have been linked to amyotrophic lateral sclerosis (ALS).

Fawzi believes these regulatory principles may apply to numerous neurodegenerative disease proteins, as they contain motifs and mutations similar to those found in hnRNPA2 and FUS. Others agreed, and said the findings point toward potential therapies. “That liquid-liquid phase transitions are regulated by posttranslational modifications is important, and suggests strategies for finding small molecules to restore normal RNA granule dynamics,” Paul Taylor at St. Jude Children’s Research Hospital in Memphis, Tennessee, wrote to Alzforum.

Droplet Dynamics.
Mutations and methylation affect the formation of liquid droplets and fibrillized aggregates. [Courtesy of Molecular Cell, Ryan et al. 2018]

This research area exploded in 2015, when scientists discovered that RNA-binding proteins linked to neurodegenerative disease undergo liquid-liquid phase separation (Sep 2015 newsOct 2015 news). Researchers have identified 29 different RNA-binding proteins that contain the low-complexity domains (LCDs) that facilitate condensation, and many of these are involved in ALS, frontotemporal dementia, Alzheimer’s disease, and Huntington’s disease (Mar 2017 news; for review, seeKing et al., 2012; Harrison and Shorter, 2017). Most recently, the list of molecules prone to liquid-liquid phase separation grew to include tau and even expanded-repeat RNAs (Oct 2015 conference newsSep 2016 newsMay 2017 conference news). However, the precise mechanism of condensation remains hazy.

To shed some light, first author Veronica Ryan in Fawzi’s group used nuclear magnetic resonance spectroscopy to study hnRNPA2 interactions in cell-free solutions. She found that the hnRNPA2 LCD had little secondary structure and formed only fleeting connections with the low-complexity domains of other hnRNPA2 molecules. Salt forced hnRNPA2 LCDs to condense into droplets, likely held together by hydrophobic bonds. However, the LCDs remained disordered, perhaps explaining how the droplets remain fluid.

This changed when hnRNPA2 contained a pathogenic variant. Ryan tested the D290V mutation that associates with multisystem proteinopathy, an ALS-like disease. D290V replaces a charged aspartic acid in the low-complexity domain with a nonpolar valine. Liquid droplets of D290V hnRNPA2 quickly converted to fibrous aggregates. Computational analysis predicted that this mutation would cause the low-complexity domains to spoon each other, forming a β-sheet, in agreement with a previous study (Thompson et al., 2006Shorter and Taylor, 2013).

By contrast, the P298L mutation associated with Paget’s disease of bone replaces a hydrophobic proline with another hydrophobic residue. Nevertheless, droplets of this variant also fibrillized, though more slowly than the D290V variant. How might this substitution promote aggregation? Fawzi noted that proline prevents β-sheet formation because it causes a kink in the protein backbone that interferes with packing. In addition, proline cannot form a hydrogen bond along the backbone, another key ingredient for β-sheet formation. The proline residue at this location is highly conserved, suggesting it is crucial for keeping protein gels liquid, Fawzi added.

What else might prevent packing? The authors examined methylation because it has been shown to affect phase separation of other proteins (Nott et al., 2015). In solution, the enzyme protein arginine methyl transferase 1 modified four arginine residues in hnRNPA2’s low-complexity domain. Methylated hnRNPA2 remained soluble at 50 percent higher protein concentrations than did wild-type hnRNPA2, indicating it is less prone to form droplets. Computer simulations led by Mittal’s group revealed that methylation disrupted contacts between the arginines and aromatic residues in the low-complexity domain, helping keep hnRNPA2 molecules separated.

Fawzi will next ask if these same interactions occur in cells, how they affect cellular function, and whether arginine methylation controls condensation of other RNA-binding proteins such as FUS and TDP-43.

Dorothee Dormann at Ludwig-Maximilians-Universität, Munich, found this intriguing. “Arginine methylation seems to be a particularly interesting modification, since it is often mis-regulated in disease, and defects have been identified in FUS-associated neurodegeneration,” she wrote to Alzforum. Arginine methylation could play a role in preventing liquid-liquid phase separation and aggregation in general, Dormann suggested (Sep 2012 conference newsThandapani et al., 2015Suárez-Calvet et al., 2016).

Granule Cleanup.
Induced pluripotent stem cells (nuclei blue) treated with an autophagy stimulator (right) accumulate fewer and smaller stress granules (green) during oxidation than do untreated cultures (left). [Courtesy of Stem Cell Reports, Marrone et al., 2018]

Sterneckert and colleagues took a different approach to combating aggregation. They investigated the FUS P525L mutation, which disrupts FUS’ nuclear localization signal such that excess FUS accumulates in cytoplasm. When first author Lara Marrone inserted the mutation into the genomes of human induced pluripotent stem cells, it led to a fourfold increase in cytoplasmic FUS. These stem cells developed bigger and more numerous stress granules in response to oxidative or heat stress.

Then the authors screened a library of about 1,000 small molecules to find those that suppressed granule formation in the iPSCs after oxidative stress. Sixty-nine compounds shrunk the total stress granule area; 13 of these were inhibitors of the mTOR signaling pathway that suppresses autophagy. Investigating five of the compounds further, the authors found a dose-dependent protection against stress granules comparable to that of the mTOR inhibitor rapamycin (see image above). The compounds seemed to affect stress granules indirectly, because there was no sign of increased autophagy of these structures. Instead, the compounds may stimulate degradation of stray cytoplasmic FUS before it incorporates into stress granules, the authors suggested. In keeping with this, the compounds only worked when administered before granules were there.

Since mTOR inhibitors suppress immune function, Marrone looked for safer ways to turn up autophagy in the brain. She screened 1,600 approved drugs in the same assay. Of 70 that inhibited stress granule formation, several, mostly antipsychotics and antidepressants, were brain-penetrant. Six of these—paroxetine, promethazine, trimipramine, penfluridol, imipramine, and chlorpromazine—had been reported previously to boost autophagy and share a similar underlying structure (Tsvetkov et al., 2010). These drugs could be a starting point to develop more effective therapies, the authors suggested.

Sterneckert is now evaluating whether these drugs lower deposits in other ALS subtypes caused by mutations in TDP-43 and C9ORF72. Other consider this a promising direction, as many autophagy genes have been linked to ALS or other neurodegenerative diseases. “Drugs that promote the degradation of misfolded proteins, and thus prevent the conversion of stress granules into aggregates, hold great promise for the development of therapeutic approaches for several types of age-related neurodegenerative diseases,” Serena Carra at the University of Modena and Reggio Emilia, Italy, wrote to Alzforum. Sterneckert is hunting for a blood-based biomarker of autophagy.

Featured Papers

Ryan VH, Dignon GL, Zerze GH, Chabata CV, Silva R, Conicella AE, Amaya J, Burke KA, Mittal J, Fawzi NL. Mechanistic View of hnRNPA2 Low-Complexity Domain Structure, Interactions, and Phase Separation Altered by Mutation and Arginine Methylation. Mol Cell. 2018 Jan 17; PubMed.

Marrone L, Poser I, Casci I, Japtok J, Reinhardt P, Janosch A, Andree C, Lee HO, Moebius C, Koerner E, Reinhardt L, Cicardi ME, Hackmann K, Klink B, Poletti A, Alberti S, Bickle M, Hermann A, Pandey U, Hyman AA, Sterneckert JL. Isogenic FUS-eGFP iPSC Reporter Lines Enable Quantification of FUS Stress Granule Pathology that Is Rescued by Drugs Inducing Autophagy. Stem Cell Reports. 2018 Jan 17; PubMed.


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King OD, Gitler AD, Shorter J. The tip of the iceberg: RNA-binding proteins with prion-like domains in neurodegenerative disease. Brain Res. 2012 Jun 26;1462:61-80. Epub 2012 Jan 21 PubMed.

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Shorter J, Taylor JP. Disease mutations in the prion-like domains of hnRNPA1 and hnRNPA2/B1 introduce potent steric zippers that drive excess RNP granule assembly. Rare Dis. 2013;1:e25200. Epub 2013 May 29 PubMed.

Nott TJ, Petsalaki E, Farber P, Jervis D, Fussner E, Plochowietz A, Craggs TD, Bazett-Jones DP, Pawson T, Forman-Kay JD, Baldwin AJ. Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles. Mol Cell. 2015 Mar 5;57(5):936-47. PubMed.

Thandapani P, Song J, Gandin V, Cai Y, Rouleau SG, Garant JM, Boisvert FM, Yu Z, Perreault JP, Topisirovic I, Richard S. Aven recognition of RNA G-quadruplexes regulates translation of the mixed lineage leukemia protooncogenes. Elife. 2015 Aug 12;4 PubMed.

Suárez-Calvet M, Neumann M, Arzberger T, Abou-Ajram C, Funk E, Hartmann H, Edbauer D, Kremmer E, Göbl C, Resch M, Bourgeois B, Madl T, Reber S, Jutzi D, Ruepp MD, Mackenzie IR, Ansorge O, Dormann D, Haass C. Monomethylated and unmethylated FUS exhibit increased binding to Transportin and distinguish FTLD-FUS from ALS-FUS. Acta Neuropathol. 2016 Apr;131(4):587-604. Epub 2016 Feb 19  PubMed.

Tsvetkov AS, Miller J, Arrasate M, Wong JS, Pleiss MA, Finkbeiner S. A small-molecule scaffold induces autophagy in primary neurons and protects against toxicity in a Huntington disease model. Proc Natl Acad Sci U S A. 2010 Sep 28;107(39):16982-7. PubMed.

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c9orf72 disease-als fibril FUS hnRNPA2 liquid droplet liquid-liquid phase transition topic-preclinical
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