ALS Dipeptides Drive Liquid-Liquid Phase Separation and Stress Granule Formation

March 18, 2017 - als

New commentary could assistance scientists explain because hexanucleotide expansions in a C9ORF72 gene are a vital means of patrimonial amyotrophic parallel sclerosis (ALS) and frontotemporal insanity (FTD). In a Mar 16 Molecular Cell, researchers led by Ludo Van Den Bosch, University of Leuven, and Peter Tompa, Vrije Universiteit Brussel, both in Belgium, news that poisonous dipeptide repeats (DPRs) coded by a enlargement curt to form glass droplets. The formula supplement DPRs to a flourishing list of ALS/FTD-related proteins that arrangement a knack for proviso separation, offer ancillary a thought that a highway to neurodegeneration runs by liquid-phase organelles such as stress granules.

In a final few years, scientists have rescued that proteins temperament disordered, low-complexity, or prion-like domains fuse into glass droplets. Called liquid-liquid proviso subdivision (LLPS), this materialisation plays a executive purpose in a pathogenesis of ALS, FTD, and other neurodegenerative diseases. In ALS/FTD, RNA contracting proteins such as hnRNP, FUS, and TDP-43 straightforwardly proviso separate, combining glass intermediates that emanate a tact belligerent for protein aggregation.

LLPS serves an vicious non-pathological duty as well. Phase subdivision underlies a arrangement of supposed membrane-less organelles, including highlight granules, a nucleolus, and Cajal bodies. These glass assemblies of RNA and RNA-binding proteins offer to boldly umpire RNA metabolism. Recent reports implicate membrane-less organelles as special targets for C9ORF72derived DPRs. Last year, dual papers from a labs of Paul Taylor of St. Jude Children’s Research Hospital in Memphis, Tennessee, and Steven McKnight, University of Texas Southwestern Medical Center in Dallas, described how dipeptides associate with highlight granule proteins, causing a routinely glass organelles to seize adult and emanate massacre for neurons (see Oct 2016 news). 

In Leuven, initial author Steven Boeynaems became meddlesome in a biophysical duty of arginine-containing C9ORF72 peptide repeats after he was means to satisfy LLPS in vitro with arginine-rich domains from several transcription factors. Encouraged, he tested 20 dipeptide glycine-arginine (GR20) and proline-arginine (PR20) repeats, and found both casually proviso distant in vitro to form round glass droplets. Given a rarely charged inlet of a peptides, Boeynaems found that a bit startling until he satisfied a phosphate in a aegis acted as a opposite ion to vacate a peptides’ charge. Subsequently, he transposed a phosphate with RNA, that is also negatively charged, and that offer extended a phase transition.

Given that glass proviso transitions set a theatre for assembly of other ALS/FTD proteins, (see Oct 2015 webinar; Sep 2015 news; Oct 2015 news), Boeynaems asked if a same was loyal for PR20. He let a droplets lay for a day, and afterwards looked for signs of fibril arrangement or aggregation. The PR20-RNA droplets didn’t change—their essence remained in a loosely disordered, energetic state, suggesting that PR20 in a glass state is not fibrillogenic. PR20 did assistance another protein aggregate, however: It extended FUS proviso subdivision and assembly in vitro.

To inspect polyPR interactions with other mobile proteins, Boeynaems churned PR30 with HeLa dungeon lysate. The PR30 proviso separated, afterwards shaped a rarely insoluble precipitate. Mass spec research of a proteins trapped with PR30 identified many that correlate with highlight granules and other glass compartments in cells. This corroborates final year’s work from a Taylor and McKnight labs, that reported a identical set of GR- and PR-interacting proteins.

What happens to PR in vital cells? When a researchers over-expressed PR100 in HeLa cells, they rescued an boost in a series of cells with PR-laden highlight granules. The granules were not wholly normal, however. Compared to granules prompted by chemical diagnosis with arsenite, a common stressor researchers use to satisfy RNA highlight granules, a PR100-induced highlight granules were some-more viscous. This jibes with prior indications that DPRs sack highlight granules and other organelles of their fluidity and deteriorate their function. Interestingly, a PR100 granules contained some-more of dual ALS-related proteins, ataxin-1 and TDP-43, than a arsenite-induced granules did.

Next, a researchers asked either a resource of DPR-induced granule arrangement concerned a physiological highlight response. Normally, highlight granules form when protein interpretation stalls. The routine requires a phosphorylation of eukaryotic arising cause 2α (eIF2α), and a participation of a granule core protein G3BP. In cells lacking phosphorylatable eIF2a or G3BP protein, Boeynaems found that PR100 countenance no longer prompted highlight granules. “This shows a outcome is not only a pristine proviso separation, though that we need a operative highlight response to get a granules to form,” pronounced Boeynaems. The formula also support targeting eIF2α kinases as a probable healing proceed in ALS, he pronounced (see Dec 2013 news). 

According to Simon Alberti, Max Planck Institute, Dresden, Germany, a paper easily shows how dipeptide repeats worry what he calls a “molecular grammar” of proviso separation, that involves formidable interactions between jumbled arginine-rich repeats, tyrosine-rich prion-like domains, and RNA. “These components like to connect dipeptide repeats, though afterwards a dipeptides change a element properties of a assemblies,” he said.

Just how that change harms cells stays to be seen, Alberti said. “This paper focuses on highlight granules, though a dipeptide targets all a glass compartments in a cell—nucleoli, Cajal bodies, and other organelles. These bodies enclose many of a same proteins. It’s not transparent that is a vicious cell that kills cells when perturbed.”

Taylor agreed. “Stress granules might be a canary in a spark mine. They are good famous and easy to study, though there are many other probable targets in neurons. Granules that ride RNAs from a iota to synapses are unequivocally vicious for neurons to function. They arrange by a really same beliefs and they are going to be equally poisoned,” he told Alzforum. In gripping with this, a new paper from Erika Holzbaur’s lab during a University of Pennsylvania, Philadelphia, reported that mutations in TDP-43 boost a flexibility of ribonucleoprotein granules and disquiet their ride down axons (see Gopal et al., 2017).—Pat McCaffrey

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News Citations

  1. ALS Research ‘Gels’ as Studies Tie Disparate Genetic Factors Together 27 Oct 2016
  2. ALS Protein Said to Liquefy, Then Freeze en Route to Disease 4 Sep 2015
  3. Do Membraneless Organelles Host Fibril Nucleation? 8 Oct 2015
  4. Stress Relief: Anti-Stress Granule Therapy Saves ALS Models 21 Dec 2013

Webinar Citations

  1. Fluid Business: Could “Liquid” Protein Herald Neurodegeneration? 8 Oct 2015

Paper Citations

  1. .
    Amyotrophic parallel sclerosis-linked mutations boost a flexibility of liquid-like TDP-43 RNP granules in neurons.
    Proc Natl Acad Sci U S A. 2017 Mar 6;

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