ALS Research ‘Gels’ As Studies Tie Disparate Genetic Factors Together

Run-on repeats of a hexanucleotide sequence in the C9ORF72 gene are the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia, but do they relate to other ALS/FTD mutations? According to two studies published in Cell on October 20, arginine-containing repeat dipeptides translated from the C9ORF72 expansions buddy up with proteins that have low-complexity domains and tend to form liquid organelles. These include the ALS/FTD culprits TDP-43 and FUS. The dipeptide repeats transformed these liquid droplets—including stress granules and the nucleolus—into molasses, preventing the free movement of proteins in and out of the organelles. This, in turn, disrupts fundamental cellular processes, including translation and protein transport, claim the authors.

“These papers are really exciting,” commented Gene Yeo of the University of California, San Diego. “There is now a convergence in the downstream molecular mechanisms that cause ALS/FTD from mutations in RNA-binding proteins and C9ORF72.”

Droplet Instigators. The nucleolar protein NPM1 (left) formed liquid droplets when mixed with polyGR (middle) or polyPR (right), two types of dipeptide repeat derived from C9ORF72 hexanucleotide repeat expansions. [Image courtesy of Lee et al., Cell 2016.]

Droplet Instigators. The nucleolar protein NPM1 (left) formed liquid droplets when mixed with polyGR (middle) or polyPR (right), two types of dipeptide repeat derived from C9ORF72 hexanucleotide repeat expansions. [Image courtesy of Lee et al., Cell 2016.]

One study, led by J. Paul Taylor at St. Jude Children’s Research Hospital in Memphis, Tennessee, uncovered how C9 dipeptide repeat proteins (DPRs) affected the dynamics and function of multiple membrane-less organelles. The other, led by Steven McKnight at University of Texas Southwestern Medical Center in Dallas, focused on the biophysical interactions between the dipeptides and their targets. Together, the studies raise the possibility of a broad pathological mechanism shared between carriers of the C9ORF72 expansion and other disease-causing mutations.

While a healthy person harbors between two and 23 repeats of the hexanucleotide sequence GGGGCC in their C9ORF72 gene, people with ALS or FTD can have hundreds to thousands. These sequences are then translated in both the sense and antisense directions to yield five different dipeptide repeats (DPRs): glycine-alanine (GA), glycine-arginine (GR), proline-arginine (PR), proline-alanine (PA), or glycine-proline (GP). Of these, the two arginine-containing DPRs—GR and PR—have the biggest wrap sheets. They reportedly clog the nucleolus and bungle RNA biogenesis, form toxic nuclear aggregates and stress granules in neurons, block traffic between the nucleus and cytoplasm, and kill flies, just to name a few (see Aug 2014 newsDec 2014 newsAug 2015 news).

Both Taylor and McKnight wanted to understand how these dipeptides might harm neurons, so both carried out screens to find proteins with which the peptides interacted. In Taylor’s lab, co-first authors Kyung-Ha Lee and Peipei Zhang and colleagues expressed 47-50 copies of each repeat sequence in HEK293T cells, immunoprecipitated the dipeptides, and analyzed any proteins that came along for the ride via liquid chromatography and mass spectrometry. While GA, GP, and PA repeats largely came up solo, the two arginine-containing dipeptides had 196 different partners, 81 of which associated with both dipeptides. Nearly 70 percent of these GR/PR associates—which included the ALS-linked proteins TDP-43, FUS, hnRNPA1, and hnRNPA2B1—contained low-complexity domains (LCDs).

Taylor and others had previously reported that these domains facilitated the formation of membrane-less organelles and might be hotbeds for protein aggregation (see Oct 2015 news; Oct 2015 webinar). Knocking down expression of most of these interactors in flies either rescued or worsened the toxic effects of the GR dipeptide repeats in the insects, indicating that the interactions played a meaningful role in facilitating DPR toxicity.

The researchers next focused in on how GR/PR dipeptide repeats affected different membrane-less organelles. A common theme emerged: Because the arginine-rich repeats interacted strongly with LCD-containing proteins in each organelle, they essentially “gelled” the organelles. For example, in the nucleolus—a structure within the nucleus where ribosomal RNA is generated, processed, and released—the polyGR/PR dipeptides associated with a protein called NPM1. By associating with other, physiological arginine-rich proteins, NPM1 normally orchestrates the formation of the liquid-like “granular component” of the nucleolus, where rRNA biogenesis takes place. When C9ORF72 GR/PRs were added to the mix, they outcompeted NPM1’s other partners, kicking droplet formation into overdrive and rendering the granular component more viscous. This essentially halted the movement of proteins in and out of the structure (see image above).

This, in turn, deprived the cell of rRNA, which remained trapped in the granular component of the nucleolus. The researchers reported a similar gelling phenomenon in stress granules. They also spotted the GR/PR peptides in other membrane-less nuclear organelles, including nuclear speckles, which are enriched with splicing and transcription factors, and in Cajal bodies, which host the spliceosome. Key proteins in each of these structures had popped up in the original GR/PR binding screen. The resulting loss of protein mobility profoundly affected the functions of these organelles.

“When GR/PR peptides infiltrate these organelles, the liquid goes from the viscosity of honey on the table to honey in the refrigerator,” Taylor explained. He pointed out that roughly a third of proteins contain LCDs, many of which may facilitate liquid organelle formation by associating with arginine-rich sequences. He speculated that such liquid phase separation likely underlies a vast array of dynamic biological processes, such as rapid clustering of receptors on membranes. “Nature would be hard-pressed to design a more potent cellular poison than arginine-rich polymers,” Taylor said.

For his part, McKnight in Dallas also screened for partners of dipeptide repeats, and pulled out many of the same hits as did Taylor’s group, including a preponderance of LCD-containing proteins. In addition, the Dallas group found intermediate filaments that bound polyPR. These filaments, which include neurofilaments and vimentin, help form the cellular cytoskeleton necessary for transport of proteins throughout the cell.

Rather than looking at specific membrane-less organelles that might be affected by these interactions, co-first authors Yi Lin and Eiichiro Mori investigated the fundamental mechanisms by which PR peptides changed the behavior of LCD-containing proteins. Previous studies from McKnight’s lab had indicated that under physiological conditions, the LCDs of numerous RNA-binding proteins, including FUS, polymerized into fibers of cross b-sheets, and that this facilitates the formation of liquid organelles (see Kato et al., 2012Han et al., 2012). Therefore, the researchers started by asking whether LCD-containing proteins needed to polymerize to bind PR dipeptides. The researchers treated liquid protein droplets or organelles with the aliphatic alcohol 1,6-hexanediol, which depolymerizes the proteins and dissolves these structures. They found that this depolymerization abolished the association between polyPR peptides and LCD containing proteins, including FUS.

Deadly Dew? Vimentin (left) bound droplets of polyPR peptides (middle) or droplets of FUS (right). Researchers proposed the PR peptides could usurp vimentin’s contacts with RNA granules. [Image courtesy of Lin et al., Cell 2016.]

Deadly Dew? Vimentin (left) bound droplets of polyPR peptides (middle) or droplets of FUS (right). Researchers proposed the PR peptides could usurp vimentin’s contacts with RNA granules. [Image courtesy of Lin et al., Cell 2016.]

Interestingly, among three RNA-binding proteins—hnRNPA1, hnRNPA2, and hnRNPDL—mutations that lead to ALS, FTD, and muscular dystrophy occur in the same aspartic acid within each protein’s LCD. The researchers found that these mutants formed more stable polymers than did the normal versions, and that in the case of the D290V mutant of hnRNPA2, the mutant bound to PR peptide repeats more strongly. Conversely, an hnRNPA2 mutant that could not form polymers failed to bind polyPR. Together, these findings indicated that PR peptides only associated with polymerized LCD-containing proteins, which would explain their uncanny ability to disrupt liquid organelles.

Finally, Lin and colleagues examined the association between polyPR peptides and intermediate filaments. These proteins contain LCDs in the amino terminal (“head”) domain, as well as their C-terminal (“tail”) domain, which flank a central α-helical portion. These LCDs polymerized with each other to form filaments, and, much like liquid organelles, were disrupted by aliphatic alcohols. The researchers reported that PR peptide repeats bound to these LCDs along polymerized filaments, forming a series of regularly spaced knobs (see image above). They speculated that these PR peptide globs could perturb the critical functions of these filaments. For example, neurofilaments near the synapse associate with RNA granules. If PR aggregates outcompeted RNA granules for binding to neurofilament, it would prevent localized protein translation near the synapse, they proposed.

Yeo believes the findings support the idea that stress granule dynamics are disrupted in these diseases. The studies also expand the list of potentially affected structures, such as the nucleolus and Cajal bodies, he said. Yeo recently reported that the D290V mutant of hnRNPA2B1 affected stress granules, but that it also triggered cell death under low stress conditions, in which those granules did not form. The mutation also fouled up alternative splicing, he reported (see Martinez et al., 2016). The new findings introduce the possibility that disruption of other membrane-less organelles, such as the spliceosome, could explain D290V toxicity, Yeo said.

“These papers raise critical questions regarding the mechanism of DPR toxicity in ALS,” commented Nicolas Fawzi of Brown University in Providence, Rhode Island. “For example, the structural details of how DPRs interact with LCDs of other disease-related RNA-binding proteins are now of prime importance,” he wrote. “It will also be interesting to examine the interaction of DPRs with TDP-43, which can self-assemble via α-helical structure present with its LCD.” Fawzi’s previous work described the formation of liquid droplets by both FUS and TDP-43 proteins (see Oct 2015 news on Burke et al., 2015; Sep 2016 news on Conicella et al., 2016). Fawzi also wondered whether disruption of LCDs could underlie cases of sporadic ALS as well.

Do these findings move researchers closer to developing treatments for ALS or FTD? Taylor thinks so. “Liquid phase separations that form membrane-less organelles are highly regulated, tunable processes,” he told Alzforum. “For every factor that promotes assembly, another promotes disassembly, so there may be antagonistic relationships we can exploit.”

Primary Reference:

Lee KH, Zhang P, Kim HJ, Mitrea DM, Sarkar M, Freibaum BD, Cika J, Coughlin M, Messing J, Molliex A, Maxwell BA, Kim NC, Temirov J, Moore J, Kolaitis RM, Shaw TI, Bai B, Peng J, Kriwacki RW, Taylor JP. C9orf72 Dipeptide Repeats Impair the Assembly, Dynamics, and Function of Membrane-Less Organelles. Cell. 2016 Oct 20;167(3):774-788.e17. [PubMed].

Lin Y, Mori E, Kato M, Xiang S, Wu L, Kwon I, McKnight SL. Toxic PR Poly-Dipeptides Encoded by the C9orf72 Repeat Expansion Target LC Domain Polymers. Cell. 2016 Oct 20;167(3):789-802.e12. [PubMed].


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disease-als disease-ftd topic-preclinical
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