Researchers Lasso TDP-43 With RNA Lariats

Out of the Wild West—well, two Northern California labs—comes a novel approach to corral the toxic RNA-binding protein that causes amyotrophic lateral sclerosis and some forms of frontotemporal dementia. Research posses led by Aaron Gitler of Stanford University in Palo Alto and Robert Farese of the Gladstone Institutes in San Francisco found that RNA lassos protect cells from overexpression of the potentially toxic protein, TDP-43. The lariats act as “decoys” that attract TDP-43, keeping other essential molecules out of harm’s way, the authors propose in an October 28 Nature Genetics paper.

TDP-43 mutations cause some forms of hereditary amyotrophic lateral sclerosis (ALS). The protein also forms neuronal inclusions in the majority of ALS cases and in many instances of frontotemporal dementia (FTD). Exactly how TDP-43, mutant or wild type, contributes to disease is still unclear, but there are two main potential mechanisms. In diseased cells, TDP-43 accumulates in the cytoplasm, limiting its natural nuclear role and precipitating pathology. Secondly, the cytoplasmic aggregates it forms are toxic. It is this second disease mechanism that the lariats might block.

Joint first authors Maria Armakola at Stanford and Matthew Higgins at the Gladstone Institutes began their projects independently, screening for mutations that suppress TDP-43 toxicity in yeast. The teams only discovered each other’s efforts later, when they sought to confirm their results in mammalian cells by enlisting the help of Steven Finkbeiner, also at the Gladstone Institutes. They then decided to pool resources and data, working with Sami Barmada at Finkbeiner’s lab.

Before then, each team found that a deletion in the RNA debranching enzyme Dbr1 was one of the most potent hits in their screens. Dbr1 acts on introns left over from pre-mRNA splicing, which are shaped like a Q. In order for exonucleases to degrade these strands, Dbr1 must first linearize the branched structure. Higgins and Farese also focused on Dbr1 because of links between RNA metabolism and TDP-43 in disease, Farese said (e.g., see ARF related news story on Kwiatkowski et al., 2009, and Vance et al., 2009; ARF related news story on Kawahara and Mieda-Sato, 2012).

Finkbeiner's group had developed an automated microscopy approach to follow hundreds of individual cells over time and calculate their risk of death (Arrasate and Finkbeiner, 2005). The researchers trained this microscope on rat primary cortical neurons that overexpressed TDP-43 along with mApple, a fluorescent marker for cell survival. Barmada treated the cultures with a small interfering RNA for Dbr1. The robotic microscope tracked the neurons individually, imaging them every 24 hours for eight days. Excess TDP-43 doubled the risk of a neuron’s demise, but knockdown of Dbr1 cut that risk by 20 percent.

Why would an RNA debranching enzyme protect from TDP-43? Gitler’s group hypothesized that the intron lariats might act as decoys, sopping up the RNA-binding protein and preventing its toxicity. To test their idea, they examined where TDP-43 and the lariats localized in yeast. They labeled the lariats by adding binding sites for the viral MS2 coat protein (MS2-CP) to introns, and expressing a gene for green fluorescent protein (GFP)-tagged MS2-CP, in the yeast cells (Haim et al., 2007). In otherwise wild-type yeast, where lariats are quickly processed, the GFP signal was faint and diffuse. In Dbr1-deficient yeast, GFP-tagged MS2 showed up as one or two bright spots in the cytoplasm. Overexpressed TDP-43 exited the nucleus and formed inclusions in the cytoplasm, as is typical for cells with too much of the RNA-binding protein. These inclusions always overlapped with the lariat-labeled spots.

Since cells with functional Dbr1 do not accumulate lariats, those quickly degraded lassos are not likely to be typical TDP-43 targets in sickness or health, Gitler said. But adding them to a cell suffering from TDP-43 toxicity might corral the protein, preventing it from running roughshod throughout the cytoplasm. Ben Wolozin of Boston University in Massachusetts, who was not involved in the work, noted that using decoys as therapeutics would be a rather contrarian approach, because it would seek to build, not dissolve, TDP-43 inclusions. It is hard to predict whether decoy lariats would help people with TDP-43 proteinopathies, since the precise pathological mechanisms remain unclear, he said. The decoys would not address any problems due to loss of TDP-43 function in the nucleus, but might complement other methods to restore TDP-43’s proper nuclear role, Gitler suggested.

What about side effects? “Yeasts seem to do just fine without Dbr1, but they do not have a lot of introns,” Farese noted. Mammal cells have plenty more introns, and thus more lariats to clear away. Plus, Farese added, not all introns are garbage—some give rise to noncoding RNAs (Mattick and Makunin, 2005), which might be lost if lariats go unprocessed. In the rat neuron experiments, high concentrations of Dbr1 antisense RNA poisoned the cells. To sidestep these concerns, Gitler is looking into building a treatment out of synthetic, Dbr1-resistant decoy lariats that would attract TDP-43 without interfering with normal lariat processing. He is also pursuing whole-animal experiments.—Amber Dance

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References

News Citations

  1. New Gene for ALS: RNA Regulation May Be Common Culprit
  2. Slicing and Dicing: TDP-43 Teams Up With Nucleases to Make MicroRNAs

Paper Citations

  1. Kwiatkowski TJ Jr, Bosco DA, Leclerc AL, Tamrazian E, Vanderburg CR, Russ C, Davis A, Gilchrist J, Kasarskis EJ, Munsat T, Valdmanis P, Rouleau GA, Hosler BA, Cortelli P, de Jong PJ, Yoshinaga Y, Haines JL, Pericak-Vance MA, Yan J, Ticozzi N, Siddique T, McKenna-Yasek D, Sapp PC, Horvitz HR, Landers JE, Brown RH Jr. Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science. 2009 Feb 27;323(5918):1205-8. PubMed.
  2. Vance C, Rogelj B, Hortobágyi T, De Vos KJ, Nishimura AL, Sreedharan J, Hu X, Smith B, Ruddy D, Wright P, Ganesalingam J, Williams KL, Tripathi V, Al-Saraj S, Al-Chalabi A, Leigh PN, Blair IP, Nicholson G, de Belleroche J, Gallo JM, Miller CC, Shaw CE. Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science. 2009 Feb 27;323(5918):1208-11. PubMed.
  3. Kawahara Y, Mieda-Sato A. TDP-43 promotes microRNA biogenesis as a component of the Drosha and Dicer complexes. Proc Natl Acad Sci U S A. 2012 Feb 28;109(9):3347-52. PubMed.
  4. Arrasate M, Finkbeiner S. Automated microscope system for determining factors that predict neuronal fate. Proc Natl Acad Sci U S A. 2005 Mar 8;102(10):3840-5. PubMed.
  5. Haim L, Zipor G, Aronov S, Gerst JE. A genomic integration method to visualize localization of endogenous mRNAs in living yeast. Nat Methods. 2007 May;4(5):409-12. PubMed.
  6. Mattick JS, Makunin IV. Small regulatory RNAs in mammals. Hum Mol Genet. 2005 Apr 15;14 Spec No 1:R121-32. PubMed.

Further Reading

Papers

  1. Jinwal UK, Abisambra JF, Zhang J, Dharia S, O'Leary JC, Patel T, Braswell K, Jani T, Gestwicki JE, Dickey CA. Cdc37/Hsp90 Protein Complex Disruption Triggers an Autophagic Clearance Cascade for TDP-43 Protein. J Biol Chem. 2012 Jul 13;287(29):24814-20. PubMed.
  2. Colombrita C, Onesto E, Megiorni F, Pizzuti A, Baralle FE, Buratti E, Silani V, Ratti A. TDP-43 and FUS RNA-binding proteins bind distinct sets of cytoplasmic messenger RNAs and differently regulate their post-transcriptional fate in motoneuron-like cells. J Biol Chem. 2012 May 4;287(19):15635-47. PubMed.
  3. Wang XN, Cui LY. TAR DNA binding protein-43 and fused in sarcoma/translocated in liposarcoma protein in two neurodegenerative diseases. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2012 Jun;34(3):286-92. PubMed.
  4. Vaccaro A, Patten SA, Ciura S, Maios C, Therrien M, Drapeau P, Kabashi E, Parker JA. Methylene blue protects against TDP-43 and FUS neuronal toxicity in C. elegans and D. rerio. PLoS One. 2012;7(7):e42117. PubMed.
  5. Da Cruz S, Cleveland DW. Understanding the role of TDP-43 and FUS/TLS in ALS and beyond. Curr Opin Neurobiol. 2011 Dec;21(6):904-19. PubMed.

Primary Papers

  1. Armakola M, Higgins MJ, Figley MD, Barmada SJ, Scarborough EA, Diaz Z, Fang X, Shorter J, Krogan NJ, Finkbeiner S, Farese RV, Gitler AD. Inhibition of RNA lariat debranching enzyme suppresses TDP-43 toxicity in ALS disease models. Nat Genet. 2012 Oct 28; PubMed.

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