San Diego: TDP-43 Targets Loom Large—But Where’s the Bull’s Eye?

Series - Society for Neuroscience Annual Meeting 2010:

TDP-43, a protein implicated in both frontotemporal lobar degeneration and amyotrophic lateral sclerosis, as well as Alzheimer’s disease (Brouwers et al., 2010), is an RNA-binding protein with an undefined role. If posters at the Society for Neuroscience (SfN) meeting and a recent publication are any indication, the field is poised to discover a wealth of clues to TDP-43 function (see also ARF related news story). Scientists are scanning the transcriptome for TDP-43 target RNAs, and early reports suggest there are quite a lot of them. A paper in the November 4 Journal of Biological Chemistry online describes more than 4,000 RNAs that interact with TDP-43 in cortical neurons, and at the SfN meeting, held November 13-17 in San Diego, California, several poster presentations described TDP-43 targets in cell lines. Scientists hope that they might link a small subset of TDP-43-binding RNAs to pathology. More specifically, altered gene expression might someday provide biomarkers for early diagnosis of ALS, suggested Shangxi Xiao of the University of Toronto, Canada, an author on one of the posters.

What RNAs does TDP-43 modify, how does it change them, and what proteins does it work with? “These are questions everybody is asking,” said Chantelle Sephton, first author on the JBC paper with senior author Gang Yu and colleagues at the University of Texas Southwestern Medical Center in Dallas. All lead up to the ultimate questions, she said: How do these processes cause pathology, and how might doctors target TDP-43 to treat disease? Therein lies the rub, since TDP-43 has so many binding partners.

“I do not believe every target plays a role in the disease process,” said Fen-Biao Gao of the University of Massachusetts Medical School in Worcester, who was not involved in the JBC paper or SfN posters. Indeed, transcriptome-scanning studies are merely hypothesis-generating tools, said Joachim Herz, another professor at Southwestern Medical Center and a coauthor on Sephton’s paper.

Foundational Studies
The JBC paper is the first published study to seek out TDP-43 targets without overexpressing the protein, the authors said. The authors isolated RNA from rat primary neuron cultures, then used TDP-43 antibodies to immunoprecipitate RNAs that interact with the protein. Collaborating with researchers in the group of Melissa Moore at the University of Massachusetts, the scientists sequenced the TDP-43 targets. “I was floored by the number of targets,” Sephton said. More than 4,000 genes appear to rely on TDP-43 to modulate their expression. “It’s really a foundation-type study,” Sephton said, noting that researchers can now pick and choose which targets they think are involved in disease for follow-up studies.

TDP-43 is bound to sequences of UG repeats in target RNAs, often with a UA in the middle, but Sephton said there may be other TDP-43 binding motifs, too. Using a standard gene ontology database, the researchers identified genetic categories that were enriched in their RNA sample. Genes involved in RNA processing, synapse function, and development were among the top hits.

Among the gene set was TDP-43 itself, suggesting it regulates its own expression via some sort of feedback loop. The authors suggest TDP-43 binds its own 3’-UTR, and alters either the stability or translational efficiency of the RNA. The exact mechanism for this feedback is unclear, Sephton said, but it dovetails nicely with animal studies. Animals heterozygous for TDP-43 often produce more than half the normal amount of the protein, suggesting the remaining allele compensates for the missing one (Sephton et al., 2010). Other TDP-43 targets readers may recognize include FUS, progranulin, tau, amyloid precursor protein, and α-synuclein, which are all related to neurodegeneration.

The researchers also identified proteins that interact with TDP-43. In collaboration with the laboratory of Junmin Peng at Emory University in Atlanta, Georgia, they used mass spectrometry to analyze proteins that co-immunoprecipitated with TDP-43 from rat brain nuclear extracts. They discovered 25 proteins that seem to buddy up with TDP-43 in the nucleus. Most were RNA-binding proteins such as splicing or translation factors, confirming previous results (Freibaum et al., 2010).

Cell Line Studies
Sephton and collaborators, as well as Clotilde Lagier-Tourenne and colleagues at the University of California in San Diego (see ARF related news story), are using brain tissue to investigate TDP-43 targets, but other scientists have started similar studies with cell lines. At the SfN meeting, Shangxi Xiao and Janice Robertson of the University of Toronto, Canada, presented work with TDP-43/RNA co-immunoprecipitation from the SH-SY5Y human neuroblastoma line. The scientists identified 102 TDP-43 targets, with UG repeat and pyrimidine-rich TDP-43 binding sequences. The majority of TDP-43 binding sites were within introns; 23 percent were outside introns. Using RT-PCR, Xiao showed that five out of 50 target genes were downregulated, or had altered splicing patterns, in the spinal cords of people who died of ALS compared to healthy control tissue.

Atsushi Shiga from Niigata University in Japan presented a poster on the gene expression and splicing changes in TDP-43-depleted cells. The researchers used short interfering RNA to dampen TDP-43 expression in HeLa human cervical cancer cells. They found that 123 genes, including several involved in inflammation, were up- or downregulated. Inflammation is a known factor in ALS as well as other neurodegenerative diseases (see ARF related news story). In addition, 892 genes were abnormally spliced in the absence of TDP-43. Many of the latter are involved in the Golgi and other endomembrane systems.

TDP-43, of course, is not the only RNA-binding protein implicated in ALS and FTLD. In a poster, Shinsuke Ishigaki and Gen Sobue reported on early studies knocking down FUS in the spinal cord neuroblastoma hybridoma NSC34. They have identified several FUS targets, Ishigaki told ARF, and hope to perform further studies with primary motor neuron cultures and knockout mice.

The multitude of TDP-43 targets indicates the complexity researchers face in untangling disease mechanisms Gao said. “All these toxic proteins affect so many pathways,” he told ARF. “It is a challenge for the field…to identify the key targets.”—Amber Dance

Comments

  1. This paper by Gang Yu and colleagues describes a very interesting study to identify the set of RNAs that bind to TDP-43, a protein that has been implicated in a wide range of neurodegenerative diseases. The authors performed an unbiased screen of TDP-43 RNA targets and found over 4,000 RNAs that were bound to TDP-43. The TDP-43-binding RNAs had diverse physiological roles, primarily involving synaptic function, RNA metabolism, and neuronal development. Importantly, several RNAs involved in neurodegeneration were identified, including those for TDP-43 itself, FUS/TLS, progranulin, tau, APP, synuclein, Cdk5, huntingtin, presenilin, PrP, sirtuin, SOD, TAR DNA binding protein, and ataxin1 and 2.

    This study is an important advance in the field and represents a first step toward a comprehensive understanding of the biological functions of TDP-43 and how this RNA binding protein is involved in neurodegenerative disease. It appears from the present study that the physiological functions of TDP-43 are quite wide and involve numerous diverse RNAs in neurons. Additional experiments will be required to fully validate the current results, but the present findings are fascinating and encouraging. Although it is not yet clear as to the level that the authors' results may inform about novel therapeutic approaches for neurodegenerative diseases, the hope is that the information provided in this study will help move toward the identification of novel targets of future drugs for the treatment or prevention of neurodegeneration.

    View all comments by Robert Vassar

  2. Two prominent messages emerge from the Sephton paper and the burgeoning literature on TDP-43: 1) TDP-43 is important for the normal functioning of cells; and 2) its actions are many and complex. This being so, is it realistic to assume that targeting one or a few of its ~4,000 interacting partners could effectively modify the course of TDP-43 proteinopathies such as ALS and FTLD?

    Maybe, but perhaps the most parsimonious target—the bull’s eye, to keep with the theme—is the same as in other neurodegenerative diseases: the seeded accumulation of the protein in the wrong place. If a normally folded TDP-43 molecule encounters a corrupted conformational variant of the protein (or possibly some other seed) outside the nucleus, the normal protein could itself become corrupted, aggregation-prone, and unable to re-enter the nucleus in its biologically active form. In addition to impairing the function of multiple RNA pathways, TDP-43 aggregates might also be transferred to other cells via normal cellular transport, release, and uptake mechanisms, thereby disseminating the disease from one region of the nervous system to another.

    If this scenario is correct, then the most direct therapeutic objective for the TDP-43 proteinopathies would be to hinder the formation of proteinaceous seeds, promote their disposal, or block their interactions with normally folded TDP-43 molecules. In short, RNA mismanagement appears to be the proximal cause of cellular dysfunction in these disorders, but the problem originates with the seeded sequestration of TDP-43 in a form that renders it unable to perform its normal duties.

    View all comments by Lary Walker

  3. Have you heard of Significance Analysis of Interactome (SAINT) (Choi et al., 2010)? It will allow researchers globally to quickly assess the reliability and accuracy of protein binding data helping to further their studies of cancer and other illnesses.

    References:

    Choi H, Larsen B, Lin ZY, Breitkreutz A, Mellacheruvu D, Fermin D, Qin ZS, Tyers M, Gingras AC, Nesvizhskii AI. SAINT: probabilistic scoring of affinity purification-mass spectrometry data. Nat Methods. 2011 Jan;8(1):70-3. PubMed.

    View all comments by Roxanne Ol

  4. This is interesting, especially the viral genome theories.

    References:
    Martin.

    View all comments by Ian Roberts

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References

News Citations

  1. ALS: T Cells Step Up

Paper Citations

  1. Brouwers N, Bettens K, Gijselinck I, Engelborghs S, Pickut BA, van Miegroet H, Montoya AG, Mattheijssens M, Peeters K, De Deyn PP, Cruts M, Sleegers K, Van Broeckhoven C. Contribution of TARDBP to Alzheimer's disease genetic etiology. J Alzheimers Dis. 2010;21(2):423-30. PubMed.
  2. Sephton CF, Good SK, Atkin S, Dewey CM, Mayer P, Herz J, Yu G. TDP-43 is a developmentally regulated protein essential for early embryonic development. J Biol Chem. 2010 Feb 26;285(9):6826-34. PubMed.
  3. Freibaum BD, Chitta RK, High AA, Taylor JP. Global analysis of TDP-43 interacting proteins reveals strong association with RNA splicing and translation machinery. J Proteome Res. 2010 Feb 5;9(2):1104-20. PubMed.

Other Citations

  1. ARF related news story

Further Reading

Papers

  1. Buratti E, De Conti L, Stuani C, Romano M, Baralle M, Baralle F. Nuclear factor TDP-43 can affect selected microRNA levels. FEBS J. 2010 May;277(10):2268-81. PubMed.
  2. Bose JK, Wang IF, Hung L, Tarn WY, Shen CK. TDP-43 overexpression enhances exon 7 inclusion during the survival of motor neuron pre-mRNA splicing. J Biol Chem. 2008 Oct 24;283(43):28852-9. PubMed.
  3. Kim SH, Shanware NP, Bowler MJ, Tibbetts RS. Amyotrophic lateral sclerosis-associated proteins TDP-43 and FUS/TLS function in a common biochemical complex to co-regulate HDAC6 mRNA. J Biol Chem. 2010 Oct 29;285(44):34097-105. PubMed.
  4. Strong MJ, Volkening K, Hammond R, Yang W, Strong W, Leystra-Lantz C, Shoesmith C. TDP43 is a human low molecular weight neurofilament (hNFL) mRNA-binding protein. Mol Cell Neurosci. 2007 Jun;35(2):320-7. PubMed.
  5. Mercado PA, Ayala YM, Romano M, Buratti E, Baralle FE. Depletion of TDP 43 overrides the need for exonic and intronic splicing enhancers in the human apoA-II gene. Nucleic Acids Res. 2005;33(18):6000-10. PubMed. Expression of concern. Editor's note.
  6. Buratti E, Dörk T, Zuccato E, Pagani F, Romano M, Baralle FE. Nuclear factor TDP-43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping. EMBO J. 2001 Apr 2;20(7):1774-84. PubMed.

Primary Papers

  1. Sephton CF, Cenik C, Kucukural A, Dammer EB, Cenik B, Han Y, Dewey CM, Roth FP, Herz J, Peng J, Moore MJ, Yu G. Identification of neuronal RNA targets of TDP-43-containing ribonucleoprotein complexes. J Biol Chem. 2011 Jan 14;286(2):1204-15. PubMed.

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