Toxic TDP-43 Truncates Point to Gain-of-Function Role in Disease

TAR DNA-binding protein-43 (TDP-43) is clearly a player in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS), glomming into insoluble inclusions along with ubiquitin and other proteins. But scientists still wonder whether it is the lack of functional TDP-43, or the presence of TDP-43 with a new, toxic ability, that causes pathology. A paper published online in PNAS this week scores points for the gain-of-function theory. But the final tallies are not yet in, and senior author Leonard Petrucelli of the Mayo Clinic in Jacksonville, Florida, notes that the loss of functional TDP-43 from its normal home in the nucleus could have pathogenic consequences as well.

The report is the first demonstration of TDP-43 aggregation and toxicity in a mammalian model, said Aaron Gitler of the University of Pennsylvania, who was not involved in the work. Gitler has made a similar demonstration in yeast (ARF related news story and Johnson et al., 2008). “A big focus in the field now is just to try to figure out how TDP-43 contributes to disease,” he said, which would guide therapeutic approaches.

Petrucelli, first author Yong-Jie Zhang and colleagues previously found that TDP-43 is chopped by caspase-3 into carboxyl-terminal fragments of 25 and 35 kD (ARF related news story and Zhang et al., 2007). In the current work, they sought the downstream effects of that truncated TDP-43, expressing the same carboxyl-terminal fragments, tagged with GFP, in cell culture. In human embryonic kidney cells, full-length TDP-43 localized to the nucleus, but the fragments moved into the cytoplasm and formed aggregates including ubiquitin, as the protein does in people with TDP-43 proteinopathies. The scientists focused further experiments on the shorter, 25-kDa piece, since it has been linked to ALS pathology (ARF related news story and Neumann et al., 2006).

The fragment was a killer. Expressed in differentiated neuroblastoma cells, the shortened TDP-43 led to fragmented nuclei and activation of caspase-3, both signs of apoptosis. Zhang and colleagues then asked whether the fragment required phosphorylation to exert its toxic effects. TDP-43 fragments from the brains of people who had ALS or FTLD with ubiquitin inclusions (the form that includes TDP-43 proteinopathy) are phosphorylated (Hasegawa et al., 2008). Using a phospho-TDP-43 antibody, the researchers discovered that in cells transfected with the truncate, the fragment was phosphorylated, but they found that a mutant 25 kD fragment lacking the serines necessary for the modification aggregated as well, showing that phosphorylation is not required for inclusion formation.

Zhang and colleagues reasoned that the fragment could cause cell death by either exerting its own toxic influence, or by binding and sequestering full-length TDP-43, dragging it away from the nucleus where it regulates transcription and RNA splicing. To distinguish between these two possibilities, they tested endogenous TDP-43 function. The scientists transfected HeLa cells with the cystic fibrosis transmembrane conductance regulator (CFTR) as a marker for normal TDP-43 activity. Wild-type TDP-43 prevents exon 9 expression in this gene. Exon 9 transcripts were not increased in cells co-transfected with the TDP-43 fragment, suggesting endogenous TDP-43 nuclear localization and activity were unaffected by the truncate. The fragment, then, must have its own toxic mechanism.

The paper provides evidence for a TDP-43 toxic gain of function, but Petrucelli noted that they only focused on a single piece of the protein. “We are not arguing that the caspase fragment is the only fragment that is found in ALS and FTLD,” he said. “Clearly other fragments are possible,” and those could have other detrimental effects.

In the course of their research, Zhang and colleagues engineered an antibody specific for the 25 kDa TDP-43 fragment. “I think that was the most exciting part of the paper, in some respects,” Petrucelli said. Like the phospho-TDP-43 antibody, this one could be useful in distinguishing FTLD with ubiquitin and TDP-43 inclusions from the tau-based form of the disease, Petrucelli suggested. That might help doctors match treatments to different kinds of FTLD, he said.

The current study seems to solidify the toxic gain of function for TDP-43. “This shows that these carboxyl-terminal fragments might be playing a direct role in the disease pathogenesis,” Gitler said, although he noted the fragment’s toxicity should be confirmed in animals. But another study, published online April 17 in FEBS Letters, found that fruit flies lacking TDP-43 had locomotion problems, abnormal neuromuscular junctions, and reduced lifespan (Feiguin et al., 2009). That work adds points to the loss of normal function column. Ultimately, there may not be a simple distinction between acquired toxicity and lost utility. “I think it’s kind of both,” Gitler said.

To further elucidate the role of TDP-43 in disease, scientists are racing to discover or create models of TDP-43 pathology in animals (ARF related news story and Tatom et al., 2009). In an April 17 paper in Neuroscience Letters, scientists report that TDP-43 aggregates with ubiquitin in a common ALS model system—mice overexpressing mutant human superoxide dismutase (see Shan et al., 2009). However, these researchers did not report carboxyl-terminal fragments or hyperphosphorylated TDP-43, even in end-stage animals, adding a new layer of complexity to the TDP-43 puzzle.—Amber Dance

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References

News Citations

  1. Heady Times for Researchers Studying TDP-43
  2. Progranulin Controls Cutting of Inclusion Protein
  3. New Ubiquitinated Inclusion Body Protein Identified
  4. TDP-43 Roundup: New Models, New Genes

Paper Citations

  1. Johnson BS, McCaffery JM, Lindquist S, Gitler AD. A yeast TDP-43 proteinopathy model: Exploring the molecular determinants of TDP-43 aggregation and cellular toxicity. Proc Natl Acad Sci U S A. 2008 Apr 29;105(17):6439-44. PubMed.
  2. Zhang YJ, Xu YF, Dickey CA, Buratti E, Baralle F, Bailey R, Pickering-Brown S, Dickson D, Petrucelli L. Progranulin mediates caspase-dependent cleavage of TAR DNA binding protein-43. J Neurosci. 2007 Sep 26;27(39):10530-4. PubMed.
  3. Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT, Bruce J, Schuck T, Grossman M, Clark CM, McCluskey LF, Miller BL, Masliah E, Mackenzie IR, Feldman H, Feiden W, Kretzschmar HA, Trojanowski JQ, Lee VM. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science. 2006 Oct 6;314(5796):130-3. PubMed.
  4. Hasegawa M, Arai T, Nonaka T, Kametani F, Yoshida M, Hashizume Y, Beach TG, Buratti E, Baralle F, Morita M, Nakano I, Oda T, Tsuchiya K, Akiyama H. Phosphorylated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Ann Neurol. 2008 Jul;64(1):60-70. PubMed.
  5. Feiguin F, Godena VK, Romano G, D'Ambrogio A, Klima R, Baralle FE. Depletion of TDP-43 affects Drosophila motoneurons terminal synapsis and locomotive behavior. FEBS Lett. 2009 May 19;583(10):1586-92. PubMed.
  6. Tatom JB, Wang DB, Dayton RD, Skalli O, Hutton ML, Dickson DW, Klein RL. Mimicking aspects of frontotemporal lobar degeneration and Lou Gehrig's disease in rats via TDP-43 overexpression. Mol Ther. 2009 Apr;17(4):607-13. PubMed.
  7. Shan X, Vocadlo D, Krieger C. Mislocalization of TDP-43 in the G93A mutant SOD1 transgenic mouse model of ALS. Neurosci Lett. 2009 Jul 17;458(2):70-4. PubMed.

Further Reading

Papers

  1. Del Bo R, Ghezzi S, Corti S, Pandolfo M, Ranieri M, Santoro D, Ghione I, Prelle A, Orsetti V, Mancuso M, Sorarù G, Briani C, Angelini C, Siciliano G, Bresolin N, Comi GP. TARDBP (TDP-43) sequence analysis in patients with familial and sporadic ALS: identification of two novel mutations. Eur J Neurol. 2009 Jun;16(6):727-32. Epub 2009 Feb 19 PubMed.
  2. Tatom JB, Wang DB, Dayton RD, Skalli O, Hutton ML, Dickson DW, Klein RL. Mimicking aspects of frontotemporal lobar degeneration and Lou Gehrig's disease in rats via TDP-43 overexpression. Mol Ther. 2009 Apr;17(4):607-13. PubMed.
  3. Igaz LM, Kwong LK, Chen-Plotkin A, Winton MJ, Unger TL, Xu Y, Neumann M, Trojanowski JQ, Lee VM. Expression of TDP-43 C-terminal Fragments in Vitro Recapitulates Pathological Features of TDP-43 Proteinopathies. J Biol Chem. 2009 Mar 27;284(13):8516-24. Epub 2009 Jan 21 PubMed.
  4. Corrado L, Ratti A, Gellera C, Buratti E, Castellotti B, Carlomagno Y, Ticozzi N, Mazzini L, Testa L, Taroni F, Baralle FE, Silani V, D'Alfonso S. High frequency of TARDBP gene mutations in Italian patients with amyotrophic lateral sclerosis. Hum Mutat. 2009 Apr;30(4):688-94. PubMed.
  5. Geser F, Martinez-Lage M, Robinson J, Uryu K, Neumann M, Brandmeir NJ, Xie SX, Kwong LK, Elman L, McCluskey L, Clark CM, Malunda J, Miller BL, Zimmerman EA, Qian J, Van Deerlin V, Grossman M, Lee VM, Trojanowski JQ. Clinical and pathological continuum of multisystem TDP-43 proteinopathies. Arch Neurol. 2009 Feb;66(2):180-9. PubMed.
  6. Mackenzie IR, Rademakers R. The role of transactive response DNA-binding protein-43 in amyotrophic lateral sclerosis and frontotemporal dementia. Curr Opin Neurol. 2008 Dec;21(6):693-700. PubMed.

Primary Papers

  1. Zhang YJ, Xu YF, Cook C, Gendron TF, Roettges P, Link CD, Lin WL, Tong J, Castanedes-Casey M, Ash P, Gass J, Rangachari V, Buratti E, Baralle F, Golde TE, Dickson DW, Petrucelli L. Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity. Proc Natl Acad Sci U S A. 2009 May 5;106(18):7607-12. PubMed.

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