There’s a new mouse in the lab for the study of amyotrophic lateral sclerosis (ALS). And there will soon be more: mice carrying a mutant version of human TAR DNA binding protein 43 (TDP-43), described in a paper posted online this week in PNAS, will likely herald a veritable herd of mice with various TDP-43 mutations. Robert Baloh and colleagues at Washington University in St. Louis, Missouri, reported on the first published transgenic model of TDP-43 proteinopathy, describing a mouse that shows features of both ALS and frontotemporal lobar degeneration with ubiquitin aggregates (FTLD-U), another TDP-43 proteinopathy that sometimes co-presents with ALS. Surprisingly, the mouse lacks one common feature of human disease: although there are ubiquitinated protein inclusions in the animal’s neurons, TDP-43 is not a component of the aggregates. This could indicate that TDP-43 aggregates are not central to disease—or simply suggest that the model is a poor facsimile of human disease.

John Trojanowski of the University of Pennsylvania in Philadelphia, who was not involved in the current research, applauded the publication. “This will energize the field,” he said. Several laboratories are working on their own TDP-43 mouse models; Trojanowski joked good-naturedly that he was “crushed” to see Baloh’s group make it into print publication first.

However, researchers warned against overinterpreting early results, particularly since the current publication lacked control mice overexpressing wild-type human TDP-43. “We have to be incredibly cautious as far as what these results mean,” said Leonard Petrucelli of the Mayo Clinic in Jacksonville, Florida, who also was not a participant in the PNAS paper. “I think it is a bit of a stretch to say that it is an ALS model per se…. It begs additional work.” Researchers should be particularly wary, he noted, given the ALS field’s history with animal models. The mouse overexpressing human superoxide dismutase 1, long a standard in ALS labs, has frequently responded to drugs that later failed in people (see ARF Live Discussion).

Baloh, first author Iga Wegorzewska, and colleagues constructed mice expressing human TDP-43-A315T under control of the mouse prion protein (PrP) promoter. This mutation was found in a St. Louis family with familial ALS. The animals made human protein in amounts approximately three times that of endogenous mouse TDP-43, and expressed the transgene most highly in the brain and spinal cord. The mice first showed motor symptoms at three to four months of age, and after four and a half months they were unable to support their own body weight, using their limbs to scoot around on their stomachs. Survival averaged 154 days, although it varied between approximately 100 days and up to 240. Necropsies of end-stage animals confirmed degeneration of motor axons, and mutant animals at end-stage possessed only 80 percent as many motor neurons as wild-type control mice. The TDP-43 mouse has more upper motor neuron involvement than the SOD1 mouse, where symptoms are primarily caused by degeneration of lower motor neurons, Baloh said.

TDP-43 is normally a nuclear protein. Although its exact function is poorly understood, it appears to be involved in several aspects of RNA regulation (for a recent review, see Geser et al., 2009). In both ALS and FTLD-U, TDP-43 exits the nucleus. Cleaved, hyperphosphorylated, and ubiquitinated, it forms inclusions in the cytoplasm (see ARF related news story on Neumann et al., 2006). Researchers have discovered several different TDP-43 mutations in people with ALS (see ARF related news story on Gitcho et al., 2008 and Sreedharan et al., 2008; and ARF related news story on Van Deerlin et al., 2008 and Kabashi et al., 2008).

In the current study, histopathology of end-stage mice showed ubiquitinated aggregates in both spinal motor neurons as well as neurons in the frontal cortex, making the pathology reminiscent of both ALS, where motor neurons are afflicted, and FTLD-U, which affects the frontal temporal lobe. As in humans, the mutant TDP-43 cleared the nucleus of brain cells. However, unlike in human disease, it did not show up in cytoplasmic aggregates. The ubiquitinated inclusions also did not stain positive for α-synuclein or tau protein, other common aggregate components. Ronald Klein of the Louisiana State University Health Sciences Center in Shreveport speculated that further examination might still yield a TDP-43 presence: “Perhaps an unknown and so far undetectable TDP-43 fragment conformation was present in the ubiquitin aggregates,” he wrote in an e-mail to ARF.

The lack of TDP-43 inclusions could be a sign that the mouse is an inadequate model for human TDP-43 proteinopathy. Alternatively, it could be a clue that nuclear clearing of TDP-43 is the key problem. “Maybe that is where all the business is really at, and these other pathologies are muddying the waters,” said Brian Kraemer of the University of Washington in Seattle, who was not involved in the current study. A crucial next step, Baloh said, will be to take a second look at human TDP-43 proteinopathy tissues to see if ubiquitinated inclusions always have TDP-43, or whether some aggregates are TDP-43-negative.

Previous work has shown that caspase-3 slices TDP-43 into 25- and 35-kilodalton carboxyl-terminal fragments (see ARF related news story on Zhang et al., 2007), and that those fragments are toxic in cell culture (see ARF related news story on Zhang et al., 2009). In the mouse model, Baloh and colleagues found that carboxyl-terminal fragments 25 and 35 kilodaltons in size appeared in brain and spinal cord lysates between one and two months of age, preceding noticeable motor symptoms, loss of nuclear TDP-43, and the majority of the ubiquitinated aggregations. The fragments could play a direct role in causing neurodegeneration, the authors suggested.

The TDP-43-A315T mouse is the latest in a diverse line of TDP-43 models. Researchers have studied TDP-43 in systems ranging from yeast (Johnson et al., 2008) and mammalian cell culture (see ARF related news story on Winton et al., 2008) to nematodes, fruit flies, and zebrafish (see ARF related news story). In mammals, researchers have used viral vectors to deliver the human TDP-43 gene into the substantia nigra of rats; the animals recapitulated some features of human disease (see ARF related news story on Tatom et al., 2009).

Adding a transgenic mouse to that panel has been difficult. “For those in the field, it is clear that generating these mouse models is a mammoth task on its own,” wrote Samir Kumar-Singh of VIB, University of Antwerp, Belgium, who also was not part of the PNAS publication, in an e-mail to ARF (see full comment below). TDP-43 is essential for development, and knockouts are not viable. Many founder mice fail to reproduce, or the phenotype weakens in successive generations. “I think TDP-43 is just very toxic,” Kraemer said. “It is always a tradeoff between getting a mouse that will live and getting a mouse that will express your toxic protein.”

Because of these factors, Baloh and colleagues were unsuccessful in engineering a wild-type counterpart to their mutant transgenic line. That leaves open questions of whether its phenotype comes from the specific mutation or simply from too much TDP-43. “It does not negate the findings, but it definitely clouds the interpretation,” Baloh said. In the rat model, overexpression of wild-type TDP-43 did cause neurodegeneration (Tatom et al., 2009).

Having a TDP-43 mutant to go along with the SOD1 mouse will strengthen translational research, Baloh suggested: “Testing in two different mouse models might give us a better chance of predicting positive correlations of drugs that work in humans.” Medicines that work in both kinds of mice, he said, should be first in line for clinical trials.—Amber Dance

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  1. This study elegantly gives a first insight on a transgenic mouse model of mutant TDP-43 (A315T) identified in familial ALS patients. For those in the field, it is clear that generating these mouse models is a mammoth task on its own. Among the many interesting findings in this paper, the first to catch my attention was that the 25-kDa TDP-43 C-terminal fragments (CTFs) were recovered from detergent-soluble fractions but not from urea fractions as observed in sporadic and familial ALS/FTLD patients. If the TDP-43 25-kDa CTFs would indeed be confirmed as the real culprit, this would yet again emphasize the importance of soluble but not aggregated protein/peptide in cellular toxicity, as has been shown for a number of other proteinopathies including Aβ, α-synuclein, polyglutamine expansion in Huntingtin, and mutant SOD1.

    Another important observation made in this paper was that ubiquitin-immunoreactive (ir) inclusions observed in select neurons including motor neurons were not TDP-43-ir. Thus, the mutant TDP-43 (A315T) mice do not completely model ALS, where ubiquitin-ir inclusions are also TDP-43-ir; nevertheless, this work does lead to a very interesting question: what are these inclusions composed of?

    Knowing earlier studies (see Tatom et al., 2009 and ARF related news story), I am also not surprised at the glaring omission of wild-type TDP-43 mice as a better control than the non-transgenic mice utilized in this study. So although clearly not all is answered yet, let's see how these and other TDP-43 mouse models currently being developed will unfold the mysteries of TDP-43-led neurodegeneration.

    References:

    . 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.

References

Webinar Citations

  1. Mice on Trial? Issues in the Design of Drug Studies

News Citations

  1. New Ubiquitinated Inclusion Body Protein Identified
  2. Gene Mutations Place TDP-43 on Front Burner of ALS Research
  3. Heady Times for Researchers Studying TDP-43
  4. Progranulin Controls Cutting of Inclusion Protein
  5. Toxic TDP-43 Truncates Point to Gain-of-Function Role in Disease
  6. ALS Research: More TDP-43, and Peripherin No Longer in Periphery?
  7. London, Ontario: TDP-43 Across the Animal Kingdom at ALS Meeting
  8. TDP-43 Roundup: New Models, New Genes

Paper Citations

  1. . Amyotrophic lateral sclerosis, frontotemporal dementia and beyond: the TDP-43 diseases. J Neurol. 2009 Aug;256(8):1205-14. PubMed.
  2. . Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science. 2006 Oct 6;314(5796):130-3. PubMed.
  3. . TDP-43 A315T mutation in familial motor neuron disease. Ann Neurol. 2008 Apr;63(4):535-8. PubMed.
  4. . TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science. 2008 Mar 21;319(5870):1668-72. Epub 2008 Feb 28 PubMed.
  5. . TARDBP mutations in amyotrophic lateral sclerosis with TDP-43 neuropathology: a genetic and histopathological analysis. Lancet Neurol. 2008 May;7(5):409-16. Epub 2008 Apr 7 PubMed.
  6. . TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nat Genet. 2008 May;40(5):572-4. Epub 2008 Mar 30 PubMed.
  7. . Progranulin mediates caspase-dependent cleavage of TAR DNA binding protein-43. J Neurosci. 2007 Sep 26;27(39):10530-4. PubMed.
  8. . 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.
  9. . 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.
  10. . Disturbance of nuclear and cytoplasmic TAR DNA-binding protein (TDP-43) induces disease-like redistribution, sequestration, and aggregate formation. J Biol Chem. 2008 May 9;283(19):13302-9. PubMed.
  11. . 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.

Further Reading

Papers

  1. . Mutations in TDP-43 link glycine-rich domain functions to amyotrophic lateral sclerosis. Hum Mol Genet. 2009 Oct 15;18(R2):R156-62. PubMed.
  2. . Frontotemporal dementia and amyotrophic lateral sclerosis-associated disease protein TDP-43 promotes dendritic branching. Mol Brain. 2009;2:30. PubMed.
  3. . TDP-43 and frontotemporal dementia. Curr Neurol Neurosci Rep. 2009 Sep;9(5):353-8. PubMed.
  4. . Cytosolic TDP-43 expression following axotomy is associated with caspase 3 activation in NFL-/- mice: support for a role for TDP-43 in the physiological response to neuronal injury. Brain Res. 2009 Nov 3;1296:176-86. PubMed.
  5. . Truncation and pathogenic mutations facilitate the formation of intracellular aggregates of TDP-43. Hum Mol Genet. 2009 Sep 15;18(18):3353-64. PubMed.
  6. . TDP-43 is intrinsically aggregation-prone and ALS-linked mutations accelerate aggregation and increase toxicity. J Biol Chem. 2009 May 22; PubMed.

Primary Papers

  1. . TDP-43 mutant transgenic mice develop features of ALS and frontotemporal lobar degeneration. Proc Natl Acad Sci U S A. 2009 Nov 3;106(44):18809-14. Epub 2009 Oct 15 PubMed.