20 Nov 2015

Neurodegeneration is usually pretty permanent—but TDP-43 mice are making a comeback. One line of mice deteriorated during six weeks when a  TDP-43 transgene was active, but improved rapidly once it was shut off, regaining the strength to grip a wire and balance on a rotating rod. Virginia Lee of the University of Pennsylvania Perelman School of Medicine in Philadelphia presented these data at RNA Metabolism in Neurological Disease, a satellite meeting held October 15-16 of the Society for Neuroscience annual meeting in Chicago. Lee published some of the findings in the July 22 Acta Neuropathologica online. Also at the meeting, Krista Spiller from Lee’s lab reported that the first motor neurons to die off in these mice match those most vulnerable in humans—the fast-fatigable motor neurons—and that more-resistant neurons can sprout additional axons and take their place once the toxic TDP-43 is gone. In the October 5 issue of the same journal, Lars Ittner and colleagues at the University of New South Wales in Sydney report on an inducible TDP-43 mouse which develops movement and memory problems, but also recovers once the transgene is silenced.

TDP-43 forms aggregates in most cases of amyotrophic lateral sclerosis, as well as some cases of frontotemporal dementia and Alzheimer’s disease. Lee’s results suggest that treatments aimed at TDP-43 proteinopathy could provide people with some level of recovery, even after neurodegeneration has commenced, said Wilfried Rossoll of Emory University in Atlanta. He did not participate in the study.

TDP-43 proteinopathy has proven difficult to model in mice (see Sep 2012 news). Part of the problem is that the protein, which shuttles between the nucleus and cytosol to manage RNAs, is required for cell viability. Lee and others suspect that in disease states it fails to perform its normal function, but that has been hard to test because knockouts do not survive (see Mar 2010 news). Since pathological TDP-43 aggregates in the cytoplasm, Lee decided to focus on that, deleting the protein’s nuclear localization sequence from the human gene before putting it into a mouse. First author Adam Walker, now at Macquarie University in Sydney, used a neurofilament heavy chain promoter, which is active in all neurons, to drive expression of the transgene. A tetracycline response element ensured the gene would remain inactive and the mice developed normally so long as their chow contained the inhibitor doxycycline.

The researchers dubbed the mice “regulatable NLS” or rNLS. When the mice reached about five weeks old, the researchers switched their diet to dox-free food and watched the disease unfold. The mice developed TDP-43 inclusions in the spinal cord and brain. In addition, the animals’ brains shrank and their motor neurons retracted from muscles and died. Researchers at the meeting found the model intriguing. Compared to other TDP-43 models, the pathology in the rNLS mice appeared more akin to that in people, commented Dieter Edbauer of Ludwig-Maximilians University of Munich, who did not participate in the work.

The animals also experienced a progressive motor neuron disease. They developed tremors in their paws and lost the ability to grip a wire or balance on a rotating rod. They lost weight, and died about 10 weeks after they stopped eating dox-laced chow. Lee thinks the disease resulted from the diminished expression of endogenous, full-length TDP-43, which she said disappeared within a week of the transgene’s activation. Because TDP-43 suppresses its own transcription, the transgene presumably turned off the regular mouse gene, suggested Lee (see Jan 2011 news).This would compromise management of RNA trafficking and translation.

The real excitement happened when Walker and colleagues returned the dox chow to some of the mice that had been dox-free for six weeks. A week later, the animals started to grip a wire more tightly, and they wobbled less on the rotating rod. They gained weight again, and lived out a normal lifespan. TDP-43 aggregates started to disappear within two weeks on dox, and were completely gone after three months. Cortical and motor neurons stopped degenerating. More than that, neurons that remained seemed to take over for those that had died. Six weeks back on dox doubled the percentage of neuromuscular junctions that were innervated by motor neurons (see image above). The researchers surmised that the motor neurons still present were able to take over for the ones that withered while the TDP-43 transgene was overexpressed.

Spiller analyzed this re-innervation in more depth. First, she figured out which motor neurons were most vulnerable to the TDP-43 toxicity, and which persisted. Previously, scientists had reported that fast-fatigable motor neurons, which innervate fast-twitch muscles and mediate quick movements such as jumping and running, were the first to degenerate in people and in ALS mice overexpressing mutant SOD1, another cause of the disease. In contrast, fast-fatigue-resistant motor neurons, which activate quick movements but are slower to tire, and slow motor neurons, which manage activity like standing or strolling, take longer to degenerate (Pun et al., 2006; Kanning et al., 2010). Though there are few markers for these motor-neuron populations, they usually can be identified by the muscles they connect to and their size. Fast-fatigable neurons link up with fast-twitch muscles and are the largest, while the slow motor neurons attach to slow-twitch muscles and are smaller. In the rNLS mice, Spiller observed that the fast-fatigable motor neurons were the ones that disappeared.

With those fast-fatigable neurons gone, how did the mice recover once their TDP-43 expression re-normalized? To label which motor neurons connected to the re-innervated muscles, Spiller injected the muscles with a fluorescent tracer. From this, she confirmed that disease-resistant neurons, the smaller fast-fatigue-resistant and slow ones, had sprouted new connections. These same fatigue-resistant or slow motor neurons also started to express MMP9, a metalloproteinase typically found only in fast-fatigable neurons. Spiller said she has not yet worked out the significance of the new MMP9 production.

“The recovery was really remarkable to see,” Spiller said. “Even after a mouse has lost a third of its motor neurons, it could still recover … and walk around the cage.” That makes targeting TDP-43 seem like a viable therapeutic strategy, she suggested, even for people who have already lost motor neurons. This kind of recovery is encouraging, agreed Paul Taylor of St. Jude Children’s Research Hospital in Memphis, Tennessee, who co-organized the symposium but did not participate in Lee’s research.

Notably, researchers working with two other lines of TDP-43 mice have observed recovery as well. In another line of repressible TDP-43 mice, this time with the CaMKIIa promoter driving expression of a cytosolic construct in the forebrain, have also observed improvements in behavior when they turn off TDP-43. With TDP-43 on, these mice have learning and memory defects and motor problems. Turning it off reversed most of the symptoms within just two weeks, so long as neurodegeneration had not progressed too far (Alfieri et al., 2014). “Both animal models represent complementary tools to address questions regarding the role of altered TDP-43 in ALS-like or FTLD-like phenotypes,” commented that study’s author, Lionel Igaz of the University of Buenos Aires in Argentina (see full comment, below). And Ittner and colleagues generated mice with an inducible TDP-43 transgene including a disease-linked mutation. Overexpression of the transgene in neurons induced neurodegeneration, leading to deficits in movement, spatial memory and disinhibition. Turning off the transgene led to significant improvements in those symptoms in just one week (Ke et al., 2015).—Amber Dance

Comments

1. • Lionel Muller Igaz
     University of Buenos Aires
   • Posted: 20 Nov 2015

   The paper by Walker and colleagues on the development and characterization of a novel TDP-43 mouse model is interesting and thorough. This research adds nicely to the increasing literature on inducible TDP-43 animal models, most notably in mice and rats, and highlights the importance of studying the consequences of post-developmental expression of TDP-43 forms. In this case, they demonstrate that widespread neuronal expression of a cytoplasmic form of TDP-43 (ΔNLS, for short) leads to several features of ALS/FTLD, including accumulation of insoluble cytoplasmic TDP-43, loss of endogenous nuclear TDP-43, motor neuron loss, muscle denervation, and progressive motor impairments. Remarkably, they show that transgene suppression decreases pathology, stops neuronal loss, improves motor deficits, and reduces mortality.

   As noted by Walker et al., we recently demonstrated the effects of short-term suppression of TDP-43- ΔNLS, but using a different promoter (CamKII) that drives expression predominantly in forebrain neurons, thus sparing lower motor neurons from transgene expression and subsequent degeneration (Alfieri et al., 2014). In these CamKII-ΔNLS inducible mice (Igaz et al., 2011), short-term transgene suppression at a young age led to recovery of behavioral deficits, including different domains affected in TDP-43 proteinopathies. Interestingly, the motor and cognitive phenotypes were recovered upon suppression, but not the social deficits, implicating differential vulnerability of underlying neuronal networks. In the NEFH-ΔNLS mice described here, non-motor phenotypes might be challenging to assess due to lower motor neuron degeneration and confounds emerging from profound motor deficits. In this light, both animal models represent complementary tools to address questions regarding the role of altered TDP-43 in ALS-like or FTLD-like phenotypes.

   It is interesting to note the convergence of some behavioral results from animal models with different initial mechanistic hypothesis. A recent mouse model, based on viral expression of C9ORF72 repeat expansions (Chew et al., 2015), develops TDP-43 cytoplasmic pathology and cortical neuron loss, and displays almost identical behavioral phenotypes to our CamKII-ΔNLS animals. In particular, decreased motor performance in the rotarod, hyperlocomotion, and increased anxiety-like behavior in the open-field test and impaired social interaction scores indicating social deficits. That these are all shared features of animals developed through quite different approaches reinforces the need for complementary animal models that will inform us, as a community, of the commonalities and specifics of etiological mechanisms. These novel NEFH-ΔNLS mice presented here also contribute to this idea.

   The results from this study from Virginia Lee’s lab also emphasize the complexity of an unresolved question in the field, i.e. the role of pathology (in the form of aggregates or inclusions) in cell function and survival.

   References:

   Alfieri JA, Pino NS, Igaz LM. Reversible behavioral phenotypes in a conditional mouse model of TDP-43 proteinopathies. J Neurosci. 2014 Nov 12;34(46):15244-59. PubMed.

   Chew J, Gendron TF, Prudencio M, Sasaguri H, Zhang YJ, Castanedes-Casey M, Lee CW, Jansen-West K, Kurti A, Murray ME, Bieniek KF, Bauer PO, Whitelaw EC, Rousseau L, Stankowski JN, Stetler C, Daughrity LM, Perkerson EA, Desaro P, Johnston A, Overstreet K, Edbauer D, Rademakers R, Boylan KB, Dickson DW, Fryer JD, Petrucelli L. Neurodegeneration. C9ORF72 repeat expansions in mice cause TDP-43 pathology, neuronal loss, and behavioral deficits. Science. 2015 Jun 5;348(6239):1151-4. Epub 2015 May 14 PubMed.

   Igaz LM, Kwong LK, Lee EB, Chen-Plotkin A, Swanson E, Unger T, Malunda J, Xu Y, Winton MJ, Trojanowski JQ, Lee VM. Dysregulation of the ALS-associated gene TDP-43 leads to neuronal death and degeneration in mice. J Clin Invest. 2011 Feb;121(2):726-38. Epub 2011 Jan 4 PubMed.

   View all comments by Lionel Muller Igaz

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References

News Citations

1. Are TDP-43 Mice Living Up to Expectations? 20 Sep 2012
2. Research Brief: TDP-43 Knockout Lethal, Hets Have Motor Symptoms 19 Mar 2010
3. TDP-43 Turns Itself Off, Inclusions a False Lead 20 Jan 2011

Paper Citations

1. Pun S, Santos AF, Saxena S, Xu L, Caroni P. Selective vulnerability and pruning of phasic motoneuron axons in motoneuron disease alleviated by CNTF. Nat Neurosci. 2006 Mar;9(3):408-19. PubMed.
2. Kanning KC, Kaplan A, Henderson CE. Motor neuron diversity in development and disease. Annu Rev Neurosci. 2010;33:409-40. PubMed.
3. Alfieri JA, Pino NS, Igaz LM. Reversible behavioral phenotypes in a conditional mouse model of TDP-43 proteinopathies. J Neurosci. 2014 Nov 12;34(46):15244-59. PubMed.
4. Ke YD, van Hummel A, Stevens CH, Gladbach A, Ippati S, Bi M, Lee WS, Krüger S, van der Hoven J, Volkerling A, Bongers A, Halliday G, Haass NK, Kiernan M, Delerue F, Ittner LM. Short-term suppression of A315T mutant human TDP-43 expression improves functional deficits in a novel inducible transgenic mouse model of FTLD-TDP and ALS. Acta Neuropathol. 2015 Nov;130(5):661-78. Epub 2015 Oct 5 PubMed.

Further Reading

Papers

1. Stribl C, Samara A, Trümbach D, Peis R, Neumann M, Fuchs H, Gailus-Durner V, Hrabě de Angelis M, Rathkolb B, Wolf E, Beckers J, Horsch M, Neff F, Kremmer E, Koob S, Reichert AS, Hans W, Rozman J, Klingenspor M, Aichler M, Walch AK, Becker L, Klopstock T, Glasl L, Hölter SM, Wurst W, Floss T. Mitochondrial dysfunction and decrease in body weight of a transgenic knock-in mouse model for TDP-43. J Biol Chem. 2014 Apr 11;289(15):10769-84. Epub 2014 Feb 10 PubMed.
2. Yang C, Wang H, Qiao T, Yang B, Aliaga L, Qiu L, Tan W, Salameh J, McKenna-Yasek DM, Smith T, Peng L, Moore MJ, Brown RH Jr, Cai H, Xu Z. Partial loss of TDP-43 function causes phenotypes of amyotrophic lateral sclerosis. Proc Natl Acad Sci U S A. 2014 Mar 25;111(12):E1121-9. Epub 2014 Mar 10 PubMed.
3. Ricketts T, McGoldrick P, Fratta P, de Oliveira HM, Kent R, Phatak V, Brandner S, Blanco G, Greensmith L, Acevedo-Arozena A, Fisher EM. A nonsense mutation in mouse Tardbp affects TDP43 alternative splicing activity and causes limb-clasping and body tone defects. PLoS One. 2014;9(1):e85962. Epub 2014 Jan 21 PubMed.
4. D'Alton S, Altshuler M, Cannon A, Dickson DW, Petrucelli L, Lewis J. Divergent phenotypes in mutant TDP-43 transgenic mice highlight potential confounds in TDP-43 transgenic modeling. PLoS One. 2014;9(1):e86513. Epub 2014 Jan 22 PubMed.
5. Zheng M, Shi Y, Fan D. Nuclear TAR DNA-binding protein 43: A new target for amyotrophic lateral sclerosis treatment. Neural Regen Res. 2013 Dec 15;8(35):3284-95. PubMed.

News

1. TDP-43 Turns Itself Off, Inclusions a False Lead 20 Jan 2011
2. Transcription Factor Nourishes Neuron-Muscle Pair in ALS Model 17 Jan 2014
3. Muscle MicroRNA Repairs Nerve-Muscle Connection in ALS Model 11 Dec 2009
4. ER Struggles in Motor Neurons That Fall to ALS 31 Mar 2009
5. Surprise Save: Excitability Protects Neurons from Lou Gehrig’s 11 Oct 2013
6. C9ORF72 Mice Point to Gain of Toxic Function in ALS, FTD 29 Oct 2015
7. Does C9ORF72 Repeat RNA Promote Protein Phase Transitions? 30 Oct 2015
8. RNA Regulator Locked Out of Nucleus by C9ORF72 Repeats 5 Nov 2015