TDP-43 was an unknown in the neurodegeneration field until four years ago, but since then has received plenty of attention. Linked to both amyotrophic lateral sclerosis and frontotemporal dementia, the misbehaving protein took its share of the limelight with more than a dozen talks and posters at the International Conference on Alzheimer’s Disease, held 10-15 July 2010 in Honolulu, Hawaii.
Perhaps the biggest take-home message from the meeting: The TDP-43 field is slowly accumulating a handy collection of animal models (see ARF related news story). But those models vary widely in their design—some overexpression, some knockouts, some knockdowns—and yield varying results. Meanwhile, stress—or rather, an affected cell’s poor ability to handle it—was another theme that bubbled up amid the wave of disparate TDP-43 data shown at ICAD.
Jada Lewis and colleagues from the Mayo Clinic College of Medicine in Jacksonville, Florida, surfed in with multiple presentations on new mice expressing human TDP-43. Bettina Schmid of the Ludwig Maximilians University in Munich, Germany, made a splash with her zebrafish loss-of-function model. Worms, too, swam in with TDP-43 data from Brian Kraemer of the University of Washington in Seattle and Peter Ash from the Mayo Clinic in Jacksonville, Florida. Blair Leavitt, from the University of British Columbia in Vancouver, Canada, sailed through the TDP-43 marina with a talk about progranulin knockout mice. These may provide a useful model for TDP-43 study since progranulin mutations are linked with frontotemporal dementia (FTD). And Ben Wolozin of Boston University visited the TDP-43 beach with a discussion of an upcoming publication on that protein’s association with stress granules. Kraemer and others cited Wolozin’s presentation as one of the most interesting of the meeting.
Scientists first identified a connection between TDP-43 and neurodegeneration in 2006 (see ARF related news story on Neumann et al., 2006) with news of its presence in inclusion bodies. The link received another boost two years later with the discovery of TDP-43 mutations in some people with amyotrophic lateral sclerosis (ALS; see ARF related news story on Sreedharan et al., 2008 and Gitcho et al., 2008). In disease, the normally nuclear protein, which is involved in RNA processing, moves into the cytoplasm and forms inclusions. Some evidence indicates that cleavage by caspases produces a toxic carboxyl-terminal fragment (see ARF related news story on Zhang et al., 2009). However, the mechanism for TDP-43 pathology remains uncertain. The question is still open if TDP-43 inclusions and mutations are loss or gain of function, observed Christian Czech of F. Hoffman-La Roche in Basel, Switzerland, in an e-mail to ARF after taking in TDP-43 presentations in Honolulu. Here, ARF rounds up TDP-43 news from ICAD.
TDP-43 Proteinopathy Spreads
TDP-43 inclusions are common in ALS and FTD, but researchers are finding that the protein’s reach extends even further than that. Frederic Calon of Universit Laval in Qubec City, Canada, examined insoluble TDP-43 in brain tissue from people who had mild cognitive impairment or Alzheimer disease. A few of the MCI samples, and the majority of AD tissues, evinced increased insoluble TDP-43 in the parietal cortex. Accumulated amyloid-β and phosphorylated tau tended to accompany the increase in insoluble TDP-43. Calon found similar pathology in triple-transgenic AD model mice, though only in aged ones (18 months old). It’s possible that TDP-43 is also a neuropathological marker of AD, Calon told ARF.
Lisa Taylor-Reinwald, working with Nigel Cairns at Washington University in St. Louis, Missouri, took that thought further with a study of brain tissue from people who had been cognitively normal at their time of death. In seven out of 50 samples, she observed abnormal TDP-43 staining coincident with amyloid-β and tau pathology. TDP-43 pathology might precede symptoms but comes after amyloid-β accumulation, Taylor-Reinwald suggested to ARF. However, she cautioned that a cross-sectional pathology study such as hers cannot, by itself, clearly delineate what happens first.
TDP-43’s Next Top Model: Candidates Strut Their Stuff
To understand TDP-43’s function from the start of disease to its end, many researchers are turning to animal models. Everybody is trying to get that [TDP-43] mouse, Taylor-Reinwald noted, and Honolulu featured several presentations related to such mouse models—not to mention worms, fish, and even monkey. Mayo’s Lewis, along with Ashley Cannon and Yafei Xu, presented soon-to-be-published mouse models, made in collaboration with Leonard Petrucelli, also at the Jacksonville Mayo Clinic. The mice express wild-type human TDP-43. The extra protein, they found, caused TDP-43 truncation, a cellular increase in ubiquitin, abnormal mitochondrial aggregates surrounding the nucleus, and reactive gliosis. These mice suffered neural degeneration and had trouble walking. They died young, their survival rate being proportional to the amount of excess TDP-43 present. However, they did not exhibit TDP-43 inclusions.
Lewis’s mice look interesting, Kraemer said. At ICAD, Kraemer discussed his own TDP-43 knockout mice (see ARF related news story on Kraemer et al., 2010), as well as new data from the study of nematodes. His C. elegans expressing wild-type TDP-43 suffer mild neural dysfunction, but no neurodegeneration. With the mutant gene, Kraemer observed neurodegeneration and toxicity. However, he found no strong evidence that this toxicity arose from TDP-43 aggregation or truncation by caspases. Either the process in worms is different from that in mammals, which is possible, or caspases are not a player in it, Kraemer told ARF. Ash presented recently published data from C. elegans, suggesting that TDP-43 causes neurotoxicity via two mechanisms: action of the full-length, nuclear protein and aggregation of cytoplasmic fragments (Ash et al., 2010).
Takanori Yokota, of the Tokyo Medical and Dental Universal University in Japan, brought news of a potential non-human primate model. He used a viral vector to express wild-type human TDP-43 gene in the spinal cord of macaques, and found that this diverted TDP-43 to the cytoplasm, as it does in human disease.
However, most of those models have in common a flaw, Schmid told ARF in an e-mail. They rely on TDP-43 overexpression. Too much TDP-43 is toxic in every species analyzed thus far, and might not have anything to do with the disease, she wrote. However, researchers have struggled to develop models that have reduced TDP-43, in part because the protein is essential for embryonic development. Other models, such as animals heterozygous for a TDP-43 knockout, showed no phenotype (Sephton et al., 2010; Wu et al., 2010).
At the meeting, Schmid presented the first vertebrate loss-of-function model to show a neuronal phenotype. Zebrafish carry two TDP-43 orthologs; knockdown of both—via a new technique based on zinc finger nucleases—caused motor neuron dysfunction in embryos that survived for only six days. The fish embryos also had circulatory problems—the heart was beating, but no blood was moving through it. Schmid hopes to rescue this phenotype by introducing human TDP-43 mRNA and then test whether various TDP-43 mutants can do the same. In addition, this model provides the opportunity to search, in vivo, for RNAs that are TDP-43 targets and relevant to disease, Schmid told ARF. The knockout approach was clever, and the analysis careful, Czech commented.
Other researchers are working with animals that have normal TDP-43 genes, but might still offer information on how this protein might go rogue. One such example is Leavitt’s mice. They are conditional progranulin knockouts that suffer subtle abnormalities in social behavior, long-term potentiation, and synaptic spine density, Leavitt told ARF. TDP-43 fans were interested because progranulin mutations can give rise to FTD with TDP-43 pathology. My guess is these mice will be very important for studying progranulin-TDP-43 interaction, said Salvatore Oddo of the University of Texas Health Science Center in San Antonio. However, the mice have not yet shown any TDP-43 pathology up until nine months of age, Leavitt said. Leavitt also provided a technical tip for researchers interested in progranulin: The Santa Cruz N-19 antibody is not specific for progranulin immunocytochemistry in mouse brain, he wrote in an e-mail to ARF.
Another such example of a TDP-43-related model is the flies by J. Paul Taylor and colleagues at St. Jude Children’s Research Hospital in Memphis, Tennessee (Ritson et al., 2010). In this study, the researchers generated Drosophila carrying mutations in valosin-containing protein (VCP). VCP mutations are associated, variously, with FTD, as well as Paget’s disease of bone or inclusion body myopathy. In a genetic screen, the Memphis group found that VCP interacts with three RNA-binding proteins including TDP-43. If either TDP-43 or VCP was mutated, then TDP-43 redistributed to the cytoplasm, causing cytotoxicity. The toxicity initiated by mutations in VCP is mediated, at least in part, by TDP-43, Taylor told ARF. He suggested that TDP-43 may be part of a common mechanism for neurodegeneration, not simply a secondary pathology, in diverse diseases.
TDP-43: Misplaced, Miscleaved, Misaggregated
Besides the numerous animal models discussed at ICAD, several scientists presented in-vitro data. For example, Janet van Eersel, working with Jrgen Gtz of the University of Sydney, Australia, is developing a cell culture model for TDP-43 proteinopathy by treating a variety of cell types with proteasome inhibitors. This means of disposal being blocked, TDP-43 aggregation and fragmentation went up, as it does in disease, suggesting that proteasome dysfunction may play a role in the proteinopathy. This simple treatment could be useful in studying TDP-43 activity and dysfunction in culture. Yonjie Zhang of the Mayo Clinic in Jacksonville is also working on a cell culture model for ALS and FTD. He created a stable human neuroblastoma line with inducible expression of the carboxyl-terminus of TDP-43. That model exhibits TDP-43 phosphorylation and aggregation in ubiquitin-positive inclusions.
Wolozin presented data from an upcoming paper linking TDP-43 and stress granules. An association between TDP-43 and stress granules in cell culture has been shown before, but Wolozin now also discovered TDP-43 in stress granules in human brain tissue. Other researchers were unable to find the association in human tissue (Colombrita et al., 2009), he suggested, because it is a weak signal easily buried by background autofluorescence. Graduate student Liqun Liu-Yesucevitz used the dye Sudan black to quelch background fluorescence, and was then able to detect TDP-43 in stress granules in brain samples from people who had had ALS or FTD.
The Boston researchers determined that TDP-43 connects with stress granules both directly and indirectly. The protein binds stress granule proteins such as TIA-1, and it also interacts with the mRNA in the complexes. Liu-Yesucevitz explored the location of four TDP-43 mutants in cells under stress. The mutations all showed dramatic two- to threefold increases in the number of inclusions that formed, and the number of cells that actually formed inclusions, Wolozin told ARF. Perhaps most dramatic was the extent to which the mutant TDP-43 left the nucleus and went to the cytoplasm. It is rare to find consistently abnormal activity among many TDP-43 mutants, but this study did, Wolozin said. Hence, the work suggests that an altered response to stress may be a key part of mutant TDP-43 pathology. Notably, Christian Haass, of Munich’s Ludwig Maximilian University, was also at ICAD presenting data on stress granules and FUS, another protein associated with ALS (see ARF related news story on Dormann et al., 2010).
For his part, Anthony White of the University of Melbourne in Australia also discussed the role of stress in TDP-43 proteinopathy. His group has found that changes in cellular metal levels, as well as oxidative stress, can alter the processing of TDP-43 (Caragounis et al., 2010). Cellular stress factors like these are common in neurodegenerative disease, and they somehow lead to TDP-43 fragmentation and relocalization to cytoplasmic aggregates. These findings indicate that in sporadic cases of ALS, changes to TDP-43 may occur through neuronal stress, White wrote in an e-mail to ARF.
Antonella Caccamo presented her work on TDP-43 and autophagy, conducted with Oddo at the University of Texas in San Antonio (Caccamo et al., 2009). These researchers found that blocking autophagy increased levels of the TDP-43 carboxyl-terminal fragment. When transfected into cells, this fragment then recruited extra TDP-43 to the cytoplasm. However, treatment with rapamycin—which bolsters autophagy—rescued the effects of the fragmented protein. The experiments were all in vitro, Oddo cautioned. If they hold up in vivo, the work would suggest that the carboxyl-terminus of TDP-43 is important for pathology, and that improving autophagy could counter its effects. On the last morning of ICAD, Oddo presented more data on rapamycin treatment in a different neurodegenerative paradigm, where driving autophagy in this way improved Aβ- and tau-related endpoints in mouse models (Caccamo et al., 2010).
The conference gave TDP-43 researchers plenty to think about, but little to be certain of. Major questions remain, Schmid noted. First and foremost among them, she wondered: What is the physiological function of TDP-43, especially in neurons Is the disease a gain of toxic function of the aggregates or oligomers, or a loss of function in the nucleus
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