Heady Times for Researchers Studying TDP-43
Two genetics papers this month add to the mounting evidence that TAR DNA binding protein (TDP-43) is a key player in amyotrophic lateral sclerosis (ALS) and not just an innocent bystander. Since 2006, when TDP-43 was identified as the aggregating protein in inclusions in ALS and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) (see ARF related news story), researchers have been trying to elucidate its role in neurodegenerative diseases. A third study out this week introduces a yeast model for mechanistic study and drug screening.
Guy Rouleau from the University of Montreal and colleagues at other Canadian and French institutions published their results on mutations in TARDBP online March 30 in Nature Genetics. Following on their heels, Vivianna Van Deerlin from the University of Pennsylvania in Philadelphia and colleagues from the University of Washington and the Department of Veterans Affairs Medical Center, Seattle, published their results online April 5 in The Lancet Neurology. The yeast work, by Aaron Gitler’s lab at the University of Pennsylvania, is in press at the Proceedings of the National Academy of Sciences online. It reports the first evidence that there is a connection between TDP-43 aggregation and toxicity.
Marc Cruts at the University of Antwerp, Belgium, who was not involved in the studies, likens the findings to the discovery of mutations in amyloid precursor protein (APP) in Alzheimer disease. “Identification of mutations is usually good proof that the protein is functionally related to the disease,” he said.
The latest papers identify new mutations in the TARDBP gene on chromosome 1. They follow a recent flurry of papers, which have identified their own mutations in the gene (see ARF related news story). Interestingly, the majority of mutations discovered in these studies cluster in the same region. “There have been several genes implicated in ALS, but this is the first new piece of the puzzle in 15 years that’s generated consensus in the field since the link between Cu/Zn superoxide dismutase 1 (SOD1) and ALS was established,” Rouleau said. Ninety percent of ALS cases are sporadic, which has made it difficult to identify genes involved in pathogenesis. Within the 10 percent of familial cases, 20 percent are linked to mutations in SOD1, which predominantly causes autosomal-dominant disease, and a few other genes have been identified in more rare forms.
Van Deerlin and colleagues screened TARDBP for mutations in 168 people with either clinical ALS or ALS combined with frontotemporal dementia, as well as in 91 autopsy brains from people who had either of these two diagnoses and TDP-43 pathology. Genomic DNA was extracted from the blood of patients or autopsy CNS tissue.
Control samples came from 705 neurologically healthy, elderly Caucasian people, and 42 brain autopsy samples without evidence of neurodegenerative disease (mean age 69 years). Another 380 Chinese participants (mean age 72) from the National Taiwan University were included in the control.
“Our paper is unique in that we were able to evaluate the neuropathology of several members of the same family with a mutation and thereby confirm the presence of TDP-43 deposits in the brain. The previous studies did not have brain tissue available to study and make a direct link between the mutation and pathology,” Van Deerlin said.
Van Deerlin and colleagues identified two heterozygous missense variants in exon 6 of TARDBP in two families with autosomal-dominant familial ALS. One mutation, glycine to alanine (G290A), was detected in a Caucasian family and the other mutation, glycine to serine (G298S), in a Chinese family. Neither mutation occurred in the corresponding control groups.
Together with the other published reports of TARDBP mutations, these new results provide evidence that these variants in the C-terminal region have a pathogenic role in ALS and that a direct link exists between the presence of a TARDBP mutation, TDP-43 pathology, and autosomal-dominant ALS.
Although the physiological role of TDP-43 is not understood, some researchers believe that TDP-43, in particular its C-terminal domain, may function in the regulation of gene expression. That four different mutations, identified by different research groups, all involve glycine residues within 11 amino acids of each other suggests that these mutations cause disease through similar mechanisms, suggest the authors.
For their part, Rouleau’s team screened TARDBP for mutations in 200 French and French Canadian individuals with ALS (120 with sporadic ALS and 80 with familial ALS) and 185 controls matched for age and ethnicity. First author Edor Kabashi and colleagues identified eight distinct heterozygous missense mutations in nine individuals: six with sporadic ALS and three with familial ALS. The researchers did not find the mutations in the 185 controls or the 175 additional controls. None of the affected individuals had a personal or family history of frontotemporal dementia.
Seven of the eight mutations clustered in the glycine-rich C-terminal region of exon 6, supporting previous results from other groups. One substitution, aspartic acid to glycine (D169G), was in the first RNA-binding motif of TDP-43 and may interfere with RNA binding to the protein. The glycine to cysteine (G348C) variant may increase the protein’s propensity to aggregate through the formation of intermolecular disulfide bridges. The authors also point out that most of the mutations identified were predicted to increase TDP-43 phosphorylation, since five of the resulting substitutions are to threonine or serine residues. This could potentially interfere with protein interactions or transport through the nuclear pore complex and lead to progressive accumulation of the aggregates seen in people with ALS.
“Greater numbers of cohorts will have to be studied so we can determine what percentage of ALS is caused by mutations in TDP-43,” Rouleau said. “At this point, there are more questions than answers. We’ve found that 5 to 6 percent of familial ALS is caused by mutations in TDP-43, but we don’t know yet how these mutations can cause the disease,” Van Deerlin said. Researchers will have to develop functional assays to determine the biochemistry in cell culture or other disease model systems.
The author of the third paper is using yeast to do exactly that. Aaron Gitler initiated these studies two years ago when TDP-43 first became a suspect in ALS. At the time, he was a postdoctoral fellow in coauthor Susan Lindquist’s laboratory at the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts.
First author Brian Johnson expressed human TDP-43 protein in yeast cells and saw that it was confined to the nucleus, which is the protein’s normal location in human cells. When the scientists drove up expression levels, they observed the same characteristic aggregates in the cytoplasm that were seen in neuronal cells from ALS patient postmortem samples. The authors also link TDP-43 aggregation to cellular toxicity, suggesting TDP-43 may have a causative role in disease pathogenesis.
Answering whether the mutations are causing a loss or gain of function will be critical to understanding TDP-43 role in disease. Gitler and colleagues used cellular structure/function analyses to reveal that only aggregating forms of the protein are toxic to yeast, suggesting TDP-43 causes a toxic gain-of-function phenotype due to protein misfolding. But the authors propose that TDP-43 cellular toxicity is not simply due to general cellular stress associated with accumulating misfolded proteins; instead it has to do with an as-yet unknown function that depends on one of the protein’s two RNA recognition motifs.
The researchers then looked for the smallest fragment of the protein that was able to cause aggregation and toxicity. Gitler was excited to find a cleaved form of the protein that was similar to a TDP-43 fragment found in the affected motor neurons of ALS patients. “Using yeast, we honed in on the C-terminal region as the part of the protein most likely associated with pathogenicity. This is the same region in which almost all of the recently identified ALS-linked TDP-43 mutations are clustered,” Gitler explained.
Studies in yeast can help define basic cellular mechanisms, which can then be tested in more relevant cell culture and animal models. “Since yeast are relatively easier to manipulate, they provide a convenient method for quickly screening genes or compounds that may mitigate the effects of a mutation,” said Gitler. In fact, yeast models have been useful in studying Parkinson’s (Cooper et al., 2006) and Huntington’s (Giorgini et al., 2005; see ARF related news story).
The implications of these findings go beyond ALS and FTLD-U, since TDP-43 has been linked pathologically to other neurodegenerative diseases including Alzheimer’s. Indeed, Dennis Dickson's and Clifford Jack's groups at the Mayo clinics in Jacksonville, Florida, and Rochester, Minnesota, respectively, report in the April 9 Neurology a closer look at a series of AD patients with or without TDP-43 pathology. In this series, 29 of 84 AD patients had TDP-43 immunoreactivity in their hippocampus or medial temporal lobe. Their test performance and their hippocampal atrophy were somewhat worse than that of AD patients without this added pathology, indicating that TDP-43 subtly affects the form of AD a person develops (Josephs et al., 2008). Future studies will lead to a better understanding of how TDP-43 fits into the larger picture of diverse neurodegenerative diseases. Undoubtedly, new groups will jump into the fray as this story unfolds.—Nadia Halim
Nadia Halim is a science writer based in Bridgewater, NJ.
- New Ubiquitinated Inclusion Body Protein Identified
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