4 October 2010. Sponsored by the Fondation André-Delambre and now in its sixth year, the Symposium on amyotrophic lateral sclerosis is rapidly becoming one of the top tickets in ALS research. The meeting, held September 24-25 at Université Laval in Québec City, Canada, attracted 80 scientists who discussed cutting-edge data on the mechanisms of the disease and progress on treatments and in the stem cell field. Speakers presented an avalanche of results that we have to think about, said Jean-Pierre Julien of Université Laval in a closing statement. Julien co-organized the meeting with Jasna Kriz, also at Laval.
A theme that stood out was TAR DNA Binding Protein 43 (TDP-43). Its role in amyotrophic lateral sclerosis (ALS) is well-accepted—but what does TDP-43 actually doβ For some time, the answer has been a vague, something having to do with RNA. In Québec, researchers presented a couple of approaches that may yield more concrete answers.
Clotilde Lagier-Tourenne of the University of California in San Diego asked which RNAs TDP-43 interacts with. Lagier-Tourenne, who works with Don Cleveland, is collaborating with Maddalini Polymenidou from the same group and the laboratory of Gene Yeo, also at UCSD. The researchers are using a relatively new technique called cross-linking immunoprecipitation and sequencing (CLIP-Seq) to identify such mRNAs in mouse brain. They treated the mouse brain samples with crosslinkers to cement any otherwise fleeting RNA-protein interactions. Then, they used TDP-43 antibodies to pull down the protein and any associated RNAs. Finally, they sequenced the nucleic acid to determine which RNAs bind TDP-43. Further, they determined specific nucleotide sequences that recruit TDP-43. Scientists suspect the protein is involved in mRNA splicing, so it was no surprise to Lagier-Tourenne to find that these binding sites are in introns and in close proximity to intron/exon splice junctions.
One particular topic of debate in this field is whether ALS pathology is related to TDP-43’s normal role. To begin to tackle this question, Lagier-Tourenne and Polymenidou, in collaboration with ISIS Pharmaceuticals Inc. in Carlsbad, California, used antisense oligonucleotides to knock down TDP-43 expression in mouse brains. By sequencing cDNA, she found changes in mRNA expression and splicing in these animals. The researchers are working to identify genes influenced by TDP-43 in the hopes of understanding how TDP-43 mutations cause ALS.
While Lagier-Tourenne is puzzling out TDP-43’s role at the molecular level, other scientists are examining its effects in the whole organism. ALS researchers are steadily accumulating a fine collection of TDP-43 model mice (for a recent summary, see ARF ICAD conference story) and the meeting brought further updates from both the Cleveland and Julien labs.
Many researchers have expressed human TDP-43 in mice under the powerful, nervous system-specific PrP or Thy1 promoters. Julien, along with his colleague Vivek Swarup, took a different tack by cloning the entire human gene, native promoter included, and inserting that into their mice. In addition to making mice with wild-type human TDP-43, the researchers used site-directed mutagenesis to create the disease-linked A315T and G348C mutations in the transgene, creating additional TDP-43 mutant lines. The transgenic animals have low human TDP-43 expression throughout the body, Julien reported.
Both the wild-and mutant animals look pretty normal, Julien said. They suffer neither paralysis nor early death, and their motor neurons remain undamaged up until 10 months, middle age for mice. They are wobbly when balancing on a rotarod, suggesting some loss of motor control. They also have memory problems that the researchers said reminded them of frontotemporal dementia, another TDP-43 proteinopathy. The mutants—particularly the G348C mouse—evince cytoplasmic aggregation and ubiquitination of TDP-43, similar to human ALS pathology. One of the most striking changes in the mice is neuroinflammation, Julien added. The animals exhibit microgliosis and astrogliosis, providing further evidence that ALS has an immune system component.
Sandrine Da Cruz presented the Cleveland lab’s latest and greatest on TDP-43 animals, covered last year on Alzforum (see ARF news story ). In addition, the lab has been making animal models overexpressing fused in sarcoma (FUS), another gene linked to ALS. In all, the Cleveland group is juggling 36 mouse lines —18 each for TDP-43 and FUS transgenes including both wild-type and mutant versions (TDP-43 Q331K and M337V and FUS R521C and R514G). The good news is there are a lot of mice, Cleveland said, adding in jest, The bad news is, there are a really a lot of mice!
The group took a two-pronged transgenic strategy for each gene. In some lines, the researchers expressed the human genomic version of FUS or TDP-43. In other animals, they used the PrP promoter and flanked the cassette with Lox sites, so they can selectively excise TDP-43 from different cells types using Cre recombinase. TDP-43 transgenic mice displayed gait abnormalities and muscle hyperactivity; FUS mice exhibited similar walking problems as well as astrogliosis and altered axon diameter.
Of all these mice, unfortunately none are quite the ideal model scientists are looking for. Echoing a long-standing difficulty in Alzheimer disease research, each strain models an aspect, or aspects, of the whole picture. In an email to ARF Julien commented: The various talks on transgenic mice illustrated the difficulty in creating models that mimic the human ALS disease well. —Amber Dance.
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