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ALS Alert Newsletter | December

TDP43 Detox

Scientists discover a novel strategy to help make TDP43 less toxic

Gitler

Mutations in the TDP43 gene have been linked to cases of familial ALS. Even in patients with sporadic ALS (thus carrying no TDP43 mutations), researchers have identified significant abnormalities in the TDP43 protein. Instead of residing in the nucleus, where TDP43 usually does its job, it clumps together in the cytoplasm of motor neurons in ALS patients. Packard scientist Aaron Gitler, a geneticist at Stanford University, and colleagues recently published a paper in Nature Genetics that identified a type of RNA molecule that might help make TDP43 aggregates less toxic.

"This interesting work validates the unbiased experimental approach taken by Dr. Gitler using yeast as a template to identify disease pathways triggered by genes and/or proteins that cause ALS. Here, the researchers have identified a gene in the path of TDP43 that would most likely never have been identified with other approaches. Although more work needs to be done to validate this gene in the context of ALS, this is a promising start," said Piera Pasinelli, Science Director at the Packard Center.

Whereas many attempts to reduce the toxic effects of TDP43 aggregates have focused on ways to keep the proteins from clumping together, Gitler and colleagues asked a slightly different question. They wanted to know if they could identify other genes that would reduce the toxicity, rather than preventing aggregation.

To do this, the researchers used a genome-wide, loss-of-function toxicity screen in yeast. This technique involved creating many different strains of yeast, each with one single gene deleted from their genome. Since the yeast don't carry the TDP43 gene, Gitler and colleagues also inserted a copy of human TDP43. Then, they watched the yeast grow and selected strains in which TDP43 was more or less toxic than in control yeast.

"This approach is useful because it allows us, in a completely unbiased way, to try and figure out why the genes are toxic in the first place," Gitler said. "The idea is that the types of genes that modify the toxicity--the genes that make it better or worse--would tell us more about what makes TDP43 so toxic."

The gene whose deletion decreased TDP43 toxicity the most was DBR1, which encodes a lariat-debranching enzyme. When the cell makes RNA, it is composed of protein-coding regions (exons) interspersed with non-coding regions (introns). After the RNA is made, but before it is used to synthesize a protein, different enzymes shape the introns into a loop and then remove them from the RNA molecule. These loops of RNA have a small tail and look like a lasso; researchers call them lariats. The lariat-debranching enzyme DBR1 turns this loop of intron RNA back into a linear molecule, which is then degraded.

To extend their findings from yeast to more relevant systems, the researchers and their colleagues at the Gladstone Institute in San Francisco decreased the expression of DBR1 in rat cortical neurons and followed these cells over time. Those cells that carried TDP43 and had decreased DBR1 activity had a 20% lower risk of death over the course of the experiment than cells carrying both genes. Interestingly, decreasing DBR1 activity didn't decrease the number of TDP43 aggregates the researchers found in the cytoplasm. The neurons had the same number regardless of whether DBR1 was active.

What the researchers did find was that an active DBR1 was required for TDP43 toxicity. Without an active DBR1, lariat RNA molecules accumulated in the cytoplasm and weren't degraded. These lariats bound to TDP43, which prevented TDP43 from binding other proteins and RNA molecules, thus preventing toxicity. So it seems that these lariat molecules act as decoys, attracting TDP43 to them and preventing it from interfering with important cellular RNA molecules.

"Our study discovered a novel potential therapeutic target for ALS. If we can inhibit DBR1 activity in human cells, that could potentially protect them against TDP43 toxicity. These results are still very preliminary, but we hope that this finding can be leveraged further to help create additional animal models and hopefully lead to the development of a new therapy," Gitler said.

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