Investigators Discover How Key Protein Leads to Neurodegenerative Diseases

The amount of cytoplasmic TDP-43 is a strong and independent predictor of neuronal death in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusion bodies (FTLDu), say researchers from the Gladstone Institute of Neurological Disease (GIND). TDP-43 is the major component of protein aggregates in patients with these diseases, and mutations in the TDP-43 gene are also associated with familial forms of ALS and FTLDu.

The researchers found that the mutant TDP-43 was toxic to neurons and that more of it was found in the cytoplasm. Using genetic manipulations, they showed that targeting wild-type TDP-43 to the cytoplasm is sufficient to recreate the toxicity associated with mutant TDP-43. Although the mutant protein formed inclusion bodies, these did not affect the risk of cell death.

The toxic effect of the mutant protein could be blunted by preventing its export from the nucleus, the scientists discovered. It appears that the toxicity of the mutation depends on cytoplasmic mislocalization of TDP-43. The study is published in the current edition of the Journal of Neuroscience.

"Our results indicate that the mutant protein is mislocalized to the cytoplasm," says GIND associate director Steven Finkbeiner, M.D., Ph.D., senior investigator and senior author on the study. "Although we don't know the underlying mechanism, the protein seems to become toxic in the cytoplasm and then causes death of the neuron."

Under normal circumstances, TDP-43 is a common protein that stays mostly in the nucleus. It has several beneficial functions including binding DNA and RNA, inhibiting retroviruses, and helping with RNA splicing and nuclear body formation. It also shuttles mRNA to the cytoplasm.

In patients with ALS and FTLDu, however, TDP-43 is redistributed from the nucleus to the cytoplasm and forms insoluble aggregates in the nucleus, cytoplasm, or neuronal processes.

Dr. Finkbeiner’s team developed a model system to find out how TDP-43 might be involved in neurodegenerative diseases. They used genetic engineering to add a fluorescent tag to normal or wild-type and mutant TDP-43 in rat neurons. The tag allowed them to easily see the intracellular location of the protein.

To determine the effects of the mutant protein, the researchers used an automated microscope that can examine hundreds of thousands of neurons individually over several days. With this large amount of data, they could use sophisticated statistical analyses to follow the fate of each individual neuron and determine its risk of death at any given time.

Their experimental system used primary neurons. These neurons are taken directly from an animal to a culture dish and provide the best cells for experiments because they retain many of the features of cells in the intact brain, the team explains. In fact, Dr. Finkbeiner's system showed many normal features of TDP-43 in neurons. For example, wild-type TDP-43 was found in the nucleus in healthy neurons. Mutant TDP-43 was also found in the nucleus, but there was more of the protein in the cytoplasm.

The system could be easily manipulated by the investigators, making it a valuable tool for dissecting the biological mechanisms underlying diseases associated with TDP-43 deposition.