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Mar 20

Optogenetics sheds light on TDP43 toxicity

In a new study, a team of Packard investigators has developed an approach that lets them use light to control the development of toxic TDP43 protein aggregates

Nearly all ALS patients have a toxic buildup of a protein called TDP43 in their degenerating neurons. In their quest to understand how the aggregation of TDP43 fits into the onset and progression of ALS and frontotemporal dementia (FTD), scientists have thus far been unable to study interactions between TDP43 and other cellular components in live cells. In a new study published in Neuron, a team led by University of Pittsburgh neuroscience graduate student Jacob Mann and advisor and Packard investigator Christopher Donnelly, along with Packard scientist Udai Pandey (also at the University of Pittsburgh), and James Shorter (of the University of Pennsylvania), has developed an approach that lets them use light to control the development of toxic TDP43 protein aggregates similar to what we see in patients. Their new strategy revealed that the presence of RNA prevents TDP43 from forming insoluble clumps, which provides clues for the development of new potential treatment strategies for ALS.

Despite the heterogeneity of people with ALS, 97% of those with the disease—and nearly half of those with FTD—have large clumps of TDP43 in the cytoplasm of degenerating motor neurons. Since TDP43 affects various aspects of RNA processing with its two RNA-binding segments, it needs to be in the nucleus to do its job. When the protein is stuck in the cytoplasm, not only can’t TDP43 do its normal job, another segment in the protein (known as the low-complexity domain, LCD) causes TDP43 proteins to misfold and bind together.

These LCD interactions occur as the molecule condense into liquid-like compartments and is known as liquid-liquid phase separation (LLPS). Besides the binding between TDP43 and itself, LLPS also occurs during periods of stress when the cell sequesters RNA and proteins into stress granules to prevent further damage. In healthy cells, the high levels of RNA in stress granules bind to TDP43, which means not enough of the protein is left in the cytoplasm to form solid aggregates. Understanding how and why these toxic clumps form would give scientists novel ways to prevent their establishment.

Mann, Donnelly, Pandey, and Shorter used a technique called optogenetics that let them use blue light to switch on TDP43 misfolding in human embryonic kidney cells. Compared to cells left in the dark, those exposed to blue light for 24 hours had less nuclear TDP43 and a greater number of cytoplasmic aggregates. These aggregates remained even after the light was switched off and showed all the hallmarks of the toxic TDP43 clumps seen in ALS patients. When the researchers repeatedly stimulated the LCD of TDP43 with blue light, they found the LCD segments condensed into liquid-like compartments that, over time, transformed from transient to persistent granules. ALS-linked mutations in TDP43 sped up this process.

The scientists couldn’t, however, use blue light to stimulate phase separation when they studied just the RNA-recognition segments of TDP43. They found that making small mutations in these segments to impede their ability to bind RNA restored that ability. Pandey, Shorter, and Donnelly found that full-length TDP43 proteins that were unable to bind RNA formed large, insoluble aggregates in the nucleus; when those proteins also had mutations in their nuclear localization signal, those aggregates formed in the cytoplasm.  

Taken together, this work shows that scientists can create ALS pathology in a dish using optogenetics, allowing us to identify the toxic pathways that lead to disease in the vast majority of patients.  Using this powerful tool enabled the team to show that the LCD drives the formation of solid TPD43 protein aggregates.  However, if TDP43 is bound to RNA or associated with stress granules, this interaction cannot occur. Using molecules that mimic TDP43’s natural binding partners prevented these pathological interactions and the formation of TDP43 solid inclusions. This raises the potential for the development of similar short nucleotide sequences for possible ALS/FTD therapeutics.