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Dec 9

Light-based strategy shows how major ALS protein causes disease

Packard scientists Christopher Donnelly and Udai Pandey are using a strategy called optogenetics to study how TDP-43 causes disease

Scientists have identified abnormal behavior of a protein called TDP-43 in 97% of those with ALS and just under half of those with frontotemporal dementia (FTD). In a new study in Neurobiology of Disease, Packard scientists Christopher Donnelly and Udai Pandey, both at the University of Pittsburgh, have used a strategy called optogenetics to study how TDP-43 causes disease. Their novel fruit fly model showed that the approach could recreate many of the pathologies seen in ALS/FTD patients.

Although ALS-causing mutations in the TDP-43 gene were discovered in 2006, scientists have yet to understand exactly how these mutations cause disease. What researchers do know is that the mutations caused TDP-43 protein to leave its usual location in the nucleus (where it functions as an RNA-binding protein) and enter the cytoplasm. Once in the cytoplasm, TDP-43 protein aggregates into dense, insoluble, toxic clumps. But individuals with sporadic forms of ALS—and thus without mutations in TDP-43—also show these pathological signs, indicating that something more than mutations are at work.

Adding to the problem, existing animal models require the overexpression of the TDP-43 gene, which leads to abnormally high levels of TDP-43 in the cell not observed in patients. What’s more, few overexpression models exhibit cytoplasmic TDP-43 inclusions and overexpression does not occur in patients. Nor does this tell scientists about the precise series of steps by which TDP-43 contributes to ALS. To remedy this problem, first author Charlie Otte and Tyler Fortuna turned to a technique called optogenetics, which lets them control protein activity using blue light. Donnelly and Pandey generated a strain of fruit fly containing TDP-43 with an optogenetic tag that allows for the control of TDP-43 aggregation with exposure to blue light.

When the researchers measured TDP-43 protein localization in larvae reared in complete darkness, they found the TDP-43 remained in the nucleus of motor neurons. After the scientists exposed the fruit fly larvae to light for 24 hours, however, they found that TDP-43 had mislocalized to the cytoplasm and formed large, insoluble aggregates. Increasing the duration of blue light exposure from 48 hours to as much as 192 hours created a corresponding increase in these aggregates. These clumps remained and even grew after the flies were returned to darkness for 72 and 144 hours. One-third of the larvae carrying optoTDP-43 that were exposed to blue light for 30 hours developed wrinkled or twisted wings. Other experiments revealed that more generalized protein clumping led to toxicity in motor neurons that decreased crawling ability in larvae.

As the light-exposed optoTDP-43 larvae aged, they began to have difficulties climbing. Based on these findings, Donnelly, Pandey, and colleagues concluded that the ability for TDP-43 to form of persistent toxic clumps creates ongoing toxicity in motor neurons, leading to the crawling and climbing problems seen in the flies. The researchers say they hope that these findings improve our understanding of TDP-43’s contribution to ALS disease and plan to use this model to identify genes that protect against TDP-43 toxicity, opening the door to better therapies.