Mechanistic analysis of TDP-43-mediated RNA localization in neurons and its misregulation in ALS

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PROJECT SUMMARY The proper functioning of essentially all cells is due in part to spatially defined regions that are populated by specific proteins to perform specific activities. Neurons, due to their elongated size and morphology, must deal with large distances when navigating protein localization with regard to axons and dendrites. This problem is solved by transporting RNA molecules to specific locations within neurons. When these RNA molecules are translated, they immediately produce correctly localized proteins. The importance of this key process is evidenced by multiple neurological diseases that are associated with its misregulation. The RNA-binding protein TDP-43 is known to be involved in RNA localization, and mutations in TDP-43 are associated with Amyotrophic Lateral Sclerosis (ALS). Yet the RNAs that depend on TDP-43 for localization, how TDP-43 recognizes these RNAs and promotes their transport, and the phenotypic consequences of their mislocalization are all currently unknown. This means that an entire regulatory modality contributing to ALS pathology may be undiscovered. Here, we propose to use our combined expertises in subcellular transcriptomics, stem cell differentiation, and live cell microscopy to engage these critical problems and provide novel insights into ALS disease mechanisms that likely will result in novel treatment modalities. To do so we have developed a novel technique to monitor neuronal RNA mislocalization transcriptome-wide, allowing us to identify transcripts that need functional TDP-43 for proper transport. By combining this approach with our expertise in stem cell differentiation, we will use this technique to probe TDP-43-mediated RNA localization in functional human motor neurons. We will identify mislocalized RNAs in engineered TDP-43-null motor neurons, motor neurons expressing ALS-associated TDP-43 mutants, and motor neurons derived from ALS patients. We will use this information to derive the RNA sequence requirements that define transcripts that depend on TDP-43 for transport and test their necessity and sufficiency with reporter transcripts. We have also developed a technique for imaging the transport of single RNA and protein molecules in live Drosophila brains. We will use this approach to visualize TDP-43-mediated RNA transport in live brains and learn about its dynamics and regulation. Drosophila TDP-43 mutants display phenotypes similar to those seen in ALS patients. However, as of now data attributing cellular or organismal phenotypes to the mislocalization of specific transcripts for ALS or any other neurological disease is missing. This may be due in part to the fact that RNA localization is often overlooked and the transcripts that are mislocalized in disease states are not known. To engage this problem, we will mislocalize specific transcripts in Drosophila brains that depend on TDP-43 for transport by removing sequences that TDP-43 recognizes within the transcript. We will then ask if these flies display the locomotor and physiological defects that characterize both TDP-43 mutant flies and ALS patients. Overall, we intend to define new connections between misregulated molecular mechanisms and ALS patient phenotypes, thus opening up new avenues of research and therapeutic development.