Despite extraordinary efforts focused on understanding the mechanisms underlying amyotrophic lateral sclerosis (ALS), a cure or treatment for this devastating disease remains elusive. Mutations in two RNA binding proteins, TAR DNA binding protein 43 (TDP43) and Fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS), cause some forms of familial ALS. In order to study the effects of mutations in FUS and TDP43 expressed at endogenous levels in a homogeneous cell population relevant to disease, the experiments proposed utilize patient-derived induced pluripotent stem cells (iPSCs) differentiated into motor neurons (iMNs). To address the hypothesis that mutations in FUS and TDP-43 alter RNA binding across the motor neuron transcriptome, that these alterations lead to changes in RNA metabolism, and ultimately to an ALS phenotype, I will assess changes in binding of the mutant compared to wild-type proteins using HiTS-CLIP (High Throughput Sequencing with Cross-Linking and ImmunoPrecipitation) technology. Preliminary data reveal that mutant TDP-43 binds differently to RNA than the wild type protein in our iMN system. Through preliminary studies, I have derived a MT FUS iPS cell line and optimized the immunoprecipitation of FUS from iMNs. I am using innovative bioinformatics tools to extract relevant binding differences from my data, in conjunction with already obtained deep sequencing data. The proposed studies will reveal how mutations in RNA binding proteins alter protein-RNA interactions, and thereby RNA metabolism. They will provide insight into motor neuron-specific mechanisms of RNA metabolism and how single base pair mutations can perturb these mechanisms.
Comparative analysis of endogenous interactions found separately for FUS and TDP-43 may identify common defects in RNA metabolism and contribute to the discovery of a common ALS disease mechanism. These common defects will represent ideal drug targets for the development of therapeutics to treat ALS. By providing new avenues for ALS treatment, we could significantly improve patients'quality of life, and alleviate the burden on the healthcare system of managing ALS patients.