The role of neuronal mRNA transport granule packaging in ALS/FTD

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PROJECT SUMMARY/ABSTRACT Neurons are highly polarized cells with processes reaching up to one meter in length that rely on mRNA transport and local translation to rapidly respond to distal stimuli. Defects in mRNA transport have been identified in a number of neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). Additionally, many ALS/FTD-associated mutations are found in RNA binding proteins (RBPs), which participate in the axonal transport of mRNA to sites of local translation. These ALS/FTD-associated RBPs undergo phase separation, a phenomenon where proteins and nucleic acids demix from the surrounding solution, forming membraneless organelles. Phase separation likely underlies mRNA transport granule formation and disease mutations alter the ability of these RBPs to undergo phase separation, leading to the pathological inclusions observed in patient tissue. Using neurons derived from induced pluripotent stem cells, live and fixed cell imaging, transcriptomics, and proteomics, I will determine if transcripts are co-transported, the effects of disease mutations on mRNA packaging, and develop antisense oligonucleotides to improve disease-associated packaging defects. Aim 1 will determine if the ALS/FTD-associated proteins FUS, TDP-43, and MATR3 transport three transcripts destined for different axonal compartments, SHANK1, ATP5B, and NTM, separately or co-package them into granules and if the proteins co-localize to the same granule. Then, I will determine the effect of ALS/FTD mutations to the RBPs on protein and mRNA packaging into transport granules. Aim 2 will identify the protein and mRNA components of FUS, TDP-43, and MATR3- containing transport granules, as well as elucidate the rules for mRNA packaging using bioinformatic and experimental techniques. Aim 3 will determine if modalities previously shown to alter RBP phase separation are able to rescue packaging defects in ALS/FTD mutant lines. Then, I will use CRISPR inhibition to determine complementary targets whose knockdown achieves a similar effect as overexpression before developing and validating antisense oligonucleotides against these targets. I hypothesize that ALS/FTD-associated RBPs will co-package transcripts destined for the same axonal compartment and that changes in phase separation due to ALS/FTD mutations alter this packaging. The proposed work will address the current knowledge gap on the role of mRNA transport in ALS/FTD, which is crucial for both understanding the basic mechanisms of mRNA transport in neurons as well as the development of effective therapeutics to treat or prevent neurodegeneration. Results from this proposal will provide critical understanding of the mechanisms of granule packaging, how they are disrupted in neurodegenerative disease, and will identify and validate therapies to rescue ALS/FTD-associated mRNA transport granule packaging defects. This fellowship will be centered around training in the technical skills required to complete the proposed project as well as professional development activities to prepare the applicant for success as an independent researcher.