Project Summary/Abstract Nuclear paraspeckles function to regulate mRNA translation. mRNAs that are sequestered in paraspeckles are translationally silent while those not entering paraspeckles can translocate from the nucleus where the can effectively be translated. There is now accumulating evidence that paraspeckles are increased in number and size in neurodegenerative diseases, including Alzheimer’s disease (AD), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS). RNA-binding proteins (RBPs) have a central role in neurodegenerative disease, but their functions in paraspeckles are not well understood, yet we confirmed a critical regulatory role of paraspeckles mediated by the stress-related RBP STAU1. In supporting studies, we discovered that STAU1 is overabundant in multiple neurodegenerative diseases including AD, FTD, and ALS, and that by normalizing STAU1 various molecular and motor phenotypes relevant to these diseases are improved or restored. Relevant to paraspeckles, STAU1 competes for mRNA binding at inverted repeat Alu sequences (IRAlus) with the paraspeckle core protein p54nrb and directs mRNA translocation to the cytoplasm, for active translation. The long noncoding RNA (lncRNA) NEAT1 is indispensable for paraspeckle formation, involving p54nrb interaction. In our preliminary studies we found that NEAT1 is strikingly downregulated in cells depleted of STAU1. NEAT1 also interacts with TDP-43 and is overabundant in AD, FTD, and ALS. Since both NEAT1 and STAU1 are overabundant in multiple neurodegenerative diseases, we hypothesize that normalization of STAU1 abundance will restore NEAT1 to normal levels, thereby restoring paraspeckle function. STAU1 will likely be a better therapeutic target than NEAT1 itself, because mice null for Neat1 have reduced cardiac function while mice null for Stau1 are viable and are characterized by no neurodegeneration. Our proposal is divided into two specific aims. The first aim will determine NEAT1 levels and paraspeckle localization as well as paraspeckle sizes and numbers in cell models that have been edited to express mutant ATXN2, that interacts with STAU1 in cytoplasmic phase-separated liquid droplets characterized by overabundant STAU1. We also evaluate Neat1 abundance and localization in a mouse model that we developed that overexpresses STAU1. In the second aim we characterize paraspeckles and NEAT1 in cultured human cortical neurons, with focus on TDP-43 pathology. The successful demonstration that NEAT1 is altered in diseases characterized by STAU1 overabundance will lead to new potential therapeutic targets for AD, FTD, ALS and other disorders with abnormal nuclear paraspeckle function, deeper understanding on the significance of NEAT1 and STAU1 to neurodegenerative disease, and will further support STAU1 as a therapeutic target.