TAR DNA binding protein – 43 (TDP-43) is a critical RNA binding protein that is intimately involved in many aspects of RNA metabolism. While primarily localized to the nucleus, TDP-43 shuttles between the nucleus and the cytoplasm performing its physiological functions. As an aggregation prone protein, TDP-43 is known to accumulate and from prion-like solid aggregates in the cytoplasm of cells leading to the sequestration of nuclear TDP-43. This behavior of TDP-43 has been well established as a pathological hallmark of a neurodegenerative disease spectrum encompassing amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) and has been described in Alzheimer’s disease and related dementias. Pathological cytoplasmic TDP-43 inclusions have been hypothesized to contribute to disease pathogenesis through both a nuclear depletion and the cytoplasmic aggregation. Despite extensive research, mechanisms that initiate this pathology under disease conditions remain elusive. Recent studies in our laboratory showed that aberrant RNA A-I editing is present in multiple brain regions of C9orf72 ALS/FTD, where we detected bidirectional changes in A-I editing. Since then, we have generated preliminary data suggesting that TDP-43 nuclear export can be regulated via Adenosine Deaminase Acting on double stranded RNA (ADAR)-mediated A-I RNA editing. We show that enhancing RNA A-I editing through ADAR2 overexpression in mammalian cell lines induces TDP-43 translocation to the cytoplasm requiring functional RNA binding domains of TDP-43. In contrast, the overexpression of catalytically inactive ADAR2 does not alter the nuclear localization of TDP-43. These findings led us to hypothesize that aberrant increases in A-I editing induces TDP-43 cytoplasmic mislocalization through an RNA dependent mechanism. To determine if this editing induced TDP-43 nuclear export also occurs in a neuronal environment, we will expand on our preliminary data and examine human induced pluripotent stem cell (iPSC) differentiated into motor neurons for A-I editing-mediated TDP-43 nuclear export. We will validate A-I RNA editing mediated cytoplasmic accumulation of TDP-43 in iPSC-MNs expressing doxycycline inducible Tet-On ADAR2 constructs: wildtype ADAR2, a catalytically inactive ADAR2 (ADAR2E396A) and a catalytically hyperactive ADAR2 (ADAR2E488Q). To address the effects of RNA-editing induced TDP-43 mislocalization on TDP-43 function, we will examine TDP-43 inclusions for disease-relevant characteristics (Aim1). To determine the identity of mRNAs bound to TDP-43 and potentially being necessary for A-I RNA editing-mediated mislocalization, we will perform eCLIP-seq on iPSC-MNs genetically altered for hypo and hyper-editing as described in Aim1. In addition, we will perform eCLIP in C9orf72 iPSC-MNs to compare RNA-editing induced TDP-43 bound transcripts to those associated with endogenous disease (Aim 2). Finally, in Aim 3, we will perform exploratory studi...