PROJECT SUMMARY Alzheimer's disease (AD) is a major neurodegenerative disease affecting millions of people with no cure. There is an urgent need to understand molecular mechanisms that contribute to AD and find novel therapeutic targets. Amyloid precursor protein (APP) is a key player in AD. A clear correlation exists between the disease phenotype and increased APP expression. G-quadruplexes (G4s) are non-canonical DNA and RNA secondary structures with important regulatory roles and have emerged as a novel class of molecular targets for drug development. Intriguingly, the 3'-untranslated region (UTR) of the APP mRNA contains a guanine-rich region that forms an rG4, which functions as a translation repressor. However, how the APP 3'-UTR rG4 regulates translation remains unknown. The nucleolin protein was found to bind the 3'-UTR of the APP mRNA. We have showed, under the current U01 support, that nucleolin binds the G4 formed in the c-Myc oncogene promoter (MycG4) to inhibit cancers. Nucleolin binds MycG4 with much higher affinity than its best-characterized NRE RNA stem-loop substrate. Notably, MycG4 adopts the same type of structure as RNA G4s. We hypothesize that nucleolin binds and stabilizes the 3'-UTR rG4 to repress APP gene translation, and the stabilizing of the nucleolin-rG4 complex can reduce APP level in AD neurons derived from human induced pluripotent stem cells (hiPSCs). Understanding how nucleolin interactions with the APP 3'-UTR rG4 impact APP translational activity is important for unveiling the potential roles of this novel structural element in AD pathogenesis, and for therapeutic intervention. The proposed research will take advantage of neurons derived from healthy and AD hiPSCs carrying APP mutations identified from AD patients. The specific aims are: 1) To determine the role of nucleolin binding to the APP 3'-UTR rG4 in translational repression. We will determine the biologically relevant rG4 formed in the 3'-UTR of APP mRNA and the role of nucleolin-rG4 interactions in APP translational repression. We will also study the effects of small molecules that stabilize the nucleolin-rG4 complex on translation repression. 2) To determine the effect of nucleolin on APP 3'-UTR rG4 formation and APP expression in AD neurons. To determine the APP 3'-UTR rG4 structure formation in AD neurons, we will test the expression of APP in neurons derived from normal and AD hiPSC cells. We will also test the effects of nucleolin siRNA and nucleolin- G4 stabilizing small molecules on the APP levels in the neurons derived from hiPSC cells carrying AD related APP mutations as well as control hiPSCs. Results generated from this Administrative Supplement are expected to uncover the role of the novel nucleolin-rG4 complex in the regulation of APP expression in human AD neurons and provide the basis for the development of novel AD therapeutics that target the nucleolin-rG4 complex. Furthermore, the results are expected to lay the groundwork for a new R0...