SUMMARY This collaborative R01 application between a neuroscience lab (led by Stuart Lipton at Scintllon Inst./UC San Diego) and a chemistry lab (led by Steve Tannenbaum at MIT) will identify the redox posttranslational modification of proteins called S-nitrosylation by developing a more effective and integrated Mass Spec-based platform to screen for the S-nitrosoproteome and resulting alterations in protein function that contribute to the pathogenesis of Alzheimer’s disease (AD). Our hypothesis is that entire biochemical pathways critical to neuronal function are affected by aberrant S-nitrosylation of multiple proteins, these aberrant redox reactions (which are located, at least in part, downstream of Aß insult) contribute to the pathogenesis of AD, and the reactions occur in both sporadic and familial cases of the disease. Chemical and functional analysis of S- nitrosylated proteins will be assessed by biochemical assays, and by imaging of cells and tissues, including human AD brain and various in vitro and in vivo models of AD, ranging from transgenic mice to hiPSC-based model systems. We will also use site-directed mutagenesis and CRISPR/Cas9 techniques to generate DNA constructs or genes encoding proteins that that cannot be S-nitrosylated (thus forming non-nitrosylatable proteins). Accordingly, our Specific Aims are as follows: AIM #1. To determine the S-nitrosoproteome in human AD brain and transgenic mouse models. We will validate our recent S-nitrosoproteome findings in the CK-p25 mouse model of AD (published in PNAS, 2016) and determine if it generalizes to human AD brain and other transgenic mouse models of AD, e.g., hAPP-J20 and Tg2576. AIM #2. To use hiPSC-derived cerebrocortical neurons generated from human AD patients or WT exposed to oligomeric Aß (as a model of sporadic AD) as an in vitro model system to study the S-nitrosoproteome and how it affects biochemical pathways. This approach will allow us to study the functional effect of SNO-proteins in AD in a human context. AIM #3. To screen the effects of various S-nitrosoproteins in hiPSC-based models for impact on potential biological functions, e.g., effect on synaptic loss or neuronal cell death. This will be accomplished by generating non-nitrosylatable constructs of proteins (e.g., substituting Ala for Cys) by replacing the underling gene by CRISPR/Cas9 technology. For selected gene products that manifest profound effects of S- nitrosylation on synaptic functions and neuronal cell survival in hiPSC-based models, the non-nitrosylatable version of the gene can also be created in mice using CRISPR/Cas9 to mechanistically test its effect in vivo.