PROJECT SUMMARY Neuroprotective actions of cystathionine γ-lyase through gasotransmitter hydrogen sulfide signaling. Hydrogen sulfide (H2S) is a gaseous signaling molecule or gasotransmitter which serves key roles in the central nervous system. However, specific targets and mechanisms of H2S action in the brain are obscure. Herein, we propose to elucidate the signaling pathways modulated by H2S in the brain that are neuroprotective to arrive at therapeutics targeting Alzheimer's disease (AD). H2S is generated from the amino acid cysteine which, in turn, is synthesized by cystathionine γ-lyase (CSE) via the transsulfuration pathway in the brain. H2S is also synthesized by two other enzymes, cystathionine β-synthase (CBS) and 3- mercaptopyruvate sulfurtransferase (3-MST), in the brain. We have demonstrated previously, that H2S and cysteine metabolism are dysregulated in AD. One of the modes by which H2S signals is via a posttranslational modification termed sulfhydration or persulfidation, wherein the reactive –SH group of cysteine residues on target proteins is converted to an –SSH group in a fashion analogous to nitrosylation by nitric oxide (NO), where the –SH groups are converted to –SNO groups. Although sulfhydration and nitrosylation modulate diverse physiological processes ranging from response to inflammation to neuroprotection, the molecular mechanisms by which cysteine and H2S/NO axes of gasotransmitter signaling affect neuronal function are yet to be deciphered. In Aim 1 of this project, we will monitor expression and activity of the three H2S biosynthetic enzymes, CSE, CBS and 3-MST in the mouse brain at various ages. Sulfhydration status in normal as well as mice lacking CSE will be assessed. The specific proteins modified by sulfhydration will be identified, and sites of sulfhydration mapped on them. The interplay of sulfhydration with nitrosylation in neuronal function will be monitored. In Aim 2, we will analyze the involvement of H2S in stress responses and behavior. In Aim 3, we will identify differentially sulfhydrated proteins in the 3xTg-AD mouse model of AD. By studying the function of CSE and H2S in the brain, we set a goal to better understand signaling mediated by cysteine, H2S and protein sulfhydration in the context of neuronal signaling in AD. Understanding the regulation of H2S signaling in the brain helps to determine the basic physiological pathways involved in neuroprotection and pins down the nodes for precision therapeutics and development of biomarkers for AD and other age-related neurodegenerative diseases involving dysregulated H2S signaling. 1