Molecular Targets of Neurosteroid Anti-depressant Action

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Project description Neuroactive steroids (NAS) are important modulators of neuronal excitability with demonstrated therapeutic efficacy as anesthetics and rapidly acting anti-depressants. While the anesthetic actions of NAS are mediated by GABAA receptors, the mechanism(s) of their anti-depressant effect remains unknown. NAS functionally modulate the activity of NMDA receptors and specifically bind several intracellular proteins that may contribute to their anti-depressant actions. Notably, NAS also localize to trans-Golgi membranes, suggesting that they are either selectively transported to Golgi or have an abundant selective binding partner in Golgi. The goals of this project are to characterize NAS binding sites on putative anti-depressant protein targets and identify novel intracellular NAS binding proteins that may contribute to the anti-depressant effects of NAS. To achieve these goals, we will utilize NAS analogue photo-affinity labeling in concert with state-of-the-art protein chemistry and expression techniques and cutting-edge mass spectrometry (MS) methods. The Evers lab has developed these methods and successfully applied them to delineating the molecular details of NAS binding to VDAC, β- tubulin and isoform-specific sites on GABAA receptors. Novel photo-affinity labeling reagents for this project will be synthesized in the Chemistry Core. The output of our labeling studies will be identification of novel NAS binding proteins and mutations in NAS binding sites that enable characterization of the role of these target proteins in the anti-depressant physiological and behavioral effects of NAS (Projects 2 and 3). The Project has two specific aims: In Aim 1, we will identify and characterize the binding sites for NAS that are negative- and positive- allosteric modulators (NAMs and PAMs) of NMDA receptors. GluN1/GluN2B NMDA receptors will be labeled with NAM and PAM NAS photo-affinity analogues and the number of labeling sites on each subunit will be determined using intact protein MS. The specific amino acids modified by the photolabeling reagents will be identified using middle-down MS. Molecular modeling will be used, in conjunction with the photolabeling data, to predict critical residues that may be necessary for NAS binding or effect. In Aim 2A we will perform an unbiased search to identify the “proteome” of NAS binding proteins in intact hippocampal neurons using photo- affinity labeling reagents with a “”click chemistry” tag to enrich binding proteins, followed by LC-MS/MS-based protein identification. In Aim 2B, we will perform a focused proteomic search to identify cytosolic neuronal proteins that selectively bind/transport NAS and may alter the lipid composition of organelle membranes and modulate cellular stress responses. The physiological and behavioral effects of identified NAS-binding proteins involved in cellular stress response pathways (Aim 2A) and NAS/lipid transport (Aim 2B) will be evaluated using CRISPR knockout methods (Projects 2 and 3).