We continued to investigate the molecular mechanisms of PS-dependent Akt activation and its biological implication. We demonstrated a novel molecular mechanism for Akt activation by finding novel PS-binding sites that are necessary for Akt conformational changes. Akt interaction with these PS-binding residues, particularly in the regulatory domain, is critical for Akt phosphorylation at S473 by mTORC2, which is known to be an important modulator of Akt activation, and therefore can serve as a new target for drug development. Based on this new target we have initiated efforts to identify inhibitors of Akt activation that differs from the well-recognized PIP3- or ATP-binding sites. For this purpose, we have first developed a cell-based assay targeting the S473 phosphorylation using homogeneous time-resolved fluorescence (HTRF) detection and tested against the MLSCN (Molecular Libraries Screening Center Network) compound collection. During this period, we have continued to refine the assay methods to extend it to the high throughput (HT) screening for approximately 400,000 MLPCN compounds in collaboration with NCGC. PI3K/Akt signaling that regulates neuronal survival has been implicated in the deleterious effects of ethanol on the central nervous system. To understand the underlying molecular mechanisms, we investigated the effect of ethanol on Akt conformational changes following Akt-membrane interaction, a prerequisite step for Akt activation processes. We quantitatively probed Akt conformational changes using chemical cross-linking, 18O labeling and mass spectrometry. We found that ethanol at pharmacologically relevant concentrations (20-170 mM) directly interacts with Akt and alters the local PH domain configuration near the PIP3-binding site. We also found that ethanol significantly impairs subsequent membrane-induced inter-domain conformational changes required for Akt activation. Alteration of Akt conformation caused by ethanol during the activation sequence may be a novel molecular basis for the impact of ethanol on Akt signaling. As PS accumulation is an important aspect of DHA- or ethanol-mediated effects on Akt signaling, we continued to characterize the neuronal PS accumulation, particularly DHA-containing PS (DHA-PS). We examined substrate preference and product inhibition in PS biosynthesis, since both processes can contribute to the accumulation of this PS species. We previously found that purified PSS2 uses DHA-containing PE as the best substrate for PS synthesis. During this period, we found that product inhibition is differentially affected by the PS fatty acyl moiety. The activity of the purified PSS2 was inhibited by DHA-PS more significantly than by OA-PS. The highest preference for DHA-species for both PS synthesis and product inhibition shown by the purified PSS2 reconstituted into PE/PC liposomes suggests that preferred PS synthesis is the primary contributor for neuronal DHA-PS accumulation. We have demonstrated that DHA metabolism to N-docosahexaenoylethanolamide (DEA, synaptamide) is a significant mechanism for hippocampal neuronal development, contributing to synaptic function. As DEA, an ethanolamide derivative of DHA, is a synaptogenic factor, we subsequently coined the term synaptamide for this compound. Synaptamide-treated neurons increased neuritogenesis, synaptogensis, and the expression of synapsins and glutamate receptor subunits and exhibited enhanced glutamatergic synaptic activity. During this period, we have extended our investigation to the differentiation of neural stem cells (NSCs). NSCs obtained from E14.5 rat embryos proliferated as neurospheres when cultured in the presence of fibroblast growth factor (FGF). The neurospheres were then dissociated, cultured repeatedly for several passages to enrich NSCs, and subsequently FGF-removed from the culture medium to initiate differentiation. We found that both DHA and synaptamide increase Tuj1 (an immature neuronal marker)- and MAP2 (immature neuronal marker)-positive cells, however, synaptamide was far more potent than DHA. Synaptamide appears to be a principal mediator for DHA-induced neuronal differentiation of NSCs as the effect of DHA was further potentiated by a fatty acid amide hydrolase inhibitor while the production of synaptamide from DHA was identified in NSCs. Along with neuron markers MAP2 and Tuj1, phosphorylation of CREB and PKA was substantially increased when NSCs were treated with synaptamide. The synaptamide-mediated increase of the neuronal markers as well as CREB and PKA phosphorylation was significantly attenuated by a PKA inhibitor, suggesting that synaptamide induces neuronal differentiation via activation of CREB/PKA signaling pathways. To understand the role of DHA in neurodevelopment and function at the molecular level, we have previously established a strategy to examine the protein composition of synaptic plasma membrane (SPM) in relation to the DHA status. The SPM proteins were obtained from DHA-adequate and deficient mouse brains by subcellular fractionation and analyzed by nanoLC- MS/MS after SDS PAGE and tryptic digestion. For quantification, the SPM proteins from DHA-adequate and deficient mice were differentially labeled with 16O/ 18O water, combined and analyzed by nanoLC-MS/MS. Using this strategy, we found lower expression of several pre- and postsynaptic proteins involved in neurotransmission in the brains of DHA-deficient animals, including Synapsin 1, PSD-95, Syntaxin-1, Munc18-1, Glutamate receptors (GluR2, NR2), Dynamin-1 and Synaptic vesicle protein. We subsequently validated the finding with western blot analysis. The protein network analysis suggested involvement of CREB and caspase-3 pathways in the DHA-dependent modulation of the synaptic proteome. Reduction of specific synaptic proteins due to brain DHA-depletion may be an important mechanism for the suboptimal brain function associated with n-3 fatty acid deficiency. During this period, we have also established a label-free quantitative approach for simultaneous comparison of multiple proteomes using Progenesis software. The results using this approach were compared with the 16O/ 18O labeling method, and validated by Western Blot analysis using specific antibodies. Using these quantitative approaches, we investigated the effects of the DHA status on synaptic proteome in aging brains, the opposite spectrum of neurodevelopment. We found that 23 synaptic proteins decreased with aging in both DHA-adequate and deficient brains. Among these, Fodrin alpha, PSD-95, SV2b, VGluT1 and Septin-3 declined significantly more in the deficient brain in comparison to the adequate brain. These data suggest a positive role of DHA not only in neurodevelopment by promoting specific synaptic protein expression but also in age-associated cognitive decline through preserving synaptic proteins during aging. These approaches are now being employed to uncover the synaptic proteome changes caused by the ethanol exposure. During this period, we have also initiated the study to develop an approach for identification of novel metabolites derived from uniformly 13C-labeled substrates using HPLC/ESI-MS/MS and software-assisted peak finding routine. As a model system, we first characterized platelet and leukocyte metabolites formed from 13C- arachidonic acid (AA) and DHA. Based on their unique isotope profile of 13C-AA- or 13C DHA-derived metabolites together with the predicted mass differences from the corresponding 12C-AA- or DHA-counterparts, automatic peak finding algorithm is being developed. The applicability of this approach to other classes of bioactive metabolites will be subsequently tested with the help of uniformly labeled 13C-isotopes.
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