Previously, we demonstrated a novel molecular mechanism for Akt activation where Akt interaction with PS-binding residues, particularly in the regulatory domain, is critical for Akt phosphorylation at S473 by mTORC2. Phosphorylation at S473 is an important modulator of Akt activation, which can serve as a new target, different from well-recognized PIP3- or ATP-binding, for drug development. As an effort to identify selective Akt inhibitors based on this new target, we performed high throughput screening (HTS) for approximately the 400,000 MLSCN (Molecular Libraries Screening Center Network) compound collections in collaboration with NCGC. Among high probability hits from an automated HTS, only compounds that showed substantial and reproducible inhibition of Akt S473 phosphorylation confirmed by western blot analysis, without an effect on PI3K, PDK1, and SGK1 were selected. During this period, we continued to evaluate these selected compounds for cell viability, proliferation and Akt activation, and narrowed down the list of potential interest to two compounds. Further testing including a conformation-based assay is in progress to determine the best candidate for lead optimization. The small molecules that inhibit specifically Akt S473 phosphorylation/mTORC2 activity thus identified will not only serve as valuable research tools but also may have significant therapeutic potential with fewer side effects, especially for conditions involving hyperactive Akt signaling such as cancer and Alzheimers disease. Separately, we demonstrated during this period a new molecular mechanism for the detachment of Akt from the plasma membrane after activation. Specifically, we found that phosphorylation of T34 in the PH domain of Akt facilitates the dissociation of Akt from the membrane to gain access to its downstream targets, which is an important step in Akt signaling. Moreover, we found that T34 can be phosphorylated by PDK1 or growth factor stimulation. This molecular mechanism offers a potential new target for controlling the Akt-dependent cellular signaling processes. We have previously demonstrated that DHA metabolism to N-docosahexaenoylethanolamine (synaptamide) is a significant mechanism for neurogenesis, neuritogenesis, synaptogensis and glutamatergic synaptic activity. We also found that synaptamide induces cAMP accumulation, CREB phosphorylation, transcriptional activity of CRE and neurite growth in cortical neurons at low nM concentrations. The synaptamide-induced neurite growth in cortical neurons as well as neurogenic differentiation of NSCs was abolished by treatment with the adenyl cyclase (AC) inhibitor, SQ22,536, suggesting that a G protein-coupled receptor (GPCR) mediates the observed synaptamide-induced bioactivities. During this period, we found the binding of an orphan GPCR to synaptamide by using affinity purification and mass spectrometry-based proteomics approach followed by competitive binding using biotinylated synaptamide as the bait. Among long chain N-acylethanolamines, synaptamide specifically exhibited such binding, and induced cAMP-dependent neurogenic differentiation and neurite outgrowth. We also found that synaptamide promotes axon outgrowth and phosphorylation and expression of neurofilaments that are major structural components of axons in cortical neuronal cultures. Synaptamide regulated the expression of sonic hedgehog (shh) while cyclopamine, an inhibitor of the shh receptor Smoothened, also promoted axon outgrowth, indicating involvement of this signaling pathway in the synaptamide-mediated effect on axon growth. During this period, we also investigated the effects of ethanol on synaptamide-induced signaling processes leading to neurogenic differentiation, and found that ethanol impairs differentiation of neural stem cells and synaptamide can reverse the ethanol effects at least in part. Prenatal exposure to ethanol is known to interfere with embryonic and fetal development and to cause abnormal neurodevelopment. Consistent with our previously findings that synaptamide is a potent neurogenic factor, synaptamide significantly increased the number of MAP2 and Tuj-1 positive neurons with concomitant induction of PKA/CREB phosphorylation. We found that chronic ethanol (25-100 mM) exposure by treating NSCs with ethanol-containing media daily for 4 days dose-dependently decreased the number of MAP2 and Tuj-1 positive neurons and PKA/CREB phosphorylation. We also found that cellular cAMP production was decreased dose-dependently by chronic ethanol treatment. Ethanol-induced cAMP reduction persisted in the presence of the adenylate cyclase (AC) inhibitor (SQ22536) or non-selective or selective phosphodiesterase (PDE) inhibitors (caffeine or rollipram), indicating that ethanol acts on multiple targets. Ethanol significantly increased the cAMP-specific PDE4 level without affecting mRNA expression. Also, chronic ethanol reduced Gs alpha protein and mRNA expression, transiently, and decreased mRNA levels of AC7 and AC8 as well as GTPγS binding. In contrast, synaptamide exerted opposite effects on cAMP production, PDE4 protein level and Gs alpha and AC expression. These results suggest that ethanol exposure impairs neuronal differentiation of NSCs while synaptamide ameliorates the adverse impact of ethanol by counter-affecting shared targets in G-protein coupled receptor (GPCR) signaling including Gs alpha, AC and PDE4. We are in the process of extending these findings to an in vivo model to confirm the significance of effects of ethanol and synaptamide on neurogenic differentiation during development. We also found that NSCs grown under chronic ethanol conditions showed no significant changes in synaptamide production from DHA. FAAH activity measured by monitoring the degradation of labeled d4-synaptamide showed no significant effect of ethanol on the rate of synaptamide hydrolysis, either. We have also extended our study to explore the role of synaptamide in neuroinflammation during this period. We found that synaptamide at low nM concentrations inhibits LPS-induced TNFαexpression in the BV-2 microglia cell line and primary microglia. Furthermore, synaptamide increased intracellular cAMP levels and inhibited LPS-induced ROS production in BV2 cells. We are now investigating the mechanisms whereby synaptamide regulates inflammatory signaling in the brain using primary microglia as a model system.
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