We have previously found that docosahexaenoic acid (DHA, 22:6n-3), a highly polyunsaturated n-3 fatty acid enriched in neuronal tissues, promotes the accumulation of phosphatidylserine (PS) and prevents apoptotic cell death in a PS- and PI3 kinase-dependent manner in neuronal cells. We have also demonstrated that n-3 fatty acid deficiency or chronic ethanol exposure markedly decreased the PS content specifically in neuronal cells, adversely affecting neuronal survival. We have also established that DHA promotes neurite outgrowth in hippocampal neurons, suggesting a role of DHA in neuronal differentiation. During this period, we investigated the signaling mechanisms underlying effects of DHA and ethanol on neuronal survival and development at the cellular and molecular level. We also extended our investigation on the involvement of PS in neuronal survival to an animal transient ischemia model. ? As we found previously that Akt-membrane interaction is a target for the DHAs antiapoptotic effect, we further examined molecular mechanism of Akt activation. Mass spectrometric probing of Akt conformation along with bio molecular interaction analyses based on surface plasmon resonance revealed that PS and PIP3 jointly regulate the Akt-membrane interaction required for inter-domain conformational changes of Akt for phosphorylation. At a given PIP3 concentration, the extent of interaction between membrane and the PH or regulatory (RD) domain as well as Akt phosphorylation at T308 and S473 was PS-dependent. Remarkably, considerable binding occurred between RD and membrane PS, enabling Akt phosphorylation at S473 by a putative PDK2 in vitro even without PIP3. Increasing PS in neuronal cells by supplementing neuronal cells with DHA facilitated Akt translocation and phosphorylation upon IGF stimulation. Our data demonstrated that PS-Akt interaction is an important modulatory mechanism in Akt signaling. The role of PS complementing PIP3 in Akt activation may be an important mechanism supporting neuronal survival, particularly under adverse conditions where PIP3 production is limited. This mechanism may provide an explanation for the PS-dependent neuronal survival affected by the DHA status and ethanol observed in our studies.? The PS localized in the cytoplasmic face of cellular membranes is particularly important since it can offer the site of interaction for cytosolic signaling molecules such as Akt and PKC. Therefore, we demonstrated the intracellular PS distribution in neuronal cells during this period. Neuroblastoma cells and hippocampal neurons expressing GFP-AnnexinV were stimulated with a calcium ionophore and localization of GFP-AnnexinV was monitored by fluorescence microscopy. Initially, GFP-AnnexinV distributed evenly in the cytosol and nucleus. Raising the intracellular calcium level with ionomycin induced translocation of cytoplasmic GFP-AnnexinV to the plasma membrane but not to the nuclear membrane, indicating that PS distributes in the cytoplasmic side of the plasma membrane. Nuclear GFP-AnnexinV subsequently translocated to the nuclear membrane, indicating PS localization in the nuclear envelope. GFP-AnnexinV also localized in a juxtanuclear organelle that was identified as the recycling endosome. However, minimal fluorescence was detected in any other subcellular organelles including mitochondria, endoplasmic reticulum, Golgi complex and lysosomes, strongly suggesting that PS distribution in the cytoplasmic face in these organelles is negligible. Similarly in hippocampal primary neurons, PS distributed in the inner leaflet of plasma membranes of cell body and dendrites, and in the nuclear envelope. To our knowledge, this is the first demonstration of intracellular PS localization in living cells, providing an insight for specific sites of PS interaction with soluble proteins involved in signaling processes.? It is well known that the hippocampal CA1 region is most susceptible to cerebral ischemia in rodent models, with significant implication in hippocampus-related functional deficits. To provide an insight into biochemical mechanisms underlying CA1-selective neuronal cell death associated with ischemia, the difference of phospholipid profile in rat CA1 and CA3 regions was evaluated. Total PS and phosphatidylethanolamine (PE) in CA1 region were 20% (p=0.02) and 14% (p=0.02) higher than those in CA3 region, respectively. When the transient cerebral ischemia was induced in rats via bilateral occlusion of carotid arteries for 20 minutes followed by reperfusion, about 79% of pyramidal cell death occurred in the CA1 region with significant inter-neuronal atrophy. Total PS and PE contents were decreased by 24% (p=0.002) and 33% (p=0.0001) in CA1 region compared to sham surgeries, respectively. The decrease was evenly distributed in most molecular species of PS and PE regardless of unsaturation status, suggesting that selective activation of PLA2, which prefers polyunsaturate release, may not be directly involved in cell death in CA1. Despite the high abundance of DHA and its susceptibility to oxidation, DHA containing species were not lost disproportionately but retained in PS or PE. In CA3 region, where no cell death was detected, no significant differences were observed in total PS and PE, either. These data suggested that the high concentration of PS in the hippocampal CA1 may play an important role in supporting neuronal survival in this susceptible region, although the reason for the vulnerability of CA1 to ischemia still remains to be answered.

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National Institute on Alcohol Abuse and Alcoholism (NIAAA)
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Huang, Bill X; Akbar, Mohammed; Kevala, Karl et al. (2011) Phosphatidylserine is a critical modulator for Akt activation. J Cell Biol 192:979-92
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Huang, Bill X; Kim, Hee-Yong (2006) Interdomain conformational changes in Akt activation revealed by chemical cross-linking and tandem mass spectrometry. Mol Cell Proteomics 5:1045-53
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Huang, Bill X; Dass, Chhabil; Kim, Hee-Yong (2005) Probing conformational changes of human serum albumin due to unsaturated fatty acid binding by chemical cross-linking and mass spectrometry. Biochem J 387:695-702

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