Alterations In Lipid Metabolism In The Nervous System By Ethanol

乙醇改变神经系统脂质代谢

• 批准号: 7591934
• 负责人: Hee-Yong Kim
• 金额: $ 201.71万
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7591934
• 关键词: 1-Phosphatidylinositol 3-Kinase; Affect; Animals; Apoptotic; Atrophic; Bilateral; Binding; Biochemical; Biological; Calcium; Carotid Arteries; Cell Death; Cell Membrane Structures; Cell Nucleus; Cell membrane; Cells; Cellular Membrane; Cerebral Ischemia; Chronic; Complement; Condition; Cytosol; Data; Dendrites; Development; Docosahexaenoic Acids; Endoplasmic Reticulum; Endosomes; Ethanol; Face; Fluorescence; Fluorescence Microscopy; Goals; Golgi Apparatus; Hippocampus (Brain); In Vitro; Investigation; Ionomycin; Ionophores; Ischemia; Knowledge; Life; Lipids; Localized; Lysosomes; Mass Spectrum Analysis; Membrane; Membrane Structure and Function; Metabolic; Mitochondria; Modeling; Modification; Molecular; Molecular Conformation; Monitor; Nervous system structure; Neurites; Neuroblastoma; Neuronal Differentiation; Neurons; Nuclear; Nuclear Envelope; Omega-3 Fatty Acids; Organelles; Personal Satisfaction; Phosphatidylethanolamine; Phosphatidylserines; Phosphoinositide-3-Kinase, Catalytic, Gamma Polypeptide; Phospholipase A2; Phospholipids; Phosphorylation; Physiological reperfusion; Play; Polyunsaturated Fatty Acids; Predisposition; Production; Proteins; Pyramidal Cells; Rattus; Recycling; Reperfusion Therapy; Research; Rodent Model; Role; Side; Signal Transduction; Signaling Molecule; Site; Somatomedins; Surface Plasmon Resonance; Techniques; Tissues; Transient Cerebral Ischemia; alcohol exposure; base; computerized data processing; insight; interest; lipid metabolism; neuron loss; neuronal cell body; neuronal survival; oxidation; phosphatidylethanolamine; prevent; sham surgery

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.

我们以前已经发现，二十六六烯酸（DHA，22：6n-3）是一种高度多不饱和的N-3脂肪酸，富含神经元组织，促进磷脂酰丝氨酸（PS）的积累，并以PS-和PI3激酶依赖性方式以神经元的方式预防凋亡细胞死亡。我们还证明，N-3脂肪酸缺乏或慢性乙醇暴露显着降低了神经元细胞中的PS含量，对神经元存活产生了不利影响。 我们还确定，DHA促进了海马神经元中神经突的生长，这表明DHA在神经元分化中的作用。 在此期间，我们研究了DHA和乙醇对细胞和分子水平上神经元存活和发育的潜在信号传导机制。 我们还扩展了对PS在神经元存活中的参与到动物瞬态缺血模型的研究。 正如我们先前发现Akt膜相互作用是DHAS抗凋亡效应的靶标，我们进一步研究了Akt激活的分子机制。 基于表面等离子体共振的Akt构象的质谱探测以及生物分子相互作用分析表明，PS和PIP3共同调节AKT AKT磷酸化的AKT构象变化所需的Akt-Mmbrane相互作用。在给定的PIP3浓度下，膜与pH或调控（RD）结构域之间的相互作用程度以及T308和S473处的AKT磷酸化依赖性PS依赖性。 值得注意的是，RD和膜PS之间发生了相当大的结合，即使没有PIP3，假定的PDK2也可以在S473处进行Akt磷酸化。 通过补充DHA的神经元细胞来增加神经元细胞中的PS，在IGF刺激后促进了Akt易位和磷酸化。 我们的数据表明，PS-AKT相互作用是AKT信号传导中重要的调节机制。 PS互补PIP3在Akt激活中的作用可能是支持神经元存活的重要机制，尤其是在PIP3产生有限的不利条件下。 这种机制可以为我们的研究中观察到的DHA状态和乙醇影响的PS依赖性神经元存活提供了解释。 在细胞膜的细胞质面中定位的PS尤其重要，因为它可以为胞质信号分子（例如Akt和PKC）提供相互作用。 因此，在此期间，我们证明了神经元细胞中细胞内PS的分布。 用钙离子载体刺激表达GFP-Annexinv的神经母细胞瘤细胞和海马神经元，并通过荧光显微镜监测GFP-ANNEXINV的定位。 最初，GFP-Annexinv均匀分布在细胞质和核中。 用离子霉素诱导细胞质GFP-Annexinv易位的细胞内钙水平，但不向核膜易位，表明PS在质膜的细胞质侧分布。 核GFP-ANNEXINV随后转移到核膜上，表明PS在核包膜中定位。 GFP-Annexinv也位于被确定为回收内体的近核细胞器中。然而，在任何其他亚细胞细胞器中都检测到最小的荧光，包括线粒体，内质网，高尔基体复合物和溶酶体，强烈表明这些细胞器中细胞质面部的PS分布忽略不计。同样，在海马原发性神经元中，PS分布在细胞体和树突的质膜内部小叶中，以及核包膜中。据我们所知，这是活细胞中细胞内PS定位的首次演示，为PS相互作用与参与信号过程的可溶性蛋白的特定位点提供了见解。 众所周知，海马CA1区域在啮齿动物模型中最容易受到脑缺血的影响，对海马相关的功能缺陷有显着影响。为了洞悉与缺血相关的CA1选择性神经元细胞死亡的生化机制，评估了大鼠CA1和CA3区域磷脂谱的差异。 CA1区域中的总PS和磷脂酰乙醇胺（PE）分别为20％（P = 0.02）和14％（P = 0.02），分别高于CA3区域。当通过双侧颈动脉闭塞20分钟，然后再灌注诱导大鼠瞬时脑缺血时，大约79％的金字塔细胞死亡发生在CA1区域中，具有明显的神经性萎缩。与假手术相比，CA1区域的总PS和PE含量分别降低了24％（P = 0.002）和33％（P = 0.0001）。无论不饱和状态如何，大多数PS和PE的分子种类均匀分布，这表明PLA2的选择性激活（偏爱多不饱和释放）可能与CA1中的细胞死亡不直接涉及。尽管DHA的丰度及其对氧化的敏感性，但含有DHA的物种的敏感性并未损失不成比例，而是保留在PS或PE中。 在未检测到细胞死亡的CA3区域，在总PS和PE中也未观察到显着差异。这些数据表明，海马CA1中PS的高浓度在支持该易感区域的神经元存活中可能起重要作用，尽管CA1脆弱性与缺血的原因仍然有待回答。