Alterations In Lipid Metabolism In The Nervous System By

神经系统脂质代谢的改变

• 批准号: 7317405
• 负责人: Hee-Yong Kim
• 金额: --
• 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/7317405
• 关键词: Alterations; Lipid; Metabolism; Nervous; System

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 continued our investigation on the signaling mechanisms underlying effects of DHA and ethanol on neuronal survival and development. We also continued to evaluate the role of DHA in development of hippocampal neurons. Analysis by immunocytochemistry indicated that DHA supplementation stimulated both dendrite growth and synapse formation in hippocampal neurons. The synapsin staining in control cells after 14 days in vitro is continuous throughout the axons, while DHA supplemented cells showed a discontinuous distribution of synapsin staining as puncta-like spots which are considered to represent synapse formation. Synapse formation at 14 DIV quantified by the number of synapsin positive dots in neurons showed that DHA significantly increased the total number of synapsis per area in comparison to the control. The effect of other fatty acids such as oleic and arachidonic are currently being evaluated. As we found that Akt was a target molecule for the DHA?s antiapoptotic effect, we examined Akt conformational changes during activation using chemical cross-linking and tandem mass spectrometry. Our cross-linking data revealed a novel Akt activation scheme where Akt undergoes stepwise inter-domain conformational changes in the sequential activation stages including membrane interaction, phosphorylation and substrate binding. During this report period, we focused on membrane-Akt interaction, especially the effect of membrane composition on Akt conformational changes and activation. Mass spectrometric analysis revealed that the presence of both PIP3 and PS in the membrane is required for Akt conformational changes needed for phosphorylation for its full activation. Consistently, in vitro Akt-phosphorylation at T308 and S473, by PDK and MAPKAP, respectively, is dependent on concentrations of both PIP3 and PS in the membrane. Moreover, PS dose-dependently enhanced the membrane interaction and phosphorylation of Akt at a given concentration of PIP3, suggesting a role of PS in complementing PIP3. Both mass spectrometric and biomolecular interaction analysis indicated that not only the PH domain but also the regulatory domain of Akt interacts with PS or PIP3. Membrane translocation of the GFP-tagged regulatory domain in response to IGF stimulation also supported the occurrence of regulatory domain-membrane interaction during Akt activation. The regulatory domain-membrane interaction was affected more prominently by PS in comparison to PIP3. In fact, the regulatory domain interacted with membranes containing PS but no PIP3 and enabled phosphorylation of S473 in the regulatory domain in vitro by MAPKAP. Our data represent the first demonstration of the distinctive molecular interaction between individual Akt domains and acidic phospholipids in cell membranes. The role of PS complementing PIP3 in Akt activation may be important in neuronal cell survival, particularly under adverse conditions where PIP3 production is compromised. This mechanism may provide an explanation for the PS-dependent neuronal survival affected by the DHA status and ethanol observed in our study.

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，我们继续研究DHA和乙醇对神经元生存和发育的信号传导机制。我们还继续评估DHA在海马神经元发展中的作用。 通过免疫细胞化学分析表明，补充DHA刺激了海马神经元中的树突生长和突触形成。体外14天后对照细胞中的突触蛋白染色在整个轴突中是连续的，而DHA补充细胞显示出突触素染色的不连续分布为untcta样斑点，这些斑点被认为代表突触的形成。通过神经元中突触蛋白阳性点数量量化的14 div的突触形成表明，与对照相比，DHA显着增加了每个区域的突触总数。目前正在评估其他脂肪酸（例如油酸和花生四环）的影响。 当我们发现AKT是DHA抗凋亡效应的靶标分子时，我们使用化学交叉链接和串联质谱法检查了在激活过程中Akt构象变化。我们的交联数据揭示了一种新型的AKT激活方案，其中AKT在顺序激活阶段进行逐步识别层间构象变化，包括膜相互作用，磷酸化和底物结合。在此报告期间，我们专注于膜 - Akt相互作用，尤其是膜组成对AKT构象变化和激活的影响。质谱分析表明，磷酸化所需的Akt构象变化需要磷酸化所需的全部激活所需的PIP3和PS的存在。一致地，PDK和MAPKAP分别在T308和S473处的体外AKT磷酸化取决于膜中PIP3和PS的浓度。此外，PS剂量依赖性地增强了在给定的PIP3下Akt的膜相互作用和磷酸化，这表明PS在补充PIP3中的作用。质谱和生物分子相互作用分析都表明，不仅pH结构域，而且AKT的调节结构域与PS或PIP3相互作用。响应于IGF刺激的GFP标记的调节结构域的膜易位也支持在AKT激活过程中发生调节域 - 膜相互作用。与PIP3相比，PS更显着地影响了调节结构域的相互作用。实际上，调节结构域与含有PS的膜相互作用，但没有PIP3并实现了MAPKAP调节域中S473的磷酸化。我们的数据代表了单个Akt结构域与细胞膜中酸性磷脂之间独特的分子相互作用的首次证明。 PS互补PIP3在AKT激活中的作用在神经元细胞存活中可能很重要，尤其是在损害PIP3产生的不利条件下。该机制可能会为受到DHA状态和我们研究中观察到的乙醇影响的PS依赖性神经元存活提供解释。