Lipid Metabolism Alteration by EtOH In Nervous System

乙醇改变神经系统中的脂质代谢

• 批准号: 7146658
• 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/7146658
• 关键词: albumins; arachidonate; brain metabolism; conformation; developmental neurobiology; developmental nutrition; dietary lipid; eicosanoid metabolism; electrospray ionization mass spectrometry; enzyme activity; enzyme structure; ethanol; fatty acid metabolism; intracellular transport; laboratory rat; neuroprotectants; nutrient drug interaction; nutrition related tag; omega 3 fatty acid; pharmacokinetics; phosphatidylcholines; phosphatidylinositol 3 kinase; phosphatidylserines; tissue /cell culture; unsaturated fatty acids

We have previously found that docosahexaenoic acid (DHA, 22:6n-3), a highly polyunsaturated n-3 fatty acid, 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 where DHA is highly enriched. We have established that docosapentaenoic acid (DPA, 22:5n-6), which replaces DHA during n-3 deficiency, is not as effective as DHA in accumulating PS or preventing apoptotic cell death, thus adversely affecting neuronal survival under n-3 deficiency conditions. Similarly, long term ethanol exposure during prenatal and developmental period depletes PS from neuronal membranes through the inhibition of PS biosynthetic activities. During this period, we continued our investigation of the biochemical and signaling mechanisms underlying effects of DHA and ethanol on cell survival. We found that PS levels were uniquely modulated by the DHA status and this DHA effect was observed only in neuronal cells. The protection against apoptotic cell death induced by serum starvation was sensitive to the PS level. Substitution of DHA with DPA resulted in less accumulation of PS and accordingly less effective in supporting neuronal survival in hippocampal cultures. Prenatal ethanol exposure also decreased PS contents, especially DHA containing PS species, in various brain regions including cerebrum, cerebellum, olfactory and hippocampus. In agreement with PS-dependent survival mechanism that we have previously reported, hippocampal neurons from ethanol treated rats also showed significantly increased susceptibility to apoptosis in cultures. These data suggest that cell survival adversely affected by prenatal ethanol exposure may contribute to neurological dysfunction associated with fetal alcohol syndrome. For mechanistic understanding of our previous finding that DHA uniquely promotes hippocampal differentiation, the role of RXR, for which DHA has been shown to be an endogenous ligand, was investigated by detecting the transcriptional activation of RXR using the dual luciferase reporter assay. We found that activation of RXR required much higher DHA concentration (over 15?M) in Neuro-2A cells than 1.5 ??M at which the promotion of neurite outgrowth was observed. To test the effect of DHA directly in hippocampal primary cells, efforts are being made to improve transfection efficiency in primary cultures. 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. The crystal structure of full-length Akt, nor its conformational changes during activation has not been demonstrated so far. We identified in inactive Akt seven cross-linked lysine pairs. Among them, two inter-domain cross-linked pairs, K30(PH)-K389(kinase) and K284(kinase)-K426(regulatory), allowed us to monitor inter-domain Akt conformational changes during activation. Upon interacting with a model membrane containing PS/PE/PC/PIP3, these cross-linked pairs were no long observed. When Akt was activated by phosphorylation at T308 and S473, K30-K389 reappeared while K284-K426 was still missing. When the active Akt was bound to the substrate and ATP K284-K426 reappeared. These results indicated that inactive Akt exists in a folded conformation with the PH and regulatory domain covering part of the kinase domain. Upon membrane interaction, the PH and regulatory domains move away from the kinase domain, presumably exposing T308 and S473 for phosphorylation. When phosphorylated, the PH domain folds back again while the regulatory domain remains open, allowing substrate entry. The regulatory domain closes after the substrate and ATP binding. Our data provide not only the first demonstration of distinctive inter-domain conformational changes of Akt at each step of activation processes, but also a strategy for further investigations on Akt-membrane, Akt-protein and/or Akt-drug interactions in solution.

我们以前已经发现，高度多不饱和的N-3脂肪酸（DHA，22：6n-3）（DHA，22：6n-3）促进磷脂酰丝氨酸（PS）的积累，并防止神经元细胞中PS-和PI3激酶依赖性方式的凋亡细胞死亡。我们还证明，N-3脂肪酸缺乏症或慢性乙醇暴露显着降低了PS含量在高度富集的神经元细胞中特别降低了PS含量。我们已经确定，在N-3缺乏期间取代DHA的Docosapentaenoic酸（DPA，22：5N-6）在积累PS或防止凋亡细胞死亡方面不如DHA有效，从而在N-3缺乏症条件下对神经元存活产生不利影响。同样，产前和发育期间的长期乙醇暴露通过抑制PS生物合成活性从神经元膜中耗尽了PS。在此期间，我们继续研究DHA和乙醇对细胞存活的生化和信号传导机制。 我们发现PS水平是由DHA状态唯一调节的，并且仅在神经元细胞中观察到这种DHA效应。血清饥饿诱导的凋亡细胞死亡的保护对PS水平敏感。 DHA用DPA替换导致PS的积累较少，因此在支持海马培养物中的神经元存活方面的有效性较低。在包括大脑，小脑，嗅觉和海马在内的各种大脑区域，产前乙醇暴露还降低了PS含量，尤其是含有PS物种的PS含量。与我们先前报道的PS依赖性生存机制一致，来自乙醇处理大鼠的海马神经元还显示出对培养物凋亡的易感性显着提高。这些数据表明，受产前乙醇暴露不利影响的细胞生存可能导致与胎儿酒精综合征相关的神经功能障碍。为了理解我们以前关于DHA独特促进海马分化的发现的理解，通过使用双荧光素酶报告基因测定法检测RXR的转录激活，研究了DHA的作用，DHA的作用已被证明是内源配体。我们发现，在神经2a细胞中，RXR的激活比观察到神经突生长的1.5？m所需的DHA浓度要高得多（超过15？m）。为了直接在海马原代细胞中测试DHA的作用，正在努力提高原发性培养物的转染效率。 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。到目前为止，尚未证明全长Akt的晶体结构，也没有在激活过程中的构象变化。我们在非活性AKT中鉴定出七个交联的赖氨酸对。其中，两个域间交联对，K30（pH）-K389（激酶）和K284（激酶）-K426（调节），使我们能够监视激活过程中域间AKT AKT构象变化。与含有PS/PE/PC/PIP3的模型膜相互作用后，这些交联对很长。当T308和S473通过磷酸化激活AKT时，K30-K389再次出现，而K284-K426仍缺失。当活动AKT与基板结合时，ATP K284-K426重新出现。这些结果表明，与pH和调节域覆盖了激酶结构域的一部分的折叠构象中存在非活性AKT。膜相互作用后，pH和调节域远离激酶结构域，大概暴露于T308和S473进行磷酸化。当磷酸化时，pH结构域在调节结构域保持打开时再次折回，从而可以进入底物。调节域在基板和ATP结合后关闭。我们的数据不仅提供了AKT在激活过程的每个步骤中的独特域间构象变化的首次演示，而且还提供了进一步研究Akt-膜，AKT-蛋白质和/或AKT-Drug相互作用的策略。