Mechanism of Action for n-3 PUFA antidepressant properties

 描述（由申请人提供）：假设G蛋白（Gs β）与脂筏相关的程度是抑郁症和治疗反应的指标。最初的观察表明，这种生物特征是在死后组织中进行的，我们已经扩展了这项工作，在体内和体外的初步数据表明，筏定位的Gs，通过简单的洗涤剂提取，是抑郁症和抗抑郁反应的生物标志物。有迹象表明，n-3 PUFA具有抗抑郁活性或可以协同某些抗抑郁药物的作用。为了验证这一点，我们将使用神经胶质细胞系以及来自抑郁症受试者的淋巴母细胞，这些受试者要么对抗抑郁药有反应，要么对抗抑郁药无反应。这将为抗抑郁药物的反应提供一个预测模型，无论该药物是鱼油，西酞普兰，或一些组合。我们将通过测量在NIMH资助的临床试验期间收集的血细胞膜中Gs蛋白的脂筏分布来测试生物标志物的适用性，该临床试验显示n-3 PUFA增强抗抑郁反应。所提出的实验还试图将机制临床前研究与临床研究相结合，以提出和测试生物标志物。临床前工作旨在确定抗抑郁药± n-3 PUFA的体外治疗是否将Gs β转移到质膜的非筏部分，在那里它更有效地激活腺苷酸环化酶。我们认为，具有抗抑郁功效的化合物集中在富含胆固醇的膜结构域（脂筏），并破坏这些结构域内的Gs的锚定。一种可能的机制是直接修饰Gs β。这将通过膜成分的生化分离、光漂白后的荧光恢复（FRAP）和细胞成像来研究。由于大多数抗抑郁治疗（除了氯胺酮）在治疗功效之前需要时滞，并且这可以在细胞中复制，因此将使用荧光Gs蛋白融合蛋白真实的监测响应于抗抑郁剂的Gs蛋白的易位。如果n-3 PUFA单独或与SSRIs联合使用时显示出抗抑郁活性的这些生物学特征，则将通过检查n-3 PUFA（± SSRIs）抗抑郁疗效临床研究中采集的血液进行翻译。初始数据 结果显示，对n3 PUFA + SSRI的抗抑郁反应比单独的安慰剂或SSRI更大。最后，我们将测试，与生化和成像研究，淋巴母细胞，来自抑郁症患者与已知的反应（或缺乏）西酞普兰。一个目标是开发一个高内容的屏幕，可能会建议，前瞻性地，一个给定的潜在治疗的有效性。成功完成拟定的研究还将表明n-3 PUFA补充剂对抗抑郁治疗的有效性，可缩短治疗起效时间和/或降低抗抑郁药物的总剂量。他们还将为建立一种低成本的血液生物标志物铺平道路，用于在广泛的药物中检测抑郁症和抗抑郁药的疗效。

 DESCRIPTION (provided by applicant): It is hypothesized that the degree to which the G protein, Gs, is associated with lipid rafts is an indicator of both depression and therapeutic response. The original observations suggesting this biosignature were made in postmortem tissue, and we have extended this work with in vivo and in vitro preliminary data suggesting that raft localization of Gs, determined by simple detergent extraction, is a biomarker of both depression and antidepressant response. There are suggestions that n-3 PUFA either have antidepressant activity or can synergize the action of some antidepressant drugs. To test this, we will use a glial cell line as well as lymphoblasts from depressed subjects who were either antidepressant responsive or unresponsive. This should provide a predictive model for the response to an antidepressant agent, whether that agent is fish oil, citalopram, or some combination. We will test the applicability of the biomarker by measuring the lipid raft distributin of Gs in membranes from blood cells collected during an NIMH-funded clinical trial showing n-3 PUFA augmentation of antidepressant response. The proposed experiments also attempt to mesh mechanistic preclinical studies with a clinical study to propose and test a biomarker. The preclinical work seeks to determine whether in-vitro treatment with antidepressants ± n-3 PUFA shifts Gs into non-raft fractions of the plasma membrane, where it more effectively activates adenylyl cyclase. We suggest that a compound with antidepressant efficacy concentrates in cholesterol-rich membrane domains (lipid rafts) and disrupts the anchoring of Gs within those domains. One possible mechanism for this is direct modification of Gs. This will be studied by biochemical fractionation of membrane components, fluorescence recovery after photobleaching (FRAP) and by cellular imaging. Since most antidepressant therapy (save ketamine) requires a time lag prior to therapeutic efficacy and this can be replicated in cells, translocation of Gs in response to antidepressant agents will be monitored in real time using a fluorescent Gs fusion protein. Should n-3 PUFA show these biological hallmarks of antidepressant activity, alone or in combination with SSRIs , this will be translated by examining blood taken in a clinical study of the antidepressant efficacy of n-3 PUFA (± SSRIs). Initial data from this study show a greater antidepressant response to n3 PUFA + SSRI than to placebo or SSRI alone. Finally, we will test, with the biochemical and imaging studies, lymphoblasts, derived from depressed patients with known response (or lack thereof) to citalopram. One goal is to develop a high content screen that might suggest, prospectively, the effectiveness of a given potential therapy. Successful completion of the proposed studies will also indicate the usefulness of n-3 PUFA supplementation to antidepressant therapy for decreasing time of therapeutic onset and/or lowering overall antidepressant dose. They will also pave the way toward establishing a low-cost blood biomarker for both depression and antidepressant efficacy over a broad range of agents.