Signal Transduction in Depression

描述（由申请人提供）：重度抑郁症（MDD）是一种常见的严重疾病，但生物学基础仍然难以捉摸。信号转导机制，包括关键酶，如蛋白激酶A（PKA）和C（PKC），似乎在MDD中发生改变，特别是在胆固醇（MEL）亚型中。我们的初步数据显示：（1）相对于对照组，MDD、MEL亚型（但非MEL）中PKA和PKC活性平行降低;（2）[3 H]环AMP与PKA结合和[3 H]PDBU与PKC结合的同时降低;（3）特异性激酶蛋白亚型水平降低。此外，我们最近已经证明了一个明显的解偶联的PKC-连接的血清素2A（5-HT 2A）受体从Gq蛋白在这一人群中。该项目将评估调查结果的潜在原因和后果。我们已经使用了培养的人成纤维细胞（FB）模型，但是现在可以将研究扩展到来自良好表征的抑郁自杀受害者和正常对照的死后脑组织（PMB）（我们也有初步数据）。我们建议比较MEL、非MEL和对照组的PMB：（1）PKA和PKC激活后CREB磷酸化水平;（2）[3 H]环AMP与PKA和[3 H]佛波醇二丁酸酯与PKC的结合;和测试（3）激酶结合和活性同时降低的可能性。我们还将在三个研究组中对比以下内容：（4）PMB和FB中PKA和PKC蛋白亚型的蛋白质和mRNA水平;（5）FB中通过脉冲追踪免疫沉淀法降解PKA和PKC亚型的速率;（6）通过抑制磷酸二酯酶IV和蛋白磷酸酶2A并评估其mRNA和蛋白质表达来比较激酶活性的关键调节剂的作用。我们还将通过对比以下组来检查MEL中5-HT 2A受体的表观解偶联：（7）FB和PMB中特异性5-HT 2A激动剂和替代性Gq偶联受体激动剂后的PI水解;（8）PMB中5-HT 2A受体活化后的GTP γ S掺入;（9）PMB中激动剂和拮抗剂结合的差异;（10）在FB和PMB中用5-HT 2A激动剂和替代性Gq偶联受体激动剂活化后的CREB-P形成。这些研究可能揭示抑郁症的重要细胞介质，并提供新的改善目标。

DESCRIPTION (provided by applicant): Major depression (MDD) is a common and serious disorder, but the biological basis remains elusive. Signal transduction mechanisms, including critical enzymes such as protein kinases A (PKA) and C (PKC), appear to be altered in MDD, particularly in the melancholic (MEL) subtype. Our preliminary data show: (1) A parallel reduction in the activity of both PKA and PKC in MDD, MEL subtype (but not in the non-melancholies [non- MEL]) relative to controls; (2) A concomitant decrease in [3H]cyclic AMP binding to PKA and [3H]PDBU binding to PKC; (3) Reduced levels of specific kinase protein isoforms. As well, we have recently demonstrated an apparent uncoupling of the PKC-linked serotonin 2A (5-HT2A) receptors from Gq proteins in this population. This project will evaluate potential causes and consequences of the findings. We have used a cultured human fibroblast (FB) model, but now can extend the investigation to post-mortem brain tissue (PMB) from well- characterized depressed suicide victims and normal controls (for which we also have preliminary data). We propose to compare in PMB from persons identified as MEL, non-MEL, and controls: (1) The level of CREB phosphorylation after PKA and PKC activation; (2) The binding of [3H] cyclic AMP to PKA and [3H]phorbol dibutyrate to PKC; and test (3) The possibility of a concomitant reduction of binding and activity of kinases. We also will contrast the following in the three study groups: (4) Protein and mRNA levels of PKA and PKC protein isoforms in PMB and FB; (5) Rates of degradation of PKA and PKC isoforms by pulse-chase immuno- precipitation in FB; (6) The comparative effects of key regulators of kinase activity by the inhibition of phosphodiesterase IV and protein phosphatase 2A and evaluating their mRNA and protein expression. We also will examine the apparent uncoupling of 5-HT2A receptors in MEL by contrasting between groups: (7) PI hydrolysis after a specific 5-HT2A agonist and alternative Gq-coupled receptor agonists in FB and PMB; (8) GTPyS incorporation following activation of 5-HT2A receptors in PMB; (9) Differential agonist and antagonist binding in PMB; (10) CREB-P formation after activation with a 5-HT2A agonist and alternative Gq-coupled receptor agonist in FB and PMB. These studies may shed light on important cellular mediators of depression and provide new targets for amelioration.