Signal Transduction in Depression

描述（由申请人提供）：重度抑郁症（MDD）是一种常见且严重的疾病，但其生物学基础尚不明确。信号转导机制，包括关键的酶，如蛋白激酶A （PKA）和C (PKC)，似乎在MDD中发生了改变，特别是在忧郁症（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的活性均平行降低；(2) [3H]环AMP与PKA和[3H]PDBU与PKC结合减少；(3)特异性激酶蛋白亚型水平降低。同样，我们最近也证明了pkc连接的5-羟色胺2A （5-HT2A）受体在该人群中与Gq蛋白明显解耦。这个项目将评估这些发现的潜在原因和后果。我们使用了培养的人类成纤维细胞（FB）模型，但现在可以将研究扩展到来自特征明确的抑郁自杀受害者和正常对照（我们也有初步数据）的死后脑组织（PMB）。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) [3H]环AMP与PKA结合，[3H]二丁酸磷与PKC结合；并测试(3)伴随降低激酶的结合和活性的可能性。我们还将在三个研究组中进行以下对比：(4)PMB和FB中PKA和PKC蛋白亚型的蛋白质和mRNA水平；(5)脉冲追踪免疫沉淀对FB中PKA和PKC亚型的降解率；(6)通过抑制磷酸二酯酶IV和蛋白磷酸酶2A比较激酶活性的关键调控因子的作用，并评估其mRNA和蛋白表达。我们还将通过对比两组之间的差异来研究MEL中5-HT2A受体的明显解耦：(7)在FB和PMB中使用特定的5-HT2A激动剂和替代gq偶联受体激动剂后，PI水解；(8) PMB中5-HT2A受体激活后GTPyS的掺入；(9) PMB中激动剂和拮抗剂的差异结合；（10） FB和PMB中5-HT2A激动剂和替代gq偶联受体激动剂激活后CREB-P的形成。这些研究可能揭示抑郁症的重要细胞介质，并为改善抑郁症提供新的靶点。

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.