Regulatory Role of Growth Hormone Secretagogue Receptor

描述（由申请人提供）：我们的目标是提供治疗干预，延长衰老期间的健康寿命。生长激素释放肽模拟物MK-0677的发现是衰老研究中的里程碑式发现。MK-0677使虚弱老年人的生长激素（GH）轴恢复活力，同时伴有骨密度、瘦体重和力量增加。似乎MK-0677挽救了内源性生长激素释放肽信号传导中的年龄依赖性缺陷;继续出现支持这一结论的证据。除了恢复调节GH轴的神经元外，GHS-R（ghrelin受体）定位于调节情绪和认知功能的神经元。多巴胺受体亚型-1（D1 R）在这些神经元中共表达，暗示ghrelin是多巴胺信号的调节剂。体外研究支持这一观点，并表明ghrelin通过形成GHS-R/D1 R异二聚体放大多巴胺诱导的cAMP积累。由于学习和记忆是通过增加cAMP在神经元中的积累来改善的，因此通过用生长素释放肽模拟物选择性地增加D1 R神经元中的cAMP来挽救受损的内源性生长素释放肽信号传导对老年人具有潜在的益处。将使用ghrelin-/-和Ghsr-/-小鼠测试ghrelin增强D1 R信号传导和形成GHS-R/D1 R异二聚体在体内发生并表现出行为效应的假设。Ghrelin具有神经保护特性，并且是解偶联蛋白-2（UCP 2）表达的正调节剂。UCP 2保护多巴胺神经元免受1-甲基-4-苯基-1，2，5，6-四氢吡啶（MPTP）诱导的氧化损伤和死亡。因此，还将检验ghrelin通过增加UCP 2表达来防止D1 R神经元损失的假设。GHS-R和D1 R之间的串扰导致共表达GHS-R和D1 R的选择性神经元中cAMP产生的放大是至关重要的。因此，所涉及的信号转导途径将被阐明，以测试这一假设，即cAMP积累的放大是由GHS-R偶联G1 i释放的23个亚基刺激的腺苷酸环化酶亚型-2活性的G1 s（来自D1 R偶联）增强介导的。具体目标：1.在野生型、生长素释放肽-/-和生长素释放肽受体敲除小鼠（Ghsr-/-）中测试生长素释放肽介导的扩增在体内发生以增强表达D1 R和GHS-R的神经元中的信号传导从而导致行为改变的假设;测试以下假设：在含有D1 R的神经元中GHS-R的表达提供了一种机制，通过该机制，生长素释放肽通过维持UCP 2表达来保护神经元免受氧化损伤; 3：定义由生长素释放肽受体（GHS-R）的共激活引起的多巴胺D1 R诱导的cAMP积累的放大的分子机制：a.）信号转导途径; B.）激动剂和G蛋白与GHS-R同源寡聚体相互作用的化学计量。这些研究具有近期的临床应用，因为口服活性长效生长激素释放肽模拟物的可用性被证明是安全和耐受性良好的。

DESCRIPTION (provided by applicant): Our objective is to provide therapeutic intervention that will extend health span during aging. The discovery of the ghrelin mimetic MK-0677 was a milestone discovery in aging research. MK-0677 rejuvenated the growth hormone (GH) axis in the frail elderly which was accompanied by increased bone density, lean mass and strength. It appears that MK-0677 rescued an age-dependent deficit in endogenous ghrelin signaling; evidence continues to emerge in support of this conclusion. Besides restoring neurons regulating the GH axis, the GHS-R (ghrelin receptor) localizes to neurons regulating mood and cognitive function. The dopamine receptor subtype-1 (D1R) is coexpressed in these neurons, implicating ghrelin as a modulator of dopamine signaling. In vitro studies support this notion and show that ghrelin amplifies dopamine-induced accumulation of cAMP by formation of GHS-R/D1R heterodimers. Because learning and memory are improved by augmentation of cAMP accumulation in neurons, rescuing impaired endogenous ghrelin signaling by selectively increasing cAMP in D1R neurons with ghrelin mimetics has potential benefit for the elderly. The hypothesis that ghrelin augmentation of D1R signaling and formation of GHS-R/D1R heterodimers occurs in vivo and manifests behavioral effects will be tested using ghrelin-/- and Ghsr-/- mice. Ghrelin has neuroprotective properties and is a positive regulator of uncoupling protein-2 (UCP2) expression. UCP2 protects dopamine neurons from oxidative damage and death induced by 1-methyl-4-phenyl-1,2,5,6 tetrahydropyridine (MPTP). Therefore, the hypothesis that ghrelin prevents loss of D1R neurons by increasing UCP2 expression will also be tested. Crosstalk between GHS-R and D1R causing amplification of cAMP production in selective neurons that coexpress GHS-R and D1R is of fundamental importance. Hence, the signal transduction pathways involved will be elucidated to test the hypothesis that amplification of cAMP accumulation is mediated by augmentation of G1s (derived from D1R coupling) stimulated adenylyl cyclase subtype-2 activity by 23 subunits liberated by GHS-R coupling to G1i. Specific Aims: 1.Test the hypothesis in wildtype, ghrelin-/- and ghrelin receptor knockout mice (Ghsr-/-) that ghrelin-mediated amplification occurs in vivo to enhance signaling in D1R and GHS-R expressing neurons resulting in behavioral changes; 2. Test the hypothesis that expression of GHS-R in D1R containing neurons provides a mechanism by which ghrelin protects neurons from oxidative damage by maintaining UCP2 expression; 3: Define the molecular mechanism of amplification of dopamine D1R-induced cAMP accumulation caused by co-activation of the ghrelin receptor (GHS-R): a.) signal transduction pathway; b.) stoichiometry of agonist and G-protein interaction with GHS-R homoligomers. These studies have near-term clinical applications because of the availability of orally active long-acting ghrelin mimetics shown to be safe and well tolerated.