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，以延长衰老期间的健康跨度。Ghrelin模拟物MK-0677的发现是衰老研究中的一个里程碑发现。MK-0677使虚弱老年人的生长激素(GH)轴恢复活力，并伴随着骨密度、瘦质量和强度的增加。MK-0677似乎挽救了内源性Ghrelin信号与年龄相关的缺陷；支持这一结论的证据继续涌现。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(D1R)在这些神经元中共表达，暗示Ghrelin是多巴胺信号的调制器。体外研究支持这一观点，并表明Ghrelin通过形成GHS-R/D1R异源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神经元中cAMP的积累来改善的，因此利用Ghrelin类似物选择性地增加D1R神经元中的cAMP来挽救受损的内源性Ghrelin信号对老年人有潜在的好处。Ghrelin增强D1R信号和形成GHS-R/D1R异二聚体的假说在体内发生，并表现出行为效应，将在Ghrelin-/-和GHSR-/-小鼠身上进行验证。Ghrelin具有神经保护作用，是解偶联蛋白-2(UCP2)表达的正调控因子。UCP2对1-甲基-4-苯基-1，2，5，6-四氢吡啶(MPTP)诱导的多巴胺神经元氧化损伤和死亡具有保护作用。因此，Ghrelin通过增加UCP2表达来防止D1R神经元丢失的假设也将得到检验。GHS-R和D1R之间的串扰导致共表达GHS-R和D1R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用来检验这一假说，即cAMP积累的放大是通过G1S(源于D1R偶联)刺激的腺苷环化酶亚型2活性增加GHS-R偶联释放到G1I的23个亚基来介导的。具体目的：1.在野生型、Ghrelin-/-和Ghrelin受体基因敲除小鼠(GHSR-/-)中测试假设，即Ghrelin介导的扩增在体内发生，以增强表达Ghs-R的神经元中的信号，导致行为变化；2.测试含有D1R的神经元中GHS-R的表达提供了一种机制，即Ghrelin通过维持UCP2的表达来保护神经元免受氧化损伤；3：确定Ghrelin共激活Ghrelin受体(GHS-R)导致多巴胺D1R扩增导致cAMP积累的分子机制：a.)B.信号转导途径；激动剂的化学计量和G蛋白与GHS-R同源低聚物的相互作用。这些研究具有近期的临床应用，因为口服有效的长效Ghrelin模拟物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.