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，以延长老年人的健康寿命。模拟生长素MK-0677的发现是衰老研究的一个里程碑式的发现。MK-0677使体弱老年人的生长激素（GH）轴恢复活力，并伴有骨密度、瘦质量和力量的增加。MK-0677似乎挽救了内源性胃饥饿素信号的年龄依赖性缺陷；支持这一结论的证据不断出现。除了恢复调节生长激素轴的神经元外，GHS-R（生长素受体）还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，暗示胃饥饿素是多巴胺信号的调节剂。体外研究支持这一观点，并表明饥饿素通过形成GHS-R/D1R异源二聚体，放大了多巴胺诱导的cAMP积累。由于学习和记忆可以通过增加神经元中cAMP的积累而改善，因此通过使用ghrelin模拟物选择性地增加D1R神经元中的cAMP来挽救受损的内源性ghrelin信号传导对老年人有潜在的益处。ghrelin增强D1R信号和GHS-R/D1R异源二聚体形成的假设发生在体内，并表现出行为效应，将在ghrelin-/-和Ghsr-/-小鼠中进行验证。Ghrelin具有神经保护作用，是解偶联蛋白2 （uncoupling protein-2, UCP2）表达的正调节因子。UCP2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，ghrelin通过增加UCP2表达来防止D1R神经元丢失的假设也将得到验证。GHS-R和D1R之间的串扰在选择性共表达GHS-R和D1R的神经元中引起cAMP产生的扩增是至关重要的。因此，我们将阐明所涉及的信号转导途径，以验证GHS-R与G1i偶联释放的23个亚基，通过增强G1s（源于D1R偶联）刺激的腺苷酸环化酶亚型-2活性来介导cAMP积累扩增的假设。具体目标：1；在野生型、ghrelin-/-和ghrelin受体敲除小鼠（Ghsr-/-）中验证ghrelin介导的体内扩增，以增强D1R和GHS-R表达神经元的信号传导，从而导致行为改变的假设；2. 验证含有D1R的神经元中GHS-R的表达提供了ghrelin通过维持UCP2表达来保护神经元免受氧化损伤的机制；3：明确ghrelin受体（GHS-R）共激活引起多巴胺d1r诱导cAMP积累扩增的分子机制：a)信号转导途径；b)激动剂和g蛋白与GHS-R同聚物相互作用的化学计量学。这些研究具有近期的临床应用，因为口服活性长效胃促生长素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.