The 5-HT1A Receptor and Brain Development

描述（由申请人提供）：5-羟色胺1A受体（5-HT 1A-R）特异性配体在治疗抑郁症、焦虑症和许多相关症状中的功效促进了对这种神经递质受体的信号传导机制的研究。它在前脑中的缺失，特别是在小鼠出生后早期发育期间，导致焦虑水平升高。本申请人的研究组已经表明，在海马神经元衍生的细胞系中刺激该受体引起促分裂原活化蛋白激酶（MARK）同工酶Erk 1/2的活化，Erk 1/2又通过蛋白激酶C α（PKC α）发出信号，引起促凋亡蛋白胱天蛋白酶-3的抑制。此外，在出生后第6天（P6）的小鼠海马脑片培养物中，Erk 1/2的激活依赖于一种未鉴定的PKC同工酶，而在P15，PKCa在5-HTiA-R信号传导中被Erk 1/2激活。由于已知MAPK促进神经元细胞的分裂以及保护和成熟，因此先前的观察表明，5-HT 1A-R依赖性MAPK途径中的这种奇怪的开关可以在脑中神经元从最初的增殖爆发到后期的有丝分裂后成熟阶段的过渡中发挥重要作用。为了检验这一假设，将用5-HT 1A激动剂处理来自P6-P20的5-HT 1A-R（+/+）和5-HT 1A-R（-/-）小鼠的培养海马切片。免疫组织化学和蛋白质印迹法将用于研究刺激的关键蛋白质，如PKCa，Erk 1/2，和CREB，这可能会引起年龄依赖性的影响分裂（通过BrdU掺入测量）和成熟（通过MAP-2和突触素染色测量）的神经元细胞。将通过使用抗活性半胱天冬酶-3抗体记录半胱天冬酶-3抑制和脱氧核苷酸转移酶介导的dUTP缺口末端标记监测DNA片段化抑制来检测5-HT 1A-R-^Erk 1/2-^PKCa途径的可能抗细胞凋亡作用。新的Erk 1/2依赖性PKCa刺激表明Erk 1/2通过直接磷酸化Threatin-638激活PKCa。为了测试这种可能性，将在体外Erk 1/2测定中使用活化的Erk 1/2和纯的PKCa。最后，我们将测试在P6和P15之间观察到的5-HTiA-R-^Erkl/2途径中PKC等级的转换是否是由于PKC同工酶表达的年龄依赖性变化。该项目将描绘一种新的途径，可能在早期大脑发育中发挥关键作用。对这一途径的了解将有助于设计更好的治疗方法来对抗大脑发育障碍。

DESCRIPTION (provided by applicant): The efficacy of the serotonin 1A receptor (5-HTiA-R)-specific ligands in treating depression, anxiety, and many related symptoms has prompted studies into the signaling mechanisms of this neurotransmitter receptor. Its absence in the forebrain, specifically during early postnatal development of mice, results in elevated anxiety levels. The applicant's groups has shown that stimulation of this receptor in a hippocampal neuron-derived cell line causes activation of the mitogen activated protein kinase (MARK) isozymes Erk1/2, which in turn signal through protein kinase C alpha (PKCa) to cause inhibition of the proapoptotic protein caspase-3. Furthermore, in postnatal day-6 (P6) hippocampal slice cultures from mice, activation of Erk1/2 was dependent on an unidentified PKC isozyme, whereas at P15, PKCa was activated by Erk1/2 in 5-HTiA- R signaling. Since MAPK is known to promote division as well as protection and maturation of neuronal cells, preceding observations suggest that this curious switch in the 5-HTiA-R-dependent MAPK pathway could play an important role in the transition from the initial burst of proliferation to the later, post-mitotic stages of maturation of neurons in the brain. To test this hypothesis cultured hippocampal slices from 5-HT1A-R (+/+) and 5-HT1A-R (-/-) mice at P6-P20 will be treated with a 5-HT1A agonist. Immunohistochemistry and Western blotting will be used to study stimulation of key proteins, like PKCa, Erk1/2, and CREB, which could elicit age-dependent effects on division (measured by BrdU incorporation) and maturation (measured by MAP-2 and synaptophysin staining) of neuronal cells. Likely anti-apoptotic effects of the 5-HT1A-R-^Erk1/2-^PKCa pathway will be tested by using an anti-active caspase-3 antibody to record caspase-3 inhibition and deoxynucleotidyl transferase-mediated dUTP nick end labeling to monitor inhibition of DNA fragmentation. The novel Erk1/2-dependent PKCa stimulation suggests that Erk1/2 activate PKCa via direct phosphorylation at Threonine-638. To test this possibility activated Erk1/2 and pure PKCa will be used in an in vitro Erk1/2 assay. Finally, we will test if the observed switch in the hierarchy of PKC in the 5-HTiA- R-^Erkl/2 pathway between P6 and P15 was due to an age-dependent change in expression of PKC isozymes. This project will delineate a novel pathway that could play a key role in early brain development. Knowledge of this pathway will help in designing better therapies to combat developmental brain disorders.