The 5-HT1A Receptor and Brain Development

描述(由申请人提供)：5-羟色胺1A受体(5-HTiA-R)特异性配体在治疗抑郁、焦虑和许多相关症状方面的有效性促使人们对这种神经递质受体的信号机制进行研究。它在前脑中的缺失，特别是在小鼠出生后的早期发育过程中，会导致焦虑水平升高。申请人的团队已经证明，刺激海马神经元衍生细胞系中的这种受体会导致丝裂原激活蛋白激酶(MARK)同工酶ERK1/2的激活，而ERK1/2又通过蛋白激酶Cα(PKCA)发出信号，导致抑制促凋亡蛋白caspase-3。此外，在出生后6天(P6)的小鼠海马片培养中，ERK1/2的激活依赖于一个未知的PKC同工酶，而在P15，5-HTiA-R信号通路中的PKCA是由ERK1/2激活的。由于MAPK被认为可以促进神经细胞的分裂以及保护和成熟，先前的观察表明，5-HTiA-R依赖的MAPK通路中的这种奇怪的开关可能在大脑中神经元从最初的增殖爆发过渡到后来的有丝分裂后成熟阶段发挥重要作用。为了验证这一假设，在P6-P20培养的5-HT1A-R(+/+)和5-HT1A-R(-/-)小鼠的海马片将用5-HT1A激动剂处理。免疫组织化学和Western blotting将用于研究对PKCA、ERK1/2和CREB等关键蛋白的刺激，这些蛋白可能对神经细胞的分裂(通过BrdU掺入)和成熟(通过MAP-2和突触素染色)产生年龄依赖性的影响。5-HT1A-R-ERK1/2-PKCA通路可能的抗凋亡作用将通过使用抗caspase-3抗体记录caspase-3抑制和脱氧核苷酸转移酶介导的dUTP缺口末端标记来监测对DNA片段的抑制来测试。这种新的依赖ERK1/2的PKCA刺激提示ERK1/2通过苏氨酸-638的直接磷酸化激活PKCA。为了测试这种可能性，激活的ERK1/2和纯的PKCA将用于体外ERK1/2检测。最后，我们将测试在P6和P15之间的5-HTiA-R-1Erk1/2通路中观察到的PKC层次结构的切换是否是由于PKC同工酶表达的年龄相关性变化。该项目将描绘一条可能在早期大脑发育中发挥关键作用的新途径。了解这一途径将有助于设计更好的治疗方法来对抗发育性大脑疾病。

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.