OMICS Analyses Unraveling Related Gene and Protein Driven Molecular Mechanisms Underlying PACAP 38 Induced Neurite Outgrowth in PC12 Cells

Recently, our group has shown that the pituitary adenylate cyclase-activating polypeptide (PACAP)-induced neurite projection elongation in rat adrenal-derived pheochromocytoma cell line (PC12) is mediated via PAC1 receptor-mediated dephosphorylation of CRMP2 majorly through PI3K/AKT and MEK/ERK pathways. However, the mechanism of neuronal outgrowth by PACAP, including CRMP2 dephosphorylation, remains unclear. In our previous report, we found that GSK-3β, CDK5, and Rho/ROCK dephosphorylated CRMP2 within 3 hours after the addition of PACAP, but the early dephosphorylation of CRMP2 by PACAP remains unclear. Thus, we considered it important to identify early factors in PACAP-induced neurite projection elongation and performed omics-based transcriptomic (whole genome DNA microarray) and proteomic (TMT-labeling in conjunction with liquid chromatography-tandem mass spectrometry) analyses of gene and protein expression profiles from 5-120 minutes after PACAP addition. Results surprisingly revealed a number of key regulators involved in neurite outgrowth, including known ones. We also identified factors that may be involved in CRMP2 dephosphorylation. Cross-referencing previous research, we tried to map these molecular components onto potential pathways. The results of this comprehensive analysis may provide important new information on the molecular mechanisms of neuronal differentiation induced by PACAP.本团队此前已证实，垂体腺苷酸环化酶激活多肽（pituitary adenylate cyclase-activating polypeptide, PACAP）可诱导大鼠肾上腺来源嗜铬细胞瘤细胞系（rat adrenal-derived pheochromocytoma cell line, PC12）发生神经突投射伸长，该过程主要通过PAC1受体（PAC1 receptor）介导CRMP2（CRMP2）去磷酸化，且依赖PI3K/AKT与MEK/ERK信号通路。然而，PACAP介导神经元生长（含CRMP2去磷酸化）的具体分子机制仍未阐明。在既往研究中，我们发现PACAP处理后3小时内，糖原合酶激酶3β（glycogen synthase kinase 3β, GSK-3β）、细胞周期蛋白依赖性激酶5（cyclin-dependent kinase 5, CDK5）及Rho/ROCK信号通路可介导CRMP2去磷酸化，但PACAP诱导CRMP2早期去磷酸化的上游调控机制仍不明确。因此，我们认为明确PACAP诱导神经突投射伸长的早期调控因子具有重要研究价值，并基于多组学（omics）技术，对PACAP处理后5至120分钟的细胞样本开展了转录组（全基因组DNA微阵列，whole genome DNA microarray）与蛋白质组学（TMT标记联合液相色谱-串联质谱，TMT-labeling in conjunction with liquid chromatography-tandem mass spectrometry）分析，以检测基因与蛋白质表达谱的动态变化。结果意外发现了多个参与神经突生长的关键调控因子，其中包括已被报道的已知因子；同时我们还鉴定出若干可能参与CRMP2去磷酸化过程的潜在调控蛋白。通过结合既往研究进行交叉比对，我们尝试将这些分子组分映射至潜在信号通路中。本研究的全面分析结果，可为PACAP诱导神经元分化的分子机制提供全新的重要理论依据。