腺苷酸环化酶3对小鼠出生后嗅觉神经细胞发育的作用及其机制

The main olfactory epithelium (MOE) is the principal tissue for sensing odors in vertebrate. Olfactory receptor neuron (ORN) replacement in the MOE is a continuous process in lifelong. It plays important roles on olfactory behaviors in animals. The postnatal neurogenesis of ORN is sculpted by cell proliferation and programmed cell death (apoptosis), and is regulated by DNA methylation. Adenylyl cyclase 3 (AC3) is a key component of the cyclic adenosine-3'-5'-monophosphate (cAMP) signaling pathway within olfactory cilia of the ORNs in the MOE. Our previous works indicated that mice with deficiency of AC3 (AC3-/-) exhibit abnormally in odorant detections and correlative behaviors. However, the detailed underlying mechanisms are elusive. Neuronal activity is essential for survival of ORNs in a competitive environment. Therefore, we speculate that postnatal neurogenesis of ORNs might be impaired in AC3-/- mice, because neuronal activities evoked by odorants are dramatically decreased in the ORNs of these animals.Furthermore,the functions of AC3 on neurogenesis of ORNs might be different dependent on the developmental stages after postnatal. In the current proposal, the functions of AC3 on postnatal neurogenesis of ORNs, as well as its proliferative，apoptotic, and the underlying DNA methylation mechanisms will be investigated, by using technical combinations consisted of immunofluorescence staining, in-situ hybridization, DNA methylation microarray, and methylation-specific PCR and so on, with MOEs from different aged AC3-/- and AC3+/+ mice. The aim of this proposal is to characterize the effect of AC3 on neurogenesis of ORNs in postnatal mice, as well as its underlying mechanisms, in order to provide basis for olfactory diagnosis for our human health and the R&D of drugs.

主要嗅觉表皮(MOE)是哺乳动物的嗅觉器官?MOE内嗅觉神经细胞发生是一个终生持续的过程,对嗅觉行为具有重要影响?嗅觉神经细胞发生与细胞增殖和凋亡及DNA甲基化密切相关?腺苷酸环化酶3(AC3)是嗅觉神经细胞内cAMP信号通路的主要成分,敲除AC3基因的小鼠气味探测及嗅觉行为失常,其机制尚待明了?推测AC3敲除小鼠因感受气味刺激活动减弱导致嗅觉神经细胞发育缺陷,且AC3的功能可能因发育时期不同而异?为了明确AC3基因对嗅觉神经细胞发育的影响及其机制,本项目拟以不同发育时期AC3基因敲除及其野生小鼠MOE组织为材料,运用免疫荧光染色?原位杂交?DNA甲基化芯片及甲基化特异PCR等技术组合,鉴定AC3对小鼠出生后MOE内嗅觉神经细胞发育的作用,并对其发育缺陷的细胞增殖和凋亡及其DNA甲基化机制进行分析,旨在阐明AC3基因对嗅觉神经细胞发育的作用及其调控机理,为嗅觉健康诊断和药物研发提供依据。

主要嗅觉表皮(MOE)是哺乳动物的嗅觉器官｡ MOE内嗅觉神经细胞再生是一个终生持续的过程, 对嗅觉行为具有重要影响｡ 腺苷酸环化酶 3(AC3)是嗅觉神经细胞内 cAMP 信号通路的主要成分, 敲除 AC3 基因的小鼠气味探测及嗅觉行为失常, 其机制尚待明了｡ 推测AC3敲除小鼠因感受气味刺激活动减弱导致嗅觉神经细胞发育缺陷｡ 为了明确AC3基因对嗅觉神经细胞发育的影响及DNA甲基化其机制, 本项目以AC3 基因敲除及其野生小鼠MOE 组织为材料, 运用免疫荧光染色､原位杂交､DNA 甲基化芯片及甲基化特PCR 等技术组合, 分析了AC3对小鼠出生后MOE内嗅觉神经细胞发育的作用, 并对其发育缺陷的细胞增殖和凋亡及其 DNA 甲基化机制进行分析。HE染色结果表明，AC3敲除小鼠MOE组织上皮层明显变薄，细胞总数减少；免疫荧光染色结果发现，非成熟神经元数目增加，成熟神经元数目减少，而且凋亡细胞数目增多，神经元寿命明显缩短。此外，敲除型小鼠MOE组织纤毛层显著变薄。扫描电镜和透射电镜所进行的超微结构分析表明，AC3缺失小鼠嗅觉神经元纤毛减少、形态异常，甚至出现没有纤毛与线粒体结构的“裸嗅泡”。DNA甲基化芯片分析结果发现，与野生小鼠相比，AC3敲除小鼠MOE组织有1978个基因启动子的甲基化程度发生了改变。这些基因功能与肿瘤、MAPK、内吞作用、内质网中蛋白的加工、转录错误调控等相关。尤其是与cAMP信号及嗅觉受体基因的启动子区甲基化水平发生了改变。总之，我们的研究结果表明，AC3基因在MOE发育和纤毛超微结构完整性的维持方面具有不可替代的作用。AC3基因对基因组DNA甲基化水平的调控可能是导致MOE嗅觉神经细胞发育缺陷的分子机制之一。