腺苷酸环化酶3缺失下调小鼠嗅觉受体基因表达北大核心CSCD

主要嗅觉表皮组织(MOE)是哺乳动物感知气味分子的重要器官,气味诱导是嗅觉受体神经元(ORN)活动的起点,嗅觉受体(OR)结合气味分子后通过环腺苷酸(c AMP)信号通路向下游传递信号。腺苷酸环化酶3(AC3)是此通路中的重要分子。为了探讨AC3缺失对小鼠MOE内ORs基因表达的影响,本文以AC3敲除型小鼠(AC3-/-)和野生型小鼠(AC3+/+)为材料,采用荧光定量PCR(qRT-PCR)、荧光原位杂交(FISH)技术分析了部分ORs基因及与其相关因子在MOE中的表达。qRT-PCR表明,3月龄AC3-/-小鼠MOE中嗅觉受体Olfr15、Olfr16、Olfr533、Olfr536、Olfr1507和Olfr642的表达量均显著下降。出生后PND7、PND30和PND90三个不同发育时期的AC3-/-小鼠MOE原位杂交显示,嗅觉受体Olfr15、Olfr536和Olfr1507表达的细胞数目均减少。进一步qRTPCR分析发现,3月龄AC3-/-小鼠嗅觉受体相关因子Rtp1、Rtp2、Reep1、Lhx2、Emx2和Ric-8b的表达也均发生显著下调。由此推测,AC3缺失导致的ORs及其相关因子的表达下调可能是嗅觉行为障碍的原因之一。     

腺苷酸环化酶3缺失对小鼠主要嗅觉表皮组织内DNA甲基化的影响

腺苷酸环化酶3 (adenylate cyclaseⅢ,AC3)是嗅觉系统中的重要成分,AC3缺失后小鼠的主要嗅觉表皮组织(main olfactory epidermal,MOE)随年龄增长逐渐变薄,MOE内基因表达谱发生改变. DNA甲基化在动物发育、基因表达调控中具有重要作用.为了探讨AC3缺失后小鼠MOE内基因启动子甲基化水平的改变以及对基因表达的影响,本文采用DNA甲基化免疫共沉淀芯片(methylated DNA immunoprecipitation chip,MeDIP-chip)筛选AC3缺失小鼠MOE内启动子区甲基化差异表达基因,利用甲基化特异PCR (methylation-specific PCR,MSP)、实时荧光定量PCR (qRT-PCR)进一步检测部分甲基化差异基因的DNA甲基化水平改变和表达差异.结果表明,AC3缺失小鼠中有1 978个基因启动子的甲基化水平发生了改变,占总探针数的9%,其中727个基因启动子甲基化水平升高,1 251个甲基化水平降低.功能分析表明,这些启动子甲基化发生改变的基因主要涉及的功能分别与嗅觉受体、神经发育、cAMP信号通路、ATP结合、钙离子调控、乙酰化修饰、转录因子等相关. MSP检测表明,嗅觉受体基因Olfr1153、Olfr231、Olfr378、Olfr651、Olfr691启动子区的甲基化水平升高,Cngb1、Pde4a和Olfr1394基因启动子区的甲基化水平降低. qRT-PCR结果显示,基因Cngb1、Hcn4、Olfm1、Olfr1394、Olfr1153、Olfr231、Olfr378、Olfr691的表达水平显著下降,而Pde4a和Olfr651基因的表达水平显著升高.总之,AC3缺失后MOE内嗅觉受体基因、神经发育相关基因、cAMP信号通路等相关基因启动子甲基化水平发生显著改变,影响核苷酸切除修复、DNA复制、错配修复等信号通路的传导,从而综合调控小鼠MOE内的基因表达数量和水平.     

腺苷酸环化酶3缺失对小鼠主要嗅觉表皮组织内相关因子及信号通路的影响

主要嗅觉表皮(main olfactory epithelium, MOE)是哺乳动物感知气味分子的主要嗅觉器官。在MOE组织内,大多数嗅觉神经元通过cAMP信号传导通路感知气味信息。作为嗅觉cAMP信号通路的主要成员之一,腺苷酸环化酶3（adenylyl cyclase 3, ac3）基因敲除小鼠嗅觉探测功能丧失。除cAMP信号传导通路外,MOE内AC3相关因子AC2和AC4,以及肌醇1,4,5-三磷酸（inositol 1,4,5-trisphosphate,IP3）信号通路和Sonic Hedgehog（Shh）信号通路均有表达。然而,敲除ac3是否会对ac2和ac4以及IP3和Shh信号通路成员产生影响,尚不清楚。本文以AC3缺失（AC3-/-）及其野生型小鼠（AC3+/+）MOE为材料,采用实时荧光定量PCR（qRT-PCR）和免疫荧光组织化学方法,发现AC3缺失后,MOE内的ac2和ac4,以及IP3信号通路中的IP3受体ip3r1及钙调蛋白calm1和calm2表达水平均明显降低。Shh信号通路中的受体patched(ptch)与smoothened(smo)、以及核转录因子gli1与gli2的表达也受到了影响。总之,AC3基因缺失不但导致小鼠MOE组织中cAMP信号通路受损,同时AC3相关因子,IP3信号通路和Shh信号通路的传导也受到抑制。本文对于阐明AC3基因敲除小鼠嗅觉丧失的原因及其嗅觉探测机制具有重要启示作用。     

利用SSH方法筛选与鉴定AC3基因缺失小鼠主要嗅觉表皮内的差异表达基因

腺苷酸环化酶3(Adenylate cyclase 3, AC3)基因在小鼠主要嗅觉表皮(Main olfactory epithelium, MOE)内的嗅觉信号传导中起着重要作用, AC3缺失是否会导致MOE内与之相关的基因发生差异表达, 尚待确定。文章利用抑制性消减杂交(Suppression subtractive hybridization, SSH)方法, 以AC3敲除(AC3^-/-)及其同窝出生的野生型(AC3^+/+)小鼠MOE为材料, 构建了正向和反向两个消减文库, 采用斑点杂交对消减文库进行初步筛选, 对筛选出的差异表达基因进行序列测定及生物信息学分析, 并利用荧光定量PCR(qRT-PCR)方法对其进行验证。斑点杂交筛选获得了386个差异表达克隆, 随机选取其中的80个进行DNA序列测定, 经序列比对后发现有62个在GenBank上获得了与之相匹配的基因信息, 其中24个上调差异表达克隆对应于kcnk3、mapk7、megf11等基因, 38个下调差异表达克隆对应于tmem88b、c-mip、skp1a、mlycd等基因。利用Gene Ontology(GO)方法对这些差异表达基因进行蛋白功能注释, 发现它们主要集中在分子结合、细胞周期、生物和细胞过程等功能方面。选取其中上调基因kcnk3和下调基因c-mip、mlycd、tmem88b及trappc5进行qRT-PCR验证。结果表明, 在AC3^-/-小鼠MOE内kcnk3的表达量显著上调, 是对照组小鼠的1.27倍, 而c-mip、mlycd、tmem88b和trappc5的表达量显著下调, 为对照组小鼠的20%、7%、32%和29%。这些基因的功能与K⁺通道、细胞发育与分化、脂肪代谢和膜蛋白转运等密切相关。推测它们可能与AC3基因共同作用, 调节小鼠MOE内的嗅觉信号传导信息。     

果蝇嗅觉分子机理研究进展

黑腹果蝇Drosophila melanogaster是生物学研究的重要模式生物,也是探索研究生物体嗅觉奥秘的理想材料。近年来,由于分子生物学技术在神经科学领域的广泛应用,黑腹果蝇嗅觉机理研究取得了许多重大突破, 对气味分子受体及其识别机理、 嗅觉神经电信号的产生和传递、嗅觉信息的加工、编码以及记忆等方面都有了深入的了解。研究表明, 果蝇约1 300个嗅神经元(olfactory receptor neurons, ORNs)共表达62种不同的气味受体蛋白（olfactory receptor proteins, ORs）, 用以检测和识别其所感受的所有化学气味分子。许多OR所识别的气味分子配体已鉴定出来,普通的气味（如水果的气味）由数种不同的OR组合来识别,而信息素（pheromone）分子则由单种特定的OR来检测。气味信息在嗅神经元内转换成神经电信号,嗅觉电信号沿嗅神经元的轴突传递到触角叶, 再经投射神经元（projection neurons, PNs）将信息送至高级中枢如蘑菇体（mushroom body, MB）和侧角（lateral horn, LH）,最终引发行为反应。在黑腹果蝇嗅觉信息传递通路中,某些蛋白如Dock,N-cadherin,Fruitless等起着重要作用,缺失这些蛋白会导致嗅觉异常。本文对这些研究进展作一综述。     

昆虫嗅觉相关蛋白的结构和功能

昆虫在长期进化的过程中形成了复杂的嗅觉系统,气味剂结合蛋白(odorant binding proteins,OBPs)、嗅觉受体(olfactory receptors,ORs)是其最主要的组分.其主要作用是结合外围挥发性的气味分子并将信号传递给细胞内的第二信使.OBPs和ORs的结构、功能、表达、进化是昆虫行为与进化关系的重要研究领域和研究热点.本文主要总结了近年来昆虫OBPs和ORs的结构特点、生理功能、表达特点、遗传进化等方面研究的最新进展,对OBPs和ORs的研究趋势进行了展望,为昆虫嗅觉系统进化研究及寻找害虫防治新途径提供参考信息.     

昆虫气味受体研究进展

嗅觉在昆虫的多种行为中发挥关键作用。气味分子与嗅觉神经元树突上气味受体的结合,参与了昆虫嗅觉识别的初始过程。昆虫的嗅觉神经元表达两类气味受体： 一是传统气味受体,该类受体同源性较低,在少部分嗅觉神经元中表达; 二是Or83b家族受体,该类受体不感受气味,在不同昆虫间较为保守且在大多数嗅觉神经元中表达。目前,对于单个传统气味受体的气味分子配体特异性所知甚少; 对于Or83b家族受体,一般认为其可能具有将传统气味受体运送至嗅觉神经元树突膜上的功能。此外,有一些实验证据不支持昆虫气味受体为G蛋白偶联受体的观点。     

棉铃虫齿唇姬蜂嗅觉受体基因 CchlOrco 的克隆及组织表达谱分析

【目的】昆虫的嗅觉受体（olfactory receptors, ORs）一般以气味分子特异的ORs与共受体（ co-Receptor, Orco）通过形成异质二聚体在嗅觉感受中发挥关键作用,其中Orco由于具有序列的保守性而受到广泛的重视。本研究旨在克隆棉铃虫齿唇姬蜂 Campoletis chlorideae 的Orco基因,并对其组织表达谱进行分析。【方法】利用RT-PCR技术和转录组分析技术克隆棉铃虫齿唇姬蜂的Orco基因,并对其编码的氨基酸序列进行生物信息学分析;利用Real-time PCR技术对该基因在该蜂成虫不同组织中的表达量进行分析。【结果】获得了棉铃虫齿唇姬蜂 Orco 的全长cDNA序列,命名为 CchlOrco（GenBank登录号：KP255444）。序列分析表明, 该基因开放阅读框全长1 437 bp,编码478个氨基酸,预测该氨基酸序列具有7个跨膜区。CchlOrco 主要在成虫触角中表达,且在雄蜂触角中的表达量最高,是雌蜂触角中表达量的8.0倍,而在其他组织中表达量极低。【结论】本研究克隆了棉铃虫齿唇姬蜂 CchlOrco 序列全长,明确了其在成虫不同组织中的表达水平,为进一步研究该基因及其他嗅觉受体基因功能奠定了基础。     

昆虫感觉气味的细胞与分子机制研究进展

昆虫作为地球上最为成功的类群,已经成功地进化了精细的化学感受系统,通过化学感受系统适应各种复杂的环境,保持种群的繁荣。自1991年在动物中发现嗅觉受体基因以来,关于昆虫感受化学信息的周缘神经系统的分子和细胞机制方面的进展十分迅速。文章主要就昆虫周缘神经系统的感受化学信息的分子和细胞机制进行综述。首先对昆虫感觉气味的细胞机制的研究进展进行简要介绍。昆虫嗅觉神经元在感受化学信息过程中起着极为重要的作用,昆虫嗅觉神经元上表达的嗅觉受体不同而执行着各异的功能。各种嗅觉神经元对于化学信息的感受谱有较大的区别;嗅觉神经元对化学信息类型、浓度、流动动态等产生相应的电生理特征反应。研究表明同一种神经原可以感受多种化学信息,而一种化学信息也可以被多种神经原所感受。由神经原对化学信息感受所形成的特征组合就是感受化学信息的编码。其次较为详细地论述与昆虫感受气味分子相关的一些蛋白质的研究进展。气味分子结合蛋白是一类分子量较小、水溶性的蛋白,主要位于化学感受器神经原树突周围的淋巴液中。在结构上的主要特征是具有6个保守的半光氨酸和由6个α螺旋组成的结合腔。自1981年发现以来,已经在40余种昆虫中发现上百种。由于研究手段的不断进步,已经对该类蛋白的表达特征、结合特性以及三维结构和结合位点进行了大量的研究,提出了多个可能的功能假说,在诸多的假说中,较为广泛接受的是气味分子结合蛋白在昆虫感觉气味的过程中,是与疏水性的气味分子相结合,并将气味分子运输到嗅觉神经原树突膜上的嗅觉受体上。这些处于树突膜上的嗅觉受体则是昆虫感觉气味过程中的另一个十分重要的蛋白质。目前,已经在果蝇、按蚊、蜜蜂和家蚕等10余个昆虫种类中发现上百个嗅觉受体蛋白基因。这类蛋白是跨膜蛋白,一般具有7个跨膜区,整个蛋白的氨基酸残基在400～600个。昆虫的嗅觉受体蛋白的N-端在胞内,而C-端在胞外,这与G耦联蛋白不同。而且,昆虫的一个嗅觉神经元可以表达1～3个嗅觉受体蛋白,也与哺乳动物的一个神经元只表达一种受体蛋白有所不同。每种嗅觉受体可以感受多种气味分子,而一种气味分子可以被多个嗅觉受体所感知,这样组成了感受化学信息的编码谱。最近采用基因敲除技术和膜片钳技术研究发现,昆虫的嗅觉受体蛋白在信号传导中也有特殊性,即嗅觉受体可以直接作为离子通道,而引起动作电位。还有近来的研究表明,神经膜蛋白对于果蝇的性信息素感受神经元感受性信息素cVA是必要的。实际上,昆虫对于化学信息的感受和信号的转导,并不是上述蛋白单独起作用完成的,而是多种蛋白相互作用的结果。论文最后对该领域研究内容进行了展望。     

Odorant receptors directly activate phospholipase C/inositol-1,4,5-trisphosphate coupled to calcium influx in Odora cells

Mechanisms by which odorants activate signaling pathways in addition to cAMP are hard to evaluate in heterogeneous mixtures of primary olfactory neurons. We used single cell calcium imaging to analyze the response to odorant through odorant receptor (OR) U131 in the olfactory epithelial cell line Odora (Murrell and Hunter 1999), a model system with endogenous olfactory signaling pathways. Because adenylyl cyclase levels are low, agents activating cAMP formation do not elevate calcium, thus unmasking independent signaling mediated by OR via phospholipase C (PLC), inositol-1,4,5-trisphosphate (IP(3)), and its receptor. Unexpectedly, we found that extracellular calcium is required for odor-induced calcium elevation without the release of intracellular calcium, even though the latter pathway is intact and can be stimulated by ATP. Relevant signaling components of the PLC pathway and G protein isoforms are identified by western blot in Odora cells as well as in olfactory sensory neurons (OSNs), where they are localized to the ciliary zone or cell bodies and axons of OSNs by immunohistochemistry. Biotinylation studies establish that IP(3) receptors type 2 and 3 are at the cell surface in Odora cells. Thus, individual ORs are capable of elevating calcium through pathways not directly mediated by cAMP and this may provide another avenue for odorant signaling in the olfactory system.     

Differential development of odorant receptor expression patterns in the olfactory epithelium: a quantitative analysis in the mouse septal organ

The rodent olfactory epithelium expresses more than 1000 odorant receptors (ORs) with distinct patterns, yet it is unclear how such patterns are established during development. In the current study, we investigated development of the expression patterns of different ORs in the septal organ, a small patch of olfactory epithelium predominantly expressing nine identified ORs. The presumptive septal organ first appears at about embryonic day 16 (E16) and it completely separates from the main olfactory epithelium (MOE) at about postnatal day 7 (P7). Using in situ hybridization, we quantified the densities of the septal organ neurons labeled by specific RNA probes of the nine abundant OR genes from E16 to postnatal 3 months. The results indicate that olfactory sensory neurons (OSNs) expressing different ORs have asynchronous temporal onsets. For instance, MOR256-17 and MOR236-1 cells are present in the septal organ at E16; however, MOR0-2 cells do not appear until P0. In addition, OSNs expressing different ORs show distinct developmental courses and reach their maximum densities at different stages ranging from E16 (e.g. MOR256-17) to 1 month (e.g. MOR256-3 and MOR235-1). Furthermore, early onset does not correlate with high abundance in adult. This study reveals a dynamic composition of the OSNs expressing different ORs in the developing olfactory epithelium.     

The role of the coreceptor Orco in insect olfactory transduction

Insects sense odorants with specialized odorant receptors (ORs). Each antennal olfactory receptor neuron expresses one OR with an odorant binding site together with a conserved coreceptor called Orco which does not bind odorants. Orco is necessary for localization of ORs to dendritic membranes and, thus, is essential for odorant detection. It forms a spontaneously opening cation channel, activated via phosphorylation by protein kinase C. Thereafter, Orco is also activated via cyclic adenosine monophosphate (cAMP). Orco forms homo—as well as heteromers with ORs with unknown stoichiometry. Contradictory publications suggest different mechanisms of olfactory transduction. On the one hand, evidence accumulates for the employment of more than one G protein-coupled olfactory transduction cascade in different insects. On the other hand, results from other studies suggest that the OR–Orco complex functions as an odorant-gated cation channel mediating ionotropic signal transduction. This review analyzes conflicting hypotheses concerning the role of Orco in insect olfactory transduction. In conclusion, in situ studies in hawkmoths falsify the hypothesis that Orco underlies odorant-induced ionotropic signal transduction in all insect species. Instead, Orco forms a metabotropically gated, slow cation channel which controls odorant response threshold and kinetics of the sensory neuron.     

cAMP and IP3 signaling pathways in HEK293 cells transfected with canine olfactory receptor genes

Olfactory receptors (ORs) expressed at the cell surface of olfactory sensory neurons lining the olfactory epithelium are the first actors of events leading to odor perception and recognition. As for other mammalian ORs, few dog OR have been deorphanized, mainly because of the absence of good methodology and the difficulties encountered to express ORs at the cell surface. Within this work, our aim was 1) to deorphanize a large subset of dog OR and 2) to compare the implication of the 2 main pathways, namely the cAMP and inositol 1,4,5-triphosphate (IP3) pathways, in the transduction of the olfactory message. For this, we used 2 independent tests to assess the importance of each of these 2 pathways and analyzed the responses of 47 canine family 6 ORs to a number of aliphatic compounds. We found these ORs globally capable of inducing intracellular calcium elevation through the IP3 pathway as confirmed by the use of specific inhibitors and/or a cAMP increase in response to aldehyde exposure. We showed that the implication of the cAMP or/and IP3 pathway was dependent upon the ligand-receptor combination rather than on one or the other partner. Finally, by exposing OR-expressing cells to the 21 possible pairs of C6-C12 aliphatic aldehydes, we confirmed that some odorant pairs may have an inhibitory or additive effect. Altogether, these results reinforce the notion that odorant receptor subfamilies may constitute functional units and call for a more systematic use of 2 complementary tests interrogating the cAMP and IP3 pathways when deorphanizing ORs.     

Identification of second messenger mediating signal transduction in the olfactory receptor cell

One of the biggest controversial issues in the research of olfaction has been the mechanism underlying response generation to odorants that have been shown to fail to produce cAMP when tested by biochemical assays with olfactory ciliary preparations. Such observations are actually the original source proposing a possibility for the presence of multiple and parallel transduction pathways. In this study the activity of transduction channels in the olfactory cilia was recorded in cells that retained their abilities of responding to odorants that have been reported to produce InsP3 (instead of producing cAMP, and therefore tentatively termed "InsP3 odorants"). At the same time, the cytoplasmic cNMP concentration ([cNMP]i) was manipulated through the photolysis of caged compounds to examine their real-time interactions with odorant responses. Properties of responses induced by both InsP3 odorants and cytoplasmic cNMP resembled each other in their unique characteristics. Reversal potentials of currents were 2 mV for InsP3 odorant responses and 3 mV for responses induced by cNMP. Current and voltage (I-V) relations showed slight outward rectification. Both responses showed voltage-dependent adaptation when examined with double pulse protocols. When brief pulses of the InsP3 odorant and cytoplasmic cNMP were applied alternatively, responses expressed cross-adaptation with each other. Furthermore, both responses were additive in a manner as predicted quantitatively by the theory that signal transduction is mediated by the increase in cytoplasmic cAMP. With InsP3 odorants, actually, remarkable responses could be detected in a small fraction of cells ( approximately 2%), explaining the observation for a small production of cAMP in ciliary preparations obtained from the entire epithelium. The data will provide evidence showing that olfactory response generation and adaptation are regulated by a uniform mechanism for a wide variety of odorants.     

Phosphoinositide and Erk signaling pathways mediate activity‐driven rodent olfactory sensory neuronal survival and stress mitigation

Olfactory sensory neurons (OSNs) are the initial site for olfactory signal transduction. Therefore, their survival is essential to olfactory function. In the current study, we demonstrated that while odorant stimulation promoted rodent OSN survival, it induced generation of reactive oxygen species in a dose‐ and time‐dependent manner as well as loss of membrane potential and fragmentation of mitochondria. The MEK‐Erk pathway played a critical role in mediating these events, as its inhibition decreased odorant stimulation‐dependent OSN survival and exacerbated intracellular stress measured by reactive oxygen species generation and heat‐shock protein 70 expression. The phosphoinositide pathway, rather than the cyclic AMP pathway, mediated the odorant‐induced activation of the MEK‐Erk pathway. These findings provide important insights into the mechanisms of activity‐driven OSN survival, the role of the phosphoinositide pathway in odorant signaling, and demonstrate that odorant detection and odorant stimulation‐mediated survival proceed via independent signaling pathways. This mechanism, which permits independent regulation of odorant detection from survival signaling, may be advantageous if not diminished by repeated or prolonged odor exposure.