Novel Transgenic Mouse Models to Analyze TRPM2 and ADP-Ribose Function

描述(由申请人提供)：钙信号的适当水平、定位和时间发展对适当和有效的免疫反应至关重要。本研究的目的是建立新的转基因小鼠模型，以探讨新的钙动员第二信使ADP-核糖(ADPR)及其效应分子ADPR门控TRPM2离子通道在先天性免疫功能中的作用。细胞电子载体NAD(P)的几种代谢物正作为一组意想不到的钙稳态调节剂出现。虽然环状ADPR和NAADP都可以耗尽细胞内的钙库，但ADPR通过结合TRPM2离子通道的门控结构域来介导钙离子进入胞浆。炎症环境中遇到的氧化应激条件预计会导致ADPR的产生，这是激活NAD-和DNA耗尽的“救援和修复”途径的结果。支持这一观点的是，在外加过氧化氢后，TRPM2介导的钙内流被触发。最近对TRPM2缺陷小鼠模型的研究表明，TRPM2对于过氧化氢介导的单核细胞细胞因子的产生是必不可少的。使用不同的TRPM2-/-小鼠品系，我们发现TRPM2是抵御李斯特菌病的宿主防御所必需的，进一步支持了TRPM2在免疫系统中发挥关键作用的发现。此外，TRPM2的基因表达水平似乎根据特定免疫细胞亚群的成熟和激活状态而受到不同的调节，这意味着通过TRPM2对ADPR积聚做出反应的能力是精心策划的。因此，我们认为强迫TRPM2在不表达它的细胞中表达可能会导致发育和功能异常，从而揭示了TRPM2/ADPR介导的信号转导。为了了解TRPM2和ADPR水平的操纵可能如何影响体内的免疫反应，我们因此提议建立两个新的小鼠品系，其靶向整合到以下结构的小鼠基因组rosa26基因座中：1)Cre诱导的高选择性ADPR-hydolase NudT9表达构建体，允许选择性地降低小鼠胞浆ADPR-水平。2)类似的构建允许Cre介导的TRPM2的表达。一旦获得，这些菌株将与表达Cre的小鼠品系杂交，例如获得髓系细胞限制性表达的转基因。初步研究将评估免疫相关器官的细胞组成和转基因菌株对LM感染的敏感性。这些研究将使我们能够评估这种类型的方法用于免疫调节和治疗目的的潜力。

DESCRIPTION (provided by applicant): The adequate level, localization, and temporal development of Ca2+-signals are crucial to an appropriate and effective immune response. The objective of this proposal is to generate novel transgenic mouse models to explore the role in innate immune functions of the novel Ca2+-mobilizing second messenger ADP-ribose (ADPR), and of its effector molecule, the ADPR-gated TRPM2 ion channel. Several metabolites of the cellular electron carrier NAD(P)+ are emerging as an unsuspected group of Ca2+-homeostasis regulators. Whereas cyclic ADPR and NAADP can both deplete intracellular Ca2+-stores, ADPR mediates Ca2+-entry into the cytosol by binding the gating domain of the TRPM2 ion channel. Conditions of oxidative stress as encountered in inflammatory environments are expected to lead to ADPR production as a consequence of the activation of NAD+- and DNA-depleting "rescue and repair" pathways. In support of this idea, TRPM2-mediated Ca2+-influx is triggered following external application of H2O2. The recent characterization of a TRPM2-deficient mouse model has shown that TRPM2 is essential for H2O2-mediated cytokine production in monocytes. Using a different TRPM2-/- mouse strain, we found that TRPM2 is required for host-defense against listeriosis, further supporting the finding that TRPM2 plays a crucial role in the immune system. Moreover, gene expression levels of TRPM2 appear to be differentially regulated in accordance to the maturation and activation status of specific immune cell subpopulations, implying that the ability to respond to ADPR-accumulation via TRPM2 is carefully orchestrated. We thus reason that forcing the expression of TRPM2 in cells that would not otherwise express it might result in developmental and functional aberrations, shedding some light on TRPM2/ADPR- mediated signaling. In order to understand how the manipulation of TRPM2 and ADPR levels might impact immune responses in vivo, we therefore propose to establish two novel mouse strains with targeted integration into the mouse genomic ROSA26 locus of the following constructs: 1) A Cre-inducible expression construct of the highly selective ADPR-hydolase NudT9 allowing to selectively reduce cytosolic ADPR-levels in mice. 2) A similar construct allowing for Cre-mediated expression of TRPM2. Once obtained, these strains will be crossed onto Cre-expressing mouse lines of choice, for example to obtain myeloid cell restricted expression of the transgenes. Preliminary studies will assess the cellular composition of immune relevant organs and the susceptibility of the transgenic stains to Lm infection. These studies will allow us to assess the potential of this type of approaches for immunomodulatory and therapeutic purposes.