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

描述（由申请人提供）：Ca2+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。本提案的目的是建立新的转基因小鼠模型，以探索新型Ca2+动员第二信使adp核糖（ADPR）及其效应分子，ADPR门控的TRPM2离子通道在先天免疫功能中的作用。细胞电子载体NAD(P)+的几种代谢物正在成为一组意想不到的Ca2+稳态调节剂。尽管环状ADPR和NAADP都可以消耗细胞内Ca2+储存，但ADPR通过结合TRPM2离子通道的门控域介导Ca2+进入细胞质。在炎症环境中遇到的氧化应激条件，由于激活NAD+和dna消耗的“拯救和修复”途径，预计会导致ADPR的产生。为了支持这一观点，trpm2介导的Ca2+内流在外用H2O2后被触发。最近对TRPM2缺陷小鼠模型的表征表明，TRPM2对于h2o2介导的单核细胞细胞因子的产生至关重要。使用不同的TRPM2-/-小鼠品系，我们发现TRPM2是宿主防御李斯特菌病所必需的，进一步支持TRPM2在免疫系统中起关键作用的发现。此外，根据特定免疫细胞亚群的成熟和激活状态，TRPM2的基因表达水平似乎受到差异调节，这意味着通过TRPM2响应adpr积累的能力是经过精心策划的。因此，我们推断，在不表达TRPM2的细胞中强迫其表达可能导致发育和功能异常，从而揭示TRPM2/ADPR介导的信号传导。为了了解操纵TRPM2和ADPR水平如何影响体内的免疫反应，我们建议建立两种新的小鼠菌株，将以下构建物靶向整合到小鼠基因组ROSA26位点：1)高选择性ADPR水解酶NudT9的re诱导表达构建物，允许选择性地降低小鼠胞质ADPR水平。2)类似的结构允许cre介导的TRPM2表达。一旦获得，这些菌株将被交叉到选择的表达cre的小鼠系上，例如获得转基因的髓细胞限制性表达。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感染的易感性。这些研究将使我们能够评估这类方法在免疫调节和治疗目的方面的潜力。

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.