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

描述（由申请方提供）：Ca 2+信号的适当水平、定位和时间发展对于适当和有效的免疫应答至关重要。本提案的目的是产生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），其效应分子，ADPR门控TRPM 2离子通道的先天免疫功能的作用。细胞电子载体NAD（P）+的几种代谢产物作为一组未被怀疑的Ca 2+稳态调节剂而出现。而环ADPR和NAADP都可以耗尽细胞内的Ca 2 +-商店，ADPR介导的Ca 2 +-进入胞质溶胶通过结合TRPM 2离子通道的门控结构域。预期在炎症环境中遇到的氧化应激条件会导致ADPR产生，这是NAD+和DNA消耗“拯救和修复”途径激活的结果。为了支持这一观点，TRPM 2介导的Ca 2+内流在外部应用H2 O2后被触发。最近的TRPM 2缺陷小鼠模型的表征表明，TRPM 2是必不可少的H2 O2介导的单核细胞细胞因子的生产。使用不同的TRPM 2-/-小鼠品系，我们发现TRPM 2是宿主防御疟原虫病所必需的，进一步支持了TRPM 2在免疫系统中起关键作用的发现。此外，TRPM 2的基因表达水平似乎根据特定免疫细胞亚群的成熟和活化状态而差异调节，这意味着通过TRPM 2响应ADPR积累的能力是精心策划的。因此，我们推断，迫使TRPM 2在原本不会表达它的细胞中表达可能导致发育和功能畸变，从而对TRPM 2/ADPR介导的信号传导提供一些线索。为了理解TRPM 2和ADPR水平的操纵如何可能影响体内免疫应答，我们因此提出建立两种新的小鼠品系，其具有靶向整合到小鼠基因组ROSA 26基因座中的以下构建体：1）允许选择性降低小鼠中胞质ADPR水平的高度选择性ADPR-水解酶NudT 9的Cre诱导型表达构建体。2)允许Cre介导的TRPM 2表达的类似构建体。一旦获得，这些品系将与所选的表达Cre的小鼠系杂交，例如以获得转基因的骨髓细胞限制性表达。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.