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

National Jewish Health, Denver CO

Investigators

Abstract

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.