Ryanodine receptors and NAADP in T cell biology

• Applicants: Professor Dr. Alexander Flügel; Professor Dr. Andreas H. Guse
• Subject Area: Molecular and Cellular Neurology and Neuropathology ; Biochemistry ; Immunology ; Cell Biology
• Term: from 2011 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 201358593

In the previous project we obtained evidence for major roles of ryanodine receptor (RyR) 1 and the 2nd messenger NAADP in T cell biology and RyR 3 in astrocyte biology. Using RyR1 and RyR3 knock-out mice we found ameliorated EAE course compared to control animals. Further, we designed and applied a novel biosensor for NFAT translocation and used high resolution Ca2+ imaging to characterize local initial Ca2+ signals.Here we aim to pursue the biochemical basis of NAADP action by identifying NAADP binding proteins and the NAADP forming enzyme. Further we will analyze in detail the role of NAADP, NAADP binding proteins and RyR1 in initial local Ca2+ signals during immune synapse initiation. T cells from a conditional knock-out model for RyR 1 will be used to study local Ca2+ signals, active experimental autoimmune encephalomyelitis (EAE) and transfer EAE, and T cell activation in the lymph node and central nervous system (CNS) by 2-photon imaging. Further, mice with RyR1 deficiency in astrocytes (GFAP-Cre), neurons (Thy1-Cre) and microglia (Cx3cr1-CreER) will be generated and their phenotyping under steady state and inflammatory conditions will be studied. In addition, we will analyze global and local Ca2+ signaling in wt vs RyR3-/- murine astrocytes and perform gene expression profiling (RNA-Seq) of Ryr3-deficient astrocytes isolated ex vivo from healthy or inflamed CNS. We expect to precisely describe the signaling axis involving NAADP and RyR1 in T cells and its role in T cell activation and CNS autoimmunity. Likewise, we expect to unravel the role of RyR3 signaling in astrocytes and their role in CNS autoimmunity.

• DFG Programme: Research Grants