A01

A01 Project Movie

A01

From Diercks, B.-P. et al. (2018). ORAI1, STIM1/2, and RYR1 shape subsecond Ca2+ microdomains upon T cell activation. Sci. Signal. 11, eaat0358.

Project A01 concentrated on the characterization of NAADP and RYR1 signaling in T cell biology. Although NAADP was discovered more than 25 years ago, neither the NAADP forming enzyme nor the NAADP receptor protein have been convincingly identified. Project A01 discovered that NADPH oxidases/dual NADPH oxidases (NOX/DUOX) convert NAADPH to NAADP under physiological conditions with correct membrane orientation (in opposite to CD38, the previous prime candidate as NAADP forming enzyme); experimental proof for NAADP formation was obtained for NOX5, DUOX1 and DUOX2. Further, glucose 6-phosphate dehydrogenase (GLC-6P-DH) was so far identified as the only enzyme catalyzing reduction of NAADP to NAADPH. In addition, project A01 also demonstrated that CD38 is not involved in acute NAADP formation in intact T cells. Thus, project A01 suggests a DUOX2- and GLC-6P-DH-catalyzed redox cycle for rapid production and degradation of NAADP via NAADPH as inactive intermediate.

As for the NAADP forming enzyme, also the NAADP receptor/binding protein has been unknown. According to our hypothesis published earlier, the NAADP receptor/binding protein HN1L/JPT2 was purified by liquid chromatography and identified by mass spectrometry in project A01. Binding of NAADP to recombinant HN1L/JPT2 was demonstrated by photoaffinity labeling. Gene knock-out of Hn1l/Jpt2 in human Jurkat and primary rat T cells resulted in decreased numbers of initial Ca2+ microdomains, and delayed onset and decreased amplitude of global Ca2+ signaling. T cell receptor (TCR)/CD3 dependent co-precipitation and co-localization data of HN1L/JPT2 with RYR suggest that HN1L/JPT2 is a major component to connect NAADP formation with RYR1 channel activation in T cells. The identification of HN1L/JPT2 as NAADP receptor/binding protein was confirmed by similar results in red blood cells, published back-to-back with our results.

Further, in close collaboration, projects A01 and A02 confirmed a major role of RYR1 in early T cell Ca2+ signaling showing marked decrease in Ca2+ microdomain numbers in Ryr1-/- T cells or upon NAADP antagonism, whereas Ryr3-/- and Tpc1-/-/Tpc2-/- T cells were not different from WT controls when analyzed for Ca2+ microdomains. The role of RYR1 and RYR3 in CNS autoimmunity was tested in project A01 in constitutive knock out (KO) mice for RYR3 and fetal liver chimeras for RYR1 (flcRYR1; constitutive KO of RYR1 is neonatally lethal). In both, Ryr3-/- and bone marrow-(bm)Ryr1-/- mice, A01 observed a moderate EAE amelioration. In Ryr3-/- mice the absence of RYR3 in brain resident cells was instrumental for the phenotype, whereas in bmRyr1-/- mice the T cell KO was relevant. For dissecting the roles of the distinct CNS resident cells for RYR3 signaling, project A01 generated a floxed Ryr3 mouse. Interestingly, neither GFAP/ALDH-cre (astrocytes), nor CX3CR1-cre (microglia), nor synapsin-cre (neurons) alone could reproduce the phenotype of the global KO, indicating a cooperative effect of RYR3 signaling.

In addition, project A01 developed a TCR transgenic Lewis rat with specificity for b-synuclein, in which the T cells attack the CNS grey matter, in particular the brain cortex. Since this part of the brain is well accessible for intravital imaging (superficial location and penetrable grey matter tissue), project A01 now can track T cell activation processes within the inflammatory lesions over time deep within the tissue (up to 800 mm). In addition, another innovative animal model was generated by project A01: a novel Cas9 transgenic Lewis rat. Using a new retroviral transduction protocol, for the first time genes can be efficiently inactivated in rat T cells, as shown for Duox1, Duox2 or Hn1l/Jpt2. Combining the newly developed rat models, in the 2nd funding period, project A01 will track and functionally analyze the roles of Ryr1, Ryr3, Duox1, Duox2, or Hn1l/Jpt2 in rat T cells during the different states of CNS autoimmunity. Finally, an innovative method for localized Ca2+ release from endo-lysosomes by photoexcitation of polymer capsules with integrated plasmonic nanoparticles was developed.

Prof. Dr. Dr. Andreas H. Guse

Selected publications (*equal contribution)

 

  1. Gu F, Krüger A, Roggenkamp HG, Alpers R, Lodygin D, Jaquet V, Möckl F, Hernandez C LC, Winterberg K, Bauche A, Rosche A, Grasberger H, Kao JY, Schetelig D, Werner R, Schröder K, Carty M, Bowie AG, Huber S, Meier C, Mittrücker HW, Heeren J, Krause KH, Flügel A, Diercks B-P*, Guse AH*. Dual NADPH oxidases DUOX1 and DUOX2 synthesize NAADP and are necessary for Ca2+ signaling during T cell activation. Science Signal. 2021; 14 eabe3800.
  2. Roggenkamp HG, Khansahib I, Hernandez C LC, Zhang Y, Lodygin D, Krüger A, Gu F, Möckl F, Löhndorf A, Wolters V, Woike D, Rosche A, Bauche A, Schetelig D, Werner R, Schlüter H, Failla AV, Meier C, Fliegert R, Walseth TF, Flügel A, Diercks B-P*, Guse AH*. HN1L/JPT2: A signaling protein that connects NAADP generation to Ca2+ microdomain formation. Science Signal. 2021; 14:eabd5647.
  3. Zhu D, Feng L, Feliu N, Guse AH, Parak WJ. Stimulation of Local Cytosolic Calcium Release by Photothermal Heating for Studying Intra- and Intercellular Calcium Waves. Adv Mater 2021; 5:e2008261.
  4. Diercks BP, Werner R, Weidemüller P, Czarniak F, Hernandez L, Lehmann C, Rosche A, Krüger A, Kaufmann U, Vaeth M, Failla AV, Zobiak B, Kandil FI, Schetelig D, Ruthenbeck A, Meier C, Lodygin D, Flügel A, Ren D, Wolf IMA, Feske S, Guse AH (2018) ORAI1, STIM1/2, and RYR1 shape subsecond Ca2+ microdomains upon T cell activation. Sci Signal 2018 Dec 18;11(561):eaat0358
  5. Fliegert R, Bauche A, Wolf Pérez AM, Watt JM, Rozewitz MD, Winzer R, Janus M, Gu F, Rosche A, Harneit A, Flato M, Moreau C, Kirchberger T, Wolters V, Potter BVL, Guse AH (2017) 2'Deoxyadenosine 5' diphosphoribose is an endogenous TRPM2 superagonist. Nat Chem Biol 13:10361044.
  6. Wolf IM, Diercks BP, Gattkowski E, Czarniak F, Kempski J, Werner R, Schetelig D, Mittrücker HW, Schumacher V, von Osten M, Lodygin D, Flügel A, Fliegert R, Guse AH (2015) Frontrunners of T cell activation: Initial, localized Ca2+ signals mediated by NAADP and the type 1 ryanodine receptor. Sci Signal 8:ra102.
  7. Cordiglieri C, Odoardi F, Zhang B, Nebel M, Kawakami N, Klinkert WE, Lodygin D, Lühder F, Breunig E, Schild D, Ulaganathan VK, Dornmair K, Dammermann W, Potter BV*, Guse AH*, Flügel A* (2010) Nicotinic acid adenine dinucleotide phosphate-mediated calcium signalling in effector T cells regulates autoimmunity of the central nervous system. Brain 133:1930-1943. *equal contribution as last authors
  8. Dammermann W, Zhang B, Nebel M, Cordiglieri C, Odoardi F, Kirchberger T, Kawakami N, Dowden J, Schmid F, Dornmair K, Hohenegger M, Flügel* A, Guse AH*, Potter BV* (2009) NAADP-mediated Ca2+ signaling via type 1 ryanodine receptor in T cells revealed by a synthetic NAADP antagonist. Proc Natl Acad Sci USA 106:10678-10683.
  9. Berg I, Potter BV, Mayr GW, Guse AH. Nicotinic acid adenine dinucleotide phosphate (NAADP+) is an essential regulator of T-lymphocyte Ca2+-signaling. J Cell Biol 2000; 150:581-588.
  10. Guse AH, da Silva CP, Berg I, Skapenko AL, Weber K, Heyer P, Hohenegger M, Ashamu GA, Schulze-Koops H, Potter BV, Mayr GW. Regulation of calcium signalling in T lymphocytes by the second messenger cyclic ADP-ribose. Nature 1999; 398:70-73.

 

 

 

Prof. Dr. Alexander Flügel

Selected publications (*equal contribution)

 

  1. Lodygin D*, Hermann M*, Schweingruber N, Flügel-Koch C, Watanabe T, Schlosser C, Merlini A, Körner H, Chang H-F, Fischer HJ, Reichardt HM, Zagrebelsky M, Mollenhauer B, Frahm J, Stadelmann C, Kügler S, Fitzner D, Haberl M, Odoardi F* & Flügel A* (2019) β Synuclein reactive T cells induce autoimmune CNS grey matter degeneration. Nature 566:503-508.
  2. Flach A*, Litke T*, Strauss J*, Haberl M, Cordero Gómez C, Reindl M, Saiz A, Fehling HJ, Wienands J, Odoardi F, Lühder F, Flügel A (2016) Autoantibody-boosted T-cell reactivation in the target organ triggers manifestation of autoimmune CNS disease. Proc Natl Acad Sci USA 113: 3323-3328.
  3. Schläger C*, Körner H*, Krueger M, Vidoli S, Haberl M, Mielke D, Brylla E, Issekutz T, Cabañas C, Nelson PJ, Ziemssen T, Rohde V, Bechmann I, Lodygin D, Odoardi F*, Flügel A*. (2016) Effector T-cell trafficking between the leptomeninges and the cerebrospinal fluid. Nature 530:349-353. *equal contribution
  4. Lodygin D, Odoardi F, Schläger C, Körner H, Kitz A, Nosov M, van den Brandt J, Reichardt HM, Haberl M, Flügel A (2013) A combination of fluorescent NFAT and H2B sensors uncovers dynamics of T cell activation in real time during CNS autoimmunity. Nat Med 19:784-790.
  5. Odoardi F, Sie C, Streyl K, Ulaganathan VK, Schläger C, Lodygin D, Heckelsmiller K, Nietfeld W, Ellwart J, Klinkert WE, Lottaz C, Nosov M, Brinkmann V, Spang R, Lehrach H, Vingron M, Wekerle H, Flügel-Koch C, Flügel A (2012) T cells become licensed in the lung to enter the central nervous system. Nature 488:675-679.
  6. Cordiglieri C, Odoardi F, Zhang B, Nebel M, Kawakami N, Klinkert WE, Lodygin D, Lühder F, Breunig E, Schild D, Ulaganathan VK, Dornmair K, Dammermann W, Potter BV*, Guse AH*, Flügel A* (2010) Nicotinic acid adenine dinucleotide phosphate-mediated calcium signalling in effector T cells regulates autoimmunity of the central nervous system. Brain 133:1930-1943. *equal contribution as last authors
  7. Bartholomäus I*, Kawakami N*, Odoardi F, Schläger C, Miljkovic D, Ellwart JW, Klinkert WE, Flügel-Koch C, Issekutz TB, Wekerle H, Flügel A (2009) Effector T cell interactions with meningeal vascular structures in nascent autoimmune CNS lesions. Nature 462:94-98. *equal contribution
  8. Dammermann W, Zhang B, Nebel M, Cordiglieri C, Odoardi F, Kirchberger T, Kawakami N, Dowden J, Schmid F, Dornmair K, Hohenegger M, Flügel A*, Guse AH*, Potter BV* (2009) NAADP-mediated Ca2+ signaling via type 1 ryanodine receptor in T cells revealed by a synthetic NAADP antagonist. Proc Natl Acad Sci USA 106:10678-10683.
  9. Odoardi F, Kawakami N, Klinkert WEF, Wekerle H, Flügel A (2007) Blood-born soluble protein antigen intensifies T cell activation in autoimmune CNS lesions and exacerbates clinical disease. Proc Natl Acad Sci USA 104:18625-18630.

 

 

 

Our Team

Prof. Dr. Dr. Andreas H. Guse

Department of Biochemistry and Molecular Cell Biology

University Medical Center Hamburg-Eppendorf (UKE)

M.Sc. Imrankhan Khansahib

Department of Biochemistry and Molecular Cell Biology

University Medical Center Hamburg-Eppendorf (UKE)

M.Sc. Feng Gu

 
Department of Biochemistry and Molecular Cell Biology

University Medical Center Hamburg-Eppendorf (UKE)

Prof. Dr. Alexander Flügel

Institute of Neuroimmunology and Multiple Sclerosis Research
University Medical Center Göttingen (UMG)
 

Dr. Marc-André Lécuyer

Institute of Neuroimmunology and Multiple Sclerosis Research
University Medical Center Göttingen (UMG)
 

Dr. Henrike Körner

 
Institute of Neuroimmunology and Multiple Sclerosis Research
University Medical Center Göttingen (UMG)
 

Dr. Dmitri Lodygin

 
Institute of Neuroimmunology and Multiple Sclerosis Research
University Medical Center Göttingen (UMG)
 

Alumni

Dr. Yunpeng Zhang

Department of Biochemistry and Molecular Cell Biology

University Medical Center Hamburg-Eppendorf (UKE)

Contact

University Medical Center Hamburg-Eppendorf 
Department of Biochemistry and Molecular Cell Biology 
Martinistrasse 52
20246 Hamburg

Scientific Coordinator

Dr. Björn-Philipp Diercks
Fon: +49  (0) 40 7410 54338
E-Mail: b.diercks©uke.de

Administration

Laura Mitsching
Fon: +49  (0) 40 7410 50301
E-Mail: l.mitsching©uke.de