Serotonylation of neuronal proteins by transglutaminases - novel mechanisms in neuronal plasticity

• Applicant: Professor Dr. Patrick Schloss
• Subject Area: Biological Psychiatry ; Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2012 to 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 228887491

• Final Report Year: 2017

Final Report Abstract

Serotonylation is a relatively new protein modification which has first been described in this millennium. It is the covalent transamidation of serotonin to selective glutamine residues and is specifically mediated by tranglutaminases (TGases). Serotonylation has been shown to regulate the activity of small GTPases and to be necessary for the cross-linking of blood platelets and the formation of blood clots. The objective of this project was to find out whether serotonylation also occurs in the CNS and whether it may play a role in neuronal plasticity. The major idea behind was that serotonylation of cell surface and/or extracellular neuronal and glial proteins could lead to cross-linked matrices which in turn stabilize neural connectivity and/or promote synaptogenesis. This would mean that serotonin in the brain does not only function as a neurotransmitter but also as kind of “neuronal glue”. Therefore we first developed a method by which we could unequivocally show that the application of recombinant TGase specifically transamidates [³H]5-HT to mouse brain homogenates and glial C6 cells. This serotonylation was inhibited by the selective TGase inhibitor cystamine and was clearly different from [³H]5-HT binding to receptors. Moreover, in these studies we could show that the TGase isoform TGase2 is endogenously expressed in brain, C6 cells and serotonergic neurons. Concurrently, we could show, that TGase-mediated transamidation of unlabeled 5-HT to C6 cells induces an aggregation of extracellular protein matrices adjacent to and between single cells. By Western blotting we identified the ECM protein fibronectin as a target for TGase-mediated serotonylation. Next we demonstrated that the catecholamines dopamine (DA) and noradrenaline (NA) inhibit serotonylation of fibronectin and that DA and NA themselves can be selectively transamidated into fibronectin by TGase. All three biogenic monoamines also block TGase-mediated transamidation of the fluorescent monoamine, monodansylacadaverine (MDC), into fibronectin, suggesting a general mechanism of TGase-mediated ‘‘monoaminylation’’. The covalent incorporation of the fluorescent MDC followed by PAGE, in-gel digestion and mass spectrometry then allowed us to identify more specific targets for monoaminylation in glial cells as well as in cultures of serotonergic neurons. These included proteins of the ECM as well as some cytosolic proteins, but no transmembrane proteins. More recently we could show that co-application of 5-HT and TGase strongly impacts on soma growth and neurite outgrowth of serotonergic neurons and increases synaptogenesis in hippocampal primary neurons. Finally, we could identify α- and β-synucleins as specific targets for monoaminylation. In summary, I think (hope) that these studies have been a starting point in neuroscience for further investigations on to which extent monoaminylation contributes to neuronal plasticity.

Publications

• (2012) Transglutaminase-mediated transamidation of serotonin, dopamine and noradrenaline to fibronectin: evidence for a general mechanism of monoaminylation. FEBS Lett. 586(19):3421-8
  Hummerich R, Thumfart JO, Findeisen P, Bartsch D, Schloss P
  
• (2013) Nongenomic, glucocorticoid receptormediated regulation of serotonin transporter cell surface expression in embryonic stem cell derived serotonergic neurons. Neurosci Lett. 554:115-20
  Lau T, Heimann F, Bartsch D, Schloss P, Weber T
  
• (2015) Differential uptake of fluorescent substrates in serotonergic neurons. ACS Chem Neurosci, 6(12):1906-12
  Matthäus F., Schloss P. and Lau T.
  
• (2015) Impact of preconditioning with retinoic acid during early development on morphological and functional characteristics of human induced pluripotent stem cell-derived neurons. Stem Cell Res.;15(1):30-41
  Horschitz S, Matthäus F, Groß A, Rosner J, Galach M, Greffrath W, Treede RD, Utikal J, Schloss P, Meyer-Lindenberg A.
  
• (2015) Monoaminylation of Fibrinogen and Glia- Derived Proteins: Indication for Similar Mechanisms in Posttranslational Protein Modification in Blood and Brain. ACS Chem Neurosci. 6(7):1130-1136
  Hummerich R, Costina V, Findeisen P, Schloss P
  
• (2015) Self-Sufficient Stem Cells: Stem Cell-Derived Serotonergic Neurons Rely on Endogenous BDNF Release to Establish Serotonergic Networks during Terminal Differentiation. Biochem. Pharmacol. (Los Angel) 4: 194
  Krause E., Schloss P. and Lau, T.
  
• (2015) Serotonin stimulates secretion of exosomes from microglia cells. Glia 63 (4) 626-634
  Glebov K, Löchner M, Jabs R, Lau T, Merkel O, Schloss P, Steinhäuser C, Walter J
  
• (2015) Visualization of neurotransmitter uptake and release in serotonergic neurons. J Neurosci Methods. 241:10-7
  Lau T, Proissl V, Ziegler J, Schloss P