The role of TGF-ß2 in the KCC2-dependent maturation of GABAergic neurotransmission
Final Report Abstract
In the immature brain, GABA is depolarizing and excitatory, whereas in the mature brain GABAA receptor-mediated responses are inhibitory and hyperpolarizing. This "developmental shift” in GABAergic neurotransmission during development is mainly controlled by the spatial and temporal expression of KCC2. The aims of the project were: (i) Elucidation of the developmental requirement for TGF-β2 in the KCC2-mediated maturation of GABAergic synapses in the hippocampus; (ii) Understanding of the biological significance of TGF-β2 in the development and function of neuronal networks; (iii) Elucidating the underlying molecular basis of TGF-β2-mediated maturation of GABAergic neurotransmission. To address these aims we have used mouse hippocampal primary neuronal cultures, mouse cortical glial cultures, and Tgf-β2-/- mice as experimental models together with of state-of-the-art methods, among them 3D stimulated emission depletion (STED) microscopy, efficacy of KCC2-mediated Cl- extrusion, measurement of intracellular H+ concentration, gain- and lossof-function experiments, co-immunoprecipitation, and ChiP. The results obtained from our work have introduced TGF-b2 as a novel regulatory factor of KCC2 functional activation. We show that TGF-β2 differentially regulates KCC2 in immature and mature neurons. TGF-β2 regulates KCC2 transcription in immature neurons, and is required for membrane trafficking and functionality of KCC2 in differentiating and mature neurons. Importantly, TGF-β2 initiates KCC2-mediated Cl- extrusion, a prerequisite for the ontogenetic change in GABAA-mediated responses from depolarizing to hyperpolarizing. We have also provided first evidence that Rab GTPases play a critical role on KCC2 trafficking and activity. We have identified the signalling pathway TGF-β2/CREB/Rab11b/KCC2 as the molecular mechanism underlying TGF-b2-dependent KCC2 trafficking and functionality. In neurons of preBötC of Tgf-β2-/- mutants KCC2 has not reached the membrane, the phenotype can be rescued in vitro by exogenous TGF-β2. Taken together, we propose a scenario, in which TGF-β2 may activate CREB by inducing its phosphorylation, which in turn induces Rab11b expression. Rab11b acting downsteam of CREB mediates TGF-β2-dependent KCC2 trafficking and incorporation in the neuronal plasma membrane, ultimately leading to activation of KCC2. In this scenario, fast functional modulation of KCC2 during physiological conditions is achieved through posttranslational events rather than regulation of the transporter at the transcriptional level. In astrocytes, activation of TGF-β signalling regulates transcription of NBCe1, the major player for establishing and maintaining extracellular pH. By controlling the ionic environment, NBCe1 modulates neuronal signalling and synaptic transmission. We have introduced TGF- β as novel regulator of NBCe1 surface expression and NBCe1 as a novel target gene of TGF-β. We propose a model in which TGF-β in the extracellular space becomes activated and acts at the autocrine and/or paracrine mode to the astrocytes by binding to the TGF-β receptor and activating TGF-β canonical and JNK signalling pathways, leading to regulation of NBCe1 functional expression. In this context TGF-β2 is necessary but not sufficient. In summary, the results of our work propose an overall requirement for TGF-β2 for the developmental shift of GABAergic transmission. Moreover the results have provided important mechanistic insights in the molecular network underlying regulation of neuronal KCC2 and glial NBCe1.
Publications
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(2017) TGF-β signaling directly regulates transcription and functional expression of the electrogenic sodium bicarbonate cotransporter 1, NBCe1 (SLC4A4), via Smad4 in mouse astrocytes. Glia 65 (8) 1361–1375
Khakipoor, Shokoufeh; Ophoven, Christian; Schrödl-Häußel, Magdalena; Feuerstein, Melanie; Heimrich, Bernd; Deitmer, Joachim W.; Roussa, Eleni
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(2016) The membrane trafficking and functionality of the K+ /Cl- cotransporter 2 KCC2 is regulated by TGF-β2. J Cell Science 129: 3485-3498
Roussa E., Speer JM, Chudotvorova I, Khakipoor S, Smirnov S, Rivera C, Krieglstein K
