Calcium and cAMP homeostasis determine network organisation of respiratory pre-Bötzinger neurons in Mecp2 null mice in vitro

Rett Syndrome (RS) is a neurodevelopmental disorder caused primarily by mutations in a Mecp2 gene on the X chromosome that encodes methyl-cytosine binding protein (MeCP2). MeCP2 acts as a transcriptional repressor and an activator for a number of genes such as DLX5/ Dlx5 and Bdnf. One of the effects of diminished Mecp2 expression activity is the appearance of a Rett-like phenotype including tremors, severe breathing irregularities, and hypoactivity. Neurons of pre-Botzinger complex (preBotC), the most important component of the complex respiratory network on which the generation of respiratory rhythm in mammals is relied were used to study possible origins of breathing disturbances. The research was focused on two major players of intracellular signal transduction, Ca2+ and cAMP, involved in various events of neuronal activity and plasticity, to establish disturbances in signal transduction, excitability, and higher vulnerability of the Mecp2-/y (KO) preBotC neurons during early postnatal development. Using recently developed methods of transduction of living neural tissue with neuron-specific fluorescent sensors, D3cpv calcium sensor and exchange protein directly activated by cAMP (Epac1-camps) were introduced into the organotypic slices from wild-type (WT) and model knockout (KO) mice. Thereby differences in the spatial organisation of neurons in preBotC and disturbances in intracellular Ca2+ ([Ca2+]i) and cAMP ([cAMP]i) homeostasis in mutant mice which appeared in RS mouse model during early postnatal development were observed. Calcium buffering in KO neurons was indicated by increased amplitude and kinetics of depolarisation-induced calcium transients related to insufficient calcium uptake into the endoplasmic reticulum. Brief hypoxia and calcium release from internal stores induced global calcium increases, after which the processes of many KO neurons were retracted. The effects were restored after a treatment with BDNF while inhibition of BDNF signalling in WT neurons produced disturbances in calcium buffering similar to those observed in KO mice. The data obtained recently point to a direct connection between calcium homeostasis and long-term changes in neuronal connectivity. It can be therefore proposed that calcium-dependent retraction of neurites in preBotC neurons can retard the development of the neural network and set up conditions for appearance of breathing irregularities in RS. Transduction of organotypic slices with a sensor based on a cAMP-dependent GTP-exchange factor (Epac1-camps) allowed us to measure the intracellular distribution of cAMP, its absolute levels and time-dependent changes in response to different physiological stimuli. After modulation by adenylate cyclase (AC), inhibition of phosphodiesterase (PDE) and activation of G-protein-coupled metabotropic receptors, [cAMP]i changes in μM range were recorded. Membrane depolarisation and Ca2+ release from internal stores slowly increased [cAMP]i levels. These effects were suppressed after AC blockade with 2′5′-dideoxyadenosine, and potentiated after inhibiting PDE with isobutylmethylxanthine or rolipram. Inhibition of proteinkinase A (PKA) with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide hydrochloride (H-89) in turn abolished these effects. This indicates the important role of phosphorylation of voltage-sensitive Ca2+ channels in the potentiation of [cAMP]i transients. A crosstalk between Ca2+ and cAMP signalling that revealed a synergism of actions of these two second messengers was investigated. Resting [cAMP]i levels in KO neurons were lower and transient [cAMP]i changes were smaller and faster, but both features were corrected by BDNF to those of the WT. The results obtained indicate that KO neurons have significant disturbances in calcium and cAMP homeostasis that is possibly responsible for higher excitability and vulnerability of neurons. Calcium-induced retraction of neurites can retard formation of properly functioning respiratory network and produce instabilities in regular breathing. An important result that BDNF corrected both calcium and cAMP homeostasis corroborates the present view of the important role of MeCP2-regulated expression and secretion of BDNF in neuronal development. This supports the current opinion about the special role of BDNF in the development of RS that has been derived from biochemical and molecular biology studies.