Regulación farmacológica de las Map Quinasas MEK1/2 y ERK1/2 en la señalización de los receptores Gabbaa y NMDAcurso temporal de sueño inducido por agentes hipnóticos/anestésicos en cerebro de ratón

Abstract

The mitogen-activated protein kinases (MAPK) are organized in protein cascades forming signaling pathways that allow them to respond to a wide variety of extracellular stimuli. The ERK1/2 MAPK pathway takes part in a large diversity of physiological processes, including cell proliferation, differentiation and survival, modulation of gene expression and memory formation. The importance of this pathway is reflected in the different disorders related to its deregulation, such as the so-called RASopathies or different types of cancer. Various studies have shown that treatments with anesthetic/hypnotic drugs lead to downregulation of p-ERK1/2 content, an effect that can be related to learning and memory deficits. In the present PhD work, an experimental paradigm of sleep induced by anesthetic/hypnotic drugs was carried out in mice in vivo. Various positive allosteric modulators of the GABAA receptor (midazolam, ethanol, chloral hydrate and isoflurane) and a non-competitive antagonist of the NMDA receptor (ketamine) were used to induce sleep (sleeping time course range: 25-160 min), during which mice were sacrificed at different times. The main objectives of the study were to evaluate how the different anesthetic/hypnotic drugs modulate the sequential phosphorylation of MEK1/2 and ERK1/2 kinases, as well as to study the role of multifunctional FADD forms and the neuroplastic p-FADD/FADD ratio, in the proposed behavioral model. All the drugs used in this study significantly reduced the activation of ERK1/2 MAPKs (ratio of phosphorylated enzyme/total enzyme). Surprisingly, the content of their activating kinases p-MEK1/2 was found increased, indicating the existence of a disruption between the sequential phosphorylation of these two kinases. The relevance of these results relies in the fact that ERK1/2 MAPKs are the only known substrates of the MEK1/2 kinases and the sequential activation between them is accepted as a well-known cellular signaling mechanism. Due to the important role of the ERK1/2 pathway in various cellular processes and the wide range of physiological responses that this pathway is able to generate, it is tightly regulated and there are numerous mechanisms for its response specificity. In this context, some of the potentially involved mechanisms in the observed disruption were assessed, such as some of the phosphatases responsible for ERK1/2 deactivation and one inactivated form of the MEK1 protein (p-Thr286 MEK1). The contents of phosphatase MKP-3 and p-MEK1 were found increased in mouse cerebral cortex during midazolam-induced sleep. Even though these results could partially explain the reduction in the activation of ERK1/2, the complex interaction observed with flumazenil showed that distinct mechanisms other than those studied might be the main responsible ones. In the case of the barbiturate pentobarbital and ketamine the contents of p-MEK1 and MKP-3 were unaltered. 14 During the time course of sleep induced by the different anesthetic/hypnotic drugs of interest, the contents of p-FADD protein in mouse cerebral cortex were significantly increased in all cases. These results, along with the reduction or non-alteration of the total FADD form, led to a very significant increase in the p-FADD/FADD ratio, which could indicate the existence of neuroplasticity mechanisms taking place during the course of anesthetic/hypnotic-induced sleep. The involvement of GABAA receptor in the effects observed during midazolam- and ketamine-induced sleep was assessed with flumazenil, a neutral ligand that behaves as an allosteric antagonist of this receptor. The pharmacological interaction with flumazenil modulated the duration of sleep induced by both drugs, indicating the involvement of GABAA receptor in the molecular systems associated with midazolam-and ketamine-induced sleep. On the other hand, the use of SL-327 (a MEK1/2 inhibitor) to induce a downregulation of p- ERK1/2 content resulted in an increase in the duration of ketamine-induced sleep. This result proves the direct involvement of the modulation of these MAPKs in the mechanisms responsible for sleep induced by this drug. The use of the metabolic inhibitor SKF525-A revealed the implication of several proteins (p-MEK1/2, p-ERK1/2, p-FADD and p-PEA-15) in the molecular processes related to pentobarbital-induced sleep, due to the noted increase in the barbiturate-induced sleep duration and the parallel and persistent regulation of the mentioned proteins. On the other hand, some neuroplasticity markers (PSD-95, NF-κB) and protein PEA-15 were studied throughout the course of drug-induced sleep. The neuroplastic marker NF-κB was increased during midazolam-, ketamine- and pentobarbital-induced sleep. Also, a significant increase in the content of p-PEA-15 was found in pentobarbital and ketamine (in a small extent) induced sleep, in line with previous studies that observed it during midazolaminduced sleep. These results indicate that PEA-15 could be involved in the molecular mechanisms adjacent to drug-induced sleep, as has been observed for FADD and ERK1/2, with which PEA-15 can interact and modulate some of its actions. In conclusion, the results obtained in this PhD work indicate the importance of the brain signaling of ERK1/2 and p-FADD in the sleep induced in mice by different drugs of very distinct characteristics. In addition, a disruption in the sequential activation of MEK1/2 and ERK1/2 kinases was discovered during the time course of sleep induced by all the anesthetic/hypnotic drugs ascertained. In turn, all treatments upregulated p-FADD content and the neuroplastic p-FADD/FADD ratio. The reduction in the activation levels of ERK1/2 together with the increase in p-FADD/FADD ratio and other neuroplastic markers could be 15 related to some of the adverse effects produced by anesthetic/hypnotic drugs, such as amnesia, tolerance or deficits in learning and memory.