Author(s): Miranda-Lourenço, Catarina
Date: 2022
Persistent ID: http://hdl.handle.net/10451/57813
Origin: Repositório da Universidade de Lisboa
Subject(s): Síndrome de Rett; Sistema adenosinérgico; Recetores A1 e A2A; Fator neurotrófico derivado do cérebro (BDNF); Terapias com aumento da adenosina; Rett Syndrome; Adenosinergic system; Adenosine A1 and A2A receptors; Adenosine augmentation therapy
Rett Syndrome (RTT) is a rare, genetically caused neurodevelopmental disorder that affects approximately 1:10000-15000 girls worldwide. It is characterized by an apparently normal development up to 6 to 18 months of age, followed by a regression phase, in which there is a loss of acquired abilities. During the progression of this disease, which takes place over four distinct stages, the following stand out: appearance of stereotyped and repetitive hand movements with progressive loss of functionality, cognitive and motor dysfunction and epilepsy. Currently, this disease has no cure and there are few therapeutic options for symptomatic control, which makes this disease devastating for both patients and caregivers. Genetic studies carried out in recent years have established that this syndrome is mainly due to mutations in the methyl-CpG-binding protein 2 (MECP2) gene, located on the X chromosome. This gene encodes the MeCP2 protein, which performs multiple functions where its role stands out as an epigenetic modulator and regulator of the structure of chromatin, controlling the expression of several other genes, making it a key protein in the development and maturation of the central nervous system (CNS). One of the proteins whose expression is controlled by MeCP2 is the brain-derived neurotrophic factor (BDNF), a neurotrophin with essential functions in cell maturation and differentiation, synaptic plasticity and neuronal survival. Consequently, alterations in MeCP2 compromise BDNF expression levels and function, and this evidence has already been demonstrated in multiple studies in RTT animal models. Those studies have also shown that the increase in BDNF expression can reverse some of the dysfunctions and symptoms present in RTT animal models. However, the therapeutic use of BDNF is not yet applicable since the blood-brain barrier (BBB) is impervious to this neurotrophic factor, preventing it from reaching the brain and performing its functions properly. In an attempt to facilitate the effects of BDNF, new strategies have been developed involving, for example, the use of molecules that cross the BBB and potentiate the neuroprotective action of BDNF. One of the molecules that has deserved particular attention is adenosine. Adenosine is a CNS neuromodulator that exerts its functions through the activation of four receptors, A1, A2A, A3 and A2B (AR). In particular, the activation of A2AR is crucial for the maintenance of BDNF and its receptor, TrkB-FL (full-length tropomyosin-related kinase B), levels as well as for its synaptic effects. It is noteworthy that the adenosinergic system, in addition to be crucial in BDNF-mediated signaling, also has a prominent role in the control of synaptic excitability through the activation of inhibitory A1 receptors, recognized as potential therapeutic targets in the control of epilepsy. These actions of adenosine, as well as the presence of some symptoms in patients with RTT overlapping with diseases with adenosinergic dysfunction already described, suggest the possibility that this neuromodulator is also affected in RTT. Thus, this project aimed to: 1) characterize in detail the adenosinergic system and BDNF-mediated signaling, through the use of models with distinct phenotypes: 1.1) animal model with an severe phenotype, Mecp2-null mutant male mice (Mecp2-/ y); 1.2) animal model with a moderate phenotype, female mice heterozygous for Mecp2 (Mecp2+/-); and 2) explore the augmentation of adenosine levels as a possible therapeutic strategy. Through Western-Blot (WB) assays it was possible to detect decreased BDNF protein levels in hippocampus, cortex, brainstem, and cerebellum homogenates obtained from Mecp2-/y symptomatic animals. The changes detected in the pre-symptomatic stage were less marked, with only a decrease in BDNF levels observed in the striatum of 3-week-old Mecp2-/y animals. Also, in Mecp2+/- animals, decreases in BDNF levels were detected both in the cortex and in the hippocampus in the symptomatic stage. Regarding protein levels of BDNF receptors, decreases in TrkB-FL were observed in the symptomatic stage in cortical and hippocampal homogenates, and in the pre-symptomatic phase in the cortex and striatum. In this brain area, an increase in TrkB-FL levels was also observed in the symptomatic phase. Analyzing the protein levels of truncated isoforms of the TrkB receptor (TrkB-Tc), negative modulators of BDNF action, an increase were detected in brainstem homogenates from 1-week and 6-week-old animals. In Mecp2+/- animals, no alterations were detected in any of the studied BDNF receptors. Electrophysiological recordings performed in the hippocampus allowed the study of synaptic plasticity, namely through the study of long-term potentiation (LTP). In Mecp2-/y animals there was a decrease in the magnitude of LTP and an absence of the facilitatory effect of BDNF upon LTP. In Mecp2+/- animals, although the basal magnitude of LTP was not affected, BDNF also lost its ability to potentiate this phenomenon associated with synaptic plasticity. The study of the adenosinergic system, analyzed by high performance liquid chromatography (HPLC), allowed to detect a decrease in the levels of adenosine and its precursor adenosine monophosphate (AMP), both in hippocampal and cortical homogenates of Mecp2-/y animals in the symptomatic stage. Simultaneously, increases in protein levels of A1R in the cortex and hippocampus and a decrease in A2AR in the cortex were found. The same changes in adenosine receptors were found in Mecp2+/- animals, but also a decrease in A2AR levels in the hippocampus. Despite the decrease in BDNF levels, in both models, it was possible to recover the facilitatory effect of BDNF upon the magnitude of hippocampal LTP, through the activation of A2AR by the selective agonist CGS21680. These data supported the hypothesis that a therapy targeting the adenosinergic system could be beneficial. Thus, 5-6 weeks old Mecp2-/y animals were administered intraperitoneally with an adenosine kinase (ADK) inhibitor drug, 5-iodotubercidin (ITU). This drug allows an increase in adenosine levels by inhibiting its metabolism. In addition to the efficacy of ADK inhibition already demonstrated in other diseases, such as epilepsy models, in Mecp2-/y animals, by WB analysis, increased ADK protein levels were detected in cortex homogenates during the pre-symptomatic stage, supporting the study of ADK as a potential therapeutic target in RTT. Through the study of ITU administration, carried out in vivo, it was possible to observe a recovery of the effect of BDNF upon LTP potentiation, in electrophysiological recordings performed in hippocampal slices, as well as a recovery of protein levels of TrkB-FL receptors in hippocampal homogenates from ITU-treated Mecp2-/y animals. Overall, the results point to a dysfunction either in signaling mediated by BDNF and in adenosinergic system, in two different phenotypes of the disease, suggesting a possible involvement of both in the pathophysiology of the disease. The positive data obtained regarding the reversal of some deficits present in the animal models studied, through the pharmacological inhibition of ADK, reinforces the importance of adenosinergic system involvement while suggesting the increase of adenosine levels as a strategy to be explored in RTT.
