Regulation of adenosine levels as a new therapeutic strategy for Rett Syndrome

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Challenges of BDNF-based therapies : from common to rare diseases

Publication . Miranda-Lourenço, Catarina; Ribeiro Rodrigues, Leonor; Fonseca-Gomes, João; Tanqueiro, Sara; Belo, Rita F.; Ferreira, Catarina B.; Rei, Nádia; Ferreira-Manso, Mafalda; de Almeida-Borlido, Carolina; Costa-Coelho, Tiago; Freitas, Céline; Zavalko, Svitlana; Mouro, Francisco; Sebastião, Ana M; Xapelli, Sara; Rodrigues, Tiago M.; Diógenes, Maria José

Neurotrophins are a well-known family of neurotrophic factors that play an important role both in the central and peripheral nervous systems, where they modulate neuronal survival, development, function and plasticity. Brain-derived neurotrophic factor (BDNF) possesses diverse biological functions which are mediated by the activation of two main classes of receptors, the tropomyosin-related kinase (Trk) B and the p75 neurotrophin receptor (p75NTR). The therapeutic potential of BDNF has drawn attention since dysregulation of its signalling cascades has been suggested to underlie the pathogenesis of both common and rare diseases. Multiple strategies targeting this neurotrophin have been tested; most have found obstacles that ultimately hampered their effectiveness. This review focuses on the involvement of BDNF and its receptors in the pathophysiology of Alzheimer's disease (AD), Amyotrophic Lateral Sclerosis (ALS) and Rett Syndrome (RTT). We describe the known mechanisms leading to the impairment of BDNF/TrkB signalling in these disorders. Such mechanistic insight highlights how BDNF signalling compromise can take various shapes, nearly disease-specific. Therefore, BDNF-based therapeutic strategies must be specifically tailored and are more likely to succeed if a combination of resources is employed.

2020Journal article

Restricted access

Unexpected short- and long-term effects of chronic adolescent HU-210 exposure on emotional behavior

Publication . Farinha Ferreira, Jorge Miguel; Rei, Nádia; Fonseca-Gomes, João; Miranda-Lourenço, Catarina; Serrão, Paula; Vaz, Sandra H.; Gomes, Joana I.; Martins, Valéria; Pereira, Beatriz de Alves; Sebastião, Ana M

Chronic adolescent cannabinoid receptor agonist exposure has been shown to lead to persistent increases in depressive-like behaviors. This has been a key obstacle to the development of cannabinoid-based therapeutics. However, most of the published work has been performed with only three compounds, namely Δ9-tetrahydrocannabinol, CP55,940 and WIN55,212-2. Hypothesizing that different compounds may lead to distinct outcomes, we herein used the highly potent CB1R/CB2R full agonist HU-210, and first aimed at replicating cannabinoid-induced long-lasting effects, by exposing adolescent female Sprague-Dawley rats to increasing doses of HU-210, for 11 days and testing them at adulthood, after a 30-day drug washout. Surprisingly, HU-210 did not significantly impact adult anxious- or depressive-like behaviors. We then tested whether chronic adolescent HU-210 treatment resulted in short-term (24h) alterations in depressive-like behavior. Remarkably, HU-210 treatment simultaneously induced marked antidepressant- and prodepressant-like responses, in the modified forced swim (mFST) and sucrose preference tests (SPT), respectively. Hypothesizing that mFST results were a misleading artifact of HU-210-induced behavioral hyperreactivity to stress, we assessed plasmatic noradrenaline and corticosterone levels, under basal conditions and following an acute swim-stress episode. Notably, we found that while HU-210 did not alter basal noradrenaline or corticosterone levels, it greatly augmented the stress-induced increase in both. Our results show that, contrary to previously studied cannabinoid receptor agonists, HU-210 does not induce persisting depressive-like alterations, despite inducing marked short-term increases in stress-induced reactivity. By showing that not all cannabinoid receptor agonists may induce long-term negative effects, these results hold significant relevance for the development of cannabinoid-based therapeutics.

2022Journal article

Open access

Adenosinergic system and BDNF signaling changes as a cross-sectional feature of RTT: characterization of Mecp2 heterozygous mouse females

Publication . Miranda-Lourenço, Catarina; Rosa, Jéssica; Rei, Nádia; Belo, Rita F.; Lopes, Ana Luísa; Silva, Diogo; Vieira, Cátia; Magalhães-Cardoso, Teresa; Viais, Ricardo; Correia-de-Sá, Paulo; Sebastião, Ana M; Diógenes, Maria José

Rett Syndrome is an X-linked neurodevelopmental disorder (RTT; OMIM#312750) associated to MECP2 mutations. MeCP2 dysfunction is seen as one cause for the deficiencies found in brain-derived neurotrophic factor (BDNF) signaling, since BDNF is one of the genes under MeCP2 jurisdiction. BDNF signaling is also dependent on the proper function of the adenosinergic system. Indeed, both BDNF signaling and the adenosinergic system are altered in Mecp2-null mice (Mecp2-/y), a representative model of severe manifestation of RTT. Considering that symptoms severity largely differs among RTT patients, we set out to investigate the BDNF and ADO signaling modifications in Mecp2 heterozygous female mice (Mecp2+/-) presenting a less severe phenotype. Symptomatic Mecp2+/- mice have lower BDNF levels in the cortex and hippocampus. This is accompanied by a loss of BDNF-induced facilitation of hippocampal long-term potentiation (LTP), which could be restored upon selective activation of adenosine A2A receptors (A2AR). While no differences were observed in the amount of adenosine in the cortex and hippocampus of Mecp2+/- mice compared with healthy littermates, the density of the A1R and A2AR subtype receptors was, respectively, upregulated and downregulated in the hippocampus. Data suggest that significant changes in BDNF and adenosine signaling pathways are present in an RTT model with a milder disease phenotype: Mecp2+/- female animals. These features strengthen the theory that boosting adenosinergic activity may be a valid therapeutic strategy for RTT patients, regardless of their genetic penetrance.

2023Journal article

Open access

Regulation of adenosine levels as a new therapeutic strategy for Rett Syndrome

Publication . Miranda-Lourenço, Catarina; Nogueira, Maria José de Oliveira Diógenes

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.

2022-01Doctoral thesis

Embargoed access

S327 phosphorylation of the presynaptic protein SEPTIN5 increases in the early stages of neurofibrillary pathology and alters the functionality of SEPTIN5

Publication . Ferreira, Catarina B.; Marttinen, Mikael; Coelho, Joana E; Paldanius, Kaisa M.A.; Takalo, Mari; Mäkinen, Petra; Leppänen, Luukas; Miranda-Lourenço, Catarina; Fonseca-Gomes, João; Tanqueiro, Sara; Vaz, Sandra H.; Belo, Rita F.; Sebastião, Ana M; Leinonen, Ville; Soininen, Hilkka; Pike, Ian; Haapasalo, Annakaisa; Lopes, Luisa V.; De Mendonça, Alexandre; Diógenes, Maria José; Hiltunen, Mikko

Alzheimer's disease (AD) is the most common form of dementia, which is neuropathologically characterized by extracellular senile plaques containing amyloid-β and intracellular neurofibrillary tangles composed of hyperphosphorylated tau protein. Previous studies have suggested a role for septin (SEPTIN) protein family members in AD-associated cellular processes. Here, we elucidated the potential role of presynaptic SEPTIN5 protein and its post-translational modifications in the molecular pathogenesis of AD. RNA and protein levels of SEPTIN5 showed a significant decrease in human temporal cortex in relation to the increasing degree of AD-related neurofibrillary pathology. Conversely, an increase in the phosphorylation of the functionally relevant SEPTIN5 phosphorylation site S327 was observed already in the early phases of AD-related neurofibrillary pathology, but not in the cerebrospinal fluid of individuals fulfilling the criteria for mild cognitive impairment due to AD. According to the mechanistic assessments, a link between SEPTIN5 S327 phosphorylation status and the effects of SEPTIN5 on amyloid precursor protein processing and markers of autophagy was discovered in mouse primary cortical neurons transduced with lentiviral constructs encoding wild type SEPTIN5 or SEPTIN5 phosphomutants (S327A and S327D). C57BL/6 J mice intrahippocampally injected with lentiviral wild type SEPTIN5 or phosphomutant constructs did not show changes in cognitive performance after five to six weeks from the start of injections. However, SEPTIN5 S327 phosphorylation status was linked to changes in short-term synaptic plasticity ex vivo at the CA3-CA1 synapse. Collectively, these data suggest that SEPTIN5 and its S327 phosphorylation status play a pivotal role in several cellular processes relevant for AD.

2022Journal article

Open access