Adenosinergic System and BDNF Signaling Changes as a Cross-Sectional Feature of RTT: Characterization of Mecp2 Heterozygous Mouse Females.

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.

Impairment of adenosinergic system in Rett syndrome: Novel therapeutic target to boost BDNF signalling.

Rett syndrome (RTT; OMIM#312750) is mainly caused by mutations in the X-linked MECP2 gene (methyl-CpG-binding protein 2 gene; OMIM*300005), which leads to impairments in the brain-derived neurotrophic factor (BDNF) signalling. The boost of BDNF mediated effects would be a significant breakthrough but it has been hampered by the difficulty to administer BDNF to the central nervous system. Adenosine, an endogenous neuromodulator, may accomplish that role since through A2AR it potentiates BDNF synaptic actions in healthy animals. We thus characterized several hallmarks of the adenosinergic and BDNF signalling in RTT and explored whether A2AR activation could boost BDNF actions. For this study, the RTT animal model, the Mecp2 knockout (Mecp2-/y) (B6.129P2 (C)-Mecp2tm1.1Bird/J) mouse was used. Whenever possible, parallel data was also obtained from post-mortem brain samples from one RTT patient. Ex vivo extracellular recordings of field excitatory post-synaptic potentials in CA1 hippocampal area were performed to evaluate synaptic transmission and long-term potentiation (LTP). RT-PCR was used to assess mRNA levels and Western Blot or radioligand binding assays were performed to evaluate protein levels. Changes in cortical and hippocampal adenosine content were assessed by liquid chromatography with diode array detection (LC/DAD). Hippocampal ex vivo experiments revealed that the facilitatory actions of BDNF upon LTP is absent in Mecp2-/y mice and that TrkB full-length (TrkB-FL) receptor levels are significantly decreased. Extracts of the hippocampus and cortex of Mecp2-/y mice revealed less adenosine amount as well as less A2AR protein levels when compared to WT littermates, which may partially explain the deficits in adenosinergic tonus in these animals. Remarkably, the lack of BDNF effect on hippocampal LTP in Mecp2-/y mice was overcome by selective activation of A2AR with CGS21680. Overall, in Mecp2-/y mice there is an impairment on adenosinergic system and BDNF signalling. These findings set the stage for adenosine-based pharmacological therapeutic strategies for RTT, highlighting A2AR as a therapeutic target in this devastating pathology.

Opposing Effects of Adenosine and Inosine in Human Subcutaneous Fibroblasts May Be Regulated by Third Party ADA Cell Providers.

Human subcutaneous fibroblasts (HSCF) challenged with inflammatory mediators release huge amounts of ATP, which rapidly generates adenosine. Given the nucleoside's putative relevance in wound healing, dermal fibrosis, and myofascial pain, we investigated the role of its precursor, AMP, and of its metabolite, inosine, in HSCF cells growth and collagen production. AMP (30 µM) was rapidly (t½ 3 ± 1 min) dephosphorylated into adenosine by CD73/ecto-5'-nucleotidase. Adenosine accumulation (t½ 158 ± 17 min) in the extracellular fluid reflected very low cellular adenosine deaminase (ADA) activity. HSCF stained positively against A2A and A3 receptors but were A1 and A2B negative. AMP and the A2A receptor agonist, CGS21680C, increased collagen production without affecting cells growth. The A2A receptor antagonist, SCH442416, prevented the effects of AMP and CGS21680C. Inosine and the A3 receptor agonist, 2Cl-IB-MECA, decreased HSCF growth and collagen production in a MRS1191-sensitive manner, implicating the A3 receptor in the anti-proliferative action of inosine. Incubation with ADA reproduced the inosine effect. In conclusion, adenosine originated from extracellular ATP hydrolysis favors normal collagen production by HSCF via A2A receptors. Inhibition of unpredicted inosine formation by third party ADA cell providers (e.g., inflammatory cells) may be a novel therapeutic target to prevent inappropriate dermal remodeling via A3 receptors activation.

Corpus cavernosum from men with vasculogenic impotence is partially resistant to adenosine relaxation due to endothelial A(2B) receptor dysfunction.

Although adenosine has been implicated in penile erection in human males, the receptor subtype responsible for adenosine regulation of human corpus cavernosum (HCC) smooth muscle tone is still a matter of debate. Using selective adenosine agonists and antagonists, we aimed at characterizing the adenosine receptors mediating relaxation of precontracted (with 1 microM phenylephrine) HCC strips. HCC specimens were collected from control subjects (organ donors) and from patients with severe vasculogenic erectile dysfunction (ED). In control subjects, adenosine and 5'-N-ethyl-carboxamide adenosine (NECA) fully relaxed HCC. The selective A(2A) receptor agonist 2-[4-(2-p-carboxy ethyl)phenylamino]-5'-N-ethylcarboxamido adenosine (CGS21680C) produced only a partial relaxation (30-50%) of HCC, which could be further enhanced by simultaneous application of 100 microM NECA. The selective A(2B) receptor antagonist N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-il)phenoxy] acetamida (MRS1706) (10 nM) attenuated NECA-induced relaxation without affecting CGS21680C action. The A(2A) receptor antagonist 4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol (ZM241385) (50 nM) consistently reduced the actions of both agonists. In contrast to CGS21680C, NECA-induced relaxation was attenuated when endothelial production of NO and prostanoids was reduced by 100 microM N(G)-nitro-l-arginine and 10 microM indomethacin, respectively. HCC strips from patients with vasculogenic ED were partially resistant to NECA but kept relaxation to CGS21680C; the remaining effect was sensitive to blockade of A(2A) receptors with 50 nM ZM241385. Data suggest that adenosine regulates HCC smooth muscle tone through the activation of two receptor populations, CGS21680C-sensitive (A(2A)) and -insensitive (A(2B)) receptors, located on smooth muscle fibers and on endothelial cells, respectively. Endothelial dysfunction may be correlated with a loss of adenosine A(2B) receptor activity in penile vessels from men with vasculogenic ED.

Fine-tuning modulation of myenteric motoneurons by endogenous adenosine: on the role of secreted adenosine deaminase.

Besides the well-characterized inhibitory effect of adenosine in the gastrointestinal tract mediated by A1 receptors, we recently demonstrated that endogenously generated adenosine facilitates [3H]acetylcholine release from myenteric neurons through preferential activation of prejunctional A2A receptors. The co-existence of both receptor subtypes on cholinergic neurons prompted the question of how does adenosine discriminate between these receptors to regulate synaptic transmission in the longitudinal muscle-myenteric plexus (LM-MP) of the rat ileum. Electrical stimulation of the LM-MP increased the outflow of adenosine, inosine and hypoxanthine. Myenteric neurons seem to be the main source of endogenous adenosine, since blockade of action potentials with tetrodotoxin (1 microM) or omission of Ca2+ (plus EGTA, 1 mM) in the buffer essentially abolished nucleosides release, while adenosine outflow remained unchanged when smooth muscle contractions were prevented by nifedipine (1 microM). Inhibition of ecto-5'-nucleotidase by concanavalin A (0.1 mg ml-1) produced only a moderate decrease (approximately 25%) on adenosine accumulation in the LM-MP, indicating that the extracellular catabolism of released ATP might not be a major source of the nucleoside. Data using the acetylcholinesterase inhibitor, physiostigmine (10 microM), and several subtype-specific muscarinic receptor antagonists, 4-DAMP (100 nM), AF-DX 116 (10 microM) and muscarinic toxin-7 (1 nM), suggest that cholinergic motoneurons are endowed with muscarinic M3 autoreceptors facilitating the outflow of adenosine. Surprisingly, bath samples collected after stimulating the LM-MP exhibited a relatively high adenosine deaminase (ADA) activity (0.60+/-0.07 U ml-1), which increased in parallel with the accumulation of adenosine and its deamination products. Our findings are in keeping with the hypothesis that ADA secretion, along with a less-efficient dipyridamole-sensitive nucleoside transport system, may restrict endogenous adenosine actions to the synaptic region channelling to facilitatory A2A receptors activation. Such a local environment may also limit diffusion of exogenously added adenosine towards the active zones, as we showed that this constrain may be overcome by inhibiting ADA activity with erythro-9(2-hydroxy-3-nonyl) adenine (50 microM).

Dual effects of adenosine on acetylcholine release from myenteric motoneurons are mediated by junctional facilitatory A(2A) and extrajunctional inhibitory A(1) receptors.

1. The coexistence of both inhibitory A(1) and facilitatory A(2) adenosine receptors in the rat myenteric plexus prompted the question of how adenosine activates each receptor subtype to regulate cholinergic neurotransmission. 2. Exogenously applied adenosine (0.3-300 microm) decreased electrically evoked [(3)H]acetylcholine ([(3)H]ACh) release. Blocking A(1) receptors with 1,3-dipropyl-8-cyclopentylxanthine (10 nm) transformed the inhibitory action of adenosine into a facilitatory effect. Adenosine-induced inhibition was mimicked by the A(1) receptor agonist R-N(6)-phenylisopropyladenosine (0.3 microm), but the A(2A) agonist CGS 21680C (0.003 microm) produced a contrasting facilitatory effect. 3. Increasing endogenous adenosine levels, by the addition of (1) the adenosine precursor AMP (30-100 microm), (2) the adenosine kinase inhibitor 5'-iodotubercidin (10 microm) or (3) inhibitors of adenosine uptake (dipyridamole, 0.5 microm) and of deamination (erythro-9(2-hydroxy-3-nonyl)adenine, 50 microm), enhanced electrically evoked [(3)H]ACh release (5 Hz for 40 s). Release facilitation was prevented by adenosine deaminase (ADA, 0.5 U ml(-1)) and by the A(2A) receptor antagonist ZM 241385 (50 nm); these compounds decreased [(3)H]ACh release by 31+/-6% (n=7) and 37+/-10% (n=6), respectively. 4. Although inhibition of ecto-5'-nucleotidase by alpha,beta-methylene ADP (200 microm) or by concanavalin A (0.1 mg ml(-1)) attenuated endogenous adenosine formation from AMP, analysed by HPLC, the corresponding reduction in [(3)H]ACh release only became evident when stimulation of the myenteric plexus was prolonged to over 250 s. 5. In summary, we found that endogenously generated adenosine plays a predominantly tonic facilitatory effect mediated by prejunctional A(2A) receptors. Extracellular deamination and cellular uptake may restrict endogenous adenosine actions to the neuro-effector region near the release/production sites.