ADAR-Mediated A>I(G) RNA Editing in the Genotoxic Drug Response of Breast Cancer.

Epitranscriptomics is a field that delves into post-transcriptional changes. Among these modifications, the conversion of adenosine to inosine, traduced as guanosine (A>I(G)), is one of the known RNA-editing mechanisms, catalyzed by ADARs. This type of RNA editing is the most common type of editing in mammals and contributes to biological diversity. Disruption in the A>I(G) RNA-editing balance has been linked to diseases, including several types of cancer. Drug resistance in patients with cancer represents a significant public health concern, contributing to increased mortality rates resulting from therapy non-responsiveness and disease progression, representing the greatest challenge for researchers in this field. The A>I(G) RNA editing is involved in several mechanisms over the immunotherapy and genotoxic drug response and drug resistance. This review investigates the relationship between ADAR1 and specific A>I(G) RNA-edited sites, focusing particularly on breast cancer, and the impact of these sites on DNA damage repair and the immune response over anti-cancer therapy. We address the underlying mechanisms, bioinformatics, and in vitro strategies for the identification and validation of A>I(G) RNA-edited sites. We gathered databases related to A>I(G) RNA editing and cancer and discussed the potential clinical and research implications of understanding A>I(G) RNA-editing patterns. Understanding the intricate role of ADAR1-mediated A>I(G) RNA editing in breast cancer holds significant promise for the development of personalized treatment approaches tailored to individual patients' A>I(G) RNA-editing profiles.

Guanosine treatment prevents lipopolysaccharide-induced depressive-like behavior in mice.

Guanosine is a purinergic nucleoside that has been shown to have neuroprotective effects, mainly through its ability to modulate the glutamatergic system. An increase in pro-inflammatory cytokine levels triggers the activation of the enzyme indoleamine 2,3-dioxygenase 1 (IDO-1), leading to glutamatergic excitotoxicity, which has important roles in the pathophysiology of depression. The aim of this study was to investigate the possible antidepressant-like effects and underlying mechanisms of action of guanosine against lipopolysaccharide (LPS)-induced depression in a mouse model. Mice were orally pre-treated with saline (0.9% NaCl), guanosine (8 or 16 mg/kg), or fluoxetine (30 mg/kg) for 7 days before LPS (0.5 mg/kg, intraperitoneal) injection. One day after LPS injection, mice were subjected to the forced swim test (FST), tail suspension test (TST), and open field test (OFT). After the behavioral tests, mice were euthanized and the levels of tumor necrosis factor-α (TNF-α), IDO-1, glutathione, and malondialdehyde in the hippocampus were measured. Pretreatment with guanosine was able to prevent LPS- induced depressive-like behaviors in the TST and FST. In the OFT, no locomotor changes were observed with any treatment. Both guanosine (8 and 16 mg/kg/day) and fluoxetine treatment prevented the LPS-induced increase in TNF-α and IDO expression and lipid peroxidation as well as decrease of reduced glutathione levels in the hippocampus. Taken together, our findings suggest that guanosine may have neuroprotective effects against LPS-induced depressive-like behavior through preventing oxidative stress and the expression of IDO-1 and TNF-α in the hippocampus.

NMDA receptor-mediated modulation on glutamine synthetase and glial glutamate transporter GLT-1 is involved in the antidepressant-like and neuroprotective effects of guanosine.

Guanosine has been reported to elicit antidepressant-like responses in rodents, but if these actions are associated with its ability to afford neuroprotection against glutamate-induced toxicity still needs to be fully understood. Therefore, this study investigated the antidepressant-like and neuroprotective effects elicited by guanosine in mice and evaluated the possible involvement of NMDA receptors, glutamine synthetase, and GLT-1 in these responses. We found that guanosine (0.05 mg/kg, but not 0.01 mg/kg, p. o.) was effective in producing an antidepressant-like effect and protecting hippocampal and prefrontocortical slices against glutamate-induced damage. Our results also unveiled that ketamine (1 mg/kg, but not 0.1 mg/kg, i. p, an NMDA receptor antagonist) effectively elicited antidepressant-like actions and protected hippocampal and prefrontocortical slices against glutamatergic toxicity. Furthermore, the combined administration of sub-effective doses of guanosine (0.01 mg/kg, p. o.) with ketamine (0.1 mg/kg, i. p.) promoted an antidepressant-like effect and augmented glutamine synthetase activity and GLT-1 immunocontent in the hippocampus, but not in the prefrontal cortex. Our results also showed that the combination of sub-effective doses of ketamine and guanosine, at the same protocol schedule that exhibited an antidepressant-like effect, effectively abolished glutamate-induced damage in hippocampal and prefrontocortical slices. Our in vitro results reinforce that guanosine, ketamine, or sub-effective concentrations of guanosine plus ketamine protect against glutamate exposure by modulating glutamine synthetase activity and GLT-1 levels. Finally, molecular docking analysis suggests that guanosine might interact with NMDA receptors at the ketamine or glycine/d-serine co-agonist binding sites. These findings provide support for the premise that guanosine has antidepressant-like effects and should be further investigated for depression management.

The antidepressant-like effect of guanosine involves the modulation of adenosine A1 and A2A receptors.

Guanosine has been considered a promising candidate for antidepressant responses, but if this nucleoside could modulate adenosine A1 (A1R) and A2A (A2AR) receptors to exert antidepressant-like actions remains to be elucidated. This study investigated the role of A1R and A2AR in the antidepressant-like response of guanosine in the mouse tail suspension test and molecular interactions between guanosine and A1R and A2AR by docking analysis. The acute (60 min) administration of guanosine (0.05 mg/kg, p.o.) significantly decreased the immobility time in the tail suspension test, without affecting the locomotor performance in the open-field test, suggesting an antidepressant-like effect. This behavioral response was paralleled with increased A1R and reduced A2AR immunocontent in the hippocampus, but not in the prefrontal cortex, of mice. Guanosine-mediated antidepressant-like effect was not altered by the pretreatment with caffeine (3 mg/kg, i.p., a non-selective adenosine A1R/A2AR antagonist), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX - 2 mg/kg, i.p., a selective adenosine A1R antagonist), or 4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo-{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl)-phenol (ZM241385 - 1 mg/kg, i.p., a selective adenosine A2AR antagonist). However, the antidepressant-like response of guanosine was completely abolished by adenosine (0.5 mg/kg, i.p., a non-selective adenosine A1R/A2AR agonist), N-6-cyclohexyladenosine (CHA - 0.05 mg/kg, i.p., a selective adenosine A1 receptor agonist), and N-6-[2-(3,5-dimethoxyphenyl)-2-(methylphenyl)ethyl]adenosine (DPMA - 0.1 mg/kg, i.p., a selective adenosine A2A receptor agonist). Finally, docking analysis also indicated that guanosine might interact with A1R and A2AR at the adenosine binding site. Overall, this study reinforces the antidepressant-like of guanosine and unveils a previously unexplored modulation of the modulation of A1R and A2AR in its antidepressant-like effect.

Guanosine as a promising target for fast-acting antidepressant responses.

Although the rapid-onset and sustained antidepressant responses elicited by ketamine have gained considerable attention in recent years, it has some knock-on effects that limit its widespread clinical use. Therefore, ketamine is considered the prototype for the new generation of glutamate-based rapid-acting antidepressants. Within this context, it has been demonstrated that guanosine, an endogenous guanine-based purine, has overlapping mechanisms of action with ketamine and is effective in eliciting fast antidepressant-like responses and even potentiating ketamine's actions in preclinical studies. Here, we review the recent findings regarding the ability of guanosine to produce rapid-acting antidepressant-like effects and we provide an overview of the molecular mechanisms underlying its antidepressant-like actions. Moreover, the neurobiological mechanisms underpinning the ability of guanosine in boosting the antidepressant-like and pro-synaptogenic effects elicited by ketamine are also reported. Taken together, this review opens perspectives for the use of guanosine alone or in combination with ketamine for the management of treatment-resistant depression.

Recombinant guanosine-5'-triphosphate (GTP)-binding protein associated with Poloxamer 407-based polymeric micelles protects against Leishmania infantum infection.

Leishmania virulence proteins should be considered as vaccine candidates against disease, since they are involved in developing infection in mammalian hosts. In a previous study, a Leishmania guanosine-5'-triphosphate (GTP)-binding protein was identified as a potential parasite virulence factor. In the present work, the gene encoding GTP was cloned and the recombinant protein (rGTP) was evaluated as a vaccine candidate against Leishmania infantum infection. The protein was associated with saponin (rGTP/Sap) or Poloxamer 407-based micelles (rGTP/Mic) as adjuvants, and protective efficacy was investigated in BALB/c mice after parasite challenge. Both rGTP/Sap and rGTP/Mic compositions induced a Th1-type immune response in vaccinated animals, with significantly higher levels of IFN-γ, IL-12, IL-2, TNF-α, GM-CSF, nitrite, specific IgG2a isotype antibody and positive lymphoproliferation, when compared to the control groups. This response was accompanied by significantly lower parasite load in the spleens, livers, bone marrows and draining lymph nodes of the animals. Immunological and parasitological evaluations indicated that rGTP/Mic induced a more polarized Th1-type response and higher reduction in the organ parasitism, and with lower hepatotoxicity, when compared to the use of rGTP/Sap. In conclusion, our preliminary data suggest that rGTP could be considered for further development as a vaccine candidate to protect against VL.

Cerebrospinal fluid purinomics as a biomarker approach to predict outcome after severe traumatic brain injury.

Severe traumatic brain injury (TBI) is associated with high rates of mortality and long-term disability linked to neurochemical abnormalities. Although purine derivatives play important roles in TBI pathogenesis in preclinical models, little is known about potential changes in purine levels and their implications in human TBI. We assessed cerebrospinal fluid (CSF) levels of purines in severe TBI patients as potential biomarkers that predict mortality and long-term dysfunction. This was a cross-sectional study performed in 17 severe TBI patients (Glasgow Coma Scale <8) and 51 controls. Two to 4 h after admission to ICU, patients were submitted to ventricular drainage and CSF collection for quantification of adenine and guanine purine derivatives by HPLC. TBI patients' survival was followed up to 3 days from admission. A neurofunctional assessment was performed through the modified Rankin Scale (mRS) 2 years after ICU admission. Purine levels were compared between control and TBI patients, and between surviving and non-surviving patients. Relative to controls, TBI patients presented increased CSF levels of GDP, guanosine, adenosine, inosine, hypoxanthine, and xanthine. Further, GTP, GDP, IMP, and xanthine levels were different between surviving and non-surviving patients. Among the purines, guanosine was associated with improved mRS (p = 0.042; r = -0.506). Remarkably, GTP displayed predictive value (AUC = 0.841, p = 0.024) for discriminating survival versus non-survival patients up to 3 days from admission. These results support TBI-specific purine signatures, suggesting GTP as a promising biomarker of mortality and guanosine as an indicator of long-term functional disability.

Guanosine boosts the fast, but not sustained, antidepressant-like and pro-synaptogenic effects of ketamine by stimulating mTORC1-driven signaling pathway.

The mTORC1-dependent dendritic spines formation represents a key mechanism for fast and long-lasting antidepressant responses, but it remains to be determined whether this mechanism may account for the ability of guanosine in potentiating ketamine's actions. Here, we investigated the ability of ketamine plus guanosine to elicit fast and sustained antidepressant-like and pro-synaptogenic effects in mice and the role of mTORC1 signaling in these responses. The combined administration of subthreshold doses of ketamine (0.1 mg/kg, i.p.) and guanosine (0.01 mg/kg, p.o.) caused a fast (1 h - 24 h), but not long-lasting (7 days) reduction in the immobility time in the tail suspension test. This behavioral effect was paralleled by a rapid (started in 1 h) and transient (back to baseline in 24 h) increase on BDNF, p-Akt (Ser473), p-GSK-3ß (Ser9), p-mTORC1 (Ser2448), p-p70S6K (Thr389) immunocontent in the hippocampus, but not in the prefrontal cortex. Conversely, ketamine plus guanosine increased PSD-95 and GluA1 immunocontent in the prefrontal cortex, but not the hippocampus after 1 h, whereas increased levels of these proteins in both brain structures were observed after 24 h, but these effects did not persist after 7 days. The combined administration of ketamine plus guanosine raised the dendritic spines density in the ventral hippocampal DG and prefrontal cortex after 24 h Rapamycin (0.2 nmol/site, i.c.v.) abrogated the antidepressant-like effect and pro-synaptogenic responses triggered by ketamine plus guanosine. These results indicate that guanosine may boost the antidepressant-like effect of ketamine for up to 24 h by a mTORC1-dependent mechanism.

Low doses of ketamine and guanosine abrogate corticosterone-induced anxiety-related behavior, but not disturbances in the hippocampal NLRP3 inflammasome pathway.

RATIONALE: Guanosine has been shown to potentiate ketamine's antidepressant-like actions, although its ability to augment the anxiolytic effect of ketamine remains to be determined. OBJECTIVE: This study investigated the anxiolytic-like effects of a single administration with low doses of ketamine and/or guanosine in mice subjected to chronic administration of corticosterone and the role of NLRP3-driven signaling. METHODS: Corticosterone (20 mg/kg, p.o.) was administered for 21 days, followed by a single administration of ketamine (0.1 mg/kg, i.p.), guanosine (0.01 mg/kg, p.o.), or ketamine (0.1 mg/kg, i.p.) plus guanosine (0.01 mg/kg, p.o.). Anxiety-like behavior and NLRP3-related targets were analyzed 24 h following treatments. RESULTS: Corticosterone reduced the time spent in the open arms and the central zone in the elevated plus-maze test and open-field test, respectively. Corticosterone raised the number of unsupported rearings and the number and time of grooming, and decreased the latency to start grooming in the open-field test. Disturbances in regional distribution (increased rostral grooming) and grooming transitions (increased aborted and total incorrect transitions) were detected in corticosterone-treated mice. These behavioral alterations were accompanied by increased immunocontent of Iba-1, ASC, NLRP3, caspase-1, TXNIP, and IL-1ß in the hippocampus, but not in the prefrontal cortex. The treatments with ketamine, guanosine, and ketamine plus guanosine were effective to counteract corticosterone-induced anxiety-like phenotype, but not disturbances in the hippocampal NLRP3 pathway. CONCLUSIONS: Our study provides novel evidence that low doses of ketamine and/or guanosine reverse corticosterone-induced anxiety-like behavior and shows that the NLRP3 inflammasome pathway is likely unrelated to this response.

A low-dose combination of ketamine and guanosine counteracts corticosterone-induced depressive-like behavior and hippocampal synaptic impairments via mTORC1 signaling.

Ketamine exhibits rapid and sustained antidepressant responses, but its repeated use may cause adverse effects. Augmentation strategies have been postulated to be useful for the management/reduction of ketamine's dose and its adverse effects. Based on the studies that have suggested that ketamine and guanosine may share overlapping mechanisms of action, the present study investigated the antidepressant-like effect of subthreshold doses of ketamine and guanosine in mice subjected to repeated administration of corticosterone (CORT) and the role of mTORC1 signaling for this effect. The ability of the treatment with ketamine (0.1 mg/kg, i.p.) plus guanosine (0.01 mg/kg, p.o.) to counteract the depressive-like behavior induced by CORT (20 mg/kg, p.o., for 21 days) in mice, was paralleled with the prevention of the CORT-induced reduction on BDNF levels, Akt (Ser473) and GSK-3ß (Ser9) phosphorylation, and PSD-95, GluA1, and synapsin immunocontent in the hippocampus. No changes on mTORC1 and p70S6K immunocontent were found in the hippocampus and prefrontal cortex of any experimental group. No alterations on BDNF, Akt/GSK-3ß, mTORC1/p70S6K, and synaptic proteins were observed in the prefrontal cortex of mice. The antidepressant-like and pro-synaptogenic effects elicited by ketamine plus guanosine were abolished by the pretreatment with rapamycin (0.2 nmol/site, i.c.v., a selective mTORC1 inhibitor). Our results showed that the combined administration of ketamine and guanosine at low doses counteracted CORT-induced depressive-like behavior and synaptogenic disturbances by activating mTORC1 signaling. This study supports the notion that the combined administration of guanosine and ketamine may be a useful therapeutic strategy for the management of MDD.

Effects of intranasal guanosine administration on brain function in a rat model of ischemic stroke.

Ischemic stroke is a major cause of morbidity and mortality worldwide and only few affected patients are able to receive treatment, especially in developing countries. Detailed pathophysiology of brain ischemia has been extensively studied in order to discover new treatments with a broad therapeutic window and that are accessible to patients worldwide. The nucleoside guanosine (Guo) has been shown to have neuroprotective effects in animal models of brain diseases, including ischemic stroke. In a rat model of focal permanent ischemia, systemic administration of Guo was effective only when administered immediately after stroke induction. In contrast, intranasal administration of Guo (In-Guo) was effective even when the first administration was 3 h after stroke induction. In order to validate the neuroprotective effect in this larger time window and to investigate In-Guo neuroprotection under global brain dysfunction induced by ischemia, we used the model of thermocoagulation of pial vessels in Wistar rats. In our study, we have found that In-Guo administered 3 h after stroke was capable of preventing ischemia-induced dysfunction, such as bilateral suppression and synchronicity of brain oscillations and ipsilateral cell death signaling, and increased permeability of the blood-brain barrier. In addition, In-Guo had a long-lasting effect on preventing ischemia-induced motor impairment. Our data reinforce In-Guo administration as a potential new treatment for brain ischemia with a more suitable therapeutic window.

8-Mercaptoguanine-based inhibitors of Mycobacterium tuberculosis dihydroneopterin aldolase: synthesis, in vitro inhibition and docking studies.

The dihydroneopterin aldolase (DHNA, EC 4.1.2.25) activity of FolB protein is required for the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) and glycolaldehyde (GA) in the folate pathway. FolB protein from Mycobacterium tuberculosis (MtFolB) is essential for bacilli survival and represents an important molecular target for drug development. S8-functionalized 8-mercaptoguanine derivatives were synthesised and evaluated for inhibitory activity against MtFolB. The compounds showed IC50 values in the submicromolar range. The inhibition mode and inhibition constants were determined for compounds that exhibited the strongest inhibition. Additionally, molecular docking analyses were performed to suggest enzyme-inhibitor interactions and ligand conformations. To the best of our knowledge, this study describes the first class of MtFolB inhibitors.

Antidepressant-like effect of guanosine involves activation of AMPA receptor and BDNF/TrkB signaling.

Guanosine is a purine nucleoside that has been shown to exhibit antidepressant effects, but the mechanisms underlying its effect are not well established. We investigated if the antidepressant-like effect induced by guanosine in the tail suspension test (TST) in mice involves the modulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, voltage-dependent calcium channel (VDCC), and brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB) pathway. We also evaluated if the antidepressant-like effect of guanosine is accompanied by an acute increase in hippocampal and prefrontocortical BDNF levels. Additionally, we investigated if the ability of guanosine to elicit a fast behavioral response in the novelty suppressed feeding (NSF) test is associated with morphological changes related to hippocampal synaptogenesis. The antidepressant-like effect of guanosine (0.05 mg/kg, p.o.) in the TST was prevented by DNQX (AMPA receptor antagonist), verapamil (VDCC blocker), K-252a (TrkBantagonist), or BDNF antibody. Increased P70S6K phosphorylation and higher synapsin I immunocontent in the hippocampus, but not in the prefrontal cortex, were observed 1 h after guanosine administration. Guanosine exerted an antidepressant-like effect 1, 6, and 24 h after its administration, an effect accompanied by increased hippocampal BDNF level. In the prefrontal cortex, BDNF level was increased only 1 h after guanosine treatment. Finally, guanosine was effective in the NSF test (after 1 h) but caused no alterations in dendritic spine density and remodeling in the ventral dentate gyrus (DG). Altogether, the results indicate that guanosine modulates targets known to be implicated in fast antidepressant behavioral responses (AMPA receptor, VDCC, and TrkB/BDNF pathway).

Adenosine A1 and A2A receptors are involved on guanosine protective effects against oxidative burst and mitochondrial dysfunction induced by 6-OHDA in striatal slices.

6-Hydroxydopamine (6-OHDA) is the most used toxin in experimental Parkinson's disease (PD) models. 6-OHDA shows high affinity for the dopamine transporter and once inside the neuron, it accumulates and undergoes non-enzymatic auto-oxidation, promoting reactive oxygen species (ROS) formation and selective damage of catecholaminergic neurons. In this way, our group has established a 6-OHDA in vitro protocol with rat striatal slices as a rapid and effective model for screening of new drugs with protective effects against PD. We have shown that co-incubation with guanosine (GUO, 100 µM) prevented the 6-OHDA-induced damage in striatal slices. As the exact GUO mechanism of action remains unknown, the aim of this study was to investigate if adenosine A1 (A1R) and/or A2A receptors (A2AR) are involved on GUO protective effects on striatal slices. Pre-incubation with DPCPX, an A1R antagonist prevented guanosine effects on 6-OHDA-induced ROS formation and mitochondrial membrane potential depolarization, while CCPA, an A1R agonist, did not alter GUO effects. Regarding A2AR, the antagonist SCH58261 had similar protective effect as GUO in ROS formation and mitochondrial membrane potential. Additionally, SCH58261 did not affect GUO protective effects. The A2AR agonist CGS21680, although, completely blocked GUO effects. Finally, the A1R antagonist DPCPX, and the A2AR agonist CGS21680 also abolished the preventive guanosine effect on 6-OHDA-induced ATP levels decrease. These results reinforce previous evidence for a putative interaction of GUO with A1R-A2AR heteromer as its molecular target and clearly indicate a dependence on adenosine receptors modulation to GUO protective effect.

Ketamine, but not guanosine, as a prophylactic agent against corticosterone-induced depressive-like behavior: Possible role of long-lasting pro-synaptogenic signaling pathway.

Ketamine has been reported to exert a prophylactic effect against stress-induced depressive-like behavior by modulating the guanosine-based purinergic system. However, the molecular pathways underlying its prophylactic effect and whether guanosine also elicits a similar effect remain to be determined. Here, we investigated the prophylactic effect of ketamine and guanosine against corticosterone (CORT - 20 mg/kg, p.o.)-induced depressive-like behavior in mice. Furthermore, we characterized if the prophylactic response may be associated with mTORC1-driven signaling in the hippocampus and prefrontal cortex. A single administration of ketamine (5 mg/kg, i.p.), but not guanosine (1 or 5 mg/kg, p.o.), given 1 week before the pharmacological stress prevented CORT-induced depressive-like behavior in the tail suspension test (TST) and splash test (SPT). Fluoxetine treatment for 3 weeks did not prevent CORT-induced behavioral effects. A single administration of subthreshold doses of ketamine (1 mg/kg, i.p.) plus guanosine (5 mg/kg, p.o.) partially prevented the CORT-induced depressive-like behavior in the SPT. Additionally, CORT reduced Akt (Ser473) and GSK-3ß (Ser9) phosphorylation and PSD-95, GluA1, and synapsin immunocontent in the hippocampus, but not in the prefrontal cortex. No alterations on mTORC1/p70S6K immunocontent were found in both regions in any experimental group. CORT-induced reductions on PSD-95, GluA1, and synapsin immunocontent were prevented only by ketamine treatment. Collectively, these findings suggest that ketamine, but not guanosine, exerts a prophylactic effect against depressive-like behavior, an effect associated with the stimulation of long-lasting pro-synaptogenic signaling in the hippocampus.

Guanosine modulates SUMO2/3-ylation in neurons and astrocytes via adenosine receptors.

SUMOylation is a post-translational modification (PTM) whereby members of the Small Ubiquitin-like MOdifier (SUMO) family of proteins are conjugated to lysine residues in target proteins. SUMOylation has been implicated in a wide range of physiological and pathological processes, and much attention has been given to its role in neurodegenerative conditions. Due to its reported role in neuroprotection, pharmacological modulation of SUMOylation represents an attractive potential therapeutic strategy in a number of different brain disorders. However, very few compounds that target the SUMOylation pathway have been identified. Guanosine is an endogenous nucleoside with important neuromodulatory and neuroprotective effects. Experimental evidence has shown that guanosine can modulate different intracellular pathways, including PTMs. In the present study we examined whether guanosine alters global protein SUMOylation. Primary cortical neurons and astrocytes were treated with guanosine at 1, 10, 100, 300, or 500 µM at four time points, 1, 6, 24, or 48 h. We show that guanosine increases global SUMO2/3-ylation in neurons and astrocytes at 1 h at concentrations above 10 µM. The molecular mechanisms involved in this effect were evaluated in neurons. The guanosine-induced increase in global SUMO2/3-ylation was still observed in the presence of dipyridamole, which prevents guanosine internalization, demonstrating an extracellular guanosine-induced effect. Furthermore, the A1 adenosine receptor antagonist DPCPX abolished the guanosine-induced increase in SUMO2/3-ylation. The A2A adenosine receptor antagonist ZM241385 increased SUMOylation per se, but did not alter guanosine-induced SUMOylation, suggesting that guanosine may modulate SUMO2/3-ylation through an A1-A2A receptor interaction. Taken together, this is the first report to show guanosine as a SUMO2/3-ylation enhancer in astrocytes and neurons.

Guanosine enhances glutamate uptake and oxidation, preventing oxidative stress in mouse hippocampal slices submitted to high glutamate levels.

Glutamate (Glu) is the main mammalian brain neurotransmitter. Concerning the glutamatergic neurotransmission, excessive levels of glutamate in the synaptic cleft are extremally harmful. This phenomenon, named as excitotoxicity is involved in various acute and chronic brain diseases. Guanosine (GUO), an endogenous guanine nucleoside, possesses neuroprotective effects in several experimental models of glutamatergic excitotoxicity, an effect accompanied by an increase in astrocytic glutamate uptake. Therefore, the objective of this study was to investigate the involvement of an additional putative parameter, glutamate oxidation to CO2, involved in ex-vivo GUO neuroprotective effects in mouse hippocampal slices submitted to glutamatergic excitotoxicity. Mice were sacrificed by decapitation, the hippocampi were removed and sliced. The slices were incubated for various times and concentrations of Glu and GUO. First, the concentration of Glu that produced an increase in L-[14C(U)]-Glu oxidation to CO2 without cell injury was determined at different time points (between 0 and 90 min); 1000 µM Glu increased Glu oxidation between 30 and 60 min of incubation without cell injury. Under these conditions (Glu concentration and incubation time), 100 µM GUO increased Glu oxidation (35%). Additionally, 100 µM GUO increased L-[3,4-3H]-glutamate uptake (45%) in slices incubated with 1000 µM Glu (0-30 min). Furthermore, 1000  µM Glu increased reactive species levels, SOD activity, and decreased GPx activity, and GSH content after 30 and 60 min; 100 µM GUO prevented these effects. This is the first study demonstrating that GUO simultaneously promoted an increase in the uptake and utilization of Glu in excitotoxicity-like conditions preventing redox imbalance.

Guanosine Neuroprotection of Presynaptic Mitochondrial Calcium Homeostasis in a Mouse Study with Amyloid-ß Oligomers.

Amyloid-ß oligomers (AßOs) toxicity causes mitochondrial dysfunction, leading to synaptic failure in Alzheimer's disease (AD). Considering presynaptic high energy demand and tight Ca2+ regulation, impairment of mitochondrial function can lead to deteriorated neural activity and cell death. In this study, an AD mouse model induced by ICV (intracerebroventricular) injection of AßOs was used to investigate the toxicity of AßOs on presynaptic function. As a therapeutic approach, GUO (guanosine) was given by oral route to evaluate the neuroprotective effects on this AD model. Following 24 h and 48 h from the model induction, behavioral tasks and biochemical analyses were performed, respectively. AßOs impaired object recognition (OR) short-term memory and reduced glutamate uptake and oxidation in the hippocampus. Moreover, AßOs decreased spare respiratory capacity, reduced ATP levels, impaired Ca2+ handling, and caused mitochondrial swelling in hippocampal synaptosomes. Guanosine crossed the BBB, recovered OR short-term memory, reestablished glutamate uptake, recovered mitochondrial Ca2+ homeostasis, and partially prevented mitochondrial swelling. Therefore, this endogenous purine presented a neuroprotective effect on presynaptic mitochondria and should be considered for further studies in AD models.

Involvement of adenosine A1 and A2A receptors on guanosine-mediated anti-tremor effects in reserpinized mice.

Parkinson's disease (PD) signs and symptoms regularly include tremor. Interestingly, the nucleoside guanosine (GUO) has already proven to be effective in reducing reserpine-induced tremulous jaw movements (TJMs) in rodent models, thus becoming a promising antiparkinsonian drug. Here, we aimed at revealing the mechanism behind GUO antiparkinsonian efficacy by assessing the role of adenosine A1 and A2A receptors (A1R and A2AR) on GUO-mediated anti-tremor effects in the reserpinized mouse model of PD. Reserpinized mice showed elevated reactive oxygen species (ROS) production and cellular membrane damage in striatal slices assessed ex vivo and GUO treatment reversed ROS production. Interestingly, while the simultaneous administration of sub-effective doses of GUO (5 mg/kg) and SCH58261 (0.01 mg/kg), an A2AR antagonist, precluded reserpine-induced TJMs, these were ineffective on reverting ROS production in ex vivo experiments. Importantly, GUO was able to reduce TJM and ROS production in reserpinized mouse lacking the A2AR, thus suggesting an A2AR-independent mechanism of GUO-mediated effects. Conversely, the administration of DPCPX (0.75 mg/kg), an A1R antagonist, completely abolished both GUO-mediated anti-tremor effects and blockade of ROS production. Overall, these results indicated that GUO anti-tremor and antioxidant effects in reserpinized mice were A1R dependent but A2AR independent, thus suggesting a differential participation of adenosine receptors in GUO-mediated effects.

Guanosine protects against behavioural and mitochondrial bioenergetic alterations after mild traumatic brain injury.

Traumatic brain injury (TBI) constitutes a heterogeneous cerebral insult induced by traumatic biomechanical forces. Mitochondria play a critical role in brain bioenergetics, and TBI induces several consequences related with oxidative stress and excitotoxicity clearly demonstrated in different experimental model involving TBI. Mitochondrial bioenergetics alterations can present several targets for therapeutics which could help reduce secondary brain lesions such as neuropsychiatric problems, including memory loss and motor impairment. Guanosine (GUO), an endogenous neuroprotective nucleoside, affords the long-term benefits of controlling brain neurodegeneration, mainly due to its capacity to activate the antioxidant defense system and maintenance of the redox system. However, little is known about the exact protective mechanism exerted by GUO on mitochondrial bioenergetics disruption induced by TBI. Thus, the aim of this study was to investigate the effects of GUO in brain cortical and hippocampal mitochondrial bioenergetics in the mild TBI model. Additionally, we aimed to assess whether mitochondrial damage induced by TBI may be related to behavioral alterations in rats. Our findings showed that 24 h post-TBI, GUO treatment promotes an adaptive response of mitochondrial respiratory chain increasing oxygen flux which it was able to protect against the uncoupling of oxidative phosphorylation (OXPHOS) induced by TBI, restored the respiratory electron transfer system (ETS) established with an uncoupler. Guanosine treatment also increased respiratory control ratio (RCR), an indicator of the state of mitochondrial coupling, which is related to the mitochondrial functionality. In addition, mitochondrial bioenergetics failure was closely related with locomotor, exploratory and memory impairments. The present study suggests GUO treatment post mild TBI could increase GDP endogenous levels and consequently increasing ATP levels promotes an increase of RCR increasing OXPHOS and in substantial improve mitochondrial respiration in different brain regions, which, in turn, could promote an improvement in behavioral parameters associated to the mild TBI. These findings may contribute to the development of future therapies with a target on failure energetic metabolism induced by TBI.