Ascorbate and its transporter SVCT2: The dynamic duo's integrated roles in CNS neurobiology and pathophysiology.

Ascorbate is a small antioxidant molecule essential for the proper development and function of the brain. Ascorbate is transported into the brain and between brain cells via the Sodium vitamin C co-transporter 2 (SVCT2). This review provides an in-depth analysis of ascorbate's physiology, including how ascorbate is absorbed from food into the CNS, emphasizing cellular mechanisms of ascorbate recycling and release in different CNS compartments. Additionally, the review delves into the various functions of ascorbate in the CNS, including its impact on epigenetic modulation, synaptic plasticity, and neurotransmission. It also emphasizes ascorbate's role on neuromodulation and its involvement in neurodevelopmental processes and disorders. Furthermore, it analyzes the relationship between the duo ascorbate/SVCT2 in neuroinflammation, particularly its effects on microglial activation, cytokine release, and oxidative stress responses, highlighting its association with neurodegenerative diseases, such as Alzheimer's disease (AD). Overall, this review emphasizes the crucial role of the dynamic duo ascorbate/SVCT2 in CNS physiology and pathology and the need for further research to fully comprehend its significance in a neurobiological context and its potential therapeutic applications.

Microglial Rac1 is essential for experience-dependent brain plasticity and cognitive performance.

Microglia, the largest population of brain immune cells, continuously interact with synapses to maintain brain homeostasis. In this study, we use conditional cell-specific gene targeting in mice with multi-omics approaches and demonstrate that the RhoGTPase Rac1 is an essential requirement for microglia to sense and interpret the brain microenvironment. This is crucial for microglia-synapse crosstalk that drives experience-dependent plasticity, a fundamental brain property impaired in several neuropsychiatric disorders. Phosphoproteomics profiling detects a large modulation of RhoGTPase signaling, predominantly of Rac1, in microglia of mice exposed to an environmental enrichment protocol known to induce experience-dependent brain plasticity and cognitive performance. Ablation of microglial Rac1 affects pathways involved in microglia-synapse communication, disrupts experience-dependent synaptic remodeling, and blocks the gains in learning, memory, and sociability induced by environmental enrichment. Our results reveal microglial Rac1 as a central regulator of pathways involved in the microglia-synapse crosstalk required for experience-dependent synaptic plasticity and cognitive performance.

RhoA balances microglial reactivity and survival during neuroinflammation.

Microglia are the largest myeloid cell population in the brain. During injury, disease, or inflammation, microglia adopt different functional states primarily involved in restoring brain homeostasis. However, sustained or exacerbated microglia inflammatory reactivity can lead to brain damage. Dynamic cytoskeleton reorganization correlates with alterations of microglial reactivity driven by external cues, and proteins controlling cytoskeletal reorganization, such as the Rho GTPase RhoA, are well positioned to refine or adjust the functional state of the microglia during injury, disease, or inflammation. Here, we use multi-biosensor-based live-cell imaging approaches and tissue-specific conditional gene ablation in mice to understand the role of RhoA in microglial response to inflammation. We found that a decrease in RhoA activity is an absolute requirement for microglial metabolic reprogramming and reactivity to inflammation. However, without RhoA, inflammation disrupts Ca2+ and pH homeostasis, dampening mitochondrial function, worsening microglial necrosis, and triggering microglial apoptosis. Our results suggest that a minimum level of RhoA activity is obligatory to concatenate microglia inflammatory reactivity and survival during neuroinflammation.

Maternal stress and vulnerability to depression: coping and maternal care strategies and its consequences on adolescent offspring.

Depressive mothers often find mother-child interaction to be challenging. Maternal stress may further impair mother-child attachment, which may increase the risk of negative developmental consequences. We used rats with different vulnerability to depressive-like behavior (Wistar and Kyoto) to investigate the impact of stress (maternal separation-MS) on maternal behavior and adolescent offspring cognition. MS in Kyoto dams increased pup-contact, resulting in higher oxytocin levels and lower anxiety-like behavior after weaning, while worsening their adolescent offspring cognitive behavior. Whereas MS in Wistar dams elicited higher quality of pup-directed behavior, increasing brain-derived neurotrophic factor (BDNF) in the offspring, which seems to have prevented a negative impact on cognition. Hypothalamic oxytocin seems to affect the salience of the social environment cues (negatively for Kyoto) leading to different coping strategies. Our findings highlight the importance of contextual and individual factors in the understanding of the oxytocin role in modulating maternal behavior and stress regulatory processes.

Src family kinases (SFKs): critical regulators of microglial homeostatic functions and neurodegeneration in Parkinson's and Alzheimer's diseases.

c-Src was the first protein kinase to be described as capable of phosphorylating tyrosine residues. Subsequent identification of other tyrosine-phosphorylating protein kinases with a similar structure to c-Src gave rise to the concept of Src family kinases (SFKs). Microglia are the resident innate immune cell population of the CNS. Under physiological conditions, microglia actively participate in brain tissue homeostasis, continuously patrolling the neuronal parenchyma and exerting neuroprotective actions. Activation of pathogen-associated molecular pattern (PAMP) and damage-associated molecular pattern (DAMP) receptors induces microglial proliferation, migration toward pathological foci, phagocytosis, and changes in gene expression, concurrent with the secretion of cytokines, chemokines, and growth factors. A significant body of literature shows that SFK stimulation positively associates with microglial activation and neuropathological conditions, including Alzheimer's and Parkinson's diseases. Here, we review essential microglial homeostatic functions regulated by SFKs, including phagocytosis, environmental sensing, and secretion of inflammatory mediators. In addition, we discuss the potential of SFK modulation for microglial homeostasis in Parkinson's and Alzheimer's diseases.

The peptide secreted at the water to land transition in a model amphibian has antioxidant effects.

In addition to the morphophysiological changes experienced by amphibians during metamorphosis, they must also deal with a different set of environmental constraints when they shift from the water to the land. We found that Pithecopus azureus secretes a single peptide ([M + H]+ = 658.38 Da) at the developmental stage that precedes the onset of terrestrial behaviour. De novo peptide and cDNA sequencing revealed that the peptide, named PaT-2, is expressed in tandem and is a member of the tryptophyllins family. In silico studies allowed us to identify the position of reactive sites and infer possible antioxidant mechanisms of the compounds. Cell-based assays confirmed the predicted antioxidant activity in mammalian microglia and neuroblast cells. The potential neuroprotective effect of PaT-2 was further corroborated in FRET-based live cell imaging assays, where the peptide prevented lipopolysaccharide-induced ROS production and glutamate release in human microglia. In summary, PaT-2 is the first peptide expressed during the ontogeny of P. azureus, right before the metamorphosing froglet leaves the aquatic environment to occupy terrestrial habitats. The antioxidant activity of PaT-2, predicted by in silico analyses and confirmed by cell-based assays, might be relevant for the protection of the skin of P. azureus adults against increased O2 levels and UV exposure on land compared with aquatic environments.

Activation of adenosine A3 receptors regulates vitamin C transport and redox balance in neurons.

Adenosine is an important neuromodulator in the CNS, regulating neuronal survival and synaptic transmission. The antioxidant ascorbate (the reduced form of vitamin C) is concentrated in CNS neurons through a sodium-dependent transporter named SVCT2 and participates in several CNS processes, for instance, the regulation of glutamate receptors functioning and the synthesis of neuromodulators. Here we studied the interplay between the adenosinergic system and ascorbate transport in neurons. We found that selective activation of A3, but not of A1 or A2a, adenosine receptors modulated ascorbate transport, decreasing intracellular ascorbate content. Förster resonance energy transfer (FRET) analyses showed that A3 receptors associate with the ascorbate transporter SVCT2, suggesting tight signaling compartmentalization between A3 receptors and SVCT2. The activation of A3 receptors increased ascorbate release in an SVCT2-dependent manner, which largely altered the neuronal redox status without interfering with cell death, glycolytic metabolism, and bioenergetics. Overall, by regulating vitamin C transport, the adenosinergic system (via activation of A3 receptors) can regulate ascorbate bioavailability and control the redox balance in neurons.

A Protocol for FRET-Based Live-Cell Imaging in Microglia.

This protocol highlights the use of FRET-based biosensors to investigate signaling events during microglia activation in real time. Understanding microglia activation has gained momentum as it can help decipher signaling mechanisms underlying the neurodegenerative process occurring in neurological disorders. Unlike more traditional methods widely employed in the microglia field, FRET allows microglia signaling events to be studied in real time with exquisite subcellular resolution. However, FRET-based live-cell imaging requires application-specific biosensors and specialized imaging systems, limiting its use in in vivo studies. For complete details on the use and execution of this protocol, please refer to Socodato et al. (2020), Portugal et al. (2017), and Socodato et al. (2018).

Daily alcohol intake triggers aberrant synaptic pruning leading to synapse loss and anxiety-like behavior.

Alcohol abuse adversely affects the lives of millions of people worldwide. Deficits in synaptic transmission and in microglial function are commonly found in human alcohol abusers and in animal models of alcohol intoxication. Here, we found that a protocol simulating chronic binge drinking in male mice resulted in aberrant synaptic pruning and substantial loss of excitatory synapses in the prefrontal cortex, which resulted in increased anxiety-like behavior. Mechanistically, alcohol intake increased the engulfment capacity of microglia in a manner dependent on the kinase Src, the subsequent activation of the transcription factor NF-κB, and the consequent production of the proinflammatory cytokine TNF. Pharmacological blockade of Src activation or of TNF production in microglia, genetic ablation of Tnf, or conditional ablation of microglia attenuated aberrant synaptic pruning, thereby preventing the neuronal and behavioral effects of the alcohol. Our data suggest that aberrant pruning of excitatory synapses by microglia may disrupt synaptic transmission in response to alcohol abuse.

Cytotoxic activity of poly-ɛ-caprolactone lipid-core nanocapsules loaded with lycopene-rich extract from red guava (Psidium guajava L.) on breast cancer cells.

The aims of this study were to produce poly-ɛ-caprolactone lipid-core nanocapsules containing lycopene-rich extract from red guava (LEG), to characterize those nanoparticles and to evaluate their cytotoxic effects on human breast cancer cells. Lipid-core nanocapsules containing the extract (nanoLEG) were produced by the method of interfacial deposition of the preformed polymer. The nanoparticles were characterized by Dynamic Light Scattering (DLS), Polydispersity Index, Zeta Potential, pH, Encapsulation Efficiency, Nanoparticle Tracking Analysis (NTA), Atomic Force Microscopy (AFM) and Transmission Electron Microscopy (TEM). Cell viability was evaluated by the MTT dye reduction method in the human breast cancer MCF-7 cell line and inhibition of ROS and NF-κB was assayed in living human microglial cell line (HMC3) by time-lapse images microscopy. A hemolytic activity assay was carried out with sheep blood. Data showed that nanoparticles average size was around 200 nm, nanoparticles concentration/mL was around 0.1 µM, negative zeta potential, pH < 5.0 and spherical shape, with low variation during a long storage period (7 months) at 5 °C, indicating stability of the system and protection against lycopene degradation. The percentage of encapsulation varied from 95% to 98%. The nanoLEG particles significantly reduced the viability of the MCF-7 cells after 24 h (61.47%) and 72 h (55.96%) of exposure, even at the lowest concentration tested (6.25-200 µg/ml) and improved on the cytotoxicity of free LEG to MCF-7. NanoLEG inhibited LPS-induced NF-kB activation and ROS production in microglial cells. The particles did not affect the membrane integrity of sheep blood erythrocytes at the concentrations tested (6.25-200 µg/mL). Thus, the formulation of lipid-core nanocapsules with a polysorbate 80-coated poly-ɛ-caprolactone wall was efficiently applied to stabilize the lycopene-rich extract from red guava, generating a product with satisfactory physico-chemical and biological properties for application as health-promoting nanotechnology-based nutraceutical, emphasizing its potential to be used as a cancer treatment.

Early-life stress affects drug abuse susceptibility in adolescent rat model independently of depression vulnerability.

The development of substance abuse problems occurs due to a diverse combination of risk factors. Among these risks, studies have reported depression and early-life stress as of importance. These two factors often occur simultaneously, however, there is a lack of understanding of how their combined effect may impact vulnerability to drug abuse in adolescence. The present study used rats with different vulnerability to depression (Wistar and Wistar-Kyoto) to investigate the impact of maternal separation (MS) on emotional state and drug addiction vulnerability during the adolescence period. Mothers and their litters were subjected to MS (180 min/day) from postnatal day 2 to 14. The offspring emotional state was assessed by observing their exploratory behavior. Drug abuse vulnerability was assessed through conditioning to cocaine. MS impacted the emotional state in both strains. Wistar responded with increased exploration, while Wistar-Kyoto increased anxiety-like behaviours. Despite the different coping strategies displayed by the two strains when challenged with the behavioural tests, drug conditioning was equally impacted by MS in both strains. Early-life stress appears to affect drug abuse vulnerability in adolescence independently of a depression background, suggesting emotional state as the main driving risk factor.

Microglia Dysfunction Caused by the Loss of Rhoa Disrupts Neuronal Physiology and Leads to Neurodegeneration.

Nervous tissue homeostasis requires the regulation of microglia activity. Using conditional gene targeting in mice, we demonstrate that genetic ablation of the small GTPase Rhoa in adult microglia is sufficient to trigger spontaneous microglia activation, producing a neurological phenotype (including synapse and neuron loss, impairment of long-term potentiation [LTP], formation of ß-amyloid plaques, and memory deficits). Mechanistically, loss of Rhoa in microglia triggers Src activation and Src-mediated tumor necrosis factor (TNF) production, leading to excitotoxic glutamate secretion. Inhibiting Src in microglia Rhoa-deficient mice attenuates microglia dysregulation and the ensuing neurological phenotype. We also find that the Rhoa/Src signaling pathway is disrupted in microglia of the APP/PS1 mouse model of Alzheimer disease and that low doses of Aß oligomers trigger microglia neurotoxic polarization through the disruption of Rhoa-to-Src signaling. Overall, our results indicate that disturbing Rho GTPase signaling in microglia can directly cause neurodegeneration.

Protein synthesis inhibition promotes nitric oxide generation and activation of CGKII-dependent downstream signaling pathways in the retina.

Nitric oxide is an important neuromodulator in the CNS, and its production within neurons is modulated by NMDA receptors and requires a fine-tuned availability of L-arginine. We have previously shown that globally inhibiting protein synthesis mobilizes intracellular L-arginine "pools" in retinal neurons, which concomitantly enhances neuronal nitric oxide synthase-mediated nitric oxide production. Activation of NMDA receptors also induces local inhibition of protein synthesis and L-arginine intracellular accumulation through calcium influx and stimulation of eucariotic elongation factor type 2 kinase. We hypothesized that protein synthesis inhibition might also increase intracellular L-arginine availability to induce nitric oxide-dependent activation of downstream signaling pathways. Here we show that nitric oxide produced by inhibiting protein synthesis (using cycloheximide or anisomycin) is readily coupled to AKT activation in a soluble guanylyl cyclase and cGKII-dependent manner. Knockdown of cGKII prevents cycloheximide or anisomycin-induced AKT activation and its nuclear accumulation. Moreover, in retinas from cGKII knockout mice, cycloheximide was unable to enhance AKT phosphorylation. Indeed, cycloheximide also produces an increase of ERK phosphorylation which is abrogated by a nitric oxide synthase inhibitor. In summary, we show that inhibition of protein synthesis is a previously unanticipated driving force for nitric oxide generation and activation of downstream signaling pathways including AKT and ERK in cultured retinal cells. These results may be important for the regulation of synaptic signaling and neuronal development by NMDA receptors as well as for solving conflicting data observed when using protein synthesis inhibitors for studying neuronal survival during development as well in behavior and memory studies.

Somuncurins: Bioactive Peptides from the Skin of the Endangered Endemic Patagonian Frog Pleurodema somuncurense.

The skin glands of amphibian species hold a major component of their innate immunity, namely a unique set of antimicrobial peptides (AMPs). Although most of them have common characteristics, differences in AMP sequences allow a huge repertoire of biological activity with varying degrees of efficacy. We present the first study of the AMPs from Pleurodema somuncurence (Anura: Leptodactylidae: Leiuperinae). Among the 11 identified mature peptides, three presented antimicrobial activity. Somuncurin-1 (FIIWPLRYRK), somuncurin-2 (FILKRSYPQYY), and thaulin-3 (NLVGSLLGGILKK) inhibited Escherichia coli growth. Somuncurin-1 also showed antimicrobial activity against Staphylococcus aureus. Biophysical membrane model studies revealed that this peptide had a greater permeation effect in prokaryotic-like membranes and capacity to restructure liposomes, suggesting fusogenic activity, which could lead to cell aggregation and disruption of cell morphology. This study contributes to the characterization of peptides with new sequences to enrich the databases for the design of therapeutic agents. Furthermore, it highlights the importance of investing in nature conservation and the power of genetic description as a strategy to identify new compounds.

Novel Ocellatin Peptides Mitigate LPS-induced ROS Formation and NF-kB Activation in Microglia and Hippocampal Neurons.

Cutaneous secretions of amphibians have bioactive compounds, such as peptides, with potential for biotechnological applications. Therefore, this study aimed to determine the primary structure and investigate peptides obtained from the cutaneous secretions of the amphibian, Leptodactylus vastus, as a source of bioactive molecules. The peptides obtained possessed the amino acid sequences, GVVDILKGAAKDLAGH and GVVDILKGAAKDLAGHLASKV, with monoisotopic masses of [M + H]± = 1563.8 Da and [M + H]± = 2062.4 Da, respectively. The molecules were characterized as peptides of the class of ocellatins and were named as Ocellatin-K1(1-16) and Ocellatin-K1(1-21). Functional analysis revealed that Ocellatin-K1(1-16) and Ocellatin-K1(1-21) showed weak antibacterial activity. However, treatment of mice with these ocellatins reduced the nitrite and malondialdehyde content. Moreover, superoxide dismutase enzymatic activity and glutathione concentration were increased in the hippocampus of mice. In addition, Ocellatin-K1(1-16) and Ocellatin-K1(1-21) were effective in impairing lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) formation and NF-kB activation in living microglia. We incubated hippocampal neurons with microglial conditioned media treated with LPS and LPS in the presence of Ocellatin-K1(1-16) and Ocellatin-K1(1-21) and observed that both peptides reduced the oxidative stress in hippocampal neurons. Furthermore, these ocellatins demonstrated low cytotoxicity towards erythrocytes. These functional properties suggest possible to neuromodulatory therapeutic applications.

Antifungal and anti-inflammatory potential of eschweilenol C-rich fraction derived from Terminalia fagifolia Mart.

ETHNOPHARMACOLOGICAL RELEVANCE: Folk knowledge transmitted between generations allows traditional populations to maintain the use of medicinal plants for the treatment of several diseases. In this context, the species Terminalia fagifolia Mart., native to Brazil, is used for the treatment of chronic and infectious diseases. Plants rich in secondary metabolites, such as this species and their derivatives, may represent therapeutic alternatives for the treatment of diseases that reduce the quality of life of people. AIM OF THE STUDY: The aim of this study was to evaluate the antifungal and anti-inflammatory potential of aqueous fraction from ethanolic extract of T. fagifolia, with in silico study of the major compound of the fraction. MATERIAL AND METHODS: The phytochemical study of the aqueous fraction was performed by HPLC, LC/MS and NMR. The antifungal activity was evaluated against yeasts, by determination of the minimum inhibitory concentration and minimum fungicidal concentration. The effect on Candida albicans was analyzed by AFM. The antibiofilm potential against biofilms of C. albicans was also tested. The anti-inflammatory potential of the aqueous fraction was evaluated in vivo by the carrageenan-induced paw edema and peritonitis. A microglial model of LPS-induced neuroinflammation was also studied. Further insights on the activation mechanism were studied using quantum chemistry computer simulations. Toxicity was evaluated in the Galleria mellonella and human erythrocytes models. RESULTS: Eschweilenol C was identified as the major constituent of the aqueous fraction of the ethanolic extract of T. fagifolia. The aqueous fraction was active against all Candida strains used (sensitive and resistant to Fluconazole) with MICs ranging from 1000 to 0.4 µg/mL. By AFM it was possible to observe morphological alterations in treated Candida cells. The fraction significantly (p < 0.05) inhibited paw edema and decreased levels of malondialdehyde induced by carrageenan. In a microglial cell model, aqueous fraction demonstrated the ability to inhibit NF-κB after induction with lipopolysaccharide. The theoretical studies showed structural similarity between eschweilenol C and indomethacin and an excellent antioxidant potential. The aqueous fraction did not present toxicity in the studied models. CONCLUSION: The results indicate that the aqueous fraction of T. fagifolia has potential for biomedical applications with low toxicity. This finding can be attributed to the predominance of eschweilenol C in the aqueous fraction.

Redox tuning of Ca2+ signaling in microglia drives glutamate release during hypoxia.

Hypoxia causes oxidative stress and excitotoxicity, culminating in neuronal damage during brain ischemia. Hypoxia also activates microglia, the myeloid resident cells of the brain parenchyma. Upon activation, microglia release high amounts of the neurotransmitter glutamate, contributing for neuronal excitotoxicity during brain insults. Here, we reveal a signaling pathway controlling glutamate release from human microglia during hypoxia. We show that hypoxia-mediated redox imbalance promotes the activation of endoplasmic reticulum inositol 1,4,5-trisphosphate (InsP3) receptors leading to Ca2+ mobilization into the cytosol. Increasing cytosolic Ca2+ signaling in microglia activates the non-receptor protein tyrosine kinase Src at the plasma membrane. Src activation enhances the permeability of microglial gap junctions promoting the release of glutamate during hypoxia. Preventing the hypoxia-triggered redox imbalance, using the dietary antioxidants neochlorogenic acid or vitamin C, inhibits InsP3-dependent Ca2+ signaling and abrogates the release of glutamate. Overall, modulating microglial Ca2+ signaling in response to changes in the redox microenvironment might be critical for controlling glutamate excitotoxicity during hypoxia.

Microglia and alcohol meet at the crossroads: Microglia as critical modulators of alcohol neurotoxicity.

Alcohol use disorders affect millions of people worldwide causing huge social and economic burden on modern society. Excessive alcohol consumption or intoxication provokes severe damage to the body inducing immune suppression, liver damage and neurological disorder. In the central nervous system (CNS), alcohol exposure can lead to neuronal loss, cognitive decline, motor dysfunction, inflammation and impairment of neuroimmune responses. Glial cells, from which microglia represent roughly 10-15%, are primary modulators of the neuroimmune responses and inflammation in the CNS. Here we overview literature relating alcohol exposure with microglia activation and brain inflammation, highlighting that microglia are critical regulators of alcohol responses in the CNS. Different studies indicate that alcohol intake alters the microglial activation spectrum, with the microglial response varying according to the dose, duration, and pattern of alcohol administration. Presently, further investigation is required to establish whether microglia dysfunction initiates or simply amplifies the neurotoxicity of alcohol in the brain. Such knowledge can be greatly facilitated by the use of microglia-specific genetic targeting in animal models and will be critical for the development of better therapeutics for mitigating the neurotoxicity induced by alcohol.

Structure and function of a novel antioxidant peptide from the skin of tropical frogs.

The amphibian skin plays an important role protecting the organism from external harmful factors such as microorganisms or UV radiation. Based on biorational strategies, many studies have investigated the cutaneous secretion of anurans as a source of bioactive molecules. By a peptidomic approach, a novel antioxidant peptide (AOP) with in vitro free radical scavenging ability was isolated from Physalaemus nattereri. The AOP, named antioxidin-I, has a molecular weight [M+H]+ = 1543.69Da and a TWYFITPYIPDK primary amino acid sequence. The gene encoding the antioxidin-I precursor was expressed in the skin tissue of three other Tropical frog species: Phyllomedusa tarsius, P. distincta and Pithecopus rohdei. cDNA sequencing revealed highly homologous regions (signal peptide and acidic region). Mature antioxidin-I has a novel primary sequence with low similarity compared with previously described amphibian's AOPs. Antioxidin-I adopts a random structure even at high concentrations of hydrophobic solvent, it has poor antimicrobial activity and poor performance in free radical scavenging assays in vitro, with the exception of the ORAC assay. However, antioxidin-I presented a low cytotoxicity and suppressed menadione-induced redox imbalance when tested with fibroblast in culture. In addition, it had the capacity to substantially attenuate the hypoxia-induced production of reactive oxygen species when tested in hypoxia exposed living microglial cells, suggesting a potential neuroprotective role for this peptide.