IL-10 and Cdc42 modulate astrocyte-mediated microglia activation in methamphetamine-induced neuroinflammation.

Methamphetamine (Meth) use is known to induce complex neuroinflammatory responses, particularly involving astrocytes and microglia. Building upon our previous research, which demonstrated that Meth stimulates astrocytes to release tumor necrosis factor (TNF) and glutamate, leading to microglial activation, this study investigates the role of the anti-inflammatory cytokine interleukin-10 (IL-10) in this process. Our findings reveal that the presence of recombinant IL-10 (rIL-10) counteracts Meth-induced excessive glutamate release in astrocyte cultures, which significantly reduces microglial activation. This reduction is associated with the modulation of astrocytic intracellular calcium (Ca2+) dynamics, particularly by restricting the release of Ca2+ from the endoplasmic reticulum to the cytoplasm. Furthermore, we identify the small Rho GTPase Cdc42 as a crucial intermediary in the astrocyte-to-microglia communication pathway under Meth exposure. By employing a transgenic mouse model that overexpresses IL-10 (pMT-10), we also demonstrate in vivo that IL-10 prevents Meth-induced neuroinflammation. These findings not only enhance our understanding of Meth-related neuroinflammatory mechanisms, but also suggest IL-10 and Cdc42 as putative therapeutic targets for treating Meth-induced neuroinflammation.

Communication defects with astroglia contribute to early impairments in the motor cortex plasticity of SOD1G93A mice.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease, involving the selective degeneration of cortical upper synapses in the primary motor cortex (M1). Excitotoxicity in ALS occurs due to an imbalance between excitation and inhibition, closely linked to the loss/gain of astrocytic function. Using the ALS SOD1G93A mice, we investigated the astrocytic contribution for the electrophysiological alterations observed in the M1 of SOD1G93A mice, throughout disease progression. Results showed that astrocytes are involved in synaptic dysfunction observed in presymptomatic SOD1G93A mice, since astrocytic glutamate transport currents are diminished and pharmacological inhibition of astrocytes only impaired long-term potentiation and basal transmission in wild-type mice. Proteomic analysis revealed major differences in neuronal transmission, metabolism, and immune system in upper synapses, confirming early communication deficits between neurons and astroglia. These results provide valuable insights into the early impact of upper synapses in ALS and the lack of supportive functions of cortical astrocytes, highlighting the possibility of manipulating astrocytes to improve synaptic function.

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.

Dopamine D1 receptor signaling and endocannabinoid cooperate to fuel striatal plasticity: An Editorial Highlight for "Cyclic AMP-dependent protein kinase and D1 dopamine receptors regulate diacylglycerol lipase-α and synaptic 2-arachidonoyl glycerol signaling" on page 334.

Endocannabinoids (eCBs) play key roles in short-term and long-term synaptic plasticity in the corticostriatal circuit. By activating cannabinoid receptors expressed in the central nervous system, eCBs regulate several neural functions and behaviors. The major eCB 2-arachidonoyl-glycerol (2-AG) is particularly important for triggering a short-term form of synaptic plasticity (depolarization-induced suppression of excitatory transmission or DSE) on cortical glutamatergic afferents inputting the striatum. The neurotransmitter dopamine, through the action of D1 and D2 receptors, is also critically involved in corticostriatal plasticity. This Editorial highlights the study by Shonesy et al., which presents evidence that activation of dopamine D1 receptor and its classical downstream target cAMP-dependent protein kinase (PKA) are involved in increasing the synthesis of 2-AG in striatal medium spiny neurons (MSN) to drive DSE in the corticostriatal circuit, as schematically outlined in Figure 1. The authors used a set of complementary approaches and identified a putative serine (Ser) residue phosphorylated by PKA in diacylglycerol lipase (DGL) alpha that is required for generating 2-AG, providing a mechanistic clue into how the canonical D1 pathway in MSN might fine-tune short-term plasticity in the corticostriatal circuit. Besides, the work by Shonesy et al. may pave the way for further studies exploring the signaling interplay between canonical dopamine D1 receptor pathway and eCBs to control other forms of synaptic plasticity in different brain circuits with possible pathological relevance.

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.

Vitamin C modulates glutamate transport and NMDA receptor function in the retina.

Vitamin C (in the reduced form ascorbate or in the oxidized form dehydroascorbate) is implicated in signaling events throughout the central nervous system (CNS). In the retina, a high-affinity transport system for ascorbate has been described and glutamatergic signaling has been reported to control ascorbate release. Here, we investigated the modulatory role played by vitamin C upon glutamate uptake and N-methyl-d-aspartate (NMDA) receptor activation in cultured retinal cells or in intact retinal tissue using biochemical and imaging techniques. We show that both forms of vitamin C, ascorbate or dehydroascorbate, promote an accumulation of extracellular glutamate by a mechanism involving the inhibition of glutamate uptake. This inhibition correlates with the finding that ascorbate promotes a decrease in cell surface levels of the neuronal glutamate transporter excitatory amino acid transporter 3 in retinal neuronal cultures. Interestingly, vitamin C is prone to increase the activity of NMDA receptors but also promotes a decrease in glutamate-stimulated [3 H] MK801 binding and decreases cell membrane content of NMDA receptor glutamate ionotropic receptor subunit 1 (GluN1) subunits. Both compounds were also able to increase cAMP response element-binding protein phosphorylation in neuronal nuclei in a glutamate receptor and calcium/calmodulin kinase-dependent manner. Moreover, the effect of ascorbate is not blocked by sulfinpyrazone and then does not depend on its uptake by retinal cells. Overall, these data indicate a novel molecular and functional target for vitamin C impacting on glutamate signaling in retinal neurons.

Methylphenidate-triggered ROS generation promotes caveolae-mediated transcytosis via Rac1 signaling and c-Src-dependent caveolin-1 phosphorylation in human brain endothelial cells.

Methylphenidate (MPH) is an amphetamine-like stimulant commonly prescribed for attention deficit hyperactivity disorder. Despite its widespread use, the cellular/molecular effects of MPH remain elusive. Here, we report a novel direct role of MPH on the regulation of macromolecular flux through human brain endothelial cells (ECs). MPH significantly increased caveolae-mediated transcytosis of horseradish peroxidase through ECs without affecting paracellular permeability. Using FRET-based live cell imaging, together with pharmacological inhibitors and lentiviral-mediated shRNA knockdown, we demonstrate that MPH promoted ROS generation via activation of Rac1-dependent NADPH oxidase (NOX) and c-Src activation at the plasma membrane. c-Src in turn was shown to mediate the phosphorylation of caveolin-1 (Cav1) on Tyr14 leading to enhanced caveolae formation and transendothelial transport. Accordingly, the inhibition of Cav1 phosphorylation by overexpression of a phosphodefective Cav1Y14F mutant or knocking down Cav1 expression abrogated MPH-induced transcytosis. In addition, both vitamin C and inhibition of NOX blocked MPH-triggered vesicular transport. This study, therefore, identifies Rac1/NOX/c-Src-dependent signaling in MPH-induced increase in transendothelial permeability of brain endothelial cell monolayers via caveolae-mediated transcytosis.

Caffeine exposure alters adenosine system and neurochemical markers during retinal development.

Evidence points to beneficial properties of caffeine in the adult central nervous system, but teratogenic effects have also been reported. Caffeine exerts most of its effects by antagonizing adenosine receptors, especially A1 and A2A subtypes. In this study, we evaluated the role of caffeine on the expression of components of the adenosinergic system in the developing avian retina and the impact of caffeine exposure upon specific markers for classical neurotransmitter systems. Caffeine exposure (5-30 mg/kg by in ovo injection) to 14-day-old chick embryos increased the expression of A1 receptors and concomitantly decreased A2A adenosine receptors expression after 48 h. Accordingly, caffeine (30 mg/kg) increased [(3) H]-8-cyclopentyl-1,3-dipropylxanthine (A1 antagonist) binding and reduced [(3) H]-ZM241385 (A2A antagonist) binding. The caffeine time-response curve demonstrated a reduction in A1 receptors 6 h after injection, but an increase after 18 and 24 h. In contrast, caffeine exposure increased the expression of A2A receptors from 18 and 24 h. Kinetic assays of [(3) H]-S-(4-nitrobenzyl)-6-thioinosine binding to the equilibrative adenosine transporter ENT1 revealed an increase in Bmax with no changes in Kd , an effect accompanied by an increase in adenosine uptake. Immunohistochemical analysis showed a decrease in retinal content of tyrosine hydroxylase, calbindin and choline acetyltransferase, but not Brn3a, after 48 h of caffeine injection. Furthermore, retinas exposed to caffeine had increased levels of phosphorylated extracellular signal-regulated kinase and cAMP-response element binding protein. Overall, we show an in vivo regulation of the adenosine system, extracellular signal-regulated kinase and cAMP-response element binding protein function and protein expression of specific neurotransmitter systems by caffeine in the developing retina. The beneficial or maleficent effects of caffeine have been demonstrated by the work of different studies. It is known that during animal development, caffeine can exert harmful effects, impairing the correct formation of CNS structures. In this study, we demonstrated cellular and tissue effects of caffeine's administration on developing chick embryo retinas. Those effects include modulation of adenosine receptors (A1 , A2 ) content, increasing in cAMP response element-binding protein (pCREB) and extracellular signal-regulated kinase phosphorylation (pERK), augment of adenosine equilibrative transporter content/activity, and a reduction of some specific cell subpopulations. ENT1, Equilibrative nucleoside transporter 1.

c-Src function is necessary and sufficient for triggering microglial cell activation.

Microglial cells are the resident macrophages of the central nervous system. Their function is essential for neuronal tissue homeostasis. After inflammatory stimuli, microglial cells become activated changing from a resting and highly ramified cell shape to an amoeboid-like morphology. These morphological changes are associated with the release of proinflammatory cytokines and glutamate, as well as with high phagocytic activity. The acquisition of such phenotype has been associated with activation of cytoplasmic tyrosine kinases, including those of the Src family (SFKs). In this study, using both in vivo and in vitro inflammation models coupled to FRET-based time-lapse microscopy, lentiviruses-mediated shRNA delivery and genetic gain-of-function experiments, we demonstrate that among SFKs c-Src function is necessary and sufficient for triggering microglia proinflammatory signature, glutamate release, microglia-induced neuronal loss, and phagocytosis. c-Src inhibition in retinal neuroinflammation experimental paradigms consisting of intravitreal injection of LPS or ischemia-reperfusion injury significantly reduced microglia activation changing their morphology to a more resting phenotype and prevented neuronal apoptosis. Our data demonstrate an essential role for c-Src in microglial cell activation.

A cytoskeleton symphony: Actin and microtubules in microglia dynamics and aging.

Microglia dynamically reorganize their cytoskeleton to perform essential functions such as phagocytosis of toxic protein aggregates, surveillance of the brain parenchyma, and regulation of synaptic plasticity during neuronal activity bursts. Recent studies have shed light on the critical role of the microtubule cytoskeleton in microglial reactivity and function, revealing key regulators like cyclin-dependent kinase 1 and centrosomal nucleation in the remodeling of microtubules in activated microglia. Concurrently, the role of the actin cytoskeleton is also pivotal, particularly in the context of small GTPases like RhoA, Rac1, and Cdc42 and actin-binding molecules such as profilin-1 and cofilin. This article delves into the intricate molecular landscape of actin and microtubules, exploring their synergistic roles in driving microglial cytoskeletal dynamics. We propose a more integrated view of actin and microtubule cooperation, which is fundamental to understanding the functional coherence of the microglial cytoskeleton and its pivotal role in propelling brain homeostasis. Furthermore, we discuss how alterations in microglial cytoskeleton dynamics during aging and in disease states could have far-reaching implications for brain function. By unraveling the complexities of microglia cytoskeletal dynamics, we can deepen our understanding of microglial functional states and their implications in health and disease, offering insights into potential therapeutic interventions for neurologic disorders.

Nitric oxide modulates sodium vitamin C transporter 2 (SVCT-2) protein expression via protein kinase G (PKG) and nuclear factor-κB (NF-κB).

Ascorbate is an important antioxidant, which also displays important functions in neuronal tissues, including the retina. The retina is responsible for the initial steps of visual processing, which is further refined in cerebral high-order centers. The retina is also a prototypical model for studying physiologic aspects of cells that comprise the nervous system. Of major importance also is the cellular messenger nitric oxide (NO). Previous studies have demonstrated the significance of NO for both survival and proliferation of cultured embryonic retinal cells. Cultured retinal cells express a high-affinity ascorbate transporter, and the release of ascorbate is delicately regulated by ionotropic glutamate receptors. Therefore, we proposed whether there is interplay between the ascorbate transport system and NO signaling pathway in retinal cells. Here we show compelling evidence that ascorbate uptake is tightly controlled by NO and its downstream signaling pathway in culture. NO also modulates the expression of SVCT-2, an effect mediated by cGMP and PKG. Kinetic studies suggest that NO increases the transport capacity for ascorbate, but not the affinity of SVCT-2 for its substrate. Interestingly, NO utilizes the NF-κB pathway, in a PKG-dependent manner, to modulate both SVCT-2 expression and ascorbate uptake. These results demonstrate that NO exerts a fine-tuned control of the availability of ascorbate to cultured retinal cells and strongly reinforces ascorbate as an important bioactive molecule in neuronal tissues.

Calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors trigger neuronal nitric-oxide synthase activation to promote nerve cell death in an Src kinase-dependent fashion.

In the retina information decoding is dependent on excitatory neurotransmission and is critically modulated by AMPA glutamate receptors. The Src-tyrosine kinase has been implicated in modulating neurotransmission in CNS. Thus, our main goal was to correlate AMPA-mediated excitatory neurotransmission with the modulation of Src activity in retinal neurons. Cultured retinal cells were used to access the effects of AMPA stimulation on nitric oxide (NO) production and Src phosphorylation. 4-Amino-5-methylamino-2',7'-difluorofluorescein diacetate fluorescence mainly determined NO production, and immunocytochemistry and Western blotting evaluated Src activation. AMPA receptors activation rapidly up-regulated Src phosphorylation at tyrosine 416 (stimulatory site) and down-regulated phosphotyrosine 527 (inhibitory site) in retinal cells, an effect mainly mediated by calcium-permeable AMPA receptors. Interestingly, experiments confirmed that neuronal NOS was activated in response to calcium-permeable AMPA receptor stimulation. Moreover, data suggest NO pathway as a key regulatory signaling in AMPA-induced Src activation in neurons but not in glial cells. The NO donor SNAP (S-nitroso-N-acetyl-DL-penicillamine) and a soluble guanylyl cyclase agonist (YC-1) mimicked AMPA effect in Src Tyr-416 phosphorylation, reinforcing that Src activation is indeed modulated by the NO pathway. Gain and loss-of-function data demonstrated that ERK is a downstream target of AMPA-induced Src activation and NO signaling. Furthermore, AMPA stimulated NO production in organotypic retinal cultures and increased Src activity in the in vivo retina. Additionally, AMPA-induced apoptotic retinal cell death was regulated by both NOS and Src activity. Because Src activity is pivotal in several CNS regions, the data presented herein highlight that Src modulation is a critical step in excitatory retinal cell death.

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.

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.

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.

Mechanical actuators in microglia dynamics and function.

Microglia are the most prominent immune resident cell population in the central nervous system (CNS). In the healthy CNS, microglia survey their surrounding microenvironment, through recurrent extension and retraction of filopodia-like membrane protrusions, without evident cell body displacement. Microglia undergo dramatic transcriptomic and shape changes upon brain insults or neurodegenerative disease states and adopt a classical immune effector function (producing an extensive array of inflammatory mediators such as cytokines, chemokines, and reactive oxygen species) to re-establish tissue homeostasis. While the biophysical principles underlying microglia morphological changes remain elusive, several recent studies have highlighted the pivotal role of the actin and non-muscle myosin II filamentous cytoskeleton in this process. In this work, we discuss how subcellular topological patterning of the actin and myosin cytoskeleton can control microglial cell shape dynamics and how it can potentially feedback on their functional specialization, which is of great importance to understanding the mechanisms of microglial action in homeostatic conditions and CNS disease states.

Profiling Microglia in a Mouse Model of Machado-Joseph Disease.

Microglia have been increasingly implicated in neurodegenerative diseases (NDs), and specific disease associated microglia (DAM) profiles have been defined for several of these NDs. Yet, the microglial profile in Machado-Joseph disease (MJD) remains unexplored. Here, we characterized the profile of microglia in the CMVMJD135 mouse model of MJD. This characterization was performed using primary microglial cultures and microglial cells obtained from disease-relevant brain regions of neonatal and adult CMVMJD135 mice, respectively. Machine learning models were implemented to identify potential clusters of microglia based on their morphological features, and an RNA-sequencing analysis was performed to identify molecular perturbations and potential therapeutic targets. Our findings reveal morphological alterations that point to an increased activation state of microglia in CMVMJD135 mice and a disease-specific transcriptional profile of MJD microglia, encompassing a total of 101 differentially expressed genes, with enrichment in molecular pathways related to oxidative stress, immune response, cell proliferation, cell death, and lipid metabolism. Overall, these results allowed us to define the cellular and molecular profile of MJD-associated microglia and to identify genes and pathways that might represent potential therapeutic targets for this disorder.

Developmental regulation of neuronal survival by adenosine in the in vitro and in vivo avian retina depends on a shift of signaling pathways leading to CREB phosphorylation or dephosphorylation.

Previous studies have shown a cAMP/protein kinase A-dependent neuroprotective effect of adenosine on glutamate or re-feeding-induced apoptosis in chick retina neuronal cultures. In the present work, we have studied the effect of adenosine on the survival of retinal progenitor cells. Cultures obtained from 6-day-old (E6) or from 8-day-old (E8) chick embryos were challenged 2 h (C0) or 1 day (C1) after seeding and analyzed after 3-4 days in vitro. Surprisingly, treatment with the selective A2a adenosine receptor agonists N(6) -[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA) or 3-[4-[2-[[6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino]ethyl]phenyl]propanoic acid (CGS21680) promoted cell death when added at E6C0 but not at E6C1 or E8C0. DPMA-induced cell death involved activation of A2a receptors and the phospholipase C/protein kinase C but not the cAMP/protein kinase A pathway, and was not correlated with early modulation of precursor cells proliferation. Regarding cyclic nucleotide responsive element binding protein (CREB) phosphorylation, cultures from E6 embryos behave in an opposite manner from that from E8 embryos, both in vitro and in vivo. While the phospho-CREB level was high at E6C0 cultures and could be diminished by DPMA, it was lower at E8C0 and could be increased by DPMA. Similar to what was observed in cell survival studies, CREB dephosphorylation induced by DPMA in E6C0 cultures was dependent on the Phospholipase C/protein kinase C pathway. Accordingly, cell death induced by DPMA was inhibited by okadaic acid, a phosphatase blocker. Moreover, DPMA as well as the adenosine uptake blocker nitrobenzyl mercaptopurine riboside (NBMPR) modulate cell survival and CREB phosphorylation in a population of cells in the ganglion cell layer in vivo. These data suggest that A2a adenosine receptors as well as CREB may display a novel and important function by controlling the repertoire of developing retinal neurons.

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.

Astrocyte-derived TNF and glutamate critically modulate microglia activation by methamphetamine.

Methamphetamine (Meth) is a powerful illicit psychostimulant, widely used for recreational purposes. Besides disrupting the monoaminergic system and promoting oxidative brain damage, Meth also causes neuroinflammation, contributing to synaptic dysfunction and behavioral deficits. Aberrant activation of microglia, the largest myeloid cell population in the brain, is a common feature in neurological disorders triggered by neuroinflammation. In this study, we investigated the mechanisms underlying the aberrant activation of microglia elicited by Meth in the adult mouse brain. We found that binge Meth exposure caused microgliosis and disrupted risk assessment behavior (a feature that usually occurs in individuals who abuse Meth), both of which required astrocyte-to-microglia crosstalk. Mechanistically, Meth triggered a detrimental increase of glutamate exocytosis from astrocytes (in a process dependent on TNF production and calcium mobilization), promoting microglial expansion and reactivity. Ablating TNF production, or suppressing astrocytic calcium mobilization, prevented Meth-elicited microglia reactivity and re-established risk assessment behavior as tested by elevated plus maze (EPM). Overall, our data indicate that glial crosstalk is critical to relay alterations caused by acute Meth exposure.