Cannabinoid regulation of neurons in the dentate gyrus during epileptogenesis: Role of CB1R-associated proteins and downstream pathways.

The hippocampal formation plays a central role in the development of temporal lobe epilepsy (TLE), a disease characterized by recurrent, unprovoked epileptic discharges. TLE is a neurologic disorder characterized by acute long-lasting seizures (i.e., abnormal electrical activity in the brain) or seizures that occur in close proximity without recovery, typically after a brain injury or status epilepticus. After status epilepticus, epileptogenic hyperexcitability develops gradually over the following months to years, resulting in the emergence of chronic, recurrent seizures. Acting as a filter or gate, the hippocampal dentate gyrus (DG) normally prevents excessive excitation from propagating through the hippocampus, and is considered a critical region in the progression of epileptogenesis in pathological conditions. Importantly, lipid-derived endogenous cannabinoids (endocannabinoids), which are produced on demand as retrograde messengers, are central regulators of neuronal activity in the DG circuit. In this review, we summarize recent findings concerning the role of the DG in controlling hyperexcitability and propose how DG regulation by cannabinoids (CBs) could provide avenues for therapeutic interventions. We also highlight possible pathways and manipulations that could be relevant for the control of hyperexcitation. The use of CB compounds to treat epilepsies is controversial, as anecdotal evidence is not always validated by clinical trials. Recent publications shed light on the importance of the DG as a region regulating incoming hippocampal excitability during epileptogenesis. We review recent findings concerning the modulation of the hippocampal DG circuitry by CBs and discuss putative underlying pathways. A better understanding of the mechanisms by which CBs exert their action during seizures may be useful to improve therapies.

Small Extracellular Vesicles in Rat Serum Contain Astrocyte-Derived Protein Biomarkers of Repetitive Stress.

BACKGROUND: Stress precipitates mood disorders, characterized by a range of symptoms present in different combinations, suggesting the existence of disease subtypes. Using an animal model, we previously described that repetitive stress via restraint or immobilization induced depressive-like behaviors in rats that were differentially reverted by a serotonin- or noradrenaline-based antidepressant drug, indicating that different neurobiological mechanisms may be involved. The forebrain astrocyte protein aldolase C, contained in small extracellular vesicles, was identified as a potential biomarker in the cerebrospinal fluid; however, its specific origin remains unknown. Here, we propose to investigate whether serum small extracellular vesicles contain a stress-specific protein cargo and whether serum aldolase C has a brain origin. METHODS: We isolated and characterized serum small extracellular vesicles from rats exposed to restraint, immobilization, or no stress, and their proteomes were identified by mass spectrometry. Data available via ProteomeXchange with identifier PXD009085 were validated, in part, by western blot. In utero electroporation was performed to study the direct transfer of recombinant aldolase C-GFP from brain cells to blood small extracellular vesicles. RESULTS: A differential proteome was identified among the experimental groups, including aldolase C, astrocytic glial fibrillary acidic protein, synaptophysin, and reelin. Additionally, we observed that, when expressed in the brain, aldolase C tagged with green fluorescent protein could be recovered in serum small extracellular vesicles. CONCLUSION: The protein cargo of serum small extracellular vesicles constitutes a valuable source of biomarkers of stress-induced diseases, including those characterized by depressive-like behaviors. Brain-to-periphery signaling mediated by a differential molecular cargo of small extracellular vesicles is a novel and challenging mechanism by which the brain might communicate health and disease states to the rest of the body.

Contribution of early Alzheimer's disease-related pathophysiology to the development of acquired epilepsy.

Aberrant epileptic activity is detectable at early disease stages in Alzheimer's disease (AD) patients and in AD mouse models. Here, we investigated in young ArcticAß mice whether AD-like pathology renders neuronal networks more susceptible to the development of acquired epilepsy induced by unilateral intrahippocampal injection of kainic acid (IHK). In this temporal lobe epilepsy model, IHK induces a status epilepticus followed after two weeks by spontaneous recurrent seizures (SRS). ArcticAß mice exhibited more severe status epilepticus and early onset of SRS. This hyperexcitable phenotype was characterized in CA1 neurons by decreased synaptic strength, increased kainic acid-induced LTP and reduced frequency of spontaneous inhibitory currents. However, no difference in neurodegeneration, neuroinflammation, axonal reorganization or adult neurogenesis was observed in ArcticAß mice compared to wild-type littermates following IHK-induced epileptogenesis. Neuropeptide Y (NPY) expression was reduced at baseline and its IHK-induced elevation in mossy fibres and granule cells was attenuated. However, although this alteration might underlie premature seizure onset, neutralization of soluble Aß species by intracerebroventricular Aß-specific antibody application mitigated the hyperexcitable phenotype of ArcticAß mice and prevented early SRS onset. Therefore, the development of seizures at early stages of AD is mediated primarily by Aß species causing widespread changes in synaptic function.

Partial inactivation of GABAA receptors containing the α5 subunit affects the development of adult-born dentate gyrus granule cells.

Alterations of neuronal activity due to changes in GABAA receptors (GABAA R) mediating tonic inhibition influence different hippocampal functions. Gabra5-null mice and α5 subunit((H105R)) knock-in mice exhibit signs of hippocampal dysfunction, but are capable of improved performance in several learning and memory tasks. Accordingly, alleviating abnormal GABAergic tonic inhibition in the hippocampal formation by selective α5-GABAA R modulators represents a possible therapeutic approach for several intellectual deficit disorders. Adult neurogenesis in the dentate gyrus is an important facet of hippocampal plasticity; it is regulated by tonic GABAergic transmission, as shown by deficits in proliferation, migration and dendritic development of adult-born neurons in Gabra4-null mice. Here, we investigated the contribution of α5-GABAA Rs to granule cell development, using retroviral vectors expressing eGFP for labeling precursor cells in the subgranular zone. Global α5-GABAA R knockout (α5-KO) mice showed no alterations in migration and morphological development of eGFP-positive granule cells. However, upregulation of α1 subunit-immunoreactivity was observed in the hippocampal formation and cerebral cortex. In contrast, partial gene inactivation in α5-heterozygous (α5-het) mice, as well as single-cell deletion of Gabra5 in newborn granule cells from α5-floxed mice, caused severe alterations of migration and dendrite development. In α5-het mice, retrovirally mediated overexpression of Cdk5 resulted in normal migration and dendritic branching, suggesting that Cdk5 cooperates with α5-GABAA Rs to regulate neuronal development. These results show that minor imbalance of α5-GABAA R-mediated transmission may have major consequences for neuronal plasticity; and call for caution upon chronic therapeutic use of negative allosteric modulators acting at these receptors.

Rheb activation in subventricular zone progenitors leads to heterotopia, ectopic neuronal differentiation, and rapamycin-sensitive olfactory micronodules and dendrite hypertrophy of newborn neurons.

Mammalian target of rapamycin (mTOR) hyperactivity in perinatal neural progenitor cells (NPCs) of tuberous sclerosis complex 1 (Tsc1) heterozygote mice leads to heterotopia and abnormal neuronal morphogenesis as seen in patients with tuberous sclerosis. Considering that pathological hyperactive mTOR also occurs in individuals carrying no genetic mutations, we examined whether increasing mTOR activity in neonatal NPCs of wild-type mice would recapitulate the above phenotypes. Electroporation of a plasmid encoding constitutively active Ras-homolog enriched in brain (Rheb(CA)) into subventricular zone NPCs increased mTOR activity in newborn cells. At 19 d post-electroporation (dpe), heterotopia and ectopic cells with a neuronal morphology were observed along the migratory path [rostral migratory stream (RMS)] and in the olfactory bulb (OB). These ectopic cells displayed action potentials and received synaptic inputs identifying them as synaptically integrated neurons. RMS heterotopias contained astrocytes, neurons, and entrapped neuroblasts. Immunostaining at 3 dpe revealed the presence of Mash1(+) Olig2(-) cells in the migratory route accompanied by ectopic neuronal differentiation and altered direction and speed of neuroblast migration at 7 dpe, suggesting a non-cell-autonomous disruption of migration. At >19 dpe, newborn Rheb(CA)-expressing neurons displayed altered distribution and formed micronodules in the OB. In addition, they displayed increased dendritic complexity along with altered membrane biophysics and increased frequency of GABAergic synaptic inputs. OB heterotopia, micronodules, and dendrite hypertrophy were notably prevented by rapamycin treatment, suggesting their mTOR dependence. Collectively, these data show that increasing mTOR activity in neonatal NPCs of wild-type mice recapitulate the pathologies observed in Tsc1 mutant mice. In addition, increased mTOR activity in individuals without known mutations could significantly impact neurogenesis and circuit formation.

Behavioral and Molecular Responses to Exogenous Cannabinoids During Pentylenetetrazol-Induced Convulsions in Male and Female Rats.

Epilepsy is a disabling, chronic brain disease,affecting ~1% of the World's population, characterized by recurrent seizures (sudden, uncontrolled brain activity), which may manifest with motor symptoms (e.g., convulsions) or non-motor symptoms. Temporal lobe epilepsies (TLE) compromising the hippocampus are the most common form of focal epilepsies. Resistance in ~1/3 of epileptic patients to the first line of treatment, i.e., antiepileptic drugs (AEDs), has been an important motivation to seek alternative treatments. Among these, the plant Cannabis sativa (commonly known as marihuana) or compounds extracted from it (cannabinoids) have gained widespread popularity. Moreover, sex differences have been proposed in epilepsy syndromes and in cannabinoid action. In the hippocampus, cannabinoids interact with the CB1R receptor whose membrane levels are regulated by ß-Arrestin2, a protein that promotes its endocytosis and causes its downregulation. In this article, we evaluate the modulatory role of WIN 55,212-2 (WIN), a synthetic exogenous cannabinoid on behavioral convulsions and on the levels of CB1R and ß-Arrestin2 in female and male adolescent rats after a single injection of the proconvulsant pentylenetetrazol (PTZ). As epilepsies can have a considerable impact on synaptic proteins that regulate neuronal toxicity, plasticity, and cognition, we also measured the levels of key proteins markers of excitatory synapses, in order to examine whether exogenous cannabinoids may prevent such pathologic changes after acute seizures. We found that the exogenous administration of WIN prevented convulsions of medium severity in females and males and increased the levels of phosphorylated CaMKII in the hippocampus. Furthermore, we observed a higher degree of colocalization between CB1R and ß-Arrestin2 in the granule cell layer.

eNOS-dependent S-nitrosylation of the NF-κB subunit p65 has neuroprotective effects.

Cell death by glutamate excitotoxicity, mediated by N-methyl-D-aspartate (NMDA) receptors, negatively impacts brain function, including but not limited to hippocampal neurons. The NF-κB transcription factor (composed mainly of p65/p50 subunits) contributes to neuronal death in excitotoxicity, while its inhibition should improve cell survival. Using the biotin switch method, subcellular fractionation, immunofluorescence, and luciferase reporter assays, we found that NMDA-stimulated NF-κB activity selectively in hippocampal neurons, while endothelial nitric oxide synthase (eNOS), an enzyme expressed in neurons, is involved in the S-nitrosylation of p65 and consequent NF-κB inhibition in cerebrocortical, i.e., resistant neurons. The S-nitro proteomes of cortical and hippocampal neurons revealed that different biological processes are regulated by S-nitrosylation in susceptible and resistant neurons, bringing to light that protein S-nitrosylation is a ubiquitous post-translational modification, able to influence a variety of biological processes including the homeostatic inhibition of the NF-κB transcriptional activity in cortical neurons exposed to NMDA receptor overstimulation.

Cannabidiol targets mitochondria to regulate intracellular Ca2+ levels.

Cannabinoids and the endocannabinoid system have attracted considerable interest for therapeutic applications. Nevertheless, the mechanism of action of one of the main nonpsychoactive phytocannabinoids, cannabidiol (CBD), remains elusive despite potentially beneficial properties as an anti-convulsant and neuroprotectant. Here, we characterize the mechanisms by which CBD regulates Ca(2+) homeostasis and mediates neuroprotection in neuronal preparations. Imaging studies in hippocampal cultures using fura-2 AM suggested that CBD-mediated Ca(2+) regulation is bidirectional, depending on the excitability of cells. Under physiological K(+)/Ca(2+) levels, CBD caused a subtle rise in [Ca(2+)](i), whereas CBD reduced [Ca(2+)](i) and prevented Ca(2+) oscillations under high-excitability conditions (high K(+) or exposure to the K(+) channel antagonist 4AP). Regulation of [Ca(2+)](i) was not primarily mediated by interactions with ryanodine or IP(3) receptors of the endoplasmic reticulum. Instead, dual-calcium imaging experiments with a cytosolic (fura-2 AM) and a mitochondrial (Rhod-FF, AM) fluorophore implied that mitochondria act as sinks and sources for CBD's [Ca(2+)](i) regulation. Application of carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and the mitochondrial Na(+)/Ca(2+) exchange inhibitor, CGP 37157, but not the mitochondrial permeability transition pore inhibitor cyclosporin A, prevented subsequent CBD-induced Ca(2+) responses. In established human neuroblastoma cell lines (SH-SY5Y) treated with mitochondrial toxins, CBD (0.1 and 1 microm) was neuroprotective against the uncoupler FCCP (53% protection), and modestly protective against hydrogen peroxide- (16%) and oligomycin- (15%) mediated cell death, a pattern also confirmed in cultured hippocampal neurons. Thus, under pathological conditions involving mitochondrial dysfunction and Ca(2+) dysregulation, CBD may prove beneficial in preventing apoptotic signaling via a restoration of Ca(2+) homeostasis.

Aberrant expression of PAR bZIP transcription factors is associated with epileptogenesis, focus on hepatic leukemia factor.

Epilepsy is a widespread neurological disease characterized by abnormal neuronal activity resulting in recurrent seizures. There is mounting evidence that a circadian system disruption, involving clock genes and their downstream transcriptional regulators, is associated with epilepsy. In this study, we characterized the hippocampal expression of clock genes and PAR bZIP transcription factors (TFs) in a mouse model of temporal lobe epilepsy induced by intrahippocampal injection of kainic acid (KA). The expression of PAR bZIP TFs was significantly altered following KA injection as well as in other rodent models of acquired epilepsy. Although the PAR bZIP TFs are regulated by proinflammatory cytokines in peripheral tissues, we discovered that the regulation of their expression is inflammation-independent in hippocampal tissue and rather mediated by clock genes and hyperexcitability. Furthermore, we report that hepatic leukemia factor (Hlf), a member of PAR bZIP TFs family, is invariably downregulated in animal models of acquired epilepsy, regulates neuronal activity in vitro and its overexpression in dentate gyrus neurons in vivo leads to altered expression of genes associated with seizures and epilepsy. Overall, our study provides further evidence of PAR bZIP TFs involvement in epileptogenesis and points to Hlf as the key player.

A Role for mir-26a in Stress: A Potential sEV Biomarker and Modulator of Excitatory Neurotransmission.

Stress is a widespread problem in today's societies, having important consequences on brain function. Among the plethora of mechanisms involved in the stress response at the molecular level, the role of microRNAs (miRNAs) is beginning to be recognized. The control of gene expression by these noncoding RNAs makes them essential regulators of neuronal and synaptic physiology, and alterations in their levels have been associated with pathological conditions and mental disorders. In particular, the excitatory (i.e., glutamate-mediated) neurotransmission is importantly affected by stress. Here, we found that loss of miR-26a-5p (miR-26a henceforth) function in primary hippocampal neurons increased the frequency and amplitude of miniature excitatory currents, as well as the expression levels of the excitatory postsynaptic scaffolding protein PSD95. Incubation of primary hippocampal neurons with corticosterone downregulated miR-26a, an effect that mirrored our in vivo results, as miR-26a was downregulated in the hippocampus as well as in blood serum-derived small extracellular vesicles (sEVs) of rats exposed to two different stress paradigms by movement restriction (i.e., stress by restraint in cages or by complete immobilization in bags). Overall, these results suggest that miR-26a may be involved in the generalized stress response and that a stress-induced downregulation of miR-26a could have long-term effects on glutamate neurotransmission.

Astrocyte-Derived Small Extracellular Vesicles Regulate Dendritic Complexity through miR-26a-5p Activity.

In the last few decades, it has been established that astrocytes play key roles in the regulation of neuronal morphology. However, the contribution of astrocyte-derived small extracellular vesicles (sEVs) to morphological differentiation of neurons has only recently been addressed. Here, we showed that cultured astrocytes expressing a GFP-tagged version of the stress-regulated astrocytic enzyme Aldolase C (Aldo C-GFP) release small extracellular vesicles (sEVs) that are transferred into cultured hippocampal neurons. Surprisingly, Aldo C-GFP-containing sEVs (Aldo C-GFP sEVs) displayed an exacerbated capacity to reduce the dendritic complexity in developing hippocampal neurons compared to sEVs derived from control (i.e., GFP-expressing) astrocytes. Using bioinformatics and biochemical tools, we found that the total content of overexpressed Aldo C-GFP correlates with an increased content of endogenous miRNA-26a-5p in both total astrocyte homogenates and sEVs. Notably, neurons magnetofected with a nucleotide sequence that mimics endogenous miRNA-26a-5p (mimic 26a-5p) not only decreased the levels of neuronal proteins associated to morphogenesis regulation, but also reproduced morphological changes induced by Aldo-C-GFP sEVs. Furthermore, neurons magnetofected with a sequence targeting miRNA-26a-5p (antago 26a-5p) were largely resistant to Aldo C-GFP sEVs. Our results support a novel and complex level of astrocyte-to-neuron communication mediated by astrocyte-derived sEVs and the activity of their miRNA content.

Presynaptic mechanisms of endocannabinoid-mediated long-term depression in the hippocampus.

Endogenous cannabinoids (eCBs) mobilized from postsynaptic cells activate presynaptic cannabinoid receptors and thereby inhibit synaptic transmitter release. The inhibition can be either brief (seconds) or long-lasting (minutes to hours). Recent studies provide insight into the presynaptic mechanisms responsible for long-lasting, eCB-mediated long-term depression. Among these, the proteins PKA and RIM1alpha appear to be crucial, although other possibilities are emerging.

Distinctions among GABAA and GABAB responses revealed by calcium channel antagonists, cannabinoids, opioids, and synaptic plasticity in rat hippocampus.

RATIONALE: Hippocampal interneurons release gamma-aminobutyric acid (GABA) and produce fast GABA(A)- and slow GABA(B)-inhibitory postsynaptic potentials (IPSPs). The regulation of GABA(B) eIPSPs or the interneurons that produce them are not well understood. In addition, while both micro-opioid receptors (microORs) and cannabinoid CB1R receptors (CB1Rs) are present on hippocampal interneurons, it is not clear how these two systems interact. OBJECTIVES: This study tests the hypotheses that: (1) all interneurons can initiate both GABA(A) and GABA(B) inhibitory postsynaptic potentials; (2) GABA(B) responses are insensitive to mGluR-triggered, endocannabinoid (eCB)-mediated inhibitory long-term depression (iLTD); (3) GABA(B) responses are produced by interneurons that express microOR; and (4) CB1R-dependent and microOR-dependent response interact. MATERIALS AND METHODS: Pharmacological and electrophysiological approaches were used in acute rat hippocampal slices. High resistance microelectrode recordings were made from pyramidal cells, while interneurons were stimulated extracellularly. RESULTS: GABA(B) responses were found to be produced by interneurons that release GABA via either presynaptic N-type or P/Q-type calcium channels but that they are insensitive to suppression by eCBs or eCB-mediated iLTD. GABA(B) IPSPs were sensitive to suppression by a microOR agonist, suggesting a major source of GABA(B) responses is the microOR-expressing interneuron population. A small eCB-iLTD (10% eIPSP reduction) persisted in conotoxin. eCB-iLTD was blocked by a microOR agonist in 6/13 slices. CONCLUSIONS: GABA(B) responses cannot be produced by all interneurons. CB1R or microOR agonists will differentially alter the balance of activity in hippocampal circuits. CB1R- and microOR-mediated responses can interact.

BDNF/NF-κB Signaling in the Neurobiology of Depression.

BACKGROUND: Mood disorders, consisting of unipolar and bipolar depression, are complex diseases characterized by depressed mood and anhedonia. These core symptoms are accompanied in a varying manner by anxiety, several neurovegetative symptoms and cognitive impairment. Mood disorders are characterized by decreases in neurogenesis, alteration in synaptic structure and synaptic transmission, all of them regulated by BDNF, a neurotrophin that performs multiple functions in the adult central nervous system. Many evidences show that BDNF is critically decreased in mood disorders and plays an essential role in most anti-depressant treatments. In turn, the transcription factor NF-kB has recently emerged as an important player in the pathophysiology of depression, with roles in neurogenesis, synaptic transmission and plasticity. METHODOLOGY: We review the bidirectional interactions between BDNF and NF-kB signaling pathways. RESULTS AND CONCLUSIONS: We discuss a potential beneficial effect of a positive feedback loop between BDNF and NF-kB activated pathways in antidepressant action. This could be transduced into the identification of downstream NF-kB gene targets able to potentiate antidepressant mechanisms, thus guiding the development of novel and faster acting antidepressant drugs.

Astrocytes at the Hub of the Stress Response: Potential Modulation of Neurogenesis by miRNAs in Astrocyte-Derived Exosomes.

Repetitive stress negatively affects several brain functions and neuronal networks. Moreover, adult neurogenesis is consistently impaired in chronic stress models and in associated human diseases such as unipolar depression and bipolar disorder, while it is restored by effective antidepressant treatments. The adult neurogenic niche contains neural progenitor cells in addition to amplifying progenitors, neuroblasts, immature and mature neurons, pericytes, astrocytes, and microglial cells. Because of their particular and crucial position, with their end feet enwrapping endothelial cells and their close communication with the cells of the niche, astrocytes might constitute a nodal point to bridge or transduce systemic stress signals from peripheral blood, such as glucocorticoids, to the cells involved in the neurogenic process. It has been proposed that communication between astrocytes and niche cells depends on direct cell-cell contacts and soluble mediators. In addition, new evidence suggests that this communication might be mediated by extracellular vesicles such as exosomes, and in particular, by their miRNA cargo. Here, we address some of the latest findings regarding the impact of stress in the biology of the neurogenic niche, and postulate how astrocytic exosomes (and miRNAs) may play a fundamental role in such phenomenon.

Endothelial Nitric Oxide Synthase Is Present in Dendritic Spines of Neurons in Primary Cultures.

Nitric oxide exerts important regulatory functions in various brain processes. Its synthesis in neurons has been most commonly ascribed to the neuronal nitric oxide synthase (nNOS) isoform. However, the endothelial isoform (eNOS), which is significantly associated with caveolae in different cell types, has been implicated in synaptic plasticity and is enriched in the dendrites of CA1 hippocampal neurons. Using high resolution microscopy and co-distribution analysis of eNOS with synaptic and raft proteins, we now show for the first time in primary cortical and hippocampal neuronal cultures, virtually devoid of endothelial cells, that eNOS is present in neurons and is localized in dendritic spines. Moreover, eNOS is present in a postsynaptic density-enriched biochemical fraction isolated from these neuronal cultures. In addition, qPCR analysis reveals that both the nNOS as well as the eNOS transcripts are present in neuronal cultures. Moreover, eNOS inhibition in cortical cells has a negative impact on cell survival after excitotoxic stimulation with N-methyl-D-aspartate (NMDA). Consistent with previous results that indicated nitric oxide production in response to the neurotrophin BDNF, we could detect eNOS in immunoprecipitates of the BDNF receptor TrkB while nNOS could not be detected. Taken together, our results show that eNOS is located at excitatory synapses where it could represent a source for NO production and thus, the contribution of eNOS-derived nitric oxide to the regulation of neuronal survival and function deserves further investigations.

Potential Therapies by Stem Cell-Derived Exosomes in CNS Diseases: Focusing on the Neurogenic Niche.

Neurodegenerative disorders are one of the leading causes of death and disability and one of the biggest burdens on health care systems. Novel approaches using various types of stem cells have been proposed to treat common neurodegenerative disorders such as Alzheimer's Disease, Parkinson's Disease, or stroke. Moreover, as the secretome of these cells appears to be of greater benefit compared to the cells themselves, the extracellular components responsible for its therapeutic benefit have been explored. Stem cells, as well as most cells, release extracellular vesicles such as exosomes, which are nanovesicles able to target specific cell types and thus to modify their function by delivering proteins, lipids, and nucleic acids. Exosomes have recently been tested in vivo and in vitro as therapeutic conveyors for the treatment of diseases. As such, they could be engineered to target specific populations of cells within the CNS. Considering the fact that many degenerative brain diseases have an impact on adult neurogenesis, we discuss how the modulation of the adult neurogenic niches may be a therapeutic target of stem cell-derived exosomes. These novel approaches should be examined in cellular and animal models to provide better, more effective, and specific therapeutic tools in the future.

MiRNAs in Astrocyte-Derived Exosomes as Possible Mediators of Neuronal Plasticity.

Astrocytes use gliotransmitters to modulate neuronal function and plasticity. However, the role of small extracellular vesicles, called exosomes, in astrocyte-to-neuron signaling is mostly unknown. Exosomes originate in multivesicular bodies of parent cells and are secreted by fusion of the multivesicular body limiting membrane with the plasma membrane. Their molecular cargo, consisting of RNA species, proteins, and lipids, is in part cell type and cell state specific. Among the RNA species transported by exosomes, microRNAs (miRNAs) are able to modify gene expression in recipient cells. Several miRNAs present in astrocytes are regulated under pathological conditions, and this may have far-reaching consequences if they are loaded in exosomes. We propose that astrocyte-derived miRNA-loaded exosomes, such as miR-26a, are dysregulated in several central nervous system diseases; thus potentially controlling neuronal morphology and synaptic transmission through validated and predicted targets. Unraveling the contribution of this new signaling mechanism to the maintenance and plasticity of neuronal networks will impact our understanding on the physiology and pathophysiology of the central nervous system.

Modulatory Effect of 2-(4-Hydroxyphenyl)amino-1,4-naphthoquinone on Endothelial Vasodilation in Rat Aorta.

The vascular endothelium plays an essential role in the control of the blood flow. Pharmacological agents like quinone (menadione) at various doses modulate this process in a variety of ways. In this study, Q7, a 2-phenylamino-1,4-naphthoquinone derivative, significantly increased oxidative stress and induced vascular dysfunction at concentrations that were not cytotoxic to endothelial or vascular smooth muscle cells. Q7 reduced nitric oxide (NO) levels and endothelial vasodilation to acetylcholine in rat aorta. It also blunted the calcium release from intracellular stores by increasing the phenylephrine-induced vasoconstriction when CaCl2 was added to a calcium-free medium but did not affect the influx of calcium from extracellular space. Q7 increased the vasoconstriction to BaCl2 (10-3 M), an inward rectifying K+ channels blocker, and blocked the vasodilation to KCl (10-2 M) in aortic rings precontracted with BaCl2. This was recovered with sodium nitroprusside (10-8 M), a NO donor. In conclusion, Q7 induced vasoconstriction was through a modulation of cellular mechanisms involving calcium fluxes through K+ channels, and oxidative stress induced endothelium damage. These findings contribute to the characterization of new quinone derivatives with low cytotoxicity able to pharmacologically modulate vasodilation.

Human erythroid cells are affected by aluminium. Alteration of membrane band 3 protein.

There is evidence that anaemia is associated with aluminium (Al). We have already reported on the sensitivity to Al, showed by erythroid cell populations of animals chronically exposed to the metal. In order to investigate whether Al could also affect human cells, experiments were carried out both on immature and mature human erythroid cells. Erythroid progenitors (CFU-E, colony-forming units-erythroid) concentrated from human peripheral blood were cultured in an Al-rich medium under erythropoietin stimulation and their development analysed. Human peripheral erythrocytes were aged in the presence of Al. Cells were examined using scanning electron microscopy, and membrane proteins analysed by polyacrylamide gel electrophoresis with sodium dodecyl sulphate and immunoblotting. The development of the Al-treated progenitors was 8750/6600-9200 CFU-E/10(6) cells, a significantly lower median value (P<0.05) than that showed by non-treated cells (12300/11200-20700 CFU-E/10(6) cells). Erythrocyte morphological changes were induced by Al during the in vitro ageing. The cells lost their typical biconcave shape, turning into acanthocytes and stomatocytes. Simultaneously, an increased membrane protein breakdown compatible with band 3 degradation was detected. Besides, Al was found within the cells and attached to the membrane. The present in vitro results suggest that Al may disturb human erythropoiesis through combined effects on mature erythrocytes and cellular metabolism in late erythroid progenitors.