Rat Cortical Astrocytes Research Articles

AhR Agonistic Components in Urban Particulate Matter Regulate Astrocytic Activation and Function.

Exposure to atmospheric particulate matter (PM) has been found to accelerate the onset of neurological disorders via the induction of detrimental neuroinflammatory responses. To reveal how astrocytes respond to urban atmospheric PM stimulation, a commercially available standard reference material (SRM1648a) was tested in this study on the activation of rat cortical astrocytes. The results showed that SRM1648a stimulation induced both A1 and A2 phenotypes in astrocytes, as characterized by the exposure concentration-dependent increases in Fkbp5, Sphk1, S100a10, and Il6 mRNA levels. Studying the functional alterations of astrocytes indicated that the neurotrophic factors of Gdnf and Ngf were transcriptionally upregulated due to astrocytic A2-type activation. SRM1648a also promoted autonomous motility of astrocytes and elevated the expressions of chemokines. The aryl hydrocarbon receptor (AhR) agonistic components, such as polycyclic aromatic hydrocarbons (PAHs), were recognized to greatly contribute to SRM1648a-induced effects on astrocytes, which was confirmed by the attenuation of PM-disturbed astrocytic effects via AhR blockage. This study, for the first time, uncovered the direct regulation of urban atmospheric PM on astrocytic activation and function and traced the containing bioactive components (e.g., PAHs) with AhR agonistic activity. The findings provided new knowledge on understanding the ambiguous neurological disturbance from ambient fine PM pollution.

Tau depletion in human neurons mitigates Aβ-driven toxicity

Alzheimer’s disease (AD) is an age-related neurodegenerative condition and the most common type of dementia, characterised by pathological accumulation of extracellular plaques and intracellular neurofibrillary tangles that mainly consist of amyloid-β (Aβ) and hyperphosphorylated tau aggregates, respectively. Previous studies in mouse models with a targeted knock-out of the microtubule-associated protein tau (Mapt) gene demonstrated that Aβ-driven toxicity is tau-dependent. However, human cellular models with chronic tau lowering remain unexplored. In this study, we generated stable tau-depleted human induced pluripotent stem cell (iPSC) isogenic panels from two healthy individuals using CRISPR-Cas9 technology. We then differentiated these iPSCs into cortical neurons in vitro in co-culture with primary rat cortical astrocytes before conducting electrophysiological and imaging experiments for a wide range of disease-relevant phenotypes. Both AD brain derived and recombinant Aβ were used in this study to elicit toxic responses from the iPSC-derived cortical neurons. We showed that tau depletion in human iPSC-derived cortical neurons caused considerable reductions in neuronal activity without affecting synaptic density. We also observed neurite outgrowth impairments in two of the tau-depleted lines used. Finally, tau depletion protected neurons from adverse effects by mitigating the impact of exogenous Aβ-induced hyperactivity, deficits in retrograde axonal transport of mitochondria, and neurodegeneration. Our study established stable human iPSC isogenic panels with chronic tau depletion from two healthy individuals. Cortical neurons derived from these iPSC lines showed that tau is essential in Aβ-driven hyperactivity, axonal transport deficits, and neurodegeneration, consistent with studies conducted in Mapt−/− mouse models. These findings highlight the protective effects of chronic tau lowering strategies in AD pathogenesis and reinforce the potential in clinical settings. The tau-depleted human iPSC models can now be applied at scale to investigate the involvement of tau in disease-relevant pathways and cell types.

Transporters involved in adult rat cortical astrocyte dopamine uptake: Kinetics, expression and pharmacological modulation.

Astrocytes, glial cells in the central nervous system, perform a multitude of homeostatic functions and are in constant bidirectional communication with neuronal cells, a concept named the tripartite synapse; however, their role in the dopamine homeostasis remains unexplored. The aim of this study was to clarify the pharmacological and molecular characteristics of dopamine transport in cultured cortical astrocytes of adult rats. In addition, we were interested in the expression of mRNA of dopamine transporters as well as dopamine receptors D1 and D2 and in the effect of dopaminergic drugs on the expression of these transporters and receptors. We have found that astrocytes possess both Na+-dependent and Na+-independent transporters. Uptake of radiolabelled dopamine was time-, temperature- and concentration-dependent and was inhibited by decynium-22, a plasma membrane monoamine transporter inhibitor, tricyclic antidepressants desipramine and nortriptyline, both inhibitors of the norepinephrine transporter. Results of transporter mRNA expression indicate that the main transporters involved in cortical astrocyte dopamine uptake are the norepinephrine transporter and plasma membrane monoamine transporter. Both dopamine receptor subtypes were identified in cortical astrocyte cultures. Twenty-four-hour treatment of astrocyte cultures with apomorphine, a D1/D2 agonist, induced upregulation of D1 receptor, norepinephrine transporter and plasma membrane monoamine transporter, whereas the latter was downregulated by haloperidol and L-DOPA. Astrocytes take up dopamine by multiple transporters and express dopamine receptors, which are sensitive to dopaminergic drugs. The findings of this study could open a promising area of research for the fine-tuning of existing therapeutic strategies.

Metabolic cross‐talk between astrocytes and neurons: implications for Alzheimer’s disease

AbstractBackgroundConverging evidence indicates that declining brain energetics contributes to the initiation and progression of neurodegenerative disorders, including Alzheimer’s disease (AD). Upstream events often characterizing AD include hypometabolism of glucose and oxygen in the brain, which may cause mitochondrial dysfunction, energetic failure, and oxidative stress (Moreira et al., 2010). Astrocytes, which have a main role in supporting neuronal function and metabolism, might contribute to the development of neurodegenerative diseases. By modulating the release of gliotransmitters (e.g. glutamate), they provide a fine control of neuronal metabolism and cell‐to‐cell communication, both of them involved in AD (Monterey et al., 2021). Impaired astrocytes metabolism may incite an inflammatory response that, in turn, negatively impacts neuronal viability.MethodPrimary rat cortical astrocytes were isolated from the cortex of Wistar rat pups. A metabolic stress was induced by using glyceraldehyde (GA) (Magi et al., 2021). We evaluated cell viability, the release of tumor necrosis factor alpha (TNFα) and the involvement of the nuclear factor‐kappa B (NF‐kB) pathway. In order to check the glutamate turnover, we analyzed the expression of GLAST and GLT1 and we evaluated the ATP content after glutamate exposure under physiological conditions. We also explored whether damaged astrocytes could elicit neuronal death in a co‐culture setting.ResultExposure to increasing concentrations of GA caused a concentration‐dependent cell injury in rat cortical astrocytes. GA (1mM) started to induce a significant cell damage after 24 h. After 48 h, GA exposure activated the NF‐kB pathway and the release of TNFα. GA treatment significantly increased GLAST and GLT1 levels after 48h. When injured astrocytes were co‐cultured with cortical neurons, neurons viability significantly decreased. Under physiological condition, glutamate significantly improved intracellular ATP levels after 4 and 8 hours of treatment, paving the way to the evaluation of its action in metabolic‐impaired setting, as induced by GA treatment.ConclusionOur results suggest that metabolically impaired astrocytes may damage neighboring neurons, suggesting astrocytes involvement in the pathophysiological development and progression of AD. Therefore, astrocytes can be considered a key therapeutic and neuroprotective target for future studies.

Ketamine Reduces the Surface Density of the Astroglial Kir4.1 Channel and Inhibits Voltage-Activated Currents in a Manner Similar to the Action of Ba2+ on K+ Currents.

A single sub-anesthetic dose of ketamine evokes rapid and long-lasting beneficial effects in patients with a major depressive disorder. However, the mechanisms underlying this effect are unknown. It has been proposed that astrocyte dysregulation of extracellular K+ concentration ([K+]o) alters neuronal excitability, thus contributing to depression. We examined how ketamine affects inwardly rectifying K+ channel Kir4.1, the principal regulator of K+ buffering and neuronal excitability in the brain. Cultured rat cortical astrocytes were transfected with plasmid-encoding fluorescently tagged Kir4.1 (Kir4.1-EGFP) to monitor the mobility of Kir4.1-EGFP vesicles at rest and after ketamine treatment (2.5 or 25 µM). Short-term (30 min) ketamine treatment reduced the mobility of Kir4.1-EGFP vesicles compared with the vehicle-treated controls (p < 0.05). Astrocyte treatment (24 h) with dbcAMP (dibutyryl cyclic adenosine 5'-monophosphate, 1 mM) or [K+]o (15 mM), which increases intracellular cAMP, mimicked the ketamine-evoked reduction of mobility. Live cell immunolabelling and patch-clamp measurements in cultured mouse astrocytes revealed that short-term ketamine treatment reduced the surface density of Kir4.1 and inhibited voltage-activated currents similar to Ba2+ (300 µM), a Kir4.1 blocker. Thus, ketamine attenuates Kir4.1 vesicle mobility, likely via a cAMP-dependent mechanism, reduces Kir4.1 surface density, and inhibits voltage-activated currents similar to Ba2+, known to block Kir4.1 channels.

Inhibition of Prolyl Oligopeptidase Restores Prohibitin 2 Levels in Psychosis Models: Relationship to Cognitive Deficits in Schizophrenia.

Cognitive impairment represents one of the core features of schizophrenia. Prolyl Oligopeptidase (POP) inhibition is an emerging strategy for compensating cognitive deficits in hypoglutamatergic states such as schizophrenia, although little is known about how POP inhibitors exert their pharmacological activity. The mitochondrial and nuclear protein Prohibitin 2 (PHB2) could be dysregulated in schizophrenia. However, altered PHB2 levels in schizophrenia linked to N-methyl-D-aspartate receptor (NMDAR) activity and cognitive deficits are still unknown. To shed light on this, we measured the PHB2 levels by immunoblot in a postmortem dorsolateral prefrontal cortex (DLPFC) of schizophrenia subjects, in the frontal pole of mice treated with the NMDAR antagonists phencyclidine and dizocilpine, and in rat cortical astrocytes and neurons treated with dizocilpine. Mice and cells were treated in combination with the POP inhibitor IPR19. The PHB2 levels were also analyzed by immunocytochemistry in rat neurons. The PHB2 levels increased in DLPFC in cases of chronic schizophrenia and were associated with cognitive impairments. NMDAR antagonists increased PHB2 levels in the frontal pole of mice and in rat astrocytes and neurons. High levels of PHB2 were found in the nucleus and cytoplasm of neurons upon NMDAR inhibition. IPR19 restored PHB2 levels in the acute NMDAR inhibition. These results show that IPR19 restores the upregulation of PHB2 in an acute NMDAR hypoactivity stage suggesting that the modulation of PHB2 could compensate NMDAR-dependent cognitive impairments in schizophrenia.

Minoxidil Regulates Aging-Like Phenotypes in Rat Cortical Astrocytes In Vitro.

Mainly due to the slanted focus on the mechanism and regulation of neuronal aging, research on astrocyte aging and its modulation during brain aging is scarce. In this study, we established aged astrocyte culture model by long-term culturing. Cellular senescence was confirmed through SA-β-gal staining as well as through the examination of morphological, molecular, and functional markers. RNA sequencing and functional analysis of astrocytes were performed to further investigate the detailed characteristics of the aged astrocyte model. Along with aged phenotypes, decreased astrocytic proliferation, migration, mitochondrial energetic function and support for neuronal survival and differentiation has been observed in aged astrocytes. In addition, increased expression of cytokines and chemokine-related factors including plasminogen activator inhibitor -1 (PAI-1) was observed in aged astrocytes. Using the RNA sequencing results, we searched potential drugs that can normalize the dysregulated gene expression pattern observed in long-term cultured aged astrocytes. Among several candidates, minoxidil, a pyrimidine-derived anti-hypertensive and anti-pattern hair loss drug, normalized the increased number of SA-β-gal positive cells and nuclear size in aged astrocytes. In addition, minoxidil restored up-regulated activity of PAI-1 and increased mitochondrial superoxide production in aged astrocytes. We concluded that long term culture of astrocytes can be used as a reliable model for the study of astrocyte senescence and minoxidil can be a plausible candidate for the regulation of brain aging.

Cytotoxic Effects of Cannabidiol on Neonatal Rat Cortical Neurons and Astrocytes: Potential Danger to Brain Development.

The influence of cannabidiol (CBD) on brain development is inadequately understood. Since CBD is considered a non-intoxicating drug, it has attracted great interest concerning its potential medical applicability, including in pregnant women and children. Here, we elucidated the response of perinatal rat cortical neurons and astrocytes to CBD at submicromolar (0.1, 0.5, 1, 5 µM) concentrations attainable in humans. The effect of CBD was concentration- and time-dependent and cell-specific. In neurons, 0.1 µM CBD induced an early and transient change in mitochondrial membrane potential (ΔΨm), ATP depletion, and caspase-8 activation, followed by rapid ATP recovery and progressive activation of caspase-9 and caspase-3/7, resulting in early apoptotic cell death with reduction and shortening of dendrites, cell shrinkage, and chromatin condensation. The decrease in neuronal viability, ATP depletion, and caspase activation due to CBD exposure was prevented by transient receptor potential vanilloid 1 (TRPV1) antagonist. In astrocytes, 0.5 µM CBD caused an immediate short-term dysregulation of ΔΨm, followed by ATP depletion with transient activation of caspase-8 and progressive activation of caspase-9 and caspase-3/7, leading to early apoptosis and subsequent necroptosis. In astrocytes, both TRPV1 and cannabinoid receptor 1 (CB1) antagonists protected viability and prevented apoptosis. Given that CBD is a non-intoxicating drug, our results clearly show that this is not the case during critical periods of brain development when it can significantly interfere with the endogenous cannabinoid system.

LncRNA BIRF Promotes Brain Ischemic Tolerance Induced By Cerebral Ischemic Preconditioning Through Upregulating GLT-1 via Sponging miR-330-5p

Long noncoding RNAs (lncRNAs) play an important regulatory role in various diseases. However, the role of lncRNAs in brain ischemic tolerance (BIT) induced by cerebral ischemic preconditioning (CIPC) is still unknown. The lncRNA profile of rat cortical astrocytes pretreated with ischemic preconditioning was analyzed by high-throughput sequencing. The results of Cell-Counting Kit-8 (CCK-8) assay showed that a novel lncRNA, NONRATT009133.2, which we referred to as brain ischemia–related factor (BIRF), was highly correlated with BIT. Through bioinformatics analysis, we predicted that BIRF, miR-330-5p, and GLT-1 (also named Slc1a2) might constitute a ceRNA regulatory network in the induction of BIT. We found that BIRF was upregulated by CIPC, which promoted GLT-1 expression and BIT induction. BIRF could directly bind to miR-330-5p. Furthermore, miR-330-5p directly targeted GLT-1, and miR-330-5p inhibited both GLT-1 expression and BIT induction in vitro and in vivo. Moreover, BIRF acts as a molecular sponge to competitively bind to miR-330-5p with GLT-1 mRNA, while the miR-330-5p inhibitor reversed all the effects of BIRF siRNA on GLT-1 expression and neuronal vitality. Taken together, our results demonstrate the important roles of the BIRF/miR-330-5p/GLT-1 axis in the induction of BIT by CIPC. BIRF may be a potentially effective therapeutic strategy against stroke injury.

Serotype-based evaluation of an optogenetic construct in rat cortical astrocytes

Optogenetics is a modern technique which has been recently expanded to non-neuronal cell types, e.g., astrocytes, and involves targeted gene delivery of light-sensitive ion channels like Channelrhodopsin-2 (ChR2). Optogenetic regulation of astrocytic activity can be used for therapeutic intervention of several neurological disorders. Astrocytic gene delivery, viz adeno-associated viral (AAV) vectors, have proven to be robust, time-, and cost-efficient contrary to the generation of transgenic animal models. When transducing astrocytes with an AAV vector, it is imperative to perform a serotype evaluation of the AAV vector due to variability in serotype transduction efficiency depending on species, target region and construct length. Rats have been a very successful animal model for studying a variety of brain disorders, from which ChR2-based intervention of astrocytes will benefit. However, the most efficient AAV capsid serotype targeting astrocytes for ChR2 expression in the in vivo rat brain cortex has not been characterized. To address this, we have evaluated AAV serotypes 1, 5, and 8 of the vector AAV-GFAP-hChR2(H134)-mCherry targeting astrocytes in the rat brain neocortex. Results show that serotype 8 exhibits promising transduction patterns, as it has demonstrated the highest tangential and radial viral spread in the rat brain. Our research will facilitate translational research for future applications of optogenetics involving the transduction of rat brain cortical astrocytes.

Hormesis – an example of astrocyte response after ethanol exposure

Purpose: In the present work we describe hormesis and review documented hormetic responses in astroglial cells after exposure to different stressors. We present an example of the astrocyte response to acute and chronic ethanol exposure with hormetic characteristics.&#x0D; Methods: As an experimental model, newborn rat cortical astrocytes in culture were used. The cells were exposed to ethanol or the primary metabolite of ethanol (acetaldehyde) for 24 h or 7 days. After treatment, the protein content was determined, and the IL–6 levels in the culture medium were measured using an enzyme– linked immunoassay.&#x0D; Results: Treatment of astroglial cells with ethanol or acetaldehyde led to enhanced protein content and stimulated IL–6 production in the low concentration zone followed by diminished protein content and an inhibition of IL–6 production at higher concentrations. Chronic exposure of astrocytes was more toxic than acute exposure for both compounds, and acetaldehyde was more toxic compared to ethanol.&#x0D; Conclusion: Ethanol and acetaldehyde represent stressors for cultured astrocytes and evoke a typical hormetic response after acute and chronic exposure.

Astrocytes Exhibit a Protective Role in Neuronal Firing Patterns under Chemically Induced Seizures in Neuron-Astrocyte Co-Cultures.

Astrocytes and neurons respond to each other by releasing transmitters, such as γ-aminobutyric acid (GABA) and glutamate, that modulate the synaptic transmission and electrochemical behavior of both cell types. Astrocytes also maintain neuronal homeostasis by clearing neurotransmitters from the extracellular space. These astrocytic actions are altered in diseases involving malfunction of neurons, e.g., in epilepsy, Alzheimer’s disease, and Parkinson’s disease. Convulsant drugs such as 4-aminopyridine (4-AP) and gabazine are commonly used to study epilepsy in vitro. In this study, we aim to assess the modulatory roles of astrocytes during epileptic-like conditions and in compensating drug-elicited hyperactivity. We plated rat cortical neurons and astrocytes with different ratios on microelectrode arrays, induced seizures with 4-AP and gabazine, and recorded the evoked neuronal activity. Our results indicated that astrocytes effectively counteracted the effect of 4-AP during stimulation. Gabazine, instead, induced neuronal hyperactivity and synchronicity in all cultures. Furthermore, our results showed that the response time to the drugs increased with an increasing number of astrocytes in the co-cultures. To the best of our knowledge, our study is the first that shows the critical modulatory role of astrocytes in 4-AP and gabazine-induced discharges and highlights the importance of considering different proportions of cells in the cultures.

Selective in vitro anticancer effect of blueberry extract (Vaccinium virgatum) against C6 rat glioma: exploring their redox status.

The aim of this study was to investigate the anticancer potential of blueberry extract (Vaccinium virgatum) against a C6 rat glioma lineage. Cultures of the C6 cells were exposed to blueberry extract at concentrations of 50 to 600µg/mL for 12, 24, 48, or 72h and then evaluated for cell viability, proliferation, migration, colony formation and oxidative stress. We also evaluated the effects of blueberry extract on primary rat cortical astrocytes. Our results show that treatment with blueberry extract did not alter the viability or proliferation of normal primary astrocytes but it did significantly reduce the viability in 21.54 % after 48h and proliferation in 8.59 % after 24h of C6 cells at 200µg/mL. We also observed a reduction in the size of the colonies of 29.99 % at 100µg/mL when compared to the control cells and cell migration was also reduced at 50µg/mL. After 72h, there was a reduction in the reactive oxygen species levels ranging from 46.26 to 34.73 %, in addition to a 380.2 % increase in total thiol content. Superoxide dismutase, catalase, and glutathione S-transferase activities were also enhanced when compared to the control. Taken together this data suggests that blueberry extract exerts some selective anticancer activity in C6 glioma cells.

Dynamic expression of homeostatic ion channels in differentiated cortical astrocytes in vitro

The capacity of astrocytes to adapt their biochemical and functional features upon physiological and pathological stimuli is a fundamental property at the basis of their ability to regulate the homeostasis of the central nervous system (CNS). It is well known that in primary cultured astrocytes, the expression of plasma membrane ion channels and transporters involved in homeostatic tasks does not closely reflect the pattern observed in vivo. The individuation of culture conditions that promote the expression of the ion channel array found in vivo is crucial when aiming at investigating the mechanisms underlying their dynamics upon various physiological and pathological stimuli. A chemically defined medium containing growth factors and hormones (G5) was previously shown to induce the growth, differentiation, and maturation of primary cultured astrocytes. Here we report that under these culture conditions, rat cortical astrocytes undergo robust morphological changes acquiring a multi-branched phenotype, which develops gradually during the 2-week period of culturing. The shape changes were paralleled by variations in passive membrane properties and background conductance owing to the differential temporal development of inwardly rectifying chloride (Cl−) and potassium (K+) currents. Confocal and immunoblot analyses showed that morphologically differentiated astrocytes displayed a large increase in the expression of the inward rectifier Cl− and K+ channels ClC-2 and Kir4.1, respectively, which are relevant ion channels in vivo. Finally, they exhibited a large diminution of the intermediate filaments glial fibrillary acidic protein (GFAP) and vimentin which are upregulated in reactive astrocytes in vivo. Taken together the data indicate that long-term culturing of cortical astrocytes in this chemical-defined medium promotes a quiescent functional phenotype. This culture model could aid to address the regulation of ion channel expression involved in CNS homeostasis in response to physiological and pathological challenges.

Distinct Effects of Escitalopram and Vortioxetine on Astroglial L-Glutamate Release Associated with Connexin43

It has been established that enhancement of serotonergic transmission contributes to improvement of major depression; however, several post-mortem studies and experimental depression rodent models suggest that functional abnormalities of astrocytes play important roles in the pathomechanisms/pathophysiology of mood disorders. Direct effects of serotonin (5-HT) transporter inhibiting antidepressants on astroglial transmission systems has never been assessed in this context. Therefore, to explore the effects of antidepressants on transmission associated with astrocytes, the present study determined the effects of the selective 5-HT transporter inhibitor, escitalopram, and the 5-HT partial agonist reuptake inhibitor, vortioxetine, on astroglial L-glutamate release through activated hemichannels, and the expression of connexin43 (Cx43), type 1A (5-HT1AR) and type 7 (5-HT7R) 5-HT receptor subtypes, and extracellular signal-regulated kinase (ERK) in astrocytes using primary cultured rat cortical astrocytes in a 5-HT-free environment. Both escitalopram and 5-HT1AR antagonist (WAY100635) did not affect basal astroglial L-glutamate release or L-glutamate release through activated hemichannels. Subchronic (for seven days) administrations of vortioxetine and the 5-HT7R inverse agonist (SB269970) suppressed both basal L-glutamate release and L-glutamate release through activated hemichannels, whereas 5-HT1AR agonist (BP554) inhibited L-glutamate release through activated hemichannels, but did not affect basal L-glutamate release. In particular, WAY100635 did not affect the inhibitory effects of vortioxetine on L-glutamate release. Subchronic administration of vortioxetine, BP554 and SB269970 downregulated 5-HT1AR, 5-HT7R and phosphorylated ERK in the plasma membrane fraction, but escitalopram and WAY100635 did not affect them. Subchronic administration of SB269970 decreased Cx43 expression in the plasma membrane but did not affect the cytosol; however, subchronic administration of BP554 increased Cx43 expression in the cytosol but did not affect the plasma membrane. Subchronic vortioxetine administration increased Cx43 expression in the cytosol and decreased it in the plasma membrane. WAY100635 prevented an increased Cx43 expression in the cytosol induced by vortioxetine without affecting the reduced Cx43 expression in the plasma membrane. These results suggest that 5-HT1AR downregulation probably increases Cx43 synthesis, but 5-HT7R downregulation suppresses Cx43 trafficking to the plasma membrane. These results also suggest that the subchronic administration of therapeutic-relevant concentrations of vortioxetine inhibits both astroglial L-glutamate and Cx43 expression in the plasma membrane via 5-HT7R downregulation but enhances Cx43 synthesis in the cytosol via 5-HT1AR downregulation. This combination of the downregulation of 5-HT1AR, 5-HT7R and Cx43 in the astroglial plasma membrane induced by subchronic vortioxetine administration suggest that astrocytes is possibly involved in the pathophysiology of depression.

Increase of glycogen storage by sodium arsenite in rat cortical astrocytes through glycogen synthase activation and its association to toxicity

Increase of glycogen storage by sodium arsenite in rat cortical astrocytes through glycogen synthase activation and its association to toxicity

Effects of ethanol-and choline-treated astrocytes on hippocampal neuron neurite outgrowth in vitro.

Exposure to ethanol in utero can result in Fetal Alcohol Spectrum Disorders, which may cause long-lasting cognitive and behavioral abnormalities. Preclinical studies indicate that choline ameliorates the behavioral effects of developmental alcohol exposure in rodents, and clinical studies on the effectiveness of choline, administered early in pregnancy, showed that the adverse effects of heavy prenatal alcohol exposure on postnatal growth, and cognition in human infants were mitigated. However, little is known on the mechanisms behind the effects of choline. We have previously reported that astrocyte pre-treatment with 75 mM ethanol, in vitro, reduces neurite outgrowth in hippocampal neurons co-cultured with the pre-treated astrocytes. Our in vitro system allows us to study the effects of chemicals on astrocyte functions, able to modulate neuronal development. The main objective was to test the hypothesis that choline can ameliorate the astrocyte-mediated effects of ethanol on neurite growth. In this study, we exposed primary rat cortical astrocytes to ethanol, choline, ethanol plus choline, or control conditions for 24 h. Culture media was then removed, replaced with fresh media containing no ethanol or choline treatments and primary rat hippocampal neurons were plated on top of the astrocyte monolayer and cultured for 16 h. Neurons were then stained for β-III Tubulin and neurite outgrowth was measured. Astrocyte exposure to ethanol (25, 50, and 75 mM) decreases neurite outgrowth in co-cultured hippocampal pyramidal neurons, while astrocyte treatment with choline had no effect. Astrocyte treatment with ethanol and choline in combination, however, prevented the effect of ethanol, leading to levels of neurite outgrowth similar the control condition. Choline prevents the inhibitory effect of ethanol-treated astrocytes on neurite outgrowth while not altering normal neuronal development. These results suggest a new, astrocyte-mediated mechanism by which choline ameliorates the effects of developmental alcohol exposure.

MiR-124 and Small Molecules Synergistically Regulate the Generation of Neuronal Cells from Rat Cortical Reactive Astrocytes.

Irreversible neuron loss caused by central nervous system injuries usually leads to persistent neurological dysfunction. Reactive astrocytes, because of their high proliferative capacity, proximity to neuronal lineage, and significant involvement in glial scarring, are ideal starting cells for neuronal regeneration. Having previously identified several small molecules as important regulators of astrocyte-to-neuron reprogramming, we established herein that miR-124, ruxolitinib, SB203580, and forskolin could co-regulate rat cortical reactive astrocyte-to-neuron conversion. The induced cells had reduced astroglial properties, displayed typical neuronal morphologies, and expressed neuronal markers, reflecting 25.9% of cholinergic neurons and 22.3% of glutamatergic neurons. Gene analysis revealed that induced neuron gene expression patterns were more similar to that of primary neurons than of initial reactive astrocytes. On the molecular level, miR-124-driven neuronal differentiation of reactive astrocytes was via targeting of the SOX9-NFIA-HES1 axis to inhibit HES1 expression. In conclusion, we present a novel approach to inducing endogenous rat cortical reactive astrocytes into neurons through co-regulation involving miR-124 and three small molecules. Thus, our research has potential implications for inhibiting glial scar formation and promoting neuronal regeneration after central nervous system injury or disease.

Long-Term Glucose Starvation Induces Inflammatory Responses and Phenotype Switch in Primary Cortical Rat Astrocytes

Astrocytes are the most abundant cell type in the brain and crucial to ensure the metabolic supply of neurons and their synapse formation. Overnutrition as present in patients suffering from obesity causes astrogliosis in the hypothalamus. Other diseases accompanied by malnutrition appear to have an impact on the brain and astrocyte function. In the eating disorder anorexia nervosa (AN), patients suffer from undernutrition and develop volume reductions of the cerebral cortex, associated with reduced astrocyte proliferation and cell count. Although an effect on astrocytes and their function has already been shown for overnutrition, their role in long-term undernutrition remains unclear. The present study used primary rat cerebral cortex astrocytes to investigate their response to chronic glucose starvation. Cells were grown with a medium containing a reduced glucose concentration (2 mM) for 15 days. Long-term glucose starvation increased the expression of a subset of pro-inflammatory genes and shifted the primary astrocyte population to the pro-inflammatory A1-like phenotype. Moreover, genes encoding for proteins involved in the unfolded protein response were elevated. Our findings demonstrate that astrocytes under chronic glucose starvation respond with an inflammatory reaction. With respect to the multiple functions of astrocytes, an association between elevated inflammatory responses due to chronic starvation and alterations found in the brain of patients suffering from undernutrition seems possible.

Establishment and Preliminary Characterization of Three Astrocytic Cells Lines Obtained from Primary Rat Astrocytes by Sub-Cloning.

Gliomas are complex and heterogeneous tumors that originate from the glial cells of the brain. The malignant cells undergo deep modifications of their metabolism, and acquire the capacity to invade the brain parenchyma and to induce epigenetic modifications in the other brain cell types. In spite of the efforts made to define the pathology at the molecular level, and to set novel approaches to reach the infiltrating cells, gliomas are still fatal. In order to gain a better knowledge of the cellular events that accompany astrocyte transformation, we developed three increasingly transformed astrocyte cell lines, starting from primary rat cortical astrocytes, and analyzed them at the cytogenetic and epigenetic level. In parallel, we also studied the expression of the differentiation-related H1.0 linker histone variant to evaluate its possible modification in relation with transformation. We found that the most modified astrocytes (A-FC6) have epigenetic and chromosomal alterations typical of cancer, and that the other two clones (A-GS1 and A-VV5) have intermediate properties. Surprisingly, the differentiation-specific somatic histone H1.0 steadily increases from the normal astrocytes to the most transformed ones. As a whole, our results suggest that these three cell lines, together with the starting primary cells, constitute a potential model for studying glioma development.