Glial Conditioned Medium Research Articles

Inflammatory Intracellular Signaling in Neurons Is Influenced by Glial Soluble Factors in iPSC-Based Cell Model of PARK2-Associated Parkinson's Disease.

Neuroinflammation is considered to be one of the driving factors in Parkinson's disease (PD). This study was conducted using neuronal and glial cell cultures differentiated from induced pluripotent stem cells (iPSC) of healthy donors (HD) and PD patients with different PARK2 mutations (PD). Based on the results of RNA sequencing, qPCR and ELISA, we revealed transcriptional and post-transcriptional changes in HD and PD neurons cultivated in HD and PD glial-conditioned medium. We demonstrated that if one or both of the components of the system, neurons or glia, is Parkin-deficient, the interaction resulted in the down-regulation of a number of key genes related to inflammatory intracellular pathways and negative regulation of apoptosis in neurons, which might be neuroprotective. In PD neurons, the stress-induced up-regulation of APLNR was significantly stronger compared to HD neurons and was diminished by glial soluble factors, both HD and PD. PD neurons in PD glial conditioned medium increased APLN expression and also up-regulated apelin synthesis and release into intracellular fluid, which represented another compensatory action. Overall, the reported results indicate that neuronal self-defense mechanisms contribute to cell survival, which might be characteristic of PD patients with Parkin-deficiency.

Melatonin Enhances Neural Differentiation of Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells (ASCs) are adult multipotent stem cells, able to differentiate toward neural elements other than cells of mesodermal lineage. The aim of this research was to test ASC neural differentiation using melatonin combined with conditioned media (CM) from glial cells. Isolated from the lipoaspirate of healthy donors, ASCs were expanded in a basal growth medium before undergoing neural differentiation procedures. For this purpose, CM obtained from olfactory ensheathing cells and from Schwann cells were used. In some samples, 1 µM of melatonin was added. After 1 and 7 days of culture, cells were studied using immunocytochemistry and flow cytometry to evaluate neural marker expression (Nestin, MAP2, Synapsin I, GFAP) under different conditions. The results confirmed that a successful neural differentiation was achieved by glial CM, whereas the addition of melatonin alone did not induce appreciable changes. When melatonin was combined with CM, ASC neural differentiation was enhanced, as demonstrated by a further improvement of neuronal marker expression, whereas glial differentiation was attenuated. A dynamic modulation was also observed, testing the expression of melatonin receptors. In conclusion, our data suggest that melatonin's neurogenic differentiation ability can be usefully exploited to obtain neuronal-like differentiated ASCs for potential therapeutic strategies.

Characterization of a Human Neuronal Culture System for the Study of Cofilin-Actin Rod Pathology.

Cofilactin rod pathology, which can initiate synapse loss, has been extensively studied in rodent neurons, hippocampal slices, and in vivo mouse models of human neurodegenerative diseases such as Alzheimer's disease (AD). In these systems, rod formation induced by disease-associated factors, such as soluble oligomers of Amyloid-β (Aβ) in AD, utilizes a pathway requiring cellular prion protein (PrPC), NADPH oxidase (NOX), and cytokine/chemokine receptors (CCR5 and/or CXCR4). However, rod pathways have not been systematically assessed in a human neuronal model. Here, we characterize glutamatergic neurons differentiated from human-induced pluripotent stem cells (iPSCs) for the formation of rods in response to activators of the PrPC-dependent pathway. Optimization of substratum, cell density, and use of glial-conditioned medium yielded a robust system for studying the development of Aβ-induced rods in the absence of glia, suggesting a cell-autonomous pathway. Rod induction in younger neurons requires ectopic expression of PrPC, but this dependency disappears by Day 55. The quantification of proteins within the rod-inducing pathway suggests that increased PrPC and CXCR4 expression may be factors in the doubling of the rod response to Aβ between Days 35 and 55. FDA-approved antagonists to CXCR4 and CCR5 inhibit the rod response. Rods were predominantly observed in dendrites, although severe cytoskeletal disruptions prevented the assignment of over 40% of the rods to either an axon or dendrite. In the absence of glia, a condition in which rods are more readily observed, neurons mature and fire action potentials but do not form functional synapses. However, PSD95-containing dendritic spines associate with axonal regions of pre-synaptic vesicles containing the glutamate transporter, VGLUT1. Thus, our results identified stem cell-derived neurons as a robust model for studying cofilactin rod formation in a human cellular environment and for developing effective therapeutic strategies for the treatment of dementias arising from multiple proteinopathies with different rod initiators.

Ghrelin peptide improves glial conditioned medium effects on neuronal differentiation of human adipose mesenchymal stem cells

The influences of ghrelin on neural differentiation of adipose-derived mesenchymal stem cells (ASCs) were investigated in this study. The expression of typical neuronal markers, such as protein gene product 9.5 (PGP9.5) and Microtubule Associated Protein 2 (MAP2), as well as glial Fibrillary Acid Protein (GFAP) as a glial marker was evaluated in ASCs in different conditions. In particular, 2 µM ghrelin was added to control ASCs and to ASCs undergoing neural differentiation. For this purpose, ASCs were cultured in Conditioned Media obtained from Olfactory Ensheathing cells (OEC-CM) or from Schwann cells (SC-CM). Data on marker expression were gathered after 1 and 7 days of culture by fluorescence immunocytochemistry and flow cytometry. Results show that only weak effects were induced by the addition of only ghrelin. Instead, dynamic ghrelin-induced modifications were detected on the increased marker expression elicited by glial conditioned media. In fact, the combination of ghrelin and conditioned media consistently induced a further increase of PGP9.5 and MAP2 expression, especially after 7 days of treatment. The combination of ghrelin with SC-CM produced the most evident effects. Weak or no modifications were found on conditioned medium-induced GFAP increases. Observations on the ghrelin receptor indicate that its expression in control ASCs, virtually unchanged by the addition of only ghrelin, was considerably increased by CM treatment. These increases were enhanced by combining ghrelin and CM treatment, especially at 7 days. Overall, it can be assumed that ghrelin favors a neuronal rather than a glial ASC differentiation.

Liver Growth Factor Induces Glia-Associated Neuroprotection in an In Vitro Model of Parkinson´s Disease.

Parkinson’s disease is a neurodegenerative disorder characterized by the progressive death of dopaminergic (DA) neurons in the substantia nigra (SN), which leads to a loss of the neurotransmitter dopamine in the basal ganglia. Current treatments relieve the symptoms of the disease, but none stop or delay neuronal degeneration. Liver growth factor (LGF) is an albumin–bilirubin complex that stimulates axonal growth in the striatum and protects DA neurons in the SN of 6-hydroxydopamine-lesioned rats. Our previous results suggested that these effects observed in vivo are mediated by microglia and/or astrocytes. To determine if these cells are LGF targets, E14 (embryos from Sprague Dawley rats of 14 days) rat mesencephalic glial cultures were used. Treatment with 100 pg/mL of LGF up-regulated the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinases 1/2 (ERK1/2) and the cyclic AMP response element binding protein (CREB) phosphorylation in glial cultures, and it increased the microglia marker Iba1 and tumor necrosis factor alpha (TNF-alpha) protein levels. The treatment of E14 midbrain neurons with a glial-conditioned medium from LGF-treated glial cultures (GCM-LGF) prevented the loss of DA neurons caused by 6-hydroxy-dopamine. This neuroprotective effect was not observed when GCM-LGF was applied in the presence of a blocking antibody of TNF-alpha activity. Altogether, our findings strongly suggest the involvement of microglia and TNF-alpha in the neuroprotective action of LGF on DA neurons observed in vitro.

Neuroprotective effect of fermented papaya preparation by activation of Nrf2 pathway in astrocytes

Objectives: Nuclear factor erythroid 2-related factor (Nrf2) in astrocyte plays important roles in brain homeostasis. Fermented papaya preparation (FPP) has anti-oxidative, anti-inflammatory, immunoregulatory properties. The present study investigated the effects of FPP on activation of Nrf2 and release of Nrf2-regulated neuroprotective antioxidants and detoxifying molecules.Methods: Primary cultured astrocytes from rat embryos were treated with FPP for 6 or 24 hours. The expression levels of nuclear Nrf2 and cytoplasmic Nrf2-regulated molecules were determined by western blot analysis and immunohistochemistry. Glutathione levels were measured in cells and medium. Dopaminergic neurons were exposed 6-hydroxydopamine (6-OHDA) with/without pre-treatment with FPP astrocytes. Mice were treated orally with FPP for 2 weeks.Results: FPP increased nuclear translocation of Nrf2 in striatal astrocytes, induced up-regulation of NAD(P)H quinine oxidoreductase-1, glutathione-S transferase and hemeoxygenase-1, and increased glutathione level and the percentage of metallothionein-expressing astrocytes. Moreover, FPP suppressed 6-OHDA-induced dopaminergic neuronal loss in not only neuron-astrocyte mixed culture, but also neuron-rich cultures pre-treated with glial conditioned medium. Two-week oral treatment of mice with FPP resulted in Nrf2 activation and increase in glutathione level in striatum.Discussion: The results indicated that FPP enhances the anti-oxidative capacity through activation of Nrf2 in astrocytes, suggesting it may provide neuroprotection in oxidative stress-related neurodegenerative diseases.

L-Theanine protects against excess dopamine-induced neurotoxicity in the presence of astrocytes.

l-Theanine (γ-glutamylethylamide), a component of green tea, is considered to have regulatory and neuroprotective roles in the brain. The present study was designed to determine the effect of l-theanine on excess dopamine-induced neurotoxicity in both cell culture and animal experiments. The primary cultured mesencephalic neurons or co-cultures of mesencephalic neurons and striatal astrocytes were pretreated with l-theanine for 72 h, and then treated with excess dopamine for further 24 h. The cell viability of dopamine neurons and levels of glutathione were evaluated. Excess dopamine-induced neurotoxicity was significantly attenuated by 72 h preincubation with l-theanine in neuron-astrocyte co-cultures but not in neuron-rich cultures. Exposure to l-theanine increased the levels of glutathione in both astrocytes and glial conditioned medium. The glial conditioned medium from l-theanine-pretreated striatal astrocytes attenuated dopamine-induced neurotoxicity and quinoprotein formation in mesencephalic neurons. In addition, replacement of l-glutamate with l-theanine in an in vitro cell-free glutathione-synthesis system produced glutathione-like thiol compounds. Furthermore, l-theanine administration (4 mg/kg, p.o.) for 14 days significantly increased glutathione levels in the striatum of mice. The results suggest that l-theanine provides neuroprotection against oxidative stress-induced neuronal damage by humoral molecules released from astrocytes, probably including glutathione.

Retinal glia promote dorsal root ganglion axon regeneration.

Axon regeneration in the adult central nervous system (CNS) is limited by several factors including a lack of neurotrophic support. Recent studies have shown that glia from the adult rat CNS, specifically retinal astrocytes and Müller glia, can promote regeneration of retinal ganglion cell axons. In the present study we investigated whether retinal glia also exert a growth promoting effect outside the visual system. We found that retinal glial conditioned medium significantly enhanced neurite growth and branching of adult rat dorsal root ganglion neurons (DRG) in culture. Furthermore, transplantation of retinal glia significantly enhanced regeneration of DRG axons past the dorsal root entry zone after root crush in adult rats. To identify the factors that mediate the growth promoting effects of retinal glia, mass spectrometric analysis of retinal glial conditioned medium was performed. Apolipoprotein E and secreted protein acidic and rich in cysteine (SPARC) were found to be present in high abundance, a finding further confirmed by western blotting. Inhibition of Apolipoprotein E and SPARC significantly reduced the neuritogenic effects of retinal glial conditioned medium on DRG in culture, suggesting that Apolipoprotein E and SPARC are the major mediators of this regenerative response.

Striatal Infusion of Glial Conditioned Medium Diminishes Huntingtin Pathology in R6/1 Mice

Huntington's disease is a neurodegenerative disorder caused by an expansion of CAG repeats in the huntingtin gene which produces widespread neuronal and glial pathology. We here investigated the possible therapeutic role of glia or glial products in Huntington's disease using striatal glial conditioned medium (GCM) from fetus mice (E16) continuously infused for 15 and 30 days with osmotic minipumps into the left striatum of R6/1 mice. Animals infused with GCM had significantly less huntingtin inclusions in the ipsilateral cerebral cortex and in the ipsilateral and contralateral striata than mice infused with cerebrospinal fluid. The numbers of DARPP-32 and TH positive neurons were also greater in the ipsilateral but not contralateral striata and substantia nigra, respectively, suggesting a neuroprotective effect of GCM on efferent striatal and nigro-striatal dopamine neurons. GCM increases activity of the autophagic pathway, as shown by the reduction of autophagic substrate, p-62, and the augmentation of LC3 II, Beclin-1 and LAMP-2 protein levels, direct markers of autophagy, in GCM infused mice. GCM also increases BDNF levels. These results suggest that CGM should be further explored as a putative neuroprotective agent in Huntington's disease.

Neuronal Per Arnt Sim (PAS) Domain Protein 4 (NPAS4) Regulates Neurite Outgrowth and Phosphorylation of Synapsin I

Neuronal Per Arnt Sim domain protein 4 (NPAS4), a brain-specific basic helix-loop-helix transcription factor, has recently been shown to regulate the development of the GABAergic inhibitory synapses and transcription program for contextual memory formation in the hippocampus. We previously reported that chronic social isolation or restriction stress in mice resulted in an impairment in memory and emotional behavior, which was associated with a decrease in Npas4 mRNA levels. In this study, we investigated the role of NPAS4 in neuronal function in vitro and in vivo. Differentiation medium-induced neurite outgrowth was inhibited in Npas4 knockdown Neuro2a cells, whereas overexpression of NPAS4 accelerated the neurite outgrowth in Neuro2a cells. Furthermore, depolarization-induced neurite outgrowth was abolished in Npas4 KO hippocampal neurons. NPAS4 overexpression increased cyclin-dependent kinase 5 (CDK5)-dependent synapsin I phosphorylation in Neuro2a cells and primary cultured hippocampal neurons. A CDK5 inhibitor, roscovitine, inhibited the neurite outgrowth and the increase in phosphorylated synapsin I (p-SYN I) levels in Npas4-overexpressed Neuro2a cells. Interaction of NPAS4 with promoters of Cdk5 and NeuN genes was demonstrated by a chromatin immunoprecipitation assay. In an in vivo study, pentylenetetrazole-induced convulsions in mice resulted in an increase in NPAS4 and p-SYN I levels in the prefrontal cortex of wild-type mice, although no changes in p-SYN I levels were observed in Npas4 knock-out mice. These results suggest that NPAS4 plays an important role in the structural and functional plasticity of neurons.

Improvement in protocol to generate homogeneous glutamatergic neurons from mouse embryonic stem cells reduced apoptosis

Obtaining a homogenous population of central nervous system neurons has been a significant challenge in neuroscience research; however, a recent study established a retinoic acid-treated embryoid bodies-based differentiation protocol that permits the effective generation of highly homogeneous glutamatergic cortical pyramidal neurons from embryonic stem cells. We were able to reproduce this protocol regarding the purity of glutamatergic neurons, but these neurons were not sufficiently healthy for long-term observation under the same conditions that were originally described. Here, we achieved a substantial improvement in cell survival by applying a simple technique: We changed the medium for glutamatergic neurons from the original complete medium to commercially available SBM (the Nerve-Cell Culture Medium manufactured by Sumitomo Bakelite Co. Ltd.) and finally succeeded in maintaining healthy neurons for at least 3weeks without decreasing their purity. Because SBM contains glial conditioned medium, we postulated that brain-derived neurotrophic factor or basic fibroblast growth factor is the key components responsible for pro-survival effect of SBM on neurons, and examined their effects by adding them to CM. As a result, neither of them had pro-survival effect on pure glutamatergic neuronal population.

Effect of Amiloride to Retinal Toxicity Induced by Tissue Plasminogen Activator

PurposeThe effects of amiloride on cellular toxicity caused by tissue plasminogen activator (tPA) in mouse primary retinal cells were investigated.MethodsPrimary retinal cell cultures were maintained using glial conditioned medium. Commercial tPA and L-arginine were added, and the level of cyclic guanosine monophosphate (cyclic-GMP) in the culture supernatant was assessed using an ELISA assay. We measured the cell viability of cultured retinal cells pretreated with three different concentrations of amiloride (1, 10, and 100 µm) in addition to commercial tPA or L-arginine treatment.ResultsAfter exposing the cultured mouse retinal cells to tPA plus L-arginine or L-arginine alone, cyclic-GMP concentrations were 61.9 ± 5.1 pmole/mL and 63.1 ± 6.1 pmole/mL, respectively. However, the control group had a significantly lower concentration of cyclic-GMP (37.2 ± 3.4 pmole/mL, p < 0.01). The cyclic GMP-dissolved solution did not cause retinal cell death. In the control group and the group treated with 1 µm amiloride and tPA containing L-arginine, the cell viability was 43.7% and 44.5%, respectively. However, cell viability increased to 70.6% with 10 µm amiloride and 78.4% with 100 µm amiloride (p = 0.015).ConclusionsL-arginine increases intracellular cyclic-GMP and may give rise to retinal cells through this mechanism. In addition, amiloride in concentrations greater than 10 µm protects against L-arginine-induced retinal cell death.

Morphine potentiates neurodegenerative effects of HIV-1 Tat through actions at -opioid receptor-expressing glia

Individuals infected with human immunodeficiency virus-1 who abuse opiates can have a higher incidence of virus-associated neuropathology. Human immunodeficiency virus does not infect neurons, but viral proteins such as transactivator of transcription and glycoprotein 120, originating from infected glia, are neurotoxic. Moreover, functional changes in glial cells that enhance inflammation and reduce trophic support are increasingly implicated in human immunodeficiency virus neuropathology. In previous studies, co-exposure with morphine enhanced transactivator of transcription neurotoxicity towards cultured striatal neurons. Since those cultures contained µ-opioid receptor-expressing astroglia and microglia, and since glia are the principal site of infection in the central nervous system, we hypothesized that morphine synergy might be glially mediated. A 60 hour, repeated measures paradigm and multiple co-culture models were used to investigate the cellular basis for opiate-enhanced human immunodeficiency virus neurotoxicity. Morphine co-exposure significantly enhanced transactivator of transcription-induced neuron death when glia were present. Synergistic effects of morphine on transactivator of transcription neurotoxicity were greatest with neuron-glia contact, but also occurred to a lesser extent with glial conditioned medium. Importantly, synergy was lost if glia, but not neurons, lacked µ-opioid receptors, indicating that opiate interactions with human immunodeficiency virus converge at the level of µ-opioid receptor-expressing glia. Morphine enhanced transactivator of transcription-induced inflammatory effectors released by glia, elevating reactive oxygen species, increasing 3-nitrotyrosine production by microglia, and reducing the ability of glia to buffer glutamate. But neuron survival was reduced even more with glial contact than with exposure to conditioned medium, suggesting that noxious elements associated with cell contact augment the toxicity due to soluble factors. Similar morphine-transactivator of transcription synergy was also observed in studies with the clade C sequence of HIV-1 transactivator of transcription, which did not cause neuron death unless morphine was present. Several paradoxical observations related to opiate effects were noted when µ-opioid receptors were specifically ablated from either glia or neurons. This suggests that µ-opioid receptor loss in isolated cell types can fundamentally distort cell-to-cell signalling, revealing opponent processes that may exist in individual cell types. Our findings show the critical role of glia in orchestrating neurotoxic interactions of morphine and transactivator of transcription, and support the emerging concept that combined exposure to opiates and human immunodeficiency virus drives enhanced pathology within the central nervous system.

Müller glia factors induce survival and neuritogenesis of peripheral and central neurons

We have examined the trophic effects of conditioned media obtained from purified murine Müller glia cells on chick purified sympathetic or dorsal root ganglia (DRG) neurons and on Retinal Ganglion Cells (RGC) from postnatal mice. Purified murine Müller glia cultures stained positively for vimentin, GFAP or S-100, but were negative for neuronal markers. Murine Müller glial conditioned medium (MMG) was concentrated and at 1:1 dilution supported 100% survival of chick or rat sympathetic neurons after 48 h compared to < 5% in controls. Partial purification of the MMG using centriprep concentrators showed that trophic activity is from molecules above 10 kDa. MMG stimulated AKT, ERK and pStat3 in sympathetic neurons. Sympathetic or DRG neuronal survival induced by MMG was blocked by anti-human NGF, but not by anti-human CNTF (sympathetic) or by anti-BDNF (DRGs) neutralizing antibodies. MMG also induced neurite outgrowth in P4 mice retinal explants and on isolated RGC. RGCs plated on top of Müller glia cells had a much better survival rate (> 80%, 96 h) compared to laminin + poly- l-lysine substrates. In conclusion, we show that purified mice Müller glia cultures secrete NGF that support peripheral neuronal survival and other unidentified trophic molecules that induce RGC survival and neuritogenesis.

Differentiation of ES cells into cerebellar neurons

The neuronal circuits of the cerebellar cortex are essential for motor and sensory learning, associative memory formation, and the vestibular ocular reflex. In children and young adults, tumors of the granule cell, the medulloblastomas, represent 40% of brain tumors. We report the differentiation of E14 ES cells into mature granule neurons by sequential treatment with secreted factors (WNT1, FGF8, and RA) that initiate patterning in the cerebellar region of the neural tube, bone morphogenic proteins (BMP6/7 and GDF7) that induce early granule cell progenitor markers (MATH1, MEIS1, ZIC1), mitogens (SHH, JAG1) that control proliferation and induce additional granule cell markers (Cyclin D2, PAX2/6), and culture in glial-conditioned medium to induce markers of mature granule neurons (GABAalpha(6)r), including ZIC2, a unique marker for granule neurons. Differentiated ES cells formed classic "T-shaped" granule cell axons in vitro, and implantation of differentiated Pde1c-Egfp-BAC transgenic ES cells into the external granule cell layer of neonatal mice resulted in the extension of parallel fibers, migration across the molecular layer, incorporation into the internal granule cell layer, and extension of short dendrites, typical of young granule cells forming synaptic connections with afferent mossy fibers. These results underscore the utility of treating ES cells with local, inductive signals that regulate CNS neuronal development in vivo as a strategy for cell replacement therapy of defined neuronal populations.

Retinal Toxicity of Commercial Tissue Plasminogen Activator Is Mediated by the Induction of Nitric Oxide in the Mouse Retinal Primary Cells

Purpose: Tissue plasminogen activator (tPA) is an efficient thrombolytic agent, but the dose-dependent retinal toxicity of intravitreal injection of commercial tPA (containing l-arginine) has been reported. Here, we sought to investigate the mechanism of tPA-induced cell death in mouse retinal cell cultures and the role of nitric oxide (NO). Methods: Primary retinal cell cultures were maintained using glial conditioned medium (GCM) solution. Mouse retinal cell death was observed by using Hoechst-propidium iodide staining. Mouse retinal cell death was also measured by lactate dehydrogenase (LDH) assay. The formation of NO was measured using Griess reagent. Results: tPA-induced cell death was detected in mouse retinal cell cultures by Hoechst-propidium iodide staining or LDH assay. l-Arginine seems to be the major factor in retinal toxicity of commercial tPA (containing l-arginine). The formation of NO was markedly increased in mouse retinal cell cultures treated with tPA (containing l-arginine) or l-arginine. NO inhibitor reduced the cell death induced by commercially available tPA or l-arginine. Conclusions: This study suggests that l-arginine from commercial tPA (containing l-arginine) induces the majority of cell death in mouse retinal cell cultures and that its cytotoxicity may depend on the induction of NO.

Glial soluble factors regulate the activity and expression of the neuronal glutamate transporter EAAC1: implication of cholesterol.

A co-ordinated regulation between neurons and astrocytes is essential for the control of extracellular glutamate concentration. Here, we have investigated the influence of astrocytes and glia-derived cholesterol on the regulation of glutamate transport in primary neuronal cultures from rat embryonic cortices. Glutamate uptake rate and expression of the neuronal glutamate transporter EAAC1 were low when neurons were grown without astrocytes and neurons were unable to clear extracellular glutamate. Treatment of the neuronal cultures with glial conditioned medium (GCM) increased glutamate uptake Vmax, EAAC1 expression and restored the capacity of neurons to eliminate extracellular glutamate. Thus, astrocytes up-regulate the activity and expression of EAAC1 in neurons. We further showed that cholesterol, present in GCM, increased glutamate uptake activity when added directly to neurons and had no effect on glutamate transporter expression. Furthermore, part of the GCM-induced effect on glutamate transport activity was lost when cholesterol was removed from GCM (low cholesterol-GCM) and was restored when cholesterol was added to low cholesterol-GCM. This demonstrates that glia-derived cholesterol regulates glutamate transport activity. With these experiments, we provide new evidences for neuronal glutamate transport regulation by astrocytes and identified cholesterol as one of the factors implicated in this regulation.

Contribution of astrocytes to hippocampal long-term potentiation through release of d -serine

Repetitive correlated activation of pre- and postsynaptic neurons induced long-term potentiation (LTP) of synaptic transmission among hippocampal neurons grown on a layer of astrocytes (mixed cultures) but not among neurons cultured in glial conditioned medium. Supplement of D-serine, an agonist for the glycine-binding site of N-methyl-D-aspartate (NMDA) receptors, enhanced NMDA receptor activation and enabled LTP induction in glial conditioned medium cultures. The induction of LTP in both mixed cultures and hippocampal slices was suppressed by NMDA receptor antagonists, glycine-binding-site blockers of NMDA receptors, or an enzyme that degrades endogenous D-serine. By providing extracellular D-serine that facilitates activation of NMDA receptors, astrocytes thus play a key role in long-term synaptic plasticity.

Glutamate-dependent glutamine, aspartate and serine release from rat cortical glial cell cultures

Glia play a pivotal role in glutaminergic excitatory neurotransmission in the central nervous system by regulating synaptic levels of glutamate and by providing glutamine as the sole precursor for the neurotransmitter pool glutamate to neurons through the glutamate–glutamine cycle. In the present investigation, we examined the influence of glutamate application on glutamine, serine and aspartate release from rat cortical glial cultures. The glial glutamate transporters rapidly cleared exogenously applied glutamate and this was accompanied by rapid increases in aspartate and glutamine, and a delayed increase in serine levels in the glial-conditioned medium. While glutamate-induced increases in glutamine and serine were sustained for up to 24 h, increases in aspartate lasted only for up to 6 h. The glutamate-induced increases in aspartate and glutamine were dependent both on the concentration and the duration of glutamate stimulus, but were largely insensitive to the inhibition of non- N-methyl- d-aspartate receptors or the metabotropic glutamate receptor 5. Inhibition of the glutamate transporter function by l- trans-pyrrolidine 2,4-dicarboxylate decreased the rate of glutamate uptake but not completely abrogated the uptake process, and this resulted in the attenuation of rate of glutamate induced glutamine synthesis. Dexamethasone treatment increased serine and glutamine levels in conditioned medium and increased glutamate induced glutamine release suggesting an upregulation of glutamine synthase activity. These results further substantiate coupling between glutamate and glutamine, and shed light on glutamate-dependent release of serine and aspartate, which may further contribute to excitatory neurotransmission.

Glial induction of blood-brain barrier-like L-system amino acid transport in the ECV304 cell line.

The blood-brain barrier (BBB) is formed by the presence of tight junction complexes between brain endothelial cells that restrict paracellular permeability. As a consequence, a number of transport proteins are expressed on cerebral endothelial cells to facilitate the transport of nutrients into the brain. Although the modulation of barrier tight junction properties by glial-conditioned medium and by second messengers is well established, little is known about the effects of these factors on carrier-mediated BBB transport processes. The ECV304 cell line shows an endothelial phenotype and can be induced to upregulate certain BBB features in the presence of glial factors. In the present study, we have examined the effect of conditioned medium derived from rat C6-glioma cells (C6CM) on the function of the L-system amino acid transporter in ECV304 cells, using L-leucine as the model substrate, and have determined whether the changes observed can be mimicked by modulating intracellular cAMP levels. ECV304 cells exposed to C6CM exhibited a significant increase in both the affinity of leucine transport and the diffusional constant (Michaelis-Menten), while the maximal transport capacity remained unchanged. Conversely, acute exposure to modulators of the PKA and PKC second messenger pathways was found to reduce significantly the maximal transport capacity and diffusion constants, while transport affinity remained unchanged. In both cases, the maximal flux of leucine was increased, indicating transport of greater efficiency. This study indicates that exposure of ECV304 cells to C6CM provides an influence inducing L-system transport properties characteristic of brain endothelial cells. Furthermore, it appears that L-system-mediated transport of amino acids can be modulated by several distinct pathways.