IFN-γ and IL-4 differentially shape metabolic responses and neuroprotective phenotype of astrocytes

Astrocytes can either exacerbate or ameliorate secondary degeneration at sites of injury in the CNS but the contextual basis for eliciting these opposing phenotypes is poorly understood. In this study, we demonstrate that the two major cytokines produced by Th1 and Th2 cells, interferon-γ (IFN-γ), and interleukin-4 (IL-4), respectively, contribute differentially to shaping a neuroprotective response in astrocytes. While IFN-γ protects the ability of oxidatively stressed murine astrocytes to clear extracellular glutamate in culture, IL-4 has no effect at any concentration that was tested (10–100 ng/mL). The enhanced release of neuroprotective thiols and lactate by astrocytes in response to T cell stimulation is mimicked by both IL-4 and IFN-γ. When co-administered, IL-4 abrogated the protective effect of low IFN-γ on the glutamate clearance function of oxidatively stressed astrocytes in a dose-dependent manner. Astrocyte-conditioned media obtained from cells cultured in the presence of IL-4 (10 or 100 ng/mL) or IFN-γ (10 ng/mL) decreased by ∼2-fold, neuronal apoptosis induced by oxidative stress in vitro . However, unlike IL-4, IFN-γ at high concentrations (100 ng/mL) was not neuroprotective. Our studies with IFN-γ and IL-4 suggest that a balanced Th1 and Th2 cytokine response might be needed for protecting two key astrocytic functions, glutamate clearance and thiol secretion and might be pertinent to neuroprotective approaches that are aimed at inhibition of an initial pro-inflammatory response to injury or its sustained boosting.     

Blood-brain barrier modeling with co-cultured neural progenitor cell-derived astrocytes and neurons

In vitro blood-brain barrier (BBB) models often consist of brain microvascular endothelial cells (BMECs) that are co-cultured with other cells of the neurovascular unit, such as astrocytes and neurons, to enhance BBB properties. Obtaining primary astrocytes and neurons for co-culture models can be laborious, while yield and heterogeneity of primary isolations can also be limiting. Neural progenitor cells (NPCs), because of their self-renewal capacity and ability to reproducibly differentiate into tunable mixtures of neurons and astrocytes, represent a facile, readily scalable alternative. To this end, differentiated rat NPCs were co-cultured with rat BMECs and shown to induce BBB properties such as elevated trans-endothelial electrical resistance, improved tight junction continuity, polarized p-glycoprotein efflux, and low passive permeability at levels indistinguishable from those induced by primary rat astrocyte co-culture. An NPC differentiation time of 12 days, with the presence of 10% fetal bovine serum, was found to be crucial for generating NPC-derived progeny capable of inducing the optimal response. This approach could also be extended to human NPC-derived astrocytes and neurons which similarly regulated BBB induction. The distribution of rat or human NPC-derived progeny under these conditions was found to be a roughly 3 : 1 mixture of astrocytes to neurons with varying degrees of cellular maturity. BMEC gene expression analysis was conducted using a BBB gene panel, and it was determined that 23 of 26 genes were similarly regulated by either differentiated rat NPC or rat astrocyte co-culture while three genes were differentially altered by the rat NPC-derived progeny. Taken together, these results demonstrate that NPCs are an attractive alternative to primary neural cells for use in BBB co-culture models.     

Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype

Gene targeting via homologous recombination in murine embryonic stem (ES) cells has been the method of choice for deciphering mammalian gene function in vivo. Despite improvements in this technology, it still remains a laborious method. Recent advances in RNA interference (RNAi) technology have provided a rapid loss-of-function method for assessing gene function in a number of organisms. Studies in mammalian cell lines have shown that introduction of small interfering RNA (siRNA) molecules mediates effective RNA silencing. Plasmid-based systems using RNA polymerase III (RNA pol III) promoters to drive short hairpin RNA (shRNA) molecules were established to stably produce siRNA. Here we report the generation of knockdown ES cell lines with transgenic shRNA. Because of the dominant nature of the knockdown, embryonic phenotypes could be directly assessed in embryos completely derived from ES cells by the tetraploid aggregation method. Such embryos, in which endogenous p120-Ras GTPase-activating protein (RasGAP), encoded by Rasa1 (also known as RasGAP), was silenced, had the same phenotype as did the previously reported Rasa1 null mutation.     

1-amino-APDC, a partial agonist of group II metabotropic glutamate receptors with neuroprotective properties.

The synthesis of the 1-amino derivative of (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylic acid (1-amino-APDC), a selective metabotropic glutamate ligand, is disclosed. This compound acts as a partial agonist of the group II mGluRs and shows pronounced neuroprotective properties in the NMDA model of cell toxicity.     

Development of a neural phenotype in differentiating ganglion cell-derived human neuroblastoma cells

Human neuroblastoma cells (clone SHSY-5Y) induced to differentiate by 12-O-tetradecanoylphorbol-13-acetate (TPA) are shown to possess properties characteristic of mature ganglion cells. Elevation of the external K+ concentration, exposure to Ca2+ ionophore A23187, and acetylcholine all stimulate the release of preloaded 3H-noradrenaline in the presence but not in the absence of added Ca2+. Acetylcholine causes a fall in the 86Rb+ or 14C-TPMP equilibrium potential across the plasma membrane and stimulates 86Rb+ efflux. These responses are prevented by atropine. Acetylcholine and muscarine but not nicotine stimulate an increase in 45Ca2+ influx, an effect blocked by atropine. None of these responses have been observed in nondifferentiating cells. Muscarinic receptors, however, as measured by the binding of tritiated quinuclidinyl benzilate (3H-QNB), were present to a similar extent in control and differentiated cells. Both cell types also exhibit an accelerated release of Ca2+ in response to acetylcholine, but the control cells were at least 1 order of magnitude more sensitive to the agonist.     

Toward a new role for plasma membrane sodium-dependent glutamate transporters of astrocytes: maintenance of antioxidant defenses beyond extracellular glutamate clearance

The primary function assigned to the sodium-dependent glutamate transporters, also known as excitatory amino acid transporters (EAATs), is to maintain the extracellular glutamate concentration in the low micromolar range, allowing glutamate to be used as a signaling molecule in the brain and preventing its cytotoxic effects. However, glutamate and cyst(e)ine, that is also a substrate of EAATs, are also important metabolites used for instance in the synthesis of the main antioxidant glutathione. This review describes the evidence suggesting that EAATs, by providing glutathione precursors, are crucial to prevent oxidative death in particular cells of the nervous system while being dispensable in others. This differential importance may depend on the way antioxidant defenses are maintained in each cell type and on the metabolic fate of transported substrates, both being probably controlled by EAAT interacting proteins. As oxidative stress invariably contributes to various forms of cell death, a better understanding of how antioxidant defenses are maintained in particular brain cells will probably help to develop protective strategies in degenerative insults specifically affecting these cells.     

T cell-mediated immunity to oncornavirus-induced tumors. I. Ly phenotype of precursor and effector cytolytic T lymphocytes

Regression of tumor induced by murine sarcoma virus (MSV) is accompanied by the formation of specific cytolytic T lymphocytes (CTL). Selection of T-cells sets by Ly phenotype determination allows separation of T sets involved in the cytolytic reaction. After MSV inoculation we demonstrate that a) Ly123+ cells contain precursors of CTL, b) direct cytolysis is mainly mediated by Ly23 cells, 3) cytolytic memory is divisible into "early" memory, carried by Ly23 cells, and "late" memory, which reverts to an Ly123 precursor population, and d) Ly1 cells are required to induce anti-MSV antibody formation.     

Functional engraftment of human ES cell-derived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes

To direct human embryonic stem (HES) cells to a dopaminergic neuronal fate, we cocultured HES cells that were exposed to both sonic hedgehog and fibroblast growth factor 8 with telomerase-immortalized human fetal midbrain astrocytes. These astrocytes substantially potentiated dopaminergic neurogenesis by both WA09 and WA01 HES cells, biasing them to the A9 nigrostriatal phenotype. When transplanted into the neostriata of 6-hydroxydopamine-lesioned parkinsonian rats, the dopaminergic implants yielded a significant, substantial and long-lasting restitution of motor function. However, although rich in donor-derived tyrosine hydroxylase-expressing neurons, the grafts exhibited expanding cores of undifferentiated mitotic neuroepithelial cells, which can be tumorigenic. These results show the utility of recreating the cellular environment of the developing human midbrain while driving dopaminergic neurogenesis from HES cells, and they demonstrate the potential of the resultant cells to mediate substantial functional recovery in a model of Parkinson disease. Yet these data also mandate caution in the clinical application of HES cell-derived grafts, given their potential for phenotypic instability and undifferentiated expansion.     

T cell-derived glucosteroid response-modifying factor (GRMFT): a unique lymphokine made by normal T lymphocytes and a T cell hybridoma

Mouse spleen cells and a murine T cell hybridoma, FS6 14.13.1, produce a glucosteroid response-modifying factor (GRMFT) after stimulation with concanavalin A. GRMFT blocks glucosteroid suppression of helper T cell function and the growth of granulocyte/macrophage progenitor cells in vitro. IL 1 also protects helper T cells and myeloid precursors from glucosteroid suppression. This suggests that GRMFT and IL 1 act congruently to ensure that an effective immune response is generated when endogenous glucosteroid levels are elevated. To understand the role of GRMFT in normal immune responses and in disease states characterized by imbalances in the immune system, we began to purify and characterize GRMFT. GRMFT appears to be distinct from other well-characterized T cell-derived factors. GRMFT is larger than IL 2 as determined by gel exclusion chromatography and is completely separated from IL 2 by isoelectric focusing. Furthermore, purified IL 2 does not have GRMFT activity. Purified IL 3 also lacks GRMFT activity, and conditions that inactivate immune interferon have no effect on GRMFT. Thus, GRMFT is different from IL 2, IL 3, and immune interferon. GRMFT also lacks activity in the thymocyte co-mitogenic assay and is therefore different from IL 1. Finally, FS6 14.13.1 reportedly does not produce TRF or CSF, which suggests that GRMFT is different from these molecules as well.     

3D Electrospun scaffolds promote a cytotrophic phenotype of cultured primary astrocytes

Astrocytes are a target for regenerative neurobiology because in brain injury their phenotype arbitrates brain integrity, neuronal death and subsequent repair and reconstruction. We explored the ability of 3D scaffolds to direct astrocytes into phenotypes with the potential to support neuronal survival. Poly‐ε‐caprolactone scaffolds were electrospun with random and aligned fibre orientations on which murine astrocytes were sub‐cultured and analysed at 4 and 12 DIV. Astrocytes survived, proliferated and migrated into scaffolds adopting 3D morphologies, mimicking in vivo stellated phenotypes. Cells on random poly‐ε‐caprolactone scaffolds grew as circular colonies extending processes deep within sub‐micron fibres, whereas astrocytes on aligned scaffolds exhibited rectangular colonies with processes following not only the direction of fibre alignment but also penetrating the scaffold. Cell viability was maintained over 12 DIV, and cytochemistry for F‐/G‐actin showed fewer stress fibres on bioscaffolds relative to 2D astrocytes. Reduced cytoskeletal stress was confirmed by the decreased expression of glial fibrillary acidic protein. PCR demonstrated up‐regulation of genes (excitatory amino acid transporter 2, brain‐derived neurotrophic factor and anti‐oxidant) reflecting healthy biologies of mature astrocytes in our extended culture protocol. This study illustrates the therapeutic potential of bioengineering strategies using 3D electrospun scaffolds which direct astrocytes into phenotypes supporting brain repair.

     

Identification of complexin II in astrocytes: a possible regulator of glutamate release in these cells

Complexins are a family of SNARE complex-binding proteins which regulate neurotransmitter release by playing a crucial role in triggering fast exocytosis at the synapse. Current evidence indicates astrocytes can release glutamate via a vesicular mechanism similar to that at nerve terminals and thereby modulate synaptic activity. In addition, components of the biochemical machinery associated with synaptic release have been identified in these cells. However, whether complexins are also present in astrocytes and may therefore participate in the vesicular release of glutamate is a key issue that is yet to be determined. In the present study we therefore examined if astrocytes express complexin I (Cpx I) and/or complexin II (Cpx II). Our results indicate these cells contain Cpx II but not Cpx I in primary culture. In addition, serum deprivation for 24 h led to a 2.6-fold increase in Cpx II, suggesting this protein is responsive to insults. These findings point to Cpx II being a likely key modulator of synaptic activity at the level of these glial cells. Given the considered involvement of complexins in neurologic and psychiatric illness, astrocytic Cpx II represents a potentially important therapeutic target for the future treatment of such maladies.     

Human astrocytes can be induced to differentiate into cells with neuronal phenotype

Several recent studies have proposed that astrocytes may contribute to neurogenesis, not only as a source of trophic substances regulating it, but also as stem cells themselves. In order to better understand these mechanisms, primary astrocyte cultures were established from human fetal brain. After 3-4 weeks in culture, astrocytes (about 95% GFAP+; neurofilament, NF-; neuro-specific enolase, NSE-) were treated with a cocktail of protein kinase activators and FGF-1. After 5 h of treatment, most cells showed morphological changes that increased progressively up to 24-48 h, exhibiting a round cell body with long processes. Immunocytochemistry showed that treatment-induced NF and NSE expression in about 40% of cells. Nestin expression increased after treatment, whereas GFAP immunostaining was not significantly modified. Western blot and RT-PCR confirmed the results. No neuronal electrophysiological properties were observed after treatment, suggesting an incomplete maturation under these experimental conditions. Understanding the regenerative capability and neurogenic potential of astrocytes might be useful in devising therapeutic approaches for a variety of neurological disorders.     

Iron uptake in quiescent and inflammation-activated astrocytes: A potentially neuroprotective control of iron burden

Astrocytes play a crucial role in proper iron handling within the central nervous system. This competence can be fundamental, particularly during neuroinflammation, and neurodegenerative processes, where an increase in iron content can favor oxidative stress, thereby worsening disease progression. Under these pathological conditions, astrocytes undergo a process of activation that confers them either a beneficial or a detrimental role on neuronal survival. Our work investigates the mechanisms of iron entry in cultures of quiescent and activated hippocampal astrocytes. Our data confirm that the main source of iron is the non-transferrin-bound iron (NTBI) and show the involvement of two different routes for its entry: the resident transient receptor potential (TRP) channels in quiescent astrocytes and the de novo expressed divalent metal transporter 1 (DMT1) in activated astrocytes, which accounts for a potentiation of iron entry. Overall, our data suggest that at rest, but even more after activation, astrocytes have the potential to buffer the excess of iron, thereby protecting neurons from iron overload. These findings further extend our understanding of the protective role of astrocytes under the conditions of iron-mediated oxidative stress observed in several neurodegenerative conditions.     

Endocrine markers of cellular immunity: defining the endocrine phenotype

The term neuroendocrine has been used to define cells that secrete their products in a regulated manner, in response to a specific stimulus. The neuroendocrine system includes neurons and endocrine cells sharing a common phenotypic program characterized by the expression of markers such as neuropeptides, chromogranins, neuropeptide processing enzymes SPC2 and SPC3 (subtilase-like pro-protein convertases) or dense core secretory granules. Various theories such as the APUD (amine precursor uptake decarboxylation) concept, the diffuse neuroendocrine system (DNES) or the paraneuron concept have been put forth to classify neuroendocrine cells as a cohesive group. Neuroendocrine characteristics have been used as evidence of a common embryological origin for normal and neoplastic cells. However, it is now recognized that neuroendocrine characteristics can be observed in various cell types, such as immunocytes, that do not share a common embryological origin with either neurons or endocrine cells. We propose to redefine previous "neuroendocrine" concepts to include the notion that activation of specific genetic switches can lead to the expression of a partial or full neuroendocrine phenotype in a variety of cell types, including immune cells.     

The neuroprotective activity of GPE tripeptide analogues does not correlate with glutamate receptor binding affinity

The influence of several modifications on the GPE tripeptide structure upon the binding to GluRs and on their neuroprotective effects has been studied. The results indicated that the prevention of neuronal death showed by GPE and some analogues is not directly related to their affinity at glutamate receptors.     

Glial cell-derived glutamate mediates autocrine cell volume regulation in the retina: activation by VEGF

Astroglial cells are a source for gliotransmitters such as glutamate and ATP. We demonstrate here that gliotransmitters have autocrine functions in the regulation of cellular volume. Hypoosmotic stress in the presence of inflammatory mediators or oxidative stress, and during blockade or down-regulation of potassium channels, induces swelling of retinal glial cells. Vascular endothelial growth factor inhibits the osmotic swelling of glial cells in retinal slices or isolated cells. This effect was mediated by a kinase domain region/flk-1 receptor-evoked calcium dependent release of glutamate from glial cells, and subsequent stimulation of glial group I/II metabotropic glutamate receptors. Activation of kinase domain region/flk-1 or glutamate receptors evoked an autocrine swelling-inhibitory purinergic signaling cascade that was calcium-independent. This cascade involved the release of ATP and adenosine, and the activation of purinergic P2Y₁ and adenosine A1 receptors, resulting in the opening of potassium and chloride channels and inhibition of cellular swelling. The glutamatergic-purinergic regulation of the glial cell volume may be functionally important in the homeostasis of the extracellular space volume during intense neuronal activation which is associated with a swelling of neuronal cell structures in the retina. However, glial cell-derived glutamate may also contribute to the swelling of activated neurons since metabolic poisoning of glial cells by iodoacetate inhibits the neuronal cell swelling mediated by activation of ionotropic glutamate receptors.     

Protection by pyruvate against glutamate neurotoxicity is mediated by astrocytes through a glutathione-dependent mechanism

Pyruvate, an endogenous metabolite of glycolysis, is an anti-toxicity agent. Recent studies have suggested possible roles for pyruvate in protecting CNS neurons from excitotoxic and metabolic insults. Utilizing cultures derived from embryonic rat cortex, the studies presented in this paper indicate that an astroglia-mediated mechanism is involved in the neuroprotective effects of pyruvate against glutamate toxicity. Glutamate-induced toxicity could be reversed by pyruvate in a mixed culture of cortex cells. Importantly, in pure neuronal cultures from the same tissue, pyruvate failed to protect against glutamate toxicity. Addition of astroglia to the pure neuronal cultures restores the ability of pyruvate to protect neurons from glutamate-induced toxicity. Our results further suggest that pyruvate can induce glia to up-regulate the synthesis of glutathione (GSH), an antioxidant that protects cells from toxins such as free radicals. Taken together, our data suggest that astroglia in mixed cultures are essential for mediating the effects of pyruvate, revealing a novel mechanism by which pyruvate, an important intermediate of tricarboxylic acid cycle in the body, may act to protect neurons from damage during insults such as brain ischemia.     

Loss of metabotropic glutamate receptor-mediated regulation of glutamate transport in chemically activated astrocytes in a rat model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurones accompanied by intense gliosis in lesioned areas of the brain and spinal cord. Glutamate-mediated excitotoxicity resulting from impaired astroglial uptake constitutes one of the current pathophysiological hypotheses explaining the progression of the disease. In this study, we examined the regulation of glutamate transporters by type 5 metabotropic glutamate receptor (mGluR5) in activated astrocytes derived from transgenic rats carrying an ALS-related mutated human superoxide dismutase 1 (hSOD1(G93A)) transgene. Cells from transgenic animals and wild-type littermates showed similar expression of glutamate-aspartate transporter and glutamate transporter 1 (GLT-1) after in vitro activation, whereas cells carrying the hSOD1 mutation showed a three-fold higher expression of functional mGluR5, as observed in the spinal cord of end-stage animals. In cells from wild-type animals, (S)-3,5-dihydroxyphenylglycine (DHPG) caused an immediate protein kinase C (PKC)-dependent up-regulation of aspartate uptake that reflected the activation of GLT-1. Although this effect was mimicked in both cultures by direct activation of PKC using phorbol myristate acetate, DHPG failed to up-regulate aspartate uptake in cells derived from the transgenic rats. The failure of activated mGluR5 to increase glutamate uptake in astrocytes derived from this animal model of ALS supports the theory of glutamate excitotoxicity in the pathogenesis of the disease.