Silica Nanoparticles Decrease Glutamate Uptake in Blood-Brain Barrier Components.

Glutamate is the major excitatory amino acid in the vertebrate brain, playing an important role in most brain functions. It exerts its activity through plasma membrane receptors and transporters, expressed both in neurons and glia cells. Overstimulation of neuronal glutamate receptors is linked to cell death in a process known as excitotoxicity, that is prevented by the efficient removal of the neurotransmitter through glutamate transporters enriched in the glia plasma membrane and in the components of the blood-brain barrier (BBB). Silica nanoparticles (SiO2-NPs) have been widely used in biomedical applications and directed to enter the circulatory system; however, little is known about the potential adverse effects of SiO2-NPs exposure on the BBB transport systems that support the critical isolation function between the central nervous system (CNS) and the peripheral circulation. In this contribution, we investigated the plausible SiO2-NPs-mediated disruption of the glutamate transport system expressed by BBB cell components. First, we evaluated the cytotoxic effect of SiO2-NPs on human brain endothelial (HBEC) and Uppsala 87 Malignant glioma (U-87MG) cell lines. Transport kinetics were evaluated, and the exposure effect of SiO2-NPs on glutamate transport activity was determined in both cell lines. Exposure of the cells to different SiO2-NP concentrations (0.4, 4.8, 10, and 20 µg/ml) and time periods (3 and 6 h) did not affect cell viability. We found that the radio-labeled D-aspartate ([3H]-D-Asp) uptake is mostly sodium-dependent, and downregulated by its own substrate (glutamate). Furthermore, SiO2-NPs exposure on endothelial and astrocytes decreases [3H]-D-Asp uptake in a dose-dependent manner. Interestingly, a decrease in the transporter catalytic efficiency, probably linked to a diminution in the affinity of the transporter, was detected upon SiO2-NPs. These results favor the notion that exposure to SiO2-NPs could disrupt BBB function and by these means shed some light into our understanding of the deleterious effects of air pollution on the CNS.

RNA-Binding Proteins: A Role in Neurotoxicity?

Despite sustained efforts to treat neurodegenerative diseases, little is known at the molecular level to understand and generate novel therapeutic approaches for these malignancies. Therefore, it is not surprising that neurogenerative diseases are among the leading causes of death in the aged population. Neurons require sophisticated cellular mechanisms to maintain proper protein homeostasis. These cells are generally sensitive to loss of gene expression control at the post-transcriptional level. Post-translational control responds to signals that can arise from intracellular processes or environmental factors that can be regulated through RNA-binding proteins. These proteins recognize RNA through one or more RNA-binding domains and form ribonucleoproteins that are critically involved in the regulation of post-transcriptional processes from splicing to the regulation of association of the translation machinery allowing a relatively rapid and precise modulation of the transcriptome. Neurotoxicity is the result of the biological, chemical, or physical interaction of agents with an adverse effect on the structure and function of the central nervous system. The disruption of the proper levels or function of RBPs in neurons and glial cells triggers neurotoxic events that are linked to neurodegenerative diseases such as spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), fragile X syndrome (FXS), and frontotemporal dementia (FTD) among many others. The connection between RBPs and neurodegenerative diseases opens a new landscape for potentially novel therapeutic targets for the intervention of these neurodegenerative pathologies. In this contribution, a summary of the recent findings of the molecular mechanisms involved in the plausible role of RBPs in RNA processing in neurodegenerative disease is discussed.

Acute Manganese Exposure Modifies the Translation Machinery via PI3K/Akt Signaling in Glial Cells.

SUMMARY STATEMENT: We demonstrate herein that short-term exposure of radial glia cells to Manganese, a neurotoxic metal, induces an effect on protein synthesis, altering the protein repertoire of these cells.

A unique snake venom neuritogenesis mechanism: A cornerstone in the treatment of neurodegenerative diseases?: An Editorial Highlight for "Transcriptomic, proteomic, and biochemical analyses reveal a novel neuritogenesis mechanism of Naja naja venom α-elapitoxin post binding to TrkA receptor of rat pheochromocytoma cells" on 612.

Neurodegenerative diseases are a worldwide health problem and are a major cause of death and disability. A progressive loss of defined neuronal populations is triggered by a diverse array of stimuli that converge in deficient neurotrophic signaling. Therefore, much effort has been placed in recent years in the characterization of the molecular mechanisms associated with the structure and function of neurotrophins, its receptors, signaling strategies, and their target genes. This Editorial highlights an impressive study by the group of Prof. Ashis K. Mukherjee, a renowned specialist in snake venoms, in which a component of the Indian Cobra N.naja venom with no significant similarity to nerve growth factor, is shown to induce sustained neuritogenesis. An elegant transcriptomic and functional analysis of this component, named Nn-α-elapitoxin, mapped novel domains in mammalian neurotrophic receptors that trigger both conventional and novel signal cascades that support neurite extension in the PC-12 neuronal model system. The authors discuss their findings in the context of the paradoxical neurite outgrowth properties of this toxin which originate in their unique receptor binding site. This study takes an important step towards a better understanding of the complexity of neuronal development and maintenance of the nervous system and provides a potential target to improve neurotrophic signaling, independent of endogenous growth factors, in the diseased brain.

Bisphenol A exposure disrupts aspartate transport in HepG2 cells.

The liver is the organ responsible for bisphenol A (BPA) metabolism, an environmental chemical agent. Exposure to this toxin is associated with liver abnormalities and dysfunction. An important role played by excitatory amino acid transporters (EAATs) of the slc1 gene family has been reported in liver injuries. To gain insight into a plausible effect of BPA exposure in the liver glutamate/aspartate transport, using the human hepatoblastoma cell line HepG2, we report a BPA-dependent dynamic regulation of SLC1A3 and SLC1A2. Through the use of radioactive [3 H]- d-aspartate uptake experiments and immunochemical approaches, we characterized time and dose-dependent regulation of the protein levels and function of these transporters after acute exposure to BPA. An increase in nuclear Yin Yang 1 was found. These results suggest an important involvement of the EAATs in liver physiology and its disruption after acute BPA exposure.

Acute Exposure to SiO2 Nanoparticles Affects Protein Synthesis in Bergmann Glia Cells.

Attractive due to an alleged high biocompatibility, silica nanoparticles have been widely used in the field of nanomedicine; however, their proven capacity to induce the synthesis and release of pro-inflammatory cytokines in several cellular models has raised concern about their safety. Glutamate, the main excitatory amino acid transmitter triggers a wide variety of signal transduction cascades that regulate protein synthesis at transcriptional and translational levels. A stimulus-dependent dynamic change in the protein repertoire in neurons and glia cells is the molecular framework of higher brain functions. Within the cerebellum, Bergmann glia cells are the most abundant non-neuronal cells and span the entire molecular layer of the cerebellar cortex, wrapping the synapses in this structure. Taking into consideration the functional role of Bergmann glia in terms of the recycling of glutamate, lactate supply to neurons, and prevention of neurotoxic insults, we decided to investigate the possibility that silica nanoparticles affect Bergmann glia and by these means alter the major excitatory neurotransmitter system in the brain. To this end, we exposed cultured chick cerebellar Bergmann glia cells to silica nanoparticles and measured [35S]-methionine incorporation into newly synthesized polypeptides. Our results demonstrate that exposure of the cultured cells to silica nanoparticles exerts a time- and dose-dependent modulation of protein synthesis. Furthermore, altered patterns of eukaryotic initiation factor 2 alpha and eukaryotic elongation factor 2 phosphorylation were present upon nanoparticle exposure. These results demonstrate that glia cells respond to the presence of this nanomaterial modifying their proteome, presumably in an effort to overcome any plausible neurotoxic effect.

Brown Seaweed Egregia menziesii's Cytotoxic Activity against Brain Cancer Cell Lines.

Brown seaweeds contain bioactive compounds that show anti-tumorigenic effects. These characteristics have been repeatedly observed in the Lessoniaceae family. Egregia menziesii, a member of this family, is distributed in the North Pacific and its properties have been barely studied. We evaluated herein the cytotoxic and anti-proliferative activity of extracts of this seaweed, through toxicity assay in Artemia salina and lymphocytes, and MTT proliferation assay, in Bergmann glia cells, 3T3-L1 and brain cancer cell lines. E. menziesii's extracts inhibited the spread of all the tested cell lines. The hexane extract showed the highest cytotoxic activity, while the methanol extract was moderately cytotoxic. Interestingly, seaweed extracts displayed a selective inhibition pattern. These results suggest that E. menziesii's extracts might be good candidates for cancer prevention and the development of novel chemotherapies due to its highest cytotoxicity in transformed cells compare to glia primary cultures.

Characterization of the cystine/glutamate antiporter in cultured Bergmann glia cells.

Glutamate, the major excitatory transmitter in the vertebrate brain is a potent neurotoxin through the over-stimulation of its specific membrane receptors. In accordance, a tight regulation of its extracellular levels by plasma membrane transporters is present. A family of excitatory amino acid transporters is expressed in neurons and glia cells and is responsible of the removal of the neurotransmitter from the synaptic cleft. Glial transporters account for more than 80% of the brain uptake activity. The cystine/glutamate antiporter is another plasma membrane-bound protein critically involved in glutamatergic transmission. Upon oxidative stress, it begins to pump out glutamate in exchange for cystine, mostly needed for glutathione production. Taking into consideration that all of these glutamate transporter proteins are present in glia cells that surround glutamatergic synapses, we reasoned that a functional coupling of them should exist to prevent an excitotoxic insult to the neighboring neuronal cells. To this end, we used the established model of chick cerebellar Bergmann glia cultures. Once we could establish the expression of the cystine/glutamate antiporter in our system, we characterized its kinetic properties and started to gain insight into its regulation and plausible coupling to other transporters. Exposure to glutamate reduces the uptake activity and favors a physical interaction with the excitatory amino acid transporter 1 and the Na+-dependent neutral amino acids transporter 3. In contrast, treatment of the cultured cells with a nitric oxide donor such as sodium nitroprussiate augments the exchanger activity. Longer sodium nitroprussiate exposure periods down-regulates the cystine/glutamate protein levels. These results suggest that a coordinated interplay between glutamate transporters and exchangers takes place in glia cells to prevent excitotoxic insults.

Coupling of glutamate and glucose uptake in cultured Bergmann glial cells.

Glutamate, the main excitatory neurotransmitter in the vertebrate brain, exerts its actions through specific membrane receptors present in neurons and glial cells. Over-stimulation of glutamate receptors results in neuronal death, phenomena known as excitotoxicity. A family of sodium-dependent, glutamate uptake transporters mainly expressed in glial cells, removes the amino acid from the synaptic cleft preventing neuronal death. The sustained sodium influx associated to glutamate removal in glial cells, activates the sodium/potassium ATPase restoring the ionic balance, additionally, glutamate entrance activates glutamine synthetase, both events are energy demanding, therefore glia cells increase their ATP expenditure favouring glucose uptake, and triggering several signal transduction pathways linked to proper neuronal glutamate availability, via the glutamate/glutamine shuttle. To further characterize these complex transporters interactions, we used the well-established model system of cultured chick cerebellum Bergmann glia cells. A time and dose-dependent increase in the activity, plasma membrane localization and protein levels of glucose transporters was detected upon d-aspartate exposure. Interestingly, this increase is the result of a protein kinase C-dependent signaling cascade. Furthermore, a glutamate-dependent glucose and glutamate transporters co-immunoprecipitation was detected. These results favour the notion that glial cells are involved in glutamatergic neuronal physiology.

Glutamate-dependent translational control through ribosomal protein S6 phosphorylation in cultured bergmann glial cells.

Glutamate (Glu) the main excitatory neurotransmitter of the central nervous system regulates gene expression at different levels through the activation of specific membrane receptors and transporters expressed in neurons and glia cells. A membrane to nucleus signaling cascade triggered by this neurotransmitter has been described in cultured cerebellar Bergmann glia cells isolated from chick embryos. Furthermore, it has also been described that Glu receptors activation is linked to a modulation of [(35)S]-methionine incorporation into newly synthesized polypeptides. In order to gain insight into the signal transduction cascades that participate in this effect, in the present study we characterized the phosphorylation of a critical component of the translational machinery, namely the ribosomal protein S6. The phosphorylation sites in rpS6 have been mapped to five clustered residues, Ser235, Ser236, Ser240, Ser244 and Ser247. Nevertheless, Ser236 phosphorylation is the primary phosphorylation site. The kinases responsible of this modification are p70(S6K) and p90(RSK). rpS6 phosphorylation increases the affinity of 40s subunit for mRNAs and thus facilitates translational initiation. Glutamate exposure of cultured cerebellar Bergmann glia cells results in a time- and dose-dependent increase in rpS6 phosphorylation. This effect is mainly observed at cytoplasm, and involves the phosphoinositol-3 kinase/protein kinase B pathway. Our results favor the notion of a continuous neuronal signaling to glia cells that regulates the proteome of these cells not only at the transcriptional level but also at the level of protein synthesis.

Fluoride exposure regulates the elongation phase of protein synthesis in cultured Bergmann glia cells.

Fluoride is an environmental pollutant present in dental products, food, pesticides and water. The latter, is the greatest source of exposure to this contaminant. Structural and functional damages to the central nervous system are present in exposed population. An established consequence of the neuronal is the release of a substantial amount of glutamate to the extracellular space, leading to an excitotoxic insult. Glutamate exerts its actions through the activation of specific plasma membrane receptors and transporters present in neurons and in glia cells and it is the over-activation of glutamate receptors and transporters, the biochemical hallmark of neuronal and oligodendrocyte cell death. In this context, taking into consideration that fluoride leads to degeneration of cerebellar cells, we took the advantage of the well-established model of cerebellar Bergmann glia cultures to gain insight into the molecular mechanisms inherent to fluoride neurotoxicity that might be triggered in glia cells. We could establish that fluoride decreases [(35)S]-methionine incorporation into newly synthesized polypeptides, in a time-dependent manner, and that this halt in protein synthesis is the result of a decrease in the elongation phase of translation, mediated by an augmentation of eukaryotic elongation factor 2 phosphorylation. These results favor the notion of glial cells as targets of fluoride toxicity and strengthen the idea of a critical involvement of glia cells in the function and dysfunction of the brain.

Glutamate regulates eEF1A phosphorylation and ribosomal transit time in Bergmann glial cells.

Glutamate, the major excitatory transmitter in the vertebrate brain, is involved in neuronal development and synaptic plasticity. Glutamatergic stimulation leads to differential gene expression patterns in neuronal and glial cells. A glutamate-dependent transcriptional control has been established for several genes. However, much less is known about the molecular events that modify the translational machinery upon exposure to this neurotransmitter. In a glial model of cerebellar cultured Bergmann cells, glutamate induces a biphasic effect on [(35)S]-methionine incorporation into proteins that suggests that the elongation phase of protein biosynthesis is the target for regulation. Indeed, after a 15 min exposure to glutamate a transient increase in elongation factor 2 phosphorylation has been reported, an effect mediated through the activation of the elongation factor 2 kinase. In this contribution, we sought to characterize the phosphorylation status of the eukaryotic elongation factor 1A (eEF1A) and the ribosomal transit time under glutamate exposure. A dose-dependent increase in eEF1A phosphorylation was found after a 60 min glutamate treatment; this phenomenon is Ca(2+)/CaM dependent, blocked with Src and phosphatidyl-inositol 3-kinase inhibitors and with rapamicyn. Concomitantly, the ribosomal transit time was increased with a 15 min glutamate exposure. After 60 more minutes, the average time used by the ribosomes to complete a polypeptide chain had almost returned to its initial level. These results strongly suggest that glutamate exerts an exquisite time-dependent translational control in glial cells, a process that might be critical for glia-neuron interactions.

Glutamate-dependent elongation factor-2 phosphorylation in Bergmann glial cells.

Glutamate, the main excitatory amino acid transmitter regulates protein biosynthesis at the transcriptional and translational levels. It is critically involved in the continuous change of the protein repertoire that is inherent to synaptic plasticity. Activity-dependent differential gene expression occurs both in neurons and glial cells. In fact, besides a membrane to nuclei signaling that leads to transcriptional control, a biphasic effect in overall protein synthesis takes place after glutamate receptors stimulation in cultured chick cerebellar Bergmann glia. Therefore, the effect of glutamate receptors activation on translation elongation was characterized. A time- and dose-dependent increase in eukaryotic elongation factor-2 phosphorylation was found. Pharmacological tools established that alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and Kainate, but not N-methyl-d-aspartate trigger this phosphorylation. The removal of external Ca2+ or the pre-treatment with a calmodulin antagonist prevented the glutamate effect. Accordingly, glutamate receptors regulate eukaryotic elongation factor-2 kinase phosphorylation through the involvement of Ca2+/calmodulin/extracellular-regulated protein kinases 1/2. These results demonstrate that glutamate receptors regulate the elongation of peptide chains in glial cells.

Glutamate activates protein kinase B (PKB/Akt) through AMPA receptors in cultured Bergmann glia cells.

Glutamate is involved in gene expression regulation in neurons and glial cells through the activation of a diverse array of signaling cascades. In Bergmann glia, Ca2+ -permeable alpha-hydroxy-5-methyl-4-isoazole-propionic acid (AMPA) receptors become tyrosine phosphorylated after ligand binding and by these means form multiprotein signaling complexes. Of the various proteins that associate to these receptors, the phosphatidylinositol 3-kinase (PI-3K) deserves special attention since D3-phosphorylated phosphoinositides are docking molecules for signaling proteins with a pleckstrin homology domain. In order to characterize the role of PI-3K in AMPA receptors signaling, in the present report we analyze the involvement of the serine/threonine protein kinase B in this process. Our results demonstrate an augmentation in protein kinase B phosphorylation and activity after glutamate exposure. Interestingly, the effect is independent of Ca2+ influx, but sensitive to Src blockers. Our present findings broaden our current knowledge of glial glutamate receptors signaling and their involvement glutamatergic neurotransmission.