Rossana C. Zepeda

GLAST/EAAT1‐induced Glutamine release via SNAT3 in Bergmann glial cells: evidence of a functional and physical coupling

Glutamate, the major excitatory transmitter in the vertebrate brain, is removed from the synaptic cleft by a family of sodium‐dependent glutamate transporters profusely expressed in glial cells. Once internalized, it is metabolized by glutamine synthetase to glutamine and released to the synaptic space through sodium‐dependent neutral amino acid carriers of the N System (SNAT3/slc38a3/SN1, SNAT5/slc38a5/SN2). Glutamine is then taken up by neurons completing the so‐called glutamate/glutamine shuttle. Despite of the fact that this coupling was described decades ago, it is only recently that the biochemical framework of this shuttle has begun to be elucidated. Using the established model of cultured cerebellar Bergmann glia cells, we sought to characterize the functional and physical coupling of glutamate uptake and glutamine release. A time‐dependent Na+‐dependent glutamate/aspartate transporter/EAAT1‐induced System N‐mediated glutamine release could be demonstrated. Furthermore, D‐aspartate, a specific glutamate transporter ligand, was capable of enhancing the co‐immunoprecipitation of Na+‐dependent glutamate/aspartate transporter and Na+‐dependent neutral amino acid transporter 3, whereas glutamine tended to reduce this association. Our results suggest that glial cells surrounding glutamatergic synapses may act as sensors of neuron‐derived glutamate through their contribution to the neurotransmitter turnover.

Glutamate-dependent translational regulation in cultured Bergmann glia cells: involvement of p70S6K.

Glutamate, the main excitatory amino acid transmitter in the vertebrate brain is involved in the dynamic changes in protein repertoire that underlie synaptic plasticity. Activity-dependent differential expression patterns occur not only in neurons but also in glial cells. In fact, a membrane to nuclei signaling has been described after ionotropic glutamate receptor stimulation in cultured chick cerebellar Bergmann glia cells. In order to characterize other levels of protein expression regulation, we explored the effect of glutamate treatment in [35S]-methionine incorporation into newly synthesized polypeptides. A time-dependent modification in protein synthesis was found. An important component of translational control is the ribosomal S6 protein kinase. Threonine phosphorylation renders the kinase active increasing translation initiation. Glutamate exposure results in ribosomal S6 protein kinase Thr389 phosphorylation in a dose and time-dependent manner that matches perfectly with the overall protein synthesis profile detected upon the excitatory amino acid. Pharmacological characterization of the receptors involved suggests the participation of both ionotropic as well as metabotropic glutamate receptors. The non-receptor tyrosine kinase Src, phosphatidylinositol 3-kinase, protein kinase B and the mammalian target of rapamycin are mediators of the glutamate effect. These results not only demonstrate that glutamate receptors activation is critically involved in translational control in glial cells adjacent to synaptic processes like cerebellar Bergmann glia cells, but also further strengthen the notion of an active participation of glial cells in synaptic transmission.

Glutamate-dependent phosphorylation of the mammalian target of rapamycin (mTOR) in Bergmann glial cells

Glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, plays an important role in neuronal development and synaptic plasticity. It activates a variety of signaling pathways that regulate gene expression at the transcriptional and translational levels. Within glial cells, besides transcription, glutamate also regulates translation initiation and elongation. The mammalian target of rapamycin (mTOR), a key participant in the translation process, represents an important regulatory locus for translational control. Therefore, in the present communication we sought to characterize the mTOR phosphorylation pattern after glutamate treatment in chick cerebellar Bergmann glia primary cultures. A time- and dose-dependent increase in mTOR Ser 2448 phosphorylation was found. Pharmacological tools established that the glutamate effect is mediated through ionotropic and metabotropic receptors and interestingly, the glutamate transporter system is also involved. The signaling cascade triggered by glutamate includes an increase in intracellular Ca2+ levels, and the activation of the p60(Src)/PI-3K/PKB pathway. These results suggest that glia cells participate in the activity-dependent change in the brain protein repertoire.

Glutamate-Dependent Transcriptional Regulation in Bergmann Glia Cells: Involvement of p38 MAP Kinase

Glutamate (Glu) is the major excitatory neurotransmitter in the Central Nervous System (CNS). Ionotropic and metabotropic glutamate receptors (GluRs) are present in neurons and glial cells and are involved in gene expression regulation. Mitogen-activated proteins kinases (MAPK) are critical for all the membrane to nuclei signaling pathways described so far. In cerebellar Bergmann glial cells, glutamate-dependent transcriptional regulation is partially dependent on p42/44 MAPK activity. Another member of this kinase family, p38 MAPK is activated by non-mitogenic stimuli through its Thr180/Tyr182 phosphorylation and phosphorylates cytoplasmic and nuclear protein targets involved in translational and transcriptional events. Taking into consideration that the role of p38MAPK in glial cells is not well understood, we demonstrate here that glutamate increases p38 MAPK phosphorylation in a time and dose dependent manner in cultured chick cerebellar Bergmann glial cells (BGC). Moreover, p38 MAPK is involved in the glutamate-induced transcriptional activation in these cells. Ionotropic as well as metabotropic glutamate receptors participate in p38 MAPK activation. The present findings demonstrate the involvement of p38 MAPK in glutamate-dependent gene expression regulation in glial cells.

Brain-derived neurotrophic factor and its receptors in Bergmann glia cells

Brain-derived neurotrophic factor is an abundant and widely distributed neurotrophin expressed in the Central Nervous System. It is critically involved in neuronal differentiation and survival. The expression of brain-derived neurotrophic factor and that of its catalytic active cognate receptor (TrkB) has been extensively studied in neuronal cells but their expression and function in glial cells is still controversial. Despite of this fact, brain-derived neurotrophic factor is released from astrocytes upon glutamate stimulation. A suitable model to study glia/neuronal interactions, in the context of glutamatergic synapses, is the well-characterized culture of chick cerebellar Bergmann glia cells. Using, this system, we show here that BDNF and its functional receptor are present in Bergmann glia and that BDNF stimulation is linked to the activation of the phosphatidyl-inositol 3 kinase/protein kinase C/mitogen-activated protein kinase/Activator Protein-1 signaling pathway. Accordingly, reverse transcription-polymerase chain reaction (RT-PCR) experiments predicted the expression of full-length and truncated TrkB isoforms. Our results suggest that Bergmann glia cells are able to express and respond to BDNF stimulation favoring the notion of their pivotal role in neuroprotection.

Anticancer activity of seaweeds

Cancer is a major health problem worldwide and still lacks fully effective treatments. Therefore, alternative therapies, using natural products, have been proposed. Marine algae are an important component of the marine environment, with high biodiversity, and contain a huge number of functional compounds, including terpenes, polyphenols, phlorotannins, and polysaccharides, among others. These compounds have complex structures that have shown several biological activities, including anticancer activity, using in vitro and in vivo models. Moreover, seaweed-derived compounds target important molecules that regulate cancer processes. Here, we review our current understanding of the anticancer activity of seaweeds.

Organochlorine Pesticide Level Differences Among Female Inhabitants from Veracruz, Puebla and Tabasco, Mexico

Organochlorine pesticides have been used in Mexico in malaria control programs and against ectoparasites. The objective of this study was to compare the levels of organochlorine pesticides: HCB, α-β-γ-HCH, pp′-DDE, op′-DDT and pp′-DDT in adipose tissue of female inhabitants from three Mexican states: Veracruz, Puebla and Tabasco. Data analyses indicated higher β-HCH levels in Puebla inhabitants. When comparing the mean values of the pp′-DDE concentrations among the three states, no statistically significant differences were noted. A trend of increasing concentrations of op′-DDT from Veracruz to Puebla and Tabasco was observed. Significantly higher pp′-DDT concentrations in Veracruz as compared to Puebla and Tabasco were determined. Using factorial analysis of three age categories (>30, 31–50, <51) organochlorine pesticide concentrations increases with age of participants, indicating time of exposure as a principal factor of organochlorine pesticides accumulation in adipose tissue.

A low density microarray method for the identification of human papillomavirus type 18 variants.

We describe a novel microarray based-method for the screening of oncogenic human papillomavirus 18 (HPV-18) molecular variants. Due to the fact that sequencing methodology may underestimate samples containing more than one variant we designed a specific and sensitive stacking DNA hybridization assay. This technology can be used to discriminate between three possible phylogenetic branches of HPV-18. Probes were attached covalently on glass slides and hybridized with single-stranded DNA targets. Prior to hybridization with the probes, the target strands were pre-annealed with the three auxiliary contiguous oligonucleotides flanking the target sequences. Screening HPV-18 positive cell lines and cervical samples were used to evaluate the performance of this HPV DNA microarray. Our results demonstrate that the HPV-18s variants hybridized specifically to probes, with no detection of unspecific signals. Specific probes successfully reveal detectable point mutations in these variants. The present DNA oligoarray system can be used as a reliable, sensitive and specific method for HPV-18 variant screening. Furthermore, this simple assay allows the use of inexpensive equipment, making it accessible in resource-poor settings.

Glutamate‐dependent transcriptional control in Bergmann glia: Sox10 as a repressor

J. Neurochem. (2009) 109, 899–910.