Astrocytes contain a vesicular compartment that is competent for regulated exocytosis of glutamate.

Astrocytes establish rapid cell-to-cell communication through the release of chemical transmitters. The underlying mechanisms and functional significance of this release are, however, not well understood. Here we identify an astrocytic vesicular compartment that is competent for glutamate exocytosis. Using postembedding immunogold labeling of the rat hippocampus, we show that vesicular glutamate transporters (VGLUT1/2) and the vesicular SNARE protein, cellubrevin, are both expressed in small vesicular organelles that resemble synaptic vesicles of glutamatergic terminals. Astrocytic vesicles, which are not as densely packed as their neuronal counterparts, can be observed in small groups at sites adjacent to neuronal structures bearing glutamate receptors. Fluorescently tagged VGLUT-containing vesicles were studied dynamically in living astrocytes by total internal reflection fluorescence (TIRF) microscopy. After activation of metabotropic glutamate receptors, astrocytic vesicles underwent rapid (milliseconds) Ca(2+)- and SNARE-dependent exocytic fusion that was accompanied by glutamate release. These data document the existence of a Ca(2+)-dependent quantal glutamate release activity in glia that was previously considered to be specific to synapses.

Effect of beta-amyloid on endothelial cells: lack of direct toxicity, enhancement of MTT-induced cell death and intracellular accumulation.

Several cerebrovascular alterations have been described in Alzheimer's disease (AD) including an accumulation of beta-amyloid (betaA) on the vascular walls in the brain. To investigate the potential toxic activity of betaA on endothelial cells (EC), two endothelial murine cell lines derived from heart and brain were exposed to betaA1-42 and the biologically active fragment betaA25-35 in the range from 5nM to 50 microM. In a low concentration range (50 nM to 2.5 microM) both peptides significantly reduced the 3-(4,5-dimethylthiazol-2y1)-2-5-diphenyltetrazolium bromide (MTT) signal in the endothelial cell lines exposed for 24h. However, microscopic examination, lactate dehydrogenase (LDH) release determination and Neutral Red assay did not confirm any toxic effect associated with inhibition of MTT formazan reduction. The effect on MTT was not susceptible to anti-oxidant treatment and did not increase the sensitivity to oxidative stress. However, when the EC were exposed to betaA and MTT for 1h, cell viability, determined by LDH release, was strongly reduced, while in normal conditions MTT-induced cell death only after 2h. An inhibitor of lysosomal ATPase activity, bafilomycin A1, completely antagonized this effect. The morphological examination showed that the functional activation by betaA in EC enhanced the production of MTT formazan crystals. To verify the accumulation of betaA in the lysosomal compartment we analyzed the subcellular distribution of betaA1-42 at different exposure times of EC to the peptide. The peptide was found in several organelles and was absent in the cytoplasmic compartment; co-treatment with bafilomycin A1 did not reduce the intracellular presence of betaA1-42. In our condition, the exposure of EC to betaA induced an intracellular accumulation of the peptide and a vasoactive effect that did not appear associated with direct toxic activity.

MAR elements regulate the probability of epigenetic switching between active and inactive gene expression.

Gene expression often cycles between active and inactive states in eukaryotes, yielding variable or noisy gene expression in the short-term, while slow epigenetic changes may lead to silencing or variegated expression. Understanding how cells control these effects will be of paramount importance to construct biological systems with predictable behaviours. Here we find that a human matrix attachment region (MAR) genetic element controls the stability and heritability of gene expression in cell populations. Mathematical modeling indicated that the MAR controls the probability of long-term transitions between active and inactive expression, thus reducing silencing effects and increasing the reactivation of silent genes. Single-cell short-terms assays revealed persistent expression and reduced expression noise in MAR-driven genes, while stochastic burst of expression occurred without this genetic element. The MAR thus confers a more deterministic behavior to an otherwise stochastic process, providing a means towards more reliable expression of engineered genetic systems.