Although cell culture studies have implicated the presence of vesicle proteins in mediating the release of glutamate from astrocytes, definitive proof requires the identification of the glutamate release mechanism and the localization of this mechanism in astrocytes at synaptic locales. In cultured murine astrocytes we show an array of vesicle proteins, including SNARE proteins, and vesicular glutamate transporters that are required to fill vesicles with glutamate. Using immunocytochemistry and single-cell multiplex reverse transcription-PCR we demonstrate the presence of these proteins and their transcripts within astrocytes freshly isolated from the hippocampus. Moreover, immunoelectron microscopy demonstrates the presence of VGLUT1 in processes of astrocytes of the hippocampus. To determine whether calcium-dependent glutamate release is mediated by exocytosis, we expressed the SNARE motif of synaptobrevin II to prevent the formation of SNARE complexes, which reduces glutamate release from astrocytes. To further determine whether vesicular exocytosis mediates calcium-dependent glutamate release from astrocytes, we performed whole cell capacitance measurements from individual astrocytes and demonstrate an increase in whole cell capacitance, coincident with glutamate release. Together, these data allow us to conclude that astrocytes in situ express vesicle proteins necessary for filling vesicles with the chemical transmitter glutamate and that astrocytes release glutamate through a vesicle-or fusion-related mechanism.During the past decade there has been increasing evidence for both integrative and dynamic roles for astrocytes in the central nervous system. Following activation of G protein-coupled receptors, astrocytes exhibit calcium oscillations, leading to the release of the chemical transmitters glutamate and ATP (1-3). Studies in vitro and in brain slices have led to the hypothesis of tripartite synaptic transmission (4); neuronal activity causes elevations of synaptically associated calcium in astrocytes, which in turn leads to the release of chemical transmitters from these glial cells to locally modulate synaptic transmission (2, 5-7).The mechanisms mediating the release of these transmitters from astrocytes are, however, ill-defined and are still the subject of intense debate. At least three distinct release pathways have been proposed as mediating the calcium-dependent release of glutamate from astrocytes: the reversal of plasma membrane glutamate transporters, anion transporter mediate release mechanisms, and calcium-dependent exocytosis (8 -10). Because the release of glutamate is stimulated by calcium elevations and is not affected by glutamate transport inhibitors and because changes in cell volume have not been detected coincident with release, it has been proposed that this transmitter is released through a vesicle-mediated exocytotic pathway.Several observations made using cultured astrocytes support such a vesicle-mediated exocytotic mechanism of glutamate release, including the calcium depend...
RNA precursors give rise to mRNA after splicing of intronic sequences traditionally thought to occur in the nucleus. Here, we show that intron sequences are retained in a number of dendritically-targeted mRNAs, using microarray and Illumina sequencing of isolated dendritic mRNA as well as in situ hybridization. Many of the retained introns contain ID elements, a class of SINE retrotransposon. A portion of these SINEs confers dendritic targeting to exogenous and endogenous transcripts showing the necessity of ID-mediated mechanisms for the targeting of different transcripts to dendrites. ID elements are capable of selectively altering the distribution of endogenous proteins, providing a link between intronic SINEs and protein function. As such, the ID element represents the first common dendritic targeting element to be found across multiple RNAs. Retention of intronic sequence is a more general phenomenon then previously thought and plays a functional role in the biology of the neuron, partly mediated by co-opted repetitive sequences.
Multiple nuclear transcription factors including E-26-like protein 1 (Elk-1) have been found in neuronal dendrites, yet the functional significance of such localization has not yet been explained. Here we use a focal transfection procedure, 'phototransfection', to introduce Elk1 mRNA into specific regions of live, intact primary rat neurons. Introduction and translation of Elk1 mRNA in dendrites produced cell death, whereas introduction and translation of Elk1 mRNA in cell bodies did not produce cell death. Elk-1 translated in dendrites was transported to the nucleus, and cell death depended upon transcription, supporting the dendritic imprinting hypothesis and highlighting the importance of the dendritic environment on protein function. Our demonstration of the utility of phototransfection for spatially controlled introduction of mRNAs opens the broader opportunity to use this method to introduce selected quantities of small molecules into discrete regions of live cells to assess their biological functions.
Dendrites are specialized extensions of the neuronal soma that contain components of the cellular machinery involved in RNA and protein metabolism. Several dendritically localized proteins are associated with the precursor-mRNA (pre-mRNA) splicing complex, or spliceosome. Although some spliceosome-related, RNA-binding proteins are known to subserve separate cytoplasmic functions when moving between the nucleus and cytoplasm, little is known about the pre-mRNA splicing capacity of intact dendrites. Here, we demonstrate the presence and functionality of pre-mRNA-splicing components in dendrites. When isolated dendrites are transfected with a chicken ␦-crystallin pre-mRNA or luciferase reporter premRNA, splicing junctions clustered at or near expected splice sites are observed. Additionally, in vitro synaptoneurosome experiments show that this subcellular fraction contains a similar complement of splicing factors that is capable of splicing chicken ␦-crystallin pre-mRNA. These observations suggest that pre-mRNAsplicing factors found in the dendroplasm retain the potential to promote pre-mRNA splicing.T he molecular properties of dendrites have been extensively examined since the discovery of components of the protein synthetic machinery in the dendroplasm, including ribosomes and membranous constituents of the endoplasmic reticulum and Golgi apparatus (1, 2). A more detailed analysis of ribosomal particles suggests that some serine-arginine (SR) proteins associate with ribosomes in the cytoplasm and may be involved in the translational regulation of associated mRNAs (3). These SR proteins, as well as select heterogeneous nuclear ribonucleoproteins (hnRNPs) (4), are known to constitutively move between the nucleus and cytoplasm in nonneuronal cells where they have roles in posttranscriptional gene expression separate from their characteristic activities in nuclear precursor mRNA (pre-mRNA) splicing as core parts of the spliceosome. The spliceosome is a multimegadalton complex of ribonucleoproteins and small nuclear RNAs (snRNAs) that catalyzes the ATP-dependent removal of introns and ligation of exons in nuclear pre-mRNA (5, 6). Some auxiliary constituents of the spliceosome [e.g., SMN (7) and SAM68 (8)] are found throughout the neuronal cytoplasm, and their localization extends into the dendrites. Although it is clear that these cytoplasmic RNA-binding proteins (RBPs) have critical roles in the postsplicing regulation of the intracellular transport, stability, nonsense-mediated decay (NMD), and translation of cellular mRNAs (9, 10), it is not known whether they retain the ability to assemble pre-mRNA splicingcompetent complexes in the dendritic milieu. Unspliced or incompletely spliced pre-mRNAs are often sequestered in the nucleus, yet removal of introns by splicing, in many cases, is not essential for mRNA export from the nucleus (11-16). It is believed that some viral mRNAs and alternatively spliced mRNAs are likely exported to the cytosol as intron-retaining transcripts (4).To determine the functional capacity ...
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