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J Eberwine

Publications and source records attributed to J Eberwine.

At least 19 recordsLinked to original sources
PubMed2005

RNA splicing capability of live neuronal dendrites.

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 delta-crystallin pre-mRNA or luciferase reporter pre-mRNA, 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 delta-crystallin pre-mRNA. These observations suggest that pre-mRNA-splicing factors found in the dendroplasm retain the potential to promote pre-mRNA splicing.

Animals↗
PubMed2001

Molecular biology of axons: "a turning point...".

Previous studies have shown that dendrites and axons contain both mRNAs and the machinery for local protein translation. While a number of studies in recent years have focused on the functional role of protein synthesis in dendrites, relatively less is know about the role of local translation in axons. Campbell and Holt (this issue of Neuron) show that local protein synthesis and degradation are required for proper chemotropic turning responses of isolated retinal growth cones.

Animals↗
PubMed2001

Identification of sites for exponential translation in living dendrites.

Neuronal processes contain mRNAs and membrane structures, and some forms of synaptic plasticity seem to require protein synthesis in dendrites of hippocampal neurons. To quantitate dendritic protein synthesis, we used multiphoton microscopy of green fluorescent protein synthesized in living isolated dendrites. Transfection of dendrites with mRNA encoding green fluorescent protein resulted in fluorescence that exponentially increased on stimulation with a glutamate receptor agonist; a reaction attenuated by the translation inhibitors anisomycin and emetine. Comparable experiments on whole neurons revealed that (RS)-3,5-dihydroxy-phenylglycine 0.5 H(2)O (DHPG)-stimulated fluorescence was linear in cell bodies relative to the exponential increase seen in dendrites. Detailed spatial analysis of the subdendritic distribution of fluorescence revealed "hotspots," sites of dendritic translation that were spatially stable. However, detailed temporal analysis of these hotspots revealed heterogeneous rates of translation. A double-label protocol counterstaining for ribosomes indicated that sites of "fastest" translation correlated with increased ribosome density, consistent with ribosome subunit assembly for initiation, the first step of translation. We propose that dendrites have specific sites specialized for fast translation.

Animals↗
PubMed2001

Local translation of classes of mRNAs that are targeted to neuronal dendrites.

The functioning of the neuronal dendrite results from a variety of biological processes including mRNA transport to and protein translation in the dendrite. The complexity of the mRNA population in dendrites suggests that specific biological processes are modulated through the regulation of dendritic biology. There are various classes of mRNAs in dendrites whose translation modulates the ability of the dendrite to receive and integrate presynaptic information. Among these mRNAs are those encoding selective transcription factors that function in the neuronal soma and ionotropic glutamate receptors that function on the neuronal membrane. Conclusive evidence that these mRNAs can be translated is reviewed, and identification of the endogenous sites of translation in living dendrites is presented. These data, as well as those described in the other articles resulting from this colloquium, highlight the complexity of dendritic molecular biology and the exquisitely selective and sensitive modulatory role played by the dendrite in facilitating intracellular and intercellular communication.

Animals↗
PubMed2001

Protein quantification from complex protein mixtures using a proteomics methodology with single-cell resolution.

We have developed an extremely sensitive technique, termed immuno-detection amplified by T7 RNA polymerase (IDAT) that is capable of monitoring proteins, lipids, and metabolites and their modifications at the single-cell level. A double-stranded oligonucleotide containing the T7 promoter is conjugated to an antibody (Ab), and then T7 RNA polymerase is used to amplify RNA from the double-stranded oligonucleotides coupled to the Ab in the Ab-antigen complex. By using this technique, we are able to detect the p185(her2/neu) receptor from the crude lysate of T6-17 cells at 10(-13) dilution, which is 10(9)-fold more sensitive than the conventional ELISA method. Single-chain Fv fragments or complementarity determining region peptides of the Ab also can be substituted for the Ab in IDAT. In a modified protocol, the oligonucleotide has been coupled to an Ab against a common epitope to create a universal detector species. With the linear amplification ability of T7 RNA polymerase, IDAT represents a significant improvement over immuno-PCR in terms of sensitivity and has the potential to provide a robotic platform for proteomics.

3T3 Cells↗
PubMed2000

Stimulation of glutamate receptor protein synthesis and membrane insertion within isolated neuronal dendrites.

The selective subcellular localization of mRNAs to dendrites and the recent demonstration of local protein synthesis have highlighted the potential role of postsynaptic sites in modulation of cell-cell communication. We show that epitope-tagged subunit 2 of the ionotopic glutamate receptor, GluR2, mRNA transfected into isolated hippocampal neuronal dendrites is translated in response to pharmacologic stimulation. Further, confocal imaging of N-terminally labeled GluR2 reveals that the newly synthesized GluR2 protein can integrate into the dendritic membrane with the N terminus externally localized. These data demonstrate that integral membrane proteins can be synthesized in dendrites and can locally integrate into the cell membrane.

Amino Acid Sequence↗
PubMed2000

Frequent lack of translation of antigen presentation-associated molecules MHC class I, CD1a and Beta(2)-microglobulin in Reed-Sternberg cells.

Epstein-Barr virus (EBV) is present in Reed-Sternberg (RS) cells of a substantial proportion of Hodgkin's lymphoma cases. Most EBV-positive cases are also MHC class I-positive, whereas the majority of EBV-negative cases lack detectable levels of MHC class I expression. Application of the SAGE technique has led to the identification of tags corresponding to MHC class I and beta(2)-microglobulin genes in the EBV- and MHC class I-negative L428 Hodgkin's cell line. Further expression studies indicated that single RS cells that do not express HLA class I also lack beta(2)-microglobulins but frequently contain mRNA coding for these proteins. Another tag was identified corresponding to CD1a, a thymocyte and Langerhans cell antigen structurally related to the MHC class I genes. CD1a expression studies revealed mRNA in all cell lines and in several of the single cells, whereas immunostaining showed a cytoplasmic signal in only 2 of the 4 cell lines and in none of the Hodgkin's lymphoma tissue samples. In conclusion, RS cells frequently lack MHC class I, beta(2)-microglobulin and CD1a protein expression but contain mRNA coding for these proteins in some of the RS cells, suggesting a common mechanism affecting the translation of these antigen presentation-associated molecules.

Antigen Presentation↗
PubMed2000

Expression of mRNAs encoding for two different olfactory receptors in a subset of olfactory receptor neurons.

Evidence has accumulated to support a model for odorant detection in which individual olfactory receptor neurons (ORNs) express one of a large family of G protein-coupled receptor proteins that are activated by a small number of closely related volatile chemicals. However, the issue of whether an individual ORN expresses one or multiple types of receptor proteins has yet to be definitively addressed. Physiological data indicate that some individual ORNs can be activated by odorants differing substantially in structure and/or perceived quality, suggesting multiple receptors or one nonspecific receptor per cell. In contrast, molecular biological studies favor a scheme with a single, fairly selective receptor per cell. The present studies directly assessed whether individual rat ORNs can express multiple receptors using single-cell PCR techniques with degenerate primers designed to amplify a wide variety of receptor sequences. We found that whereas only a single OR sequence was obtained from most ORNs examined, one ORN produced two distinct receptor sequences that represented different receptor gene families. Double-label in situ hybridization studies indicated that a subset of ORNs co-express two distinct receptor mRNAs. A laminar segregation analysis of the cell nuclei of ORNs labeled with the two OR mRNA probes showed that for one probe, the histogram of the distribution of the cell nuclei along the depth of the epithelium was bimodal, with one peak overlapping the (unimodal) histogram for the other probe. These results are consistent with co-expression of two OR mRNAs in a population of single ORNs.

Amino Acid Sequence↗
PubMed1999

Preparation of cDNA from single cells and subcellular regions.

Phenotypic characterization of cells in conjunction with single-cell mRNA analysis, which yields information regarding expression of multiple genes in individual neurons, facilitates a detailed and comprehensive view of neuronal cell biology. More specifically, the aRNA amplification method has provided an approach to analyze mRNA levels in single cells that have been phenotypically characterized on the basis of electrophysiology, morphology, and/or protein expression. In this way, relative mRNA abundances can be directly assayed from a well-defined population of neurons. The concept of expression profiling led to the development of robotics methods for arraying thousands of cDNAs on microarrays. These cDNA arrays can be screened with labeled aRNA or cDNA to generate a molecular fingerprint of a specific cell type, disease state, or therapeutic efficacy. A broad view of how gene expression is altered in single neurons affected by a particular disease process may provide clues to pathogenetic disease mechanisms or avenues for therapeutic interventions. The use of mRNA profiles to produce diagnostics and therapeutics is called transcript-aided drug design (TADD). When coupled with single-cell resolution, TADD promises to be an important tool in diagnosis of disease states, as well as provide a blueprint on which to develop therapeutic strategies. For example, mRNA abundances in an individual diseased cell may increase, decrease, or remain constant, and thus it is possible that a pharmaceutical alone or in combination with other drugs may be specifically designed to restore mRNA abundances to a normal state. Alternatively, if functional protein levels parallel the mRNA level changes, then drugs targeting the function of the proteins translated from these altered mRNAs may prove to be therapeutic. One promise of such an approach is that information about mRNA abundances that are altered in a diseased cell may provide new therapeutic indications for existing drugs. For example, if the abundance of mRNA for the beta-adrenergic receptor is altered as shown by the microarrays for a particular disease, already available adrenergic receptor agonists or antagonists that had not previously been used in this particular disease paradigm may prove to be therapeutically efficacious. The expression profile of a given cell is a measure of the potential for protein expression. Proteins are generally the functional entities within cells and differences in protein function often result in disease. The ability to monitor the coordinate changes in gene expression, in single phenotypically identified cells, that correlate with disease will provide unique insight into the expressed genetic variability of cells and will likely furnish unforeseen insight into the underlying cellular mechanisms that produce disease etiology.

Animals↗
PubMed1999

Cytoplasmic proteins interact with a translational control element in the protein-coding region of proopiomelanocortin mRNA.

Previous studies have indicated that proopiomelanocortin (POMC) is translationally regulated. We proposed that the regulatory mechanism involves an interaction between trans-acting protein factors and a cis-acting stem-loop structure in the coding region of POMC mRNA. Functional interactions were tested by examining the translation of mouse POMC mRNA in a rabbit reticulocyte system. Specific binding was demonstrated with ultraviolet-crosslinking and RNA gel mobility shift assays. The evidence presented supports our hypothesis that the translational regulation of POMC gene expression involves recognition of the stem-loop by RNA-binding proteins. Furthermore, POMC stem-loop RNA-binding proteins specifically recognized a predicted stem-loop found in the coding region of corticotropin-releasing hormone, suggesting a novel mechanism of gene regulation that may extend to other neuropeptides as well.

Animals↗
PubMed1998

Presence and phosphorylation of transcription factors in developing dendrites.

In screening amplified poly(A) mRNA from hippocampal dendrites and growth cones in culture to determine candidates for local translation, we found that select transcription factor mRNAs were present. We hypothesized that synthesis of transcription factor proteins within dendrites would provide a direct signaling pathway between the distal dendrite and the nucleus resulting in modulation of gene expression important for neuronal differentiation. To evaluate this possibility, radiolabeled amplified antisense RNA was used to probe slot blots of transcription factor cDNAs as well as arrayed blots of zinc finger transcription factors. The mRNAs encoding the cAMP response element binding protein (CREB), zif 268, and one putative transcription factor were detected. We expanded upon these results showing that CREB protein is present in dendrites, that translation of CREB mRNA in isolated dendrites is feasible and that CREB protein found in dendrites can interact with the cis-acting cyclic AMP reponse element DNA sequence by using an in situ Southwestern assay. Further, CREB protein in dendrites is not transported to this site from the cell body because fluorescently tagged CREB microperfused into the soma did not diffuse into the dendrites. In addition, CREB protein microperfused into dendrites was rapidly transported to the nucleus, its likely site of bioactivity. Lastly, by using the isolated dendrite system we show that phosphorylation of Ser-133 on CREB protein can occur in isolated dendrites independent of the nucleus. These data provide a regulatory pathway in which transcription factors synthesized and posttranslationally modified in dendrites directly alter gene expression bypassing the integration of signal transduction pathways that converge on the nucleus.

Animals↗
PubMed1997

Cellular and molecular basis of cerebral dysgenesis.

Maldevelopment of the cerebral cortex, cortical dysgenesis (CD), may be associated with epilepsy, mental retardation (MR), and focal or widespread neurologic deficits. The histologic hallmark of CD is disrupted cytoarchitecture, including disorganized lamination, malpositioned neurons with respect to their normal radial orientation, abnormal dendritic arborization, and heterotopic neurons within the white matter. Seizures in these patients are particularly difficult to control with conventional anti-epileptic drugs (AEDs) and may require epilepsy surgery to remove these abnormal foci. Focal CD has been reported in up to 30% of epilepsy surgery specimens and are believed to provide the central pathologic substrate responsible for seizures in these patients. How and why CD results in epileptiform activity is unknown. Advances in understanding the pathogenesis of some types of CD have occurred recently with the cloning genes responsible for a few types of X-linked and autosomal CD. This review will outline the major subtypes of CD, the pathologic findings, and the molecular etiologies for a variety of CD. We will also address recent experimental advances in studying the pathogenesis of CD.

Animals↗
PubMed1997

Fragile X mental retardation protein is translated near synapses in response to neurotransmitter activation.

Local translation of proteins in distal dendrites is thought to support synaptic structural plasticity. We have previously shown that metabotropic glutamate receptor (mGluR1) stimulation initiates a phosphorylation cascade, triggering rapid association of some mRNAs with translation machinery near synapses, and leading to protein synthesis. To determine the identity of these mRNAs, a cDNA library produced from distal nerve processes was used to screen synaptic polyribosome-associated mRNA. We identified mRNA for the fragile X mental retardation protein (FMRP) in these processes by use of synaptic subcellular fractions, termed synaptoneurosomes. We found that this mRNA associates with translational complexes in synaptoneurosomes within 1-2 min after mGluR1 stimulation of this preparation, and we observed increased expression of FMRP after mGluR1 stimulation. In addition, we found that FMRP is associated with polyribosomal complexes in these fractions. In vivo, we observed FMRP immunoreactivity in spines, dendrites, and somata of the developing rat brain, but not in nuclei or axons. We suggest that rapid production of FMRP near synapses in response to activation may be important for normal maturation of synaptic connections.

Amino Acid Sequence↗