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J Eberwine

Publications and source records attributed to J Eberwine.

31 records · Page 2Linked to original sources
PubMed1997

Internexin, MAP1B, and nestin in cortical dysplasia as markers of developmental maturity.

Cortical dysplasias (CD) are characterized histologically by disorganized cortical lamination and abnormally shaped neurons. We hypothesized that neurons within CD have failed to differentiate fully and may express proteins such as cytoskeletal elements characteristic of immature cells. Disrupted expression of certain cytoskeletal proteins, which have been implicated in neuronal polarity, process outgrowth, and migration, could result in disorganized cortical lamination. Thus, we probed two CD subtypes, focal CD (FCD) and hemimegalencephaly (HME), with antibodies specific for cytoskeletal proteins that are developmentally regulated in neural progenitor cells and neurons to define more fully the developmental phenotype of neurons within CD. Microtubule-associated protein 1B (MAP1B) and the intermediate filament (IF) protein nestin are enriched in neural progenitors, whereas MAP2B, phosphorylated and non-phosphorylated forms of medium (NFM) and high (NFH) molecular weight neurofilament (NF) proteins, as well as the light NF subunit (NFL) and IF protein alpha internexin are expressed in developing and mature neurons. Immunolabeling for internexin and MAP1B was more abundant in the most abnormally shaped neurons that populated dysplastic regions than in adjacent regions exhibiting milder cytoarchitectural abnormalities or control cortex. Nestin immunoreactivity was noted in large dysplastic and heterotopic neurons within the deeper cortical layers of CD specimens but not in normal cortex. In contrast, neurons in CD specimens also expressed cytoskeletal markers characteristic of differentiated neurons such as NF subunits and MAP2B. These findings suggest that the cytoarchitectural abnormalities in CD may reflect pathophysiological changes in the developing brain that disrupt expression of several key components of the neuronal cytoskeleton and may contribute to impaired migration of cortical neurons.

Antibodies, Monoclonal↗
PubMed1996

Embryonic neuronal markers in tuberous sclerosis: single-cell molecular pathology.

One hallmark of tuberous sclerosis (TSC) is the presence of highly epileptogenic dysplastic cerebral cortex (tubers) composed of abnormally shaped neurons and giant cells. Mutation of the TSC gene (TSC2) may disrupt differentiation and maturation of neuronal precursors, since the TSC2 gene product tuberin is believed to regulate cellular proliferation. To test the hypothesis that cells in tubers may retain the molecular phenotype of embryonic or immature neurons, tubers from five TSC patients were probed with antibodies to proteins expressed in neuronal precursors (nestin, Ki-67, and proliferating cell nuclear antigen). Many dysmorphic neurons and giant cells in tubers were stained by these antibodies, while neurons in adjacent normal and control cortex were not labeled. To further characterize the molecular phenotype of cells in tubers, we developed a methodology in which poly(A)+ mRNA was amplified from immunohistochemically labeled single cells in paraffin-embedded brain specimens. This approach enabled us to detect mRNAs encoding nestin, and other cytoskeletal elements, cell cycle markers, and synthetic enzymes present in individual nestin-stained cells by means of reverse Northern blotting. We conclude that the presence of immature phenotypic markers (mRNAs and proteins) within tubers suggests disruption of cell cycle regulation and neuronal maturation in TSC during cortical development. Characterization of multiple mRNAs within fixed, immunohistochemically labeled cells provides a powerful tool for studying gene expression and the molecular pathophysiology of many neurologic diseases.

Biomarkers↗
PubMed1996

Presence of mRNA for glutamic acid decarboxylase in both excitatory and inhibitory neurons.

Neurons in very low density hippocampal cultures that are physiologically identified as either GABAergic inhibitory or glutamatergic excitatory all contain mRNA for the gamma-aminobutyric acid (GABA) synthetic enzyme, glutamic acid decarboxylase (GAD), as detected by single cell mRNA amplification and PCR. However, consistent with the physiology, immunocytochemistry revealed that only a subset of the neurons stain for either GAD protein or GABA. A similar fraction hybridize with RNA probes for GAD65 and GAD67. Hippocampal CA1 pyramidal neurons in slice preparations, which are traditionally thought to be excitatory, also contain mRNA for GAD65 and GAD67. Hippocampal neurons in culture did not contain mRNA for two other neurotransmitter synthesizing enzymes, tyrosine hydroxylase, and choline acetyl transferase. These data suggest that in some neurons, presumably the excitatory neurons, GAD mRNA is selectively regulated at the level of translation. We propose that neurotransmitter phenotype may be posttranscriptionally regulated and neurons may exhibit transient phenotypic plasticity in response to environmental influences.

Animals↗
PubMed1996

Molecular characterization of the dendritic growth cone: regulated mRNA transport and local protein synthesis.

The molecular mechanisms that regulate growth cone guidance of dendrite outgrowth remain to be elucidated. We hypothesized that mRNA localization in dendritic growth cones and their local protein synthesis may be important for growth cone functioning. The appearance of 23 of 31 growth cone mRNAs was developmentally regulated. Also, alteration of growth cone morphology affected the relative levels of three mRNAs. Finally, using single dendrite transfection, it was shown that local protein synthesis occurs in dendrites and growth cones. A heterogeneous population of mRNAs exists in dendritic growth cones of cultured hippocampal neurons whose relative abundances are developmentally regulated and can vary with changes in growth cone physiology. The demonstration of protein synthesis in growth cones suggests that translation of the localized mRNAs may contribute to regulation of growth cone motility and dendrite outgrowth.

Animals↗
PubMed1996

CAG trinucleotide RNA repeats interact with RNA-binding proteins.

Genes associated with several neurological diseases are characterized by the presence of an abnormally long trinucleotide repeat sequence. By way of example, Huntington's disease (HD), is characterized by selective neuronal degeneration associated with the expansion of a polyglutamine-encoding CAG tract. Normally, this CAG tract is comprised of 11-34 repeats, but in HD it is expanded to > 37 repeats in affected individuals. The mechanism by which CAG repeats cause neuronal degeneration is unknown, but it has been speculated that the expansion primarily causes abnormal protein functioning, which in turn causes HD pathology. Other mechanisms, however, have not been ruled out. Interactions between RNA and RNA-binding proteins have previously been shown to play a role in the expression of several eukaryotic genes. Herein, we report the association of cytoplasmic proteins with normal length and extended CAG repeats, using gel shift and UV crosslinking assays. Cytoplasmic protein extracts from several rat brain regions, including the striatum and cortex, sites of neuronal degeneration in HD, contain a 63-kD RNA-binding protein that specifically interacts with these CAG-repeat sequences. These protein-RNA interactions are dependent on the length of the CAG repeat, with longer repeats binding substantially more protein. Two CAG repeat-binding proteins are present in human cortex and striatum; one comigrates with the rat protein at 63 kD, while the other migrates at 49 kD. These data suggest mechanisms by which RNA-binding proteins may be involved in the pathological course of trinucleotide repeat-associated neurological diseases.

Aged↗
PubMed1995

Mechanisms of neuronal plasticity as analyzed at the single cell level.

This chapter has highlighted how correlates of neuronal plasticity such as electrophysiological responsiveness and changes in gene expression may be examined in defined CNS regions as well as in single cells. The ability to simultaneously measure the mRNA levels for hundreds of different genes, to clone novel genes, and to characterize the physiology and morphology of the cell promises to provide insight into molecular mechanisms of plasticity. The importance of understanding how one gene product changes relative to another (coordinated changes) as well as subcellular distribution of mRNAs cannot be overstated. It is only through an analysis of both the molecular and cellular processes associated with plasticity that a thorough understanding of the mechanisms of neuronal plasticity can be gained.

Animals↗
PubMed1994

On the nature and differential distribution of mRNAs in hippocampal neurites: implications for neuronal functioning.

Neurons are highly polarized cells with a mosaic of cytoplasmic and membrane proteins differentially distributed in axons, dendrites, and somata. In Drosophila and Xenopus, mRNA localization coupled with local translation is a powerful mechanism by which regionalized domains of surface or cytoplasmic proteins are generated. In neurons, there is substantial ultrastructural evidence positing the presence of protein synthetic machinery in neuronal processes, especially at or near postsynaptic sites. There are, however, remarkably few reports of mRNAs localized to these regions. We now present direct evidence that an unexpectedly large number of mRNAs, including members of the glutamate receptor family, second messenger system, and components of the translational control apparatus, are present in individual processes of hippocampal cells in culture.

Animals↗
PubMed1994

Cellular and molecular characterization of Ca2+ currents in acutely isolated, adult rat neostriatal neurons.

Ca2+ currents in acutely isolated, adult rat neostriatal neurons were studied with whole-cell voltage-clamp techniques. In the vast majority of neurons (approximately 90%, n > 250), currents were exclusively of the high-voltage-activated (HVA) type. HVA currents activated near -40 mV and reached their maximum amplitude near 0 mV. Quasi-steady-state inactivation curves in many neurons were well fitted only with a sum of Boltzmann functions, suggesting that the HVA currents were heterogeneous. Although the block of whole-cell current by Cd2+ was well fitted with a single isotherm having an IC50 of near 1 microM, experiments with organic channel antagonists suggested that at least four types of HVA channels were expressed by most cells. On average, the L-channel antagonist nifedipine (5-10 microM) blocked 31 +/- 10% of the whole-cell current (n = 20), the N-channel antagonist omega-conotoxin GVIA (omega-CgTx) (2-5 microM) blocked 27 +/- 11% (n = 20), and the P-channel antagonist omega-agatoxin IVA (100-500 nM) blocked 21 +/- 10% (n = 18). In many neurons, the block by omega-CgTx was partially or completely reversible. In cells tested with a combination of these antagonists, 34 +/- 17% of the peak Ca2+ current remained unblocked (n = 13). Single-cell expression profiling of medium-sized neurons revealed the presence of rbA and rbB Ca2+ channel alpha 1 subunit mRNAs but low or undetectable levels of rbC mRNA (n = 12). These findings suggest that although adult neostriatal projection neurons do not express significant levels of LVA Ca2+ current, they do express a pharmacologically and structurally heterogeneous population of HVA currents.

Animals↗
PubMed1992

Dopamine receptor subtypes colocalize in rat striatonigral neurons.

Dopaminergic neurons of the substantia nigra provide one of the major neuromodulatory inputs to the neostriatum. Recent in situ hybridization experiments have suggested that postsynaptic dopamine receptors are segregated in striatonigral and striatopallidal neurons. We have tested this hypothesis in acutely isolated, retrogradely labeled striatonigral neurons by examining the neuromodulatory effects of selective dopaminergic agonists on Na currents and by probing single-cell antisense RNA populations with dopamine receptor cDNAs. In most of the neurons examined (20/31), the application of the D1 dopamine receptor agonist SKF 38393 reduced evoked whole-cell Na+ current. The D2 agonists quinpirole and bromocriptine had mixed effects; in most neurons (23/42), whole-cell Na+ currents were reduced, but in others (8/42), currents were increased. In cell-attached patch recordings, bath application of SKF 38393 decreased currents as in whole-cell recordings, whereas quinpirole consistently (6/10) enhanced currents--suggesting that D2-like receptors could act through membrane delimited and non-delimited pathways. Changes in evoked current were produced by modulation of peak conductance and modest shifts in the voltage dependence of steady-state inactivation. Antisense RNA probes of dopamine receptor cDNA Southern blots consistently (5/5) revealed the presence of D1, D2, and D3 receptor mRNA in single striatonigral neurons. These findings argue that, contrary to a strict receptor segregation hypothesis, many striatonigral neurons colocalize functional D1, D2, and D3 receptors.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1992

Analysis of gene expression in single live neurons.

We present here a method for broadly characterizing single cells at the molecular level beyond the more common morphological and transmitter/receptor classifications. The RNA from defined single cells is amplified by microinjecting primer, nucleotides, and enzyme into acutely dissociated cells from a defined region of rat brain. Further processing yields amplified antisense RNA. A second round of amplification results in greater than 10(6)-fold amplification of the original starting material, which is adequate for analysis--e.g., use as a probe, making of cDNA libraries, etc. We demonstrate this method by constructing expression profiles of single live cells from rat hippocampus. This profiling suggests that cells that appear to be morphologically similar may show marked differences in patterns of expression. In addition, we characterize several mRNAs from a single cell, some of which were previously undescribed, perhaps due to "rarity" when averaged over many cell types. Electrophysiological analysis coupled with molecular biology within the same cell will facilitate a better understanding of how changes at the molecular level are manifested in functional properties. This approach should be applicable to a wide variety of studies, including development, mutant models, aging, and neurodegenerative disease.

Animals↗
PubMed1992

Complementary DNA synthesis in situ: methods and applications.

In situ transcription is the synthesis of cDNA within cells. This chapter has illustrated some of the application of IST to the study of gene expression in complex cell environments. While the importance of transcription in modulating cellular activity has been long appreciated, the role of translational control mechanisms in regulating central nervous system functioning is just beginning to be recognized. Previous limitations in the availability of tissue have made it difficult to construct cDNA libraries from defined cell populations, to examine translational control, and to quantitate differences in the amount of mRNA for many distinct mRNAs in the same sample. In situ transcription facilitates all of these procedures, making it possible to characterize aspects of gene regulation that were previously difficult. Indeed, taken to its furthest extreme it is now possible to characterize gene expression in single live cells. This level of analysis allows basic questions, such as How different morphologically identical cells are at the level of gene expression, and How synaptic connectivity and glial interactions influence gene expression in single cells, to be experimentally approached. The ability to characterize gene expression in small amounts of tissue and single cells is critical to gaining an understanding of the contribution of specific cell types to the physiology of the central nervous system.

Animals↗
PubMed1986

Acromegaly and pheochromocytoma: a multiple endocrine syndrome caused by a plurihormonal adrenal medullary tumor.

A 42-yr-old man with congestive heart failure and diabetes mellitus was found to have acromegaly and a pheochromocytoma. Serum GH-releasing hormone (GHRH) levels were elevated (2.34 ng/dl; normal, less than 0.02 ng/dl), suggesting that the acromegaly was caused by ectopic secretion of GHRH. Postmortem examination revealed that the right adrenal gland contained a pheochromocytoma in which GHRH was demonstrated by immunohistochemical studies. Gel permeation chromatography combined with the use of two GHRH antisera showed that GHRH-(1-44)-NH2 was a predominant form of the hormone. When the RNA from the tumor was extracted and analyzed by Northern gel blotting, two mRNA species were identified, with transcripts corresponding to 1600 and 780 base pairs. The pituitary gland was enlarged, but no distinct adenoma was found. Diffuse and nodular hyperplasia of somatotrophs in some areas resembling adenoma was identified on histological examination. These findings indicate that GH excess accompanied by somatotroph hyperplasia and acromegaly were secondary to a pheochromocytoma which secreted not only catecholamines but also GHRH.

Acromegaly↗