Expression of the 70 kdalton neurofilament protein in clonal lines of mouse neuroblastoma

The present study measures the content of dynorphin B in the rat hippocampus, and localizes the dynorphins within the intrinsic hippocampal neuronal circuitry. The level of dynorphin B, which is representative of the prodynorphin-derived peptides, was markedly depleted by intrahippocampal injection of colchicine, which destroyed the great majority of the hippocampal granule cells and the associated mossy fiber pathway. The hippocampus contralateral to the injection demonstrated a slight, non-significant rise in dynorphin B levels after colchicine. Entorhinal cortical lesions ablating the perforant pathway input to the hippocampus did not significantly alter dynorphin B levels in the hippocampus. Unilateral fimbrial transection caused a small but significant increase in dynorphin B on the side of the lesion relative to the unlesioned side, but neither side was significantly different from control.     

Induction and quantitation of the RI cAMP-binding protein in clonal mouse neuroblastoma cell lines: evidence that the increase in RI is not linked to neurite outgrowth

This is a study of the regulation of expression of the RI cAMP-binding protein in mouse neuroblastoma cells as it relates to neurotransmitter phenotype and neurite outgrowth. Dibutyryl cAMP was used to promote differentiation of the cholinergic NS-20, the adrenergic N1E-115, the neurotransmitter-inactive N-18, and the neurite-minus N1A-103 mouse neuroblastoma cells. The amount of the RI cAMP-binding protein was quantitated by photoaffinity labeling of the 47,000-dalton RI protein with 8-N3-[32P]cAMP and by Western blot, ELISA, and immunocytochemistry. Our results showed that dibutyryl cAMP induced the RI cAMP-binding protein by three to fivefold in each of the four neuroblastoma cell lines examined. The increased expression of the RI cAMP-binding protein was not linked to neurite outgrowth, a parameter of morphological differentiation in the neuroblastoma cells. Thus, the RI cAMP-binding protein can be induced in the neurite-minus N1A-103 neuroblastoma round cells; further, 8-bromo-cAMP effected neurite outgrowth without inducing the RI cAMP-binding protein in the neurite-positive cell lines. Indirect immunocytochemistry of RI showed a cytoplasmic localization with little evidence of nuclear staining. The increase in RI cAMP-binding protein coincided with an increase in the cAMP-phosphodiesterase and a decrease in cAMP-dependent phosphotransferase activity in the mouse neuroblastoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphine activates calcium channels in cloned mouse neuroblastoma cell lines

Membrane effects of morphine on cloned neuroblastoma cells (strains NG108-CC15 and N1E-115), were studied using intracellular recording and ion flux techniques. Morphine (10 microM) induced a depolarization that was affected by changes in the concentration of calcium, but not in sodium. Further, morphine induced an uptake of 45Ca2+ which was blocked by naloxone (1 microM). The simplest explanation for the findings is that morphine activates calcium channels via mu-receptors.     

Regulation of peripherin in mouse neuroblastoma and rat PC 12 pheochromocytoma cell lines

Peripherin (Formerly the Y protein) is found in the peripheral nervous system. This Triton-insoluble protein is characterized by its isoelectric point (5.6), its apparent molecular weight (56,000 daltons) and its peptide map. Peripherin was also observed in a mouse neuroblastoma cell line, NIE 115, where its expression appeared regulated by the presence of an inducer of morphological differentiation. In order to analyze more precisely this control, the presence of peripherin was investigated in several neuroblastoma cell lines which exhibit different morphological patterns of differentiation and in the rat pheochromocytoma PC 12 cell line. Differentiation of these cells was induced with 1-methylcyclohexane carboxylic acid (CCA) and nerve growth factor (NGF), respectively. Peripherin was found in these different cell lines. Moreover, the cellular amount of peripherin appraised by [35S]-methionine incorporation was significatively increased in differentiated cells. In contrast, other cytoskeletal components did not undergo a similar raise. The level at which the control of the peripherin content takes place was studied in a cell-free translation system. Poly(A)-rich RNAs extracted from growing or differentiated NIE 115 cells directed the synthesis of similar amounts of peripherin in a reticulocyte lysate. In contrast, polysomes prepared from differentiated cells and the corresponding polysomal RNA programmed in vitro the synthesis of twice more peripherin than polysomes or polysomal RNA from growth-phase cells. Since peripherin synthesis is enhanced 5 times in living cells, it seems probable that the cellular amount of peripherin is controlled partly at the translational level and partly at the turn-over level.     

Induction of epitopes associated with neurofibrillary tangles in clonal mouse neuroblastoma (S20Y) cells

Summary Accumulation of paired helical filaments (PHF) in neurofibrillary tangles is a key neuropathological hallmark in Alzheimer's disease (AD). To date, PHF have been found primarily in humans. Cultured murine cholinergic neuroblastoma (S20Y) cells, following exposure to a serum-free medium or a differentiation medium, developed immunoreactivity to anti-PHF antibodies, and to the Alz-50 by immunocytochemical and immunoblot analyses. Electron microscopic examination revealed abundant fascicles of 10-nm filaments coursing tortuously amongst organelles, such as mitochondria, endoplasmic reticulum and dense-core vesicles, in perikarya and in neuritic extensions. However, subcellular structures identical or similar to PHF could not be found in these non-human cells. This convenient cell culture model may prove to be useful for studying certain aspects of the mechanisms underlying the abnormal cytoskeletal alterations which are characteristic of AD and related neurodegenerative disorders.Supported by grants from the Overbrook Foundation, the Will Rogers Institute, the Dr. I. Fund Foundation, the Winifred Masterson Burke Relief Foundation, the Alzheimer's Disease Research Program of the American Health Assistance Foundation and the National Institute of Aging (AG03853)     

Expression of GABAA receptor polypeptides in clonal rat cell lines.

The neuronal-like cell lines, B35, B65, B103, and B104, previously reported to possess high affinity GABA binding, were analyzed for various cellular properties. They possessed peripheral but lacked central benzodiazepine binding. Only B65 cells possessed [3H-]muscimol binding; none bound [35S]TBPS. None of the cells exhibited GABA-stimulated chloride conductance with patch clamp recordings. By Western blots the cells possessed alpha subunits. Northern blot and polymerase chain reaction analysis showed that out of alpha 1, alpha 4, beta 1 and gamma 2 subunits, only alpha 1 subunit mRNA was present. Thus, GABAA-receptor binding without associated central benzodiazepine receptor sites and without functional chloride channels appears to result from expression of an incomplete subunit composition.     

Neurofilament protein in cerebrospinal fluid: a marker of white matter changes.

The objective of this study was to compare cerebrospinal fluid (CSF) levels of the light subtype of the neurofilament proteins (NFL), tau, and beta-amyloid42 (Abeta42) in individuals with moderate or severe white matter changes (WMC) and in those with mild or no WMC. Twenty-two patients with Alzheimer's disease (AD), nine patients with subcortical vascular dementia (SVD), and 20 normal controls were included in the study. The occurrence of WMC was evaluated by a neuroradiologist using the Blennow-Wallin scale. Thirty-seven subjects had no or only punctate WMC; 14 had moderate to severe WMC. Both diagnostic group and WMC, but not gender or apolipoproteinE E4 inheritance, contributed to the variance in the CSF levels of tau, NFL, and Abeta42. In patients with moderate to severe WMC, CSF NFL (P < 0.01), but not CSF tau or CSF Abeta42, was increased also after correction for age, gender, and degree of cognitive impairment. A comparison between patients and controls with any signs of WMC and those without such signs yielded a similar result: CSF NFL (P < 0.001) was increased in the group with signs of WMC. As in numerous previous studies, we found that CSF tau was increased in AD (P < 0.001) compared with controls. Furthermore, CSF NFL was increased in both AD and SVD compared with controls (P < 0.001 for both). Although diagnostic group seems to be a stronger predictor of the variance found in CSF NFL, a clear association between the presence of WMC and increased CSF NFL was found. Because NFL is located mainly in large myelinated axons, increased CSF NFL in individuals with WMC probably reflects axonal degeneration.     

Neurofilament protein levels in CSF are increased in dementia

The neurofilament is the major cytoskeletal structure of myelinated axons. In this study, CSF levels of the light subunit of the neurofilament protein (NFL) were increased in patients with vascular dementia (VAD), AD, and frontotemporal dementia (FTD) compared with neurologically healthy individuals. Because NFL is localized mainly in myelinated axons, these results suggest that the degeneration of white matter in these disorders causes the increased CSF NFL levels.     

Expression in murine and human neuroblastoma cell lines of VGF, a tissue specific protein

Screening by different means has demonstrated the presence, in human and murine neuroblastoma cell lines, of VGF, a gene product identified in a limited number of neuronal and endocrine cells. Indirect immunofluorescence and Western and Northern blot analyses have shown the presence of this protein in some of the tested lines, confirming that VGF is not an ubiquitous molecule. Further studies, using human SK-N-BE and murine N18TG2 lines, showed that VGF expression is upregulated during differentiation, suggesting that various species, including man, express VGF and regulate it in a similar manner. The subcellular localization of the protein, which is associated with vesicles, its electrophoretic molecular profile and its specific release under different conditions are all consistent with results reported in other cells. Neuroblastomas are thus added to the class of VGF-positive cells and provide a new in vitro model for investigation of the structural and functional properties of this protein.     

Ionic excitation of a clone of mouse neuroblastoma.

The electrical properties and the possible regulation of these properties were studied by means of intracellular microelectrode recordings in cells of mouse neuroblastoma clone N1E-115. This clone has high levels of tyrosine hydroxylase and regulates this enzyme. Cells treated for 24 h with 4 muM aminopterin followed by at least 5 days in culture developed rhythmic discharge of action potentials when superfused with phosphate-buffered saline containing less than 0.2 mM calcium or less than 0.2 mM calcium and zero potassium. This ionic excitation occurred in no cells at less than 5 days after treatment with aminopterin but at 5 days or more after treatment, 20% of cells responded to low calcium while 52% responded to low calcium and zero potassium. Concomitant with the development of a susceptibility to ionic excitation was an increase in the average resting membrane potential and morphologic maturation. This ionic excitation of cultured mouse neuroblastoma cells may be useful for studying biochemical events associated with repetitive discharge of action potentials.     

Lithium induces morphological differentiation of mouse neuroblastoma cells.

Neuroblastoma cells are used as a model system to study neuronal differentiation. Here we describe the induction of morphological differentiation of mouse neuroblastoma Neuro 2a (N2a) cells by treatments with either chemical inhibitors of cyclin-dependent kinases or lithium, which inhibits glycogen synthase kinase-3. Cyclin-dependent kinase inhibitors cause a rapid cell cycle block as well as the extension of multiple neurites per cell. These multipolar differentiated cells then undergo a massive death. However, lithium promotes a delayed mitotic arrest and the extension of one or two long neurites per cell. This differentiation is maximal after 48 hours of lithium treatment and the differentiated cells remain viable for long periods of time. Neuronal differentiation in lithium-treated cells is preceded by the accumulation of beta-catenin, a protein which is efficiently proteolyzed when it is phosphorylated by glycogen synthase kinase-3. Both neuronal differentiation and beta-catenin accumulation are observed in lithium-treated cells either in the absence or in the presence of supraphysiological concentrations of inositol. The results are consistent with the hypothesis that inhibition of glycogen synthase kinase-3 by lithium triggers the differentiation of neuroblastoma N2a cells.     

Acetylcholine-activated currents in mouse neuroblastoma cells

The nicotinic and muscarinic responses of differentiated mouse neuroblastoma cells from the clonal line N1E 115 to applied cholinergic agents were recorded using single channel and whole cell patch clamp techniques. An inward macroscopic current induced by acetylcholine (ACh) at the resting potential was blocked by curare; cell-attached recordings revealed a single channel conductance of 18 pS and a lifetime of 36 ms at 30°C, with 200 nM ACh. The zero current potential was close to 0 mV. The kinetics of these nicotinic currents were described by multiexponential functions for both the open and closed time distributions. An outward single channel current, present at resting and slightly depolarized potentials, was also observed and has been tentatively described as being dependent on muscarinic receptor activation, as it was usually blocked by atropine. Under our conditions of whole cell clamp, no macroscopic outward current sensitive to ACh was observed.     

Altered insulin receptor processing and function in scrapie-infected neuroblastoma cell lines.

The underlying neurochemical changes contributing to prion-induced neurodegeneration remain largely unknown. This study shows that scrapie infection induced a 2-fold increase of insulin receptor (IR) protein and aberrantly processed IR beta-chain in scrapie-infected N2a neuroblastoma cells (ScN2a) as measured by Western blot of immunoprecipitated IR, in the absence of increased IR mRNA. Elevated IR protein level was further confirmed in an independently scrapie-infected neuroblastoma cell line N1E-115 (ScN1E-115). Proliferation studies showed that the increased IR level in ScN2a did not result in an increased insulin-mediated cell growth compared to normal N2a cells. Binding studies indicated that this apparent paradox was due to a 65% decrease in specific [(125)I]insulin binding sites in ScN2a when compared to the amount of immunoreactive IR, although the IR binding affinity was unchanged. Analysis of insulin stimulated IR tyrosine phosphorylation showed a slight but not significant reduction in ScN2a, when related to the increased level of immunoreactive IR. However, comparing the IR tyrosine phosphorylation to the loss of binding sites in ScN2a, we demonstrated an increased IR tyrosine phosphorylation of the remaining functional IR. In addition to these differences in IR properties, the basal extracellular signal regulated kinase-2 (ERK2) phosphorylation detected by Western blot, was significantly elevated and the insulin stimulated ERK2 phosphorylation was subsequently decreased in ScN2a. Together, these data show that scrapie infection affects the level and processing of the IR and signal transduction mediated by the IR in neuroblastoma cells, as well as induces an elevated basal ERK2 phosphorylation. Aberrant regulation of neuroprotective receptors may contribute to neurodegeneration in prion diseases.