Neuron-derived Cell Line Research Articles

Redistribution of secretory granule components precedes that of synaptic vesicle proteins during differentiation of a neuronal cell line in serum-free medium

Incubation of the rat sensory neuron-derived cell line ND7 in serum-free medium results in the arrest of mitosis and the appearance of numerous neuronal processes. During this differentiation event, secretory granule components such as chromogranins, neuropeptide Y and the C-flanking peptide of pro-neuropeptide Y move to the tips of the majority of the neuronal processes regardless of process length. In contrast, the synaptic vesicle component, synaptophysin, is found only at the tips of the very long processes which appear following prolonged periods of culture in serum-free medium. A similar restriction of synaptophysin to long processes is also observed following differentiation and process formation induced by other treatments such as incubation in reduced serum or treatment with cyclic AMP or phorbol myristate acetate. Hence the regulated secretory pathway associated with the chromogranins and neuropeptides appears to be segregated into the processes at an earlier stage of ND7 differentiation than the synaptophysin-associated synaptic vesicle pathway. ND7 cells therefore provide a model system for studying the processes regulating these pathways and the redistribution of their components during neuronal differentiation.

Suppressive effect of carbachol on forskolin-stimulated neurite outgrowth in human neuroblastoma NB-OK1 cells

We have used human neuroblastoma NB-OK1 cells to investigate the regulation of neurite outgrowth. Carbachol suppressed forskolin-stimulated neurite outgrowth in NB-OK1 cells although forskolin-stimulated cAMP levels were enhanced. The dose-response curve for this suppression was very similar to that for stimulation of inositol monophosphate (IP1) formation and for stimulation of the initial rise of [Ca2+]i elicited by carbachol. Carbachol-mediated changes in neurite outgrowth, IP1 formation and [Ca2+]i displayed high sensitivity for pirenzepine but low sensitivity for AF-DX116. Inhibition of intracellular calcium release with TMB-8 prevented the suppressive effect of carbachol on forskolin-stimulated neurite outgrowth. Hence we describe for the first time a relationship between neurite outgrowth and inositol triphosphate-triggered calcium release mediated by carbachol in the human neuron-derived cell line.

Carbachol enhances forskolin-stimulated cyclic AMP accumulation via activation of calmodulin system in human neuroblastoma SH-SY5Y cells

We have investigated the modulatory action of carbachol on intracellular cAMP levels in human neuroblastoma SH-SY5Y cells. Carbachol enhanced forskolin-stimulated cAMP levels in a dose-dependent manner (EC 50=3 μM). The enhancing effect of carbachol was completely inhibited by pirenzepine and atropine. Pertussis toxin treatment of the cells partially affected the ability of carbachol. Furthermore, carbachol also enhanced the effect of vasoactive intestinal peptide (EC 50=3 μM)-, adenosine- and prostaglandin E 1-stimulated cAMP levels. The enhancing response of carbachol was sensitive to trifluoperazine but insensitive to calphostin C. These results suggest that the mechanism for carbachol-induced cAMP levels may act, at least in part, through the activation of calmodulin system in SH-SY5Y cells. Hence we describe for the first time a synergistic interaction between calmodulin- and cAMP-dependent signal transduction pathway mediated by carbachol in neuron-derived cell line.

Activity of herpes simplex virus type 1 latency-associated transcript (LAT) promoter in neuron-derived cells: evidence for neuron specificity and for a large LAT transcript

By using chloramphenicol acetyltransferase (CAT) assays in neuron-derived cell lines, we show here that promoter activity associated with the herpes simplex virus type 1 latency-associated transcript (LAT) had neuronal specificity. Promoter activity in these transient CAT assays coincided with a DNA region containing excellent RNA polymerase II promoter consensus sequences. Primer extension analysis in a LAT promoter-CAT plasmid construct placed the start of transcription about 28 nucleotides from the first T in the consensus TATA box sequence. Neuronal specificity of this promoter was suggested by examining the effect of sequences upstream of the promoter on CAT activity in neuronal versus nonneuronal cells. In nonneuronal cells, promoter activity was decreased 3- to 12-fold with the addition of upstream sequences. In contrast, in neuron-derived cells, the addition of upstream sequences did not decrease promoter activity. The LAT promoter predicted by our transient CAT assays was located over 660 nucleotides upstream from the 5' end of the previously mapped 2-kilobase (kb) LAT. This unusual location was explained by in situ and Northern (RNA) blot hybridization analyses that suggested that LAT transcription began near the promoter detected in our CAT assays, rather than near the 5' end of the 2-kb LAT. In situ hybridization with neurons from latently infected rabbits detected small amounts of LAT RNA within 30 nucleotides of the consensus TATA box sequence. This suggested that LAT transcription began near this TATA box. Northern blot hybridization of RNA from ganglia of latently infected rabbits revealed a faint 8.3-kb band of the same sense as LAT. We conclude that (i) the LAT promoter has neuronal specificity, (ii) the LAT promoter is located over 660 nucleotides upstream of the 5' end of the previously characterized stable 2-kb LAT, (iii) LAT transcription begins about 28 nucleotides from the first T of the consensus TATA box sequence and extends to near the first available polyadenylation site approximately 8.3 kb away, and (iv) this 8.3-kb RNA may be an unstable precursor of the more stable 2- and 1.3-kb LATs.

Complementary DNAs for choline acetyltransferase from spinal cords of rat and mouse: nucleotide sequences, expression in mammalian cells, and in situ hybridization

Complementary DNA clones containing the entire coding region of choline acetyltransferase (ChAT) were isolated from the spinal cords of rat and mouse. The cDNAs of rat and mouse coded for 640 and 641 amino acids, respectively, and showed 95% mutual homology and 80% homology with the cDNA of porcine ChAT. Northern blot analysis revealed a single band of 4.4 kb in the spinal cord and brain in each species. Introduction of the cDNAs into Chinese hamster ovary cells and neuron-derived cell lines, N1E115 and NG108-15, expressed a high ChAT activity, which was inhibited by a specific ChAT inhibitor. In situ hybridization using the rat cRNA probe revealed specific labeling of the motoneurons in the spinal cord and neurons in various forebrain nuclei of the rat where the existence of cholinergic neurons has been demonstrated immunohistochemically.

The human medulloblastoma cell line TE671 expresses a muscle-like acetylcholine receptor Cloning of the α-subunit cDNA

Nicotinic acetylcholine receptors (AChRs) from muscle bind α-bungarotoxin (αBgt) and are composed of four kinds of subunits, whereas AChRs from mammalian brains do not bind αBgt and are composed of two kinds of subunits. αBgt-binding proteins whose function is unknown are also found in brain. All these proteins belong to the same gene family. The human medulloblastoma cell line TE671 expresses a functional AChR which binds αBgt. Surprisingly, the AChR of this neuron-derived cell line has electrophysiological, immunological and biochemical properties different from neuronal AChRs and very similar to muscle AChRs. The TE671 AChR binds αBgt, but is different from αBgt-binding proteins in brain. Here we show that TE671 expresses the α-subunit mRNA coding for the muscle AChR, thereby proving that TE671 expresses a muscle-type AChR that is not expressed in adult brain. The isolated cDNA clones should prove useful for expression of large amounts of human muscle-type AChR α-subunit protein for studies of the autoimmune response to muscle AChRs in human myasthenia gravis.