RN46A Cells Research Articles

Antisense oligonucleotides reach mRNA targets via the RNA matrix: downregulation of the 5-HT1A receptor

Successful application of antisense oligonucleotides (ODNs) in cell biology and therapy will depend on the ease of design, efficiency of (intra)cellular delivery, ODN stability, and target specificity. Equally essential is a detailed understanding of the mechanism of antisense action. To address these issues, we employed phosphorothioate ODNs directed against specific regions of the mRNA of the serotonin 5HT1A receptor, governed by sequence and structure. We demonstrate that rather than various intracellular factors, the gene sequence per se primarily determines the antisense effect, since 5HT1a autoreceptors expressed in RN46A cells, postsynaptic receptors expressed in SN48 cells, and receptors overexpressed in LLP-K1 cells are all efficiently downregulated following ODN delivery via a cationic lipid delivery system. The data also reveal that the delivery system as such is a relevant parameter in ODN delivery. Antisense ODNs bound extensively to the RNA matrix in the cell nuclei, thereby interacting with target mRNA and causing its subsequent degradation. Antisense delivery effectively diminished the mRNA pool, thus resulting in downregulation of newly synthesized 5HT1A proteins, without the appearance of truncated protein fragments. In conjunction with the selected mRNA target sequences of the ODNs, the latter data indicated that effective degradation rather than a steric blockage of the mRNA impedes protein expression. The specificity of the antisense approach, as described in this study, is reflected by the effective functional downregulation of the 5-HT1A receptor.

Characterization of reproductive steroid receptors and response to estrogen in a rat serotonergic cell line

Study of the cellular and molecular consequences of steroid hormone action in the serotonin neural system will provide new avenues for pharmacotherapeutic intervention in mental illness related to reproductive function. However, it is difficult to probe intracellular mechanisms with whole animal models. We sought the steroid receptor compliment and estrogen response of two rat serotonin cell lines in order to determine if they could be of future assistance in this matter. Immunohistochemistry with a panel of antibodies, RT-PCR and a serotonin ELISA were utilized to characterize the RN46A-V1 cells (herein called RN46A), and the subclone RN46A-B14 (herein called B14) that is stably transfected with brain derived neurotrophic factor (BDNF). RN46A and B14 cells express estrogen receptor beta (ERβ), androgen receptors (AR) and nuclear factor kappa B (NFκB) but not estrogen receptor alpha (ERα) or progestin receptors (PR). RT-PCR confirmed the presence of ERβ and the absence of ERα and PR in both cell lines. B14 cells contain more immunodetectable BDNF and serotonin than the RN46A parent line. In addition, immunofluorescence for the serotonin reuptake transporter (SERT) was observed in the cell body region of undifferentiated B14 cells. After differentiation at a nonpermissive temperature, SERT immunostaining was observed in both the cell body region and along the extent of the axons. Serotonin content as determined by ELISA was higher in B14 than RN46A cells. Estrogen (0.1 and 1.0 nM) stimulated serotonin in the B14 cells in serum free medium. In summary, the RN46A cells and the B14 subclone contain the same compliment of nuclear steroid receptors as rat raphe serotonin neurons and thus may provide a convenient in vitro model for study of intracellular mechanisms of action of steroid hormones in the context of a serotonin neuron.

Divergent regulation of GAP-43 expression and CNS neurite outgrowth by cyclic AMP.

Robust process outgrowth and high expression of the growth-associated protein GAP-43 seem to be intrinsic features of neurons, but both are down-regulated after axonal contact of target cells. We report that chronic exposure of the serotonergic CNS cell line RN46A to cyclic AMP analogs, forskolin, or cholera toxin represses GAP-43 expression in a dose dependent manner. Thus, cAMP could mediate a GAP-43 repressive signal that is initiated extracellularly. Activation of the cyclic AMP pathway by these same reagents, however, enhances the rate that RN46A cells extend neurites. This stimulation of neurite growth can occur during inhibition of new transcription, and in the absence of high levels of GAP-43. These findings demonstrate that a GAP-43-repressing intracellular signaling pathway exists, that repression of GAP-43 expression by cAMP is not directly coupled to inhibition of neurite growth, and that acceleration of growth cone advancement by cAMP is not dependent on the presence of GAP-43.

TATA-driven transcriptional initiation and regulation of the rat serotonin 5-HT1A receptor gene.

The transcriptional initiation and regulation of the rat serotonin 5-HT1A receptor gene were characterized. By three types of analyses, a single brain-specific site of transcriptional initiation was localized to -967 bp upstream of the translation initiation codon that is utilized both in hippocampus and in the rat raphe RN46A cell line. This major site of transcriptional initiation was located 58 bp downstream from a consensus TATA element, suggesting TATA-driven transcription of the rat 5-HT1A receptor. To identify the promoter activity of the receptor gene, progressive 5' deletions of the -2,719/-117-bp fragment of the 5-HT1A promoter linked to luciferase gene were transfected into 5-HT1A-negative (pituitary GH4C1, L6 myoblast, and C6 glioma) and 5-HT1A-positive (septal SN-48 and raphe RN46A) cell lines. Enhancer regions were identified within a fragment between nucleotides -426 and -117 that selectively enhanced transcription in 5-HT1A-positive cells. A nonselective enhancer/promoter that mediated expression in all cell lines was located upstream between -1,519 and -426 bp in a DNA segment containing consensus TATA, CCAAT, SP-1, and AP-1 elements as well as a poly-GT26 dinucleotide repeat. Strong repression of transcription in all cell lines was conferred by the region upstream of -1,519 bp that contains a 152-bp DNA segment with >80% identity to RANTES, tumor necrosis factor-beta, and other immune system genes. Our results indicate that TATA-driven expression of the 5-HT1A receptor is regulated by a novel proximal tissue-specific enhancer region, a nonselective promoter, and an upstream repressor region that is distinct from previously identified neuron-specific repressors.

Functional analysis of a novel human serotonin transporter gene promoter in immortalized raphe cells

To investigate the structural basis for genetic regulation of the human serotonin transporter gene, a 1.8 kb fragment upstream to the cap site was cloned and sequenced. The promoter possesses a polymorphic repeat region with 16 and 14 repeats, respectively. Both were cloned and characterized. The promoter sequence revealed an internal 379 bp fragment not reported in previous publications. This novel fragment contains consensus sequences for several transcription factors including SpI and GATA. DNA from 48 unrelated individuals was PCR amplified, in this region, to test for allelic variations. All were found to possess the additional 379 bp fragment. The integrity of the promoter was furthermore confirmed by genomic Southern blotting. The promoter activity was analyzed by reporter gene assays in neuronal and non-neuronal serotonergic cell lines. In immortalized serotonergic raphe neurons, RN46A, three cis-acting, cell specific, activating elements and a silencer were located. One of the activators and the silencer are located in the repeat region and one activator is positioned in the novel fragment. A fourth activating element was found to be active in both RN46A cells and in a non-neuronal serotonergic cell line, JAR. A 3.5 kb fragment from intron 1 was cloned and found to possess cell specific activity in JAR cells indicating the presence of an alternative promoter in intron 1.

Changes in GAD- and GABA- immunoreactivity in the spinal dorsal horn after peripheral nerve injury and promotion of recovery by lumbar transplant of immortalized serotonergic precursors

We have utilized RN46A cells, an immortalized neuronal cell line derived from E13 brainstem raphe, as a model for transplant of bioengineered serotonergic cells. RN46A cells require brain-derived neurotrophic factor (BDNF) for increased survival and serotonin (5HT) synthesis in vitro and in vivo. RN46A cells were transfected with the rat BDNF gene, and the 46A-B14 cell line was subcloned. These cells survive longer than 7 weeks after transplantation into the subarachnoid space of the lumbar spinal cord and synthesize 5HT and BDNF. Chronic constriction injury (CCI) of the sciatic nerve was used to induce chronic neuropathic pain in the affected hindpaw in rats. Transplants of 46A-B14 cells placed 1 week after CCI alleviated chronic neuropathic pain, while transplants of 46A-V1 control cells, negative for 5HT and without the BDNF gene, had no effect on the induction of thermal and tactile nociception. When endogenous cells of the dorsal horn which contain the neurotransmitter γ-aminobutyric acid (GABA) and its synthetic enzyme glutamate decarboxylase (GAD) were immunohistochemically quantified in the lumbar spinal cord 3 days and 1–8 weeks after CCI, the number of GABA- and GAD-immunoreactive (ir) cells decreased bilateral to the nerve injury as soon as 3 days after CCI. At 1 week after CCI, the number of GABA-ir cells continued to significantly decline bilaterally, returning to near normal numbers on the side contralateral to the nerve injury by 8 weeks after the nerve injury. The number of GAD-ir cells began to increase bilaterally to the nerve injury at 1 week after CCI and continued to significantly increase in numbers over normal values by 8 weeks after the nerve injury. When examined 2 and 8 weeks after CCI plus cell transplants, the transplants of 46A-B14 cells reversed the increase in GAD-ir cell numbers and the decrease in GABA-ir cells by 1 week after transplantation, while 46A-V1 control cell transplants after CCI had no effect on the changes in numbers of GAD-ir or GABA-ir cells. Collectively, these data suggest that altered 5HT levels, and perhaps BDNF secretion, related to the transplants ameliorate chronic pain and reverse the induction and maintenance of an endogenous pain mechanism in the dorsal horn. This induction mechanism is likely dependent on altered GAD regulation and GABA synthesis, initiated by CCI.

Amyloid β neurotoxicity in the cholinergic but not in the serotonergic phenotype of RN46A cells

Neuropathological examination of brains from AD patients has documented that distinct areas and nuclei are differently affected by the disease. It is unknown as to what extent the neurochemical phenotype plays a role in this process, but particularly acetylcholine (Ach) neurons in the basal forebrain are lost during the progress of the disease. The exact molecular mechanism by which the neuronal death is induced remains unclear, but the amyloid β peptide (A β) is cytotoxic in vitro and may be important for the neuronal cell death in vivo. Previous reports have demonstrated that an immortalized neuronal cell line (RN46A) derived from rat raphe nucleus differentiate in the presence of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) to a cholinergic and a serotonergic phenotype, respectively [J.S. Rudge et al., Mol. Cell Neurosci. 7 (1996) 204–221]. This study takes advantage of the RN46A cell line to investigate whether the sensitivity to A β is dependent on cell differentiation and neurochemical phenotype. We found that cellular reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) could be inhibited 30–40% by A β in undifferentiated cells. The cholinergic phenotype induced by CNTF remained sensitive to A β whereas the serotonergic phenotype induced by BDNF was unaffected by concentrations of A β up to 10 μM. These findings suggest that differentiation and neurochemical phenotype may play a role for A β induced lesions in Alzheimer's disease.

Lumbar transplants of immortalized serotonergic neurons alleviate chronic neuropathic pain

The RN46A cell line was derived from embryonic day 13 rat medullary raphe cells by infection with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen. This cell line is neuronally restricted and constitutively differentiates following a shift to non-permissive temperature. Brain-derived neurotrophic factor (BDNF) induced the serotonergic phenotype and increased the survival of RN46A cells in vitro. After transfection of the rat BDNF gene into RN46A cells, an autocrine BDNF-secreting cell line, 46A-B14, was isolated and transplanted into the rat CNS. Transplanted 46A-B14 cells had increased survival and enhanced serotonin (5HT) synthesis compared to 46A-V1 cells, RN46A cells transfected with vector-alone. When 46A-B14 cells were transplanted in the lumbar subarachnoid space of the spinal cord 1 week after a chronic constriction injury (CCI) of the sciatic nerve, they survived longer than 6 weeks on the pia mater. Furthermore, the tactile and cold allodynia and thermal hyperalgesia induced by CCI was significantly reduced during a 4–6- week period. The maximal effect occurred 1 week after transplantation. 46A-V1 cells, transplanted after CCI, did not survive beyond 2–3 weeks and had no effect on the allodynia and hyperalgesia induced by CCI. Acute intrathecal injection of the 5HT receptor antagonist methysergide decreased the antinociceptive effects of the 46A-B14 cells to pre-transplant levels. These data suggest that a chronically applied, low local dose of serotonin near the dorsal horn was able to reverse the development of chronic neuropathic pain following CCI. The use of neural cell lines that are able to deliver inhibitory neurotransmitters such as serotonin, in a model of chronic pain offers a novel approach to pain management.

Transcriptional regulation of neurofilament expression by protein kinase A.

RN46A cells, a conditionally immortalized neuronal cell line derived from E12 rat medullary raphe nucleus, upregulate low M(r) (68 kDa, neurofilament [NF]-L) and medium M(r) (160 kDa, NF-M) neurofilament protein expression upon activation of protein kinase A (PKA). To examine possible transcriptional regulation of neurofilament protein expression by PKA, two cell lines were used; RN46A cells and C alpha EV6 cells, a cell line derived from RN46A cells that stably expresses the catalytic subunit of PKA under the control of the metallothionein promoter. Treatment of RN46A cells with dbcAMP resulted in an increase in the steady-state levels of both NF-L and NF-M, but not high M(r) (200 kDa, NF-H) neurofilament mRNA. These increases were both time and dose dependent and were sensitive to treatment with the protein synthesis inhibitor cycloheximide. In C alpha EV6 cells, activation of PKA by 80 microM ZnSO4 upregulated the expression of C alpha mRNA with maximal levels reached 8 hr post-treatment and maintained at 24 hr. Reporter gene assays in C alpha EV6 cells following transfection with increasing lengths of the NF-L promoter demonstrated that both a putative Sp1-like and a cAMP response (CRE), but not a NGFI-A, element were likely involved in PKA-dependent activation of the NF-L promoter. Electrophoretic mobility shift assays confirmed these results but showed that the nuclear proteins induced by PKA which bound to the NF-L promoter Sp1-like sequence were not Sp1. Collectively, these data suggest that constitutively expressed Sp1 may be involved in basal NF-L promoter activity, and newly synthesized, PKA-dependent nuclear proteins may synergistically activate the rat NF-L promoter.

Autocrine BDNF Secretion Enhances the Survival and Serotonergic Differentiation of Raphe Neuronal Precursor Cells Grafted into the Adult Rat CNS

RN46A cells are a temperature-sensitive neuronal cell line derived from the E13 rat raphe nucleus. RN46A cells grafted into the adult rat hippocampus and cerebral cortex do not survive beyond 2 weeks. Brain-derived neurotrophic factor (BDNF) regulates thein vitrosurvival and serotonergic phenotype of RN46A cells, and we hypothesized that expression of BDNF in RN46A cells would potentiate their survival and serotonin (5HT) expressionin vivo.The gene encoding rat BDNF was transfected into RN46A cells and the clonal 46A-B14 cell line isolated. 46A-B14 cells synthesize and secrete biologically active BDNFin vitroand synthesize 5HT following partial membrane depolarization. Two weeks following 46A-B14 cell transplantation into the adult rat cortex and hippocampus, there is a threefold increase in survival of 46A-B14 cells compared to RN46A cells transfected with the vector alone. The grafted 46A-B14 cells immunohistochemically stain for BDNF and 5HT, while RN46A cells transfected with vector only are negative for both BDNF and 5HT. In addition, 46A-B14 cells attain more morphologically complex phenotypes, indicating enhanced neuronal differentiation. Autocrine secretion of BDNF by RN46A cells thus potentiates survival and can be used to deliver both BDNF and 5HTin vivo.

CNTF Induces Raphe Neuronal Precursors to Switch from a Serotonergic to a Cholinergic Phenotypein Vitro

Ciliary neurotrophic factor (CNTF) is a multifunctional cytokine that mediates survival and differentiation of neurons as well as many other cell types. In this study, CNTF and leukemia inhibitory factor (LIF) reduced the apparent number of primary serotonergic neurons in E14 raphe culture by 90% as determined by immunocytochemistry for serotonin (5HT). The reduction in 5HT cell number was not due to neuronal loss as removal of CNTF after 4 days in culture resulted in a partial restitution of the serotonergic phenotype. In the RN46A serotonergic cell line which is induced to become serotonergic by brain-derived neurotrophic factor (BDNF), the addition of CNTF suppressed tryptophan hydroxylase and 5HT synthesis and increased choline acetyl transferase (ChAT) expression by 6-fold and ChAT activity by 20- to 30-fold over 12 days. As with the primary neurons, removal and replacement of CNTF with BDNF after 4 days resulted in a partial restitution of 5HT expression. Moreover, other members of the CNTF-cytokine family that use gp130 and/or LIF receptor β as their signal transducing receptors—LIF, oncostatin M, interleukin 6, and interleukin 11—had similar effects on increasing ChAT activity and reducing 5HT expression in RN46A cells. Analysis of 5HT levels showed no significant difference in the amount of serotonin between wild-type and CNTFRα knockout mice at birth, suggesting that the potential to switch phenotype mediated through CNTFRα is a latent property of neuroepithelial precursors in the raphe nucleus.

Adrenocorticotropic hormone activation of adenylate cyclase in raphe neurons: Multiple regulatory pathways control serotonergic neuronal differentiation

The RN46A cell line was derived from embryonic day 13 rat medullary raphe cells by infection with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen (tsT-ag). The RN46A cell line is neuronally restricted and constitutively differentiates following a shift to nonpermissive temperature. Differentiated RN46A cells express low levels of tryptophan hydroxylase (TPH) but no detectable levels of serotonin (5-HT). Treatment of cultures with the adrenocorticotropic hormone peptide ACTH4-10 up-regulates the expression of TPH immunoreactivity in differentiated RN46A cells, but 5-HT synthesis requires initial treatment with ACTH4-10, followed by partial membrane depolarizing conditions. Up-regulation of TPH by ACTH4-10 is apparently due to activation of adenylate cyclase, whereas the increased 5-HT synthesis with membrane depolarization can be blocked with the voltage-sensitive Ca(2+)-channel blockers nifedipine and omega-conotoxin. ACTH4-10 treatment also markedly up-regulates the expression of the 5-HT reuptake transporter, as do dibutyryl cyclic AMP and forskolin; chronic membrane depolarization has no effect on 5-HT reuptake. The expression of the high-affinity 5-HT1A receptor is increased threefold by ACTH4-10 treatment during differentiation and fivefold by differentiation under partial membrane depolarizing conditions. Combining ACTH4-10 treatment and membrane depolarization does not increase expression of the 5-HT1A receptor further. 5-HT release is constitutive in ACTH-treated RN46A cells and linked to spontaneous synaptic vesicle fusion in RN46A cells. Considered with previous results, these data indicate that multiple effectors, ACTH, brain-derived neurotrophic factor, and membrane depolarization, have both distinct and overlapping effects that regulate specific elements of the serotonergic neuronal phenotype during differentiation and maturation.

Developmental Regulation of Early Serotonergic Neuronal Differentiation: The Role of Brain-Derived Neurotrophic Factor and Membrane Depolarization

The RN46A cell line was derived from Embryonic Day 13 rat medullary raphe cells by infection with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen. This cell line is neuronally restricted and constitutively differentiates following a shift to nonpermissive temperature. Undifferentiated RN46A cells express low levels of tryptophan hydroxylase (TPH), low-affinity neurotrophin receptor (p75 NTR), and trkB immunoreactivities, but no detectable levels of serotonin (5HT) immunoreactivity. TrkB, p75 NTR, and TPH, but not 5HT, expressions increase with differentiation and treatment with brain-derived neurotrophic factor (BDNF). 5HT synthesis in RN46A cells requires initial treatment with BDNF, followed by growth under partial membrane depolarizing conditions. Embryonic raphe cultures treated similarly with BDNF and partial depolarizing conditions also demonstrate increased 5HT synthesis. The sodium-dependent transporter for 5HT reuptake is present in undifferentiated RN46A cells, and the apparent K m and B max are unchanged by differentiation or BDNF treatment and membrane depolarization. The high-affinity 5HT 1A receptor is present in both undifferentiated and differentiated RN46A cells, and while the K d is unaffected by differentiation or BDNF/membrane depolarization, the B max increases 20-fold after differentiation and 3.5-fold further with BDNF under depolarizing conditions. The expression of the synaptic vesicular monoamine transporter, as determined by the binding of [ 125I]iodovinyltetrabenazine, also increases in RN46A cells with differentiation. However, 5HT release is constitutive and is independent of acute membrane depolarization. Collectively these data indicate that distinct aspects of serotonin metabolism are differentially regulated during development and suggest that 5HT may function as a developmental signal in an autocrine loop during early serotonergic differentiation.

Phenotypic Diversity in Neuronal Cell Lines Derived from Raphe Nucleus by Retroviral Transduction

Two neuronal cell lines, RN33B and RN46A, were derived from E12 rat medullary raphe neuroepithelial cells by infection with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen (T-ag). At permissive temperature (33°C), both cell lines express nuclear T-ag immunoreactivity, low levels of neuron-specific enolase (NSE), and low (NF-L) and medium (NFM), but not high (NF-H), MW neurofilament proteins. Once differentiated, both cell lines cease dividing, take on a neuronal morphology, and express enhanced levels of NSE and all three NF proteins. The complex interaction of variables which mediates the differentiation of these neuronal cell lines is discussed. RN33B and RN46A cells show both similar and different physiological properties, and those features are highlighted to demonstrate the utility of these cell lines in studying neuronal differentiation, both in vitro and in vivo. For example, the expression of NF-L and NF-M in differentiating RN33B and RN46A cells can be potentiated by activation of protein kinase A. RN33B cells are glutamatergic while RN46A cells are serotonergic. 5-HT uptake and synthesis in RN46A cells are differentially regulated, as synthesis is maximal only in differentiated RN46A cells which have been treated with BDNF followed by partial depolarization. 5-HT uptake is seen at similar levels in both undifferentiated and differentiated RN46A cells and is independent of neurotrophin regulation. Following transplantation into the CNS, RN33B cells survive ≥6 months and differentiate according to local environmental signals; RN46A cells survive ≤2 months and do not show similar plasticity in vivo. These data are modeled to provide a conceptual framework as to the generation and differentiation capacity of neuronal cell lines derived by retroviral transduction.

Distinct regulatory pathways control neurofilament expression and neurotransmitter synthesis in immortalized serotonergic neurons

Following infection of dissociated embryonic day 13 rat medullary raphe cells with a retrovirus encoding the temperature-sensitive mutant of SV40 large T-antigen (T-ag), a neuronal cell line, RN46A, was cloned by serial dilution. At 33 degrees C, RN46A cells express nuclear T-ag immunoreactivity and divide with a doubling time of 9 hr. Undifferentiated RN46A cells express low levels of neuron-specific enolase (NSE) and low (NF-L)-and medium (NF-M)- but not high (NF-H)-molecular-weight neurofilament proteins. Under differentiation conditions, RN46A cells cease dividing, take on a neuronal morphology, and express enhanced levels of NSE and all three NF proteins. Elevation of intracellular cAMP levels increases neurofilament protein expression, whereas activators of various other intracellular second messenger systems have no effect. Differentiated RN46A cells express low-affinity nerve growth factor (NGF) receptor (p75NGFR) and are immunoreactive using an antibody that recognizes the carboxy-terminal 13 amino acids of all three trk proteins (pan-trk). Both immunoreactivities could be potentiated by treatment with brain-derived neurotrophic factor (BDNF), NGF, and adrenocorticotropic hormone, fragment 4-10 (ACTH4-10). Differentiated RN46A cells express low levels of tryptophan hydroxylase (TPH) immunoreactivity, which could be enhanced by treatment with ACTH4-10, BDNF, or NGF. Low levels of serotonin immunoreactivity are detected in differentiated RN46A cells, and this was potentiated by differentiating RN46A cells with BDNF for 8 d and 40 mM KCl for days 4-8. HPLC analysis confirmed these immunohistochemical data. RN46A cells should prove useful to elucidate intracellular mechanisms that control neurofilament assembly and 5-HT expression in differentiating raphe neurons.