ND7 Cells Research Articles

Regulation of T-type Ca2+ channel expression by herpes simplex virus-1 infection in sensory-like ND7 cells.

Infection of sensory neurons by herpes simplex virus (HSV)-1 disrupts electrical excitability, altering pain sensory transmission. Because of their low threshold for activation, functional expression of T-type Ca2+ channels regulates various cell functions, including neuronal excitability and neuronal communication. In this study, we have tested the effect of HSV-1 infection on the functional expression of T-type Ca2+ channels in differentiated ND7-23 sensory-like neurons. Voltage-gated Ca2+ currents were measured using whole cell patch clamp recordings in differentiated ND7-23 neurons under various culture conditions. Differentiation of ND7-23 cells evokes a significant increase in T-type Ca2+ current densities. Increased T-type Ca2+ channel expression promotes the morphological differentiation of ND7-23 cells and triggers a rebound depolarization. HSV-1 infection of differentiated ND7-23 cells causes a significant loss of T-type Ca2+ channels from the membrane. HSV-1 evoked reduction in the functional expression of T-type Ca2+ channels is mediated by several factors, including decreased expression of Cav3.2 T-type Ca2+ channel subunits and disruption of endocytic transport. Decreased functional expression of T-type Ca2+ channels by HSV-1 infection requires protein synthesis and viral replication, but occurs independently of Egr-1 expression. These findings suggest that infection of neuron-like cells by HSV-1 causes a significant disruption in the expression of T-type Ca2+ channels, which can results in morphological and functional changes in electrical excitability.

Anticancer drug oxaliplatin induces acute cooling-aggravated neuropathy via sodium channel subtype Na(V)1.6-resurgent and persistent current.

Infusion of the chemotherapeutic agent oxaliplatin leads to an acute and a chronic form of peripheral neuropathy. Acute oxaliplatin neuropathy is characterized by sensory paresthesias and muscle cramps that are notably exacerbated by cooling. Painful dysesthesias are rarely reported for acute oxaliplatin neuropathy, whereas a common symptom of chronic oxaliplatin neuropathy is pain. Here we examine the role of the sodium channel isoform Na(V)1.6 in mediating the symptoms of acute oxaliplatin neuropathy. Compound and single-action potential recordings from human and mouse peripheral axons showed that cooling in the presence of oxaliplatin (30-100 μM; 90 min) induced bursts of action potentials in myelinated A, but not unmyelinated C-fibers. Whole-cell patch-clamp recordings from dissociated dorsal root ganglion (DRG) neurons revealed enhanced tetrodotoxin-sensitive resurgent and persistent current amplitudes in large, but not small, diameter DRG neurons when cooled (22 °C) in the presence of oxaliplatin. In DRG neurons and peripheral myelinated axons from Scn8a(med/med) mice, which lack functional Na(V)1.6, no effect of oxaliplatin and cooling was observed. Oxaliplatin significantly slows the rate of fast inactivation at negative potentials in heterologously expressed mNa(V)1.6r in ND7 cells, an effect consistent with prolonged Na(V) open times and increased resurgent and persistent current in native DRG neurons. This finding suggests that Na(V)1.6 plays a central role in mediating acute cooling-exacerbated symptoms following oxaliplatin, and that enhanced resurgent and persistent sodium currents may provide a general mechanistic basis for cold-aggravated symptoms of neuropathy.

Epigenetic regulation of sensory neurogenesis in the dorsal root ganglion cell line ND7 by folic acid

The epigenetic mechanism of folic acid (FA) action on dorsal root ganglion (DRG) cell proliferation and sensory neuron differentiation is not well understood. In this study, the ND7 cell line, derived from DRG cells, was used to elucidate this mechanism. In ND7 cells differentiated with dbcAMP and NGF, Hes1 and Pax3 levels decreased, whereas Neurog2 levels showed a modest increase. Chromatin immunoprecipitation (ChIP) assays examining epigenetic marks at the Hes1 promoter showed that FA favored increased H3K9 and H3K19 acetylation and decreased H3K27 methylation. Hence, FA plays a positive role in cell proliferation. In differentiated ND7 cells, H3K27 methylation decreased, whereas H3K9 and H3K18 acetylation increased at the Neurog2 promoter. FA did not favor this phenotypic outcome. Additionally, in differentiated ND7 Neurog2 associated with the NeuroD1 promoter, FA decreased this association. The results suggest that the switch from proliferation to sensory neuron differentiation in DRG cells is regulated by alterations in epigenetic marks, H3K9/18 acetylation and H3K27 methylation, at Hes1 and Neurog2 promoters, as well as by Neurog2 association with NeuroD1 promoter. FA although positive for proliferation, does not appear to play a role in differentiation.

Folic Acid Remodels Chromatin on Hes1 and Neurog2 Promoters during Caudal Neural Tube Development

The mechanism(s) behind folate rescue of neural tube closure are not well understood. In this study we show that maternal intake of folate prior to conception reverses the proliferation potential of neural crest stem cells in homozygous Splotch embryos (Sp(-/-)) via epigenetic mechanisms. It is also shown that the pattern of differentiation seen in these cells is similar to wild-type (WT). Cells from open caudal neural tubes of Sp(-/-) embryos exhibit increased H3K27 methylation and decreased expression of KDM6B possibly due to up-regulation of KDM6B targeting micro-RNAs such as miR-138, miR-148a, miR-185, and miR-339-5p. In our model, folate reversed these epigenetic marks in folate-rescued Sp(-/-) embryos. Using tissue from caudal neural tubes of murine embryos we also examined H3K27me2 and KDM6B association with Hes1 and Neurog2 promoters at embryonic day E10.5, the proliferative stage, and E12.5, when neural differentiation begins. In Sp(-/-) embryos compared with WT, levels of H3K27me2 associated with the Hes1 promoter were increased at E10.5, and levels associated with the Neurog2 promoter were increased at E12.5. KDM6B association with Hes1 and Neurog2 promoters was inversely related to H3K27me2 levels. These epigenetic changes were reversed in folate-rescued Sp(-/-) embryos. Thus, one of the mechanisms by which folate may rescue the Sp(-/-) phenotype is by increasing the expression of KDM6B, which in turn decreases H3K27 methylation marks on Hes1 and Neurog2 promoters thereby affecting gene transcription.

Cell-specific regulation of the pro-survival Brn-3b transcription factor by microRNAs

We have previously shown that the Brn-3b transcription factor is subjected to post-transcriptional gene regulation by specific microRNAs (mir-23 and mir-214) in the ND7 and SHSY-5Y neuronal cell lines (Calissano et al., 2007). As Brn-3b plays an essential role in the survival of retinal ganglion cells in the rat (Erkman et al., 1996; Gan et al., 1996; Gan et al., 1999; Erkman et al., 2000), we wanted to investigate whether mir-23 and mir-214 are expressed and target Brn-3b mRNA in a retinal ganglion cell line (RGC-5) thus potentially killing the cells expressing it.Here we show that, possibly due to its pro-survival role, Brn-3b is protected from degradation by microRNAs in RGC-5 cells in contrast to its fate in other cell types. This seems to be accomplished by i) the lack of expression of one of the two microRNAs targeting its 3′UTR and by ii) the requirement of at least two distinct microRNAs to mediate its down-regulation in retinal ganglion cells.We speculate that this mechanism could have a widespread role in the regulation of mRNAs encoding for essential proteins.

Interactions between the L1 cell adhesion molecule and ezrin support traction‐force generation and can be regulated by tyrosine phosphorylation

An Ig superfamily cell-adhesion molecule, L1, forms an adhesion complex at the cell membrane containing both signaling molecules and cytoskeletal proteins. This complex mediates the transduction of extracellular signals and generates actin-mediated traction forces, both of which support axon outgrowth. The L1 cytoplasmic region binds ezrin, an adapter protein that interacts with the actin cytoskeleton. In this study, we analyzed L1-ezrin interactions in detail, assessed their role in generating traction forces by L1, and identified potential regulatory mechanisms controlling ezrin-L1 interactions. The FERM domain of ezrin binds to the juxtamembrane region of L1, demonstrated by yeast two-hybrid interaction traps and protein binding analyses in vitro. A lysine-to-leucine substitution in this domain of L1 (K1147L) shows reduced binding to the ezrin FERM domain. Additionally, in ND7 cells, the K1147L mutation inhibits retrograde movement of L1 on the cell surface that has been linked to the generation of the traction forces necessary for axon growth. A membrane-permeable peptide consisting of the juxtamembrane region of L1 that can disrupt endogenous L1-ezrin interactions inhibits neurite extension of cerebellar cells on L1 substrates. Moreover, the L1-ezrin interactions can be modulated by tyrosine phosphorylation of the L1 cytoplasmic region, namely, Y1151, possibly through Src-family kinases. Replacement of this tyrosine together with Y1176 with either aspartate or phenylalanine changes ezrin binding and alters colocalization with ezrin in ND7 cells. Collectively, these data suggest that L1-ezrin interactions mediated by the L1 juxtamembrane region are involved in traction-force generation and can be regulated by the phosphorylation of L1.

Post-transcriptional regulation of the Brn-3b transcription factor in differentiating neuroblastoma cells

The post-transcriptional control of mRNA levels is a very powerful mechanism which allows cells to quickly change the amount of specific proteins. In this study, we wanted to analyze whether the Brn-3b transcription factor, essential for the proper development of mouse retinal ganglion cells, is subjected to such post-transcriptional regulation. In particular, due to its conservation amongst different species, we wanted to study the role of its 3′ untranslated region (3′UTR). We show that the 3′UTR of the Brn-3b mRNA does indeed contain regulatory sequences that mediate mRNA degradation upon serum starvation-induced differentiation of ND7 neuroblastoma cells. The specific region mediating this effect has been characterized and two different microRNAs that potentially regulate the stability of Brn-3b have been identified. Moreover we show that Dicer, one of the key enzymes in the production of microRNAs, is strongly up-regulated in ND7 cells subjected to differentiation.

Cloning of a novel human caspase-9 splice variant containing only the CARD domain

Caspase-9 plays a key role in the intrinsic apoptotic pathway and currently two splice variants (caspase-9-α and -β) have been identified. The present study cloned and characterized a novel caspase-9 splice variant, hereby designated Casp9-γ. Casp9-γ is generated from an additional alternative 3′ splice site in the fourth exon of caspase-9, resulting in a 58-nucleotide fragment insertion compared with the full-length caspase-9-α. The fragment introduces an in-frame stop codon, and the resulting open reading frame (ORF) is preterminated. The Casp9-γ comprises the deduced 154 amino acid residues containing only the caspase recruitment domain (CARD) and does not contain the large and small subunits. The Casp9-γ does not promote apoptosis when overexpressed in mammalian cells. Moreover, it inhibits the cleavage of procaspase-3 mediated by proapoptotic member Bax or apoptosis inductor staurosporine. Therefore, Casp9-γ may function as an endogenous apoptotic inhibitor by interfering with the CARD–CARD interaction between Apaf-1 (apoptotic protease activating factor-1) and procaspase-9. In addition, Casp9-γ does not enhance NF-κB activation in transfected 293T cells, conflicting with previous evidence that the isolated CARD of caspase-9 activates NF-κB in ND7 cells. This suggests that the procaspase-9-mediated NF-κB activation in response to cellular stresses is cell type-specific through an unidentified mechanism.

Brn-3a Transcription Factor Blocks p53-mediated Activation of Proapoptotic Target Genes Noxa and Bax in Vitro and in Vivo to Determine Cell Fate

The Brn-3a POU transcription factor is associated with survival and the differentiation of sensory neuronal cells during development. Brn-3a mediates its effects either by the direct regulation of target genes or indirectly upon interaction with proteins such as p53. Brn-3a differentially regulates p53-mediated gene expression and modifies its effect on cell fate. Here we show that, like Bax, Brn-3a antagonizes p53-mediated transcription of another proapoptotic target, Noxa, significantly reducing transactivation of the Noxa promoter by p53. This effect requires the p53 binding site, and electrophoretic mobility shift assay studies suggest that Brn-3a is associated with p53 when it is bound to its site in the Noxa promoter. The wild type but not the mutant promoter can be immunoprecipitated with Brn-3a in chromatin immunoprecipitation assays. Thus, Brn-3a may act by preventing the recruitment of cofactors required for p53 to transactivate this promoter. The co-expression of Brn-3a and p53 results in decreased endogenous Noxa protein in the neuronal cell line, ND7, suggesting a direct functional effect of this interaction. Moreover, there is a significant elevation of both proapoptotic Bax and Noxa proteins in sensory neuronal tissue taken from Brn-3a-/- embryos during development, compared with wild type controls. Striking changes occurred at embryonic day 14.5, a time that precedes a significant loss of specific neurons in the mutant embryos, but not at embryonic day 16.5 when Brn-3a-expressing cells are already lost by apoptosis. Therefore, the lack of antagonism by Brn-3a on activation of proapoptotic p53 target genes may contribute to the increased apoptosis seen in the Brn-3a-/- embryos. These results support a crucial role for Brn-3a in determining the pathway taken by p53 when co-expressed during development and thus in controlling the fate of these cells.

Varicella-zoster virus IE63 protein represses the basal transcription machinery by disorganizing the pre-initiation complex

Using transient transfection assays, regulation properties of varicella-zoster virus (VZV)-encoded IE63 protein were analyzed on several VZV immediate early (ORF4), early (ORF28) and late (ORF67) promoters. IE63 was shown to repress the basal activity of most of the promoters tested in epithelial (Vero) and neuronal (ND7) cells to various extents. Trans-repressing activities were also observed on heterologous viral and cellular promoters. Since a construct carrying only a TATA box sequence and a series of wild-type or mutated interleukin (IL)-8 promoters was also repressed by IE63, the role of upstream regulatory elements was ruled out. Importantly, the basal activity of a TATA-less promoter was not affected by IE63. Using a series of IE63 deletion constructs, amino acids 151-213 were shown to be essential to the trans-repressing activity in Vero cells, while in ND7 cells the essential region extended to a much larger carboxy-terminal part of the protein. We also demonstrate that IE63 is capable of disrupting the transcriptional pre-initiation complex and of interacting with several general transcription factors. The central and carboxy-terminal domains of IE63 are important for these effects. Altogether, these results demonstrate that IE63 protein is a transcriptional repressor whose activity is directed towards general transcription factors.

Mitochondrial Injury and Caspase Activation by the Local Anesthetic Lidocaine

Lidocaine, a local anesthetic, can be neurotoxic. However, the cellular mechanisms of its neurotoxicity at concentrations encountered during spinal anesthesia remain unclear. The authors examined the mechanisms of lidocaine neurotoxicity in the ND7 cell line derived from rat dorsal root ganglion. Individual neurons were assayed by flow cytometry or microscopy using fluorescent probes of plasma membrane integrity, mitochondrial membrane potential, caspase activity, phospholipid membrane asymmetry, and mitochondrial cytochrome c release. In the ND7 cell line, lidocaine at 185 mm x 10 min to 2.3 mm x 24 h caused necrosis or late apoptosis. Equimolar Tris buffer and equipotent tetrodotoxin controls were not toxic, indicating that neither osmotic nor Na-blocking effects explain lidocaine neurotoxicity. The earliest manifestation of lidocaine neurotoxicity was complete loss of mitochondrial membrane potential within 5 min after exposure to lidocaine at a concentration of 19 mm or greater. Consistent with these data, 37 mm lidocaine (1%) induced release of mitochondrial cytochrome c into the cytoplasm, as well as plasma membrane blebbing, loss of phosphatidylserine membrane asymmetry, and caspase activation, with release of mitochondrial cytochrome c to the cytoplasm within 2 h. Treatment with z-VAD-fmk, a specific inhibitor of caspases, prevented caspase activation and delayed but did not prevent neuronal death, but did not inhibit the other indicators of apoptosis. Collectively, these data indicate that lidocaine neurotoxicity involves mitochondrial dysfunction with activation of apoptotic pathways.

Neuroprotective effects of copper/zinc-dependent superoxide dismutase against a wide variety of death-inducing stimuli and proapoptotic effect of familial amyotrophic lateral sclerosis mutations

Superoxide dismutase plays a key role in cell protection against the damaging effects of superoxide. Mutations in the copper/zinc dependent intracellular form of superoxide dismutase (SOD1) are associated with a subset of cases of familial amyotrophic lateral sclerosis (FALS). In this study we have investigated the effects of over-expressing wild-type SOD1 and two mutant forms of SOD1 found in FALS, G93A and G93R, on cell survival using stably transfected neuronal cells. G93R is associated with early age of onset and severely reduced erythrocyte SOD1 enzyme activity. Overexpression of wild-type SOD1 in ND7 cells significantly enhanced cell survival and reduced apoptosis after serum deprivation. Conversely, cells expressing the G93R mutation of SOD1 exhibited significantly increased cell death and increased number of TUNEL-positive cells, having a more profound effect than G93A SOD1 expressing cells, thus reflecting the relative clinical severity of these mutations. The effects of three further apoptotic and nonapoptotic death-inducing paradigms were investigated, hypoxia with reperfusion, staurosporine and γ-interferon induced cell death. With each paradigm, cell death was significantly reduced by overexpression of wild-type SOD1 and increased by overexpression of the SOD1 mutations G93A and G93R. We further used these SOD1 constructs to develop a virus expressing either wild type SOD1 or the SOD1 mutant G93R and found a similar protective effect against serum withdrawal following infection with an HSV vector expressing wild-type SOD1 which offers a potential tool for neuroprotective gene delivery in vivo.

Effect of local anesthetic on neuronal cytoplasmic calcium and plasma membrane lysis (necrosis) in a cell culture model.

To investigate the mechanism by which rare cases of spinal local anesthetic (LA) neurotoxicity occur, we have tested the hypotheses that LAs elevate cytoplasmic calcium (Ca2+(cyt)), that this is associated with a neurotoxic effect, and that lidocaine and bupivacaine differ in their neurotoxicity. Neurons of the ND7 cell culture line, derived from dorsal root ganglion, were loaded with fura-2 and analyzed by digitized video fluorescence microscopy during 60 min LA exposure, allowing determination of Ca2+(cyt) and time of necrotic cell death (plasma membrane lysis) at the single neuron level. Lidocaine 0.1% and bupivacaine 0.025% caused minimal changes in Ca. Lidocaine 0.5-5% and bupivacaine 0.125-0.625% caused an early, small (less than threefold), concentration-dependent increase in Ca2+(cyt) that was transient and returned to near baseline within 10 min. Lidocaine 2.5% and 5% then caused a sustained, greater than ten-fold increase in Ca2+(cyt) and death in some neurons during the 60 min exposure period. Pretreatment with thapsigargin eliminated the initial transient increase in Ca2+(cyt), consistent with endoplasmic reticulum (ER) as its source, and increased neuronal death with 5% lidocaine, suggesting that lidocaine neurotoxicity can be increased by failure of ER to take up elevated Ca2+(cyt). The later sustained increase in Ca2+(cyt) seen with 2.5 and 5% lidocaine was prevented in Ca2+ -free medium, and restored when Ca2+ was added back to the buffer in the presence of lidocaine, suggesting that higher concentrations of lidocaine increase influx of Ca2+ through the plasma membrane. In this model, lidocaine greater than 2.5% elevates Ca2+(cyt) to toxic levels. Bupivacaine and lower concentrations of lidocaine transiently alter Ca2+(cyt) homeostasis for several minutes, but without an immediate neurotoxic effect within 60 min.

Functional interaction between Brn-3a and Src-1 co-activates Brn-3a-mediated transactivation

The Brn-3a POU domain transcription factor is able to regulate the transcription of promoters containing a Brn-3 response element via its POU domain. In addition, the POU domain of Brn-3a has been shown to functionally interact with the estrogen receptor and regulate transcription from estrogen responsive promoters. The steroid receptor coactivator, Src-1, enhances transcription with a variety of steroid receptors. Here we describe a functional interaction between Brn-3a and Src-1. In glutathione S-transferase pull-down assays Src-1 was shown to specifically interact with Brn-3 proteins. Moreover, Src-1 co-immunoprecipitated from intact cells with Brn-3a. The transactivation potential of the Brn-3a/Src-1 complex was tested on both the Brn-3 responsive SNAP-25 promoter and the estrogen responsive vitellogenin promoter, in each of two different cell lines, the neuronal ND7 cell line, and the kidney BHK21 cell line. Src-1 consistently and strongly potentiated the activation of Brn-3a on the SNAP promoter construct in both the ND7 and BHK21 cell lines. The vitellogenin promoter construct, however, was only weakly activated by the Brn-3/Src-1 complex in the ND7 cells and there was even less effect on this promoter in the BHK21 cells. These results suggest a functional role for Src-1 in enhancing Brn-3a mediated transactivation, seemingly independent of nuclear hormone receptors, thus broadening the transcriptional repertoire of both Brn-3a and Src-1.

Induction of antisense Pax-3 expression leads to the rapid morphological differentiation of neuronal cells and an altered response to the mitogenic growth factor bFGF.

Mutations within the Pax-3 gene lead to a range of developmental abnormalities in both humans and mice. In this report, we have investigated the role that Pax-3 plays in neuronal cell development by specifically downregulating Pax-3 expression within a neuronal cell line. This was achieved by stably transfecting the neuronal cell line ND7 with an expression vector in which antisense Pax-3 RNA was produced under the control of the inducible MMTV promoter. In the stable transfectants, we found that the addition of dexamethasone led to the induction of antisense Pax-3 RNA and a rapid downregulation in endogenous Pax-3 protein expression. The decrease in endogenous Pax-3 protein expression corresponded with a dramatic change in the morphology of the cell: the normally rounded ND7 cells exhibited increased cell to substrate adhesion, extended long neurite processes and expressed genes such as snap-25 that are characteristic of a mature neuron. The morphological differentiation induced by a reduction in Pax-3 expression was followed 24-48 hours later by a cessation in cell proliferation. Interestingly the morphological differentiation and cessation in cell proliferation inducted in the cell lines lacking Pax-3 could be reversed by the addition of the mitogenic growth factor EGF but not by bFGF, whose receptor was downregulated in these cells. These results suggest that the expression of Pax-3 is essential to maintain the undifferentiated phenotype of these immature neuronal cells, and in its absence the cells acquire many of the characteristics of a mature neuronal cell. The slow onset of cell cycle arrest in the cells lacking Pax-3 argues against this transcription factor playing a direct role in the regulation of neuronal cell proliferation.

Nerve Growth Factor Up-regulates the Transcriptional Activity of CBP through Activation of the p42/p44MAPK Cascade

Cyclic AMP response element-binding protein-binding protein (CBP) functions as a transcriptional coactivator through interactions with a number of cellular and viral transcription factors. It has been suggested to play a central integrative role in gene regulation. However, little is known about signal cascades that can regulate CBP activity. Here we show that either nerve growth factor (NGF) or cAMP treatment led to enhanced activity of CBP in PC12 cells. The C-terminal glutamine-rich activation domain of CBP was shown to be responsible for induction by NGF and cAMP. NGF-induced enhancement of CBP activity was also observed in protein kinase A (PKA)-deficient PC12 cells, whereas cAMP failed to increase the transcriptional activity of CBP in these cells. Moreover, the specific PKA inhibitor H-89 blocked cAMP-induced but not NGF-induced up-regulation of CBP activity. The up-regulation of CBP transcriptional activity in response to NGF was, however, prevented by the specific inhibitor of mitogen-activated protein kinase (p42/44(MAPK)) activation, PD98059, which had no effect on the up-regulation induced by cyclic AMP, indicating that activation of the mitogen-activated protein kinase signal pathway is specifically involved in the NGF-induced activation of CBP. In addition, expression of a dominant-negative interfering mutant of p42/44(MAPK) can prevent the NGF-mediated induction of the CBP activity, whereas expression of a p42/44(MAPK) constitutively active mutant can enhance the transcriptional activity of CBP. These data indicate that activation of the p42/p44(MAPK) cascade mediates the up-regulation of the transcriptional activity of CBP by NGF, whereas the similar up-regulation induced by cyclic AMP is mediated by PKA activation.

Bcl-2 Transcription from the Proximal P2 Promoter Is Activated in Neuronal Cells by the Brn-3a POU Family Transcription Factor

The BCL-2 protein is able to protect neuronal and other cell types from apoptotic programmed cell death and plays a key role in regulating the rate of apoptosis during development of the nervous system. We have previously demonstrated that the Brn-3a POU domain transcription factor protects sensory neurons from apoptotic programmed cell death induced by nerve growth factor withdrawal. We report here that Bcl-2 transcription is predominantly initiated from the Bcl-2 P2 promoter in both the ND7 neuronal cell line and primary dorsal root ganglion neurons, in contrast to the predominant use of the Bcl-2 P1 promoter in other cell types. Moreover, Bcl-2 transcription initiated from the P2 region increases in ND7 cells stably overexpressing Brn-3a, resulting in enhanced BCL-2 protein levels. Similarly, the Bcl-2 P2 promoter is directly activated by Brn-3a in co-transfection assays in both ND7 cells and dorsal root ganglion neurons. Analysis of the Bcl-2 regulatory sequence revealed a binding site for Brn-3a that is required for maximal activation by Brn-3a both in transfected cells and during differentiation of ND7 cells. Together these data identify Brn-3a as the first transcription factor regulating Bcl-2 activity specifically in neuronal cells and indicate that the anti-apoptotic effect of Brn-3a is likely to be mediated, at least in part, via the up-regulation of Bcl-2 expression.

Coordinate Induction of the Three Neurofilament Genes by the Brn-3a Transcription Factor

The POU domain transcription factor Brn-3a is able to stimulate neurite outgrowth when overexpressed in the neuronal ND7 cell line, whereas the closely related Brn-3b factor does not have this effect. We show that Brn-3a overexpression also enhances the expression of the three neurofilament genes at both the mRNA and protein levels, whereas Brn-3b overexpression has no effect. In addition Brn-3a activates the three neurofilament gene promoters in co-transfection assays in both neuronal and non-neuronal cells. As observed for enhanced neurite outgrowth, the stimulation of neurofilament gene expression and activation of the neurofilament gene promoters is observed with the isolated POU domain of Brn-3a. A single amino acid change in the POU homeodomain of Brn-3a to the equivalent amino acid in Brn-3b abolishes its ability to activate the neurofilament promoters, whereas the reciprocal change converts Brn-3b to an activator of these promoters.

The Brn-3a transcription factor induces neuronal process outgrowth and the coordinate expression of genes encoding synaptic proteins.

The Brn-3a POU family transcription factor is expressed only in posmitotic neurons in the central nervous system and identifies the first differentiated neurons to appear in the midbrain, hindbrain, and spinal cord during development. This factor is also induced when undifferentiated proliferating ND7 cells cease dividing and differentiate to a mature neuronal-like phenotype bearing numerous neurite processes. We show that overexpression of Brn-3a in undifferentiated ND7 cells induces a mature neuronal phenotype characterized by process outgrowth and the induction of genes encoding synaptic proteins, although the cells continue to proliferate. In contrast, the closely related factors Brn-3b and Brn-3c do not have this effect. Although the N-terminal activation domain of Brn-3a is required for maximum induction of neurite outgrowth and gene expression, these effects are primarily dependent on the DNA binding POU domain, which also acts as an activation domain. Overexpression of the isolated POU domain of Brn-3a is sufficient to induce neurite outgrowth, while the ability of full-length Brn-3a to do so is abolished by mutating a single amino acid in the Brn-3a POU homeodomain to its equivalent in Brn-3b. Thus, Brn-3a appears to play a critical role in the specification of the mature neuronal phenotype, acting by stimulating the expression of genes whose products are required for process outgrowth and synapse formation.

Immediate upstream promoter regions required for neurospecific expression of SNAP-25.

The promoter structure and regulation of Snap, a gene encoding the presynaptic t-SNARE SNAP-25 implicated in synaptic vesicle docking and fusion, was studied. Transcription start-site analysis revealed two major start sites located 42 nucleotides apart. Nucleotide sequence of a promoter region 2073 nucleotides upstream of the first transcription site contains three AP-1, one CRE sequence, and three Sp1-like sites close to the TATA box. Further upstream of these sites two TG repeats were found. The ability of regions within the 5' upstream sequence to promote basal neural-specific expression in tissue culture cells was evaluated using a series of constructs containing both Snap gene start sites with progressively restricted 5' sequence linked to the chloramphenicol acetyl transferase (CAT) reporter gene. CAT expression was maximal in neuron-like undifferentiated ND7 and PC12 cells transfected with constructs containing Snap sequences up to 127 bp from the start site. In contrast, nonneuronal fibroblast cell lines did not express significant amounts of CAT, suggesting that this short 127-bp sequence is sufficient to drive neural specific expression of SNAP-25. Band shift analysis of oligonucleotides spanning from -127 to -41 bp of the Snap promoter revealed three distinct DNA-protein complexes generated by brain nuclear extracts and one by liver nuclear extracts, indicating that transcription factors that bind to this 86-bp sequence located just upstream of the TATA box are involved in regulation of basal neurospecific expression of this gene.