Nerve growth factor-induced differentiation of PC12 cells is accompanied by elevated adenylyl cyclase activity.

Rat pheochromocytoma (PC12) cells characteristically undergo differentiation when cultured with nerve growth factor (NGF). Here we show that NGF dramatically increased the adenylyl cyclase-activating property of forskolin in PC12 cells. This effect of NGF was well maintained even when NGF was removed after 4 days, even though the morphological features of neuronal differentiation were rapidly lost on removal of NGF. The enhanced cAMP production in response to forskolin could be due to a synergistic interaction between forskolin and endogenously released agonists acting on G(s)-coupled receptors. However, responses to forskolin were not attenuated by antagonists of adenosine A2 receptors or pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, suggesting that adenosine and PACAP were not involved. Adenylyl cyclases 3, 6 and 9 were the predominant isoforms expressed in PC12 cells, but we found no evidence for NGF-induced changes in expression levels of any of the 9 adenylyl cyclase isoforms, nor in the expression of Gα(s). These findings highlight that NGF has a subtle influence on adenylyl cyclase activity in PC12 cells which may influence more than the neurite extension process classically associated with neuronal differentiation.

Transcripts for the acetylcholine receptor and acetylcholine esterase show distribution differences in cultured chick muscle cells.

In situ hybridization of chick cultured muscle cells using exonic DNA probes for both AChR alpha-sub-unit and the catalytic subunit of AChE, revealed major differences in the distribution of label both over nuclei and in their surrounding cytoplasm, although some overlap in these distributions exists. For the AChR alpha-subunit there is a highly skewed distribution of labeled nuclei, with 35% of the nuclei being relatively inactive (less than 0.25 times the mean label) and approximately 10% being very heavily labeled (greater than 2.5 times the mean label). In contrast the nuclei labeled with the exonic probe for the AChE transcripts had a more Gaussian distribution, yet with some slight skewness in the direction of a few heavily labeled nuclei. There was also a difference in the cytoplasmic distribution of the label. The AChR alpha-subunit mRNA was mainly within 4 microns of labeled nuclei while the AChE mRNA was more widely distributed throughout the cytoplasm, possibly within a 10 microns rim around labeled nuclei. An intronic probe for the AChE gave the identical distribution of nuclear label to that of the exonic probe (but without any cytoplasmic label). In addition, calibration of the technique indicated that per myotube the AChE transcript is about sixfold more abundant than the AChR alpha-subunit transcript.

cDNA that encodes active agrin.

Agrin is thought to mediate the motor neuron-induced aggregation of AChRs and AChE on the surface of muscle fibers at neuromuscular junctions. We have isolated a cDNA from a chick brain library that, based on sequence homology and expression experiments, codes for active agrin. Examination of the sequence reveals considerable similarity to homologous cDNAs previously isolated from ray and rat libraries. A conspicuous difference is an insertion of 33 bp in chick agrin cDNA, which endows the encoded protein with AChR/AChE aggregating activity. Homologous transcripts having the 33 bp insertion were detected in the ray CNS, which indicates that an insertion of similar size is conserved in agrin in many, if not all, vertebrate species. Results of in situ hybridization studies and PCR experiments on mRNA isolated from motor neuron-enriched fractions of the spinal cord indicate that, consistent with the agrin hypothesis, motor neurons contain transcripts that code for active agrin.

The agrin gene codes for a family of basal lamina proteins that differ in function and distribution.

We isolated two cDNAs that encode isoforms of agrin, the basal lamina protein that mediates the motor neuron-induced aggregation of acetylcholine receptors on muscle fibers at the neuromuscular junction. Both proteins are the result of alternative splicing of the product of the agrin gene, but unlike agrin, they are inactive in standard acetylcholine receptor aggregation assays. They lack one (agrin-related protein 1) or two (agrin-related protein 2) regions in agrin that are required for its activity. Expression studies provide evidence that both proteins are present in the nervous system and muscle and that, in muscle, myofibers and Schwann cells synthesize the agrin-related proteins while the axon terminals of motor neurons are the sole source of agrin.

Cdk5 is involved in neuregulin-induced AChR expression at the neuromuscular junction.

Here we describe an important involvement of Cdk5/p35 in regulating the gene expression of acetylcholine receptor (AChR) at the neuromuscular synapse. Cdk5 and p35 were prominently expressed in embryonic muscle, and concentrated at the neuromuscular junction in adulthood. Neuregulin increased the p35-associated Cdk5 kinase activity in the membrane fraction of cultured C2C12 myotubes. Co-immunoprecipitation studies revealed the association between Cdk5, p35 and ErbB receptors in muscle and cultured myotubes. Inhibition of Cdk5 activity not only blocked the NRG-induced AChR transcription, but also attenuated ErbB activation in cultured myotubes. In light of our finding that overexpression of p35 alone led to an increase in AChR promoter activity in muscle, Cdk5 activation is sufficient to mediate the up-regulation of AChR gene expression. Taken together, these results reveal the unexpected involvement of Cdk5/p35 in neuregulin signaling at the neuromuscular synapse.

Ginsenoside Re attenuate beta-amyloid and serum-free induced neurotoxicity in PC12 cells.

AIM: To study the effect of ginsenoside Re on PC12 cell damage induced by serum deprivation and beta-amyloid peptide. METHODS: PC 12 cell survival was measured by MTT and lactate dehydrogenase (LDH) assay. Results Serum-free medium and beta-amyloid peptide (10-100 microM) induced cytotoxicity in PC 12 cells. Ginsenoside Re (0.1-100 microM) attenuated the cytotoxic effects of serum-free medium and beta-amyloid peptide (50 microM) in a concentration-dependent manner. CONCLUSION: Ginsenoside Re prevented PC 12 cells from lesion induced by serum-free medium and beta-amyloid peptide.

Anxiety and depression with neurogenesis defects in exchange protein directly activated by cAMP 2-deficient mice are ameliorated by a selective serotonin reuptake inhibitor, Prozac.

Intracellular cAMP and serotonin are important modulators of anxiety and depression. Fluoxetine, a selective serotonin reuptake inhibitor (SSRI) also known as Prozac, is widely used against depression, potentially by activating cAMP response element-binding protein (CREB) and increasing brain-derived neurotrophic factor (BDNF) through protein kinase A (PKA). However, the role of Epac1 and Epac2 (Rap guanine nucleotide exchange factors, RAPGEF3 and RAPGEF4, respectively) as potential downstream targets of SSRI/cAMP in mood regulations is not yet clear. Here, we investigated the phenotypes of Epac1 (Epac1(-/-)) or Epac2 (Epac2(-/-)) knockout mice by comparing them with their wild-type counterparts. Surprisingly, Epac2(-/-) mice exhibited a wide range of mood disorders, including anxiety and depression with learning and memory deficits in contextual and cued fear-conditioning tests without affecting Epac1 expression or PKA activity. Interestingly, rs17746510, one of the three single-nucleotide polymorphisms (SNPs) in RAPGEF4 associated with cognitive decline in Chinese Alzheimer's disease (AD) patients, was significantly correlated with apathy and mood disturbance, whereas no significant association was observed between RAPGEF3 SNPs and the risk of AD or neuropsychiatric inventory scores. To further determine the detailed role of Epac2 in SSRI/serotonin/cAMP-involved mood disorders, we treated Epac2(-/-) mice with a SSRI, Prozac. The alteration in open field behavior and impaired hippocampal cell proliferation in Epac2(-/-) mice were alleviated by Prozac. Taken together, Epac2 gene polymorphism is a putative risk factor for mood disorders in AD patients in part by affecting the hippocampal neurogenesis.

Expression of the P2Y1 nucleotide receptor in chick muscle: its functional role in the regulation of acetylcholinesterase and acetylcholine receptor.

In vertebrate neuromuscular junctions, ATP is stored at the motor nerve terminals and is co-released with acetylcholine during neural stimulation. Here, we provide several lines of evidence that the synaptic ATP can act as a synapse-organizing factor to induce the expression of acetylcholinesterase (AChE) and acetylcholine receptor (AChR) in muscles, mediated by a metabotropic ATP receptor subtype, the P2Y(1) receptor. The activation of the P2Y(1) receptor by adenine nucleotides stimulated the accumulation of inositol phosphates and intracellular Ca(2+) mobilization in cultured chick myotubes. P2Y(1) receptor mRNA in chicken muscle is very abundant before hatching and again increases in the adult. The P2Y(1) receptor protein is shown to be restricted to the neuromuscular junctions and colocalized with AChRs in adult muscle (chicken, Xenopus, and rat) but not in the chick embryo. In chicks after hatching, this P2Y(1) localization develops over approximately 3 weeks. Denervation or crush of the motor nerve (in chicken or rat) caused up to 90% decrease in the muscle P2Y(1) transcript, which was restored on regeneration, whereas the AChR mRNA greatly increased. Last, mRNAs encoding the AChE catalytic subunit and the AChR alpha-subunit were induced when the P2Y(1) receptors were activated by specific agonists or by overexpression of P2Y(1) receptors in cultured myotubes; those agonists likewise induced the activity in the myotubes of promoter-reporter gene constructs for those subunits, actions that were blocked by a P2Y(1)-specific antagonist. These results provide evidence for a novel function of ATP in regulating the gene expression of those two postsynaptic effectors.

Prenatal cocaine exposure revealed minimal postnatal changes in rat striatal dopamine D2 receptor sites and mRNA levels in the offspring.

It has been reported from this laboratory that prenatal cocaine exposure results in the postnatal transient alterations of rat striatal dopamine uptake sites examined from postnatal 0-32 wk. The present study aims to examine whether this will result in a direct/indirect stimulation of dopamine D2 receptors. Pregnant rats were dosed orally with cocaine hydrochloride (60 mg/kg/d) from gestational day (GD) 7-21. Control animals received an equivalent volume of water. The striatum from the offspring at postnatal 0-32 wk was examined. The radioligand [3H]sulpiride was used for the Scatchard analysis of the D2 receptors, and the changes in the levels of mRNA for the D2 receptor were studied using Northern blot analysis. Results from the present study revealed that in the control group, there was an age-dependent increase in the number of D2 receptor sites (Bmax: 44.00 +/- 2.12 to 178.00 +/- 45.10 fmol/mg protein) and in the levels of D2 mRNA from PN0-32 wk with the most rapid increase occurring during the first 4 wk of postnatal development. Prenatal cocaine exposure resulting in only a significant decrease (p < 0.001) in the number of D2 receptor sites at PN0 wk and in a 10% increase in mRNA levels at PN3, 4, and 12 wk. It was concluded from this study that prenatal cocaine exposure resulted in minimal postnatal changes in the dopamine D2 receptor.

NG108-15 cells express neuregulin that induces AChR alpha-subunit synthesis in cultured myotubes.

A cholinergic neuroblastoma x glioma hybrid cell line NG108-15 is able to form functional synapses, and contains both AChR-aggregating and AChR-inducing activities when cocultured with myotubes. Several lines of evidence indicate that the AChR-inducing activity of NG108-15 cells is derived from neuregulin. The conditioned medium of cultured NG108-15 cells induced the expression of AChR alpha-subunit as well as the tyrosine phosphorylation of erbB-3 receptor. NG108-15 cells expressed neuregulin with a protein of approximately 100 kDa in size and transcripts of approximately 6.8 kbp, approximately 2.6 kbp and approximately 1.8 kbp; mRNAs encoding beta1 and alpha2 isoforms of neuregulin were revealed. NG108-15 cells were induced to differentiate by chemicals, and the chemical-induced differentiation of NG108-15 cells increased the level of neuregulin mRNA expression approximately 3-fold while the expression of a housekeeping gene remained relatively unchanged. The activity of neuregulin in the conditioned medium of NG108-15 cells was reduced by treating the medium with heparin and anti-neuregulin antibody. In addition, NG108-15 cells were transfected with antisense neuregulin cDNA and its expression of neuregulin was reduced, while its neuregulin-induced tyrosine phosphorylation activity was markedly decreased. This is the first direct demonstration that the NG108-15 cell-induced AChR upregulation on cultured myotubes is mediated by neuron-derived neuregulin.

Cerebellar granule cells express a specific isoform of agrin that lacks the acetylcholine receptor aggregating activity.

Agrin is a synapse-organizing molecule that mediates nerve-induced aggregation of acetylcholine receptors and other postsynaptic components at the developing and regenerating vertebrate neuromuscular junctions. Several lines of evidence indicate that agrin might play a similar role in directing the organization of postsynaptic specifications of neuron-neuron synapse formation. Here we used immunological methods and polymerase chain reaction to identify the expression of agrin protein and alternatively spliced mRNA isoforms in the culture of rat granule cells. Anti-agrin polyclonal antibody labeled the cultured granule cells and it detected a protein of over 200 kDa in size from the lysate of the cultured cells. Analysis by polymerase chain reaction showed that the granule cells in culture expressed predominantly the B0 isoform of agrin mRNA. When granule cells were co-cultured with primary chick myotubes, there was no detectable effect on the aggregation of acetylcholine receptors on the surface of the myotubes. These results show that the cerebellar granule cells, similar to motor neurons in vitro, express and secrete agrin but it lacks the acetylcholine receptor aggregating activity.

The EGF-like domain of chick acetylcholine receptor-inducing activity (ARIA) contains its full biological activity.

Acetylcholine receptor-inducing activity (ARIA) is a glycoprotein initially purified from chick brain based on its ability to increase the synthesis of acetylcholine receptor (AChR) on cultured myotubes. cDNA encoding ARIA contains different domains and the functions of each domain in ARIA activity are not known. We used molecular genetic methods to construct a chimeric fusion protein, designated ARIA(S136-K205)-Fc, that contained the leader sequence, the EGF-like domain of chick ARIA (S136 to K205) and the Fc region of human immunoglobulin. The ARIA(S136-K205)-Fc cDNA was transfected into HEK 293 cells and stable cell lines secreting soluble ARIA(S136-K205)-Fc were obtained. The secreted ARIA(S136-K205)-Fc has a molecular mass of approximately 60 kDa and can be purified by protein G chromatography. The purified ARIA(S136-K205)-Fc retained its full biological activity of chick ARIA that included: (i) induction of tyrosine phosphorylation of erbB 3 receptor in C2C12 myotubes; and (ii) approximately 12-fold stimulation of AChR alpha-subunit mRNA synthesis when applied onto cultured chick myotubes. This Fc-tagged ARIA could be rapidly purified and provides a very useful ligand for identifying its true receptor(s) on muscle cell surface.

The role of neonatal and pubertal gonadal hormones in regulating the sex dependence of the hepatic microsomal testosterone 5-reductase activity in the rat.

Heptic microsomal testosterone 5-reductase activity was approximately fourfold higher in adult female rats than in males. This discrepancy was only partly androgen-dependent since gonadectomy of male rats at 68 days of age resulted in only a partial increase of the enzyme activity. This increase was reversible by the administration of testosterone. Similar treatment, however, produced no effect in the female rat, indicating that there is a sex difference in testosterone responsivity. Castration of newborn male rats resulted in a marked increase in the basal enzyme activity. This increase was not affected by treating the adults with testosterone. Giving testosterone to male rats immediately after neonatal gonadectomy, or to newborn female rats, did not produce the male pattern of both the basal enzyme activity and the testosterone responsivity in adulthood. These results suggest that a brief exposure to neonatal androgen is not critical for the expression of the male type of enzyme activity, but that the continuous presence of the male gonads up to and including the pubertal period is essential. Exposure of pubescent female rats to testosterone during the period from 35 to 50 days of age resulted in a significant increase in testosterone sensitivity when tested at 90 days of age, suggesting that pubertal exposure to androgen is important for the expression of testosterone responsivity in adulthood. The sensitivity was potentiated when the animals were ovariectomized before puberty. Furthermore, the enzyme activity in prepubertally ovariectomized female rats was significantly lower than that in adult gonadectomized animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for the involvement of essential sulphydryl group in rat hepatic lactogenic receptor but not in somatogenic receptor.

Incorporation of p-chloromercuribenzene sulphonate (PCMBS) (1mM) in the assay medium for rat hepatic lactogenic receptor produced complete inhibition of binding of [125I]oPRL to the membrane. However, the presence of the thiol-reactive agent produced no effect on the binding of [125I]bGH to rat hepatic somatogenic receptor. Pretreatment of rat hepatic membrane with PCMBS inhibited the binding of [125I]oPRL but not that of [125I]bGH. The lactogenic receptor binding inhibition by PCMBS pretreatment was both concentration- and time-dependent, with complete inhibition at 0.5 mM for 60 min at 0 degrees C. Scatchard analysis of [125I]oPRL binding to membrane at 50% inhibition by PCMBS (0.11 mM) revealed that the binding capacity was decreased rather than the binding affinity. Furthermore, the inhibition of lactogenic receptor binding by PCMBS could be reversed by dithioerythritol (DTE) treatment. Following 80% inhibition by 0.2 mM PCMBS, full recovery of receptor binding was achieved at 6 mM DTE for 60 min at 0 degrees C. The 'recovered' membrane showed no difference from the control membrane in terms of binding capacity and affinity.

Prolactin receptor in rat liver: sex difference in estrogenic stimulation and imprinting of the responsiveness to estrogen by neonatal androgen in male rats.

Rat hepatic prolactin receptor is regulated by sex steroids. A high level of the receptor was found in female rats but the level was nearly undetectable in males. Gonadectomy reduced the receptor level in females but increased the level in males. Administration of estradiol benzoate (0.05 mumoles/kg on alternate days subcutaneously for 9 days) to adult gonadectomized females increased the receptor level by 473% whereas the same treatment in adult gonadectomized males produced a more modest 276% increase. This sexually dimorphic pattern in the responsiveness to estrogen stimulation in adult rats appeared to be determined neonatally. Neonatal gonadectomy of male rats changed the hepatic response system to a more female pattern in adulthood. Replacement of testosterone (1.45 mumoles at days 1 and 3 after birth) to these neonatally gonadectomized male rats restored the male pattern. Diethylstilbestrol replacement (1.45 mumoles at days 1 and 3 after birth) to the neonatally gonadectomized male rats showed the same effect as neonatally administered testosterone. Scatchard analysis revealed that the observed changes in binding are related to changes in binding capacity but not affinity. Desaturation by 4 M MgCl2 indicated that the amount of endogenously bound hormone was negligible in our membrane preparations.

Cloning of cDNAs encoding xenopus neuregulin: expression in myotomal muscle during embryo development.

Neuregulin has diverse functions in neural development, and one of them is the up regulation of acetylcholine receptors (AChRs) at the muscle fiber during the formation of neuromuscular junctions. Although the primary source of neuregulin is derived from motor neuron, the expression in muscle has also been demonstrated. The precise role of neuron-derived and muscle-derived neuregulin during the early stages of development is not known. In order to study the role of neuregulin during early embryo development, we isolated the cDNAs encoding Xenopus neuregulin by cross-hybridization with its chick homologue. The amino acid sequence of Xenopus protein is 50 to 70% identical to members of the neuregulin family. The cDNAs encoding different isoforms of Xenopus neuregulin were identified, and these isoforms have two variation sites: (i) the spacer domain with either 0 or 43 amino acid insertion; and (ii) the C-terminus of EGF-like domain to derive either alpha or beta isoform. When the EGF-like domain of Xenopus neuregulin was expressed in mammalian cells, the recombinant protein was able to induce the expression of AChR and the tyrosine phosphorylation of erbB receptors in cultured myotubes. An approximately 6.5 kb transcript corresponding to neuregulin was detected in RNA isolated from brain and muscle. Various splicing variants were expressed in different Xenopus tissues. In situ hybridization showed a strong expression of neuregulin in developing brain and spinal cord of Xenopus embryo. In addition, it was also prominently expressed in the myotomal muscle. These data suggest that in addition to motor neurons, the postsynaptic muscle cells can also contribute neuregulin for synaptogenesis.

The cAMP-dependent protein kinase mediates the expression of AChE in chick myotubes.

Calcitonin gene-related peptide (CGRP), a neuropeptide synthesized by motor neurons, stimulates the expression of AChR and AChE at the vertebrate neuromuscular junctions. The signaling mechanism of CGRP-induced AChE expression in muscle was determined both in vitro and in vivo. In cultured chick myotubes, the intracellular cAMP-dependent protein kinase (PKA) activity increased to approximately 2-fold after the application of CGRP or PKA activators; the induction was blocked by PKA inhibitors. in vivo transfection analysis on chick gastrocnemius muscles showed that the transfection of cDNA encoding constitutively active mutant Galphas increased the expression of AChE mRNA and protein to approximately 2-fold, while the constitutively active mutant Galphai cDNA transfection showed an opposite effect. The induced catalytic subunit of AChE at approximately 105 kDa was determined by specific antibody. These findings indicate that the CGRP-induced AChE expression in chick muscle is mediated by a PKA-dependent pathway.

Muscle-derived neurotrophin-3 increases the aggregation of acetylcholine receptors in neuron-muscle co-cultures.

Neurotrophins, a group of protein ligands that are structurally related to the prototype nerve growth factor (NGF), are prominently expressed in the skeletal muscle during the critical period of synapse formation. In the present study, we utilized a co-culture system of NG108-15 cells expressing the Trk receptors and C2C12 myotubes expressing the individual neurotrophins to examine whether these factors can act in a target-derived manner to influence the postsynaptic specializations. Our findings demonstrated that muscle-derived neurotrophin-3 (NT-3) has the unique ability to enhance the aggregation of acetylcholine receptors (AChRs) on the myotubes following co-culture with NG108-15 cells expressing TrkC. Taken together, our findings suggest that NT-3 can act as a retrograde factor to modulate the postsynaptic specializations.

Truncated form of pro-acetylcholine receptor-inducing activity (ARIA) induces AChR alpha-subunit but not AChE transcripts in cultured chick myotubes.

Acetylcholine receptor-inducing activity (ARIA) is a glycoprotein initially purified from chick brain based on its ability to increase the synthesis of acetylcholine receptor (AChR). We used reverse transcription-polymerase chain reaction (RT-PCR) to obtain a partial pro-ARIA cDNA clone from methonine-1 to serine-358 including the full functional sequence of ARIA. Northern blot analysis of mRNAs from the embryonic chick brain and muscle showed a transcript with a size of approximately 7.5 kb. The cloned cDNA was subcloned into an eukaryotic expression vector and stably transfected into human embryonic kidney 293 cells. The conditioned medium of the transfected cells was found to increase the level of transcript encoding for the alpha-subunit of AChR by approximately 4.4-fold, but not for acetylcholinesterase (AChE), in the cultured chick myotubes.

Identification of a 17 S asymmetric butyrylcholinesterase in chick muscle by monoclonal antibodies.

A 20 S asymmetric (non-globular) form of acetylcholinesterase (AChE, E.C. 3.1.1.7) has been purified from 1-day chick muscle. This form is a hybrid molecule containing both AChE and butyrylcholinesterase (BuChE, E.C. 3.1.1.8) catalytic subunits, linked through a collagenous tail. However, the 20 S hybrid AChE/BuChE could not account for the total enzyme activities of AChE and BuChE in a high-salt/Triton X-100 extract of 1-day chick muscle. By applying AChE- and BuChE-specific monoclonal antibodies for immunoadsorption, homogeneous asymmetric AChE and BuChE forms were also identified in that extract. The homogeneous BuChE accounts for 20% of the total activity of the asymmetric BuChE present and sediments at 17 S. About 6% of the asymmetric AChE present is, likewise, in a homogeneous, instead of the hybrid, form. The 17 S asymmetric BuChE does not react with monoclonal antibodies specific for the collagenous tail of the hybrid 20 S AChE/BuChE molecule, suggesting that the collagenous subunit differs between these two forms.