Molecular characterization of the neuronatin gene in the porcine placenta

Imprinted genes play important roles in placental and embryonic development. Neuronatin (NNAT), first identified as an imprinted gene in human and mouse brains, played important roles in neuronal differentiation in the brain and in glucose-mediated insulin secretion in pancreatic β cells. In the pig, NNAT was reported to be imprinted in eleven tissues. Our previous microarray hybridization study showed that NNAT was differentially expressed in Yorkshire and Meishan pig placentas, but the imprinting status and function of NNAT in the placenta have not been investigated. We demonstrated for the first time that NNAT was monoallelically expressed in the placenta. Immunochemistry analysis showed that NNAT was located in the uterine luminal and glandular epithelium in placentas. We also confirmed the differential expression of NNAT in Meishan and Yorkshire pig placentas by qPCR. Using IPA software and the published literature, we created a model network of the possible relationships between NNAT and glucose transporter genes. A dual luciferase reporter assay demonstrated that the crucial promoter region of NNAT contained a CANNTG sequence in the +210 to +215 positions, which corresponded to the E-box. Our findings demonstrated important roles of NNAT in placenta function.     

Functional characterization of the mammalian mRNA decapping enzyme hDcp2

Regulation of decapping is a critical determinant of mRNA stability. We recently identified hDcp2 as a human decapping enzyme with intrinsic decapping activity. This activity is specific to N(7)-methylated guanosine containing RNA. The hDcp2 enzyme does not function on the cap structure alone and is not sensitive to competition by cap analog, suggesting that hDcp2 requires the RNA for cap recognition. We now demonstrate that hDcp2 is an RNA-binding protein and its recognition and hydrolysis of the cap substrate is dependent on an initial interaction with the RNA moiety. A biochemical characterization of hDcp2 revealed that a 163 amino acid region containing two evolutionarily conserved regions, the Nudix fold hydrolase domain and the adjacent Box B region contained methyl-cap-specific hydrolysis activity. Maximum decapping activity for wild-type as well as truncation mutants of hDcp2 required Mn(2+) as a divalent cation. The demonstration that hDcp2 is an RNA-binding protein with an RNA-dependent decapping activity will now provide new approaches to identify specific mRNAs that are regulated by this decapping enzyme as well as provide novel avenues to control mRNA decapping and turnover by influencing the RNA-binding property of hDcp2.     

Stimulus-induced coordinate changes in mRNA abundance in single postsynaptic hippocampal CA1 neurons.

The molecular effects of use-dependent changes in synaptic transmission were studied in individual CA1 pyramidal neurons from rat hippocampal slices. Potentiation of excitatory postsynaptic currents was associated with coordinate changes in the relative abundance of several mRNAs 30 min to 3 hr after stimulation. There was a 300% increase in calcium/calmodulin-dependent protein kinase II mRNA levels concordant with a 50% decrease in protein kinase C beta 1 isoform mRNA. A 2-fold increase in zif-268 mRNA was seen, while increases in c-fos and c-jun mRNA levels were inconsistent, gamma-Aminobutyric acid A receptor beta 1 subunit mRNA levels increased 3-fold. Potentiation-induced changes were prevented by N-methyl-D-aspartate receptor blockade. Changes in mRNA abundance in individual cells, with synaptic and glial interactions intact, combine to produce a molecular fingerprint of a potentiated CA1 neuron.     

Cytoplasmic polyadenylation element-like sequences are involved in dendritic targeting of BDNF mRNA in hippocampal neurons

Several mRNAs are known to be targeted to dendrites in hippocampal neurons. In this study, we show that brain-derived neurotrophic factor (BDNF) mRNA has two distinct cis-acting dendritic targeting elements in the short 3′ untranslated region (UTR): a constitutive element and an activity-dependent one. Moreover, deletion of serial cytoplasmic polyadenylation element (CPE)-like sequences in the short 3′UTR suppressed both constitutive and activity-dependent dendritic targeting. In addition to the interaction with cytoplasmic polyadenylation element binding protein-1 (CPEB-1), depolarization enhanced CPEB-1 recruitment to the activity-dependent targeting element. These results suggest that CPE-like sequences are involved in the activity-dependent as well as constitutive dendritic targeting of BDNF mRNA.     

Localization of FMRP-associated mRNA granules and requirement of microtubules for activity-dependent trafficking in hippocampal neurons

Fragile X syndrome is caused by the absence of the fragile X mental-retardation protein (FMRP), an mRNA-binding protein, which may play important roles in the regulation of dendritic mRNA localization and/or synaptic protein synthesis. We have recently applied high-resolution fluorescence imaging methods to document the presence, motility and activity-dependent regulation of FMRP granule trafficking in dendrites and spines of cultured hippocampal neurons. In this study, we show that FMRP granules distribute to F-actin-rich compartments, including filopodia, spines and growth cones during the staged development of hippocampal neurons in culture. Fragile X mental-retardation protein granules were shown to colocalize with ribosomes, ribosomal RNA and MAP1B mRNA, a known FMRP target, which encodes a protein important for microtubule and actin stabilization. The levels of FMRP within dendrites were reduced by disruption of microtubule dynamics, but not by disruption of F-actin. Direct measurements of FMRP transport kinetics using fluorescence recovery after photobleaching in living neurons showed that microtubules were required to induce the mGluR-dependent translocation into dendrites. This study provides further characterization of the composition and regulated trafficking of FMRP granules in dendrites of hippocampal neurons.     

Kinetic characterization and immunodetection of ecto-ATP diphosphohydrolase (EC 3.6.1.5) in cultured hippocampal neurons

Extracellular ATP and adenosine modulate synaptic transmission in hippocampal neurons. ATP released from neural cells is hydrolyzed to adenosine by a chain of ecto-nucleotidases. ATP diphosphohydrolase hydrolyses ATP and ADP nucleotides to AMP and 5'-nucleotidase hydrolyses AMP to adenosine. In this work, we investigated the ATPase and ADPase activities of ATP diphosphohydrolase in cultured hippocampal neurons. The apparent Michaelis-Menten constant (K(m)) was 233.9 +/- 14.6 and 221.8 +/- 63.6 microM, with a calculated maximal velocity (V(max), approximately) of 49.2 +/- 10.7 and 10.9 +/- 5.2 nmol Pi/mg protein/min for ATP and ADP, respectively. The horizontal straight line obtained in the competition plot indicated that only one active site is able to hydrolyze both substrates. Furthermore, we detected the presence of this enzyme using anti-CD39 antibody, which strongly stained the soma of pyramidal and bipolar neurons, but the neurites connecting the cell clusters were also immunopositive. This antibody recognized three bands with a molecular mass close to 95, 80 and 60kDa in immunoblotting analysis. The present results show, for the first time, the kinetic and immunocytochemical characterization of an ATP diphosphohydrolase in cultured hippocampal neurons. Probably, the widespread distribution of this enzyme on the surface of neurons in culture could reflect its functional importance in studies of synaptic plasticity hippocampal.     

Long-term potentiation in cultured hippocampal neurons

Studies performed on low-density primary neuronal cultures have enabled dissection of molecular and cellular changes during N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP). Various electrophysiological and chemical induction protocols were developed for the persistent enhancement of excitatory synaptic transmission in hippocampal neuronal cultures. The characterisation of these plasticity models confirmed that they share many key properties with the LTP of CA1 neurons, extensively studied in hippocampal slices using electrophysiological techniques. For example, LTP in dissociated hippocampal neuronal cultures is also dependent on Ca(2+) influx through post-synaptic NMDA receptors, subsequent activation and autophosphorylation of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and an increase in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor insertion at the post-synaptic membrane. The availability of models of LTP in cultured hippocampal neurons significantly facilitated the monitoring of changes in endogenous postsynaptic receptor proteins and the investigation of the associated signalling mechanisms that underlie LTP. A central feature of LTP of excitatory synapses is the recruitment of AMPA receptors at the postsynaptic site. Results from the use of cell culture-based models started to establish the mechanism by which synaptic input controls a neuron's ability to modify its synapses in LTP. This review focuses on key features of various LTP induction protocols in dissociated hippocampal neuronal cultures and the applications of these plasticity models for the investigation of activity-induced changes in native AMPA receptors.     

Transforming growth factor-alpha in the mammalian brain. Immunohistochemical detection in neurons and characterization of its mRNA

In this communication, we demonstrate that adult mammalian brain neurons express transforming growth factor-alpha (TGF-alpha). We used the anti-TGF-alpha monoclonal antibody, MF9, to immunohistochemically localize TGF-alpha in human and rat brain. We found specific immunoreactivity in neurons throughout the brain which was not a result of cross-reactivity of MF9 with the neuropeptide, synenkephalin. Northern blot analysis of bovine and rat brain RNA using human and rat TGF-alpha cDNA probes, respectively, revealed a single 4.8-kilobase pair mRNA with approximately equal abundance in the bovine brainstem, cerebellum, hypothalamus, and cerebral cortex. Fetal rat brain had about 2-fold more TGF-alpha mRNA than did adult rat. The brain TGF-alpha cDNA was cloned from a human neonatal brainstem library. Four identical clones were isolated after screening 10(6) recombinant lambda gt11 phage. The sequence of the 894-base pair cDNA was virtually identical with the cDNA isolated from a human renal cell carcinoma. A single alanine codon was deleted in the brain cDNA at an exon-exon junction. The alanine deletion is within the amino-terminal region of the TGF-alpha precursor that is thought to be removed by proteolytic processing of the precursor to the mature growth factor. These studies indicate that the normal mammalian brain neurons express TGF-alpha.     

Characterization of rebound depolarization in hippocampal neurons

Rebound depolarization (RD) following hyperpolarizing pulses is found in several neuronal cell types where it takes part in the regulation of neuronal firing behavior. During whole-cell current and voltage clamp recordings in slice preparations, we investigated the modulation of RD by different stimulation patterns and its underlying ionic currents in rat CA1 pyramidal cells. RD was mainly carried by the hyperpolarization-activated cation current I(h) (about two-third) and T-type calcium currents (about one-third), respectively. RD increased with increasing hyperpolarizing amplitude and stimulation frequency, whereas RD substantially decreased with longer pulse duration and, less pronounced, with increasing pulse number. The pulse duration-related decrease of RD was due to a decrease of the driving force of I(h). In conclusion, we showed that RD is differentially modulated by precedent hyperpolarization. Since RD amplitude was high enough to generate action potentials, RD may serve, even under physiologic conditions, as an inhibition-excitation converter.     

Myelination of rodent hippocampal neurons in culture

Axons of various hippocampal neurons are myelinated mainly postnatally, which is important for the proper function of neural circuits. Demyelination in the hippocampus has been observed in patients with multiple sclerosis, Alzheimer's disease or temporal lobe epilepsy. However, very little is known about the mechanisms and exact functions of the interaction between the myelin-making oligodendrocytes and the axons within the hippocampus. This is mainly attributable to the lack of a system suitable for molecular studies. We recently established a new myelin coculture from embryonic day (E) 18 rat embryos consisting of hippocampal neurons and oligodendrocytes, with which we identified a novel intra-axonal signaling pathway regulating the juxtaparanodal clustering of Kv1.2 channels. Here we describe the detailed protocol for this new coculture. It takes about 5 weeks to set up and use the system. This coculture is particularly useful for studying myelin-mediated regulation of ion channel trafficking and for understanding how neuronal excitability and synaptic transmission are regulated by myelination.     

Role of hippocampal neurons in theta-wave generation

Temporal relations between hippocampal unit activity and phases of the theta-waves were studied in unanesthetized rabbits immobilized with tubocurarine. When spontaneous thetarhythm potentials were recorded layer by layer from the dorsal hippocampus, a change in their polarity was observed 0.15–0.2 mm deeper than the pyramidal layer (i.e., in the radial layer). Most hippocampal neurons fired synchronously with the extracellular thetawaves. The numerous pyramidal cells, in which the spontaneous activity was inhibited during stimulation of the contralateral hippocampus and sciatic nerve and a hyperpolarization potential developed, were excited mainly during the positive phase of the theta-waves. Intracellular recording showed that their membrane potential decreased during the positive phase and increased during the negative phase. The less numerous basket cells, whose response to stimulation of the contralateral hippocampus consisted of a short high-frequency volley of spikes, and whose response to sciatic nerve stimulation was marked by a prolonged increase of frequency of the spontaneous discharges, were activated mainly during the negative phase of the theta-waves. It is concluded from these findings that inhibitory mechanisms play a role in the theta-activity of the hippocampus. It is postulated that the extracellular theta-waves are integral EPSPs of the basal dendrites and of the proximal segments of the apical dendrites, and that IPSPs of the pyramidal cell bodies play the principal role in the generation of the intracellular theta-rhythm.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 4, No. 5, pp. 531–539, September–October, 1972.     

Molecular and functional characterization of the electroneutral Na/HCO3 cotransporter NBCn1 in rat hippocampal neurons

We examined molecular and electrophysiological properties of the electroneutral sodium/bicarbonate cotransporter (NBCn1) that is present in rat hippocampal neurons. By PCR, a deletion variant (NBCn1-E) that lacks 123 amino acids in the cytoplasmic N-terminal domain was found in adult neurons. The previously characterized NBCn1-B, which does not have the deletion, was detected in embryonic neurons. In Xenopus oocytes, NBCn1-E raised the intracellular pH in the presence of HCO(3) without significantly affecting the membrane potential. Despite this electroneutral cotransport activity, the transporter mediated a steady-state current that positively shifted the resting potential by almost 30 mV. The mean reversal potential of the steady-state current was -21.2 mV, close to the resting potential of -21.4 mV. The reversal potential shifted 26 mV in response to a 10-fold increase of external Na(+) for concentrations above 10 mm. The current activity mediated by the transporter was unaffected by K(+), Mg(2+), Ca(2+), or Cl(-). Stable expression of NBCn1-E in human embryonic kidney cells also evoked an inward current that shifted the resting potentials more positive compared with the sham-transfected controls. In primary cultures of embryonic hippocampal neurons, the NBCn1 protein was localized in somatodendrites and synapses. NBCn1 protein was partially colocalized with the postsynaptic density protein PSD-95. Single-cell PCR showed that NBCn1 mRNA expression was present in both gamma-aminobutyric acid (GABA)ergic and non-GABAergic neurons. We propose that NBCn1 in hippocampal neurons may affect neuronal activity by regulating local pH as well as steady-state inward currents at synapses.