The alpha(1A) subunits of rat brain calcium channels are developmentally regulated by alternative RNA splicing.

Calcium influx through voltage-gated calcium channels governs important aspects of CNS development. Multiple alternative splicings of the pore-forming alpha(1) subunits have been evidenced in adult brain but little information about their expression during ontogenesis is presently available. The aim of this study was to focus on the expression of three rat voltage-gated calcium channel alpha(1A) splice variants (alpha(1A-a), alpha(1A-b) and alpha(1A-EFe)) during brain ontogenesis in vivo. Using a reverse transcription-polymerase chain reaction strategy, we found that the three isoforms have different timings of development throughout the brain: alpha(1A-b) is expressed from embryonic to the adult stage, alpha(1A--EFe) is restricted to the embryonic period whereas alpha(1A-a) is expressed only postnatally. In situ hybridization indicated that alpha(1A-a) and alpha(1A-b) isoforms develop with different regional and cellular patterns. In hippocampus and cerebellum, alpha(1A-b) represented the predominant isoform at all developmental stages. Taken together, these data reveal that alternative RNA splicing may modulate the alpha(1A) calcium channel properties during development.

Disruption of the mouse Necdin gene results in hypothalamic and behavioral alterations reminiscent of the human Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a complex neurogenetic disorder with considerable clinical variability that is thought in large part to be the result of a hypothalamic defect. PWS results from the absence of paternal expression of imprinted genes localized in the 15q11-q13 region; however, none of the characterized genes has so far been shown to be involved in the etiology of PWS. Here, we provide a detailed investigation of a mouse model deficient for NECDIN: Linked to the mutation, a neonatal lethality of variable penetrance is observed. Viable NECDIN: mutants show a reduction in both oxytocin-producing and luteinizing hormone-releasing hormone (LHRH)-producing neurons in hypothalamus. This represents the first evidence of a hypothalamic deficiency in a mouse model of PWS. NECDIN:-deficient mice also display increased skin scraping activity in the open field test and improved spatial learning and memory in the Morris water maze. The latter features are reminiscent of the skin picking and improved spatial memory that are characteristics of the PWS phenotype. These striking parallels in hypothalamic structure, emotional and cognitive-related behaviors strongly suggest that NECDIN is responsible for at least a subset of the multiple clinical manifestations of PWS.

ARP3beta, the gene encoding a new human actin-related protein, is alternatively spliced and predominantly expressed in brain neuronal cells.

A cDNA encoding a new human actin-related protein (ARP) was cloned. The corresponding protein is highly conserved with the previously described ARP3 protein, suggesting that it represents a second isoform of the human ARP3 subfamily. This new actin-related protein was subsequently named ARP3beta and represents the second example of multiple isoforms of an actin-related protein in a single organism. The ARP3beta gene was mapped to chromosome band 7q34, centromeric to Sonic Hedgehog. Gene structure analysis revealed that at least part of the observed ARP3beta mRNA heterogeneity is caused by alternative splicing resulting in exon skipping. Transcripts produced after exon 2 skipping are predicted to encode truncated products, whose functionality is still unclear. An ARP3beta pseudogene was detected on chromosome 2p11 by database searching. Several ARP3beta mRNA species were detected by Northern blotting and their abundance varied importantly among tissues: the highest expression levels were detected in fetal and adult brain, whereas lower levels were observed in liver, muscle and pancreas. In contrast, ARP3 mRNAs were detected in all tissues tested. Using in situ hybridization, the expression of ARP3beta in brain was shown to be restricted to neurons and epithelial cells from choroid plexus. This suggests a specific function for ARP3beta in the physiology of the development and/or maintenance of distinct subsets of nerve cells.

Regulation of calcium channel alpha(1A) subunit splice variant mRNAs in kainate-induced temporal lobe epilepsy.

P/Q-type voltage-gated Ca(2+) channels (VGCC) regulate neurotransmitter release in the hippocampus and molecular alterations of their alpha(1A) pore-forming subunits are involved in various animal and human CNS diseases. We evaluated, using RT-PCR and in situ hybridization, the spatio-temporal activation of two alpha(1A) subunits splice variants (alpha(1A-a) and alpha(1A-b)) in control and kainic acid (KA)-treated rats. Six hours after KA treatment, alpha(1A-a) and alpha(1A-b) mRNAs increased, decreased or remained unchanged with area specific patterns. These changes were evidenced in the hippocampus and the dentatus gyrus and absent in the cerebellum. The alpha(1A) mRNA upregulation lasted for at least 7 days after KA treatment. Altogether, these results indicate that alpha(1A-a) and alpha(1A-b) mRNAs following seizure onset exhibit a complex and specific spatio-temporal pattern. The long-lasting changes in alpha(1A) subunit mRNA contents suggests that VGCC may be involved in the mechanisms generating chronic focal hyperexcitability and/or cellular damage in temporal lobe epilepsy.

Two Nkx-3-related genes are expressed in the adult and regenerating central nervous system of the urodele Pleurodeles waltl.

We report the isolation and characterization of two NK-3-related genes (PwNkx-3.2 and PwNkx-3.3) and their expression patterns during embryonic development, in the adult CNS, and during tail regeneration in the urodele Pleurodeles waltl. PwNkx-3.2 is the ortholog of the mouse and Xenopus genes, Bapx 1 and Xbap, but PwNkx-3.3 has no known homologue in any other vertebrate. We demonstrate that PwNkx-3.2 and PwNkx-3.3 exhibit graded axial expression patterns in adult spinal cord. During tail regeneration, the two genes are expressed in the wound epidermis, the regenerating muscle masses, the regenerating neural tube, the spinal ganglia, and the cartilage rod. The spatial distribution of transcripts in the CNS suggests that these genes could participate in maintaining the position information along the anteroposterior axis and may explain the ability of the adult CNS to regenerate. During tail regeneration, both genes could be implicated in the reformation of the axial skeleton.

Effect of gamma knife radiosurgery on rat brain sodium channel subunit mRNA expression.

Several lines of evidence underscore a possible and delayed antiepileptic effect of Gamma Knife irradiation. This effect could be related to structural and molecular changes. Since voltage-gated Na+ channels (NaChs) play a crucial role in neuron excitability, we studied the effect of Gamma Knife (GK) irradiation on the distribution of Na+ channel (NaCh) subunit mRNAs in rat brains. A left side irradiation was performed in rats using a stereotactic device adapted for GK radiosurgery. A dose of 100 Gy was administered with the 4 mm collimator. The left dentate gyrus and thalamus coordinates were based on De Groot's rat stereotactic atlas. The isodose curve distribution was calculated with the dose planning software used in Gamma Knife and superimposed on the target. Na+ channels alpha unit and mRNAs (subtype II and subtype III) expression was studied 1 hour, 30 days and 60 days later. We used non-radioactive in situ hybridization with subtype-specific digoxigenin-labeled cRNA probes. Labeling intensity was evaluated with a densitometric analysis of digitized images from the control side (right) and lesioned side (left) in each rat. No morphological changes were observed one hour after GK irradiation. 30 days later, the upper thalamic nuclei exhibited a few necrotic regions associated with gliosis. In contrast, no lesions were observed in the hippocampus. 60 days later, the necrotic region involving thalamic nuclei was enlarged. NaCh II and III mRNAs expression did not appear to be modified after GK at the three times studied here. In particular, neurons surrounding the GK necrosis continued to express high levels of NaCh mRNAs. Thus, regulation of NaCh II and III subtypes do not appear to explain the functional antiepileptic effect of GK.

mRNA coding for voltage-gated sodium channel beta2 subunit in rat central nervous system: cellular distribution and changes following kainate-induced seizures.

The cellular distribution of sodium channel beta2 subunit mRNA was examined in the central nervous system from adult Wistar rats using a non-radioactive in situ hybridization method with digoxigenin-labeled cRNA probes. The expression of the subunit was strong in cerebral and cerebellar cortex, in medulla oblongata and in the spinal cord whereas heterogeneous in hippocampus. The distribution was evaluated in hippocampus and cerebral cortex from 1 to 72 h after kainate injection and compared to control rats using densitometric analysis. In these areas, a transient increase was seen 1 h after the drug administration, followed, in the hippocampus, by a significant decrease. These variations differ from those we previously reported for alpha subunits and might play a role in cellular excitability changes occurring in the course of seizures.

Changes in the mRNAs encoding subtypes I, II and III sodium channel alpha subunits following kainate-induced seizures in rat brain.

Several lines of evidence underscore a possible role of voltage-gated Na+ channels (NaCH) in epilepsy. We compared the regional distribution of mRNAs coding for Na+ channel alpha subunit I, II and III in brains from control and kainate-treated rats using non-radioactive in situ hybridization with subtype-specific digoxigenin-labelled cRNA probes. Labelling intensity was evaluated by a densitometric analysis of digitized images. Heterogeneous distribution of the three Na+ channel mRNAs was demonstrated in brain from adult control rats, which confirmed previous studies. Subtype II mRNAs were shown to be abundant in cerebellum and hippocampus. Subtype I mRNAs were also detected in these areas. Subtype III mRNAs were absent in cerebellar cortex, but significantly expressed in neurons of the medulla oblongata and hippocampus. The three subtypes were differentially distributed in neocortical layers. Subtype II mRNAs were present in all of the layers, but mRNAs for subtypes I and III were concentrated in pyramidal cells of neocortex layers IV-V. During kainate-induced seizures, we observed an increase in Na+ channel II and III mRNA levels in hippocampus. In dentate gyrus, subtype III mRNAs increased 3 h after KA administration to a maximum at 6 h. At this latter time, a lower increase in NaCh III mRNAs was also recorded in areas CA1 and CA3. NaCh III overexpression in dentate gyrus persisted for at least 24 h. In the same area, NaCh II mRNAs were also increased with a peak 3 h after KA injection and a return to control levels by 24 h. No changes in NaCh I mRNAs were seen. The KA-induced up-regulation in NaCh mRNAs probably resulted in an increase in hippocampal neuronal excitability.

The human necdin gene, NDN, is maternally imprinted and located in the Prader-Willi syndrome chromosomal region.

Prader-Willi syndrome (PWS) is a neurogenetic disorder that results from the absence of a normal paternal contribution to the 15q11-13 region. The clinical manifestations of PWS are a transient severe hypotonia in the newborn period, with mental retardation, hypogonadism and obesity observed later in development. Five transcripts with exclusive expression from the paternal allele have been isolated, but none of these has been shown to be involved in PWS. In this study, we report the isolation and characterization of NDN, a new human imprinted gene. NDN is exclusively expressed from the paternal allele in the tissues analysed and is located in the PWS region. It encodes a putative protein homologous to the mouse brain-specific NECDIN protein, NDN; as in mouse, expression in brain is restricted to post-mitotic neurons. NDN displays several characteristics of an imprinted locus, including allelic DNA methylation and asynchronous DNA replication. A complete lack of NDN expression in PWS brain and fibroblasts indicates that the gene is expressed exclusively from the paternal allele in these tissues and suggests a possible role of this new gene in PWS.

NHE-3 isoform of the Na+/H+ exchanger in human gallbladder. Localization of specific mRNA by in situ hybridization.

BACKGROUND/AIMS: Electroneutral absorption of NaCl by the gallbladder mucosa is likely to depend at least in part on a Na+/H+ exchanger. In intestine and colon, absorption due to Na+/H+ exchanger is explained by the presence of specific isoforms of the exchanger, the NHE-3 isoform and possibly the NHE-2 isoform. The aim of the present work was to determine whether the mRNAs coding for NHE-2 and NHE-3 are expressed in epithelial cells of human gallbladder. METHODS: Total RNAs from human gallbladder were subjected to reverse transcription-polymerase chain reaction using specific primers. No message was observed with NHE-2 specific primers, showing that NHE-2 isoform plays no role in gallbladder absorption. With NHE-3 specific primers, a 239 bp cDNA fragment was obtained and showed a high homology with the NHE-3 isoform, confirming the presence of NHE-3 in the gallbladder wall. This fragment was cloned in a pLitmus vector in order to produce cRNA probes by in vitro transcription. Cellular localization of the NHE-3 mRNA was studied on cryostat sections using the cRNA probes labeled with Digoxigenin-11-UTP, controls included assays with sense probe, antibodies without probe and RNaseA treated tissue. A specific staining of the NHE-3 mRNAs was found to be strictly localized to the gallbladder epithelial cells. RESULTS/CONCLUSIONS: Expression of NHE-3 in the gallbladder was found only in the absorptive epithelial cells. The NHE-3 isoform of the Na+/H+ exchanger is likely to be involved in water and electrolyte absorption from bile.

Increase in mRNAs encoding neonatal II and III sodium channel alpha-isoforms during kainate-induced seizures in adult rat hippocampus.

Subtypes I, II and III of sodium channel alpha-subunit mRNAs were analyzed in adult rat brain areas after kainate-induced seizures. Tissue samples were microdissected from occipital neocortex, CA1 and CA3 hippocampus areas and dentate gyrus. Three reverse transcriptase-polymerase chain reaction (RT-PCR) protocols were undertaken to amplify these mRNAs. Amplification products were then distinguished after digestion by restriction enzymes, electrophoresis separation and densitometric analysis of gel profiles. PCR 1 evidenced the relative percentage of mRNAs I, II and III as well as neonatal II and III subtype isoforms, which resulted from an alternative splicing. PCR 2 and 3 were performed to focus on the neonatal vs. adult ratio in II and III subtypes, respectively. Seizures were shown to induce an increase in both neonatal subtypes, which suggested an alteration at the splicing level. These changes exhibited a peculiar brain regional distribution, the maximal effect being observed in dentate gyrus and hippocampus CA1 area. In situ hybridization experiments, using a digoxigenin-labeled oligonucleotide probe-specific for neonatal II and III mRNAs, confirmed this increase in neonatal mRNA subtypes. These changes were transient, reaching a maximum 6 h after drug injection, then disappearing between 12 and 48 h. They were prevented by a pre-treatment of animals by MK-801, a non-competitive antagonist of NMDA receptors. This work, thus, suggested that KA-induced seizures can be accompanied by transient alteration in the splicing pattern of sodium channel alpha-subunit mRNAs which resulted in an increase in expression of their neonatal isoforms within localized areas of adult rat brain.

Characterization of the human jumonji gene.

While constructing a cDNA library of human embryos, we have isolated a clone homologous to jumonji, a mouse gene required for neural tube formation. We have determined the complete coding sequence of the human homologue (JMJ) and deduced the amino acid sequence of the putative protein. We show here that human and mouse jumonji putative proteins are homologous and present 90% identity. During human embryogenesis, JMJ mRNAs are predominantly expressed in neurons and particularly in dorsal root ganglion cells. They are also expressed in neurons of human adult cerebral cortex. In view of these observations, we propose JMJ as a candidate gene for developmental defects of the central nervous system in the human. The human JMJ gene maps at position 6p24-6p23.

A Distal-less-like gene is induced in the regenerating central nervous system of the urodele Pleurodeles waltl.

We report the cloning of a Distal-less-like gene (PwDlx-3) and its pattern of expression during embryonic development and adult tail regeneration in the urodele Pleurodeles waltl. Using RT-PCR and in situ hybridization experiments we determined that, during regeneration, PwDlx-3 is expressed in the epidermis, the cells associated with muscle masses and in the ventrolateral parts of the ependymal tube. PwDlx-3 localization in the muscle masses and in cells of the ependymal tube, which give rise during regeneration to the ventral roots and the spinal ganglia, suggests that this gene might be expressed in cells which have some neural crest cell potentialities. PwDlx-3 is the first homeobox gene shown to be expressed in the regenerating spinal cord but not in the adult one and could thus be involved in the regeneration of the nervous system.

Detection by in situ hybridization of hepatitis C virus positive and negative RNA strands using digoxigenin-labeled cRNA probes in human liver cells.

In situ hybridization was performed using cRNA probes on human liver biopsies to localize both positive and negative RNA strands of hepatitis C virus. From the 5' non-coding region of the viral genome, 210 bp, were amplified by reverse transcriptase-polymerase chain reaction and cloned in a plasmid. Probes were produced by in vitro transcription, and labeled using digoxigenin-11-UTP. Positive HCV-RNA strands were detected in all 20 of the patients analyzed, whereas negative strands were detected in only nine patients, as confirmed using computerized image analysis. Both probes labeled the cytoplasm of hepatocytes with a perinuclear intensification. Few of the mononuclear cells infiltrating the portal connective space contained positive HCV-RNA strands only. Stacks of dilated endoplasmic reticulum cisternae were observed by electron microscopy and their relationship with the infection was discussed. This study confirmed that non-radioactive in situ hybridization represents a useful tool to analyze the localization and replication of hepatitis C virus in liver tissue.

Tissue-specific expression and methylation of the human CYP2E1 gene.

The level and number of CYP2E1 gene transcripts were investigated by northern blot analysis in various human adult tissues including liver, lung, placenta, skin and neurinoma. Three transcripts of 1.8, 2.6 and 4 Kb were expressed in a tissue-specific manner. The origin of the various transcripts was studied and showed that both 4 and 2.6 Kb mRNAs contained sequences from the 3' non-translated region of the gene and that the 4 Kb also contained region localized in the 5' non-translated region. Furthermore, it clearly appeared that a catalytically active CYP2E1 enzyme (as proved by NDMA demethylase activity) was only detected in tissues expressing the 1.8 Kb. The human CYP2E1 was also identified through immunohistochemical techniques. Finally, we observed a relation between the hypomethylation of the human CYP2E1 gene and the hypoexpression of the corresponding protein.

Voltage-sensitive Na+ channels in mammalian peripheral nerves detected using scorpion toxins.

The localization of voltage-sensitive sodium channels was investigated in mouse, rat and rabbit sciatic nerves using iodinated alpha- and beta-Scorpion toxins (ScTx) as specific probes. Saturable specific binding for a beta-ScTx was detected in mouse sciatic nerve homogenates (Kd = 90 pM, binding site capacity = 90 fmol mg-1 protein). LM autoradiographic studies demonstrated that the two types of ScTx stained the Ranvier nodes of the myelinated fibres, and also showed a clear but weaker labelling of the unmyelinated Remak bundles. In the sciatic nerve, which is widely considered as a model 'myelinated nerve', the nodal membrane represented only a small fraction of the total axonal membranes (0.2% and 0.05% for mouse and rabbit sciatic nerves respectively). Therefore, despite their high channel density, nodal membranes contribute only a small proportion of the total labelling by beta-ScTx (15% and 2.3% for mouse and rabbit sciatic nerves respectively), with the major contribution to labelling arising from unmyelinated axons. The distribution of specific binding sites for a beta-Scorpion toxin was then analysed in cross-sections of rabbit sciatic nerve at the EM level. The quantitative analysis of autoradiograms involved three methods, the 50% probability circle method, and two cross-fire analyses using either systematically distributed hypothetical sources or hypothetical sources only located on the plasma membranes of axons and of Schwann cells associated with unmyelinated Remak bundles. No specific beta-Scorpion toxin binding sites were detected at the plasma membrane of Schwann cells from either myelinated fibres or unmyelinated bundles, or at the internodal surface of myelinated axons. Sites were only detected at the surface of unmyelinated axons and at nodal axolemma. Their density in unmyelinated axons was found to be in the range of 1-6 per micron2 of plasma membrane surface area by combining quantitative EM autoradiography and stereological measurements.

Quantitative analysis of fetal rat brain neurons developing in primary cultures. I. Stereological study of the neuronal differentiation.

An ultrastructural stereological analysis was performed to analyze the morphological differentiation of primary cultures of fetal rat brain neurons, growing for two weeks in a serum-free medium. The number of neurons and of gliofibrillary acidic protein (GFAP)-positive glial cells was estimated by light microscopy counting in the culture wells. These cultures provided a quasi-pure neuronal population, since the number of GFAP-positive glial cells was found to be 1% (day 7) and 2% (day 14) respectively of the total number of cultured cells. Cell counts and the stereological measurements were related to the surface area of the culture well. The neuronal differentiation was characterized by an increase in the plasma membrane surface area (x9) and volume (x8) of neurites, contrasting with the decrease in the perikarya surface area and volume. These primary stereological data were combined with the number of neurons to obtain parameters characterizing an average neuron. The increase in membrane surface area of an average neuron was found to be a linear function of time, 29 micron 2 and 445 micron 2 of new membrane being added per day of culture to perikarya and neurites respectively. The number of chemical synapses was also counted and compared to the changes in the plasma membrane surface area. After 7 days in vitro they increased in number more rapidly than the increase in the plasma membrane surface area of neurons.

Quantitative analysis of rat brain neurons developing in primary cultures. II. Changes in the distribution of N-CAM associated to neuronal cell surfaces.

Cultured rat fetal brain cells underwent morphological differentiation, as quantitatively described in the companion paper. In the same system, biochemical and immunolabeling studies were performed to analyze the developmental changes in neural cell adhesion molecule (N-CAM) distribution and quantity at the cell surface of neurons. The cell surface-associated N-CAM, related to the culture protein content, remained stable during the two-week period under study, as demonstrated by 125I-protein A binding assays. Immunogold labeling experiments, both in transmission and scanning electron microscopy, indicated a dramatic decrease in N-CAM site density in each membrane compartment, perikarya and neurites. This temporal variation of N-CAM distribution was not accompanied by differences in N-CAM site density between these two membrane compartments. On the other hand, individual perikarya, observed in scanning electron microscopy, showed various levels of labeling. In addition, immunoblot experiments demonstrated the absence of chemical modulation of N-CAM during the period under study, since the high molecular weight (embryonic) form remained dominant. Moreover, an increase in the total N-CAM amount was detected, contrasting with the stable quantity of cell surface-associated N-CAM. This suggested the existence of an N-CAM intracellular pool in cultured neurons. Finally, since the neurite membrane surface area increased 9-fold (companion paper) and since only a 5-fold decrease in N-CAM site density was observed in this compartment, N-CAM supply to neurite membranes was postulated.

Photoaffinity labeling of ANF receptor in cultured brain neurones.

A monoiodo derivative of rat atrial natriuretic factor (rANF) was shown to specifically bind to rat brain neurones in culture with low binding site capacity (10-20 fmoles per mg of protein) and high affinity (Kd = 50-100 pM). Several analogs of both rat and human ANF competed with 125I-rANF. No change in the number of binding sites was detected upon morphological differentiation of neurones in vitro. Finally a photoreactive derivative of 125I-rANF was prepared and photoaffinity labeling experiments carried out on cultured neurones. After reduction of disulfide bridges, a single band of Mr 60,000 was specifically labeled whereas without reduction, two labeled components of Mr 60,000 and 117,000 were detected.

The correlation between Na+ channel subunits and scorpion toxin-binding sites. A study in rat brain synaptosomes and in brain neurons developing in vitro.

Photoreactive derivatives of alpha- and beta-scorpion toxins have been used to analyze the subunit composition of Na+ channels in rat brain. In synaptosomes, both types of toxins preferentially labeled (greater than 85%) a component of 34,000 Da and, at a lower level, another component of 300,000 Da. Reduction of disulfide bridges shifted this latter band from 300,000 Da to 272,000 Da but did not modify the migration of the 34,000-Da component. Similarly, two bands were labeled in cultured brain neurons, one at 259,000 Da by alpha-scorpion toxins and the other at 34,000 Da by both alpha- and beta-scorpion toxins. Contrary to what was observed in synaptosomes, in cultured brain neurons reduction of disulfide bridges had no effect on the migration of the labeled high molecular weight component. Labeling of the smaller polypeptide was obtained even when cells were solubilized with sodium dodecyl sulfate immediately after cross-linking which proves that the 34,000-Da component is not a product of proteolysis. Binding sites for alpha- and beta-scorpion toxins, respectively, did not develop in parallel during neuronal maturation in culture: the increase in beta-scorpion toxin-binding site density was lower and later than that for alpha-scorpion toxin. When related to morphological development, the increase in alpha-scorpion toxin-binding sites was correlated to neurite growth, whereas the increase in beta-scorpion toxin-binding sites was associated with the development of chemical synapses. Finally, in cultured neurons, but not in synaptosomes, both the binding of beta-scorpion toxin and the labeling of the 34,000-Da component by beta-scorpion toxin were enhanced by depolarization of the cell membrane.