Oncogenes and signal transduction pathways involved in the regulation of Na+ channel expression.

Recent progress in neurobiology has revealed that proteins called 'neurotrophic factors' influence development, maintenance of function, and regeneration of neurons in vertebrate nervous system. These factors include the neurotrophin family, epidermal growth factor (EGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF), which are expressed in the nervous system. Effects of the neurotrophic factors are mediated through signal transduction pathways in which several cellular protooncogenes play intrinsic roles. Furthermore, studies on mechanisms coupling membrane events to gene activation have demonstrated that transsynaptic input via action potential and neurotransmitters, and membrane depolarization play an important role in the regulation of electrical activities in neurons during and after maturation. Voltage-dependent sodium (Na+) channels mediate an increase in permeability of Na+ during the initial, rapid phase of the action potential in neurons, and are considered to be important determinants of neuronal functions. Their synthesis and expression, therefore, are crucial aspects of neural differentiation and functions. In mammals, an array of functionally distinct Na+ channels arise, at least in part, through transcriptional regulation of the multiple genes that encode distinct Na+ channel alpha subunits (Na alpha). In this review, we discuss the potential roles of the protooncogenes in the nervous system, with particular emphasis on dynamic expression of the Na+ channel gene family.

Downregulation of Na+ channel mRNA in olfactory bulb tufted cells following deafferentation.

Unilateral naris cauterization blocks odorant access to ipsilateral olfactory receptor cells and results in functional deafferentation of the olfactory bulb (OB). We used naris cauterization on postnatal day 2 (P2) to study the effects of deafferentation on the expression of Na+ channel subunits in OB. In situ hybridization at P18 showed that expression of Na+ channel alpha II and beta I mRNA was dramatically downregulated in tufted cells, while signals in mitral cells showed no detectable changes. Our observations suggest that during critical periods of development in some neurons, Na+ channel expression may be modulated by physiological activity. The differential response in the tufted and mitral cells may reflect varying degrees of dependence on afferent input or fundamental differences in cell properties.

Orphan nuclear receptor ROR alpha gene: isoform-specific spatiotemporal expression during postnatal development of brain.

We analyzed expression of mouse orphan nuclear receptor ROR alpha during postnatal development of rodent brain. Using a riboprobe corresponding to the 3'-end of mROR alpha cDNA a peak of ROR alpha expression was observed at postnatal 16 day (P16) in the Purkinje cells of cerebellum, neurons of the thalamus and the olfactory bulb. The hippocampus was also shown to express ROR alpha with an earlier peak at P7. Expression in cell types other than the Purkinje cells appeared transient. On the other hand, when a probe to the 5'-end of mROR alpha cDNA was used, we observed patterns of ROR alpha expression that are different from those observed with the 3'-probe. No specific transcripts of ROR alpha were detected with the 5'-probe in the Purkinje cells until P16. Additionally, the relative level of the hybridization signals with the 5'-probe and the 3'-probe were different among the various brain regions. Together with the previous findings that ROR alpha comprises at least four isoforms which differ from one another in their N-terminal regions, these observations suggest that the spatiotemporal expression of ROR alpha is under isoform-specific regulation. The timing of its expression suggests that ROR alpha may be involved in regulation of postnatal maturation of specific classes of neurons.

Cell-specific differential expression of Na(+)-channel beta 1-subunit mRNA in the olfactory system during postnatal development and after denervation.

Activity-dependent mechanisms have been implicated in olfactory system development but, although such activity requires ion channels, few reports have described their expression in the olfactory system. We investigated the developmental and denervation-induced regulation of the Na(+)-channel beta 1 subunit (Na beta 1) in rat olfactory bulb (OB) and piriform cortex (PC). In situ hybridization shows that Na beta 1 mRNA expression is upregulated developmentally, but with different time courses in mitral, tufted, and pyramidal cells. In mitral cells, label was detected at postnatal day 4 (P4) and gradually increased to P14. Tufted cells were devoid of Na beta 1 mRNA before P14, when most cells expressed adult levels. In pyramidal cells of PC, Na beta 1 expression was not detectable clearly until P14, with maximal expression at P28. To examine the regulation of Na beta 1 mRNA, we surgically deafferented the OB at P30 and compared the effects on Na beta 1 with those for Na(+)-channel alpha-subunit (Na alpha) mRNAs. Within 5 d of surgery, the Na beta 1 and Na alpha II signals within tufted cells disappeared almost completely. Na beta 1 and Na alpha II expression was decreased in mitral cells to low-to-moderate levels. In pyramidal cells, Na beta 1 mRNA expression was decreased moderately without significant changes in Na alpha II mRNA. Deafferentation had no detectable effects on Na alpha I or III mRNAs in either OB or PC. These data indicate that Na beta 1 mRNA is expressed differentially in subpopulations of cells in the olfactory system during development and after deafferentation and suggest that the expression of Na beta 1 is regulated independently of Na alpha mRNAs via cell-specific and pathway-specific mechanisms.

Na+ channel beta 1 subunit mRNA expression in developing rat central nervous system.

The sodium channel beta 1 subunit (Na beta 1) is a component of the rat brain voltage-dependent sodium channel. We have used nonradioactive in situ hybridization cytochemical techniques to demonstrate that transcript levels of Na beta 1 are differentially upregulated during postnatal development of several CNS regions, with selective labeling of specific neuronal populations. In the hippocampus, labeling of the pyramidal cell layer (particularly in the CA3 region) and dentate granule cells was initially observed at postnatal day 2 (P2) and P10, respectively, and became progressively more intense with maturation. Labeled cells were first observed in the hilus at P10. In the developing cerebellum, transient labeling was observed in the external granule cell layer beginning at P1 while label increased in the internal granule cell layer up to P21. Purkinje cells showed significant label beginning at P4 and increasing up to P21. Weak signal was seen in neurons of deep nuclei at P1 and increased up to P21. Na beta 1 labeling in the spinal cord was first observed in the ventral horn at P2, and the intensity of labeling in these large motoneurons gradually increased. In addition, there was a ventral-dorsal gradient in this region, with label appearing subsequently in neurons of Rexed laminae IX, VII and VIII, and in the dorsal horn (Rexed laminae I-VI). In these regions, the labeling reached a plateau within the first 2-3 weeks after birth and persisted into the adult rat. The time course and regional heterogeneity of Na beta 1 expression are consistent with the hypothesis that the expression of mature Na+ channels, including Na beta 1, contributes to the development of circuitry that supports complex patterns of electrogenesis.

An orphan nuclear receptor, mROR alpha, and its spatial expression in adult mouse brain.

We have cloned cDNA encoding a mouse nuclear receptor mROR alpha which is a homolog of human retinoic acid receptor-related orphan receptor (hROR alpha). Cotransfection experiments revealed that mROR alpha activates transcription through a retinoic acid responsive element of the laminin B1 gene (lamRARE), but not through a RARE of RAR beta gene (beta RARE) or a synthetic palindromic thyroid hormone responsive element (TREpal). The most distal AGGTCA half-site among the three half-sites of lamRARE was sufficient for binding of mROR alpha and consequently for activation of transcription. Transactivation by mROR alpha was dependent on serum in culture medium after transfection, suggesting the presence of a possible ligand. Northern hybridization and in situ hybridization analyses revealed that mROR alpha is expressed in specific areas of the brain including thalamus and olfactory bulb as well as cerebellum where it is present at highest levels in Purkinje cells. In addition to regionally heterogeneous expression in brain, its expression was temporally regulated during differentiation of P19 cells into neural cells, but not into muscle cells. These observations suggest that mROR alpha plays important roles as a transcription factor not only in differentiation of neural cell lineages but also in the mature brain.

Na+ channel beta 1 subunit mRNA: differential expression in rat spinal sensory neurons.

The brain Na+ channel beta 1 subunit (Na beta 1) mRNA has recently been localized within rat central nervous system where it is expressed at differing levels in different types of neurons. In the present study, we have studied the expression pattern of Na beta 1 mRNA in rat dorsal root ganglion (DRG) neurons using non-radioactive in situ hybridization histochemistry. Na beta 1 mRNA is differentially expressed in adult DRG, with higher levels in intermediate-to-large (> approximately 25 microns in diameter) DRG neurons than in small (< 25 microns) DRG neurons. This cell body size-related Na beta 1 mRNA expression is consistently observed beginning at postnatal day 4 and continues throughout development to adulthood. The present results indicate that (i) Na beta 1 mRNA is expressed in neurons in the peripheral nervous system and (ii) Na beta 1 gene expression is differentially regulated in DRG neurons in relation to their cell body sizes.

Tissue-specific distribution of a novel C-terminal truncation retinoic acid receptor mutant which acts as a negative repressor in a promoter- and cell-type-specific manner.

A cDNA clone which encodes a truncation form of the gamma subtype of the retinoic acid receptor (RAR gamma) has been isolated. The mutant RAR gamma (RAR gamma Bm382) has lost its 65 C-terminal amino acids, thus truncating a part of the dimerization and activation domains. By using a reverse transcription-coupled PCR technique, it was shown that RAR gamma Bm382 is expressed at different levels in various mouse tissues and that the level of its expression does not correlate with that of normal RAR gamma B. Cotransfection studies revealed that RAR gamma Bm382 acts as a repressor of normal RARs in a promoter- and cell-type-specific manner. Transcription of beta RARE and TREinv promoters was inhibited by RAR gamma Bm382 in both HeLa and F9 cells. Unlike these two promoters, however, RAR gamma Bm382 did not inhibit transcription of the TREpal promoter in HeLa cells but did so in F9 cells. Moreover, while transcription of the lamRARE promoter was inhibited by RAR gamma Bm382 in both HeLa and F9 cells, the inhibition was not observed when F9 cells were induced to differentiate with retinoic acid and dibutyryl cyclic AMP. DNA-binding analysis revealed that RAR gamma Bm382 is able to form a heterodimer with the retinoid X receptor and bind to the different types of retinoic acid response elements with almost the same efficiency as normal RAR. By comparison with effects of other truncation mutants created in vitro, it was suggested that the C-terminal end of the ligand binding domain of RAR is crucial for determining the specificity of transactivation by RAR. Given these observations, we discuss the possibility that protein factors which mediate retinoic acid response element- and cell-type-specific transactivation by RAR are present.

Differential up-regulation of voltage-dependent Na+ channels induced by phenytoin in brains of genetically seizure-susceptible (E1) and control (ddY) mice.

We investigated the effect of in vivo administration of an antiepileptic drug, phenytoin, on the saxitoxin binding capacity of receptor site 1 of the Na+ channel alpha-subunit, and the expression activity of the channel messenger RNA in epileptic El mouse brains, as compared with parental ddY mice. Subchronic treatment with phenytoin (25 mg/kg per day) for 14 days increased the [3H]saxitoxin binding to brain-derived synaptic membranes of both El and control ddY mice in a time dependent manner. This increase plateaued at 21 +/- 4% in El mice and 28 +/- 3% in ddY control mice after administration of phenytoin for seven days. After cessation of treatment with phenytoin, [3H]saxitoxin binding capacity returned to the basal level within two weeks in both ddY and El brains. Scatchard plot analysis revealed that the phenytoin treatment caused a 20-30% increase in maximum binding capacity of [3H]saxitoxin binding without any change in equilibrium dissociation constant in the brain cortical synaptic membranes of both epileptic El and control ddY mice. A single injection of phenytoin (25 mg/kg) elevated the level of Na+ channel messenger RNA within 1 h in ddY mouse brains. The increase in Na+ channel messenger RNA reached a peak (about 80% increase) after 5 h of phenytoin administration in a concentration-dependent manner (6.25-50 mg/kg). On the other hand, in El mouse brains, Na+ channel messenger RNA was not elevated until more than 5 h after phenytoin injection, and was increased by only about 33%.(ABSTRACT TRUNCATED AT 250 WORDS)

In situ hybridization localization of the Na+ channel beta 1 subunit mRNA in rat CNS neurons.

Localization of Na+ channel beta 1 subunit (Na beta 1) mRNA was examined in adult rat hippocampus, cerebellum and spinal cord by in situ hybridization histochemistry. In hippocampus, Na beta 1 mRNA was strongly expressed by CA3 followed by CA1 pyramidal cells and dentate granule cells. In cerebellum, strong Na beta 1 mRNA expression was observed in Purkinje cells and moderate expression in granule cells and scattered cells of the molecular layer. In spinal cord, neurons in gray matter exhibited moderate to strong expression of Na beta 1 mRNA. These results provide the first localization study of Na beta 1 mRNA in the CNS, demonstrating a differential expression in different neurons.

Phenytoin, an antiepileptic drug, competitively blocked non-NMDA receptors produced by Xenopus oocytes.

The blocking effect of phenytoin (PHT) and other antiepileptic agents on the glutamate receptors was investigated under voltage-clamp conditions using Xenopus oocytes that translated ddY-stock mouse brain mRNA. PHT shifted the concentration-response curve for kainate to right without significant block on the maximum response obtained, indicating that PHT competitively blocks the non-NMDA receptors. An apparent dissociation constant of PHT was 1.9 x 10(-4) M. The block was voltage-independent. Other antiepileptic agents examined hardly blocked the kainate responses except phenobarbital that blocked the responses. The possibility that the competitive block constitutes a part of the antiepileptic action of PHT was discussed.

Overproduction of voltage-dependent Na+ channels in the developing brain of genetically seizure-susceptible E1 mice.

We used E1 mice, a ddY mouse-derived, autosomal mutant strain and a model of hereditary sensory-precipitated epilepsy, to test the hypothesis that epileptic susceptibility may be associated with the activity of voltage-dependent ion channels. We examined the saxitoxin binding capacity of the receptor site 1 of the Na+ channel alpha-subunit, the expression activity of the Na+ channel mRNA, the veratridine-induced 22Na+ influx in the brain synaptosomes, and the regional distribution of Na+ channels in the brain. Compared with control ddY mice, in E1 mice which have not experienced seizures, the number of Na+ channels in the brain synaptosomes increased by approximately 20% starting at the fourth postnatal week through the adult stage as determined by [3H]saxitoxin binding assay. Northern blot hybridization analysis showed excess expression of Na+ channel mRNA (by 30-40%) coincidentally with Na+ channel increases. Regional analysis using the saxitoxin binding assay demonstrated approximately 1.3-fold denser distribution of Na+ channels in the cortex and cerebellum but not the hippocampus and midbrain including thalamus of E1 mice compared to ddY mice. Scatchard plot analysis for saxitoxin binding in the cortex of E1 mouse brains revealed higher maximum binding capacity (Bmax) values (ddY, 4.43 +/- 0.28 pmol/mg protein; E1, 5.43 +/- 0.25 pmol/mg protein) without a change in Kd (ddY, 1.05 +/- 0.03 nM; E1, 1.03 +/- 0.01 nM). Lastly, veratridine-evoked 22Na+ influx, sensitive to tetrodotoxin, was increased approximately 45% in the cortical synaptosomes in six-week-old E1 mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Unusual biochemical development of genetically seizure-susceptible El mice.

Looking for the 'epilepsy gene', we used ddY derived, genetically seizure-susceptible El mice. To find biochemical abnormalities, we examined the amino acid metabolism and gene activity, including poly(A)+ RNA and sodium channel mRNA expressions, in the developmental growth of El mice. At the early postnatal stage, abnormalities in amino acid metabolism were aberrant free amino acid fluctuations. Almost all free amino acids in the liver of newborn El mice showed considerably lower levels than did ddY mice. Among those amino acids, Asp, Glu and Tyr were extremely low, but rapidly recovered to the ddY level within a week. During the successive growth period, we observed no significant difference in hepatic amino acid levels between El and ddY mice. No such drastic changes were noted in the amino acid levels in the brains of ddY and El mice; only the Gly level was greater in El mice than in ddY mice on the day of birth. Rotatory stimulation which evokes convulsions in El mice but not in ddY mice was applied to adult mice and changes in the amino acid level were assessed. The level of Glu and Tyr in seizure-induced El mice was approximately twice that noted in the liver and brain of El mice, which did not experience seizures. It was also somewhat increased in ddY mice subjected to rotational stress which did not induce seizures in that strain. Gene activity that expresses poly(A)+ RNAs, including sodium channel mRNA, was determined by Northern blot analysis, which reveals unscheduled mRNA synthesis by the appearance of an extra band approximately 3 kb in size.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical abnormalities in developing E1 mouse.

Complex biochemical abnormalities were found in the early developmental stage of the E1 mouse. First, the E1 mouse has abnormal levels of specific amino acid concentration within a week from birth. Second, an unusual expression of poly(A)+ RNA from the one-day newborn liver of the E1 mouse was detected by use of Cot 100 DNA as a probe. Third, sodium channels are increased in synaptosomes and at the mRNA expression level of the 3 or 4-week-old E1 mouse brains, compared with the ddY mouse. These results suggest that the biochemical abnormalities described in this study may affect greatly the epileptogenesis of E1 mouse.