The 5'-flanking region of the RP58 coding sequence shows prominent promoter activity in multipolar cells in the subventricular zone during corticogenesis.

Pyramidal neurons of the neocortex are produced from progenitor cells located in the neocortical ventricular zone (VZ) and subventricular zone (SVZ) during embryogenesis. RP58 is a transcriptional repressor that is strongly expressed in the developing brain and plays an essential role in corticogenesis. The expression of RP58 is strictly regulated in a time-dependent and spatially restricted manner. It is maximally expressed in E15-16 embryonic cerebral cortex, localized specifically to the cortical plate and SVZ of the neocortex, hippocampus, and parts of amygdala during brain development, and found in glutamatergic but not GABAergic neurons. Identification of the promoter activity underlying specific expression patterns provides important clues to their mechanisms of action. Here, we show that the RP58 gene promoter is activated prominently in multipolar migrating cells, the first in vivo analysis of RP58 promoter activity in the brain. The 5.3 kb 5'-flanking genomic DNA of the RP58 coding region demonstrates promoter activity in neurons both in vitro and in vivo. This promoter is highly responsive to the transcription factor neurogenin2 (Ngn2), which is a direct upstream activator of RP58 expression. Using in utero electroporation, we demonstrate that RP58 gene promoter activity is first detected in a subpopulation of pin-like VZ cells, then prominently activated in migrating multipolar cells in the multipolar cell accumulation zone (MAZ) located just above the VZ. In dissociated primary cultured cortical neurons, RP58 promoter activity mimics in vivo expression patterns from a molecular standpoint that RP58 is expressed in a fraction of Sox2-positive progenitor cells, Ngn2-positive neuronal committed cells, and Tuj1-positive young neurons, but not in Dlx2-positive GABAergic neurons. Finally, we show that Cre recombinase expression under the control of the RP58 gene promoter is a feasible tool for conditional gene switching in post-mitotic multipolar migrating young neurons in the developing cerebral cortex.

A newly modified SCG10 promoter and Cre/loxP-mediated gene amplification system achieve highly specific neuronal expression in animal brains.

We designed a new promoter that drives transgene expression in an exclusively neuron-specific manner. The promoter of superior cervical ganglion10 (SCG10), expressed in neurons, was further modified to enhance its neuron specificity and activity by changing its length and fusing a multiple neuronal restrictive silencer element (NRSE) to its upstream or downstream regions. The promoter, which contained 2 kb original promoter length and two extra NRSEs in its downstream region, eventually exhibited remarkable neuron specificity as well as strong activity. To further amplify the promoter activity, the promoter was introduced into a Cre recombinase (Cre)-expressing adenovirus, and subsequent combination with Cre-inducible enhanced green fluorescence protein (EGFP)-expressing adenovirus vector, which has much stronger general promoter, resulted in a remarkably strong gene expression exclusively in neuronal cells of mixed cultures and in an animal model. This system is also applicable to astrocyte-specific expression; for instance, by changing the Cre promoter cassette to an astrocyte-specific promoter. The present relatively compact promoter combined with Cre/loxP system could be useful for a wide range of transgene experiments in vivo as well as for clinical applications.

Doping of magic nanoclusters in the submonolayer In/Si(100) system.

Si(100)4 x 3-In reconstruction is essentially a superlattice of magic (identical-size) Si7In6 nanoclusters. Using scanning tunneling microscopy (STM) observations, we have found that under appropriate growth conditions up to 35% of these clusters can be modified; namely, two Si atoms in the cluster can be replaced by two In atoms, thus forming a Si5In8 cluster. This modification can be considered as a doping of the magic cluster, as it changes the electronic properties of the cluster from semiconducting towards metallic. The doped cluster is less rigid than the ordinary one and swings in the electrical field of the STM tip. The atomic structure and stability of the doped magic cluster have been examined using first-principles total-energy calculations.

Rapid redistribution of the postsynaptic density protein PSD-Zip45 (Homer 1c) and its differential regulation by NMDA receptors and calcium channels.

PSD-Zip45 (Homer 1c) and PSD-95 are postsynaptic density (PSD) proteins containing distinct protein-interacting motifs. Green fluorescent protein (GFP)-tagged PSD-Zip45 and PSD-95 molecules were targeted to the PSD in hippocampal neurons. We analyzed dynamic behavior of these GFP-tagged PSD proteins by using time-lapse confocal microscopy. In contrast to the less dynamic properties of PSD-95, PSD-Zip45 showed rapid redistribution and a higher steady-state turnover rate. Differential stimulation protocols were found to alter the direction of PSD-Zip45 assembly-disassembly. Transient increases in intracellular Ca(2+) by voltage-dependent Ca(2+) channel activation induced PSD-Zip45 clustering. In contrast, NMDA receptor-dependent Ca(2+) influx resulted in the disassembly of PSD-Zip45 clusters. Thus, neuronal activity differentially redistributes a specific subset of PSD proteins, which are important for localization of both surface receptors and intracellular signaling complexes.

Primary structure, developmental expression and functional properties of an inward rectifier K+ channel of the tunicate.

A cDNA encoding for a tunicate inward rectifier K+ channel (TuIRK) was isolated. TuIRK exhibited the highest similarity (approximately 50%) with mammalian Kir2 (IRK) subfamily. Maternal RNA of TuIRK was detected by RT-PCR in unfertilized eggs. By in situ hybridization, the transcript was observed at the 32-cell stage, restricted at the 64-cell stage in anterior epidermal cells of a4-2 blastomere lineage, and disappeared at the late gastrula stage. Therefore, TuIRK was identified to be the inward rectifier whose expression was previously reported to change dramatically upon the neural/epidermal cell fate selection. In Xenopus oocytes, TuIRK expressed a strongly inward rectifying K+ current. The basic electrophysiological properties of TuIRK were similar to those of the mouse IRK1 (mIRK1), except that the sensitivity to the block by extracellular Mg2+ was much lower than that of mIRK1. To identify the structural determinant, we made mutants of the pore region, and then of the extracellular loop (N226 of TuIRK, and E125 of mIRK1). In E125N mutant of mIRK1, the sensitivity to the Mg2+ block was decreased significantly, whereas N226E of TuIRK1 did not acquire the sensitivity. These results demonstrate the contribution of this site to the Mg2+ block and the presence of additional determinant(s).

In vitro and in vivo transfer of bcl-2 gene into keratinocytes suppresses UVB-induced apoptosis.

Bcl-2 is a member of the large Bcl-2 family and protects cells from apoptosis. Ultraviolet B (UVB) irradiation induces apoptosis of keratinocytes that is known as "sunburn cells." Previously we reported that UVB irradiation induces apoptosis accompanied by sequential activation of caspase 8, 3 and 1 in keratinocytes, and that the process is inhibited by various caspase inhibitors. Using bcl-2-expressing adenovirus vector we investigated the effect of Bcl-2 on UVB-induced apoptosis. Adenovirus vector efficiently introduced bcl-2 gene in cultured normal mouse keratinocytes (NMK cells); almost all NMK cells (1 x 10(6)) were transfected at 1 x 10(8) plaque-forming unit (PFU)/mL. Bcl-2-transfected NMK cells were significantly resistant to UVB-induced apoptosis with the suppressive effect dependent on the Bcl-2 expression level. Following UVB irradiation caspase 8, 3 and 9 activities were stimulated in NMK cells, whereas in bcl-2-transfected cells only caspase 8, but not caspase 3 or 9, activity was stimulated. In order to investigate the effect of Bcl-2 in vivo topical application of Ad-bcl-2 on tape-stripped mouse skin was performed. Following the application Bcl-2 was efficiently overexpressed in almost all viable keratinocytes. The expression was transient with the maximal expression of Bcl-2 on the first day following the application of 1 x 10(9) PFU in 200 microL. The introduced Bcl-2 remained at least for 6 days. UVB irradiation (1250 J/m2) induced apoptosis within 12 h and the maximal effect was observed at 24 h in control mouse skin. Both bcl-2-transfected and topical caspase 3 inhibitor-treated mice skin were resistant to UVB-induced apoptosis. The suppressive effect of Bcl-2 was more potent than that of caspase 3 inhibitor application. Topical application of empty adenovirus vector alone had no effect on Bcl-2 expression or UVB-induced apoptosis. These results indicate that adenovirus vector is an efficient gene delivery system into keratinocytes and that Bcl-2 is a potent inhibitor of UVB-induced apoptosis both in vitro and in vivo.

Callosal commissural neurons of Dab1 deficient mutant mouse, yotari.

The yotari mouse is an autosomal recessive mutant mouse, caused by mutation of disabled homolog 1 (Dab1) gene. The mutant mouse is recognized by unstable gait and tremor and by early deaths around at the time of weaning. The cytoarchitectures of cerebeller and cerebral cortices and hippocampal formation of the yotari mouse are abnormal. These malformations strikingly resemble those of reeler mouse. In the present study we examined the callosal commissural (CC) neurons of yotari, reeler and normal mice with the injection of recombinant adenovirus into the frontal area 1 (Fr1) to find some possible phenotypes specific for the yotari mouse. The distribution pattern of CC neurons of the yotari was similar to that of the reeler: retrogradely labeled CC neurons were seen throughout all depths of the contralateral Fr1. However, the present statistical analysis revealed that the difference of the mean intracortical position of the CC neurons between the yotari and the reeler is significantly different (Student's t-test), suggesting that the phenotype of the yotari is clearly different from that of the reeler.

Spine formation and correlated assembly of presynaptic and postsynaptic molecules.

Hippocampal pyramidal neurons in culture showed a developmental shift in synapse distribution from dendritic shafts to spines. Using dual wavelength time-lapse fluorescence microscopy, we analyzed the morphogenesis of three synaptic components: dendritic spines, postsynaptic densities (PSDs), and presynaptic vesicles. Local assembly of a major PSD protein, PSD-95, was spatially and temporally correlated with spine morphogenesis. Clustering of postsynaptic PSD-95 and that of a predominant synaptic vesicle protein, synaptophysin, were also correlated. In contrast, pre-existing PSD-95 clusters in dendritic shafts were preferentially eliminated without promoting spine formation. The local and stepwise assembly of synaptic components at the contact sites between dendritic protrusions and axons explains the developmental remodeling of excitatory synapses.

Bcl-2 expression by retrograde transport of adenoviral vectors with Cre-loxP recombination system in motor neurons of mutant SOD1 transgenic mice.

We investigated genes expression by retrograde axonal transport of replication-defective adenoviruses carrying genes for LacZ (AdLacZ) and Bcl-2 in motor neurons of transgenic mice expressing mutant human Cu/Zn superoxide dismutase (SOD1) gene containing a substitution of alanine for glycine at position 93. We found that intramuscular injection of AdLacZ into the tongue of mutant SOD1 transgenic mice and their wild-type littermates at various ages results in high expression of the transgene and similar time course of expression in hypoglossal cranial nerve nuclei, suggesting no difference in the behavior of the transgene expression between the two groups. Subsequently, we employed a molecular switching cassette for Bcl-2 designed to express Bcl-2 by Cre-loxP recombination using adenoviral vectors, and examined the COS7 and primary neuronal cells with the mutant SOD1 gene. The overexpression of Bcl-2 in both cells and the neuronal protection against staurosporine-induced apoptosis were observed, after dual infection of adenoviral vectors with cassette for Bcl-2 (AxCALNLBcl-2) and Cre recombinase (AxCANCre). After inoculation of AxCALNLBcl-2 followed by AxCANCre into the tongue of both mutant SOD1 transgenic mice and wild-type littermates, Bcl-2 was detected in both the injection site and the hypoglossal nuclei of brainstems, suggesting that this was the result of retrograde transport of AxCALNLBcl-2 and AxCANCre and expression of Bcl-2 by Cre recombinase in the hypoglossal nuclei. This strategy for delivery of exogenous genes such as Bcl-2 will be useful for studying neuronal death/survival and introducing foreign genes into postmitotic motor neurons, and in gene therapy for motor neuron diseases such as ALS.

Tracing axons in the peripheral nerve using lacZ gene recombinant adenovirus and its application to regeneration of the peripheral nerve.

The usefulness of recombinant adenovirus with LacZ to trace axons in the peripheral nervous system was investigated. Recombinant adenovirus with LacZ was applied to the cut end of the tibial nerve in rats. The LacZ gene product (B-galactosidase) filled axons of the tibial nerve, which permitted the continuous long-range tracing of axons. Further, the branching and the direction of the branches could also be examined. Labeled axons in the tibial nerves ran parallel to each other without branching and kept this relative position in the tibial and the sciatic nerve. When the virus was introduced to the regenerating nerve using a silicon tube, the regenerating fibers grew tortuously with short branches in the bulge at the proximal end of the silicon tube. The fibers grew straight in the tube and passed through the bulge at the distal end of the tube without branching. These observations indicate that the LacZ gene recombinant adenovirus is a useful tracer for the study of the peripheral nervous system and of the regeneration processes.

Glia-synapse interaction through Ca2+-permeable AMPA receptors in Bergmann glia.

Glial cells express a variety of neurotransmitter receptors. Notably, Bergmann glial cells in the cerebellum have Ca2+-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) assembled without the GluR2 subunit. To elucidate the role of these Ca2+-permeable AMPARs, we converted them into Ca2+-impermeable receptors by adenoviral-mediated delivery of the GluR2 gene. This conversion retracted the glial processes ensheathing synapses on Purkinje cell dendritic spines and retarded the removal of synaptically released glutamate. Furthermore, it caused multiple innervation of Purkinje cells by the climbing fibers. Thus, the glial Ca2+-permeable AMPARs are indispensable for proper structural and functional relations between Bergmann glia and glutamatergic synapses.

Extension of glial processes by activation of Ca2+-permeable AMPA receptor channels.

AMPA type-glutamate receptor channels (AMPARs) assembled without the GluR2 (GluR-B) subunit are characterized by high Ca2+ permeability, and are expressed abundantly in cerebellar Bergmann glial cells. Here we show that the morphology of cultured Bergmann glia-like fusiform cells derived from the rat cerebellum was changed by manipulating expression of Ca2+-permeable AMPARs using adenoviral vector-mediated gene transfer. Converting endogenous Ca2+-permeable AMPARs into Ca2+-impermeable channels by viral-mediated transfer of GluR2 gene induced retraction of glial processes. In contrast, overexpression of Ca2+-permeable AMPARs markedly elongated glial processes. The process extension was blocked by 2,3-Dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX), a specific antagonist of AMPAR. These results indicate that glutamate regulates the morphology of glial processes by activating Ca2+-permeable AMPARs.

Characterization of heteromultimeric G protein-coupled inwardly rectifying potassium channels of the tunicate tadpole with a unique pore property.

Two cDNAs that encode the G protein-coupled inwardly rectifying K(+) channel (GIRK, Kir3) of tunicate tadpoles (tunicate G protein-coupled inwardly rectifying K(+) channel-A and -B; TuGIRK-A and -B) have been isolated. The deduced amino acid sequences showed approximately 60% identity with the mammalian Kir3 family. Detected by whole mount in situ hybridization, both TuGIRK-A and -B were expressed similarly in the neural cells of the head and neck region from the tail bud stage to the young tadpole stage. By co-injecting cRNAs of TuGIRK-A and G protein beta(1)/gamma(2) subunits (Gbetagamma) in Xenopus oocytes, an inwardly rectifying K(+) current was expressed. In contrast, coinjection of TuGIRK-B with Gbetagamma did not express any current. When both TuGIRK-A and -B were coexpressed together with Gbetagamma, an inwardly rectifying K(+) current was also detected. The properties of this current clearly differed from those of TuGIRK-A current, since it displayed a characteristic decline of the macroscopic conductance at strongly hyperpolarized potentials. TuGIRK-A/B current also differed from TuGIRK-A current in terms of the lower sensitivity to the Ba(2+) block, the higher sensitivity to the Cs(+) block, and the smaller single channel conductance. Taken together, we concluded that TuGIRK-A and -B form functional heteromultimeric G protein-coupled inwardly rectifying K(+) channels in the neural cells of the tunicate tadpole. By introducing a mutation of Lys(161) to Thr in TuGIRK-B, TuGIRK-A/B channels acquired a higher sensitivity to the Ba(2+) block and a slightly lower sensitivity to the Cs(+) block, and the decrease in the macroscopic conductance at hyperpolarized potentials was no longer observed. Thus, the differences in the electrophysiological properties between TuGIRK-A and TuGIRK-A/B channels were shown to be, at least partly, due to the presence of Lys(161) at the external mouth of the pore of the TuGIRK-B subunit.

Expression of AMPA receptors in rat superior colliculus and effect of orbital enucleation.

We analyzed the distribution and the morphological characteristics of neurons expressing AMPA-type glutamate receptor subunits (GluR1 and GluR2) in the superficial partition (stratum zonale (SZ), stratum griseum superficiale (SGS) and stratum opticum (SO)) of the rat superior colliculus. GluR1-expressing neurons had round or ovoid somata in SGS and round or fusiform somata and primary dendrites extending tangential or horizontal side in SO. On the other hand, GluR2-expressing neurons mainly corresponded to vertical fusiform cells with vertically oriented dendrites in SGS and medium-sized stellate or ovoid cells with many primary dendrites in SO. The results suggest that the expressions of GluR1 and GluR2 are differentially regulated in individual neurons of the superficial partition. To analyze the effect of retinal deafferentation on the expression of the GluRs, we performed unilateral orbital enucleations in rats within a week after birth. Thirty days after retinal lesioning, lower expression of GluR2 mRNA was observed in the neurons of the contralateral side as compared with that of the ipsilateral side in SO, but not in SGS. These results indicate that GluR2 expression in the SO neurons is regulated by the correct afferentation from the retina.

A high GluR1 : GluR2 expression ratio is correlated with expression of Ca2+-binding proteins in rat forebrain neurons.

alpha-Amino-3-hydroxy-5-methyl-4-isoxazle propionic acid (AMPA) receptors are ubiquitously expressed; however, their subtypes and abundance vary from region to region. We classified the neurons in various forebrain regions (hippocampus, striatum, amygdala, piriform cortex and somatosensory cortex) into six types: [R1+/R2+], [R1-/R2+], [R1+/R2-], [R1-/R2-], [R1++/R2+] and [R1++/R2-], and analysed the expression of Ca2+-binding proteins, such as parvalbumin and calbindin-D28k, using a triple-staining method. The neurons showing a high GluR1 : GluR2 expression ratio, [R1+/R2-], [R1++/R2+] and [R1++/R2-] neurons, comprised 13-30% of the total neuronal population. In addition, the expression of Ca2+-binding proteins was mainly observed in these three types of neurons. The results suggest that Ca2+-binding protein-positive neurons express Ca2+-permeable AMPA receptors, because the Ca2+-permeability of AMPA receptors is enhanced by the relative scarcity of the GluR2 subunit. To directly test the possibility that Ca2+-binding protein-positive neurons express Ca2+-permeable AMPA receptors, we performed Ca2+-imaging experiments in cultured cortical neurons. Ca2+ influx through AMPA receptors was measured selectively by addition of AMPA together with cyclothiazide in the presence of blockers of other Ca2+ influx routes. More than half of the calbindin-D28k-positive neurons showed a large increase in the intracellular Ca2+ concentration ([Ca2+]i), whilst most of the calbindin-D28k-undetectable neurons exhibited only a slight rise in [Ca2+]i after AMPA addition. These results suggest that the expression of calbindin-D28k is related to the expression of Ca2+-permeable AMPA receptors.

A pathway of neuronal apoptosis induced by hypoxia/reoxygenation: roles of nuclear factor-kappaB and Bcl-2.

As a model of the reperfusion injury found in stroke, we have exposed neurons to hypoxia followed by reoxygenation. Neurons treated with hypoxia/reoxygenation (H/R) respond by activating nuclear factor-kappaB (NFkappaB), releasing cytochrome c from their mitochondria, and ultimately dying. Further supporting an apoptotic mechanism, expression of the antiapoptotic Bcl-2 and Bcl-x proteins was increased following H/R. In this model, adenoviral-mediated transduction of lkappaB expression inhibited NFkappaB activation and significantly accelerated cytochrome c release and caspase-dependent neuronal death. At the same time, expression of mutated lkappaB prevented the increased expression of endogenous Bcl-2 and Bcl-x. In the presence of mutated lkappaB, singular overexpression of only Bcl-2 by adenoviral-mediated transduction significantly inhibited cytochrome c release, caspase-3-like activation, and cell death in response to H/R. These findings suggest a pathway where NFkappaB activation induces overexpression of Bcl-2 and Bcl-x, which function to prevent apoptotic cell death following H/R treatments.

Regulation of GluR2 promoter activity by neurotrophic factors via a neuron-restrictive silencer element.

The AMPA glutamate receptor subunit GluR2, which plays a critical role in regulation of AMPA channel function, shows altered levels of expression in vivo after several chronic perturbations. To evaluate the possibility that transcriptional mechanisms are involved, we studied a 1254-nucleotide fragment of the 5'-promoter region of the mouse GluR2 gene in neural-derived cell lines. We focused on regulation of GluR2 promoter activity by two neurotrophic factors, which are known to be altered in vivo in some of the same systems that show GluR2 regulation. Glial-cell line derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) both induced GluR2 promoter activity. This was associated with increased expression of endogenous GluR2 immunoreactivity in the cells as measured by Western blotting. The effect of GDNF and BDNF appeared to be mediated via a NRSE (neuron-restrictive silencer element) present within the GluR2 promoter. The response to these neurotrophic factors was lost upon mutating or deleting this site, but not several other putative response elements present within the promoter. Moreover, overexpression of REST (restrictive element silencer transcription factor; also referred to as NRSF or neuron restrictive silencer factor), which is known to act on NRSEs in other genes to repress gene expression, blocked the ability of GDNF to induce GluR2 promoter activity. However, GDNF did not alter endogenous levels of REST in the cells. Together, these findings suggest that GluR2 expression can be regulated by neurotrophic factors via an apparently novel mechanism involving the NRSE present within the GluR2 gene promoter.

Akt/protein kinase B prevents injury-induced motoneuron death and accelerates axonal regeneration.

Motoneurons require neurotrophic factors for their survival and axonal projection during development, as well as nerve regeneration. By using the axotomy-induced neuronal death paradigm and adenovirus-mediated gene transfer, we attempted to gain insight into the functional significances of major growth factor receptor downstream cascades, Ras-extracellular signal-regulated kinase (Ras-ERK) pathway and phosphatidylinositol-3 kinase-Akt (PI3K-Akt) pathway. After neonatal hypoglossal nerve transection, the constitutively active Akt-overexpressing neurons could survive as well as those overexpressing Bcl-2, whereas the constitutively active ERK kinase (MEK)-overexpressing ones failed to survive. A dominant negative Akt experiment demonstrated that inhibition of Akt pathway hastened axotomy-induced neuronal death in the neonate. In addition, the dominant active Akt-overexpressing adult hypoglossal neurons showed accelerated axonal regeneration after axotomy. These results suggest that Akt plays dual roles in motoneuronal survival and nerve regeneration in vivo and that PI3K-Akt pathway is probably more vital in neuronal survival after injury than Ras-ERK pathway.

Alternative splicing of the C-terminal domain regulates cell surface expression of the NMDA receptor NR1 subunit.

Subcellular localization of the NMDA receptor NR1 splice forms was studied by expressing individual splice variants and their epitope-tagged derivatives in mouse fibroblasts and in hippocampal neurons. When NR1 splice variants were expressed in fibroblasts, the amount of NR1 molecules expressed on the cell surface varied among forms with different C-terminal cytoplasmic domains. The splice forms with the longest C-terminal cytoplasmic tail (NR1-1a and NR1-1b) showed the lowest amount of cell surface expression, and the splice forms with the shortest C-terminal cytoplasmic tail (NR1-4a and NR1-4b) showed the highest cell surface expression. Cell surface expression of NR1 was enhanced by the coexpression of the NR2 subunit. We measured the glutamate-induced increase of calcium concentration in fibroblasts expressing one of the NR1 splice forms and the NR2B subunit. The increase of calcium concentration after glutamate application had a positive correlation with the amount of NR1 splice forms expressed on the cell surface. When epitope-tagged NR1 splice variants were expressed in primary hippocampal neurons using recombinant adenoviruses, we also observed the differential expression on the cell surface between splice variants. These results suggest that the splicing of the C-terminal domain of the NR1 subunit regulates the cell surface expression of the functional NMDA receptors.