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.

Continual remodeling of postsynaptic density and its regulation by synaptic activity.

A postsynaptic density (PSD) protein, PSD-95, was tagged with green fluorescent protein (GFP-PSD-95) and expressed in cultured hippocampal neurons using recombinant adenoviruses. GFP-PSD-95 was selectively localized to excitatory postsynaptic sites. Time-lapse fluorescence imaging of hippocampal neurons revealed that >20% of GFP-PSD-95 clusters turned over within 24 hours. The appearance rate of clusters was higher than the disappearance rate, and this difference accounted for the gradual increase of the cluster density observed in culture. Dynamics of PSD-95 clusters were also inhibited by blockers of excitatory synaptic transmission. Continual PSD turnover and its regulation by synaptic activity may be important in activity-dependent remodeling of neuronal connections.

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.

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.

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.

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.

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.

Adenovirus-mediated expression of AMPA-type glutamate receptor channels in PC12 cells.

The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor channels are expressed ubiquitously in brain neurons and mediate fast excitatory neurotransmission. They are composed of four subunits, GluR1, GluR2, GluR3 and/or GluR4. We constructed recombinant adenoviruses encoding rat AMPA receptor subunit cDNAs, GluR1 (AxCAGluR1) and silently mutated GluR2 (AxCAGluR2X) with modified chicken beta-actin promoter and cytomegalovirus immediate-early enhancer. Using these adenoviral vectors, we transferred the GluR1 and GluR2 genes into PC12 cells that possess no functional AMPA receptor channels. PC12 cells infected with these viruses expressed GluR1 and GluR2 RNAs. Immunoblot analysis indicated that the expressed GluR1 and GluR2 proteins were equivalent to those of the rat brain. Functional expression of the AMPA receptor channels was examined using the whole-cell patch clamp technique. In AxCAGluR1-infected cells, the current-voltage (I-V) relationship of response to kainate, a non-desensitizing agonist of AMPA receptors, exhibited a strong inward rectification, indicating the formation of functional GluR1-homomeric channels. In cells infected with both AxCAGluR1 and AxCAGluR2X, the I-V relationship of kainate responses exhibited an outward rectification, indicating the formation of heteromeric GluR1/R2 channels. Immunocytochemical analysis revealed that the AMPA receptor subunit genes were transferred in more than 95% of the infected PC12 cells.

Postsynaptic expression of Ca2+-permeable AMPA-type glutamate receptor channels by viral-mediated gene transfer.

The ability to artificially express a particular receptor protein in the postsynaptic sites of neurons in the central nervous system (CNS) would be useful for the study of synaptic function of cloned receptor genes as well as for gene therapy of neurological disorders caused by dysfunction of postsynaptic receptors. In this study, we aimed to express the cDNA of unedited GluR2 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor that forms inwardly rectifying and Ca2+-permeable channel in CNS neurons by using adenoviral-mediated gene transfer. For this purpose, we have constructed a recombinant adenovirus bearing an expression-switching unit, where the unedited GluR2 cDNA can be activated by the Cre recombinase-mediated excisional deletion of a stuffer DNA interposed between the promotor and the coding region. When PC12 cells were infected with this recombinant adenovirus together with an adenovirus expressing Cre recombinase, the inwardly rectifying and Ca2+-permeable AMPA receptor channels were expressed in nearly 100% of infected cells. Two days after co-infection of cultured rat hippocampal neurons with these adenoviruses, fast excitatory neurotransmission in the glutamatergic synapse was mediated predominantly by the inwardly rectifying and Ca2+-permeable AMPA receptor channels. This indicates that the native AMPA receptors in the postsynaptic sites of the glutamatergic synapse are replaced rapidly with recombinant receptors newly produced by the viral-mediated gene transfer.

Expression of recombinant NMDA receptors in hippocampal neurons by adenoviral-mediated gene transfer.

N-methyl-d-aspartate (NMDA) receptors have attracted a great deal of attention because they are intimately involved in brain development, synaptic plasticity and a variety of neurological disorders. The ability to artificially alter the properties of NMDA receptors in central nervous system (CNS) neurons would be useful for elucidating the physiological roles of these receptors. It would also raise the possibility of gene therapy of neurological diseases caused by malfunction of NMDA receptors. In this study, we constructed three recombinant adenoviruses encoding rat NMDA receptor subunit cDNAs, NMDAR1 (NR1), NMDAR2B (NR2B) and mutant NR1(N598R) in which the asparagine (N) site of the wild-type NR1 was replaced with arginine (R) by site-directed mutagenesis. PC12 cells co-infected with recombinant adenoviruses bearing NR1 and NR2B cDNAs expressed conventional NMDA receptors that were permeable to Ca2+ and sensitive to Mg2+, whereas those with viruses bearing NR1(N598R) and NR2B cDNAs expressed Ca2+-impermeable and Mg2+-insensitive receptors. When rat hippocampal neurons in culture were infected with NR1(N598R) and NR2B viruses, both Ca2+ permeability and Mg2+ sensitivity of NMDA receptors were markedly reduced in the infected neurons. Excitatory postsynaptic currents (EPSCs) mediated by NMDA receptors also became much less sensitive to Mg2+. Thus, the NR1(N598R)/NR2B receptors were more dominant than the native NMDA receptors in the infected neurons, and the former receptors introduced by the adenoviral vectors functioned as postsynaptic receptors. These results indicate that the functional properties of postsynaptic NMDA receptors can be manipulated by gene transfer technology using adenoviral vectors.

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.

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.

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.

Ambiguus nucleus neurons innervating the abdominal esophagus are malpositioned in the reeler mouse.

To examine whether the migration of ambiguus nucleus (NA) neurons is affected in the reeler mouse, recombinant replication-deficient adenoviral vector carrying E. coli-galactosidase gene (lacZ) was injected into the abdominal esophagus of the reeler mouse and normal control at two months of age prior to 5 days of sacrifice of the animals. In the normal control, lacZ-positive neurons were found in the compact formation of the NA, whereas, in the reeler, they were scattered from the base of the fourth ventricle to the ventro-lateral margin of the medulla. The present study confirmed that NA neurons are malpositioned in the reeler mouse, suggesting that the migration of NA neurons is guided by the reelin-related protein (Reelin).

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.

Retrograde and anterograde labeling of cerebellar afferent projection by the injection of recombinant adenoviral vectors into the mouse cerebellar cortex.

Adenoviral vectors have recently been recognized as highly efficient systems for gene delivery into various tissues. We show that a reporter gene introduced into nerve terminals via an adenovirus can be used to label cell bodies retrogradely and then label the axons and nerve terminals of the infected neurons anterogradely in vivo. We injected a replication-defective recombinant adenovirus carrying the E. coli beta-galactosidase gene (lacZ) into the cerebellar cortex of the adult mouse. The first evidence of retrograde labeling was obtained at 2 days after the infection when neurons in the pontine nuclei and the reticulotegmental nucleus of the pons weakly expressed beta-galactosidase, and at 3 days post-infection when neurons in all precerebellar nuclei, known to project to the cerebellar cortex, were strongly stained with X-gal in a Golgi-like manner. Anterograde transport of lacZ gene products was recognized at 3 days post-infection; beta-galactosidase-positive axons arose from somata or dendrites of retrogradely labeled neurons, passed through the middle or inferior cerebellar peduncles, and entered the cerebellum. Anterogradely labeled mossy terminals were recognized on the injection side at 8 days post-infection, and on the contralateral side at 14 days post-infection. Beta-galactosidase expression persisted for up to two months, with a decrease in the total number of labeled cells over time. We could not find any signs of anterograde or retrograde transsynaptic labeling in the nuclei synaptically linked to the cerebellar cortex at any time point after injection up to 58 days post-infection.

An immunocytochemical technique for analysis of regulation of genes encoding early differentiation marker antigens in an oocyte translation system.

Monoclonal antibodies are useful probes for analyzing cells at the molecular level at various developmental stages. Although identification of the genes encoding tissue- and stage-specific antigens could be informative for further molecular analysis, gene cloning is usually a time-consuming step, particularly when a monoclonal antibody is the only probe available. We describe here an immunocytochemical method for preliminary and immediate analysis of the regulation of antigen-coding genes. mRNAs purified from stage 27 and 38 Xenopus tadpoles were fractionated by size and injected into newt oocytes, from which frozen sections were prepared for immunostaining with tissue-specific monoclonal antibodies. Both of the antigens we tested, which are early markers for differentiating epidermal cells of Xenopus tadpoles, were detected in mRNA injected oocytes, but not in control oocytes. Immunostaining for each of the antigens showed that their relative levels in stage 27 and 38 tadpole tissue were reflected in those oocytes injected with mRNA purified from tadpoles of the respective stages. We suggest that this oocyte translation system combined with immunostaining provides for rapid analysis of changes in levels of antigen coding mRNAs throughout development.

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.