Kensuke Nakahira

Purification and Characterization of P400 Protein, a Glycoprotein Characteristic of Purkinje Cell, from Mouse Cerebellum

Abstract: P400 protein is a concanavalin A (Con A)‐binding, 250–kilodalton glycoprotein characteristic of cerebellum. Extraction conditions for P400 protein were investigated, and complete solubilization of P400 protein from a submicrosomal fraction (P31 fraction) of mouse cerebellum was attained by the combination of 4% Zwittergent 3–14 and 4 M guanidinium chloride. The solubilized P400 protein was purified using Sepharose CL‐4B and Con A‐Sepharose chromatography. A monoclonal antibody (18A10) was prepared against P400 protein. Endo‐β‐N‐acetylglucosaminidase F digestion of P400 protein revealed that P400 protein has a small number of asparagine‐linked oligosaccharide chains and that the epitope that is recognized by 18A10 monoclonal antibody is not on the asparagine‐linked oligosaccharide portion. Tissue distribution of P400 protein was investigated by immunoblot analysis using 18A10 monoclonal antibody. P400 protein was abundant in the cerebellum, but a very small amount of P400 protein or related antigen was also detected in other parts of the nervous system and in nonneural tissues. Immunohis‐tochemical studies indicated that P400 protein was distributed abundantly in the soma, the dendritic arborization, and the axon of the Purkinje cell. No immunoreaction was observed in the other types of cells.

Completion of myelin compaction, but not the attachment of oligodendroglial processes triggers K(+) channel clustering.

The characteristic localization of ion channels is crucial for the propagation of saltatory conduction in myelinated nerves. Voltage‐gated Na+ channels are located at nodes of Ranvier while voltage‐gated K+ channels are mainly found at juxtaparanodal regions. Recently, a humoral factor secreted by oligodendrocytes has been reported to induce clustering of Na+ channels in CNS axons. However, the molecular mechanisms for K+ channel clustering as well as the role of oligodendrocytes are still uncertain. To clarify whether myelin sheath itself can induce the distinct distribution of K+ channels, we have investigated the localization of K+ channels in adult and developing mouse optic nerves. The CNS axons from chronic demyelinating and hypomyelinating mice were also examined to determine if myelin sheaths were required for the maintenance of clusters. In all cases, the K+ channel clustering correlated well with compact myelin, but not with the presence of oligodendrocytes, suggesting that, in contrast to Na+ channel clustering, the formation of compact myelin is required for initiation as well as maintenance of K+ channel clustering. In addition, postsynaptic density protein‐95 (PSD‐95) or its highly related protein was found colocalized with K+ channels, suggesting that it may interact with K+ channels to form clusters at juxtaparanodal regions. J. Neurosci. Res. 58:752–764, 1999.

Pleiotrophin mRNA is highly expressed in neural stem (progenitor) cells of mouse ventral mesencephalon and the product promotes production of dopaminergic neurons from embryonic stem cell-derived nestin-positive cells

Neural stem cells are promising candidates for donor cells in neural transplantation. However, the mechanism by which neural stem cells differentiate into neurons is not well understood. In the present study, a serial analysis of gene expression (SAGE) was carried out to generate a gene file of neural stem (progenitor) cells from the mouse ventral mesencephalon. Among the 15,815 tags investigated, the mRNA of the housekeeping genes (elongation factor 1‐α, ATPase subunit 6, GAPDH, actin), laminin receptor 1, HSP 70, pleiotrophin, and nestin were highly expressed. Because pleiotrophin (PTN) exhibits mitogenic and trophic effects on neural development and exhibits trophic effects on survival of dopaminergic (DAergic) neurons, we investigated the role of PTN in neurogenesis, especially to DAergic neurons. Here, we show that PTN increased the production of tyrosine hydroxylase (TH)‐positive neurons from embryonic stem (ES) cell‐ derived nestin‐positive cells. The expression of Nurr1 mRNA was enhanced by PTN. L‐dopa in the culture medium was increased by PTN. This effect was as strong as with sonic hedgehog. Data suggest that PTN mRNA is highly expressed in neural stem (progenitor) cells of mouse ventral mesencephalon, and PTN promotes the production of DAergic neurons from ES cell‐ derived nestin‐positive cells.

Novel Isoforms of Mouse Myelin Basic Protein Predominantly Expressed in Embryonic Stage

Abstract: Myelin basic protein (MBP), a major protein of myelin, is thought to play an important role in myelination, which occurs postnatally in mouse. Here we report that the MBP gene is expressed from the 12th embryonic day in mouse brain and that most of the predominant embryonic isoforms are not those reported previously. These isoforms have a deletion of a sequence encoded by exon 5 from the well‐known isoforms. These isoforms show a unique developmental profile, i.e., they peak in the embryonic stage and decrease thereafter. In jimpy, a dysmyelinating mutant, the level of these isoforms remains high even in the older ages. These results suggest that MBPs have heretofore unknown functions unrelated to myelination before myelinogenesis begins. The possible presence of 18 isoforms of MBP mRNA, which are classified into at least three groups with different developmental profiles, is also reported here.

Expression of Kv3.1 and Kv4.2 Genes in Developing Cerebellar Granule Cells

The expression of voltage-gated potassium channels plays an important role in the acquisition of membrane excitability in neurons. We examined the expression pattern of genes in developing cerebellar granule neurons in vivo and in vitro. In situ hybridization of Kv3.1 mRNA demonstrated that the gene was expressed at high levels in the external granule layer (EGL) as well as in the internal granule layer (IGL) at all postnatal stages (P) examined (from P3 to P10). In contrast, Kv4.2 mRNA was detected in the premigratory zone (PMZ) of the EGL, but not in the proliferative zone (PLZ), in addition to the IGL. This indicates that Kv4.2 gene expression initiates in the postmitotic migrating neurons. We also examined the expression of the channel genes in microexplant culture systems. Kv3.1 polypeptide was detected in parallel fibers of granule cells at 2 days in vitro, and the expression continued in later stages. The signal of Kv4.2 protein was very low at 2 days in vitro; however, the number of positive cells and the intensity of the signals were increased at 6 days in vitro. These in vitro observations matched those in vivo and our previous electrophysiological studies in which we demonstrated that delayed- rectifier-type current was predominant in the immature granule cells followed by the later appearance of A-type current. The patterns of K+ channel expression suggest that sequential expression of these channel genes primarily determines the membrane excitability.

Mossy fibre contact triggers the targeting of Kv4.2 potassium channels to dendrites and synapses in developing cerebellar granule neurons

Compartmentalization of neuronal function is achieved by highly localized clustering of ion channels in discrete subcellular membrane domains. Voltage‐gated potassium (Kv) channels exhibit highly variable cellular and subcellular patterns of expression. Here, we describe novel activity‐dependent synaptic targeting of Kv4.2, a dendritic Kv channel, in cerebellar granule cells (GCs). In vivo, Kv4.2 channels are highly expressed in cerebellar glomeruli, specializations of GC dendrites that form synapses with mossy fibres. In contrast, in cultured GCs, Kv4.2 was found localized, not to dendrites but to cell bodies. To investigate the role of synaptic contacts, we developed a co‐culture system with cells from pontine grey nucleus, the origin of mossy fibres. In these co‐cultures, synaptic structures formed, and Kv4.2 was now targeted to these synaptic sites in a manner dependent on synaptic activity. Activation of NMDA‐ and/or AMPA‐type glutamate receptors was necessary for the targeting of Kv4.2 in co‐cultures, and activation of these receptor systems in GC monocultures induced dendritic targeting of Kv4.2 in the absence of synapse formation. These results indicate that the proper targeting of Kv4.2 channels is dynamically regulated by synaptic activity. This activity‐dependent regulation of Kv4.2 localization provides a crucial yet dynamic link between synaptic activity and dendritic excitability.

Retrovirus-mediated gene transfer targeted to malignant glioma cells in murine brain

A murine model for meningeal metastasis of malignant glioma was developed to study selective gene transfer into tumor cells and to establish a reliable means of determining the rate of tumor cell infection. A murine ecotropic retroviral vector was created in which the Escherichia coliβ‐galactosidase gene served as a marker for gene expression from the integrated retrovirus. This retrovirus exhibited a high rate of infectivity in RSV‐M mouse glioma cells in vitro. The recombinant retrovirus was injected directly into the cisterna magna of the mice. Staining of β‐galactosidase showed that the rate of gene integration was high in the disseminated glioma cells. These results suggest the possibility of retrovirus‐mediated gene therapy for meningeal dissemination of malignant glioma.

PROXIMAL PROMOTER REGION IS SUFFICIENT TO REGULATE TISSUE-SPECIFIC EXPRESSION OF UDP-GALACTOSE : CERAMIDE GALACTOSYLTRANSFERASE GENE

UDP‐galactose:ceramide galactosyltransferase (CGT) is the enzyme which catalyzes the final step of the biosynthesis of galactocerebroside (GalC), the most abundant glycolipid in myelin. We identified regulatory elements which are related to the tissue‐specific expression of the mouse CGT gene by promoter assay using chimeric CGT‐luciferase constructs. By comparing promoter activity in oligodendroglial CG4 cells and NIH3T3 fibroblasts, only a few hundred base pairs spanning from −309 to −98 were shown to be necessary for the tissue‐specific activity of CGT promoter. A negative regulatory element was found in a more distal region, from −709 to −527, and it also worked in tissue‐specific manner. Sequence analysis suggests that several known elements found commonly in myelin‐related genes may explain these tissue‐specific regulations of the transcriptional activity. J. Neurosci. Res. 52:757–765, 1998.

Neuron-specific expression of a chicken gicerin cDNA in transient transgenic zebrafish

Gicerin, a novel cell adhesion molecule which belongs to the immunoglobulin superfamily, is expressed temporally and spatially in the developing chick brain and retina. The previous in vitro experiments using transfected cells showed that gicerin can function as a cell adhesion molecule which has both homophilic and heterophilic binding activities. For the in vivo analyses of gicerin in neural development, we tried to utilize a zebrafish system, a vertebrate suitable for studying early development. We generated transient transgenic animals by microinjecting DNA constructs into zebrafish embryos. Chicken gicerin, under control of the neurofilament gene promoter, was preferentially expressed in neuronal cells and gicerin-expressing neurons exibited a fasciculation formation with neighboring gicerin-positive axons, which may be partly due to homophilic cell adhesion activity of gicerin. These experimental results suggest that this fast and efficient transgenic animal system is useful for studying the functional roles of neuron-specific genes during the development.

Structural characterization and chromosomal localization of the MAGE-E1 gene

Genes of the melanoma-associated antigen (MAGE) family are characterized by the expression of tumor antigens on a malignant melanoma recognized by autologous cytolytic T lymphocytes. We have previously identified novel members of the MAGE gene family expressed in human glioma and named them MAGE-E1a-c. In the present study, we have revealed the genomic structure of MAGE-E1 by sequence analysis of a human chromosome bacterial artificial chromosome clone containing the MAGE-E1 gene. The MAGE-E1 gene is composed of 13 exons, and three of these (exon 2, exon 3 and exon 12) are alternatively spliced in each variant (E1a-c). The open reading frame encoding the MAGE-E1 peptides initiates in exon 2 and ends in exon 13. We have also demonstrated that the MAGE-E1 gene is located in Xp11 through the analysis of radiation hybrid panels. The genomic structure of MAGE-E1 is markedly similar to that of MAGE-D and its chromosomal locus is also identical to that of MAGE-D, but these features contrast with those of other MAGEs. These results suggest that MAGE-D and -E1 may be evolutionarily distant from other members of the MAGE family, and the two may be ancestral genes for the others.