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.

Glial fibrillary acidic protein (GFAP) is expressed exclusively in astrocytes in the central nervous system. In order to characterize individual cultured cells in which the GFAP promoter is active and to identify the regulatory mechanisms of GFAP expression in these cells, we have developed a unique assay system for promoter activity using retrovirus vectors. Retrovirus containing the mouse GFAP promoter fused to the lacZ gene were used to infect mixed glial cultures. The infected cells, in which the GFAP promoter was active, were visualized by X-Gal staining. From these experiments, we found that a 256 bp fragment 5'' of the transcription initiation site was sufficient to confer astrocyte-specific expression of GFAP. The GFAP promoter became active about 3 days before GFAP protein can be detected immunohistochemically, which indicates that detection of GFAP promoter activity can be used to identify astrocyte progenitors. We have also established immortalized astrocyte cell lines in which we detect GFAP promoter activity. Immorto mouse is a transgenic mouse generated by the introduction of thermolabile SV40 T Ag, tsA58. A mixed glial culture prepared from 2-day-old Immorto mouse brain was incubated at 32° C, at which temperature most of the cells expressed T Ag. The culture was then infected with retrovirus containing GFAP promoter-lacZ, and the infected cells were selected. Using the fluorescence-activated cell sorter with fluorescein di-β-D-galactopyranoside as a substrate (FDG-FACS), these cells were separated into two groups: FDG(+), in which the GFAP promoter was active, and FDG(–), in which it was inactive. Mature astrocyte cell lines were established from the FDG(+) cells by colony isolation. The FDG(–) cells were cloned by colony isolation and cultured at 32 or 39°C. At the latter temperature the expression of T Ag was suppressed and cell differentiation was induced in most cells. The cells which became positive for X-Gal staining only after switching to 39°C were collected as immature astrocyte cell lines. These immortalized cell lines should be useful to investigate the molecular mechanisms of astrocyte differentiation.

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.

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We have searched for suitable promoters to regulate the expression of suicide genes for use in gene therapy. We have shown that the 1.3‐kb fragment of the mouse myelin basic protein (MBP) promoter region initiates transcription in mouse glioma cells more efficiently than glial flbrillary acidic protein (GFAP) or myelin proteolipid protein (PLP) promoter. Among three different lengths of the MBP promoter, the shortest (256‐bp) core promoter region initiates transcription as efficiently as 650‐bp or 1.3‐kh MBP promoter lengths in RSV‐M glioma cells. To assess the suitability of the MBP promoter for use in clinical trials of malignant glioma gene therapy, we also had to show that it (the 1.3‐kb length in this case) Is effective in human glioma cells, as well as in murine glioma cells. The activity of the MBP promoter is much higher than that of GFAP or PLP promoter in most human glioma cells, suggesting that the MBP promoter would be best for directing toxic gene expression in gene therapy for patients with malignant glioma. Human glioma cells in which the MBP promoter was strongly active were sensitive to ganciclovir when they were transduced with MBP promoter/herpes simplex virus thymidine kinase gene‐bearing retroviruses. In conclusion, retrovirus‐targeted gene therapy for malignant glioma using this MBP promoter is a promising candidate for clinical trials.