Biomedical subjects

K Ikenaka

Publications and source records attributed to K Ikenaka.

At least 37 records · Page 2Linked to original sources

PubMed1999

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.

Animals↗

PubMed1999

Cancer gene therapy with HSV-tk/GCV system depends on T-cell-mediated immune responses and causes apoptotic death of tumor cells in vivo.

To examine the immunological mechanisms involved in cancer gene therapy using the herpes simplex virus thymidine kinase (HSV-tk) gene and ganciclovir (GCV), murine hepatocellular carcinoma (HCC) cells, BNL1ME A.7R.1, were transduced retrovirally with the HSV-tk gene. HSV-tk-transduced cells exhibited a more than 2,000-fold higher sensitivity to GCV compared with untransduced parental cells. When HSV-tk-transduced HCC cells were mixed with parental cells at a 50% ratio and implanted subcutaneously into immunocompetent syngeneic mice, complete inhibition of tumor formation was achieved by GCV treatment. Conversely, no significant inhibitory effects on tumor formation were observed in athymic nude mice. When established solid tumors in immunocompetent mice containing HSV-tk-transduced cells at an only 5% ratio were treated with GCV, marked infiltration by lymphocytes including CD4(+) and CD8(+) ones, and apoptotic death of tumor cells were induced, and significant reduction or even complete regression of tumors was achieved. Furthermore, such cured mice rejected rechallenge with parental HCC cells into the contraflank regions. Our results indicate that cancer gene therapy with the HSV-tk/GCV system can indeed induce efficient antitumor effects and protective immunity in immunocompetent mice but not in nude mice, indicating that T-cell-mediated immune responses may be a critical factor for achieving successful gene therapy against cancer using the HSV-tk/GCV system.

Animals↗

PubMed1999

Molecular and functional characterization of a novel mouse transient receptor potential protein homologue TRP7. Ca(2+)-permeable cation channel that is constitutively activated and enhanced by stimulation of G protein-coupled receptor.

Characterization of mammalian homologues of Drosophila transient receptor potential protein (TRP) is an important clue to understand molecular mechanisms underlying Ca(2+) influx activated in response to stimulation of G(q) protein-coupled receptors in vertebrate cells. Here we have isolated cDNA encoding a novel seventh mammalian TRP homologue, TRP7, from mouse brain. TRP7 showed abundant RNA expression in the heart, lung, and eye and moderate expression in the brain, spleen, and testis. TRP7 recombinantly expressed in human embryonic kidney cells exhibited distinctive functional features, compared with other TRP homologues. Basal influx activity accompanied by reduction in Ca(2+) release from internal stores was characteristic of TRP7-expressing cells but was by far less significant in cells expressing TRP3, which is structurally the closest to TRP7 in the TRP family. TRP7 induced Ca(2+) influx in response to ATP receptor stimulation at ATP concentrations lower than those necessary for activation of TRP3 and for Ca(2+) release from the intracellular store, which suggests that the TRP7 channel is activated independently of Ca(2+) release. In fact, TRP7 expression did not affect capacitative Ca(2+) entry induced by thapsigargin, whereas TRP7 greatly potentiated Mn(2+) influx induced by diacylglycerols without involvement of protein kinase C. Nystatin-perforated and conventional whole-cell patch clamp recordings from TRP7-expressing cells demonstrated the constitutively activated and ATP-enhanced inward cation currents, both of which were initially blocked and then subsequently facilitated by extracellular Ca(2+) at a physiological concentration. Impairment of TRP7 currents by internal perfusion of the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid revealed an essential role of intracellular Ca(2+) in activation of TRP7, and their potent activation by the diacylglycerol analogue suggests that the TRP7 channel is a new member of diacylglycerol-activated cation channels. Relative permeabilities indicate that TRP7 is slightly selective to divalent cations. Thus, our findings reveal an interesting correspondence of TRP7 to the background and receptor stimulation-induced cation currents in various native systems.

Amino Acid Sequence↗

PubMed1999

Cytosine deaminase/5-fluorocytosine gene therapy can induce efficient anti-tumor effects and protective immunity in immunocompetent mice but not in athymic nude mice.

Murine hepatocellular carcinoma cells were retrovirally transduced with the bacterial cytosine deaminase (CD) gene. CD-transduced cells exhibited more than 120-fold higher sensitivity to 5-fluorocytosine (5-FC) compared with parental cells. When syngeneic immunocompetent mice were inoculated s.c. with parental hepatocellular carcinoma cells containing as little as 5% CD-transduced cells, significant inhibition of tumor formation was induced by 5-FC treatment. Furthermore, established solid tumors in immunocompetent mice containing only 5% CD-transduced cells were infiltrated markedly with CD4- and CD8+ T lymphocytes and macrophages by 5-FC treatment, such that significant reduction or even complete regression of tumors was observed. These tumor-free mice resisted subsequent rechallenge with wild-type tumor. Conversely, when athymic nude mice were inoculated with a cell mixture containing CD-transduced cells and parental cells at a ratio of 40:60, all developed tumors despite 5-FC treatment. Our results indicate that gene therapy using the CD/5-FC system can induce efficient anti-tumor effects and protective immunity in immunocompetent mice but not in athymic immunodeficient mice, suggesting that the host's immunocompetence may be a critical factor for achieving successful gene therapy against cancer.

Animals↗

PubMed1999

Proteolipid protein gene product can be secreted and exhibit biological activity during early development.

A gene encoding myelin proteolipid protein (PLP) and its smaller isoform DM20 is expressed at least 1 week before myelination. Mutations within the gene cause abnormalities in the development of premyelinating oligodendrocytes, resulting in hypomyelinating disorders. These findings suggest a premyelinating function of the PLP gene products. We previously demonstrated that PLP gene expression is directly associated with secretion of a factor that increases the number of oligodendrocytes. Here we show that this activity is mediated by a secreted fragment containing the C-terminal portion of PLP. This factor increased the bromodeoxyuridine incorporation rate in both oligodendrocyte and astrocyte lineage cells; a synthetic peptide (PLP 215-232) exhibited a similar activity. Dose-response curves of PLP and PLP peptide showed maximum activities at a concentration in the picomolar range, which decreased at higher concentrations. These observations demonstrate that a secreted PLP gene product exerts biological activity at a premyelinating stage before the major induction of the gene.

3T3 Cells↗

PubMed1999

Systematic analysis of N-linked sugar chains from whole tissue employing partial automation.

A partially automated technique for the isolation and characterization of N-linked sugar chains from glycoproteins of crude tissue samples is established. The N-linked sugar chains from the acetone-extracted tissues are made free by a process of hydrazinolysis and subsequently N-acetylated by GlycoPrep 1000 (Oxford Glycosystems). These free sugar chains are further converted to pyridylamino derivatives by GlycoTag (Takara). Characterization of these sugar chains is achieved by a combination of HPLC columns using a highly sensitive fluorescence detector at femtomole levels. Tissue sample can be successfully pyridylaminated and analyzed to give highly reproducible results with consistent yield, requiring fewer purification steps, minimum skills, and less time. Moreover, fixed tissues can also be analyzed employing this technique, giving a similar sugar chain pattern compared to normal tissue samples. Using this method we show that the pattern of N-linked sugar chains present in human sera or in one small region of brain is strikingly similar among the different individuals. However, the absence of a highlighted peak in one of the samples suggests this method can be extrapolated to identify changes, if any, associated with disorders such as inflammation or cancer. Furthermore, this two-dimensional display of sugar chains would discover the function-specific molecules as we see in proteins.

Animals↗

PubMed1999

Intrathecal 5-fluoro-2'-deoxyuridine (FdUrd) for the treatment of solid tumor neoplastic meningitis: an in vivo study.

To evaluate the possible intrathecal use of 5-fluoro-2'-deoxyuridine (FdUrd) for neoplastic meningitis, its antitumor activity and neurotoxicity in vivo were assessed. FdUrd at doses in the range 5-100 microg/animal was effective against meningeal carcinomatosis using Walker 256 carcinoma cells in rats and MM46 mammary cancer cells in mice and against meningeal gliomatosis using 203 glioma cells in mice. After four intrathecal injections, FdUrd at these doses also showed minimal neurotoxicity in the C57BL/6 mouse brain. To estimate the mechanism of FdUrd efficacy, thymidine phosphorylase (TPase) and thymidine kinase (TK), key enzymes in the metabolism of FdUrd, were measured in rat, mouse and normal human brain tissue, and in human brain tumor tissues and cerebrospinal fluid (CSF) from patients with malignant brain tumors including meningeal carcinomatosis. TPase levels were lower in brain and malignant brain tumors than in other organs and their tumors. Moreover, the activity of TPase in the gray matter of human brain, which faces the cerebrospinal fluid across the cortical surface and into which malignant cells invade in meningeal carcinomatosis, was lower than that in the white matter. TK was undetectable, and TPase was detected (at very low concentrations) in only 4 of 56 patients with brain tumors or meningeal carcinomatosis. These findings indicate that brain tissue and CSF are favorable sites for FdUrd chemotherapy because the rate of conversion of FdUrd to 5-FU would be minimal. In conclusion, FdUrd is potentially useful for intrathecal treatment of neoplastic meningitis from primary brain tumors and systemic cancer.

Animals↗

PubMed1999

Clinical trial of intrathecal administration of 5-fluoro-2'-deoxyuridine for treatment of meningeal dissemination of malignant tumors.

Intrathecal administration of 5-fluoro-2'-deoxyuridine (FdUrd) was performed in patients with meningeal dissemination of malignant tumors during the period from January 1996 to September 1998, and they were followed up until February 1999. The study population consisted of 23 patients: 12 with lung cancer, 4 with breast cancer, 2 with colon cancer, 1 with malignant lymphoma, 2 with glioblastoma and 2 with metastatic brain tumors of unknown origin. FdUrd was administered intrathecally through an Ommaya reservoir placed in the lateral ventricle initially at a dose of 1 microg twice per week, and the dose was increased to 10 microg and administration schedule was also increased every day. Headache and nuchal pain were relieved in all patients regardless of responsiveness to intrathecal FdUrd therapy as determined from the findings in the cerebrospinal fluid (CSF). Patients showed no side effects during the course of intrathecal chemotherapy except for slight nausea in two patients and dull headache in one. Sixteen of the 23 patients showed decreased cell number in the cerebrospinal fluid (CSF). Positive cytological findings in CSF became negative in 6 of the 23 patients, and the levels of CSF tumor markers were decreased in 14. Responsiveness to intrathecal administration of FdUrd was defined as 'response' when both the cell number and tumor markers were decreased in both ventricular and spinal CSF or when the cell number was decreased in cases in which the tumor markers were not detected. Overall, 16 of the 23 patients (70%) showed complete or partial responses to intrathecal FdUrd therapy as determined from CSF findings. These results demonstrated the efficacy of intrathecal FdUrd chemotherapy without apparent neurotoxicity for treatment of meningeal dissemination of malignant tumors.

Aged↗

PubMed1999

Programmed cell death without DNA fragmentation in the jimpy mouse: secreted factors can enhance survival.

Jimpy is one of many related mutations affecting the myelin proteolipid protein gene that causes severe hypomyelination in the central nervous system (CNS). Underlying the hypomyelination is a failure of oligodendrocytes (OLs) to differentiate, and the premature death of large numbers of OLs during the developmental period. Previous light and electron microscopic evidence suggested that jimpy OLs die in a manner consistent with programmed cell death. We have used TUNEL staining as a biochemical marker for apoptosis in conjunction with immunostaining for OL and myelin markers. At 13 - 14 days postnatal, a time when the number of dying OLs in jimpy CNS is increased more than five times normal, there are only modest increases (70% in spinal cord; 20% in cerebral cortex) in TUNEL labeled cells in mutant CNS tissues. The results in vitro are similar, and only a small per cent of TUNEL labeled cells have the antigenic phenotype of OLs. The discrepancy between numbers of dying and TUNEL labeled cells suggests either that most jimpy OLs do not undergo programmed cell death or that the biochemical pathways leading to their death do not involve DNA fragmentation which is detected by the TUNEL method. We also present evidence that jimpy OLs show increased survival and enhanced differentiation when they are grown in vitro in medium conditioned by cells lines which express products of the proteolipid protein gene. Cell lines expressing proteolipid protein and the alternatively spliced DM20 protein have differential effects on cell numbers and production of myelin-like membranes.

Animals↗

PubMed1999

Complete cure of established murine hepatocellular carcinoma is achievable by repeated injections of retroviruses carrying the herpes simplex virus thymidine kinase gene.

Although xenotransplantation of retrovirus-producing cells into a tumor has been shown to be effective for the treatment of cancer, injections of recombinant retroviruses are much more feasible for clinical applications. We established a clone producing retroviruses carrying the herpes simplex virus thymidine kinase (HSVtk) gene with titers of up to 4 x 10(7) colony-forming units/ml, and examined the effectiveness of in vivo gene therapy against cancer. Syngeneic mice were inoculated subcutaneously with murine hepatocellular carcinoma (HCC) cells, BNL1ME A.7R.1, and the treatment was initiated after tumors were established. When mice were given an intratumoral injection of HSVtk-carrying retroviruses or their producing cells followed by ganciclovir (GCV) treatment, significantly prolonged survival periods were observed. When mice were treated with repeated intratumoral injections of HSVtk-carrying retrovirus-producing cells, significant antitumor responses and some cures were induced by GCV treatment. Furthermore, repeated intratumoral injections of HSVtk-carrying retroviruses and GCV treatment resulted in complete regression of established HCC tumors in all animals used in the experiment. Mice that completely eradicated tumors exhibited protective immunity against wild-type HCC tumors. These results suggest that repeated injections of HSVtk-carrying retroviruses followed by GCV treatment is a potent modality for the treatment of solid tumors.

Animals↗

PubMed1999

Abnormal expression of neurofilament proteins in dysmyelinating axons located in the central nervous system of jimpy mutant mice.

Myelination in the peripheral nervous system is considered to increase the phosphorylation level of neurofilament proteins in the axon, resulting in an increase in axonal calibre. To understand the relationship between myelination and neurofilament proteins in axons, we examined jimpy mutant mice with a point mutation in the proteolipid protein gene and dysmyelination in the central nervous system. The jimpy mice exhibited a characteristic similarity in neurofilament nature to the myelin-deficient mice in the peripheral nervous system reported previously. The following novel results were obtained in the jimpy mice: dysmyelinated axons, in which the amount of non-phosphorylated neurofilament-H was drastically increased without a significant reduction of the phosphorylated form, compared with the control myelinated axons, did not suffer any decrease in their diameters. Expression levels of all neurofilament subunit proteins and their mRNAs were enhanced in the central nervous system tissue. Because the above biochemical data were obtained from the cytoskeletal fraction, at least some of the increased neurofilament-H and -M proteins appeared to be coassembled into neurofilaments but remained non-phosphorylated. Axonal neurofilaments of the jimpy were, probably due to this abnormal stoichiometry and phosphorylation state in neurofilaments, more compact and random in alignment with less prominent cross-bridges than those of the control, providing possible evidence for disturbing the axonal transport of other organelles. These results suggest that myelination regulates both the expression and phosphorylation of neurofilament proteins, and is essential for the cytoplasmic organization of myelinated axons.

Animals↗

PubMed1999

Conversion of brain-specific complex type sugar chains by N-acetyl-beta-D-hexosaminidase B.

The N-linked sugar chains, GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-4)(Manalpha1++ +-3)Manbeta1-4GlcNAcb eta1-4(Fucalpha1-6)GlcNAc (BA-1) and GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-4)(GlcNAcbeta1 -2Manalpha1-3)Manb eta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc (BA-2), were recently found to be linked to membrane proteins of mouse brain in a development-dependent manner [S. Nakakita, S. Natsuka, K. Ikenaka, and S. Hase, J. Biochem. 123, 1164-1168 (1998)]. The GlcNAc residue linked to the Manalpha1-3 branch of BA-2 is lacking in BA-1 and the removal of this GlcNAc residue is not part of the usual biosynthetic pathway for N-linked sugar chains, suggesting the existence of an N-acetyl-beta-D-hexosaminidase. Using pyridylaminated BA-2 (BA-2-PA) as a substrate the activity of this enzyme was found in all four subcellular fractions obtained. The activity was much greater in the cerebrum than in the cerebellum. To further identify the N-acetyl-beta-D-hexosaminidase, BA-1 and BA-2 in brain tissues of Hex gene-disrupted mutant mice were detected and quantified. PA-sugar chains were liberated from the cerebrum and cerebellum of the mutant mice by hydrazinolysis-N-acetylation followed by pyridylamination. PA-sugar chains were separated by anion-exchange HPLC, size-fractionation, and reversed-phase HPLC. Each peak was quantified by measuring the peaks at the elution positions of authentic BA-1-PA and BA-2-PA. BA-2-PA was detected in all the PA-sugar chain fractions prepared from Hexa, Hexb, and both Hexa and Hexb (double knockout) gene-disrupted mice, but BA-1 was not found in the fractions from Hexb gene-disrupted and double knockout mice. These results indicate that N-acetyl-beta-D-hexosaminidase B encoded by the Hexb gene hydrolyzed BA-2 to BA-1.

Animals↗

PubMed1999

beta1-4Galactosyltransferase activity of mouse brain as revealed by analysis of brain-specific complex-type N-linked sugar chains.

We previously reported two brain-specific agalactobiantennary N-linked sugar chains with bisecting GlcNAc and alpha1-6Fuc residues, (GlcNAcbeta1-2)(0)(or)(1)Manalpha1-3(GlcNAcbeta1-2M analpha1-6)(GlcNA cbeta1-4)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)Glc NAc [Shimizu, H., Ochiai, K., Ikenaka, K., Mikoshiba, K., and Hase, S. (1993) J. Biochem. 114, 334-338]. Here, the reason for the absence of Gal on the sugar chains was analyzed through the detection of other complex type sugar chains. Analysis of N-linked sugar chains revealed the absence of Sia-Gal and Gal on the GlcNAc residues of brain-specific agalactobiantennary N-linked sugar chains. We therefore investigated the substrate specificity of galactosyltransferase activities in brain using pyridylamino derivatives of agalactobiantennary sugar chains with structural variations in the bisecting GlcNAc and alpha1-6Fuc residues as acceptor substrates. While the beta1-4galactosyltransferases in liver and kidney could utilize all four oligosaccharides as substrates, the beta1-4galactosyltransferase(s) in brain could not utilize the agalactobiantennary sugar chain with both bisecting GlcNAc and Fuc residues, but could utilize the other three acceptors. Similar results were obtained using glycopeptides with agalactobiantennary sugar chains and bisecting GlcNAc and alpha1-6Fuc residues as substrates. The beta1-4galactosyltransferase activity of adult mouse brain thus appears to be responsible for producing the brain-specific sugar chains and to be different from beta1-4galactosyltransferase-I. The agalactobiantennary sugar chain with bisecting GlcNAc and alpha1-6Fuc residues acts as an inhibitor against "brain type" beta1-4galactosyltransferase with a K(i) value of 0.29 mM.

Animals↗

PubMed1999

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.

Aging↗

PubMed1999

Retroviruses prepared from human DAF expressing murine packaging cells acquire resistance against human serum.

The complement system of the human body inactivates the infectious ability of retroviruses injected as an artificial gene transfer vector. We established new murine leukemia virus (MuLV) packaging cell lines; D2SS and D7S which express decay-accelerating factor (DAF) on their surface. Both D2SS and D7S were resistant against incubation with fresh human serum. Moreover, the retroviruses produced from these packaging cell lines were also resistant to serum treatment. This resistance can be inhibited by DAF neutralizing antibody 1C6. These data demostrate that DAF induces a partial protection of MuLV infection from the human complement system.

3T3 Cells↗

PubMed1998

Multiple restricted origin of oligodendrocytes.

The plp gene encodes the proteolipid protein and its alternatively spliced product DM-20, major proteins of CNS myelin. In the mouse, plp/dm-20 transcripts are expressed beginning at embryonic day 9.5 (E9.5) by restricted foci of germinative neuroepithelial cells. To determine the identity of the neural precursors expressing plp/dm- 20, a zeomycin resistance gene fused to the lacZ reporter was expressed in transgenic mice under the control of the plp regulatory sequences. In the three different lines generated, the pattern of beta-galactosidase expression was similar and superimposable on the expression pattern of endogenous plp/dm-20. Both in vivo and in vitro, the transgene was expressed by O4(+) pre-oligodendrocytes, and later by RIP+ differentiated oligodendrocytes, but not by neuronal cells, astrocytes, or radial glial cells. After zeomycin selection, a dramatic enrichment in O4(+) pre-oligodendrocytes was observed in cultures derived from E12.5 transgenic embryos. This enrichment indicates the oligodendroglial specification of neural precursors that continuously express plp/dm-20. Early plp/dm-20-expressing precursors, however, appear to be a separate population from previously described PDGFRalpha oligodendrocyte precursors, as shown by the striking differences in their (1) patterns of distribution and (2) responsiveness to PDGF. These data suggest that oligodendrocytes have a plural origin and that early plp/dm-20 defines one of the neural lineages generating oligodendrocytes.

Animals↗

PubMed1998

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.

Animals↗

PubMed1998

Metabolic labeling of a subset of glial cells by UDP-galactose: implication for astrocyte lineage diversity.

Astrocytes are implicated in many aspects of brain function; however, it remains unclear whether astrocytes arise from a single cell lineage. It is therefore important to obtain new markers for the astrocyte cell lineage. We show that exogenously added UDP-galactose (UDP-Gal) can be used to metabolically label a subset of glial fibrillary acidic protein-positive (GFAP+) cells. UDP-Gal was incorporated into the cultured embryonic mouse brain slices in a time-dependent manner. Surprisingly, the transferred sugar moiety was no longer Gal but was mainly glucose. Most of the radioactivity was transferred to a polymer of glucose, most likely to be glycogen, and also to glucosyl ceramide. In the slice culture, the reaction products were distributed densely in the ventricular zone and also on process-like structures extending to the pial surface. In dissociation culture, UDP-Gal labeled some of the GFAP+ cells and some of the vimentin+ cells. Because radial glial cells (RGCs) contain glycogen and change from vimentin+ to GFAP+, it is strongly suggested that UDP-Gal labeled RGCs and their descendants. Only 27% of the GFAP+ cells were labeled with UDP-Gal, which suggests that only a subset of astrocytes are derived from RGCs and that there is a discrete group of GFAP+ cells that is not generated from RGCs.

Animals↗