Functional central nervous system myelin repair in an adult mouse model of demyelination caused by proteolipid protein overexpression.

Two types of interventions to remyelinate the adult demyelinated central nervous system were investigated in heterozygous transgenic mice overexpressing the proteolipid protein gene. 1) A cocktail of trophic factors, "TS1," was directed toward the activation of the endogenous pool of neural progenitors to increase the number of myelinating oligodendrocytes (OL) in the brain. 2) A combinatorial approach in which OL progenitors were coinjected with TS1 into the corpus callosum of wild-type and He4e transgenic mice that displayed hindlimb paralysis. The levels of locomotor ability in these mice were evaluated after a single treatment. The data showed that a single administration of either one of the interventions had similar therapeutic effects, alleviating the symptoms of demyelination and leading to the recovery of hindlimb function. Histological and immunofluorescent examination of brain sections showed extensive remyelination that was sufficient to reverse hindlimb paralysis in transgenic mice. When the interventions were administered prior to hindlimb paralysis, He4e mice were able to walk up to 1 year of age without paralysis.

Immunohistochemical and western analyses of protein arginine N-methyltransferase 3 in the mouse brain.

The distribution of protein arginine N-methyltransferase 3 (PRMT3) was investigated in the mouse brain using indirect immunofluorescence. PRMT3 was observed to be localized in the cell bodies and dendrites of neurons but not in the axons and glial cells, indicating that PRMT3 is involved in neuronal function. The distribution of the immunoreactive neurons in the brain was uneven, indicating that PRMT3 plays a role in specific neuronal systems such as the motor and limbic systems, as well as functions related to the cerebellum. The present ontogenetic analysis of PRMT1 and PRMT3 using Western blot methodology clearly revealed that PRMT3 develops during the perinatal stage and its expression is maintained even in adulthood. PRMT1, on the other hand, is expressed transiently during the early embryonic stage. These findings indicate that PRMT3 is related with neuronal function in both young and adult brains, while PRMT1 has roles in the immature brain, such as the formation of neural circuits.

Brain malformations caused by retroviral vector-mediated gene transfer of hippocampal cholinergic neurostimulating peptide precursor protein into the CNS via embryonic mice ventricles.

Hippocampal cholinergic neurostimulating peptide precursor protein (HCNP-pp) is a unique multifunctional protein, being not only the precursor of HCNP, which promotes the phenotype development of septo-hippocampal cholinergic neurons, but also the binding protein of phosphatidylethanolamine, ATP, Raf-1 kinase (known as "Raf-1 kinase inhibitory factor" in peripheral organs), and serine protease. We obtained a high-titer retroviral vector harboring HCNP-pp cDNA by the use of a modified packaging cell line and centrifugation, and by injecting it into embryonic mouse ventricles, we investigated the function of its gene product within the central nervous system (CNS). We found that efficient transduction into hippocampal pyramidal neurons can be achieved by injecting the vector into embryonic brain ventricles on embryonic day 14 (E14). Three days after receiving the intraventricular injection of the high-titer HCNP-pp retrovirus vector on E14, the tissues around the ventricles showed an overexpression of HCNP-pp. This was accompanied by a reduced amount of activated MEK and Erk (as analyzed by histochemical and Western blot methods), suggesting that HCNP-pp also regulates the MAP-kinase cascade within the CNS. Surprisingly, mouse brains that received the HCNP-pp retroviral vector showed massive malformation of the hippocampus and cerebellum when examined 30 days after birth. This shows that strictly regulated HCNP-pp gene expression is necessary for the normal development of the mouse brain, and that the moderate overexpression achieved by retroviral vector-mediated gene transfer is sufficient to cause severe abnormality of entire brain structures.

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.

Mutant Plp/DM20 cannot be processed to secrete PLP-related oligodendrocyte differentiation/survival factor.

Most of the mutations within the PLP gene result in degeneration of oligodendrocytes and this is believed to be caused by intracellular trafficking defects. Previous studies have demonstrated that cells expressing the wild type PLP gene release a factor promoting differentiation/survival of oligodendrocyte and that this factor is the C-terminal portion of the protein itself. In this study we asked how the naturally occurring mutations of the PLP gene (jimpy, jimpy msd, and rumpshaker) affect this activity. We developed a transient expression system for retroviral production and transduction that enabled the expression of mutant PLP/DM20 cDNAs in NIH3T3 cells. None of the NIH3T3 cells producing mutant PLP/DM20s secreted the PLP-related factor that increases the number of oligodendrocytes. Since it has been shown that rumpshaker DM20 can be transported to the cell surface, but its folding is incorrect, absence of secretion of this factor is more heavily attributable to incorrect protein folding than to the defect in the PLP/DM20 trafficking.

Remyelination of the adult demyelinated mouse brain by grafted oligodendrocyte progenitors and the effect of B-104 cografts.

The 4e transgenic mouse is characterized by overexpression of the PLP gene. Heterozygous littermates containing three PLP gene copies develop and myelinate normally. However, a progressive CNS demyelination begins at 3-4 months of age. Despite focal demyelination, these animals survive for one year with hind limb paralysis. We used this CNS demyelination model to determine if grafts of CG4 oligodendrocyte progenitors would survive and myelinate the adult CNS. Either CG4 cells, or co-grafts of CG4/B 104 cells 11:1 ratio respectively) were performed. Grafted cells survived and migrated in the normal and transgenic brain. Non-treated transgenic animals revealed extensive lack of myelin. Three months post-transplant hosts with CG4 or co-transplants displayed a near normal myelin pattern. Double immunofluorescence for neurofilament and myelin basic protein revealed the presence of many naked axons in non-grafted transgenic animals. Those grafted with progenitor CG4 cells or cografts displayed a clear increase in remyelination. This data provides a new direction for the development of cell replacement therapies in demyelinating diseases.

Treatment of glioblastoma by direct inoculation of concentrated high titer-recombinant retrovirus carrying the herpes simplex virus thymidine kinase gene.

We prepared retroviruses carrying the lacZ gene or herpes simplex virus thymidine kinase (HTK) gene with titers of 1.4-2.5 x 10(11) colony-forming units (cfu)/ml, and stereotaxically inoculated only 3 microliters of the retroviruses into a mouse glioma model. This resulted in highly efficient transduction in vivo. The transduced glioma cells migrated far from the implantation site, potentiating the induction of the remarkable bystander effect. Following repetitive ganciclovir (GCV) intraperitoneal injection, effective killing of glioma cells in the mouse brain was observed. The transduction efficiency was nearly as high as that observed for the implantation of high-titer retrovirus-producing fibroblasts. Eighty per cent of brain tumor-bearing mice were completely cured by our treatment protocol using concentrated HTK-harboring retroviruses. Our results suggest that repeated inoculations of high-titer retroviruses carrying the HTK gene followed by GCV treatment may be a promising strategy for the clinical treatment of malignant gliomas. To achieve further safety in the gene therapy of glioma, genes abundantly expressed in human glioblastoma were searched by the Serial Analysis of Gene Expression (SAGE) technique. Among the top-147 most expressed tags in glioblastoma, we found a tag, TTTTGGGTAT, originated from an unidentified gene, which was not detected in human astrocyte cultures. Real-time quantitative RT-PCR showed that MAGE-E1 expression was 2.6-15 fold enriched in glioblastoma relative to human astrocytes. Expressed Sequence Tags (ESTs) containing this tag were homologous to melanoma-associated antigen gene (MAGE) family, and this new cDNA, named MAGE-E1, was cloned by 5'-rapid amplification of cDNA ends (RACE) technique. MAGE-E1 expression was enriched in glioblastoma and low in other cancers, and MAGE-E1 expression was detected only in brain and ovary among normal tissues. These results indicate that MAGE-E1 is a novel and glioma-specific member of MAGE family, which can be applied to glioma-specific gene transduction.

MAGE-E1, a new member of the melanoma-associated antigen gene family and its expression in human glioma.

To unearth glioma-specific genes in human glioblastoma, the serial analysis of gene expression technique was applied to a primary glioblastoma, using cultured human astrocytes as a normal control. Among the top 147 most-expressed tags in glioblastoma, we found a tag, TTTTGGGTAT, that originated from an unidentified gene and which was not detected in human astrocyte cultures. Real-time quantitative reverse transcription-PCR showed that MAGE-E1 expression was 2.6-15-fold enriched in glioblastoma relative to human astrocytes. Expressed sequence tags containing this tag were homologous to the melanoma-associated antigen gene (MAGE) family, and this new cDNA, named MAGE-E1, was cloned by the 5'-rapid amplification of cDNA ends technique. Three alternatively spliced variants (MAGE-E1a-c) were found, and deduced amino acid sequence showed that MAGE-E1a and -E1b shared the MAGE-conserved region, whereas -E1c did not. This suggests that although MAGE-E1c is expressed from one of the MAGE family, it has distinct functions from other members. Tissue distribution analysis showed that MAGE-E1 was distinct from other MAGEs. MAGE-E1 expression was detected only in brain and ovary among normal tissues. Interestingly, MAGE-E1a and/or -E1b were specifically expressed in glioma cells among cancer cells. These results indicate that MAGE-E1 is a novel and glioma-specific member of MAGE family.

Eradication of murine brain tumors by direct inoculation of concentrated high titer-recombinant retrovirus harboring the herpes simplex virus thymidine kinase gene.

Implantation of retrovirus-producing cells within a tumor has been demonstrated to eliminate malignant brain tumors effectively in animal models. In our previous study, the implantation of high-titer retrovirus-producing fibroblasts into tumors resulted in highly efficient transduction in vivo. The transduced glioma cells migrated far from the implantation site, potentiating the induction of the remarkable bystander effect. It is also possible, however, that the implantation of murine fibroblast-derived virus-producing cells may induce an immune response in patients. In this study, we prepared retroviruses carrying the herpes simplex virus thymidine kinase (HTK) gene with titers of 1.4--2.5 x 10(11) colony-forming units (c.f.u.)/ml, and stereotactically inoculated only 3 microl of the HTK-bearing retroviruses into the brain tumors of mice. Following repetitive ganciclovir (GCV) intraperitoneal injection, effective killing of glioma cells in the mouse brain was observed. The transduction efficiency was nearly as high as that observed for the implantation of high-titer retrovirus-producing fibroblasts. Eighty percent of brain tumor-bearing mice were completely cured by our treatment protocol using concentrated HTK-harboring retroviruses. Our results suggest that repeated inoculations of high-titer retroviruses carrying the HTK gene followed by GCV treatment may be a promising strategy for the clinical treatment of malignant gliomas.

Amelioration of retrovirus-mediated gene transfer into hepatocellular carcinoma cells.

Recombinant retroviruses are by far the most frequently used vehicle in clinical gene therapy. No serious side-effects have been reported so far in clinical gene therapy trials using recombinant retroviral systems. Low titers of recombinant retroviruses, however, have limited the usefulness of recombinant retroviruses. To improve the efficiency of retrovirus-mediated gene transfer, we previously introduced the polyomavirus early region into amphotropic PA317 cells and established a modified retroviral packaging cell line, PAMP51. We demonstrate here that recombinant retroviruses produced by PAMP51-derived retroviral producing cells have approximately 10-fold higher titers compared with those produced by conventional PA317-derived retroviral producing cells. Importantly, recombinant retroviruses produced by PAMP-derived retroviral producing cells could infect hepatocellular carcinoma cells much more efficiently and could induce much stronger expression of a lacZ reporter gene in HCC cells compared with those produced by PA317-derived ones. These results indicate that recombinant retroviruses prepared from PAMP51-derived retroviral producing cells are much more useful than those prepared from PA317-derived ones and that the use of PAMP51 retroviral packaging cells may open up new avenues for the treatment of various types of cancer including hepatocellular carcinoma.

Isolation and characterization of an N-linked oligosaccharide that is significantly increased in sera from patients with non-small cell lung cancer.

The structures of N-linked oligosaccharides present in human sera from 12 healthy volunteers and from 14 patients with non-small cell lung cancer (NSCLC) were analyzed by our recently developed partially automated systematic method. Thirty different structures of oligosaccharides were deduced, and these accounted for 84.1% of the total N-linked oligosaccharides present in human sera. All of the quantified oligosaccharide levels in healthy human sera were within twice the standard deviation. The amount of a triantennary trigalactosylated structure with one outer arm fucosylation (A3G3Fo) was found to be markedly increased in NSCLC patients in comparison to that in healthy volunteers (p < 0.01). No significant positive correlation with other clinical data was found. Serum A3G3Fo levels can thus be a novel marker for the diagnosis of NSCLC.

Regulation of oligodendrocyte development.

Oligodendrocytes are myelinating cells in the central nervous system. Recent studies demonstrated that oligodendrocyte progenitor cells are generated from a restricted region in the ventricular zone. In the rodent spinal cord, progenitor cells appear from narrow and bilateral longitudinal columns in the ventral ventricular zone, and then migrate dorsally. This ventral-specific appearance of oligodendrocyte progenitors may be controlled along the dorso-ventral axis in the spinal cord by extrinsic signals secreted from both the dorsal and ventral cords. The combined action of the Notch signaling pathway and a basic helix-loop-helix class of transcription factors may modulate this early specification of spinal oligodendrocytes and also be involved in multiple steps of oligodendrocyte differentiation.

Prognostic significance of polysialic acid expression in resected non-small cell lung cancer.

Polysialic acid (PSA) is a carbohydrate attached mainly to the neural cell adhesion molecule. Because PSA is composed of a linear homopolymer of alpha-2-8-linked sialic acid residues and has a large negative charge, the presence of PSA attenuates the adhesive property of neural cell adhesion molecule and increases cellular motility. In an earlier study, we demonstrated that PSA and STX, a polysialyltransferase, were associated with tumor progression in non-small cell lung cancer (NSCLC) (F. Tanaka et al., Cancer Res., 60: 3072-3080, 2000). Therefore, in the present study, to assess the prognostic significance of PSA in resected NSCLC, a total of 236 patients who underwent complete resection for pathological (p)-stage I-IIIa disease were reviewed retrospectively. PSA was expressed in 44 of 236 (18.6%) patients, and the expression was correlated with p-stage disease. For all p-stage patients, 5-year survival rates for those with PSA-positive and PSA-negative tumors were 52.1% and 71.3%, respectively, demonstrating a significantly worse prognosis for the PSA-positive patients (P = 0.012). Analysis for only p-stage I patients also demonstrated a significantly worse prognosis for the PSA-positive patients; 5-year survival rates of the PSA-positive and the PSA-negative patients were 45.1% and 83.5%, respectively, (P < 0.001). In addition, there proved to be no difference in the postoperative survival among p-stage I, II, and IIIa patients when PSA expression was positive. Multivariate analysis confirmed that PSA expression was an independent factor to predict poor prognosis in resected NSCLC. These results suggested that PSA could be an important clinical marker and that preoperative induction and/or postoperative adjuvant therapies should be performed for PSA-positive NSCLC, even if the disease is classified as p-stage I.

Change of oligosaccharides of rat brain microsomes depending on dietary fatty acids and learning task.

We have analyzed oligosaccharide chains in brain microsomes of rats fed an n-3 polyunsaturated fatty acid-deficient (safflower oil group; S group) or -rich (perilla oil group; P group) diet before and after brightness-discrimination learning tasks. The amount of concanavalin A-binding sites (mainly mannoside) of the brain microsomes was found to be significantly less in the S group than the P group before the learning task. Detailed analysis of glycoprotein glycans demonstrated that high mannose type oligosaccharides were dominant in brain microsomes before the learning task in both dietary groups, whereas multiantennary complex-type oligosaccharides became dominant after the learning task and especially a tetra-antennary glycan, that had a core structure of the glycan of neural cell adhesion molecule, was more increased in the S-group than the P group. When polysialylated glycans were analyzed on serotonin-conjugated HPLC column, the glycans in the S-group microsomes before the learning task contained larger amount of higher affinity-polysialylated glycans to serotonin column than those in the P-group, and also contained larger amount of phosphoglycans that showed also high affinity to serotonin column than the P-group. Removal of mannoside from microsomes by alpha-mannosidase-treatment changed the membrane surface physical property, especially permittivity, as revealed by analysis of the interaction with 1-anilinonaphthalene-8-sulfonate. These results suggest that high mannose content and several multiantennary glycans including polysialylated and phospho-glycans were changed by dietary n-3 fatty acid deficiency and learning task in rat brain microsomal glycoproteins and that these changes may affect membrane functions through changes of membrane surface physical properties and reactivity against serotonin.

Dorsal spinal cord inhibits oligodendrocyte development.

Oligodendrocytes are the myelinating cells of the mammalian central nervous system. In the mouse spinal cord, oligodendrocytes are generated from strictly restricted regions of the ventral ventricular zone. To investigate how they originate from these specific regions, we used an explant culture system of the E12 mouse cervical spinal cord and hindbrain. In this culture system O4(+) cells were first detected along the ventral midline of the explant and were subsequently expanded to the dorsal region similar to in vivo. When we cultured the ventral and dorsal spinal cords separately, a robust increase in the number of O4(+) cells was observed in the ventral fragment. The number of both progenitor cells and mature cells also increased in the ventral fragment. This phenomenon suggests the presence of inhibitory factor for oligodendrocyte development from dorsal spinal cord. BMP4, a strong candidate for this factor that is secreted from the dorsal spinal cord, did not affect oligodendrocyte development. Previous studies demonstrated that signals from the notochord and ventral spinal cord, such as sonic hedgehog and neuregulin, promote the ventral region-specific development of oligodendrocytes. Our present study demonstrates that the dorsal spinal cord negatively regulates oligodendrocyte development.

Expression of polysialic acid and STX, a human polysialyltransferase, is correlated with tumor progression in non-small cell lung cancer.

Polysialic acid (PSA) is a carbohydrate composed of a linear homopolymer of alpha-2-8-linked sialic acid residues and is mainly attached to the neural cell adhesion molecule (NCAM). Because of the large negative charge of PSA, presence of PSA attenuates the adhesive property of NCAM and increases the cellular motility. PSA expression on NCAM is developmentally regulated, and PSA plays important roles in formation and remodeling of the neural system through regulation of the adhesive property of NCAM. Expression of the polysialated form of NCAM has been also demonstrated in some malignant tumors, such as Wilms' tumor and small cell lung cancer. Despite the possible importance as an onco-developmental antigen, however, significance of PSA expression in most malignant tumors has not been revealed. Therefore, PSA expression in non-small cell lung cancer was assessed in the present study. PSA was expressed only in 5 (20.8%) of 24 pathological stage I cases, whereas it was expressed in most stage IV cases (76.8%, 11 of 14 cases). PSA expression was correlated with nodal metastasis and distant metastasis, but not with local extent of the primary tumor. Next, expression of polysialyltransferase genes (PST and STX genes) which controlled formation of PSA, was examined. The PST gene was constantly expressed in both normal lung tissue and tumor tissue of all cases. In contrast, the STX gene was not expressed in normal lung tissue of any case, and STX gene expression in tumor tissue was closely correlated with tumor progression. The STX gene was expressed only in 1 (4.2%) of 24 stage I cases, whereas it was expressed in most stage IV cases (85.7%, 12 of 14 cases). These results suggested that the PSA and STX genes could be new targets of cancer therapy as well as important clinical markers.

A-type K+ current mediated by the Kv4 channel regulates the generation of action potential in developing cerebellar granule cells.

During neuronal differentiation and maturation, electrical excitability is essential for proper gene expression and the formation of synapses. The expression of ion channels is crucial for this process; in particular, voltage-gated K(+) channels function as the key determinants of membrane excitability. Previously, we reported that the A-type K(+) current (I(A)) and Kv4.2 K(+) channel subunit expression increased in cultured cerebellar granule cells with time. To examine the correlation between ion currents and the action potential, in the present study, we measured developmental changes of action potentials in cultured granule cells using the whole-cell patch-clamp method. In addition to an observed increment of I(A), we found that the Na(+) current also increased during development. The increase in both currents was accompanied by a change in the membrane excitability from the nonspiking type to the repetitive firing type. Next, to elucidate whether Kv4.2 is responsible for the I(A) and to assess the effect of Kv4 subunits on action potential waveform, we transfected a cDNA encoding a dominant-negative mutant Kv4.2 (Kv4.2dn) into cultured cells. Expression of Kv4.2dn resulted in the elimination of I(A) in the granule cells. This result demonstrates that members of the Kv4 subfamily are responsible for the I(A) in developing granule cells. Moreover, elimination of I(A) resulted in shortening of latency before the first spike generation. In contrast, expression of wild-type Kv4.2 resulted in a delay in latency. This indicates that appearance of I(A) is critically required for suppression of the excitability of granule cells during their maturation.

Astrocyte differentiation of fetal neuroepithelial cells by interleukin-11 via activation of a common cytokine signal transducer, gp130, and a transcription factor, STAT3.

The interleukin (IL)-6 family cytokines utilize membrane glycoprotein gp130 in common as a critical signal-transducing receptor component. IL-11, a cytokine initially identified as a plasmacytoma growth factor, belongs to this family. We show here that IL-11 and its cognate receptor components are expressed in fetal mouse neuroepithelial cells. We also show that after 4 days of culture with IL-11, cells with typical astrocytic morphologies expressing glial fibrillary acidic protein (GFAP; a marker for astrocytes) come out. This differentiation process is totally dependent on the gp130-mediated signal-transduction pathway involving activation of a latent cytoplasmic transcription factor, STAT3 (for signal transducer and activator of transcription 3), because (a) IL-11-induced astrocyte differentiation is not observed when neuroepithelial cells prepared from gp130-deficient mice were used, (b) stimulation of neuroepithelial cells by IL-11 rapidly induces tyrosine-phosphorylation of STAT3, and (c) transfection of neuroepithelial cells with a dominant-negative form of STAT3 inhibits IL-11-induced activation of the GFAP gene promoter. We have further identified, in the GFAP promoter region, a STAT3 site at which nucleotide substitutions almost completely abolished the IL-11-induced GFAP promoter activation. Taken together, it is suggested that IL-11 contributes to astrocytogenesis in fetal brain via activation of gp130 and STAT3.