Tohru Yamakuni

Development and migration of Purkinje cells in the mouse cerebellar primordium

SummaryThe mode of Purkinje cell migration in the mouse cerebellar primordium was examined immunohistochemically, by marking Purkinje cells with anti-spot 35 antibody and labeling them with 5′-bromodeoxyuridine. The cells migrated radially from the neuroepithelium of the fourth ventricle towards the cortical surface between the 13th and 17th days (E13–E17) of gestation. Regional differences in the migratory process were evident: the final settlement of the Purkinje cells proceeded earlier in the lateral and posterior parts of the primordium, exhibiting latero-medial and posteroventral-anterodorsal diminishing sequences. To elucidate the factors involved in the migration, the arrangement of radial glial fibers, and expression of the cell adhesion molecule, tenascin, were examined immunohistochemically with the monoclonal antibody 1D11, a marker for both immature and mature astroglia, and an anti-tenascin antibody. At E14, 1D11-immunopositive fibers were seen to extend from the ventricle to the pial surface, and the cell bodies of immature glia migrated after E15 towards the cortex, shortening the radial processes whose end-feet were attached to the pia mater. Tenascin, which possesses a neuron-glial adhesiveness, was also expressed on the radial fibers during the migration of the Purkinje cells. The fibers were closely apposed to the migratory Purkinje cells, and their arrangement and orientation accorded with the migratory direction of the Purkinje cells. Further, changes in the molecular species of antigens detected by both the 1D11 and anti-tenascin antibodies were observed by immunoblotting analysis during the course of cerebellar development. These findings suggest that the arrangement of radial glia and expression of adhesion molecules may be involved in the control and guidance of Purkinje cell migration.

Presynaptic impairment of cerebellar inhibitory synapses by an autoantibody to glutamate decarboxylase

Glutamic acid decarboxylase (GAD), the enzyme responsible for converting glutamate to gamma-aminobutyric acid (GABA), is a target of humoral autoimmunity in stiff-man syndrome and subacute cerebellar ataxia. Recently, we found that an anti-GAD autoantibody in the CSF of an ataxic patient selectively suppressed GABA-mediated transmission on cerebellar Purkinje cells without affecting glutamate-mediated transmission. Here, we examine the mechanism by which the autoantibody impaired the inhibitory transmission, using immunohistochemistry and whole-cell recording in rat cerebellar slices. The present results indicate that CSF immunoglobulins prepared from an ataxic patient acted on the presynaptic terminals of GABAergic interneurons and decreased GABA release onto Purkinje cells.

Expression of β-nerve growth factor mRNA in rat glioma cells and astrocytes from rat brain

A 50‐base synthetic oligodeoxynucleotide complementary to a portion of mouse nerve growth factor (NGF) mRNA was used as a probe for analysis of the expression of NGF gene. Northern blot analysis showed the presence of a major 1.3 kb transcript, which was identical in size to mouse NGF mRNA, in both C6Bu1 cells and rat astrocytes cultured from newborn rat brain. Further, the rearrangement of DNA sequence in and around the NGF gene locus of C6Bu1 cells was not detected by Southern blot analysis. These results indicate the expression of NGF mRNA in both C6Bu1 cells and astrocytes from rat brain, suggesting that astrocytes may produce NGF protein in the rat brain, especially in developing rat brain.

Production of nerve growth factor in rat skeletal muscle.

Production of the nerve growth factor (NGF) was confirmed by Northern blot hybridization using a specific probe of synthetic cDNA. In normal rat skeletal muscle, this probe clearly showed a band equivalent to 1.3 kilobases (kb) of messenger RNA (mRNA) of NGF of male mouse submaxillary gland. By denervation, the density of the bands derived from muscles increased by a factor of more than 3 at 4 and 6 days later compared to the control. The synthesis of mRNA of NGF in muscle was also confirmed following tetrodotoxin (TTX) blockade of sciatic nerve without denervation.

Differential gene expression of fibroblast growth factor receptor isoforms in rat ovary.

The gene expression of four fibroblast growth factor receptors (flg, bek, FGFR-3, and FGFR-4) in rat ovary cells was studied. Northern blot hybridization revealed that flg and bek mRNAs were detectable during all stages except a diestrus stage, whereas FGFR-3 and FGFR-4 mRNAs were almost undetectable throughout the cycles. In situ hybridization also demonstrated that only flg and bek gene expression was detectable. A modest flg mRNA signal was detected in developing antral follicles and it was more prominent in the theca-interstitial cells than in the granulosa cells. A modest to weak flg mRNA signal was seen in the hypertrophied theca-interstitial cells of atretic follicles and a very weak flg mRNA signal was observed in the corpora lutea. On the other hand, a weak bek mRNA signal was seen in granulosa and theca-interstitial cells in developing follicles and also hypertrophied theca-interstitial cells of atretic follicles, but not in the corpora lutea. Intense signals for both flg and bek mRNAs were unexpectedly found in the epithelium of paroophoron at the hilus. These results demonstrate that the bFGF receptor isoforms are expressed differentially in the rat ovary cells.

Down-regulation of an ankyrin repeat-containing protein, V-1, during skeletal muscle differentiation and its re-expression in the regenerative process of muscular dystrophy

Using Western blot analysis and immunohistochemical methods, we examined the expression of V-1, a member of the ankyrin repeat-containing protein family, during differentiation and regeneration of skeletal muscle. The expression of V-1 was high in cultured myoblasts and decreased during their differentiation into myotubes, while high expression was maintained when muscle differentiation was inhibited by treatment with basic fibroblast growth factor. Down-regulation of V-1 also occurred during in vivo muscle differentiation from embryonic to postnatal stages, reaching an undetectable level in mature skeletal muscle. In contrast, strong V-1 immunoreactivity was detected again in myoblasts and regenerating muscle fibers with a small diameter, which were observed in Duchenne muscular dystrophy and its animal model, mdx mouse. Thus, it seems that V-1 is a good marker for early stage of muscle regeneration and changes of its expression suggest that V-1 plays a role in prenatal muscle differentiation and postnatal muscle regeneration.

Murine central neurons express a novel member of the cdc10/SWI6 motif-containing protein superfamily

V-1 protein is a novel member of the cdc10/SWI6 motif-containing protein superfamily several members of which have been demonstrated to play crucial roles in the regulation of intracellular signaling. In the present study we examined the distribution of V-1 mRNA in the murine central nervous system (CNS). Northern analysis revealed the expression of V-1 mRNA in various regions of the brain with the following rank order: hippocampus, cerebellum > cerebral cortex, olfactory bulb, medulla oblongata, pons > thalamus. In situ hybridization also showed that V-1 mRNA is widely distributed in various regions of the brain, with parallel expression levels to those revealed by Northern analysis. Immunohistochemical analysis revealed that the V-1 protein exists in various types of neurons, mainly in cell bodies but also in dendrites, axons and possibly in synaptic areas. These expression patterns of the V-1 gene in the murine CNS suggest that the V-1 protein performs some common function in different classes of neurons. We found no significant difference in the expression level of V-1 mRNA in cerebellar granule cells between the control and mutant mice of Purkinje cell degeneration (pcd). In comparison with our previous data obtained in another mutant, staggerer, we discussed the effects of target deprivation on the expression of V-1 mRNA in cerebellar granule cells.

Production and secretion of nerve growth factor by clonal striated muscle cell line, G8-1.

Two specific methods, Northern blot analysis using a 50 nucleotides probe to the conserved region of the nerve growth factor (NGF) gene, and enzyme immunoassay using a monoclonal biotinylated rat anti-NGF IgG-avidin conjugated peroxidase system, were used to demonstrate the production and secretion of NGF by mouse striated muscle cell line G8-1. Calcium ionophore, A23187 (0.1-1 microM), forskolin (0.1-100 microM) and dibutyryl cyclic AMP (0.1-10 mM) strongly decreased the secretion of ir-NGF. The level of NGF mRNA was decreased by veratridine, A23187, forskolin and cyclic AMP but not by cyclic GMP. Consequently, we conclude that the secretion of NGF molecules paralleled the changes of NGF mRNA levels in the cells induced by all agents tested. Carmamylcholine also decreased the level of NGF mRNA. Immunoblot analysis suggested that denatured ir-NGF molecules exist in a higher molecular weight form (22 KDa) than those of mouse submaxillary gland (13 KDa). Both Ca(2+)- and cAMP mediated mechanisms contribute to the decreased production of NGF mRNA in the cells and the consequent inhibition of secretion of NGF molecules. Finally, molecular cloning of NGF of G8-1 cells was conducted and confirmed the structure of the gene that consists of 1, 3, and 4 exons deleting exon 2. Thus, G8-1 NGF is derived from transcript B.

Enhancement of Noradrenergic Phenotype Expression in Transgenic Mice Overexpressing V-1, A Cytoplasmic Ankyrin Repeat Protein

Catecholaminergic cells exhibit three types of transmitter phenotypes: dopaminergic, noradrenergic and adrenergic. The catecholaminergic phenotypes are thought to be determined by a phenotype-specific coordinate regulation of gene expression of the four catecholamine synthesizing enzymes, i.e. tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). However, molecular mechanisms that control the coordinate transcriptional regulation of these genes remain to be fully elucidated, although the second messenger systems and transcription factors involved in catecholaminergic cell-specific gene expression for catecholamine synthesizing enzymes have been so far identified and extensively investigated (Zetterstrom et al., 1997; Morin et al., 1997; Lo et al., 1999). Recently, an ankyrin repeat protein designated V-1 has been suggested to act as a possible coordinate regulator for the expression of genes encoding catecholamine synthesizing enzymes, since it has been demonstrated that stable overexpression of V-1 protein in the catecholaminergic cell line, PC12D cell, augments the expression of transcripts encoding TH, AADC and DBH, and as a resuit, catecholamine production is upregulated (Yamakuni et al., 1998). V-1 is a 12 kDa protein containing 2.5 copies of the ankyrin repeat, which has been originally isolated as a developmentally regulated protein from murine cerebellum (Taoka et al., 1992).

A New Regulatory Protein of Catecholamine Synthesizing-Enzyme Expression

Publisher Summary Direct protein sequencing and cDNA cloning revealed that V-1 is a novel protein that consists of 117 amino acids and contains 2.5 contiguous repeats of the cdc10/SWI6 motif, alternatively called the cell cycle motif or ankyrin repeat. It was initially tested if there are any changes in neuron-like properties, including neurotransmitter biosynthesis, ion channel currents, and nerve growth factor-inducible neurite elongation and branching among these stable transfectants. By screening, an increase in dopamine content in the V-1-overexpressing clones compared with in the control clones was found. Further, tyrosine hydroxase (TH) enzyme activities and repetitively quantified dopamine contents were determined. The results of biochemical and neurochemical assays indicated that changes in TH enzyme activity appear to be responsible for the increases in dopamine content observed in the two V-1-overexpressing clones. It is reasonable to suggest that upregulation of mRNA expression of three catecholamine biosynthesizing enzymes, observed in the V-1-overexpressing clones, results from the cooperative action of the V-1 protein with a partner protein that influences transcription factors or transcription regulators that regulate the TH, AADC, and DBH genes. To generate PC12 cell lines that overexpress the V-1 gene in a stable manner, PC12D cells with a human elongation factor 1 gene promoter-driven expression vector carrying the rat V-1 cDNA was transfected. Following neomycin selection, neomycin-resistant clones were screened by Western blot analysis with anti-V-1 antibody. On the other hand, there were no significant differences in V-1 expression levels among control clones transfected with the vector alone, C-7 and C-9. These stable clones were used for further phenotype analyses.