Yuhei Miyata

Sensitive assay of RNA interference in Drosophila and Chinese hamster cultured cells using firefly luciferase gene as target.

A sensitive cellular assay system for RNA interference was developed using the firefly luciferase gene as target. RNA interference was noted not only in Drosophila cultured cells but Chinese hamster cells (CHO‐K1) as well, although double‐stranded RNA required for the latter was 2500 times more than for the former. Cognate double‐stranded RNA as short as 38 bp was found to be still capable of inducing RNA interference in Drosophila cultured cells.

Newly established cell lines from Drosophila larval CNS express neural specific characteristics.

SummaryFrom the central nervous system ofDrosophila melanogaster 3rd instar larvae, eight continuous cell lines have been established (named ML-DmBG1 to 8). Using ML-DmBG2, single colony isolation was carried out and six colonial clones were obtained. All reacted to the antibody to horseradish peroxidase, which is a neuronal marker in insects. Acetylcholine, a known neurotransmitter inDrosophila, was detected in three of the colonial clones by high performance liquid chromatography. Therefore, it is concluded that the established colonial clones are neural cells originating in the larval central nervous system. Among them, some variation was observed with respect to morphology, acetylcholine content, and reactivity to anti-HRP. The variation may reflect the heterogeneity of cells composing the central nervous system.

Schwann cell myelination occurred without basal lamina formation in laminin α2 chain-null mutant (dy3K/dy3K) mice

The laminin α2 chain is a major component of basal lamina in both skeletal muscle and the peripheral nervous system. Laminin α2 chain deficiency causes merosin‐deficient congenital muscular dystrophy, which affects not only skeletal muscles, but also the peripheral and central nervous systems. It has been reported that the formation of basal lamina is required for myelination in the peripheral nervous system. In fact, the spinal root of dystrophic mice (dy/dy mice), whose laminin α2 chain expression is greatly reduced, shows lack of basal lamina and clusters of naked axons. To investigate the role of laminin α2 chain and basal lamina in vivo, we examined the peripheral nervous system of dy3K/dy3Kmice, which are null mutants of laminin α2 chain. The results indicate the presence of myelination although Schwann cells lacked basal lamina in the spinal roots of dy3K/dy3K mice, suggesting that basal lamina is not an absolute requirement for myelination in vivo. Immunohistochemically, the expression of laminin α4 chain was increased and laminin α5 chain was preserved in the endoneurium of the spinal root. Laminin α4 and α5 chains may play the critical role in myelination instead of laminin α2 chain in dy3K/dy3Kmice. In addition, the motor conduction velocity of the sciatic nerve was significantly reduced compared with that of wild‐type littermate. This reduction in conduction velocity may be due to small axon diameter, thin myelin sheath and the patchy disruption of the basal lamina of the nodes of Ranvier in dy3K/dy3Kmice. GLIA 35:101–110, 2001.

Prominent expression of glial cell line-derived neurotrophic factor in human skeletal muscle

Glial cell line–derived neurotrophic factor (GDNF) has been shown to exert neurotrophic effects on motor neurons as well as mesencephalic dopaminergic neurons. Because GDNF promotes survival of motor neurons in vivo and in vitro and rescues motor neurons from naturally occurring cell death, the potential use of GDNF for treatment of motor neuron diseases has been a major focus of recent research. The expression of GDNF in humans, however, has not been fully examined. In the present study, we examined the expression of GDNF in adult human muscle by Northern blot, reverse transcriptase polymerase chain reaction (RT‐PCR), and immunohistochemical analyses to address physiological roles of GDNF in humans. Northern blot analysis demonstrated high expression of GDNF mRNA in human skeletal muscle when compared to that of mouse. Intense GDNF immunoreactivity was observed in the vicinity of plasma membranes of skeletal muscle, particularly at neuromuscular junctions. GDNF immunoreactivity was also observed within the axons and surrounding Schwann cells of peripheral nerves. However, RT‐PCR detected expression of GDNF mRNA only in skeletal muscle, and not within the anterior horn cells of human spinal cord. These results suggest that GDNF is produced by skeletal muscle and taken up at the nerve terminals for retrograde transport by axons. Thus, GDNF in human skeletal muscle may be involved in promoting motor neuron survival as a target‐derived neurotrophic factor. J. Comp. Neurol. 402:303–312, 1998.

A novel gene derived from developing spinal cords, SCDGF, is a unique member of the PDGF/VEGF family

We isolated a novel gene designated spinal cord‐derived growth factor (SCDGF). Its expression was increased in chick spinal cords with embryonic development and decreased after hatching. The amino acid sequences of chick and human SCDGFs revealed a putative signal sequence followed by a CUB domain and a region homologous to the members of the platelet‐derived growth factor/vascular endothelial growth factor family. Furthermore, human SCDGF secreted from the cells showed a mitogenic activity for 10T1/2 cells in vitro. These results led us to speculate that SCDGF plays an important role in the development of the spinal cord.

Up-regulation of glial cell line-derived neurotrophic factor (GDNF) expression in regenerating muscle fibers in neuromuscular diseases

Glial cell line-derived neurotrophic factor (GDNF) has been shown to exert a target-derived trophic factor for motor neurons. Immunohistochemical analyses revealed that expression of GDNF in regeneration muscle fibers was up-regulated in polymyositis (PM) and Duchenne type muscular dystrophy (DMD). Reverse transcriptase polymerase chain reaction (RT-PCR) analyses showed that the full length GDNF was up-regulated in PM and DMD muscle; normal muscle exhibited mostly truncated GDNF. The results indicate that the GDNF expression is regulated in regeneration of human skeletal muscle.

The expression of SCDGF/PDGF-C/fallotein and SCDGF-B/PDGF-D in the rat central nervous system.

We examined the expression patterns of the two homologous genes, spinal cord-derived growth factor (SCDGF)/platelet-derived growth factor (PDGF)-C/fallotein and SCDGF-B/PDGF-D in the rat central nervous system. In the spinal cord, SCDGF/PDGF-C/fallotein was expressed in the floor plate at embryonic day (E) 11 and also in the ventricular zone at E16 but not in adult. However, SCDGF-B/PDGF-D was prominently expressed in the adult motoneurons, although faint expression was observed in the ventral ventricular zone at E16. Also in the brain, the expression of SCDGF/PDGF-C/fallotein was more remarkable at E16 than at adult. It was highly expressed in the cortex, pontine area and choroid plexus at E16. Contrary to SCDGF/PDGF-C/fallotein, SCDGF-B/PDGF-D expression was notable in several nuclei at adult.

H-7-induced apoptosis in the cells of a Drosophila neuronal cell line through affecting unidentified H-7-sensitive substance(s)

The present study was undertaken to reveal underlying mechanisms of apoptosis in neurons using clonal neuronal cells, ML-DmBG2-c2, derived from Drosophila larval central nervous system 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7), a protein kinase inhibitor, induced cell death with typical features of apoptosis such as internucleosomal DNA fragmentation, nuclear condensation and apoptotic bodies in the cells. Though H-7 is known to inhibit cAMP-dependent protein kinase (PKA), protein kinase C (PKC), cGMP-dependent protein kinase (PKG), myosin light chain kinase (MLCK), and casein kinase I (CKI), specific inhibitors for these kinases such as H-89, calphostin C, ML-9, or CKI-7 did not induce apoptosis in the cells. Other kinases such as tyrosine kinase. PI3-kinase and Ca2+/CaM kinase II so far examined in the present study were interpreted not to be involved in the apoptotic cascade. Therefore, it is concluded that an H-7-sensitive substance(s) other than these kinases is responsible for the apoptosis in the neuronal cells. Caspase inhibitors prevented apoptosis in the cells treated with H-7. These results suggest that caspase(s) is involved downstream of the H-7-sensitive point in the cascade of the apoptosis.

Chemical analysis of neurotransmitter candidates in clonal cell lines from Drosophila central nervous system. I. ACh and l-DOPA

To characterize neuronal phenotypes, aminergic neurotransmitters, i.e. acetylcholine (ACh), catecholamine (CA)s and other biogenic amines (serotonin, octopamine), were surveyed in 10 colonial clones from one cell line of Drosophila larval CNS using HPLC-ECD system. ACh, which is a neurotransmitter in Drosophila, was found in 7 out of 10 clones. CAs, their metabolites, and other amines were not detected in any clones. However, all the 10 clones expressed L-dopa, a precursor of CAs. Consequently, seven clones expressed ACh and L-dopa. L-dopa as a novel neurotransmitter candidate was discussed.

Calmodulin-dependent and -independent apoptosis in cells of a Drosophila neuronal cell line

This study was undertaken to reveal apoptotic pathways in neurons using a Drosophila neuronal cell line derived from larval central nervous system. We could induce apoptotic cell death in the cells by a Ca2+ ionophore (A23187), a protein kinase inhibitor (H-7), an RNA synthesis inhibitor (actinomycin D) and a protein synthesis inhibitor (cycloheximide). All the apoptosis induced by each chemical required Ca2+ ions, although the origin of Ca2+ ions were different: apoptosis induced by A23187 was dependent on extracellular Ca2+ ions whereas those by the other three chemicals utilized intracellular Ca2+ ions. Furthermore, different reactions to W-7, a calmodulin inhibitor, were found: W-7 prevented the cell death by each of the three chemicals but not by A23187. Based on the results, we proposed that the apoptotic pathways are classified into two types in individual cells. One pathway induced by H-7, actinomycin D or cycloheximide is calmodulin-dependent (pathway H), and another induced by A23187 is calmodulin-independent (pathway A).