Chihiro Akazawa

Involvement of P2X4 and P2Y12 receptors in ATP-induced microglial chemotaxis

We previously reported that extracellular ATP induces membrane ruffling and chemotaxis of microglia and suggested that their induction is mediated by the Gi/o‐protein coupled P2Y12 receptor (P2Y12R). Here we report discovering that the P2X4 receptor (P2X4R) is also involved in ATP‐induced microglial chemotaxis. To understand the intracellular signaling pathway downstream of P2Y12R that underlies microglial chemotaxis, we examined the effect of two phosphatidylinositol 3′‐kinase (PI3K) inhibitors, wortmannin, and LY294002, on chemotaxis in a Dunn chemotaxis chamber. The PI3K inhibitors significantly suppressed chemotaxis without affecting ATP‐induced membrane ruffling. ATP stimulation increased Akt phosphorylation in the microglia, and the increase was reduced by the PI3K inhibitors and a P2Y12R antagonist. These results indicate that P2Y12R‐mediated activation of the PI3K pathway is required for microglial chemotaxis in response to ATP. We also found that the Akt phosphorylation was reduced when extracellular calcium was chelated, suggesting that ionotropic P2X receptors are involved in microglial chemotaxis by affecting the PI3K pathway. We therefore tested the effect of various P2X4R antagonists on the chemotaxis, and the results showed that pharmacological blockade of P2X4R significantly inhibited it. Knockdown of the P2X4 receptor in microglia by RNA interference through the lentivirus vector system also suppressed the microglial chemotaxis. These results indicate that P2X4R as well as P2Y12R is involved in ATP‐induced microglial chemotaxis.

Selective expression of Gi/o-coupled ATP receptor P2Y12 in microglia in rat brain

Extracellular nucleotides, including ATP, have been demonstrated to transmit important physiological signals in the brain through either G‐protein‐coupled P2Y receptors or P2X receptors, which are ligand‐gated ion channels. In this study, we performed a detailed analysis of the expression of the Gi/o‐coupled receptor P2Y12 in the brain. Northern blot analysis demonstrated that P2Y12 is expressed predominantly in the brain, and to a lesser extent in the spleen. The cellular localization of P2Y12 was investigated by in situ hybridization, and P2Y12 mRNA was detected in small cells distributed throughout the brain, including the hippocampus. Expression of P2Y12 was also observed in naive and axotomized facial nuclei, and the number of P2Y12‐expressing cells increased following facial nerve axotomy. Selective expression of P2Y12 mRNA in microglia was confirmed by double‐label in situ hybridization and immunohistochemistry with antibodies against NeuN and Iba1 as an immunohistochemical marker for neurons and microglia, respectively. Hardly any P2Y12 mRNA was detected in macrophages obtained from the spleen and abdominal cavity, which share many surface molecules with microglia.

Expression of mRNAs of l-AP4-sensitive metabotropic glutamate receptors (mGluR4, mGluR6, mGluR7) in the rat retina

Expression patterns of mRNAs of L-AP4-sensitive metabotropic glutamate receptors (mGluR4, mGluR6, mGluR7) in the rat retina were examined by northern blot analysis and in situ hybridization histochemistry. Expression patterns of mGluR4 and mGluR7 mRNAs were quite different from that of mGluR6 mRNA which was expressed at the outer part of the inner nuclear layer. The mGluR4 mRNA was expressed on the cell bodies of the ganglion cells, but not in the inner or outer nuclear layer. The expression of mGluR7 mRNA was observed throughout the entire region of the inner nuclear layer and on the cell bodies of the ganglion cells.

The Upregulated Expression of Sonic Hedgehog in Motor Neurons after Rat Facial Nerve Axotomy

Nerve injury leads to the induction of a large number of genes to repair the damage and to restore synaptic transmission. We have attempted to identify molecules whose mRNA expression is altered in response to facial nerve axotomy. Here we report that facial nerve axotomy upregulates Sonic hedgehog (Shh) and its receptor Smoothened (Smo) in facial motor neurons of adult rats, whereas facial nerve axotomy does not upregulate mRNA of Shh or Smo in neonatal rats. We tested whether overexpression of Shh in facial motor neurons of axotomized neonatal rats may promote neuronal survival. Adenovirus-mediated overexpression of Shh, but not that ofβ-galactosidase, transiently rescues axotomy-induced neuronal cell death for 3-5 d after axotomy. Finally, the pharmacological inhibitor of Shh signaling, cyclopamine, induces motor neuron death in adult rats after axotomy. These results suggest that Shh plays a regulatory role in nerve injury.

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.

Distribution of the galectin-1 mRNA in the rat nervous system: its transient upregulation in rat facial motor neurons after facial nerve axotomy

Galectin-1 is a member of the animal lectin family that displays conserved consensus sequences and similar carbohydrate binding specificities. Recent analyses revealed that galectin-1 plays an important role in the process of nerve regeneration. We analyzed the topological expression of galectin-1 mRNA in adult rat nervous system. Galectin-1 mRNA was predominantly observed in the cell bodies of neurons such as oculomotor nucleus (III), trochlear nucleus (IV), trigeminal motor nucleus (V), abducens nucleus (VI), facial nucleus (VII), hypoglossal nucleus (XII), red nucleus, and locus ceruleus. Neurons in pineal gland and dorsal root ganglia expressed galectin-1 mRNA. We next tested whether the axotomy of facial nerve altered the expression of galectin-1 mRNA in motor neurons. In the adult rats, the axotomy of facial nerve induced transient upregulation of galectin-1 mRNA around 6 h after axotomy. These results indicate that galectin-1 may play roles in the early event of the nerve injury and regeneration through the transient change of its expression level.

Ubiquitylation and Degradation of Serum-inducible Kinase by hVPS18, a RING-H2 Type Ubiquitin Ligase

Serum-inducible kinase (SNK) is a member of polo-like kinases that serve as regulators of multiple events during cell division. Rapid changes in the activity and abundance of SNK were reported after the serum stimulation and after the activation of synaptic transmission in the brain. Yet the detailed mechanisms that control the level of SNK protein have not been fully elucidated. In this report, we show that the RING-H2 domain of hVPS18 (human vacuolar protein sorting 18) has a genuine ubiquitin ligase (E3) activity. Using the yeast two-hybrid screening, we identify SNK as a candidate substrate of hVPS18. The half-life of SNK is increased in HeLa cells that down-regulated hVPS18 by lentivirus-mediated small hairpin RNA interference. Furthermore, the delayed entry into S phase is observed in HeLa cells overexpressing hVPS18. These results suggest that hVPS18 may play an important role in regulation of SNK activity through its ubiquitin ligase.

Expression of human GFRα-1 (GDNF receptor) at the neuromuscular junction and myelinated nerves

Motor neurons have been known to require a wide variety of neurotrophic factors for their survival. As one of the target-derived trophic factors, glial cell line-derived neurotrophic factor (GDNF) has been shown to exert its effects on motor neurons via a receptor complex including GDNF receptor alpha 1 (GFRα-1). Immunoreactivity of GFRα-1 was observed at myelinated peripheral nerves and neuromuscular junction (NMJ) of human skeletal muscles. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses showed that mRNA of GFRα-1 existed in the ventral horn of human spinal cord, but not in the skeletal muscles. The results suggested that GFRα-1 might play a key role for uptake and internalization of GDNF at the human NMJ.

Expression of the mammalian homologue of vacuolar protein sorting 16 (Vps16p) in the mouse and rat brain

Synaptic vesicle fusion events are essential for synaptic transmission. Membrane docking and fusion events are highly regulated processes requiring the participation of a large number of soluble N-ethylmaleimide-sensitive factors (SNAREs) and SNARE-interacting proteins. We report the neuronal expression of mammalian homologue of vacuolar protein sorting 16 (mVps16p) which exhibits a high homology to the yeast Vps16p, a component of Class C Vps. Western blot and immunohistochemical analyzes revealed that mVps16p is highly expressed in the various brain areas and developmental stages tested. The immunoreactivities of mVps16p colocalized with microtubule associated protein 2, but not glial fibrillary acidic protein. In the primary culture of cortical neurons, mVps16p immunoreactivities were observed in the cell body and the neuronal processes, and highly enriched in the axonal outgrowths.

Characterization of neural crest-derived cells and oligodendrocyte using Sox10-VENUS mice

we report the dynamic, autonomous formation of the optic cup (retinal primordium) structure from a three-dimensional culture of mouse ES cell aggregates. ES cell-derived retinal epithelium spontaneously formed hemispherical epithelial vesicles that become patterned along its proximal–distal axis. While the rigid proximal portion differentiated into pigment epithelium, the flexible distal portion progressively folded inward to form a shape reminiscent of the embryonic optic cup, exhibited interkinetic nuclear migration and generated stratified neural retinal tissue, just as seen in vivo. We demonstrate that optic-cup morphogenesis in this simple cell culture depends on an intrinsic self-organizing program involving stepwise and domain-specific regulations in local epithelial properties. Research fund: KAKENHI40415097.