Satoru Arata

Tlx3 and Tlx1 are post-mitotic selector genes determining glutamatergic over GABAergic cell fates

Glutamatergic and GABAergic neurons mediate much of the excitatory and inhibitory neurotransmission, respectively, in the vertebrate nervous system. The process by which developing neurons select between these two cell fates is poorly understood. Here we show that the homeobox genes Tlx3 and Tlx1 determine excitatory over inhibitory cell fates in the mouse dorsal spinal cord. First, we found that Tlx3 was required for specification of, and expressed in, glutamatergic neurons. Both generic and region-specific glutamatergic markers, including VGLUT2 and the AMPA receptor Gria2, were absent in Tlx mutant dorsal horn. Second, spinal GABAergic markers were derepressed in Tlx mutants, including Pax2 that is necessary for GABAergic differentiation, Gad1/2 and Viaat that regulate GABA synthesis and transport, and the kainate receptors Grik2/3. Third, ectopic expression of Tlx3 was sufficient to suppress GABAergic differentiation and induce formation of glutamatergic neurons. Finally, excess GABA-mediated inhibition caused dysfunction of central respiratory circuits in Tlx3 mutant mice.

Rnx deficiency results in congenital central hypoventilation.

The genes Tlx1 (Hox11), Enx (Hox11L1, Tlx-2 ) and Rnx (Hox11L2, Tlx-3) constitute a family of orphan homeobox genes. In situ hybridization has revealed considerable overlap in their expression within the nervous system, but Rnx is singularly expressed in the developing dorsal and ventral region of the medulla oblongata. Tlx1-deficient and Enx-deficient mice display phenotypes in tissues where the mutated gene is singularly expressed, resulting in asplenogenesis and hyperganglionic megacolon, respectively. To determine the developmental role of Rnx, we disrupted the locus in mouse embryonic stem (ES) cells. Rnx-deficient mice developed to term, but all died within 24 hours after birth from a central respiratory failure. The electromyographic activity of intercostal muscles coupled with the C4 ventral root activity assessed in a medulla-spinal cord preparation revealed a high respiratory rate with short inspiratory duration and frequent apnea. Furthermore, a coordinate pattern existed between the abnormal activity of inspiratory neurons in the ventrolateral medulla and C4 motorneuron output, indicating a central respiratory defect in Rnx−/− mice. Thus, Rnx is critical for the development of the ventral medullary respiratory centre and its deficiency results in a syndrome resembling congenital central hypoventilation.

Pleiotropic Functions of PACAP in the CNS

Abstract:  Pituitary adenylate cyclase‐activating polypeptide (PACAP) is a pleiotropic neuropeptide that belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP) family. PACAP prevents ischemic delayed neuronal cell death (apoptosis) in the hippocampus. PACAP inhibits the activity of the mitogen‐activated protein kinase (MAPK) family, especially JNK/SAPK and p38, thereby protecting against apoptotic cell death. After the ischemia‐reperfusion, both pyramidal cells and astrocytes increased their expression of the PACAP receptor (PAC1‐R). Reactive astrocytes increased their expression of PAC1‐R, released interleukin‐6 (IL‐6) that is a proinflammatory cytokine with both differentiation and growth‐promoting effects for a variety of target cell types, and thereby protected neurons from apoptosis. These results suggest that PACAP itself and PACAP‐stimulated secretion of IL‐6 synergistically inhibit apoptotic cell death in the hippocampus. The PAC1‐R is expressed in the neuroepithelial cells from early developmental stages and in various brain regions during development. We have recently found that PACAP, at physiological concentrations, induces differentiation of mouse neural stem cells into astrocytes. Neural stem cells were prepared from the telencephalon of mouse embryos and cultured with basic fibroblast growth factor. The PAC1‐R immunoreactivity was demonstrated in the neural stem cells. When neural stem cells were exposed to PACAP, about half of these cells showed glial fibrillary acidic protein (GFAP) immunoreactivity. This phenomenon was significantly antagonized by a PAC1‐R antagonist (PACAP6‐38), indicating that PACAP induces differentiation of neural stem cell into astrocytes. Other our physiological studies have demonstrated that PACAP acts on PAC1‐R in mouse neural stem cells and its signal is transmitted to the PAC1‐R‐coupled G protein Gq but not to Gs. These findings strongly suggest that PACAP plays very important roles in neuroprotection in adult brain as well as astrocyte differentiation during development.

Transgenic expression of group V, but not group X, secreted phospholipase A2 in mice leads to neonatal lethality because of lung dysfunction.

In an effort to elucidate the functions of secreted phospholipase A2 (sPLA2) enzymes in vivo, we generated transgenic (Tg) mice for group V sPLA2 (sPLA2-V) and group X sPLA2 (sPLA2-X), which act potently on phosphatidylcholine in vitro.We found that sPLA2-V Tg mice died in the neonatal period because of respiratory failure. The lungs of sPLA2-V Tg mice exhibited atelectasis with thickened alveolar walls and narrow air spaces, accompanied by infiltration of macrophages and only modest changes in eicosanoid levels. This severe pulmonary defect in sPLA2-V Tg mice was attributable to marked reduction of the lung surfactant phospholipids, phosphatidylcholine and phosphatidylglycerol. Given that the expression of sPLA2-V is greatly elevated in human lungs with severe inflammation, our present results raise the intriguing possibility that this isozyme may contribute to ongoing surfactant hydrolysis often observed in the lungs of patients with respiratory distress syndrome. In contrast, sPLA2-X Tg neonates displayed minimal abnormality of the respiratory tract with normal alveolar architecture and surfactant composition. This unexpected result was likely because sPLA2-X protein existed as an inactive zymogen in most tissues. The active form of sPLA2-X was detected in tissues with inflammatory granulation in sPLA2-X Tg mice. These results suggest that sPLA2-X mostly remains inactive under physiological conditions and that its proteolytic activation occurs during inflammation or other as yet unidentified circumstances in vivo.

Depletion of selenoprotein GPx4 in spermatocytes causes male infertility in mice.

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids. GPx4 is strongly expressed in the mitochondria of testis and spermatozoa. We previously found a significant decrease in the expression of GPx4 in spermatozoa from 30% of infertile human males diagnosed with oligoasthenozoospermia (Imai, H., Suzuki, K., Ishizaka, K., Ichinose, S., Oshima, H., Okayasu, I., Emoto, K., Umeda, M., and Nakagawa, Y. (2001) Biol. Reprod. 64, 674–683). To clarify whether defective GPx4 in spermatocytes causes male infertility, we established spermatocyte-specific GPx4 knock-out mice using a Cre-loxP system. All the spermatocyte-specific GPx4 knock-out male mice were found to be infertile despite normal plug formation after mating and displayed a significant decrease in the number of spermatozoa. Isolated epididymal GPx4-null spermatozoa could not fertilize oocytes in vitro. These spermatozoa showed significant reductions of forward motility and the mitochondrial membrane potential. These impairments were accompanied by the structural abnormality, such as a hairpin-like flagella bend at the midpiece and swelling of mitochondria in the spermatozoa. These results demonstrate that the depletion of GPx4 in spermatocytes causes severe abnormalities in spermatozoa. This may be one of the causes of male infertility in mice and humans.

Group III secreted phospholipase A2 regulates epididymal sperm maturation and fertility in mice

Although lipid metabolism is thought to be important for the proper maturation and function of spermatozoa, the molecular mechanisms that underlie this dynamic process in the gonads remains incompletely understood. Here, we show that group III phospholipase A2 (sPLA2-III), a member of the secreted phospholipase A2 (sPLA2) family, is expressed in the mouse proximal epididymal epithelium and that targeted disruption of the gene encoding this protein (Pla2g3) leads to defects in sperm maturation and fertility. Although testicular spermatogenesis in Pla2g3-/- mice was grossly normal, spermatozoa isolated from the cauda epididymidis displayed hypomotility, and their ability to fertilize intact eggs was markedly impaired. Transmission EM further revealed that epididymal spermatozoa in Pla2g3-/- mice had both flagella with abnormal axonemes and aberrant acrosomal structures. During epididymal transit, phosphatidylcholine in the membrane of Pla2g3+/+ sperm underwent a dramatic shift in its acyl groups from oleic, linoleic, and arachidonic acids to docosapentaenoic and docosahexaenoic acids, whereas this membrane lipid remodeling event was compromised in sperm from Pla2g3-/- mice. Moreover, the gonads of Pla2g3-/- mice contained less 12/15-lipoxygenase metabolites than did those of Pla2g3+/+ mice. Together, our results reveal a role for the atypical sPLA2 family member sPLA2-III in epididymal lipid homeostasis and indicate that its perturbation may lead to sperm dysfunction.

Pbx3 Deficiency Results in Central Hypoventilation

Pbx proteins comprise a family of TALE (three amino acid loop extension) class homeodomain transcription factors that are implicated in developmental gene expression through their abilities to form hetero-oligomeric DNA-binding complexes and function as transcriptional regulators in numerous cell types. We demonstrate here that one member of this family, Pbx3, is expressed at high levels predominantly in the developing central nervous system, including a region of the medulla oblongata that is implicated in the control of respiration. Pbx3-deficient mice develop to term but die within a few hours of birth from central respiratory failure due to abnormal activity of inspiratory neurons in the medulla. This partially phenocopies the defect in mice deficient for Rnx, a metaHox homeodomain transcription factor, that we demonstrate here is capable of forming a DNA-binding complex with Pbx3. Rnx expression is unperturbed in Pbx3-deficient mice, but its ability to enhance transcription in vitro as a complex with TALE proteins is compromised in the absence of Pbx3. Thus, Pbx3 is essential for respiration and, like its DNA-binding partner Rnx, is critical for proper development of medullary respiratory control mechanisms. Pbx3-deficient mice provide a model for congenital central hypoventilation syndrome and suggest that Pbx3 mutations may promote the pathogenesis of this disorder.

Reduced postischemic apoptosis in the hippocampus of mice deficient in interleukin-1

The cytokine interleukin‐1 (IL‐1) has been implicated in ischemic brain damage, because the IL‐1 receptor antagonist markedly inhibits experimentally induced neuronal loss. However, to date, no studies have demonstrated the involvement of endogenous IL‐1α and IL‐ 1β in neurodegeneration. We report here, for the first time, that mice lacking IL‐1α/β (double knockout) exhibit markedly reduced neuronal loss and apoptotic cell death when exposed to transient cardiac arrest. Furthermore, we show that, despite the reduced neuronal loss, phosphorylation of JNK/SAPK (c‐Jun NH2‐ terminal protein kinase/stress activated protein kinase) and p38 enzymes remain elevated in IL‐1 knockout mice. In contrast, the inducible nitric oxide (iNOS) immunoreactivity after global ischemia was reduced in IL‐1 knockout mice as compared with wild‐type mice. The levels of nitrite (NO2−) and nitrate (NO3−) in the hippocampus of wild‐type mice were increased with time after ischemia‐reperfusion, whereas the increase was significantly inhibited in IL‐1 knockout mice. These observations strongly suggest that endogenous IL‐1 contributes to ischemic brain damage, and this influence may act through the release of nitric oxide by iNOS. J. Comp. Neurol. 448:203–216, 2002.

Expression of the receptor for pituitary adenylate cyclase-activating polypeptide (PAC1-R) in reactive astrocytes

We generated transgenic mice that express an enhanced green fluorescent protein (EGFP) under the control of the mouse glial fibrillary acidic protein (GFAP) promoter. In one of the transgenic lines, the green fluorescence of EGFP was undetectable in almost all of the brain regions, including the neocortex, in untreated animals. However, when reactive astrogliosis was induced by cortical stab wounding, the strong fluorescence of EGFP was observed around the needle track but was not found in the corresponding area of the contralateral hemisphere. The EGFP-expressing cells had the morphological features of reactive astrocytes such as thick processes. The EGFP-expressing cells were found to overlap with the astroglial marker GFAP, but not with the microglial marker CD11b or the neuronal marker NeuN. Furthermore, there were some EGFP-expressing cells that expressed vimentin-like immunoreactivity, the specific marker for reactive astrocytes. These results strongly suggest that the EGFP-expressing cells are reactive astrocytes, but not resting astrocytes. Using these transgenic mice, immunostaining for the PAC1 receptor (PAC1-R) was performed. PAC1-R, which is a pituitary adenylate cyclase-activating polypeptide (PACAP)-specific receptor, binds PACAP, which is known to have a wide variety of functions. An immunohistochemical study revealed the localization of PAC1-R in reactive astrocytes visualized with EGFP around the needle track at 5 days postsurgery.

Transient Increase in Plasma Oxidized LDL During the Progression of Atherosclerosis in Apolipoprotein E Knockout Mice

Background—Plasma level of oxidized low-density lipoprotein (OxLDL) is a risk marker for cardiovascular diseases. The behavior of plasma OxLDL before disease progression has not been studied previously. Methods and Results—In this study, we developed a sensitive ELISA procedure for detecting mouse circulating OxLDL using a monoclonal antibody that recognizes oxidized phosphatidylcholine and a rabbit antimouse apolipoprotein B-48 polyclonal antibody. Apolipoprotein E knockout mice were fed on a chow diet for 40 weeks. Oil red O–positive lesions developed gradually by 20 weeks, and the percentage area covered by the lesions increased dramatically after 28 weeks; it covers 33.4% of the surface area by 40 weeks. The OxLDL level, measured after LDL fraction was isolated from each mouse, at 10 weeks was 0.015 ng/&mgr;g LDL. It increased 3-fold at 20 weeks of age and then decreased to the basal level by 40 weeks of age, suggesting that OxLDL appears before the development of atherosclerotic lesions. The occurrence of lipid peroxidation products, acrolein and oxidized phosphatidylcholines, in aortic tissue were revealed by immunohistochemical staining as early as 10 weeks. Conclusion—These results suggest that OxLDL might be involved in the early stages of progression of atherosclerotic lesions.