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Interaction between miRNA-132 function and ERK signalings in BDNF-mediated synaptic function
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Abstracts / Neuroscience Research 71S (2011) e108–e415
microglia in vitro. Thus, this study suggested that microglia are involved in the regulation of energy metabolism in the CNS. doi:10.1016/j.neures.2011.07.973
P3-c20 Microenvironment for neurogenesis and angiogenesis in the circumventricular organs Shinri Ukai , Natsuki Iimori, Shoko Morita, Seiji Miyata Dept. of Appl. Biol., Kyoto Inst. Tech., Kyoto Adult brain vasculature has the blood-brain barrier (BBB) that restricts the movement of molecules between the blood and brain, while the circumventricular organs (CVOs) lack the BBB. The fenestrated microvessels allow neurons and/or glial cells to sense blood ions, pathogenic organisms, toxins, and hormones in sensory circumventricular organs, such as the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO) and area postrema (AP). Moreover, fenestrated microvessels enable neuronal terminals to secrete a variety of peptide hormones into the blood in secretory circumventricular organs, such as the median eminence (ME) and neurohypophysis (NH). In our previous study, we have demonstrated dynamic vascular reconstruction in the adult CVOs. The present study revealed that many VEGF-positive puncta were scattered within the cytoplasm of somatodendrites in the OVLT and SFO, axonal terminals in the ME, and astrocytes in the NH. In the AP, however, VEGF-positive puncta were seen in both astrocytes and neurons of the AP. VEGF-positive puncta were considered as secretory vesicles, since secretory proteins are stored in Glogi-derived vesicles and released extracellularly via exocytosis of the secretory or consecutive pathway. Therefore, it is evident that the CVOs possess sufficient sources of VEGF in neurons and/or astrocytes for angiogenesis. In next experiment, we revealed that the expression of serine-proteases, tissue-type plasminogen activator (tPA) and plasminogen, was prominently higher at neurons in the adult CVOs as compared with adjacent hypothalamic and medulla regions. These data indicate that these extracellular proteases are responsible for rearrangement of neuro-vascular architectures. Research fund: KAKENHI 21500323. doi:10.1016/j.neures.2011.07.974
P3-c21 The mechanism of vascular reconstruction in the adult circumventricular organs Shoko Morita , Tetsuya Mannari, Seiji Miyata Dep. of Appl. Biol., Kyoto Inst. of Tech., Kyoto, Japan The blood–brain barrier (BBB) is a barrier that prevents free access of circulating substances to the brain and maintains a specialized brain environment to protect the brain from blood-derived bioactive and toxic molecules, ions, and hormones. However, the circumventricular organs (CVOs) lack the BBB and therefore are fenestrated even in adult. The fenestrated microvessels allow neurons and/or glial cells to sense blood-derived ions, pathogenic organisms, cytokines, toxins, and hormones in the sensory CVOs, the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), and area postrema (AP). Moreover, they enable neuronal terminals to secrete a wide variety of peptide hormones into the blood, from the secretory CVOs, the median eminence (ME) and neurohypophysis (NH). In the present study, we revealed that angiogenesis-related proteins were highly expressed at microvascular pericytes of the adult mouse CVOs. The quantitative analysis showed significantly higher number of proliferating endothelial cells in the CVOs as compared with the cerebral cortex. Filopodia and bridges of endothelial cells were frequently seen to emerge from existing microvessels. Furthermore, apoptosis was often seen at microvascular endothelial cells in the CVOs, which is compensatory mechanism to adjust the vascular density at constant. It seems reasonable to conclude that dynamic vascular reconstruction occurs in the adult CVOs. In this study, moreover, we found that Notch ligand delta-like 4 (Dll4) was highly expressed synapses and endothelial cells in the adult CVOs. It is possible that Dll4-Notch1 signaling regulates the new formation of neuro- and glial-vascular units in the adult CVOs. Research fund: KAKENHI (21500323). doi:10.1016/j.neures.2011.07.975
P3-d01 Effects of blood-derived parameters on vascular reconstruction in the circumventricular organs Atsushi Hourai , Shoko Morita, Seiji Miyata Dept. of Appl. Biol., Kyoto Inst. of Tech., Kyoto The circumventricular organs (CVOs) lack the BBB and therefore their microvessels permit neurons and/or glial cells to sense blood ions, pathogenic
organisms, toxins and hormones in the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), and area postrema (AP), and neuronal terminals to secrete peptide hormones into the blood in the median eminence (ME) and neurohypophysis (NH). We have demonstrated dynamic vascular reconstruction of the adult CVOs in our previous study. The present study aimed to examine whether or not peripheral parameters, osmotic and immunological challenges, affect vascular reconstruction of the CVOs by using the expression of NG2. NG2 proteoglycan binds to FGF-2, PDGF, and collagen type IV, and has important roles in vascular development, since postnatal angiogenesis is reduced in NG2 null mice. Chronic osmotic stimulation (2 and 5 days) by 2% NaCl drinking progressively increased vascular expression of NG2 in the OVLT, SFO, and ME, and slightly increased it in the AP, but had no effect in the NH. The increase of NG2 expression was reversed to normal control level after the cessation of the osmotic stimulation. On the other hand, vascular expression of NG2 was significantly decreased in the OVLT, SFO, and ME 1–3 days after systemic administration of LPS, but its expression was not changed in the AP and NH. These data indicate that vascular reconstruction of the CVOs is controlled by blood-derived parameters and possibly contributes to central control to sense peripheral blood parameters and release of peptide hormones. Research fund: KAKENHI 21500323. doi:10.1016/j.neures.2011.07.976
P3-d03 Interaction between miRNA-132 function and ERK signalings in BDNF-mediated synaptic function Soichiro Kishi 1 , Tadahiro Numakawa 2,3 , Naoki Hideya Mizuno 4 , Hiroshi Kunugi 2,3 , Kazuo Hashido 1
Adachi 2,3 ,
1
Administrative Section of Radiation Protection, NIN, NCNP, Tokyo, Japan Dept. of Mental. Disorder Res., NIN, NCNP, Tokyo, Japan 3 CREST, JST, Saitama, Japan 4 First Dept. of Biochem., Sch. of Pharm. Sci., Mukogawa Women’s Univ., Hyogo, Japan
2
It is well established that microRNAs (miRs) regulate gene expression via influence on translation from target mRNAs. Brain-specific miRs may have multiple roles in neuronal functions. Indeed, we previously reported that transient transfection of double-strand miR-132, one of the brain-specific miRs, resulted in increasing levels of synaptic proteins in cultured cortical neurons. Interestingly, we found that BDNF induced upregulation of miR-132 via ERK signalings, in turn, the ERK signaling is also reinforced by transfection of the exogenous miR-132, implying possible involvement of phosphatase. In the present study, we confirmed that overexpression of miR132 by using coding DNA enhanced the expression of glutamate receptors including NR2A, and GluR1. In addition, we investigated several phosphatase candidates as the target of miR-132 with bioinformatic algorithms, and performed luciferase analysis to confirm direct miR-132 interaction to the target. Research fund: Grants-in-Aid for Young Scientists (A) (21680034) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. doi:10.1016/j.neures.2011.07.977
P3-d04 Brain-derived neurotrophic factor regulates localization of microRNA-720 in growth cones Yukio Sasaki 1,2 , Lei Xing 2 , Christina Gross 2 , Yoshio Goshima 1 , Gary J. Bassell 2 1
Dept Mol Phamacol Neurobiol, Yokohama City Univ Grad Sch Med 2 Dept. Cell Biol., Emory Univ. Sch. Med There is increasing evidence that localized mRNAs in axons and growth cones play an important role in axon extension and pathfinding via local translation. Recently, microRNAs (miRNAs), approximately 21-nucleotide-long non-coding RNAs, have been demonstrated to bind their target mRNAs to regulate the translation and stability of mRNAs for variety of cellular functions. Although miRNAs must co-exist with their target mRNAs in same cellular compartment to regulate translation and metabolism, no study have yet done to identify localization of miRNAs in axons and growth cones. In this study, we have examined localization of miRNAs in axons and growth cones using a novel culture method, “neuron ball” culture. This new culture method that we have developed is suitable to prepare axonal miRNAs and mRNAs with high yield and purity. Axonal miRNAs prepared from the neuron ball culture of mouse cortical neurons were analyzed by quantitative real-time RT-PCR based on a 386-well plate format. Among 380 miRNAs we analyzed, 105 miRNAs were detected in axons, and 6 miRNAs were significantly enriched in axonal fractions compared to cell body fractions. Fluorescent in situ hybridization (FISH) using locked nucleic acid (LNA) probes revealed that microRNA-720 (miR-720), one of enriched miRNAs
Abstracts / Neuroscience Research 71S (2011) e108–e415
microglia in vitro. Thus, this study suggested that microglia are involved in the regulation of energy metabolism in the CNS. doi:10.1016/j.neures.2011.07.973
P3-c20 Microenvironment for neurogenesis and angiogenesis in the circumventricular organs Shinri Ukai , Natsuki Iimori, Shoko Morita, Seiji Miyata Dept. of Appl. Biol., Kyoto Inst. Tech., Kyoto Adult brain vasculature has the blood-brain barrier (BBB) that restricts the movement of molecules between the blood and brain, while the circumventricular organs (CVOs) lack the BBB. The fenestrated microvessels allow neurons and/or glial cells to sense blood ions, pathogenic organisms, toxins, and hormones in sensory circumventricular organs, such as the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO) and area postrema (AP). Moreover, fenestrated microvessels enable neuronal terminals to secrete a variety of peptide hormones into the blood in secretory circumventricular organs, such as the median eminence (ME) and neurohypophysis (NH). In our previous study, we have demonstrated dynamic vascular reconstruction in the adult CVOs. The present study revealed that many VEGF-positive puncta were scattered within the cytoplasm of somatodendrites in the OVLT and SFO, axonal terminals in the ME, and astrocytes in the NH. In the AP, however, VEGF-positive puncta were seen in both astrocytes and neurons of the AP. VEGF-positive puncta were considered as secretory vesicles, since secretory proteins are stored in Glogi-derived vesicles and released extracellularly via exocytosis of the secretory or consecutive pathway. Therefore, it is evident that the CVOs possess sufficient sources of VEGF in neurons and/or astrocytes for angiogenesis. In next experiment, we revealed that the expression of serine-proteases, tissue-type plasminogen activator (tPA) and plasminogen, was prominently higher at neurons in the adult CVOs as compared with adjacent hypothalamic and medulla regions. These data indicate that these extracellular proteases are responsible for rearrangement of neuro-vascular architectures. Research fund: KAKENHI 21500323. doi:10.1016/j.neures.2011.07.974
P3-c21 The mechanism of vascular reconstruction in the adult circumventricular organs Shoko Morita , Tetsuya Mannari, Seiji Miyata Dep. of Appl. Biol., Kyoto Inst. of Tech., Kyoto, Japan The blood–brain barrier (BBB) is a barrier that prevents free access of circulating substances to the brain and maintains a specialized brain environment to protect the brain from blood-derived bioactive and toxic molecules, ions, and hormones. However, the circumventricular organs (CVOs) lack the BBB and therefore are fenestrated even in adult. The fenestrated microvessels allow neurons and/or glial cells to sense blood-derived ions, pathogenic organisms, cytokines, toxins, and hormones in the sensory CVOs, the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), and area postrema (AP). Moreover, they enable neuronal terminals to secrete a wide variety of peptide hormones into the blood, from the secretory CVOs, the median eminence (ME) and neurohypophysis (NH). In the present study, we revealed that angiogenesis-related proteins were highly expressed at microvascular pericytes of the adult mouse CVOs. The quantitative analysis showed significantly higher number of proliferating endothelial cells in the CVOs as compared with the cerebral cortex. Filopodia and bridges of endothelial cells were frequently seen to emerge from existing microvessels. Furthermore, apoptosis was often seen at microvascular endothelial cells in the CVOs, which is compensatory mechanism to adjust the vascular density at constant. It seems reasonable to conclude that dynamic vascular reconstruction occurs in the adult CVOs. In this study, moreover, we found that Notch ligand delta-like 4 (Dll4) was highly expressed synapses and endothelial cells in the adult CVOs. It is possible that Dll4-Notch1 signaling regulates the new formation of neuro- and glial-vascular units in the adult CVOs. Research fund: KAKENHI (21500323). doi:10.1016/j.neures.2011.07.975
P3-d01 Effects of blood-derived parameters on vascular reconstruction in the circumventricular organs Atsushi Hourai , Shoko Morita, Seiji Miyata Dept. of Appl. Biol., Kyoto Inst. of Tech., Kyoto The circumventricular organs (CVOs) lack the BBB and therefore their microvessels permit neurons and/or glial cells to sense blood ions, pathogenic
organisms, toxins and hormones in the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), and area postrema (AP), and neuronal terminals to secrete peptide hormones into the blood in the median eminence (ME) and neurohypophysis (NH). We have demonstrated dynamic vascular reconstruction of the adult CVOs in our previous study. The present study aimed to examine whether or not peripheral parameters, osmotic and immunological challenges, affect vascular reconstruction of the CVOs by using the expression of NG2. NG2 proteoglycan binds to FGF-2, PDGF, and collagen type IV, and has important roles in vascular development, since postnatal angiogenesis is reduced in NG2 null mice. Chronic osmotic stimulation (2 and 5 days) by 2% NaCl drinking progressively increased vascular expression of NG2 in the OVLT, SFO, and ME, and slightly increased it in the AP, but had no effect in the NH. The increase of NG2 expression was reversed to normal control level after the cessation of the osmotic stimulation. On the other hand, vascular expression of NG2 was significantly decreased in the OVLT, SFO, and ME 1–3 days after systemic administration of LPS, but its expression was not changed in the AP and NH. These data indicate that vascular reconstruction of the CVOs is controlled by blood-derived parameters and possibly contributes to central control to sense peripheral blood parameters and release of peptide hormones. Research fund: KAKENHI 21500323. doi:10.1016/j.neures.2011.07.976
P3-d03 Interaction between miRNA-132 function and ERK signalings in BDNF-mediated synaptic function Soichiro Kishi 1 , Tadahiro Numakawa 2,3 , Naoki Hideya Mizuno 4 , Hiroshi Kunugi 2,3 , Kazuo Hashido 1
Adachi 2,3 ,
1
Administrative Section of Radiation Protection, NIN, NCNP, Tokyo, Japan Dept. of Mental. Disorder Res., NIN, NCNP, Tokyo, Japan 3 CREST, JST, Saitama, Japan 4 First Dept. of Biochem., Sch. of Pharm. Sci., Mukogawa Women’s Univ., Hyogo, Japan
2
It is well established that microRNAs (miRs) regulate gene expression via influence on translation from target mRNAs. Brain-specific miRs may have multiple roles in neuronal functions. Indeed, we previously reported that transient transfection of double-strand miR-132, one of the brain-specific miRs, resulted in increasing levels of synaptic proteins in cultured cortical neurons. Interestingly, we found that BDNF induced upregulation of miR-132 via ERK signalings, in turn, the ERK signaling is also reinforced by transfection of the exogenous miR-132, implying possible involvement of phosphatase. In the present study, we confirmed that overexpression of miR132 by using coding DNA enhanced the expression of glutamate receptors including NR2A, and GluR1. In addition, we investigated several phosphatase candidates as the target of miR-132 with bioinformatic algorithms, and performed luciferase analysis to confirm direct miR-132 interaction to the target. Research fund: Grants-in-Aid for Young Scientists (A) (21680034) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. doi:10.1016/j.neures.2011.07.977
P3-d04 Brain-derived neurotrophic factor regulates localization of microRNA-720 in growth cones Yukio Sasaki 1,2 , Lei Xing 2 , Christina Gross 2 , Yoshio Goshima 1 , Gary J. Bassell 2 1
Dept Mol Phamacol Neurobiol, Yokohama City Univ Grad Sch Med 2 Dept. Cell Biol., Emory Univ. Sch. Med There is increasing evidence that localized mRNAs in axons and growth cones play an important role in axon extension and pathfinding via local translation. Recently, microRNAs (miRNAs), approximately 21-nucleotide-long non-coding RNAs, have been demonstrated to bind their target mRNAs to regulate the translation and stability of mRNAs for variety of cellular functions. Although miRNAs must co-exist with their target mRNAs in same cellular compartment to regulate translation and metabolism, no study have yet done to identify localization of miRNAs in axons and growth cones. In this study, we have examined localization of miRNAs in axons and growth cones using a novel culture method, “neuron ball” culture. This new culture method that we have developed is suitable to prepare axonal miRNAs and mRNAs with high yield and purity. Axonal miRNAs prepared from the neuron ball culture of mouse cortical neurons were analyzed by quantitative real-time RT-PCR based on a 386-well plate format. Among 380 miRNAs we analyzed, 105 miRNAs were detected in axons, and 6 miRNAs were significantly enriched in axonal fractions compared to cell body fractions. Fluorescent in situ hybridization (FISH) using locked nucleic acid (LNA) probes revealed that microRNA-720 (miR-720), one of enriched miRNAs