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Involvement of microglia in glucose metabolism in the CNS
Abstracts / Neuroscience Research 71S (2011) e108–e415
P3-c15 Pathway analysis of single-microglia geneexpression profiles reveals the new molecular targets of minocycline treatment Haruyuki Suzuki 1 , Yoshihisa Sugimura 1 , Shintaro Iwama 1 , Hiromi Suzuki 2 , Hiroshi Takagi 1 , Atsushi Kiyota 1 , Kazuki Fukuoka 1 , Makoto Sawada 2 , Yutaka Oiso 1 1 Dept. of Endocrinology and Diabetes, Nagoya Univ. Grad. Sch. of Med, Nagoya, Japan 2 Dept. of Brain Func., Res. Ins. of Env. Med., Nagoya Univ., Nagoya, Japan
Osmotic demyelination syndrome (ODS) is a central demyelinating disease associated with a rapid correction of hyponatremia. We previously reported that the microglia that accumulate in the demyelinative lesions may play a detrimental role in the pathogenesis of ODS by producing proinflammatory cytokines and also reported that minocycline prevents ODS by inhibiting the activation and accumulation of microglia at the site of demyelinative lesions. On the other hand, microglia has also reported to confer neuroprotection. The role of microglia seems to depend on the pathological condition and injury severity. To further understand the role of microglia and the effect of minocycline on the characteristics of microglia in ODS, we analyzed single-microglia gene-expression profiles using high-throughput microarray assays. We used laser-capture microdissection to isolate single microglia that are located inside and outside of the lesion with or without minocycline treatment in ODS. Pathway analysis revealed significant enrichment of integrin-mediated cell adhesion, prostaglandin synthesis and regulation, nuclear receptors in lipid metabolism and toxicity, matrix metalloproteinase, and blood clotting cascade signaling pathways in microglia inside the area of demyelination when compared with those from outside the demyelinated area. In addition, the enrichment of matrix metalloproteinase and blood clotting cascade signaling pathways in microglia inside of the demyelinative lesion was significantly reduced with minocycline treatment. Our data provide a new insight into the characteristic of microglia associated with injured oligodendrocytes and new molecular targets of minocycline treatment. doi:10.1016/j.neures.2011.07.969
P3-c16 The expression of myeline-related molecules in KLK6-deficient mouse Koichi Murakami , Yoshio Bando, Shigetaka Yoshida Department of Anatomy, Asahikawa Medical College, Asahikawa, Japan Spinal cord injury (SCI) is one of the major central nervous system damages, in which oligodendrocytes play important roles in protecting axons and functional recovery. Studying myelinating process may thus be important in understanding functional recovery of axons. Kallikrein 6 (KLK6), a serin protease, is expressed by oligodendrocyte in the central nervous system and the expression is changed in several physiological and pathological conditions, especially in spinal cord injury. We previously reported that the number of NG2 positive cells in the white matter of spinal cord was more increased in KLK6-knockout (KO) mice than wild type (WT) mice and the amount of myelin basic protein (MBP) in the injured spinal cord of KLK6-KO mice was significantly more decreased than that of WT mice. In this study, we investigated the cellular population of oligodendrocyte lineage in the developing spinal cord KLK6-KO mice. At postnatal day 7 (P7), the number of CC1 positive cells in the white matter of spinal cord was less than WT mice. In contrast, the number of NG2 positive cell was more than WT mice at P14 and P21. We also examined the expression of myeline-related molecules in the developing spinal cord of KLK6-KO mice. At P7, the expression of MBP and OSP/claudin11 in KLK6-KO spinal cord was lower than those in WT. OSP/claudin-11 was still lower at P21. Myelin gene regulatory factor (MRF), a master gene for myelin-related molecules such as MBP and MOG, was also lower at P1, P4 and P7. Moreover, the expression of myelin-related molecules in the oligodendrocytes cultured from KLK6-KO mice was markedly less than that from WT mice. These results suggest that KLK6 is involved in the regulation of maturation and myelination of oligodendrocyte. doi:10.1016/j.neures.2011.07.970
P3-c17 Glia–glia communication under inflammation causes the down-regulation of astrocyte l-glu transporter Takeshi Suzuki 1 , Junpei Takaki 1 , Koki Fujimori 1 , Kaoru Sato 2 1
Div Basic Biol Sci, Fac Pharm, Keio Univ, Tokyo, Japan 2 Div Pharmacol, Natl Inst Health Sci, Tokyo, Japan
Astrocyte l-glutamate (l-glu) transporters play an important role for the maintenance of low concentration of extracellular l-glu synaptically released
e223
as an excitatory neurotransmitter. Recent studies have reported the impairment of l-glu transporters in the various CNS diseases. It has been also reported that inflammation plays an important roles in the pathology of these CNS diseases. In this study, we focused on the cell-cell communication under inflammation and investigated precisely the functional changes of astrocyte l-glu transporters. We made in vitro inflammation model by treating lipopolysaccharide (LPS) with co-culture comprised of astrocytes, microglia and neurons. LPS decreased l-glu transport by down-regulation of GLAST in this co-culture. We also clarified that LPS induced continuous l-glu release from activated microglia and the resulting high concentration of extracellular l-glu caused the decrease in l-glu transport. Our results indicate that glia–glia communication through l-glu under inflammation caused the down-regulation of l-glu transporter. Research fund: KAKENHI (21700422). doi:10.1016/j.neures.2011.07.971
P3-c18 Paroxetine prevents the decrease in l-glutamate transport activity under inflammatory condition via novel mechanisms Kaoru Sato 1 , Junpei Takaki 1,2 , Koki Fujimori 1,2 , Takeshi Suzuki 2 , Yuko Sekino 1 1
Div Pharmacol, Natl Inst Hlth Sci, Tokyo, Japan 2 Div Basic Biol Sci, Fac Pharm, Keio Univ, Tokyo, Japan
Aberrant function of l-glu transporters under inflammatory conditions has been reported. Recently we clarified the precise mechanisms underlying the decrease in l-glu transporter activity in in vitro inflammation model: activated microglia release l-glu and the resulting high concentration of extracellular l-glu down-regulated l-glu transporter expression. Because some antidepressants have been shown to suppress neuroinflammation, we here examined the effects of five antidepressants, i.e., fluvoxamine, paroxetin, sertraline, milnacipran, and amitriptyline, on the functional impairment of l-glu transporters in in vitro inflammation model. Among these antidepressants, only Paroxetine inhibited the LPS-induced l-glu release from activated microglia and prevented the decrease in the l-glu transport activity. The effects of Paroxetine were independent of the potential as SSRI, because l-glu transport was not affected by the increased concentrations of extracellular serotonin. Recent reports have suggested that various CNS diseases are associated with the aberrant function of l-glu transporters. Our results revealed the novel effect of Paroxetine independent of the potential as SSRI and suggest that Paroxetine might be effective in various CNS diseases. Research fund: This work was partly supported by a Grant-in-Aid for Young Scientists from MEXT, Japan (KAKENHI 21700422), the Program for Promotion of Fundamental Studies in Health Sciences of NIBIO, Japan, a Health and Labor Science Research Grant for Research on Risks of Chemicals, a Labor Science Research Grant for Research on New Drug Development from the MHLW, Japan, awarded to K.S. doi:10.1016/j.neures.2011.07.972
P3-c19 Involvement of microglia in glucose metabolism in the CNS Yousuke Takezawa 1 , Shinichi Kohsaka 2 , Kazuyuki Nakajima 1,2 1
Dept. of Bioinformatics, Faculty of Engineering, Soka Univ, Tokyo 2 Dept. of Neurochemistry, National Institute of Neuroscience, Tokyo Glucose is a major energy source in brain parenchyma, and its concentration has been believed to be elaborately regulated around neurons through the uptake from blood and the degradation of glycogen in the central nervous system (CNS). To date, astrocytes have been described to be the main regulatory cell in the CNS. However, the implication of microglia in the regulation is uncertain. In the present study, we investigated the ability of microglia to support neurons by supplying glucose derived from glycogen and/or by producing lactate. At first, we tried to determine the amounts of glycogen in microglia in vitro according to alkali extraction and ethanol precipitation method. The results indicated that microglial cells as well as astrocytes contain some amounts of glycogen, suggesting the presence of glycogen synthesizing enzyme. In favor of the results, glycogen synthetase (GS) was detected in highly purified microglia. We next examined a possibility that microglia are able to produce and secrete lactate. Immunoblotting analysis revealed that lactate dehydrogenase (LDH), an essential enzyme for producing lactate from pyruvic acid, and monocarboxylate transporter (MCT) for transporting lactate from cytosol to extracellular space are present in
e224
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
P3-c15 Pathway analysis of single-microglia geneexpression profiles reveals the new molecular targets of minocycline treatment Haruyuki Suzuki 1 , Yoshihisa Sugimura 1 , Shintaro Iwama 1 , Hiromi Suzuki 2 , Hiroshi Takagi 1 , Atsushi Kiyota 1 , Kazuki Fukuoka 1 , Makoto Sawada 2 , Yutaka Oiso 1 1 Dept. of Endocrinology and Diabetes, Nagoya Univ. Grad. Sch. of Med, Nagoya, Japan 2 Dept. of Brain Func., Res. Ins. of Env. Med., Nagoya Univ., Nagoya, Japan
Osmotic demyelination syndrome (ODS) is a central demyelinating disease associated with a rapid correction of hyponatremia. We previously reported that the microglia that accumulate in the demyelinative lesions may play a detrimental role in the pathogenesis of ODS by producing proinflammatory cytokines and also reported that minocycline prevents ODS by inhibiting the activation and accumulation of microglia at the site of demyelinative lesions. On the other hand, microglia has also reported to confer neuroprotection. The role of microglia seems to depend on the pathological condition and injury severity. To further understand the role of microglia and the effect of minocycline on the characteristics of microglia in ODS, we analyzed single-microglia gene-expression profiles using high-throughput microarray assays. We used laser-capture microdissection to isolate single microglia that are located inside and outside of the lesion with or without minocycline treatment in ODS. Pathway analysis revealed significant enrichment of integrin-mediated cell adhesion, prostaglandin synthesis and regulation, nuclear receptors in lipid metabolism and toxicity, matrix metalloproteinase, and blood clotting cascade signaling pathways in microglia inside the area of demyelination when compared with those from outside the demyelinated area. In addition, the enrichment of matrix metalloproteinase and blood clotting cascade signaling pathways in microglia inside of the demyelinative lesion was significantly reduced with minocycline treatment. Our data provide a new insight into the characteristic of microglia associated with injured oligodendrocytes and new molecular targets of minocycline treatment. doi:10.1016/j.neures.2011.07.969
P3-c16 The expression of myeline-related molecules in KLK6-deficient mouse Koichi Murakami , Yoshio Bando, Shigetaka Yoshida Department of Anatomy, Asahikawa Medical College, Asahikawa, Japan Spinal cord injury (SCI) is one of the major central nervous system damages, in which oligodendrocytes play important roles in protecting axons and functional recovery. Studying myelinating process may thus be important in understanding functional recovery of axons. Kallikrein 6 (KLK6), a serin protease, is expressed by oligodendrocyte in the central nervous system and the expression is changed in several physiological and pathological conditions, especially in spinal cord injury. We previously reported that the number of NG2 positive cells in the white matter of spinal cord was more increased in KLK6-knockout (KO) mice than wild type (WT) mice and the amount of myelin basic protein (MBP) in the injured spinal cord of KLK6-KO mice was significantly more decreased than that of WT mice. In this study, we investigated the cellular population of oligodendrocyte lineage in the developing spinal cord KLK6-KO mice. At postnatal day 7 (P7), the number of CC1 positive cells in the white matter of spinal cord was less than WT mice. In contrast, the number of NG2 positive cell was more than WT mice at P14 and P21. We also examined the expression of myeline-related molecules in the developing spinal cord of KLK6-KO mice. At P7, the expression of MBP and OSP/claudin11 in KLK6-KO spinal cord was lower than those in WT. OSP/claudin-11 was still lower at P21. Myelin gene regulatory factor (MRF), a master gene for myelin-related molecules such as MBP and MOG, was also lower at P1, P4 and P7. Moreover, the expression of myelin-related molecules in the oligodendrocytes cultured from KLK6-KO mice was markedly less than that from WT mice. These results suggest that KLK6 is involved in the regulation of maturation and myelination of oligodendrocyte. doi:10.1016/j.neures.2011.07.970
P3-c17 Glia–glia communication under inflammation causes the down-regulation of astrocyte l-glu transporter Takeshi Suzuki 1 , Junpei Takaki 1 , Koki Fujimori 1 , Kaoru Sato 2 1
Div Basic Biol Sci, Fac Pharm, Keio Univ, Tokyo, Japan 2 Div Pharmacol, Natl Inst Health Sci, Tokyo, Japan
Astrocyte l-glutamate (l-glu) transporters play an important role for the maintenance of low concentration of extracellular l-glu synaptically released
e223
as an excitatory neurotransmitter. Recent studies have reported the impairment of l-glu transporters in the various CNS diseases. It has been also reported that inflammation plays an important roles in the pathology of these CNS diseases. In this study, we focused on the cell-cell communication under inflammation and investigated precisely the functional changes of astrocyte l-glu transporters. We made in vitro inflammation model by treating lipopolysaccharide (LPS) with co-culture comprised of astrocytes, microglia and neurons. LPS decreased l-glu transport by down-regulation of GLAST in this co-culture. We also clarified that LPS induced continuous l-glu release from activated microglia and the resulting high concentration of extracellular l-glu caused the decrease in l-glu transport. Our results indicate that glia–glia communication through l-glu under inflammation caused the down-regulation of l-glu transporter. Research fund: KAKENHI (21700422). doi:10.1016/j.neures.2011.07.971
P3-c18 Paroxetine prevents the decrease in l-glutamate transport activity under inflammatory condition via novel mechanisms Kaoru Sato 1 , Junpei Takaki 1,2 , Koki Fujimori 1,2 , Takeshi Suzuki 2 , Yuko Sekino 1 1
Div Pharmacol, Natl Inst Hlth Sci, Tokyo, Japan 2 Div Basic Biol Sci, Fac Pharm, Keio Univ, Tokyo, Japan
Aberrant function of l-glu transporters under inflammatory conditions has been reported. Recently we clarified the precise mechanisms underlying the decrease in l-glu transporter activity in in vitro inflammation model: activated microglia release l-glu and the resulting high concentration of extracellular l-glu down-regulated l-glu transporter expression. Because some antidepressants have been shown to suppress neuroinflammation, we here examined the effects of five antidepressants, i.e., fluvoxamine, paroxetin, sertraline, milnacipran, and amitriptyline, on the functional impairment of l-glu transporters in in vitro inflammation model. Among these antidepressants, only Paroxetine inhibited the LPS-induced l-glu release from activated microglia and prevented the decrease in the l-glu transport activity. The effects of Paroxetine were independent of the potential as SSRI, because l-glu transport was not affected by the increased concentrations of extracellular serotonin. Recent reports have suggested that various CNS diseases are associated with the aberrant function of l-glu transporters. Our results revealed the novel effect of Paroxetine independent of the potential as SSRI and suggest that Paroxetine might be effective in various CNS diseases. Research fund: This work was partly supported by a Grant-in-Aid for Young Scientists from MEXT, Japan (KAKENHI 21700422), the Program for Promotion of Fundamental Studies in Health Sciences of NIBIO, Japan, a Health and Labor Science Research Grant for Research on Risks of Chemicals, a Labor Science Research Grant for Research on New Drug Development from the MHLW, Japan, awarded to K.S. doi:10.1016/j.neures.2011.07.972
P3-c19 Involvement of microglia in glucose metabolism in the CNS Yousuke Takezawa 1 , Shinichi Kohsaka 2 , Kazuyuki Nakajima 1,2 1
Dept. of Bioinformatics, Faculty of Engineering, Soka Univ, Tokyo 2 Dept. of Neurochemistry, National Institute of Neuroscience, Tokyo Glucose is a major energy source in brain parenchyma, and its concentration has been believed to be elaborately regulated around neurons through the uptake from blood and the degradation of glycogen in the central nervous system (CNS). To date, astrocytes have been described to be the main regulatory cell in the CNS. However, the implication of microglia in the regulation is uncertain. In the present study, we investigated the ability of microglia to support neurons by supplying glucose derived from glycogen and/or by producing lactate. At first, we tried to determine the amounts of glycogen in microglia in vitro according to alkali extraction and ethanol precipitation method. The results indicated that microglial cells as well as astrocytes contain some amounts of glycogen, suggesting the presence of glycogen synthesizing enzyme. In favor of the results, glycogen synthetase (GS) was detected in highly purified microglia. We next examined a possibility that microglia are able to produce and secrete lactate. Immunoblotting analysis revealed that lactate dehydrogenase (LDH), an essential enzyme for producing lactate from pyruvic acid, and monocarboxylate transporter (MCT) for transporting lactate from cytosol to extracellular space are present in
e224
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