Regulatory effect of NGF on apoptosis and autophagy induced by tunicamycin with chloroquine in PC12 cells

Abstracts / Neuroscience Research 68S (2010) e109–e222

generated Mlc1 over-expressing mice. Mlc1 is predominant expressed in astrocyte-lineage cells and its mutation causes “Megalencephalic leukoencephalopathy with subcortical cyst” in human. We found the leukodystrophy of cerebral white matter in the Mlc1 over-expression mice as seen in the human case. In addition to this phenotype, we also found the disturbance of Purkinje cell and granule cell alignments in the cerebellum, which was caused by Mlc1 over-expression in Bergmann glia. In the Mlc1 over-expression mice, cerebellar phenotype appeared at first postnatal week and persisted adulthood. Moreover, some translocated Bergmann glia are observed. We propose that this line is a good model to study neuron-glia interaction in neural cell migration doi:10.1016/j.neures.2010.07.2196

P1-e24 Effect of aging and exercise on expression of neurotrophin in skeletal muscle Hiroshi Maejima 1,2 , Ryo Kunishi 3 , Ayumi Hamazaki 3 , Takuya Otani 2 , Tomoyuki Kurose 2 , Masataka Deie 2 1

Faculty of Medical Sciences, Teikyo University of Science 2 Graduate School of Health Sciences, Hiroshima University, Hiroshima, Japan 3 Department of Health Sciences, Hiroshima University School of Medicine, Hiroshima, Japan Neurotrophins play a crucial role in the regulation of survival and maintenance of neurons. The expression of various neurotrophins (i.e., BDNF, NT4/5, NT3, and NGF) and their Trk receptors were detected in skeletal muscle. Physical exercise enhances BDNF expression in skeletal muscle, which is thought to prevent neural retardation or aging. However, the effect of aging on neurotrophin expression in the skeletal muscle was poorly understood. The objective of this study was to clarify the effects of aging, exercise, and their interaction on the expression of neurotophins in skeletal muscle. Twenty senescence-accelerated mice (SAM) were used. Ten adult mice (10 weeks of age) were assigned to sedentary control group (five mice) and exercise group (five mice). Also, ten senescent mice (35 weeks of age) were assigned similarly. The mice in exercise group performed treadmill running at a speed of 6.4m/min for 40 min every day. The term of intervention was four weeks. After the intervention, the soleus muscles were deprived for quantitative PCR analyses based on real time PCR. In quantitative PCR, mRNA expression of neurotrophins and Trk receptors were measured. A twoway ANOVA was performed to detect the significant effects of aging and exercise on mRNA expressions. There were main effects of aging and treadmill exercise on the expression of BDNF and NGF. There was no two-factor interaction. The expressions of both neurotrophins in senescent mice were significantly higher compared with adult mice. Also, the expressions of both neurotrophins in exercise group were significantly higher compared with sedentary control group. Thus, the increase of both neurotrophins induced by exercise was extremely higher in senescent mice. There was no significant effect of aging and exercise on other mRNA expressions. Our data suggest expressions of some neurotrophins are enhanced by aging, and the enhanced expression following exercise was higher especially in the elderly. doi:10.1016/j.neures.2010.07.2197

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P1-e26 bFGF reversed a suppression in BDNF-stimulated glutamate release caused by glucocorticoid exposure Tadahiro Numakawa 1,2 Hiroshi Kunugi 1,2

, Naoki Adachi 1,2 , Emi Kumamaru 1 ,

1 Department of Mental Disorder Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP) 2 Core Research for Evolutional Science and Technology (CREST)

Increased glucocorticoid and decrease in expression/function of BDNF (brainderived neurotrophic factor) may be involved in the pathophysiology of depression. Recently, we have demonstrated possible crosstalk between glucocorticoid- and BDNF-mediated neuronal functions in the central nervous system (CNS). We examined whether chronic glucocorticoid exposure influences BDNF-stimulated intracellular signaling to induce release of glutamate, an excitatory neurotransmitter, and found that chronic exposure with dexamethasone (DEX, a synthetic glucocorticoid) decreased the BDNF-induced glutamate release via weakening the activation of the PLC␥ (phospholipase C␥)/Ca2+ system in cultured cortical neurons (Numakawa et al., 2009, PNAS). Here, we investigated whether several growth factors, including basic fibroblast growth factor (bFGF), exert protective effect on the DEX-reduced BDNF function and found that bFGF reversed the reduction in BDNF-stimulated glutamate release following DEX exposure. doi:10.1016/j.neures.2010.07.2199

P1-e27 Neurotrophin-3 alters the laminar fate of cortical neurons via activating MAP kinase pathway Hidefumi Fukumitsu , Masanari Ohtsuka, Masami Hanai, Hitomi Somiya, Shoei Furukawa Dep of Biofunctional Analysis, Lab of Molecular BIology, Gifu Pharmaceutical University Diverse neurons composing the neuronal layers of the cerebral cortex are sequentially generated from common cortical progenitors, and the postmitotic neurons then migrate to their destinations within the cortical plate. Although recent investigations have clarified important roles of intrinsic factors such as transcription factors and regulators of the cell cycle for the maturation of cortical progenitors, growth factors and neurotrophic factors environmentally supplied by the cerebral cortex are thought to regulate proliferation and neural development, and specify neuronal subtypes, in the cerebral cortex. To test the possible involvement of neurotrophin-3 (NT3) in the specification processes of neuronal subtypes, we examined the effect of NT3 directly administered into the telencephalic ventricular space of 13.5day-old mouse embryos. NT-3 altered the position, and gene-expression properties of neurons otherwise destined for layer IV to those of neurons for upper layers II/III of the cortex. Using cell culture system, we revealed that NT3 stimulated phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in the cortical progenitors, and the effects of NT3 on the alteration of neuronal laminar fate were cancelled by a MEK inhibitor, suggesting the involvement of MEK-mediated ERK signal transduction in the NT3 actions. doi:10.1016/j.neures.2010.07.2200

P1-e25 Biological roles of the BDNF pro-peptide in the nervous system Toshiyuki Mizui 1,3 , Koichi Uegaki 1,3 , Yasuyuki Ishikawa 2,3 , Tomoko Hara 1,3 , Sadao Shiosaka 2,3 , Masami Takahashi 3,4 , Haruko Kumanogoh 1,3,4 , Masami Kojima 1,3 1

AIST, Ikeda, Japan 2 Division of Structural Cell Biology, Nara Institute of Science and Technology, Ikoma, Japan 3 CREST, JST, Kawaguchi, Japan 4 Department of Biochemistry, Kitasato University School of Medicine, Sagamihara, Japan The discovery of a mutation in the human proBDNF gene correlating with decreased memory performance focused attention on the BDNF pro-peptide, but its biological role has remained unclear. This work finds that the BDNF pro-peptide binds BDNF and interferes with the ability of BDNF to modulate LTD. Conversely, the BDNF pro-peptide facilitates LTD on its own and decreases the surface levels of GluR1. The BDNF pro-peptide is thus a new modulator of synaptic plasticity. doi:10.1016/j.neures.2010.07.2198

P1-e28 Regulatory effect of NGF on apoptosis and autophagy induced by tunicamycin with chloroquine in PC12 cells Hiromi Irifune 1 , Koji Shimoke 1,2 , Yosuke Nakatani 1 , Harue Sasaya 1,2 , Kensuke Sugihara 1 , Michimasa Nobuhara 1 , Toshihiko Ikeuchi 1,2 1

Graduate School of Science and Engineering, Kansai University Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University 2

It has been elucidated that autophagy-lysosome system is important for the maintenance of the homeostatic balance in mammalian cells. Autophagy involves the degradation of long-lived proteins or organelles concomitantly with the drastic membrane traffick leading to a huge encompassment. Various stress such as amino acid starvation or hormone stimulation induces autophagy. It has been reported that endoplasmic reticulum (ER) stress is one of the key phenomena for induction of specific autophagy. In the present study, we induced ER stress and autophagy using specific chemical inducers in PC12 cells. We found that coaddition of chloroquine (CQ), a lysosome enzyme inactivator, and tunicamycin (Tm), a inhibitor of glycosylation in the ER, resulted in the accumulation of LC3-II more than addition of CQ alone

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Abstracts / Neuroscience Research 68S (2010) e109–e222

when detected by Western blotting in PC12 cells. Furthermore, addition of nerve growth factor (NGF) which is known as an attenuator of ER stress, with CQ and Tm resulted in the decrease of LC3-II in PC12 cells. It has been reported that NGF inhibits ER stress-mediated apoptosis through up-regulated expression of GRP78. Taken together, these results suggest that NGF contributes not only to the attenuation of ER stress-mediated apoptosis but also to the attenuation of autophagy in PC12 cells.

P1-e31 AMPK regulates BDNF-induced activation of mTOR signaling and enhancement of translation in cortical neurons

doi:10.1016/j.neures.2010.07.2201

Neuregulin1 (NRG1) is one of the EGF family ligands and interacts to the ErbB1-4 receptors. Differential promoter choice and alternative splicing of NRG1 genome generate a large variety of NRG1 isoforms and variants, although their difference in receptor binding and signaling is not fully understood. The major classification of NRG1 isoforms is termed as Type I-V. In the present study, we compared their activity on phosphorylation of ErbB3 and ErbB4 as well as their binding to the receptors. First, we generated recombinant proteins of soluble NRG1 Type I, II and III isoforms using the mammalian in vitro transcription/translation system, which enables their natural protein refolding. Each isoform was labeled with 35S-Met or His-Tag. MDA453 cells (ErbB2/3 dominant) and ErbB4-transfected L929 cells were stimulated with Type I, II or III NRG1 in the range of 0.1-3pmol/ml. Type I, II and III NRG1 trigged ErbB3 phosphorylation in MDA453 cells in the same degree. The concentrations of Type I and III induced ErbB4 phosphorylation of L929 cells whereas those of Type II hardly increased ErbB4 phosphorylation. In agreement, 35S-Met-labeled Type II NRG1 showed a markedly lower binding to L929-ErbB4 cells than Type I NRG1. These results indicate significant difference in the affinity and signaling of NRG1 isoforms.

AMPK is known to a cellular “energy sensor”, as its activity is sensitively changed by the cellular energy state. An increase in the AMP: ATP ratio activates AMPK. In contrast to the amino acids sufficiency is essential for growth factor-induced activation of mTOR signaling in fibroblasts, glucosedeprivation (but not amino acid-deprivation) cancels BDNF-induced mTOR signaling activation in neurons. Thus we examined the roles of AMPK on mTOR signaling and succeeding translation that were activated by BDNF, insulin and serum in cultured rat cortical neurons. Glucose-deprivation changed phosphorylation level of translation regulators such as p70S6Kinase and eEF2 (eukaryotic elongation factor 2), to decrease translation efficiency. As an end point of biological response, glucosedeprivation markedly suppresses protein synthesis and the re-addition of glucose restored it. The effect of growth factors, BDNF and Insulin on the activation of translation factors and protein synthesis under the presence or absence of glucose were examined. Although they activated translation in neurons under the presence of glucose, BDNF and insulin had no effects on protein synthesis, as well as the activation of translation regulators anymore under the glucose-deprived condition. The inhibition of phosphorylation of p70S6K was specific on its Thr389, a target site of mTORC1, but not on MAPK target Thr421/Ser424. The result suggests glucose-deprivation that used in this study does not cause general ATP-deficiency but rather specific for inhibition of AMPK. In fact, similar results on both p70S6K phosphorylation and protein synthesis were obtained by the treatment of AMPK activators, metformin and resveratrol in neurons. Furthermore, overexpression of constitutive-active AMPK␣ (AMPK␣ 1. 1-312 subunit) decreased p70S6K phosphorylation and protein synthesis. These results strongly suggest that AMPK dominantly regulates of the mTOR signaling downstream of receptor tyrosine kinase.

doi:10.1016/j.neures.2010.07.2202

doi:10.1016/j.neures.2010.07.2204

P1-e30 Activation of RAW264.7 macrophages by oxidized galectin-1 Yuya Koizumi , Yu Etigo, Hiroko Inoue

P1-e32 Pigment epithelium-derived factor (PEDF) induces glutamate transporters expression in rat cultured astrocytes

Graduate School of Advanced Science and Engineering, Waseda Univ, Tokyo, Japan

Kentaro Hata 1 Yamada 1,2,3

P1-e29 Distinct ErbB receptor binding and signaling of neuregulin-1 splicing isoforms Ran Wang 1 , Yuriko Iwakura 1 , Nobuyuki Takei 1 , Yuta Ishizuka 1 , Yingjun Zheng 1 , Taisuke Kato 1 , Shigeki Higashiyama 2 , Hiroyuki Nawa 1 1

Mol. Neurobiol. Brain Res. Inst. Niigata Univ, Niigata Univ Grad Sch Med, Ehime

2

Mol Genet, Ehime

Yuta Ishizuka , Hiroyuki Nawa, Nobuyuki Takei Department of Molecular neurobiology, Brain Research Institute, Niigata University

, Tomomi Sanagi 1 , Takeshi Yabe 1,2,3 , Haruki

1

Galectin-1 (Gal-1), a member of the;␤-galactoside-binding lectin family, is differentially expressed by various normal and pathologic tissues and appears to be functionally multivalent, with a wide range of biological functions in development and adult tissues. Gal-1 exists in both reduced and oxidized states, but shows lectin activity only in its reduced state. In adult dorsal root ganglion and motor neurons, Gal-1 is intensely expressed in axons and Schwann cells, and only oxidized Gal-1 (Gal-1/Ox), and not reduced Gal-1, has been shown to promote axonal regeneration. After axonal injury, cytosolic reduced Gal-1 is likely to be externalized from axons and reactive Schwann cells to the extracellular space, where some of the molecules become oxidized. Gal-1/Ox is suggested to activate macrophages to release factors that enhance Schwann cell migration and axonal elongation. However, the mechanism of macrophage activation by Gal-1/Ox remains unknown. We previously reported that Gal-1/Ox stimulated migration of RT4-D6P2T Schwann cells directly and by activating RAW264.7 macrophages to release factors that promoted cell migration. Here, to reveal the signal transduction pathways involved in Gal-1/Ox-mediated activation of macrophages, we examined whether Gal-1/Ox activated ERK1/2 in RAW264.7 macrophages. Phosphorylation of ERK1/2 was transiently increased by application of Gal-1/Ox, but the ERK1/2 pathway was found not to be involved in release of factors that promoted Schwann cell migration. On the other hand, RT4-D6P2T Schwann cell migration induced by Gal-1/Ox and Gal-1/Ox-activated RAW264.7 macrophage conditioned medium appeared to be mediated by the ERK1/2 pathway. Furthermore, we analyzed proteins released from RAW264.7 macrophages by Gal-1/Ox using antibody array. doi:10.1016/j.neures.2010.07.2203

Grad. Sch. Infect. Contr., Kitasato Univ. Tokyo 2 Kitasato Inst. Life Sci., Kitasato Univ. Tokyo 3 Orient. Med. Res. Cnt., Kitasato Univ. Tokyo

Pigment Epithelium-Derived Factor (PEDF) is a 50-kDa glycoprotein and a member of the serine protease inhibitor gene family. PEDF is a potent and broadly acting neurotrophic factor that protects various types of cultured neurons against glutamate excitotoxicity and induced-apoptosis. We have recently also reported that PEDF is effective in protecting CNS neurons from ischemic insults in vivo (Sanagi et al., J. Neurochem. 2008). Therefore, we hypothesized that attenuation of glutamate toxicity is closely associated with in vivo neuroprotective effect of PEDF. It is suggested that astrocytes play an important role in the efficient clearance of glutamate from the synaptic cleft through the glutamate transporters such as glutamate aspartate transporter (GLAST) and glutamate transporter 1 (GLT-1), however the effects of PEDF on the expression and function of glutamate transporters in astrocytes are not fully understood. When cultured rat astrocytes were incubated with recombinant PEDF (1-50 nM) for 3 hr, induction of mRNA level of glutamate transporters (GLAST and GLT-1) was observed in PEDF-treated astrocytes. Western blotting and immunocytochemical analysis revealed that protein levels of GLAST and GLT-1 were increased 24 hr after PEDF treatment. Likewise, an increase in glutamate clearance of externally added glutamate was observed in PEDF treated astrocyte cultures. Furthermore, adenovirally gene transfer of PEDF in rat striatum resulted in the induction of glutamate trasnsporters (GLAST and GLT-1) in vivo. These results suggest that PEDF could modulate the glutamate clearance by induction of glutamate transporters. Our findings also suggest that glial cells are are targets for PEDF, and that modulation of glial function may be part of the mechanism of PEDF-mediated neuroprotection against glutamate toxicity. doi:10.1016/j.neures.2010.07.2205