Extracellular administration of protein kinase A promotes recovery from an experimental spinal cord injury model

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Abstracts / Neuroscience Research 71S (2011) e108–e415

Schwann cells and regenerating axon. However, little is known about the mechanisms underlying reutilization of cholesterol by regenerating axons. We therefore examined the expressions of genes associated with cholesterol uptake by using hypoglossal nerve injury model of rodents. RT-PCR and in situ hybridization revealed that the expression of mRNA for the lowdensity lipoprotein receptor (LDLR), which functions to uptake LDL including abundant cholesterol and its ester into plasma membrane, was prominently up-regulated in the motor neurons after nerve injury, while other lipid receptor mRNAs for VLDLR, ApoER2, LRP1b and LRP10 were unchanged. These data may suggest that the induction of LDLR expression in nerve-injured motor neurons is crucial for axon to reutilize cholesterol from macrophage and to promote proper regeneration of axon. doi:10.1016/j.neures.2011.07.1489

P4-f19 Extracellular administration of protein kinase A promotes recovery from an experimental spinal cord injury model Kenji Suehiro 1 , Yoshinori Takei 2 , Gozoh Tsujimoto 1 1

Dept. Genomic Drug Discovery Sci., Grad. Sch. of Pharm. Sci., Kyoto Univ., Kyoto, Japan 2 Dept. Nanobio Drug Discovery, Grad. Sch. of Pharm. Sci., Kyoto Univ., Kyoto, Japan

serious than pair housed rats. Motor activity was significantly increased by TMT injection on pair housed and enrichment rats. Motor activity of isolated rats was sufficiently high even before TMT injection, and not altered by TMT treatment. Conclusion: Through histochemical and motor activity analysis, it is suggested that housing condition at rearing period affects neurotoxicity of TMT in rat brain. Research fund: LRI. doi:10.1016/j.neures.2011.07.1491

P4-f21 Analyses of the proliferative and neuroprotective functions of oversulfated chondroitin sulfate in the developing cortex Mutsuki Kuraoka 1 , Yukari Komuta 2 , Keisuke Hitoshi Kawano 2 , Nobuaki Maeda 1

Kamimura 1 ,

1

Dep. of Dev. Neurosci., Tokyo Metro. Inst. of Med. Sci., Tokyo, Japan 2 Dep. of Dev. Morphol., Tokyo Metro. Inst. of Med. Sci., Tokyo, Japan Chondroitin sulfate (CS) is the major constituent of the extracellular matrix and cell surface in the CNS. CS is involved in the various cellular events such as proliferation and differentiation of neural stem cells and neuronal migration. It has been shown that CS polysaccharides rich in highly sulfated disaccharide (D and E units) bind with various growth factors, and participate in proliferation and survival of cultured neural stem cells. However, the in vivo functions of oversulfated CS have been poorly understood. Herein, using in utero electroporation, we knocked down the two CS sulfotransferases, uronyl 2-O-sulfotransferase (UST) and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (4,6-ST), which are responsible for the biosynthesis of D and E units, respectively. The embryonic mouse cortices were electroporated with the shRNA constructs of UST and 4,6-ST at E12 and harvested 2 days later. Knocked down cells were immunohistochemically analyzed for the BrdU incorporation, and the expression of PH3 for cells at the cell-cycle and cleaved caspases 3 and 9 for apoptotic cells. In UST- and 4,6ST-knocked down cortices, the BrdU-, PH3- and cleaved caspase 9-positivity rates were totally similar to those in the control cortex throughout coronal slices. Cleaved caspase 3-positivity was slightly increased in 4,6-ST-knocked down cells, compared to the control and UST-knocked down cells. These results suggested that CS E unit is potentially, but not drastically, involved in neuroprotection. In our knocked down model, proliferative and neuroprotective functions of oversulfated CS might be compensated for by the large amount of CS proteoglycans secreted by the surrounding normal cells.

The mammalian adult central nervous system (CNS) can be hardly regenerated after traumatic damage. This inability prevents recovery not only from traumatic CNS damages, such as spinal cord injury (SCI) and stroke, but also from neurodegenerative disorders, such as Alzheimer’s disease and Parkinson’s disease. Therefore, therapeutic methods to promote CNS regeneration are strongly desired. The inability of the CNS to regenerate is, at least a part, due to endogenous inhibitors for regeneration, such as chondroitin sulphate proteoglycans in the extracellular matrix and myelin-associated proteins in oligodendrocytes. Nogo-A is one of the myelin-associated proteins that inhibit new formation of synapses in the CNS, thereby stabilizing the CNS neuronal network. Inhibition of Nogo-A signaling is favorable for recovery from SCI and thus, Nogo-A is proposed as a target for CNS regeneration. Nogo-A can collapse axonal growth cones in vitro. However, the in vivo mechanism of which Nogo-A contributes to the inhibition of CNS regeneration is not fully understood. Previously we reported that both casein kinase 2 and protein kinase A (PKA) phosphorylate a Nogo-A receptor NgR1, inhibiting interaction between Nogo-A and NgR1. CNS neurons can extend neurites on the surface coated with a Nogo-A fragment, when NgR1 is phosphorylated. Here, we examined the effects of the extracellular kinase treatment on CNS regeneration in vivo. Female Wistar rats (8 weeks) received dorsal hemisection injuries at T9 level. Marked behavioral improvement was observed in the lesioned animals treated with both ATP and PKA, but not treated with either ATP or PKA. We will discuss the mechanism of CNS regeneration induced by the extracellular kinase treatment.

P4-g01 Deterioration of barrier function of nuclear pore complexes in aging neurons–Immunohistochemical evaluation using antibodies against scaffold nucleoporins

doi:10.1016/j.neures.2011.07.1490

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P4-f20 Evaluation of neurotoxicity of trimethyltin in rat brain under various housing conditions Tsutomu Ooyama , Takao Mukuda, Takeshi Yamazaki, Atsuhiko Ishida, Shogo Sakata, Yuya Sakimoto Hiroshima Univ Grad School of Intergrated Arts and Sci Trimethyltin (TMT), an organotin compound, is a potent neurotoxicant for hippocampus. Housing condition at rearing period affects brain function. For example, socially isolated rats exert hyperemotionality, while rats living in an enriched environment are extremely active and intelligent. Estradiol is a neuroprotective steroid, and its synthesis in hippocampus was significantly stimulated in socially isolated rat. To examine effect of housing condition at rearing period on neurotoxicity of exogenous chemicals, here we analyzed neurotoxicity of TMT on differently housed rats. Wister male rats on postnatal day 28 were divided into three groups: isolated, pair housed, and enrichment housed group. Isolated rats were housed individually in clear plastic cages while pair housed rats were housed two per cage. Enriched rats were housed ten in a big cage equipped with running wheels, tunnels, hinding place, etc. Rats were housed for 6 weeks, and motor activity was analyzed by open field test. After the test, rats were injected TMT (8 mg/kg) i.p. Seven days after the injection, rats were analyzed for their motor activity again, and then sacrificed by transcardiac perfusion. Brain slices were (20 ␮m thickness) immunostained with anti NeuN. TMT treatment induced pyramidal cell loss in the hippocampal CA3 region. The damage in the CA3 region was most significant in the isolated rats. The damage of enriched rats was more

doi:10.1016/j.neures.2011.07.1492

Akinori Matsuo 1 , Jean-Pierre Bellier 1 , Shinichiro Nakamura 2 Mol. Neurosci. Res. Center, Shiga Univ. of Med. Sci., Otsu, Japan 2 Res. Center. Animal Life Sci., Shiga Univ. of Med. Sci., Otsu, Japan Nucleus is the largest organelle in eukaryotic cells, and isolated by double layered nuclear envelope from cytoplasm. Nuclear pore complexes (NPCs) play key roles to control transportation of substances through the nuclear envelope. Stability of NPCs seems to be a very important factor to keep cells healthy. Since NPCs consisted of more than 50 types of proteins, nucleoporins (Nups), their total renewal can be done only during mitosis. It means that maintenances of NPCs are kept by partial exchange of parts in postmitotic cells. Recently, some of scaffold Nups in postmitotic cells were reported to be not changeable throughout the entire lifetime, by D’Angelo et al. We have been also interested in subcellular localization of biologically active molecules, such as choline acetyltransferase (ChAT), especially functions of intranuclear ChAT. The purpose of this study is establishment of index for evaluation of cellular deterioration from the point of view of breaking down of NPCs’ barrier function. We studied rat (Wistar, SHR and WKY) brains of different ages (12 weeks, 24 weeks and more than 1 year) by immunohistochemistry using antibodies against Nup93, Nup107 and other Nups. We found that the Nup93-like immunoreactivity decreases according to aging, while the intensity of Nup107 staining is stable in all the tested rat brains of different ages. Furthermore, semi-quantitative immunoblot indicated the same tendency. We started to evaluate differences of staining of Nups in neuron groups in the rat brains. Additionally, ectopic intranuclear existence of class III beta-tubulin, which is normally a cytoplasmic protein, was studied by confocal laser microscopy to show loose of nuclear envelope permeability barrier. We also examined non-human primates, Macaca fascicularis brains of different ages as well as those of rodents. The ratio between Nup93 inten-