Oral Sessions

Oral Sessions 神経系の発生・再生、神経画像

1O2-1 Signaling of the Unfolded Protein Response is Critical for Axon Regeneration after Nerve Injury Yosuke Ohtake^1,2，Atsushi Saito²，Koji Matsuhisa²，Kazunori Imaizumi¹ ¹Dept Biochem, Inst Biomed & Health Sci, Hiroshima Univ，²Dept Stress Prot Processing, Inst Biomed & Health Sci, Hiroshima Univ Multiple cellular malfunctions perturb endoplasmic reticulum (ER) functions (ER stress). ER stress transducers IRE1, PERK and ATF6 sense ER stress and transduce signals to maintain biological functions (unfolded protein response: UPR). Recent studies indicate that the UPR is activated in response to axon injury, suggesting the prospective association of the UPR in the developed axonal ER network with axon regeneration and degeneration. We examined the mechanisms for inducing ER stress after axon injury and the roles of UPR in regulating axon regeneration in sensory neurons. To assess the effect of the UPR signaling on axon regrowth, we used axotomized neurons of mouse dorsal root ganglion (DRG) at E13 and a mouse model of sciatic nerve crush. The phosphorylation levels of IRE1 and PERK were upregulated in cultured DRG after axon injury. The inhibition of calcium ion release from ER by 2-APB and dantrolene reduced the phosphorylation of those transducers. These data suggest that at least one of the underlying factors for the activation of UPR after the injury is calcium ion release from ER and its depletion in axonal ER lumen. We observed the activation of IRE1 and PERK in distal axons in response to sciatic nerve crush. The attenuation of these phosphorylation was exhibited by the pretreatment of axons with 2-APB and dantrolene, consistent with those in cultured DRG neurons. Axon regrowth of DRG neurons and sciatic nerve was reduced by the pretreatment of axons with specific inhibitors of ER stress transducers. We found that the blocking of UPR signaling promoted the ER fragmentation in distal axons. Consequently, axon injury-induced UPR signaling at an axonal segment has a potential to promote axon regeneration through the proper manipulation of axonal ER dynamics.		1O2-2 Promotion of mTOR signaling for neurogenesis facilitated by the green tea amino acid theanine in neural progenitor cells Nobuyuki Kuramoto¹，Maiko Tsujiai¹，Airi Yoshimura¹，Hiroshi Higashi¹，Toshihiko Kinjo¹，Yukio Yoneda² ¹Lab Mol Pharmacol, Fac Pharm Sci, Setsunan Univ，²Sec Prophylactic Pharmacol, Kanazawa Univ, VBL Theanine is an amino acid ingredient in green tea with a chemical structure analogous to glutamine rather than glutamate, in terms of the absence of a free carboxylic acid moiety from the gamma carbon position. In neural progenitor cells isolated from embryonic rodent neocortex, theanine markedly promoted proliferation and subsequent commitment to a neuronal lineage. In cultured progenitor cells from the hippocampus of adult nestin-GFP mice, theanine increased the size of neurospheres composed of clustered proliferating cells after exposure for a long period. Marked upregulation was seen in expression of the glutamine transporter Slc38a1 transcript in rodent progenitors exposed to theanine. Stable overexpression of Slc38a1 accelerated proliferation and neuronal commitment in murine embryonic carcinoma P19 cells. In these stable Slc38a1 transfectants, marked upregulation was seen in transcript expression of particular repressor- and activator-type bHLH transcription factors essential for the regulation of cellular properties of neural progenitors. Theanine was found to promote the phosphorylation of mTOR and downstream proteins in undifferentiated neurospheres prepared from embryonic mouse neocortex after long-term exposure. Although stable overexpression of the glutamine transporter Slc38a1 facilitated the phosphorylation of mTOR-relevant proteins in undifferentiated P19 cells, theanine failed to additionally accelerate the increased phosphorylation in these stable transfectants. These results suggest that theanine activates the mTOR signaling pathway for self-renewal together with accelerated neurogenesis through a mechanism relevant to upregulation of bHLH gene expression in undifferentiated neural progenitor cells.

1O2-3 Microglial phagocytosis of dying neurons promotes new neuron addition in the adult olfactory bulb Masato Sawada¹，Hiroyuki Inada²，Shinichi Kohsaka³，Junichi Nabekura²，Kazunobu Sawamoto^1,4 ¹Dept Dev Regen Biol, Nagoya City Univ Grad Sch Med Sci, Nagoya, Japan，²Div Hom Dev, NIPS, Okazaki, Japan，³Dept Neurochem, NIN, Tokyo, Japan，⁴Div Neural Dev Regen, NIPS, Okazaki, Japan New neurons are continuously added and old ones eliminated throughout life in the adult mouse olfactory bulb (OB). Previous reports suggest that olfactory input regulates the integration of new neurons into the OB circuits. We have previously reported that the turnover of OB neurons is regulated in spatiotemporal and olfactory-input-dependent manners, by using in vivo two-photon laser-scanning microscopy (2PLSM) (Sawada et al., J. Neurosci., 2011). Here, we investigated the role of microglial phagocytosis in neuronal turnover in the adult OB. Using in vivo 2PLSM of microglia in Iba1-GFP mice, we observed the dynamic movement of resting microglia for the formation of a phagocytic pouch in the adult OB. Immunohistological analyses revealed that microglia efficiently phagocytose dying OB neurons, which is affected by olfactory input and aging. To investigate the role of microglial phagocytosis in neuronal turnover in the adult OB, we inhibited phagocytosis of dying neurons by microglia, and analyzed its effect on new neuron addition in the OB. Collectively, our results suggest that phagocytosis of dying neurons by resting microglia promotes neuronal turnover in the adult OB, leading to the maintenance of homeostasis in the OB circuitry.		1O2-4 Prdm16 is crucial for progression of the multipolar phase during neural differentiation of the developing neocortex. Ken-ichi Mizutani^1,2,3，Ryota Iwai^1,2，Mayuko Inoue²，Hidenori Tabata⁴，Daijiro Konno⁵，Mariko Suzuki^1,2，Chisato Watanabe²，Fumio Matsuzaki⁵，Koh-ichi Nagata⁴ ¹Lab Stem Cell Biol. Grad Sch Pharm Sci, Kobe Gakuin Univ，²Grad Sch Brain Sci, Doshisha Univ，³JST PRESTO，⁴Dept Mol Neurobio, Inst Dev Bio，⁵Riken CDB The precise control of neuronal migration and morphological changes during differentiation is essential for neocortical development. We hypothesized that the transition of progenitors through progressive stages of differentiation involves dynamic changes in levels of mitochondrial reactive oxygen species (mtROS), depending on cell requirements. We found that progenitors had higher levels of mtROS, but that these levels were significantly decreased with differentiation. The Prdm16 gene was identified as a candidate modulator of mtROS using microarray analysis, and was specifically expressed by progenitors in the ventricular zone. However, Prdm16 expression declined during the transition into NeuroD1-positive multipolar cells. Subsequently, repression of Prdm16 expression by NeuroD1 on the periphery of ventricular zone was crucial for appropriate progression of the multipolar phase and was required for normal cellular development. Furthermore, time-lapse imaging experiments revealed abnormal migration and morphological changes in Prdm16-overexpressing and -knockdown cells. Reporter assays and mtROS determinations demonstrated that PGC1α is a major downstream effector of Prdm16 and NeuroD1, and is required for regulation of the multipolar phase and characteristic modes of migration. Taken together, these data suggest that Prdm16 plays an important role in dynamic cellular redox changes in developing neocortex during neural differentiation.

1O2-5 3D-brain mapping with electron microscopy Shinsuke Shibata^1,2，Mana Yoshimura^1,2，Toshihiro Nagai¹，Tetsuya Yano¹，Hideyuki Okano^1,2 ¹Keio Univ Sch of Med, Electron Microscope lab，²Keio Univ Sch of Med, Dept of Physiol To understand the whole-brain neural circuitry, connectomics analysis is actively curried out with using newly invented biotechnologies all over the world. Here, we demonstrate the recent progress of our connectomics analysis in the brain. A whole neural network in the brain was revealed manually for the first time with nematode, Caenorhabditis elegans (C. elegans). Nematodes have a limited number of neurons and the transparent body, allowing us to visualize whole brain using light and electron microscopy (EM). It is very difficult to adopt it for human or mice because of the size of the central nervous system. For revealing the comprehensive neural connection with the wide range of species including mammals and primates, three-dimensional imaging with EM is magnificent approach for the micro-connectomics with the brain. In our study, images from high throughput multi-beam scanning EM reveal the whole neural circuitry of the brain and will help us to understanding the mechanism of the neuropsychiatric disorders.