Wakate Dojo

Wakate Dojo 神経細胞死、神経ネットワーク、その他

1DJ2-1 The inhibitory effects of accumbal TMEM168-regulated osteopontin system on the methamphetamine-induced pharmacological actions in mice Kequan Fu¹，Yoshiaki Miyamoto¹，Kazuya Otake¹，Kazuyuki Sumi¹，Eriko Saika¹，Naoki Sato¹，Kyosuke Uno¹，Shin-ichi Muramatsu^2,3，Atsumi Nitta¹ ¹Dept Pharmaceutical Therapy and Neuropharmacology, Grad Pharm Sch, Toyama Univ，²Division of Neurology, Dept Med, Jichi Medical Univ，³Center for Gene & Cell Therapy, Institute of Medical Science, Tokyo Univ Chronic exposure to methamphetamine causes adaptive changes in the brain, which underlie dependence symptoms. We have found that the transmembrane protein 168 (TMEM168) is overexpressed in the nucleus accumbens of mice upon repeated methamphetamine administration. Here, we demonstrate the inhibitory effects of TMEM168 on methamphetamine-induced behavioral changes in mice, and attempt to elucidate the mechanism of this inhibition. In this study, we overexpressed TMEM168 in the nucleus accumbens of mice by using an adeno-associated virus vector (NAc-TMEM mice). Methamphetamine-induced hyperlocomotion and conditioned place preference were attenuated in NAc-TMEM mice. Additionally, methamphetamine-induced extracellular dopamine elevation was suppressed in the nucleus accumbens of NAc-TMEM mice. Next, we identified extracellular matrix protein osteopontin as an interacting partner of TMEM168, by conducting immunoprecipitation in cultured COS-7 cells. TMEM168 overexpression in COS-7 cells induced the enhancement of extracellular and intercellular osteopontin. Similarly, osteopontin enhancement was also observed in the nucleus accumbens of NAc-TMEM mice, in in vivo studies. Furthermore, the infusion of osteopontin proteins into the nucleus accumbens of mice was found to inhibit methamphetamine-induced hyperlocomotion. Our studies suggest that the TMEM168-regulated osteopontin system is a novel target pathway for the therapy of methamphetamine dependence, via regulating the dopaminergic function in the nucleus accumbens.		1DJ2-2 Nitration of TRPM2 as a Molecular Switch Regulates Zinc Oxide Nanoparticles-Induced Autophagy in Brain Pericytes Cheng-Kun Wang¹，Quan Jiang¹，Yinping Gao¹，Rongrong Tao¹，Ying-mei Lu²，Feng Han¹ ¹Coll Pharm Sci, Zhejiang Univ，²Sch Med, Zhejiang Univ City Coll There is growing evidence that exposure to zinc oxide nanoparticles (ZnO-NP) could be harmful to neurovascular system, but the molecular mechanisms are largely unknown. In this study, we evaluated the role of the autophagy and TRPM2 channel during ZnO-NP-mediated brain pericyte injury in vitro and in vivo. ZnO-NP stimulated pericyte autophagic flux, as shown by an increase in autophagosome number, a rise in LC3-II levels and LC3 puncta formation in the presence of lysosomal inhibitors. A rapid induction in ER stress and autophagy was paralleled by an increase in the expression of TRPM2-S truncated isoform, which was abolished by treatment with a nitric oxide synthase inhibitor and peroxynitrite scavenger. Furthermore, Y1485 residue in TRPM2 was identified as tyrosine nitration substrate by mass spectrometry. Overexpression of the TRPM2 Y1485S mutant reduced ZnO-NP-mediated LC3-II accumulation and pericyte injury. Increased ER stress and autophagy elicited by ZnO-NP were reduced by siRNA-mediated depletion of TRPM2 or inhibition of nitrosative stress. In patch-clamp recording, the TRPM2 channel currents were decreased by ZnO-NP treatment, which were restored by L-NAME or uric acid. Consistently, LC3 accumulation was reduced and pericytes were better preserved in intact brain microvessels of the TRPM2 KO mice following ZnO-NP injection. Together, our present study has revealed a previously unrecognized mechanism of ZnO-NP-mediated neurovascular toxicity. Pericytes exposure to ZnO-NP produces a nitrosative stress response that involves the tyrosine nitration of TRPM2, increased autophagy, and eventually leads to pericytes and neurovascular injury.

1DJ2-3 Isoform-independent and-dependent phosphorylation of microtubule-associated protein tau in mouse brain during postnatal development Dilina Tuerde¹，Taeko Kimura¹，Tomohiro Miyasaka²，Masato Hasegawa³，Kanae Ando¹，Shin-ichi Hisanaga¹ ¹Department of Biological Sciences, Tokyo Metropolitan University，²Neuropathology, Faculty of Life and Medical Sciences, Doshisha University，³Tokyo Metropolitan Institute of Medical Science The microtubule-associated protein tau is a principal component of NFTs in brains of Alzheimer's disease (AD). Tau in NFTs is hyperphosphorylated, but it is not known why and how those tau are hyperphosphorylated. There are 6 isoforms in tau, which are produced by alternative splicing. The isoforms of tau in aggregates are different depending on tauopathies. Interestingly, both phosphorylation and isoforms of tau are changed during development. Highly phosphorylated 3R tau is replaced with less phosphorylated 4R tau. However, it is not addressed how the isoform and phosphorylation changes are regulated during neuronal development and how it contributes mechanistically to development of AD or tauopathies. Here, we investigated regulation of the isoform and phosphorylation changes during early postnatal development in mouse. Detailed analysis of developing brains revealed that the switch from 3R to 4R tau occurred during postnatal days 9 (P9) to P18 under the same time course as the conversion of phosphorylation from high to low. However, hypothyroidism, which is known to delay brain development, delayed the timing of tau dephosphorylation, but not the exchange of isoforms, indicating that isoform switching and phosphorylation are not necessarily linked. Furthermore, we confirmed this finding by using mouse brains that expressed a single isoform of human tau. Human tau, either 3R or 4R, reduced phosphorylation levels during development, even though the isoform did not change. We also found that 3R and 4R tau were phosphorylated differently in vivo even at the same developmental days. These results show for first time that the phosphorylation and isoform alteration of tau are regulated differently during mouse development.		1DJ2-4 Nrp2 is sufficient to instruct circuit formation to mediate odor-induced attractive social responses Kasumi Inokuchi^1,2,4，Fumiaki Imamura³，Haruki Takeuchi¹，Ryang Kim⁴，Hiroyuki Okuno⁴，Hirofumi Nishizumi^1,2，Haruhiko Bito⁴，Takefumi Kikusui⁵，Hitoshi Sakano¹ ¹Dept Brain Function, Grad Sch Med Sci, Univ of Fukui，²Dept Biophys and Biochem, Grad Sch Science, The Univ of Tokyo，³Dept Pharmacol, Penn State College of Medicine，⁴Dept Neurochem, Grad Sch Med, The Univ of Tokyo，⁵Sch Veterinary Med, Azabu Univ Odor information induces various innate responses that are critical to the survival of the individual and for the species. An axon guidance molecule, Neuropilin 2 (Nrp2), is known to mediate targeting of olfactory sensory neurons to the posteroventral main olfactory bulb (PV MOB) in mice. Here, we report that Nrp2-positive (Nrp2+) mitral cells (MCs) play crucial roles in transmitting attractive social signals from the PV MOB to the anterior part of medial amygdala (MeA). Semaphorin 3F, a repulsive ligand to Nrp2, regulates both migration of Nrp2+ MCs to the PV MOB and their axonal projection to the anterior MeA. In the MC-specific Nrp2 knockout mice, circuit formation of Nrp2+ MCs and odor-induced attractive social responses are impaired. In utero electroporation demonstrates that activation of the Nrp2 gene is sufficient to determine the functional lineage of MCs and instruct their circuit formation from the PV MOB to the anterior MeA.

1DJ2-5 Deciphering Ca²⁺ signaling during radial migration of immature cortical neurons Shin-ichiro Horigane^1,2，Sayaka Takemoto-Kimura^1,2，Aki Adachi-Morishima²，Satoshi Kamijo²，Hajime Fujii²，Haruhiko Bito² ¹Dept of Neurosci I. Res Inst of Environ Med, Nagoya Univ，²Dept Neurochem. Grad Sch Med. Univ of Tokyo During cortical circuit formation, spontaneous Ca²⁺ transients are believed to occur even before sensory inputs stimulate neuronal firings and preceding studies showed Ca²⁺ signaling regulates several aspects of neuronal development in the embryonic brain. Consistent with these findings, we previously reported that distinct limbs of the CaMKK-CaMKI cascade were specifically implicated in determining the extent of either dendritic or axonal growth. After specification of axons and dendrites, cortical neurons start glial fiber guided migration in the upper layer of intermediate zone (IZ), which is a part of radial migration and called "locomotion mode". To further study the involvement of Ca²⁺ signaling in cortical circuit formation, we investigated whether Ca²⁺ signaling regulates radial migration. At first, we measured Ca²⁺ dynamics of migrating neurons in the cortical plate (CP). Live imaging study using GCaMP6s, genetically encoded Ca²⁺ indicator, showed migrating neurons have intracellular Ca²⁺ transients and these transients are modulated in a layer-specific manner. We next monitored Ca²⁺ transients in parallel with neuronal migration over 10hrs, intriguingly migrating neurons showed low-frequency of Ca²⁺ transients during fast migrating phase, while in slow migrating phase high-frequency of Ca²⁺ transients are observed. Furthermore, pharmacological studies revealed intracellular Ca²⁺ influx is L-type Ca²⁺ channel dependent and activation of L-type channel caused migration defect. Finally, chelation of intracellular free Ca²⁺ using EGTA-AM impaired radial migration. Taken together, these results suggest cyclic change of Ca²⁺ dynamics facilitates radial migration via Ca²⁺ dependent signaling molecules.		1DJ2-6 Cereblon promotes mitochondrial degradation via autophagy in human neuroblastoma cells Kosuke Kataoka¹，Toru Asahi^1,2，Naoya Sawamura^1,2 ¹Faculty of Science and Engineering, Waseda University，²Research Organization for Nano-Life innovation, Waseda University Cereblon (CRBN) is a causative gene for autosomal recessive type of intellectual disability and its product CRBN binds to teratogenic drug thalidomide. Our previous studies have indicated CRBN is present in multiple subcellular compartments and responsive to extracellular stresses. Specifically, CRBN in mitochondria protects cells from oxidative stress, suggesting a putative role in mitochondrial quality control. Mitochondrial quality control is an essential process for normal neural functions and its dysfunction is associated with neurodegenerative and mental disorders, such as Parkinson’s disease and autism. Recent studies have highlighted the significance of autophagy-mediated removal of damaged mitochondria (mitophagy) in neurons. Here we show CRBN promotes mitophagy under mitochondrial depolarization to protect SH-SY5Y cells, human neuroblastoma. First, CRBN transcript was upregulated by mitochondrial uncoupler CCCP. Moreover, endogenous CRBN was recruited to mitochondria in response to CCCP. Notably endogenous CRBN was also co-localized with CCCP-induced autophagosomes, double membrane structures that deliver the enclosed contents to the lysosome for degradation. Knockdown of endogenous CRBN prevented CCCP-induced autophagosomal formation. To manipulate dosage of mitochondrial pool of CRBN, we mitochondrially expressed CRBN. As a result, overexpression of mitochondrially targeted-CRBN enhanced formation of autophagosomes and clearance of mitochondria under CCCP treatment. Finally, mitochondrial-targeted CRBN supported cell viability under the CCCP-induced mitochondrial depolarization. These findings suggest CRBN is a novel protein which promotes mitophagy to alleviate toxic effect of damaged mitochondria.