公募シンポジウム

公募シンポジウム4【オルガネラ動態から迫る脳機能制御と疾患】

2021/9/30　14:00～16:00　ZOOM A会場 S4-1 小胞体膜局在転写因子OASIS を介したグリオブラストーマの細胞老化制御 Regulations of cellular senescence in glioblastoma mediated by ER-resident transcription factor OASIS ^○齋藤敦¹,吉丸哲郎²,松下洋輔²,片桐豊雅²,今泉和則¹ 1.広島大学大学院医系科学研究科分子細胞情報学,2.徳島大学先端酵素学研究所ゲノム制御学分野 ^○Atsushi Saito¹Tetsuro Yoshimaru²,Yosuke Matsushita²,Toyomasa Katagiri²,Kazunori Imaizumi¹ 1.Department of Biochemistry, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan,2.Division of Genome Medicine, Institute of Advanced Medical Sciences, Tokushima University, Tokushima, Japan Cellular senescence is a stable cell cycle arrest induced by various cellular stress including DNA damage. p53 and its downstream p21 play a critical role in its control. The cell cycle arrest by this phenomenon not only facilitates the repair of DNA damage, but also suppresses tumorigenesis. We found that ER-resident transmembrane transcription factor OASIS, specifically expressed in astrocytes in central nervous system, is activated by DNA damage. The activated OASIS induces cellular senescence by the transcriptional induction of p21. This pathway does not show any crosstalk with that of p53. We also revealed that several human glioblastoma including U251MG cells showed the attenuation of OASIS expression due to hypermethylation of its promoter. BrdU and SA-β-gal assays showed the inhibited proliferation and the acceleration of p21-mediated cellular senescence in U251MG cells stably expressing OASIS in a p53-independent manner. Xenograft experiments using U251MG cells expressing OASIS exhibited the inhibited tumor development with the induction of p21 and cellular senescence. The removal of hypermethylation in Oasis promoter by CRISPR-based epigenomic engineering using a construct collectively expressing inactive Cas9 nuclease, ten-eleven translocation methylcytosine dioxygenase 1 (TET1: responsible for DNA demethylation) and guide RNA (gRNA) targeting Oasis promoter as an all-in-one system (Oasis-gRNA) induced p21-mediated cellular senescence. Delivery of Oasis-gRNA with nanoparticles into tumors efficiently suppressed its development. Since the epigenomic editing we showed can regulate cellular senescence without genome editing processes, our approach may lead to a novel and safety therapeutic strategies for glioblastoma irrespective of the p53 activity.		2021/9/30　14:00～16:00　ZOOM A会場 S4-2 神経系におけるゴルジ体酸性環境維持の重要性について Importance of the pH homeostasis of the Golgi apparatus in the central nervous system ^○小池正人順天堂大学大学院医学研究科神経機能構造学 ^○Masato Koike Department of Cell Biology and Neuroscience, Juntendo University Graduate School of Medicine The Golgi apparatus plays an indispensable role in posttranslational modification and transport of proteins to their target destinations. Morphological alteration of the Golgi apparatus is associated with several neurodegenerative disorders, including Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. It is well established that the Golgi apparatus requires an acidic luminal pH for optimal activity. Perturbation of the luminal pH of the Golgi apparatus has marked effects on optimal activity but also causes morphological abnormalities. However, relationships between perturbations in luminal pH of the Golgi apparatus and morphological and functional abnormalities in the central nervous system are not fully understood. Here, by taking advantage of Purkinje cell (PC) and basket cell (BC) -specific Golgi pH regulator (GPHR) deficient mice, we performed morphological and functional characterization of cerebellar circuits. PCs from the mutant mice exhibited vesiculation and fragmentation of the Golgi apparatus, followed by axonal degeneration and progressive cell loss. Morphological analysis provided evidence for the disruption of terminals of the BCs around PC soma. Electrophysiological recordings showed selective loss of large amplitude responses, suggesting BC terminal disassembly. In addition, the innervation of mutant PCs was altered such that climbing fiber terminals abnormally synapsed on the somatic spines of mutant PCs in the mature cerebellum. The combined results describe an essential role for luminal acidification of the Golgi apparatus in maintaining proper neuronal morphology and neuronal circuitry.

2021/9/30　14:00～16:00　ZOOM A会場 S4-3 ALS 関連小胞体タンパク質VAP の細胞内及び細胞外における生理機能 Intra- and extracellular functions of ALS-related ER protein VAP ^○千原崇裕広島大学大学院統合生命化学研究科 ^○Takahiro Chihara Graduate School of Integrated Sciences for Life, Hiroshima University VAP, an Amyotrophic Lateral Sclerosis (ALS)-related endoplasmic reticulum (ER) protein functions as a tethering protein of the membrane contact sites between ER and various intracellular organelles. Recent studies suggested that VAP is cleaved, and its N-terminal MSP domain is secreted to the extracellular space in C. elegans, Drosophila, mouse, and human. The secreted MSP domain is known to bind to a variety of axon guidance receptors, implying that VAP could function non-cell autonomously. However, how the intra- and extracellular functions of VAP are implicated in disease onset and progression is not well understood. To investigate the physiological functions of VAP, we utilized Drosophila, enabling us to perform the genetic analyses for VAP. We found that vap^-/- neuronal clones exhibited severe morphological defects in their dendrite but not axon, which are rescued by the cell-autonomous expression of vap cDNA, suggesting that VAP cell-autonomously and preferentially regulates dendrite morphology. Next, to examine the roles of the secreted MSP domain, we biochemically identified the site for VAP cleavage and generated the uncleavable vap mutant whose VAP is not cleaved and secreted but can function intracellularly. The uncleavable vap mutant exhibited organismal lethality, indicating that the secreted MSP domain is essential for animal viability. Interestingly, the lethality of the uncleavable vap mutant was rescued by the overexpression of the MSP domain in the motor neuron. We will discuss the intra- and extracellular functions of VAP for a better understanding of ALS.		2021/9/30　14:00～16:00　ZOOM A会場 S4-4 小胞体プロテオスタシスを介した脳内コレステロール生合成の制御 ER proteostasis-regulated cholesterol biosynthesis in the brain ^○西頭英起宮崎大学医学部機能生化学 ^○Hideki Nishitoh Lab. of Biochemistry and Molecular Biology, Dept. of Medical Sciences, Univ. of Miyazaki Derlin family members (Derlins) are primarily known as components of the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway that eliminates misfolded proteins. Here we report an unexpected function of Derlins in the regulation of brain cholesterol metabolism. Deletion of Derlin-1 or Derlin-2 in the central nervous system of mice impaired postnatal brain development, particularly of the cerebellum and striatum, and induced motor control deficits. Derlin-1 or Derlin-2 deficiency reduced neurite outgrowth in vitro and in vivo, and surprisingly also inhibited sterol regulatory element binding protein 2 (SREBP-2)-mediated brain cholesterol biosynthesis. In addition, reduced neurite outgrowth due to Derlin-1 deficiency was rescued by SREBP-2 pathway activation. Overall, our findings demonstrate that Derlins sustain brain cholesterol biosynthesis, which is essential for appropriate postnatal brain development and function. In this session, I would like to discuss the relationship between ER protein homeostasis (proteostasis) and cholesterol homeostasis (cholesterostasis) in the brain.