一般口演
自閉スペクトラム症 / 神経変性疾患
Autism Spectrum Disorder / Neurodegenerative Disorders
座長：桑原 知樹（東京大学大学院医学系研究科 脳神経医学専攻基礎神経医学講座） |
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| 2022年7月3日 11:00～11:15 沖縄コンベンションセンター 会議場B3・4 第6会場
4O06a1-01
Identification of regulatory noncoding mutations associated with autism spectrum disorder
*Joon-Yong An(1,2,3), Il Bin Kim(4), Junehawk Lee(5), Yu-Jin Kim(1,2), Jae-Hyun Kim(1,2), Soo-Whee Kim(1,2), Jung Kyoon Choi(6), Jeong Ho Lee(7), Eunjoon Kim(6,7), Hee Jeong Yoo(8,9)
1. Department of Integrated Biomedical and Life Science, Korea University, Seoul, 02841, Republic of Korea, 2. BK21FOUR R&E Center for Learning Health Systems, Korea University, Seoul 02841, Korea, 3. School of Biosystem and Biomedical Science, College of Health Science, Korea University, Seoul 02841, Korea, 4. Department of Psychiatry, Hanyang University Guri Hospital, Guri 11923, Republic of Korea, 5. Center for Supercomputing Applications, Division of National Supercomputing, Korea Institute of Science and Technology Information, Daejeon 34141, Republic of Korea, 6. Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea, 7. Center for Synaptic Brain Dysfunctions, Institute for Basic Science, Daejeon 34141, Republic of Korea, 8. Department of Psychiatry, Seoul National University Bundang Hospital, Bundang 13620, Republic of Korea, 9. Department of Psychiatry, Seoul National University College of Medicine, Seoul 03080, Republic of Korea
Keyword: Autism Spectrum Disorder, Transcription Factor, Whole Genome Sequencing
De novo mutations play an important role in human disorders that impair reproductive fitness, including autism spectrum disorder (ASD), severe developmental delay, epileptic encephalopathy, and a spectrum of congenital anomalies. Next generation sequencing technology has successfully estimated the contribution of de novo mutations to ASD and resulted in remarkable advances in our understanding of the genetic architecture of risk for ASD. Recent advance in whole genome sequencing (WGS) has enabled the comprehensive analysis of genetic variants in the noncoding genome, where various regulatory elements determine the location of a gene in the nervous system and the cell type that expresses a gene during development. Here we examine noncoding variation in ASD using whole genome sequencing and discuss the latest findings in the study of the Korean ASD-WGS cohort, including 600 families (2,000 individuals) of ASD families, and prioritize the risk variants underlying ASD development. We apply the category-wide association study (CWAS) method, a novel analytic framework to estimate a risk of noncoding mutations across multiple functional annotation categories related to noncoding biology. We demonstrate noncoding mutation associated with promoter regions and conserved transcription factor binding sites distal to the transcription start site. We then show the common regulatory trajectory of ASD-associated mutations, acting act as components of an intricate molecular mechanism necessary for sufficient expression of genes in developing cells of the neuronal lineage. Our data suggest that de novo mutations in the noncoding genome contribute to ASD and may disrupt gene transcription via their interaction with enhancer elements in the promoter region. | | 2022年7月3日 11:15～11:30 沖縄コンベンションセンター 会議場B3・4 第6会場
4O06a1-02
ミクログリア置換は一次性アストロサイト疾患であるアレキサンダー病に対する新たな治療戦略となり得る。
Microglia replacement could be the new therapeutic strategy on Alexander disease, the primary astrocyte disease.
*小林 憲司(1,2)、繁冨 英治(1,2)、パラジュリ ビージェイ(1,2)、久保田 友人(1,2)、齋藤 光象(1,2)、田中 謙二(3)、池中 一裕(4)、小泉 修一(1,2)
1. 山梨大学院医薬理学、2. 山梨大学院医GLIAセンター、3. 慶應義塾大学医学部精神神経科学、4. 生理学研究所分子神経生理部門
*Kenji Kobayashi(1,2), Eiji Shigetomi(1,2), Bijay Parajuli(1,2), Yuto Kubota(1,2), Kozo Saito(1,2), Kenji F Tanaka(3), Kazuhiro Ikenaka(4), Schuichi Koizumi(1,2)
1. Dept Neuropharmacol, Interdiscipl Grad Sch Med, Univ Yamanashi, Yamanashi, Japan, 2. GLIA center, Interdiscipl Grad Sch Med, Univ Yamanashi, Yamanashi, Japan, 3. Dept Neuropsych, Keio Univ Sch Med, Tokyo, Japan, 4. Div Neurobiol and Bioinfo, NIPS, Aichi, Japan
Keyword: Microglia replacement, Astrocyte, Neurodegenerative disease, Neuroinflammation
Alexander disease (AxD) is caused by a gain-of-dysfunction mutation in the Glial Fibrillary Acidic Protein (GFAP) gene, the major intermediate filament in astrocytes. AxD is a rare neurodegenerative disease characterized by the degeneration of white matter and the formation of Rosenthal fiber (RF), the abnormal aggregates within astrocytes which is mainly composed of GFAP. AxD shows severe symptoms such as intractable epilepsy, but currently there is no effective treatment. Thus, AxD is a primary astrocyte disease and AxD researchers have mainly focused on astrocytes. However, recent studies have shown inflammation with microglia activation occurs in AxD brains. Here, we focus on microglia, and elucidate the molecular pathogenesis of AxD from the point of view of microglia and develop new therapeutic strategies. We have previously reported the protective role of microglia on AxD pathology using the AxD model mice with over-expression of human GFAP with R239H mutation (60TM). This finding motivated us to manipulate the profile of microglia by replacing it with a new one to enhance the protective role of microglia. To accomplish this, we used an ON/OFF protocol of PLX5622 (PLX), a selective Colony Stimulating Factor-1 Receptor (CSF-1R) antagonist. Since CSF-1R signaling is necessary for microglia survival, PLX-ON for 7 days depleted about 94 % of microglia in the 60TM hippocampus. Subsequent withdrawal of PLX (PLX-OFF) for 14 days resulted in a dramatic recovery in the number of microglia to ~110 % of that of age-matched control 60TM. To assess the influence of microglia replacement on AxD astrocytes, we evaluated the RF stained with Fluoro-Jade B (FJB). The percentage of GFAP and FJB double-positive astrocytes was significantly decreased after microglia replacement. To further confirm, we conducted RNA-seq using whole hippocampal tissues. RNA-seq showed that the replacement of microglia significantly altered the gene expression profile of ~300 genes. Among them, we found significant decreases in the top 4 upregulated inflammatory genes in the 60TM hippocampus, Lcn2, Cxcl10, Ccl2, and C3. These results showed that the microglia replacement ameliorated proinflammatory status of the 60TM brain. Taken together, it is suggested that although AxD is a primary astrocyte disease, microglia are involved in its pathogenesis and that interventions in microglia could be an effective and potential new therapeutic strategy on AxD. | | 2022年7月3日 11:30～11:45 沖縄コンベンションセンター 会議場B3・4 第6会場
4O06a1-03
ミクログリアにおけるLRRK2-Rab10経路を介したαシヌクレイン分泌機構の解明
Mechanism of α-synuclein release from microglia via the LRRK2-Rab10 pathway
*桑原 知樹(1)、阿部 哲郎(1)、末長 祥一(1)、櫻井 まりあ(1)、吉井 元(1)、小森 禎之(1)、岩坪 威(1)
1. 東京大学大学院医学系研究科
*Tomoki Kuwahara(1), Tetsuro Abe(1), Shoichi Suenaga(1), Maria Sakurai(1), Gen Yoshii(1), Tadayuki Komori(1), Takeshi Iwatsubo(1)
1. Grad Sch Med, Univ of Tokyo, Tokyo, Japan
Keyword: Parkinson's disease, alpha-synuclein, LRRK2, microglia
alpha-Synuclein and LRRK2 are two major genes associated with both familial and idiopathic Parkinson’s disease (PD). alpha-Synuclein aggregates, as observed in PD lesions, has been shown to cause lysosomal stress upon exposure to cells, whereas LRRK2, a Rab kinase, is upregulated when lysosomes are stressed. We have previously shown that treating cells with lysosomotropic drugs such as chloroquine causes the recruitment of LRRK2 and its substrates Rab10 onto overloaded lysosomes, leading to the extracellular release of lysosomal contents via Rab10 phosphorylation. Here we reveal that lysosomal overload stress causes extracellular release of insoluble alpha-synuclein aggregates from microglial cells that had taken up alpha-synuclein pre-formed fibrils (PFFs). This release was fully dependent on LRRK2 and its substrate Rab10, and was not observed in neuronal cells. On the other hand, the internalization of alpha-synuclein PFFs significantly upregulated LRRK2 phosphorylation of Rab10. This upregulation was accompanied by lysosomal localization of incorporated alpha-synuclein as well as the increased proximity of LRRK2/Rab10 to lysosomal surface, as observed upon lysosomal overload. In addition, the increased proximity of LRRK2 to lysosomes was detected in the substantia nigra of PD patients, as compared with healthy controls. These results suggest that alpha-synuclein aggregates enhance LRRK2 phosphorylation of Rab10, and the enhanced LRRK2-Rab10 pathway in turn promotes extracellular release of alpha-synuclein aggregates, forming a positive feedback loop that leads to pathogenic alpha-synuclein transmission. This loop may form especially in microglia and under lysosomal stress, and thus could be a novel pathomechanism of PD. | | 2022年7月3日 11:45～12:00 沖縄コンベンションセンター 会議場B3・4 第6会場
4O06a1-04
プログラニュリン発現低下によるオートファジーフラックスの抑制は易凝集性のTDP-43蓄積を増加する
The suppression of autophagic flux from progranulin insufficiency increases aggregate-prone TDP-43 accumulation
*田中 良法(1)、松原 叶実(1)、日野 浩嗣(2)、本間 優希(1)、楠本 竣也(1)、竹谷 浩介(1)、江藤 真澄(1)
1. 岡山理科大学 獣医学部、2. 日本大学医学部 機能形態学系
*Yoshinori Tanaka(1), Kanami Matsubara(1), Hirotsugu Hino(2), Yuki Honma(1), Shun-ya Kusumoto(1), Kosuke Takeya(1), Masumi Eto(1)
1. Dept. of Veterinary Medicine, Okayama University of Science, 2. Dept. of Anatomical Scinece, Nihon University School of Medicine
Keyword: progranulin, TDP-43, autophagy flux, vacuolation
Progranulin (PGRN) haploinsufficiency resulting from the loss-of-function mutations of the PGRN gene causes frontotemporal lobar degeneration, which is characterized by the cytoplasmic inclusion predominantly containing a nuclear protein TDP-43. Macroautophagy (hereafter autophagy) is a cellular degradation system, and an essential machinery to suppress the aggregate-prone TDP-43 accumulation. Autophagy process is mainly comprised of two parts; an autophagosome formation and an autolysosome formation via autophagosome-lysosome fusion. Cellular TDP-43 is sequestered in autophagosome and degraded in autolysosome. We previously showed that PGRN insufficiency causes lysosomal dysfunction, suggesting outstanding roles in the regulation of lysosomes. Therefore, we hypothesized that PGRN insufficiency influences autophagosome-lysosome fusion, which contributes to aggregate-prone TDP-43 accumulation. We firstly investigated the role of PGRN in autophagy. PGRN insufficiency did not influence phagophore and autophagosome formation in the ordinary cultural condition. On the other hand, we found that PGRN insufficiency suppressed autophagic flux evidenced by studies using an endogenous LC3II immunoblotting, an autophagic flux marker GFP-LC3-RFP-LC3-ΔG, and an autolysosome marker DALGreen. Fluorescence imaging using the cell expressing mRFP-GFP-LC3 showed that PGRN insufficiency increases autophagosome accumulation. Next, we investigated the role of PGRN insufficiency on aggregate-prone TDP-43 accumulation. Live cell confocal imaging showed that aggregate-prone C-terminal fragment of TDP-43 tagged with GFP (162-414 residues; 162C) was co-localized with mCherry-LC3. Knockdown of PGRN increased 162C aggregation and accumulation. To investigate whether suppression of autophagic flux due to PGRN insufficiency increases aggregate-prone TDP-43 accumulation, we focused on Abemaciclib (Abe) or Vacuolin-1 (Vac) as an accelerator for autophagosome-lysosome fusion. In fact, Abe and Vac treatment facilitated autophagic flux, and could induce the vacuole-like enlarged autolysosome formation autophagy-dependently. Abe and Vac treatment decreased autophagosome accumulation and increased autolysosome formation in PGRN insufficient cells. Moreover, Abe and Vac treatment reduced 162C accumulation and aggregation in PGRN insufficient cells. Consequently, these results suggest that PGRN insufficiency suppresses autophagic flux, which contributes to aggregate-prone TDP-43 accumulation. | |
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