[보고서]뇌손상과 재생 관련 astrocyte의 기능변화 규명과 뇌기능 조절

김성현

보고서 정보

주관연구기관

경희대학교
Kyung Hee University

연구책임자

김성현

참여연구자

양해걸

보고서유형

최종보고서

발행국가

대한민국

언어

한국어

발행년월

2021-02

과제시작연도

2021

주관부처

과학기술정보통신부
Ministry of Science and ICT

연구관리전문기관

한국연구재단
National Research Foundation of Korea

등록번호

TRKO202100010517

과제고유번호

1711129298

사업명

뇌과학원천기술개발(R&D)

DB 구축일자

2022-04-09

키워드

뇌손상.재생.성상교세포.교세포기능 조절.뇌기능조절.뇌염증.지속적 억제.세포부착.brain demage.Regeneration.Astrocyte.Regulation of Astrocyte function.Regulation of Brain Function.Neuroinflammation.Tonic inhibition.cell adhesion.

표 Astrogliosis in response to injury is attenuated in the DJ-1-KO brain. Brain damage was induced by stereotaxic injection of ATP (400 nmole) into the striatum of WT and DJ-1-KO mice. (A) GFAP protein levels at the indicated times after ATP injection were measured by Western blot, and quantified. Values are means±SEMs of 3 mouse at indicated time points. (B) Sections (40 μm) obtained at 3 and 7 d after ATP injection were stained with GFAP antibodies. The most damaged sections are shown. *, injection sites. Dotted lines indicate damaged region. Astrocytes close to the damage core (P1, arrows) and adjacent to the core (P2, arrowheads). (C) The morphology of GFAP-positive astrocytes in WT and DJ-1-KO brains was analyzed 3 and 7 d after ATP injection. The morphology of astrocytes was reconstructed by Neurolucida 3D modeling. At the indicated times, lengths of processes were measured by Sholl analysis. Values are means±SEMs of 50 cells in 4 mice. n.s, nonsignificant; **, P<0.005; ***, P<0.0001 from Two-way ANOVA (A) and unpaired two tailed t-test (C). Scale bar, 100 μm (B)
표 BDNF and GDNF expression are attenuated in the DJ-1-KO brain. (A, B) Brain sections obtained 3 d after ATP injection were stained with antibodies for BDNF, GFAP, NeuN and/or GDNF as indicated, and visualized using Alexa Fluor 488- or Alexa Fluor 555-conjugated secondary antibodies. Arrows and arrowheads denote neurons and astrocytes, respectively. Intensities were measured using Image J (A, B right). Values are means±SEMs of 20 cells from 3 mice. (C, D) BDNF and GDNF mRNA levels in WT and KO brains at the indicated times after ATP injection (C) or slices at the indicated times after slicing (D) were analyzed by qPCR. (E) BDNF and GDNF protein levels in WT and KO SCM were analyzed by ELISA. Values are means±SEMs of 3 mice (C), 3 (D) or 6 (E) independent slice cultures. n.s, nonsignificant; *, P<0.05; **, P<0.005; ***, P<0.0001 from Two-way ANOVA (A-D) and from unpaired two tailed t-test (E). Scale bars, 20 μm (A, B)
표 A DJ-1 deficiency delays repair of the injury in the striatum. Brain damage was induced by stereotaxic injection of ATP into the striatum of WT and DJ-1-KO mice. (A) Damage sites were imaged by 9.4T MRI at the indicated times after ATP injection. Serial coronal images were taken every 250 μm. Yellow lines indicate damaged regions. (B) Damaged regions (red) were reconstructed by Neurolucida 3D modeling based on MRI images. Damage volumes were calculated. Values are means±SEMs of 3 mice. (C) Brain sections (40 μm) were prepared at the indicated times after ATP injection and stained with TH antibodies. The most damaged sections are shown. (D) TH-negative regions (red) were reconstructed by Neurolucida 3D modeling based on TH staining, and volumes were quantified. Values are means±SEMs of 4 mice (D). n.s, nonsignificant from unpaired two tailed t-test (C, E); **, P<0.005; ***, P<0.0001 from Two-way ANOVA (C, E). Scale bar, 1 mm (C)
표 DJ-1 deficiency attenuates STAT3 activation. (A, B) Levels of pSTAT3 and GFAP were analyzed by Western blotting in acutely prepared WT and KO slices at the indicated times after slicing (A), or in KO slices transfected with Flag-DJ-1 at the indicated times after ATP treatment (B). Band intensities were analyzed; GAPDH was used as a loading control. Values are means±SEMs of 3 independent acute slice tissues. (c) Acutely prepared slices were incubated with or without the STAT3 inhibitor, DPP (50 mM). BDNF, GDNF, and GFAP mRNA levels were analyzed by qPCR at the indicated times after slicing. Values are means±SEMs of 6 independent acute slice tissue. *,P<0.05;**,P<0.005;***,P<0.0001 from Two-way ANOVA (A, B) and One-way ANOVA (C)
표 DJ-1 positively regulates Sox9 expression. (A-D) Sox9 expression in WT and DJ-1 KO brain (A, C) and cultured astrocytes (B, D) was analyzed by Western blot (A, B) and double-immunostaining using GFAP and Sox9 specific antibodies (C, D). Sox9 intensities were quantified by Image J. The numbers in (A) indicates three different animals. Values are means±SEMs of 6 mice (A) of 6 independent culture (B), of 18 cells in 3 mice (C), of 6 independent culture (D). (E) DJ-1 KO astrocytes were transfected with Mock or Flag-hDJ-1 for 3day. Sox9 expression was analyzed by Western blot. Values are means±SEMs of 4 independent culture. ***, P<0.0001 from unpaired two tailed t-test (A-E). Scale bars, 10 μm (C, D)
표 Overexpression of Sox9 restores GFAP expression in DJ-1 KO slices. Slices were prepared from KO mouse cortices and incubated for 7 d, and transfected with empty vector (Mock), or myc-Sox9. Astrogliosis was induced by ATP (50 mM) for 48 h. GFAP expression was analyzed by immunostaining (left panel) or western blot (righht panel). Fluorescence intensities or band intensities were quantified using Image J (graphs). GAPDH was used as a loading control. Values are means±SEMs of 3 independent experiments. *, P<0.05; **, P<0.005; ***, P<0.0001 from Two-way ANOVA. Scale bar, 50 ㎛
표 Overexpression of Sox9 rescues growth factor expression in DJ-1 KO brain slices and astrocytes. KO slices cultured for 7 d (left panel), and astrocytes cultured from KO brains (right panel) were transfected with empty vector (Mock) or myc-Sox9. BDNF and GDNF mRNA expression were measured by qPCR at the indicate times after 50 mM ATP treatment. Values are means±SEMs of 3 independent experiments. *, P<0.05; **, P<0.005; ***, P<0.0001 from Two-way ANOVA
표 Different expression of astrocyte markers in different brain regions. (A) Protein samples were obtained from different brain regions. GFAP and ALDH1L1 levels were analyzed with Western blotting. GAPDH was used as a loading control. Data are representative of at least 3 animals. (B) GFAP was analyzed with ALDH1L1 of cortex, striatum and hippocampus, respectively. Location of each protein was visualized with Alexa 488- and 594-conjugated secondary antibodies. DAPI marked nuclei in blue. Arrows, GFAP+ cells; white arrowheads, ALDH1L1+ cells; yellow arrowheads, ALDH1L1+ GFAP- cells. Confocal images were obtained. Scale bars 50 ㎛
표 Time-dependent behavior of GFAP+ astrocytes in ATP-injured cortex, striatum and hippocampus. (A) Coronal sections obtained at 1-30 d after stereotaxic injection of ATP (400 nmol) were processed for GFAP immunostaining. Photographs of the most damaged sections were obtained. Asterisks (*) indicate injection sites. cc, copus callosum; v, ventricle. Higher magnification images of boxed areas in the left column of each region were shown in right columns. Blue arrows, hypertrophic and polarized GFAP+ cells found in the penumbra regions next to the damage site (P1); blue arrowheads, hypertrophic GFAP+ cells found in the penumbra regions next to P1 (P2); blue bold arrows, glia scar formed near pia mater. (B) GFAP negative areas at the indicated times after ATP injection were quantified as damage areas. Values are means ± SEMs of more than three animals (*p < 0.001; Student′s t-test)
표 Sox9 expression in different brain regions
표 Blood vessel formation play critical roles for astroglial scar formation in the cortex and the striatum
표 Effect of Sox9 siRNA and over-expression on astrocyte CCL2 expression (a, b, c) and effect on macrophage migration of slice culture conditioned media obtained after Sox9 or Mock transfection
표 monocyte infiltration in injured brains of WT, DJ-1 KO, and CCR2 KO mice
표 Attenuated repair of neural functions in injured brains of WT, DJ-1 KO, and CCR2 KO mice
표 유전자 전달 시스템 구축
표 astrocyte 에서 기원한 세포 내, 외부의 전달물질 분석
표 astrocyte 유래 GABA가 매개하는 지속적 억제 (tonic inhibition) 차이 관찰
표 GIT1 KO ADHD mouse model에서 glial GABA와 tonic inhibition
표 astrocyte에서 분비된 GABA에 의한 신경활성 조절
표 astrocyte에서 분비된 GABA의 생리기능조절
표 viral injection for Astrocyte specific GABA-T regulation in vivo
표 Tonic current change in the presence of GABA-T antagonist (left), the screening for astrocyte specific GABA-T expression (right)
표 타겟단백질 (CADM3) shRNA모식도(위), shRNA를 세포에 주입하여 타겟단백질의 효과적 발현저해를 면역형광법으로 확인함 (아래)
표 타겟단백질 (CADM3) shRNA 발현 후 신경활성에 따른 신경시냅스트랜스미션을 나타낸 그래프 (왼쪽). 컨트롤과 shRNA발현 세포에서 신경시냅스트랜스미션의 평균량 비교 (가운데), 컨트롤과 shRNA발현 세포의 신경시냅스트랜스미션의 양 비교 사진 (오른쪽)
표 타겟단백질 (CADM3) shRNA 발현 후 신경말단의 생리적 기능 확인을 위해 기능성 시냅스의 크기 확인한 그래프 (정상: 왼쪽, 실험군: 오른쪽). 기능성 시냅스의 평균 크기 측정 결과 (오른쪽)
표 타겟단백질 (CADM3) shRNA 발현 후 신경말단의 칼슘다니마믹스 확인. 1 Action Potentil (1AP)에 의한 신경말단 칼슘 다이나믹스 측정 (위). 100 AP에 의한 칼슘 다이나믹스 측정 (아래). 모두 변화가 없음
표 타겟단백질 (CADM3) 발현저해 후 재발현을 통해 신경시냅스트랜스미션 기능조절 확인. 신경활성에 따른 신경트랜스미션의 조절 양상에 대한 그래프 (왼쪽), 타겟단백질 (CADM3)의 발현 저해 및 재발현에 따른 신경시냅스트랜스미션의 평균량(가운데)과 그에 상응은 하는 실시간 반응 사진 (오른쪽)
표 타겟단백질 (CADM3)의 도메인 발현을 통한 신경시냅스트랜스미션 담당 지역 규명. 각 도메인으로 구성된 지역 그림 (왼쪽). 각 도메일의 발현에 의해 나타나는 신경시냅스트랜스미션 그래프 (가운데)와 그 평균 히스토그램 (오른쪽)
표 타겟단백질 (CADM3) 발현을 통해 구조적 시냅스 형성에 참여하는지 기능확인. 교세포에 타겟단백질을 발현하고 신경세포를 함께 발현하여 시냅스형성에 구조/기능적으로 참여하는가를 확인 (왼쪽, 가운데). 사진의 정량분석 히스토그램 (오른쪽)
표 DJ-1의 세포분포를 위해 면역형광법 (왼쪽), 전자현미경 (가운데), 초고해상도 이미징 (오른쪽)을 이용하여 세포 특이지역에 분포함을 규명함
표 DJ-1의 결핍 시냅스에서 전자현미경을 통해 미세구조의 변화를 분석 (왼쪽) 하고 그 미세 구조 사진 (오른쪽)
표 Localization of CADM3 WT and deletion mutant in astrocyte (left) and in dendrite of neurons (right)
표 Suppression of synaptic transmission in astrocytic CADM3 deleted tripartite synapses
표 alteration of synaptic transmission in Caveolin-1 KD synapses

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[국가R&D연구보고서] 뇌손상과 재생 관련 astrocyte의 기능변화 규명과 뇌기능 조절
Astrocyte functional changes and regulation of brain function during brain damage and restoration 원문보기

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[국가R&D연구보고서] 뇌손상과 재생 관련 astrocyte의 기능변화 규명과 뇌기능 조절 Astrocyte functional changes and regulation of brain function during brain damage and restoration 원문보기

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[국가R&D연구보고서] 뇌손상과 재생 관련 astrocyte의 기능변화 규명과 뇌기능 조절
Astrocyte functional changes and regulation of brain function during brain damage and restoration 원문보기

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