[보고서]Tg 및 KO 마우스를 이용한 PLPP 매개성 신경세포 수용체 조절 및 세포사멸기전 연구

강태천

보고서 정보

주관연구기관

한림대학교
HalLym University

연구책임자

강태천

보고서유형

최종보고서

발행국가

대한민국

언어

한국어

발행년월

2018-05

과제시작연도

2017

주관부처

과학기술정보통신부
Ministry of Science and ICT

등록번호

TRKO201900023257

과제고유번호

1711051707

사업명

개인기초연구(미래부)

DB 구축일자

2020-08-01

키워드

세포사멸.신경세포흥분도.PLPP/CIN.CSEN.ULK1.F-actin.Cofilin.P2X7R.Heat shock protein 25.

표 Profiles of seizure activity in PLPP/CINTg and PLPP/CIN-/-mice induced by KA. (A-B) Characteristics of dendritic spine morphology in PLPP/CINTg and PLPP/CIN-/-mice. (C-E) Seizure activity in response to KA. PLPP/CINTg mice demonstrate increases in seizure intensity, duration and the latency of seizure on-set, as compared to PLPP/CIN-/-mice. (F-G) KA-induced neuronal death. One day after KA injection, neuronal death in PLPP/CINTg mice is more severe than that in PLPP/CIN-/- mice, as compared to WT animals
표 The role of F-actin dynamics in seizure activity induced by KA. (A-C) Changed PLPP/CIN expression and cofilin phosphorylation 2 h after KA injection. (D-F) The effect of F-actin dynamics on seizure activity in response to KA. Jasplakinolide (a F-actin stabilizer) decreases seizure intensity (total EEG power) in PLPP/CINTg mice, but latrunculin A( a F-actin depolymerizer) enhances it in PLPP/CIN-/- animals. However, neither jasplakinolide nor latrunculin Aa ffects the latency of seizure on-set in both groups
표 PLPP/CIN-mediated CSEN dephosphorylation. (A) Representative double immunofluorescent photos for PLPP/CIN and CSEN. PLPP/CIN is colocalized with CSEN in hippocampal neurons in PLPP/CINTgmice. (B-C) Changed CSEN phosphorylation 2h after KA injection. Western blot shows the CSEN phosphorylation is lower in PLPP/CINTg mice, but is higher in PLPP/CIN-/- mice. In addition, KA reduces CSEN phosphorylation without altered CSEN expression in WT animals. The reduced CSEN phosphorylation in PLPP/CINTg mice is more efficient, while that in PLPP/CIN-/- mice is less, as compared to WT animals. (D-F) In vitro assay using recombinant proteins. Co-immnoprecipitation and western blot reveal that PLPP/CIN binds to CSEN, and dephosphorylates CSEN
표 The effect of seizure activity on PLPP/CIN-mediated CSEN dephosphorylation invivo. (A-B) Changed PLPP/CIN-CSEN bindings 2 h after KA injection. Western blot shows that PLPP/CIN-CSEN co-precipitation in PLPP/CINTg mice is higher than WT animals under physiological condition. KA increases the binding of PLPP/CIN to CSEN in WT and PLPP/CINTg mice. (C-D) Colocalization of PLPP/CIN and CSEN. KA increases the area of colocalization of PLPP/CIN and CSEN in CA1 neurons
표 The PLPP/CIN-mediated CSEN dephosphorylation in Kv4.2 functionality in vivo and in vitro. (A-B) Changed Kv4.2-CSEN bindings 2h after KA injection. Under physiological condition, western blot shows that Kv4.2-CSEN co-precipitation is higher in PLPP/CINTg mice, but is lower in PLPP/CIN-/- mice, as compared to WT animals. KA increases the binding of CSEN to Kv4.2 in WT and PLCC.CINTg mice. (C-D) In vitro assay using crude extract obtained from PLPP/CIN-/- mouse brain. PLPP/CIN treatment increases the Kv4.2-CSEN binding, as compared to vehice. (E-G) The comparison of Kv4.2 functionality in response to 4-AP. As compared to WT animals, PLPP.CINTg mice show the higher responsiveness to 4-AP, but PLPP/CIN-/- animals reveal lower responsiveness, as compared to WT animals
표 The roles of PLPP/CIN in GluN1-CSEN binding and CK1-mediated CSEN phosphorylation in vivo and in vitro. (A-B)Changed GluN1-CSEN bindings 2 h after KA injection. Under physiological condition, western blot shows that GluN1-CSEN co-precipitation is lower in PLPP/CINTg mice, but is higher in PLPP/CIN-/- mice, as compared to WT animals. KA decreases the binding of CSEN to GluN1 in all groups. (C-D) In vitro assay using crude extract obtained from PLPP/CIN-/- mouse brain. PLPP/CIN treatment decreases the GluN1-CSEN binding, as compared to vehicle. (E-I) The competitive role of PLPP/CIN in the binding of CK1 to CSEN in vitro. The interactions between PLPP/CIN and CSEN are in competition with CK1
표 Effect of CK1 inhibitors on CSEN phosphorylation, CSEN interaction and seizure activity in PLPP/CIN-/-mice. (A-C) The effect of CK1 inhibitors on CSEN phosphorylation and CSEN interaction with GluN1 and Kv4.2. IC261 and CKI-7 cannot change pCofilin level. Both CK1 inhibitors decrease pCSEN level, and CK1- and GluN1-CSEN bindings. However, CK1 inhibitiors increase Kv4.2-CSEN co-precipitation. (D-F) The effect of CK1 inhibitors on seizure activity in response to KA. CK1 inhibitors increase the latency of seizure on-set and the seizure duration in response to KA. (G-H) Effect of CK1 inhibitors on KA-induced neuronal death. One day after KA injection, both CK1 inhibitors exacerbate neuronal death, as compared to vehicle
표 Summary of the role of PLPP/CIN in seizure activity in response to KA
표 Effect of P2X7R deletion on HSP25-mediated autophagy in response to KA. (A) Effect of P2X7R deletion on seizure susceptibility in response to KA. P2X7R deletion does not affect the seizure susceptibility and its severity in response to KA. (B) Effect of P2X7R deletion on HSP25 mRNA expression in response to KA. P2X7R deletion significantly elevates HSP25 mRNA expression. (C-E) Effect of P2X7R deletion on SE-induced HSP25 and LAMP1 protein expressions. As compared to WT mice, both HSP25 and LAMP1 protein expression levels are higher in KO mice. (F) Representative photos demonstrating HSP25 and LAMP1 expression in astrocytes 7 days after KA injection. P2X7R deletion increases HSP25 and LAMP1 expressions. Bar = 12.5 μm
표 Role of P2X7R in HSP25 and LAMP1 expression following KA injection. (A) Representative photos demonstrating HSP25 expression in astrocytes 3 days after KA injection. Bar = 25 μm. (B) Representative photos demonstrating LAMP1 expression in astrocytes of control animals. Bar = 25 μm. (C) Effect of P2X7R agonist and antagonists on HSP25 and LAMP1 protein expressions in WT mice 7 days after KA injection. (D) Quantifications of effect of P2X7R agonist and antagonists on HSP25 and LAMP1 protein expressions in WT mice 7 days after KA injection (*p < 0.05 vs. vehicle)
표 HSP25-mediated ER stress in KO mice following KA injection. (A) Representative western blot of ER stress-related molecules. P2X7R deletion induces ER stress following KA injection. (B) Effect of HSP25 knockdown in HSP25-mediated ER stress. HSP25 siRNA attenuates HSP25-mediated ER stress in KO mice following SE. (C) Quantifications of ER stress-related molecules in KO mice following KA injection. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (*,#p < 0.05 vs. control and WT mice, respectively). (D) Quantifications of effect of HSP25 siRNA on SE-induced ERstress (*p < 0.05 vs. control siRNA)
표 (A-B) Effect of P2X7R deletion on CHOP and ATF6 expressions and IRE1 phosphorylation. There is no difference in p-IRE1, CHOP and ATF6 levels between WT and KO mice. (A) Representative western blot of CHOP and ATF6 expressions and IRE1 phosphorylation. (B) Quantifications of CHOP and ATF6 expressions and IRE1 phosphorylation. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (*p < 0.05 vs. vehicle, respectively). (C) Effect of P2X7R deletion on CMA in KO mice. Co-immunoprecipitation reveals that HSP25 cannot bind with HSC70
표 Effect of HSP25 knockdown on HSP25-mediated astroglial ER stress and autophagy in KO mice. HSP25 siRNA abrogates HSP25 induction, ER stress and autophagy in KO mice following SE. (A) Representative photos demonstrating astroglial HSP25, ATF4 and LAMP1 expression in KOmice 7days after KA injection. Bar=6.25μm. (B) Quantifications of effect of HSP25 siRNA on astroglial ER stress and autophagy. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (*p < 0.05 vs. control siRNA;n = 7, respectively)
표 AMPK/ULK1-mediated astroglial autophagy in KO mice following KA injection. (A) Representative western blot of ULK1-related molecules. KA injection increases ULK1-S555, AMPK-T173 and AKT-S473 phosphorylation in KO mice without altered their expressions. (B) Quantifications of expressions of ULK1-related molecules. Open circles indicate each individual value. (C) Quantifications of phosphorylations of ULK1-related molecules. (D) Representative western blot of effect of HSP25 knockdown on ULK1-related molecules in KO mice. HSP25 siRNA decreases ULK1-S555 and AMPK-T173 phosphorylations in KO mice without altered their expressions 7 days after KA injection. (E) Quantifications of effect of HSP25 knockdown on ULK1-related molecules. (F) Representative western blot of effect of AMPK inhibition on ULK1-related molecules in KO mice. Compound C (Comp C) decreases ULK1-S555 phosphorylation in KO mice without altered its expression 7 days after KA injection. (G) Quantifications of effect of Comp C on ULK1-related molecules. (H) Representative photos demonstrating astroglial ULK1-S555phosphorylation in KO mice 7days after KA injection. Both HSP25 siRNA and Comp C mitigate ULK1-S555 phosphorylation in astrocytes. Bar = 6.25 μm
표 Effect of P2X7R deletion on PI3K, PRAS40, mTOR and p70S6K expressions and their phosphorylations. PI3K-Y458 phosphorylation is reduced in both WT and KO mice following KA treatment. In addition, mTOR expression and its phosphorylation are reduced in both WT and KO mice 3 days after KA injection. However, there is no difference in PI3K, PRAS40, mTOR and p70S6K expressions and their phosphorylations between WT and KO mice. (A) Representative western blot of PI3K, PRAS40, mTOR and p70S6K expressions and their phosphorylations. (B) Quantifications of PI3K, PRAS40, mTOR and p70S6K expressions and their phosphorylations. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (*p < 0.05 vs. control). (C) QuantificationsofPI3K, PRAS40, mTOR and p70S6K phosphorylations (*p < 0.05 vs. control)
표 AKT-GSK3β/Bif-1-mediated astroglial autophagy in KO mice following KA injection. (A)Representative western blot of GSK3β, Bif-1 and HSF1 expression and their phosphorylations. KA injection increases GSK3β-S9 phosphorylation and Bif-1 expression in KO mice without altered HSP1 expression and its phosphorylation. (B) Quantifications of GSK3β, Bif-1 and HSF1 expressions. (C) Quantifications of GSK3β and HSF1 phosphorylations. (D) Representative western blot of effect of HSP25 knockdown on GSK3β phosphorylation and Bif-1 expression in KO mice. HSP25 siRNA decreases GSK3β-S3 phosphorylation and Bif-1 expression in KO mice 7 days after KA injection. (E) QuantificationsofeffectofHSP25knockdownonGSK3β phosphorylation and Bif-1 expression. (F) Representative western blot of effect of AKT inhibition on GSK3β, Bif-1, HSP25 and HSF1 expression and their phosphorylations. 3CAI decreases GSK3β-S3 phosphorylation and Bif-1 expression in KO mice without altered HSP25 expression and HSF1 phosphorylation 7 days after KA injection. (G) Quantifications of effect of 3CAI on GSK3β, Bif-1, HSP25 and HSF1 expression and their phosphorylations. (H) Representative photos demonstrating astroglial Bif-1 accumulation phosphorylation in KO mice 7 days after KA injection. Both HSP25 siRNA and 3CAI mitigate Bif-1 accumulation in astrocytes. Bar = 3.5μm
표 ERK-SP1-HSP25-mediated astroglial autophagy in KO mice following KA injection. (A) Representative western blot of ERK1/2 and SP1 expression and their phosphorylations. KA injection reduces ERK1/2 and SP1-T739 phosphorylations in KO mice without altered their expressions 7 days after SE. (B) Quantifications of ERK1/2 and SP1 expressions and their phosphorylations. (C) Representative western blot of effect of SP1 inhibition on AMPK- and AKT-mediated astroglial autophagy in KO mice. Mithramycin A (MMA) effectively inhibits AMPK- and AKT-mediated signaling pathways following KA injection. (D) Quantifications of effect of MMA on AMPK- and AKT-mediated signaling pathways. (E) Representative photos demonstrating the effect of MMA on HSP25 and LAMP1 expression in KOmice 7days after KA injection. MMA abolishes both HSP25 and LAMP1 expression in astrocytes. Bar = 25 μm
표 Effect of ERK1/2 inhibition on HSP25 expression in WT mice following KA injection. (A) Representative western blot of astroglial autophagy-related molecules. U0126 injection leads to HSP25-mediated astroglial autophagy in WT mice 7 days after SE. (B) Quantifications of effect of U0126 on HSP25-mediated astroglial autophagy in WT mice. (C) Representative photos demonstrating the effect of U0126 on HSP25 and LAMP1 expression in WT mice 7 days after KA injection. U0126 increases both HSP25 and LAMP1 expression in astrocytes. Bar = 25μm
표 Scheme of inhibitory role of P2X7R in astroglial autophagy induced by KA injection. SE increases HSP25 expression, and activates P2X7R, which inhibits prolonged HSP25 expression via ERK1/2-mediated SP1 phsophorylation. However, P2X7R deletion or its antagonists impair this inhibitory signaling pathway, which leads to sustained HSP25 expression and ER stress. Subsequently, ER stress activates AMPK/ULK1-mediated astroglial autophagy. Sustained HSP25 expression also activates AKT/GSK3β-mediated Bif-1 accumulation and triggers astroglial autophagy. Thus, P2X7R may regulate ER stress and autophagy by the fine-tuning of HSP25 expression in astrocytes

과제명(ProjectTitle) :	-
연구책임자(Manager) :	-
과제기간(DetailSeriesProject) :	-
총연구비 (DetailSeriesProject) :	-
키워드(keyword) :	-
과제수행기간(LeadAgency) :	-
연구목표(Goal) :	-
연구내용(Abstract) :	-
기대효과(Effect) :	-

내보내기 구분	파일저장 인쇄 메일전송
구성항목	기본정보 상세정보 관리번호, 제목(한글), 저자명(한글), 발행일자, 전자원문, 초록(한글), 초록(영문) 관리번호, 제목(한글), 제목(영문), 저자명(한글), 저자명(영문), 주관연구기관(한글), 주관연구기관(영문), 발행일자, 총페이지수, 주관부처명, 과제시작일, 보고서번호, 과제종료일, 주제분류, 키워드(한글), 전자원문, 키워드(영문), 입수제어번호, 초록(한글), 초록(영문), 목차
저장형식	Text(ASCII format) Excel format
메일정보	받는사람 (필수) @ 보내는사람 (선택) @ 제목 내용 KISTI 검색결과 이메일 서비스
안내	총 건의 자료가 검색되었습니다. 다운받으실 자료의 인덱스를 입력하세요. (1-10,000) 검색결과의 순서대로 최대 10,000건 까지 다운로드가 가능합니다. 데이타가 많을 경우 속도가 느려질 수 있습니다.(최대 2~3분 소요) 다운로드 파일은 UTF-8 형태로 저장됩니다. 파일의 내용이 제대로 보이지 않을실 때는 웹브라우저 상단의 보기 -> 인코딩 -> 자동선택 여부를 확인하십시오. ~ Text(ASCII format) Excel format

연합인증

Tg 및 KO 마우스를 이용한 PLPP 매개성 신경세포 수용체 조절 및 세포사멸기전 연구
The mechanism of PLPP-mediated neuronal receptor regulation and cell death using Tg and KO mice 원문보기

초록 ▼

목차 Contents

표/그림 (19)

표/그림 (19)

참고문헌 (25)

연구과제 타임라인

관련 콘텐츠

원문 보기

이 보고서와 함께 이용한 콘텐츠

AI-Helper ※ AI-Helper는 오픈소스 모델을 사용합니다.

선택된 텍스트

연합인증

Tg 및 KO 마우스를 이용한 PLPP 매개성 신경세포 수용체 조절 및 세포사멸기전 연구 The mechanism of PLPP-mediated neuronal receptor regulation and cell death using Tg and KO mice 원문보기

초록 ▼

목차 Contents

표/그림 (19) 모든 표/그림 보기

표/그림 (19) 슬라이드로 보기

참고문헌 (25)

연구과제 타임라인

전체(0) 논문(0) 특허(0) 보고서(0)

전체(0) 논문(0) 특허(0) 보고서(0)

관련 콘텐츠

원문 보기

이 보고서와 함께 이용한 콘텐츠

AI-Helper ※ AI-Helper는 오픈소스 모델을 사용합니다.

선택된 텍스트

Tg 및 KO 마우스를 이용한 PLPP 매개성 신경세포 수용체 조절 및 세포사멸기전 연구
The mechanism of PLPP-mediated neuronal receptor regulation and cell death using Tg and KO mice 원문보기

표/그림 (19)

표/그림 (19)