[보고서]P2X7 receptor/PDI 활성조절에 의한 세포내 산화스트레스 유발기전 규명

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보고서 정보

주관연구기관

한림대학교
HalLym University

연구책임자

김지은

보고서유형

최종보고서

발행국가

대한민국

언어

한국어

발행년월

2021-03

과제시작연도

2020

주관부처

과학기술정보통신부
Ministry of Science and ICT

연구관리전문기관

한국연구재단
National Research Foundation of Korea

등록번호

TRKO202100017288

과제고유번호

1711107409

사업명

개인기초연구(과기정통부)(R&D)

DB 구축일자

2022-02-12

키워드

피2엑스7 수용체.피디아이.산화스트레스.에스-나이트로실레이션.글루타치온.P2X7 receptor.PDI.oxidative stress.S-nitrosylation.Glutathione.

표 The role of seizure activity in NO generation in response to pilocarpine
표 The effects of L-NAME on SNO-PDI, SNO-DRP1, and mitochondrial fusion in CA1 neurons under normal condition
표 The effects of PDI knockdown on SNO-PDI, SNO-DRP1, and mitochondrial fusion in CA1 neurons under normal condition
표 The effects of P2X7 receptor on GSH synthesis and the expressions of ASCT1, ASCT2, GCLC and GSS
표 The effects of L-NAME on the amounts of SNO-thiol- and total thiol-P2X7R following SE. As compared to vehicle (V), L-NAME (L) reduces the amount of SNO-thiol-P2X7R, and increased that of total thiol-P2X7R without altering P2X7R expression. (A) Representative western blot for S-nitrosylation and thiolization on P2X7R and its expression. (B) Quantification of analyses of S-nitrosylation and thiolization on P2X7R and its expression
표 The effects of PDI knockdown on the amounts of SNO-thiol and total thiol-P2X7R following SE. As compared to control siRNA, PDI siRNA abolishes S-nitrosylation ratio of P2X7R following SE. (A) Representative western blot for S-nitrosylation and thiolization on P2X7R and its expression. (F) Quantification of analyses of S-nitrosylation and thiolization on P2X7R and its expression
$The effects of L-NAME and PDI knockdown on the surface expression of P2X7R. As compared to control animals, SE increases the membrane P2X7R expressions, which are abrogated by both L-NAME and PDI siRNA (A) Representative western blot for surface expression of P2X7R following SE (B) Quantification of analyses of P2X7R expression in membrane fractions. (C) Scheme of the role of PDI-mediated S-nitrosylation of P2X7R in its trafficking to cell membrane. SE elevates NO synthesis, which S-nitrosylates PDI. SNO-PDI transfers NO to the reduced (immature) P2X7R. In turn, SNO-P2X7R is oxidized by denitrosylases (presumablyTrx-1), which facilitates its trafficking to cell surface$ 표 The effects of L-NAME and PDI knockdown on the surface expression of P2X7R. As compared to control animals, SE increases the membrane P2X7R expressions, which are abrogated by both L-NAME and PDI siRNA (A) Representative western blot for surface expression of P2X7R following SE (B) Quantification of analyses of P2X7R expression in membrane fractions. (C) Scheme of the role of PDI-mediated S-nitrosylation of P2X7R in its trafficking to cell membrane. SE elevates NO synthesis, which S-nitrosylates PDI. SNO-PDI transfers NO to the reduced (immature) P2X7R. In turn, SNO-P2X7R is oxidized by denitrosylases (presumablyTrx-1), which facilitates its trafficking to cell surface
표 Effects of P2X7R deletion on expression of GS, GLS and ASCT2. P2X7R deletion increases GS and ASCT2, but not GLS, expressions. (A) Representative Western blots of GS, GLS and ASCT2 expressions. (B-F) Quantification of GS (B), GLS (C), glycosylated ASCT2 (Glycol-ASCT2, D), intact ASCT2 (E) and (F) total ASCT2 levels based on Western blot data
표 GSH assay in the hippocampus. P2X7R deletion decreases basal GSH level. NAC increases GSH level in WT animals, but not P2X7R KO mice. Although SIN-1 does not affect GSH level in both animals, it reduces GSH concentration to basal level in NAC-treated WT animals, but not P2X7R KO mice
표 Effects of P2X7R deletion on xCT expression. P2X7R deletion does not affect xCT expression. (A) Representative Western blots of xCT expression. (B) Quantification of xCT level based on Western blot data
표 Scheme of roles of P2X7R in basal GSH level based on the present data and a previous report. Under physiological condition, P2X7R activation increases Ca2+influx, which inhibits GS expression by PKC activation without altering GLS expression. This mode of regulation maintains the yield of glutamate (Glu)-glutamine (GlN) cycle and xCT-mediated uptake of cysteine (Cys) precursors such as cystine/NAC, which increases GSH level. However, P2X7R deletion leads to the up-regulation of GS expression, which inhibits xCT activity due to reduced glutamate level. Instead, the elevated glutamine level accelerates ACST2-mediated glutamine-cysteine exchanges. The reductions in both glutamate and cystine level diminish GSH level
표 The role of seizure activity in NO generation in response to pilocarpine using real-time simultaneous monitoring. (A) Representative NO concentration (top), EEG trace (middle) and frequency-power spectral temporal maps (bottom). (B) Quantification of NO level and total EEG power in response to PILO (mean ± S.E.M.; *p < 0.05 vs. basal level; n = 7, respectively). (C) Quantitative values (mean ± S.E.M) of nitrate/nitrite levels in the hippocampus 3 days after SE. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (*p < 0.05 vs. non-SE animals; n = 7, respectively)
표 The effects of L-NAME on the amounts of SNO-thioland total thiol-P2X7R following SE. As compared to vehicle (V), L-NAME (L) reduces the amount of SNO-thiol-P2X7R, and increased that of total thiol-P2X7R without altering P2X7R expression under physiological condition. Thus, S-nitrosylation ratio of P2X7R is decreased. SE up-regulates P2X7R expression and S-nitrosylation ratio of P2X7R due to the reduced the amount of total thiol without the altered that of SNO-thiol-P2X7R. L-NAME inhibits these alterations induced by SE, except P2X7R expression. (A) Representative western blot for S-nitrosylation and thiolization on P2X7R and its expression. (B-E) Quantification of analyses of S-nitrosylation and thiolization on P2X7R and its expression. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (*,#p < 0.05 vs. control animals and vehicle, respectively; n = 7, respectively). (F) Representative photos of the localization of P2X7R in the CA1 region following SE. Up-regulation of P2X7R expression induced by SE is restricted to microglia
표 The effects of L-NAME on neuronal death and microglial activation following SE. L-NAME attenuates microglial activation by inhibiting p65‐S276 NF‐κB phosphorylation, while it does not affect CA1 neuronal death, following SE. (A) Representative photos for FJB-positive degenerating CA1 neurons induced by SE. (B) Quantification of analyses of the effect of L-NAME on SE-induced CA1 neurons. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (n = 7, respectively). (C) Representative photos for microglial p65‐S276 NF‐κB phosphorylation in the CA1 region following SE. (D-E) Quantification of analyses of the effect of L-NAME on microglial activation (D) and p65‐S276 NF‐κB phosphorylation in microglia (E). Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (*,#p < 0.05 vs. control animals and vehicle, respectively; n = 7, respectively)
표 The effects of L-NAME on astroglial death and reactive astrogliosis in the hippocampus following SE. SE leads to astroglial apoptosis in the molecular layer of the dentate gyrus and reactive astrogliosis in the CA1 region, respectively. L-NAME ameliorates SE-induced astroglial apoptosis, but not reactive astrogliosis. (A) Representative photos for TUNEL-positive astrocytes in the dentate gyrus following SE. (B) Quantification of analyses of the effect of L-NAME on GFAP positive area following SE. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (*,#p < 0.05 vs. control animals and vehicle, respectively; n = 7, respectively). (C) Representative photos for double immunostaining for TUNEL and GFAP in the CA1 region following SE. Reactive CA1 astrocytes do not show TUNEL signals following SE
표 The effects of L-NAME and/or PDI knockdown on the amounts of SNO-thiol and total thiol-PDI or -P2X7R, and PDI-P2X7R bindings following SE. As compared to vehicle (V), L-NAME (L) reduces the amount of SNO-thiol-PDI and PDI-P2X7R bindings without affecting PDI expression and the amount of total thiol-PDI under physiological condition. Thus, S-nitrosylation ratio of PDI is decreased. L-NAME abrogates SE-induced alterations in PDI-P2X7R bindings and S-nitrosylation ratio of PDI, except PDI expression. As compared to control siRNA (C), PDI siRNA (P) abolishes the changes in PDI-P2X7R bindings and S-nitrosylation ratio of P2X7R following SE, except its expression. (A) Representative western blot for S-nitrosylation and thiolization on PDI and its expression. (B) Quantification of analyses of S-nitrosylation and thiolization on PDI and its expression. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (*,#p < 0.05 vs. control animals and vehicle, respectively; *p < 0.05 vs. vehicle; n = 7, respectively). (C) Representative western blot for the PDI-P2X7R bindings following SE. (D) Quantification of analyses of PDI-P2X7R bindings. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (*,#p < 0.05 vs. control animals and vehicle or control siRNA, respectively; n = 7, respectively). (E) Representative western blot for S-nitrosylation and thiolization on P2X7R and its expression. (F) Quantification of analyses of S-nitrosylation and thiolization on P2X7R and its expression. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (*,#p < 0.05 vs. control animals and control siRNA, respectively; n = 7, respectively)
표 The effects of PDI knockdown on neuronal death, microglial activation and astroglial apoptosis following SE. PDI siRNA inhibits microglial activation by inhibiting p65‐S276 NF‐κB phosphorylation, and attenuates astroglial apoptosis in the dentate gyrus following SE, while it does not affect SE-induced neuronal death. (A) Representative photos for FJB-positive degenerating CA1 neurons following SE. (B) Quantification of analyses of the effect of PDI siRNA on SE-induced CA1 neuronal degeneration. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (n = 7, respectively). (C) Representative photos for p65‐S276 NF‐κB phosphorylation in microglia in the CA1 region (left panels) and TUNEL-positive astrocytes in the dentate gyrus (right panels) following SE. (D-F) Quantification of analyses of the effect of PDI siRNA on microglial activation (D), p65‐S276 NF‐κB phosphorylation in microglia (E) and astroglial apoptosis in the dentate gyrus (F). Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (*,#p < 0.05 vs. control animals and control siRNA, respectively; n = 7, respectively)
$The effects of L-NAME and PDI knockdown on the surface expression of P2X7R. As compared to control animals, SE increases the membrane P2X7R expressions, which are abrogated by both L-NAME and PDI siRNA (A) Representative western blot for surface expression of P2X7R following SE. (B) Quantification of analyses of P2X7R expression in membrane fractions. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (*,#p < 0.05 vs. control animals and vehicle or control siRNA, respectively; n = 7, respectively)$ 표 The effects of L-NAME and PDI knockdown on the surface expression of P2X7R. As compared to control animals, SE increases the membrane P2X7R expressions, which are abrogated by both L-NAME and PDI siRNA (A) Representative western blot for surface expression of P2X7R following SE. (B) Quantification of analyses of P2X7R expression in membrane fractions. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (*,#p < 0.05 vs. control animals and vehicle or control siRNA, respectively; n = 7, respectively)
표 Scheme of the role of PDI-mediated S-nitrosylation of P2X7R in its trafficking to cell membrane. SE elevates NO synthesis, which S-nitrosylates PDI. SNO-PDI transfers NO to the reduced (immature) P2X7R. In turn, SNO-P2X7R is oxidized by denitrosylases (presumably Trx-1 [8]), which facilitates its trafficking to cell surface

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[국가R&D연구보고서] P2X7 receptor/PDI 활성조절에 의한 세포내 산화스트레스 유발기전 규명
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