Microglia are the first immune cells that are activated in the brain following ischemic stroke.Mitochondrial dysfunction exacerbates microglia-mediated neuroinflammation post-stroke.Caspase activation and recruitment ...Microglia are the first immune cells that are activated in the brain following ischemic stroke.Mitochondrial dysfunction exacerbates microglia-mediated neuroinflammation post-stroke.Caspase activation and recruitment domain 19(CARD19)is involved in innate immune response and inflammatory response,which are also important functions of microglia.However,the role of CARD19 in microglial biology and ischemic stroke remains unknown.Here,we observed that CARD19 expression was significantly elevated in microglia in the penumbra after ischemic stroke via analyzing the spatial transcriptomic sequencing data of ischemic brain tissue,as well as in an in vitro model of microglial activation.Remarkably,conditional knockdown of Card19 in microglia promoted post-stroke neuroinflammation and worsened neurological outcomes in a mouse model of ischemic stroke.Mechanistically,we found that CARD19 localized to mitochondria and promoted the assembly of mitochondrial intermembrane bridge components,while CARD19 deficiency in microglia caused ultrastructural and functional damage to the mitochondrial cristae,leading to an exaggerated pro-inflammatory response.Thus,our findings suggest that preserving mitochondrial cristae,by targeting CARD19 could be a novel therapeutic strategy for ameliorating neuroinflammation post-stroke and decreasing the volume of the ischemic penumbra.展开更多
Preprotein import into chloroplasts depends on macromolecular machineries in the outer and inner chloroplast envelope membrane （TOC and TIC）. It was suggested that both machineries are interconnected by components o...Preprotein import into chloroplasts depends on macromolecular machineries in the outer and inner chloroplast envelope membrane （TOC and TIC）. It was suggested that both machineries are interconnected by components of the intermembrane space （IMS）. That is, amongst others, Tic22, of which two closely related isoforms exist in Arabidopsis thaliana, namely atTic22-III and atTic22-IV. We investigated the function of Tic22 in vivo by analyzing T-DNA insertion lines of the corresponding genes. While the T-DNA insertion in the individual genes caused only slight defects, a double mutant of both isoforms showed retarded growth, a pale phenotype under high-light conditions, a reduced import rate, and a reduction in the photosynthetic performance of the plants. The latter is supported by changes in the metabolite content of mutant plants when compared to wild-type. Thus, our results support the notion that Tic22 is directly involved in chloroplast preprotein import and might point to a particular importance of Tic22 in chloroplast biogenesis at times of high import rates.展开更多
Mammalian mitochondrial genome encodes a small set of tRNAs, rRNAs, and mRNAs. The RNA synthesis process has been well characterized. How the RNAs are degraded, however, is poorly understood. It was long assumed that ...Mammalian mitochondrial genome encodes a small set of tRNAs, rRNAs, and mRNAs. The RNA synthesis process has been well characterized. How the RNAs are degraded, however, is poorly understood. It was long assumed that the degradation happens in the matrix where transcription and translation machineries reside. Here we show that contrary to the assumption, mammalian mitochondrial RNA degradation occurs in the mitochondrial intermembrane space （IMS） and the IMS- localized RNASET2 is the enzyme that degrades the RNAs. This provides a new paradigm for understanding mitochondrial RNA metabolism and transport.展开更多
In this study,the release of mitochondrial proapoptotic intermembrane space proteins induced by ex-ogenous C2-ceramide in human colon carcinoma(HT-29)cell line was investigated.HT-29 cells were treated with 12.5,25 an...In this study,the release of mitochondrial proapoptotic intermembrane space proteins induced by ex-ogenous C2-ceramide in human colon carcinoma(HT-29)cell line was investigated.HT-29 cells were treated with 12.5,25 and 50μmol/L C2-ceramide in vitro.Flow cytometer was used to detect the mito-chondrial membrane potential(ΔΨm).Subcellular fractions were extracted by Mitochondrial/Cytosol Fractionation Kit after C2-ceramide treatment for 24 h.SDS-PAGE was used to determine the level of cytochrome c(Cyt c),high temperature requirement A2(HtrA2)and second mitochondrial-derived ac-tivator of caspases(Smac)released from mitochondria,the expression of X-linked inhibitor of apop-tosis protein(XIAP)and caspase-3 for 24 h.The results showed thatΔΨm began to decrease from 6 h after 25 and 50μmol/L C2-ceramide treatment(P<0.05)and cyclosporin A(CsA)could inhibit the col-lapse ofΔΨm through regulating mitochondrial membrane permeability transition pore.There was no effect of C2-ceramide on the expression of Cyt c,HtrA2 and Smac in the total levels.12.5,25 and 50μmol/L C2-ceramide could induce Cyt c,HtrA2 and Smac to release from mitochondria to cytosol and down-regulate the expression of XIAP(P<0.05).Also there was expression of cleaved caspase-3 with C2-ceramide treatment.After the treatment with caspase inhibitor,C2-ceramide still induced the release of Cyt c and HtrA2,but Smac did not.Therefore,C2-ceramide could induce apoptosis of HT-29 cells through the mitochondria pathway.The release of Cyt c,HtrA2 and Smac from mitochondria did not occur via the same mechanism,the release of Cyt c and HtrA2 was caspase-independent and the re-lease of Smac was caspase-dependent.展开更多
基金National Natural Science Foundation of China,Nos.81920108017(to YX),82401546(to HL)Jiangsu Province Key Medical Discipline,No.ZDXK202216(to YX)the Key Research and Development Program of Jiangsu Province of China,No.BE2020620(to YX).
文摘Microglia are the first immune cells that are activated in the brain following ischemic stroke.Mitochondrial dysfunction exacerbates microglia-mediated neuroinflammation post-stroke.Caspase activation and recruitment domain 19(CARD19)is involved in innate immune response and inflammatory response,which are also important functions of microglia.However,the role of CARD19 in microglial biology and ischemic stroke remains unknown.Here,we observed that CARD19 expression was significantly elevated in microglia in the penumbra after ischemic stroke via analyzing the spatial transcriptomic sequencing data of ischemic brain tissue,as well as in an in vitro model of microglial activation.Remarkably,conditional knockdown of Card19 in microglia promoted post-stroke neuroinflammation and worsened neurological outcomes in a mouse model of ischemic stroke.Mechanistically,we found that CARD19 localized to mitochondria and promoted the assembly of mitochondrial intermembrane bridge components,while CARD19 deficiency in microglia caused ultrastructural and functional damage to the mitochondrial cristae,leading to an exaggerated pro-inflammatory response.Thus,our findings suggest that preserving mitochondrial cristae,by targeting CARD19 could be a novel therapeutic strategy for ameliorating neuroinflammation post-stroke and decreasing the volume of the ischemic penumbra.
文摘Preprotein import into chloroplasts depends on macromolecular machineries in the outer and inner chloroplast envelope membrane （TOC and TIC）. It was suggested that both machineries are interconnected by components of the intermembrane space （IMS）. That is, amongst others, Tic22, of which two closely related isoforms exist in Arabidopsis thaliana, namely atTic22-III and atTic22-IV. We investigated the function of Tic22 in vivo by analyzing T-DNA insertion lines of the corresponding genes. While the T-DNA insertion in the individual genes caused only slight defects, a double mutant of both isoforms showed retarded growth, a pale phenotype under high-light conditions, a reduced import rate, and a reduction in the photosynthetic performance of the plants. The latter is supported by changes in the metabolite content of mutant plants when compared to wild-type. Thus, our results support the notion that Tic22 is directly involved in chloroplast preprotein import and might point to a particular importance of Tic22 in chloroplast biogenesis at times of high import rates.
基金We thank Haiteng Deng for the help on Mass Spec and Zhi Lu and Hongwei Wang for discussion. This research was supported by the Priority Research Program of the Ministry of Science and Technology 2017YFA0504600, the National Natural Science Foundation of China （Grant Nos. 31371439 and 91649103）, and Ministry of Education 1000 youth program.
文摘Mammalian mitochondrial genome encodes a small set of tRNAs, rRNAs, and mRNAs. The RNA synthesis process has been well characterized. How the RNAs are degraded, however, is poorly understood. It was long assumed that the degradation happens in the matrix where transcription and translation machineries reside. Here we show that contrary to the assumption, mammalian mitochondrial RNA degradation occurs in the mitochondrial intermembrane space （IMS） and the IMS- localized RNASET2 is the enzyme that degrades the RNAs. This provides a new paradigm for understanding mitochondrial RNA metabolism and transport.
基金Supported by the National Natural Science Foundation of China(Grant No.30471447)Special-Purpose Scientific Research Foundation of Ph.D Subject in the Colleges and Universities of China(Grant No.20060226004)
文摘In this study,the release of mitochondrial proapoptotic intermembrane space proteins induced by ex-ogenous C2-ceramide in human colon carcinoma(HT-29)cell line was investigated.HT-29 cells were treated with 12.5,25 and 50μmol/L C2-ceramide in vitro.Flow cytometer was used to detect the mito-chondrial membrane potential(ΔΨm).Subcellular fractions were extracted by Mitochondrial/Cytosol Fractionation Kit after C2-ceramide treatment for 24 h.SDS-PAGE was used to determine the level of cytochrome c(Cyt c),high temperature requirement A2(HtrA2)and second mitochondrial-derived ac-tivator of caspases(Smac)released from mitochondria,the expression of X-linked inhibitor of apop-tosis protein(XIAP)and caspase-3 for 24 h.The results showed thatΔΨm began to decrease from 6 h after 25 and 50μmol/L C2-ceramide treatment(P<0.05)and cyclosporin A(CsA)could inhibit the col-lapse ofΔΨm through regulating mitochondrial membrane permeability transition pore.There was no effect of C2-ceramide on the expression of Cyt c,HtrA2 and Smac in the total levels.12.5,25 and 50μmol/L C2-ceramide could induce Cyt c,HtrA2 and Smac to release from mitochondria to cytosol and down-regulate the expression of XIAP(P<0.05).Also there was expression of cleaved caspase-3 with C2-ceramide treatment.After the treatment with caspase inhibitor,C2-ceramide still induced the release of Cyt c and HtrA2,but Smac did not.Therefore,C2-ceramide could induce apoptosis of HT-29 cells through the mitochondria pathway.The release of Cyt c,HtrA2 and Smac from mitochondria did not occur via the same mechanism,the release of Cyt c and HtrA2 was caspase-independent and the re-lease of Smac was caspase-dependent.
