Mitochondria undergo morphological changes during spermatogenesis in some animals.The mechanism and role of mitochondrial morphology regulation,however,remain somewhat unclear.In this study,we analyzed the molecular c...Mitochondria undergo morphological changes during spermatogenesis in some animals.The mechanism and role of mitochondrial morphology regulation,however,remain somewhat unclear.In this study,we analyzed the molecular characteristics,expression dynamics and subcellular localization of optic atrophy protein 1(OPA1),a mitochondrial fusion and cristae maintenance-related protein,to reveal the possible regulatory mechanisms underlying mitochondrial morphology in Phascolosoma esculenta spermiogenesis.The full-length cDNA of the P.esculenta opa1 gene(Pe-opa1)is 3743 bp in length and encodes 975 amino acids.The Pe-OPA1 protein is highly conservative and includes a transmembrane domain,a GTPase domain,two helical bundle domains,and a lipid-interacting stalk.Gene and protein expression was higher in the coelomic fluid(a site of spermatid development)of male P.esculenta and increased first and then decreased from March to December.Moreover,their expression during the breeding stage was significantly higher than during the non-breeding stage,suggesting that Pe-OPA1 is involved in P.esculenta reproduction.The Pe-OPA1 protein was more abundant in components consisting of many spermatids than in components without,indicating that Pe-OPA1 mainly plays a role in the spermatid in coelomic fluid.Moreover,Pe-OPA1 was mainly detected in the spermatid mitochondria.Immunofluorescence experiments showed that the Pe-OPA1 are constitutively expressed and co-localized with mitochondria during spermiogenesis,suggesting its involvement in P.esculenta spermiogenesis.These results provide evidence for Pe-OPA1's involvement in the regulation of mitochondrial morphology during spermiogenesis.展开更多
Dominant optic atrophy(DOA)is an inherited optic neuropathy and more than 75%of DOA patients harbor pathogenic mutations in OPA1.We reported a 39-year-old female harboring c.2119G>T mutation of OPA1 and manifested ...Dominant optic atrophy(DOA)is an inherited optic neuropathy and more than 75%of DOA patients harbor pathogenic mutations in OPA1.We reported a 39-year-old female harboring c.2119G>T mutation of OPA1 and manifested progressive visual impairment after hydroxychloroquine(HCQ)therapy.The patient’s visual impairment remained stable for 10 years until she began to take HCQ 13 months ago.She complained about progressively decreased vision in both eyes.Bilateral pale temporal optic disc was similar with that of 11 years ago.Optical coherence tomography showed bilateral moderate retinal nerve fiber layer thinning other than the nasal quadrant and general thinning of the inner retina in the macular.Microcystic macular edema was noted in nasal macular in both eyes.Visual field testing showed paracentral scotoma and microperimetry showed decrease sensitivity in the macular in both eyes.After the patient stopped taking HCQ,her functional tests including visual acuity,field testing and microperimetry testing was stable compared with those of 2 years ago.However,progressive inner macular and RNFL thinning was shown by OCT.OPA1 c.2119 G>T found in this patient was a mutation that had been rarely reported in previous studies.The patient has been followed up for over 10 years and her visual acuity stayed stable for decades long until she took HCQ for 13 months.Her vision decline terminated after she stopped taking HCQ.Although HCQ toxicity is highly related to the duration and daily dose,HCQ may aggravate visual impairment in certain individuals harboring OPA1 mutation.Patients with DOA should avoid using neurotoxic HCQ and other medications that may interfere mitochondrial metabolism.展开更多
目的:探讨缺氧-复氧(hypoxia/reoxygenation,H/R)后人近曲肾小管上皮细胞(HK-2)内动力相关蛋白1(Dynamin related protein 1,DRP1)及视神经萎缩症蛋白1(optic atrophy,OPA1)表达的变化。方法:以人近曲肾小管上皮细胞株为研究对象,将培...目的:探讨缺氧-复氧(hypoxia/reoxygenation,H/R)后人近曲肾小管上皮细胞(HK-2)内动力相关蛋白1(Dynamin related protein 1,DRP1)及视神经萎缩症蛋白1(optic atrophy,OPA1)表达的变化。方法:以人近曲肾小管上皮细胞株为研究对象,将培养细胞随机分为正常对照组和H/R组。正常对照组常规培养,H/R组先缺氧24h,然后复氧培养6h。光镜观察细胞形态变化,CCK-8检测细胞活力,透射电子显微镜观察线粒体形态改变,免疫组织化学法检测细胞内DRP1和OPA1蛋白表达。结果:H/R组与对照组相比,细胞活力下降,线粒体出现凋亡相关改变,OPA1表达减少而DRP1的表达增加。结论:缺氧-复氧可导致HK-2细胞发生凋亡相关改变,其机制可能与诱导线粒体形态相关蛋白DRP1、OPA1的表达改变相关。展开更多
目的:基于O P A1介导的线粒体稳态明确不同浓度姜黄素双向调节人脐静脉血管内皮细胞(HUVECs)增殖、迁移的药理作用及机制。方法:高糖DMEM完全培养基培养HUVECs,CD31荧光染色鉴定;不同浓度姜黄素干预HUVECs,CCK8检测增殖活性,结晶紫染色...目的:基于O P A1介导的线粒体稳态明确不同浓度姜黄素双向调节人脐静脉血管内皮细胞(HUVECs)增殖、迁移的药理作用及机制。方法:高糖DMEM完全培养基培养HUVECs,CD31荧光染色鉴定;不同浓度姜黄素干预HUVECs,CCK8检测增殖活性,结晶紫染色检测克隆增殖状态;细胞划痕实验及Transwell实验检测其迁移能力;RT-qPCR检测HUVECs的VEGFα、HIF-1αmRNA水平;ROS、JC-1试剂盒检测HUVECs ROS水平及线粒体膜电位;Western Blotting检测HUVECs OPA1及其下游蛋白表达。结果:20、30、50μmol/L姜黄素显示显著的HUVECs增殖抑制效果(P<0.01),1μmol/L姜黄素能够促进HUVECs的克隆增殖;与Ctrl组比较,1、10μmol/L姜黄素能够显著促进HUVECs迁移(P<0.01),20、30μmol/L姜黄素能够显著抑制HUVECs细胞迁移(P<0.01);20、30μmol/L姜黄素能够显著抑制HIF-1αmRNA水平(P<0.05);与Ctrl组比较,20μmol/L姜黄素显著上调HUVECsROS水平(P<0.05),20、30μmol/L姜黄素能够显著降低HUVECs线粒体活性(P<0.05);与Ctrl组比较,1、10μmol/L姜黄素均能够显著上调OPA1蛋白表达(P<0.01,P<0.05)。结论:以10μmol/L为区分,不同浓度的姜黄素对HUVECs的增殖、迁移发挥双向调节作用,并且这一作用与OPA1介导的线粒体稳态密切相关。展开更多
The purpose of this study is to construct OPA1 overexpression plasmid and transfected into human neuroblastoma SH-SY5Y cells to test the overexpression efficiency.Human neuroblastoma SH SY5Y cells were transfected wit...The purpose of this study is to construct OPA1 overexpression plasmid and transfected into human neuroblastoma SH-SY5Y cells to test the overexpression efficiency.Human neuroblastoma SH SY5Y cells were transfected with the OPA1 overexpression plasmid by Lipo3000 transfection reagent in liposome transduction technology.A total of 3 groups were set up:the control group,negative control group,and OPA1 overexpression model group.RT-PCR technique was used to detect the expression of OPA1 at mRNA level.In addition,Western blot technique was used to detect the expression of OPA1 at protein level.We found that the mRNA and protein expression of OPA1 were significantly increased after transfection with OPA1 overexpression plasmid.These results highlight using liposome transduction technology,the OPA1 overexpression plasmid was successfully transfected into SH-SY5Y cells by Lipo3000 and the cell model of OPA1 gene overexpression was successully established.展开更多
基金the Ningbo Science and Technology Plan Projects(Nos.2019B10016,2016C10004)the Major Science and Technology Projects in Zhejiang Province(No.2011C12013)+1 种基金the Natural Science Foundation of Zhejiang Province(No.LY18C190007)the Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy Aquaculture,the K.C.Wong Magna Fund in Ningbo University。
文摘Mitochondria undergo morphological changes during spermatogenesis in some animals.The mechanism and role of mitochondrial morphology regulation,however,remain somewhat unclear.In this study,we analyzed the molecular characteristics,expression dynamics and subcellular localization of optic atrophy protein 1(OPA1),a mitochondrial fusion and cristae maintenance-related protein,to reveal the possible regulatory mechanisms underlying mitochondrial morphology in Phascolosoma esculenta spermiogenesis.The full-length cDNA of the P.esculenta opa1 gene(Pe-opa1)is 3743 bp in length and encodes 975 amino acids.The Pe-OPA1 protein is highly conservative and includes a transmembrane domain,a GTPase domain,two helical bundle domains,and a lipid-interacting stalk.Gene and protein expression was higher in the coelomic fluid(a site of spermatid development)of male P.esculenta and increased first and then decreased from March to December.Moreover,their expression during the breeding stage was significantly higher than during the non-breeding stage,suggesting that Pe-OPA1 is involved in P.esculenta reproduction.The Pe-OPA1 protein was more abundant in components consisting of many spermatids than in components without,indicating that Pe-OPA1 mainly plays a role in the spermatid in coelomic fluid.Moreover,Pe-OPA1 was mainly detected in the spermatid mitochondria.Immunofluorescence experiments showed that the Pe-OPA1 are constitutively expressed and co-localized with mitochondria during spermiogenesis,suggesting its involvement in P.esculenta spermiogenesis.These results provide evidence for Pe-OPA1's involvement in the regulation of mitochondrial morphology during spermiogenesis.
基金supported by grants from the Guangdong Basic and Applied Basic Research Foundation(2023A1515011250).
文摘Dominant optic atrophy(DOA)is an inherited optic neuropathy and more than 75%of DOA patients harbor pathogenic mutations in OPA1.We reported a 39-year-old female harboring c.2119G>T mutation of OPA1 and manifested progressive visual impairment after hydroxychloroquine(HCQ)therapy.The patient’s visual impairment remained stable for 10 years until she began to take HCQ 13 months ago.She complained about progressively decreased vision in both eyes.Bilateral pale temporal optic disc was similar with that of 11 years ago.Optical coherence tomography showed bilateral moderate retinal nerve fiber layer thinning other than the nasal quadrant and general thinning of the inner retina in the macular.Microcystic macular edema was noted in nasal macular in both eyes.Visual field testing showed paracentral scotoma and microperimetry showed decrease sensitivity in the macular in both eyes.After the patient stopped taking HCQ,her functional tests including visual acuity,field testing and microperimetry testing was stable compared with those of 2 years ago.However,progressive inner macular and RNFL thinning was shown by OCT.OPA1 c.2119 G>T found in this patient was a mutation that had been rarely reported in previous studies.The patient has been followed up for over 10 years and her visual acuity stayed stable for decades long until she took HCQ for 13 months.Her vision decline terminated after she stopped taking HCQ.Although HCQ toxicity is highly related to the duration and daily dose,HCQ may aggravate visual impairment in certain individuals harboring OPA1 mutation.Patients with DOA should avoid using neurotoxic HCQ and other medications that may interfere mitochondrial metabolism.
文摘目的:探讨缺氧-复氧(hypoxia/reoxygenation,H/R)后人近曲肾小管上皮细胞(HK-2)内动力相关蛋白1(Dynamin related protein 1,DRP1)及视神经萎缩症蛋白1(optic atrophy,OPA1)表达的变化。方法:以人近曲肾小管上皮细胞株为研究对象,将培养细胞随机分为正常对照组和H/R组。正常对照组常规培养,H/R组先缺氧24h,然后复氧培养6h。光镜观察细胞形态变化,CCK-8检测细胞活力,透射电子显微镜观察线粒体形态改变,免疫组织化学法检测细胞内DRP1和OPA1蛋白表达。结果:H/R组与对照组相比,细胞活力下降,线粒体出现凋亡相关改变,OPA1表达减少而DRP1的表达增加。结论:缺氧-复氧可导致HK-2细胞发生凋亡相关改变,其机制可能与诱导线粒体形态相关蛋白DRP1、OPA1的表达改变相关。
文摘The purpose of this study is to construct OPA1 overexpression plasmid and transfected into human neuroblastoma SH-SY5Y cells to test the overexpression efficiency.Human neuroblastoma SH SY5Y cells were transfected with the OPA1 overexpression plasmid by Lipo3000 transfection reagent in liposome transduction technology.A total of 3 groups were set up:the control group,negative control group,and OPA1 overexpression model group.RT-PCR technique was used to detect the expression of OPA1 at mRNA level.In addition,Western blot technique was used to detect the expression of OPA1 at protein level.We found that the mRNA and protein expression of OPA1 were significantly increased after transfection with OPA1 overexpression plasmid.These results highlight using liposome transduction technology,the OPA1 overexpression plasmid was successfully transfected into SH-SY5Y cells by Lipo3000 and the cell model of OPA1 gene overexpression was successully established.
