Increased endogenous αB-crystallin protein levels have been shown to reduce cell apoptosis, although the effects of exogenous aB-crystallin protein remain poorly understood. The present study established an acute ocu...Increased endogenous αB-crystallin protein levels have been shown to reduce cell apoptosis, although the effects of exogenous aB-crystallin protein remain poorly understood. The present study established an acute ocular hypertension model in the right eye of Sprague-Dawley rats. Fluorogold retrograde tracing and immunofluorescence methods showed that the number of retinal ganglion cells decreased in the right eyes and caspase-3 expression increased following acute ocular hypertension. Intravitreal injection of aB-crystallin in the right eye increased the number of retinal ganglion cells and reduced caspase-3 expression. Results demonstrated that exogenous αB-crystallin protein inhibited caspase-3 expression and improved retinal ganglion cell survival following acute ocular hypertension.展开更多
Background:Parkinson’s disease(PD)is characterized by a chronic loss of dopaminergic neurons and the presence of proteinaceous inclusions(Lewy bodies)within some remaining neurons in the substantia nigra.Recently,ast...Background:Parkinson’s disease(PD)is characterized by a chronic loss of dopaminergic neurons and the presence of proteinaceous inclusions(Lewy bodies)within some remaining neurons in the substantia nigra.Recently,astroglial inclusion body has also been found in some neurodegenerative diseases including PD.However,the underlying molecular mechanisms of how astroglial protein aggregation forms remain largely unknown.Here,we investigated the contribution ofαB-crystallin(CRYAB),a small heat shock protein,inα-synuclein inclusion formation in astrocytes.Methods:Small interfering RNA(siRNA)-mediated CRYAB(siCRYAB)knockdown or CRYAB overexpression was performed to investigate the impact of CRYAB on the autophagy in human glioblastoma cell line U251 cells.Coimmunoprecipitation(co-IP)and immunoblotting were used to dissect the interaction among multiple proteins.The clearance ofα-synuclein in vitro was evaluated by immunocytochemistry.CRYAB transgenic mice and transgenic mice overexpressing A30P mutant form of humanα-synuclein were used to examine the influence of CRYAB toα-synuclein accumulation in vivo.Results:We found that knockdown of CRYAB in U251 cells or primary cultured astrocytes resulted in a marked augmentation of autophagy activity.In contrast,exogenous CRYAB disrupted the assembly of the BAG3-HSPB8-HSC70 complex via binding with BAG3,thereby suppressing the autophagy activity.Furthermore,CRYAB-regulated autophagy has relevance to PD pathogenesis.Knockdown of CRYAB remarkably promoted cytoplasmic clearance ofα-synuclein preformed fibrils(PFFs).Conversely,selective overexpression of CRYAB in astrocytes markedly suppressed autophagy leading to the accumulation of α-synuclein aggregates in the brain of transgenic mice expressing humanα-synuclein A30P mutant.Conclusions:This study reveals a novel function for CRYAB as a natural inhibitor of astrocytic autophagy and shows that knockdown of CYRAB may provide a therapeutic target against proteinopathies such as synucleinopathies.展开更多
AIM:To evaluate if topical use of αB-crystallin minipeptides supports corneal healing following flap surgery.METHODS:Cultured corneal cells were treated with fluorescent taggedαB-crystallin mini-peptides to assess i...AIM:To evaluate if topical use of αB-crystallin minipeptides supports corneal healing following flap surgery.METHODS:Cultured corneal cells were treated with fluorescent taggedαB-crystallin mini-peptides to assess its internalization.Cultured corneal cells pre-treated with or without the mini-peptides were exposed to H_(2)O_(2) and cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.Elongation of neurites of cultured trigeminal neurones was examined following treatment either withαB-crystallin mini-peptides or protein.Cultured trigeminal neurones were pre-treated either with αB-crystallin mini-peptides or crystallin protein and exposed to H_(2)O_(2) and presence of beading in the dendrites and axons was assessed.Corneal flap surgery was conducted on rabbit cornea and treated topically either withαB-crystallin peptide(0.5 mg/mL thrice daily for 14d)or phosphate-buffered saline(PBS).Corneal healing was evaluated under slit-lamp biomicroscope,mRNA expression of inflammatory cytokines were assessed and the corneas were evaluated by histopathology.RESULTS:Internalization ofαB-crystallin mini-peptides was ascertained by the detection of fluorescence within the corneal cells.The MTT assay revealed that treatment withαB-crystallin mini-peptide reduced cell death induced by H_(2)O_(2) treatment.The mini-peptides did not influence the elongation of trigeminal neurites,but significantly(P<0.05)reduced beading in the neurites.In rabbit eye,the treated corneas showed reduced hyper-reflective zones(P<0.05)and suppression in the expression of inflammatory cytokines.Histopathological examination also revealed reduction of inflammatory response in treated corneas.CONCLUSION:TheαB-crystallin mini-peptides restrict the damage to corneal cells and neurons and aids in corneal healing.展开更多
Protein misfolding and aggregation are crucial pathogenic factors for cataracts,which are the leading cause of visual impairment worldwide.α-crystallin,as a small molecular chaperone,is involved in preventing protein...Protein misfolding and aggregation are crucial pathogenic factors for cataracts,which are the leading cause of visual impairment worldwide.α-crystallin,as a small molecular chaperone,is involved in preventing protein misfolding and maintaining lens transparency.The chaperone activity of α-crystallin depends on its oligomeric state.Our previous work identified a natural compound,celastrol,which could regulate the oligomeric state of αB-crystallin.In this work,based on the UNcle and SEC analysis,we found that celastrol induced𝛼αB-crystallin to form large oligomers.Large oligomer formation enhanced the chaperone activity of αB-crystallin and prevented aggregation of the cataract-causing mutant αA3-G91del.The interactions between𝛼αB-crystallin and celastrol were detected by the FRET(Fluorescence Resonance Energy Transfer)technique,and verified by molecular docking.At least 9 binding patterns were recognized,and some binding sites covered the groove structure of αB-crystallin.Interestingly,αB-R120G,a cataract-causing mutation located at the groove structure,and celastrol can decrease the aggregates of αB-R120G.Overall,our results suggested celastrol not only promoted the formation of large αB-crystallin oligomers,which enhanced its chaperone activity,but also bound to the groove structure of its α-crystallin domain to maintain its structural stability.Celastrol might serve as a chemical and pharmacological chaperone for cataract treatment.展开更多
AIM: To evaluate αB-crystallin malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) changes in X-ray irradiated rat lens. METHODS: Eight-week-old Sprague-Dawley male rats received X-ray ...AIM: To evaluate αB-crystallin malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) changes in X-ray irradiated rat lens. METHODS: Eight-week-old Sprague-Dawley male rats received X-ray irradiation to the head with rest of the body protected. The exposure dose ranged from 2 to 25 Grays (Gy). The cataract status were examined by slit lamp and rated with 'four-grade systems' post-irradiation. The lens MDA level, and the activities of SOD and GPx were measured in a short-term experiment post-irradiation, and αB-crystallin protein levels were quantified. RESULTS: The lenses of normal control and the X-ray irradiated groups with the dose up to 10 Gy remained transparent throughout the experiment. The lens first appeared tiny scatters, and even lamellar opacities in the posterior capsule 45 days post-irradiation with the dose of 15 Gy, and progressed slowly to the advance stage of cataract; while, for the higher dose (25 Gy), the opacity of lens appeared much earlier, and progressed more rapidly to mature stage of cataract within 1 month. At the end of the observation (90 days post-irradiation), almost all lenses became complete opacity with the higher dose (25 Gy). The degree of lens opacity was rated accordingly. The lens MDA level was increased, and SOD and GPx activities were decreased with a dose-dependent manner post-irradiation. The αB-crystallin protein level was decreased dose-dependently at the end point of observation. CONCLUSION: Oxidative events and αB-crystallin may play important roles in the pathogenesis of cataract in X-ray irradiated rat lens.展开更多
基金supported by the China Post-doctoral Foundation(The protective effect of αB-crystallin on retinal ganglial cells in glaucoma),No.20080430444
文摘Increased endogenous αB-crystallin protein levels have been shown to reduce cell apoptosis, although the effects of exogenous aB-crystallin protein remain poorly understood. The present study established an acute ocular hypertension model in the right eye of Sprague-Dawley rats. Fluorogold retrograde tracing and immunofluorescence methods showed that the number of retinal ganglion cells decreased in the right eyes and caspase-3 expression increased following acute ocular hypertension. Intravitreal injection of aB-crystallin in the right eye increased the number of retinal ganglion cells and reduced caspase-3 expression. Results demonstrated that exogenous αB-crystallin protein inhibited caspase-3 expression and improved retinal ganglion cell survival following acute ocular hypertension.
基金This work was supported by grants from the Natural Science Foundation of China(31430036,91742116,U1801681)National Key Basic Research Program of China(2015CB553500)+3 种基金Key Research Program of Frontier Sciences(QYZDJ-SSW-SMC002)Strategic Priority Research Program of Chinese Academy of Science(XDB32020100)Shanghai Municipal Science and Technology Major Project(2018SHZDZX05)the Shanghai Municipal Science and Technology Commission(17ZR1435300 to SZZ).
文摘Background:Parkinson’s disease(PD)is characterized by a chronic loss of dopaminergic neurons and the presence of proteinaceous inclusions(Lewy bodies)within some remaining neurons in the substantia nigra.Recently,astroglial inclusion body has also been found in some neurodegenerative diseases including PD.However,the underlying molecular mechanisms of how astroglial protein aggregation forms remain largely unknown.Here,we investigated the contribution ofαB-crystallin(CRYAB),a small heat shock protein,inα-synuclein inclusion formation in astrocytes.Methods:Small interfering RNA(siRNA)-mediated CRYAB(siCRYAB)knockdown or CRYAB overexpression was performed to investigate the impact of CRYAB on the autophagy in human glioblastoma cell line U251 cells.Coimmunoprecipitation(co-IP)and immunoblotting were used to dissect the interaction among multiple proteins.The clearance ofα-synuclein in vitro was evaluated by immunocytochemistry.CRYAB transgenic mice and transgenic mice overexpressing A30P mutant form of humanα-synuclein were used to examine the influence of CRYAB toα-synuclein accumulation in vivo.Results:We found that knockdown of CRYAB in U251 cells or primary cultured astrocytes resulted in a marked augmentation of autophagy activity.In contrast,exogenous CRYAB disrupted the assembly of the BAG3-HSPB8-HSC70 complex via binding with BAG3,thereby suppressing the autophagy activity.Furthermore,CRYAB-regulated autophagy has relevance to PD pathogenesis.Knockdown of CRYAB remarkably promoted cytoplasmic clearance ofα-synuclein preformed fibrils(PFFs).Conversely,selective overexpression of CRYAB in astrocytes markedly suppressed autophagy leading to the accumulation of α-synuclein aggregates in the brain of transgenic mice expressing humanα-synuclein A30P mutant.Conclusions:This study reveals a novel function for CRYAB as a natural inhibitor of astrocytic autophagy and shows that knockdown of CYRAB may provide a therapeutic target against proteinopathies such as synucleinopathies.
基金Supported by the DST Nano-mission,Govt of India,Grant No DST No.SR/NM/NS-1067/2016Facilities were provided by the West Bengal University of Animal&Fishery Sciences and CSIR-IICB for conducting this research。
文摘AIM:To evaluate if topical use of αB-crystallin minipeptides supports corneal healing following flap surgery.METHODS:Cultured corneal cells were treated with fluorescent taggedαB-crystallin mini-peptides to assess its internalization.Cultured corneal cells pre-treated with or without the mini-peptides were exposed to H_(2)O_(2) and cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.Elongation of neurites of cultured trigeminal neurones was examined following treatment either withαB-crystallin mini-peptides or protein.Cultured trigeminal neurones were pre-treated either with αB-crystallin mini-peptides or crystallin protein and exposed to H_(2)O_(2) and presence of beading in the dendrites and axons was assessed.Corneal flap surgery was conducted on rabbit cornea and treated topically either withαB-crystallin peptide(0.5 mg/mL thrice daily for 14d)or phosphate-buffered saline(PBS).Corneal healing was evaluated under slit-lamp biomicroscope,mRNA expression of inflammatory cytokines were assessed and the corneas were evaluated by histopathology.RESULTS:Internalization ofαB-crystallin mini-peptides was ascertained by the detection of fluorescence within the corneal cells.The MTT assay revealed that treatment withαB-crystallin mini-peptide reduced cell death induced by H_(2)O_(2) treatment.The mini-peptides did not influence the elongation of trigeminal neurites,but significantly(P<0.05)reduced beading in the neurites.In rabbit eye,the treated corneas showed reduced hyper-reflective zones(P<0.05)and suppression in the expression of inflammatory cytokines.Histopathological examination also revealed reduction of inflammatory response in treated corneas.CONCLUSION:TheαB-crystallin mini-peptides restrict the damage to corneal cells and neurons and aids in corneal healing.
基金supported by the National Natural Science Foundation of China(31872724,and 81900837)the Natural Science Foundation of Zhejiang Province(LR21H120001).
文摘Protein misfolding and aggregation are crucial pathogenic factors for cataracts,which are the leading cause of visual impairment worldwide.α-crystallin,as a small molecular chaperone,is involved in preventing protein misfolding and maintaining lens transparency.The chaperone activity of α-crystallin depends on its oligomeric state.Our previous work identified a natural compound,celastrol,which could regulate the oligomeric state of αB-crystallin.In this work,based on the UNcle and SEC analysis,we found that celastrol induced𝛼αB-crystallin to form large oligomers.Large oligomer formation enhanced the chaperone activity of αB-crystallin and prevented aggregation of the cataract-causing mutant αA3-G91del.The interactions between𝛼αB-crystallin and celastrol were detected by the FRET(Fluorescence Resonance Energy Transfer)technique,and verified by molecular docking.At least 9 binding patterns were recognized,and some binding sites covered the groove structure of αB-crystallin.Interestingly,αB-R120G,a cataract-causing mutation located at the groove structure,and celastrol can decrease the aggregates of αB-R120G.Overall,our results suggested celastrol not only promoted the formation of large αB-crystallin oligomers,which enhanced its chaperone activity,but also bound to the groove structure of its α-crystallin domain to maintain its structural stability.Celastrol might serve as a chemical and pharmacological chaperone for cataract treatment.
基金Scientific Research Foundation for Returned Scholars, the Second Hospital Affiliated to Soochow University (No.SDFEY-2007-10)National Natural Science Foundation of China (No.81000383)+2 种基金Research Fund for the Doctoral Program of Higher Education of China (No.20100072120051)Program of Tongji University (No.1500219024 No.2010QH04 and No. 2010YF02)
文摘AIM: To evaluate αB-crystallin malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) changes in X-ray irradiated rat lens. METHODS: Eight-week-old Sprague-Dawley male rats received X-ray irradiation to the head with rest of the body protected. The exposure dose ranged from 2 to 25 Grays (Gy). The cataract status were examined by slit lamp and rated with 'four-grade systems' post-irradiation. The lens MDA level, and the activities of SOD and GPx were measured in a short-term experiment post-irradiation, and αB-crystallin protein levels were quantified. RESULTS: The lenses of normal control and the X-ray irradiated groups with the dose up to 10 Gy remained transparent throughout the experiment. The lens first appeared tiny scatters, and even lamellar opacities in the posterior capsule 45 days post-irradiation with the dose of 15 Gy, and progressed slowly to the advance stage of cataract; while, for the higher dose (25 Gy), the opacity of lens appeared much earlier, and progressed more rapidly to mature stage of cataract within 1 month. At the end of the observation (90 days post-irradiation), almost all lenses became complete opacity with the higher dose (25 Gy). The degree of lens opacity was rated accordingly. The lens MDA level was increased, and SOD and GPx activities were decreased with a dose-dependent manner post-irradiation. The αB-crystallin protein level was decreased dose-dependently at the end point of observation. CONCLUSION: Oxidative events and αB-crystallin may play important roles in the pathogenesis of cataract in X-ray irradiated rat lens.
