| Glaucoma, as a neurodegenerative disease of the optic nerve, is the leading cause of irreversible blindness worldwide. The accelerated death of retinal ganglion cells (RGCs) and their axons ultimately leads to progressive visual field loss and eventual blindness. Elevated intraocular pressure (IOP) is a major risk and initiating factor for glaucoma, however, simply lowering intraocular pressure can only alleviate the development of this illness to a certain extent and would not stop secondary RGCs death and axon of collapse, making the clinically effective control means of optic neuropathy development limited.People are paying more and more attention to the role of mitochondrial dysfunction in glaucoma as glaucoma and mitochondria optic neuropathy share many similarities in the pathological features and clinical manifestations. In addition, it has been confirmed that mitochondrial dysfunction play an important role in neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD).Glaucoma also belongs to neurodegenerative diseases and has the common characteristic of these diseases:age-related, loss of neuron is selective and progressive. All of these evidences suggest the possibility that mitochondria play an important role in the development of glaucoma. Then what had happened to the mitochondria in the process of RGCs progressive die? If it can explain the phenomenon that some clinical patients continue to lose sight despite well controlled IOP? These problems have not been reported.Mitochondria which are called the cells’power plants play a vital role in regulating cell survival and death. Mitochondrial DNA (mtDNA) is the only genetic material outside the nucleus, it is particularly vulnerable to injury and has relatively high mutation rate compared to nDNA, because of their special composition structure and positioning inside the cell. Importantly, there are almost no non-coding regions in mtDNA, any locus mutation may be transcribed and lead protein expression changes, thus cause serious consequences. RGCs, because of their high energy requirement, own a high content of mitochondria in cell bodies and axons and that determines the RGC sensitivity to mitochondrial dysfunction. Recently some scholars found that mtDNA mutations increased and ATP synthesis disordered in the peripheral blood of glaucoma patients, prompting that mtDNA damage and the decline in mitochondrial function is one of the high-risk factors of the pathogenesis of glaucoma. However, these studies were limited to patients with peripheral blood, they cannot explain the reason why RGCs and optic nerve was progressively damaged and why visual impairment continued to aggravate even after intraocular pressure was effectively controlled in the process of glaucoma optic neuropathy. The problems about the mutations of mtDNA in RGCs, possible mechanisms of these mutations and the role of mtDNA mutations in progressive cell death in the process of glaucoma optic neuropathy have not been reported.Therefore, we take chronic high intraocular pressure model in Wistar rats as research objects, to study the changes of the mtDNA, mtDNA repair enzyme, mitochondrial complex function in and after high intraocular pressure. And we also discuss the possible mechanisms of mtDNA mutations in high intraocular pressure and the relationship between mtDNA mutation and mitochondrial dysfunction by vitro cell culture experiments. In addition, we further discuss the possible mechanisms of progressive death of retinal ganglion cells caused by mtDNA mutation or damage. Part I The changes of mitochondrial DNA and functions in the retinal ganglion cells following chronic ocular hypertension rat modelPurpose:To investigate alterations of mtDNA in RGCs using a well-established rat glaucoma model and explore the mechanisms underlying the progressive loss of RGCs.Methods:Ocular chronic hypertension was induced by episcleral vein cauterization in Wistar rat. Retrograde labeling of RGCs with FG and flat mount were performed to count RGCs. Histological assessment of the survival of optic nerve axons by SMI32immunofluoresence. RGCs was isolated by immunopanning and then mitochondria, mtDNA, nDNA, mitochondrial protein, cytoplasmic protein, total mRNA were extracted. mtDNA damages were determined using long-extension PCR and mtDNA mutations were detected by random capture assay. The mRNA and protein of OGG1, MYH and POLG were analysis by western blotting and Real-time PCR, respectively. Mitochondrial complex I and III activities, ATP production rate, and ROS were detected by biochemistry methods.Results:IOP in80%of EVC-eyes was increased1day after surgery (25.3±1.6mmHg),35%of which sustained IOP elevation till6weeks (18.7±1.1mmHg). The IOP in sham-operation control was12.16±0.89mmHg. RGCs loss not only during the period of IOP elevation, but also elevated IOP returned to normal. RGCs loss in the EVC-eyes was24.6±0.17%(p<0.05),30.4±0.78%(p<0.01) and34.8±0.15%(p<0.01) at2,4,6months, respectively. SMI32staining for axons further confirmed this. mtDNA damages were detected2weeks after EVC and continue to damage after, reaching to1.5-folds (p<0.05),2.8-folds (p<0.01) and3.4-folds (p<0.01) than controls at2,4,6months respectively. mtDNA mutations were detected2-4weeks after EVC and became more worsen even when IOP returned to normal. At2,4,6months, mtDNA mutations at Taql1427site was7.6-folds (p<0.05),10.3-folds (p<0.01) and16.8-folds (p<0.01) than the controls, respectively. mtDNA mutations at Taql8835site was9.9-folds(p<0.01)and12.7-folds(p<0.01)at6weeks and6months, respectively. The protein of OGG1, MYH and POLG in mitochondria decreased after IOP elevation and remained decreased even when IOP returned to normal. However, the mRNA expressions of these three enzymes increased shortly after IOP elevation and then gradually decreased, with OGG1and MYH to normal level, and POLG lower than normal. Mitochondrial complex I activity decreased by 36%,(p<0.05)、39%(p<0.05)、41%(p<0.05) and43%(p<0.05) at6weeks,2,4and6months after EVC. The reduction of mitochondrial complex III activity was more obviously than complex III. The mtDNA encoded protein, cyt B and ND5decreased at6months after EVC. The ATP production rate shortly increased after IOP elevation (5.2-folds than the control, p<0.05), and then decreased till6months after EVC, reaching to48%reduction(p<0.05). ROS increased immediately after IOP elevation, reaching a peak (1.6-folds than control, p<0.05). After that, ROS decreased to normal value. Interestingly, re-increased of ROS was detected at4and6months after EVC.Conclusions:Progressive loss of RGCs and their axons after elevated IOP returns to normal in an EVC-induced glaucomatous rat model. Progressive RGC death accompanies accumulated mtDNA alterations (both damage and mutation) that compromise the mtDNA repair system and accelerate mitochondrial dysfunction even when IOP has returned to normal for an extended period of time.Part Ⅱ High pressure causes mtDNA damages and mitochondrial dysfunctionPurpose:To investigate whether high pressure can cause mtDNA damages and mutations and the relationship of mtDNA alterations and mitochondrial function abnormality.Methods:Rat astrocytes were cultured under30mmHg pressure to mimic ocular chronic hypertension. Cells were collected at12,24,48,72,96and120hours after high pressure. mtDNA damages and mutations were detected by Long-extension PCR and random mutation capture assay. OGG1, MYH, and POLG were analyzed using Real-time PCR and western blotting. ROS was determined by DCF-DA method. Lenti-shPOLG was transducted to astrocytes to induced mtDNA damages and mutations. mtDNA encoded proteins, ND4. ND5、ND6and cyt B, were detected by western blotting. In addition, mitochondrial complex I and III activities, ATP production rate were detected by biochemistry methods. Mitochondrial membrane potential was determined by JC-1.Results:mtDNA damages were detected at24hours after high pressure and increased more than40%(p<0.05),compared to the controls at72hours. mtDNA mutations increased and2.2-folds (p<0.05) and5.1-folds (p<0.001) more than controls at48,72hours, respectively at Taql1427site. Similar to Taql1427, mtDNA mutations at Taq18335site showed4.9-folds (p<0.001) than the control. No detectable changes were observed in ROS under high pressure. The mRNA expression of OGG1, MYH, and POLG increased sharply, reaching a peak at12hour after high pressure. Subsequently, OGG1and MYH mRNA expressions gradually decreased to normal value till the end, while POLG continued to decrease and lower than normal. The protein expressions of OGG1, MYH, and POLG decreased48hours after high pressure, with the reduction of56%(p<0.05)、60%(p<0.01);38%(p<0.05).63%(p<0.01)'39%(p<0.05),37%(p<0.05) at72and120hours, respectively. mtDNA damages increased by16%(p>0.05)and46%(p<0.01) at5,10days after transduction with Lenti-shPOLG, relative to the control. mtDNA-encoded protein ND5、ND6and cyt B decreased by43%(p<0.05)、32%(p<0.05)'34%(p<0.05). Mitochondrial complex I and III decreased by22.6%(p<0.05) and40.8%(p<0.05) at10days after transduction. Mitochondrial membrane potential decreased by63%(p<0.001). The MAPR in the cells transfected with shPOLG decreased in a time-dependent manner, with levels amounting to67.9%of those in the SC-transfected cells at9days (p<0.05).Conclusions:High pressure causes mtDNA alterations and that the latter directly lead to mitochondrial dysfunction.Part III RGC susceptibility to apoptosisPurpose:To investigate the susceptibility to apoptosis of RGCs harboring increased mtDNA damages and mutations in a rat model with mtDNA mutations in RGCs.Methods:shPOLG1, shPOLG2, shPOLG3, and control plasmid was constructed using pSilencer1.0-U6vector. POLG mRNA and protein expressions were detected by Real-time PCR and western blot to test which shRNA worked best. Then the DNA containing the shRNA sequence and the U6promoter was cut from the shPOLG3or the shCTL plasmid and cloned into the pAAV-CMV-GFP vector followed by purification. Intravitreous injection of AAV2-shPOLG was performed to induce RGCs mtDNA rat model. At2,6, and12months after injection, POLG protein expression was detected using western blot, mtDNA mutations and damages was tested using Long-extension PCR and random mutation capture assay, ROS was determined by DCF-DA. At12months after intravitreous injection of AAV2-shPOLG, the rat divided into three groups randomly. EVC operation was performed to induce IOP elevation in one group, intravitreous injection of glutamate was performed in the second group, and the third group considered as control. In situ TUNEL assay in retina and Dil retro-grade labeling were performed for quantitative analysis of RGCs. Cleaved caspase-3was detected using western blot analysis. Results:mtDNA damages increased by21%、31%(p<0.01) and63%(p<0.01) at2,6,and12months after intravitreous injection of AAV2-shPOLG, respectively, compared to the control group. mtDNA mutations at Taq11427site showed6.5-folds (p<0.001)、9.3-folds (p<0.001), and17.1folds (p<0.001) increased than the control. The apoptosis of RGCs in the rAAV2-shPOLG group showed2.3-folds (p<0.05) increased than the control. The apoptosis of RGCs in the rAAV2-shPOLG/EVC and rAAV2-shPOLG/glutamate increased by21%(p<0.05) and35%(p<0.01) relative to control groups. The expression of cleaved caspase-3increased by43%(p<0.05)、41%(p<0.05) and48%(p<0.05)in the rAAV2-shPOLG, rAAV2-shPOLG/EVC and rAAV2-shPOLG/glutamate, respectively, compared to the corresponding control group.Conclusions:Cumulative mtDNA damages and mutations results in RGCs apoptosis and vulnerability to secondary insults such as IOP elevation, high glutamate level. |
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