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Proteasomic Basis of Protein Degradation Dysfunction after Transient Cerebral Ischemia

Author: GePengFei
Tutor: LuoYiNan
School: Jilin University
Course: Surgery
Keywords: transient cerebral ischemia proteasome neuron death
CLC: R743.31
Type: PhD thesis
Year: 2007
Downloads: 128
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Abstract


Objective: To investigate the alterations of proteasome after transient cerebral ischemia and its significance in the protein degradation dysfunction.Methods: Two vessels occlusion transient global ischemia rat model was used. According to different reperfusion period, the rats were divided into sham group, 0.5h recovery group, 4h recovery group, 24h recovery group, and 72h recovery group. Photo-microscope, transmission electronic microscope, laser scanning confocal microscope, differential centrifuge, sucrose gradient density centrifuge, gel filtration, westernblot, enzyme activity assay, and statistical analysis were used to study this project.Results:1. There were no morphological alterations in the cortex neurons from 0.5h, 4h, 24h recovery group compared with sham group. However, after 72h reperfusion, pink cells with polygonal nucleus were observed in the hippocampus CA1 region and cortex under photo-microscope, indicating that these cells died.2. Suc-LLVY-Amc was selected as substrate for assaying proteasome activity. The results showed, the activity of the proteasome from sham group was 54601.6±1602.4, and decreased to 42035.9±1465.2(p<0.01)after 0.5h reperfusion following ischemia. Although the activity transiently recovered to 47536.2±2532.3 after 4h reperfusion, it was still below the value of the sham group (p<0.05) . After 24h reperfusion, the activity decreased again to 45450.1±648.5 (compared with sham group, p<0.01) , then decreased continuously to 43108.4±994.5(compared with sham group, p<0.01) , which was after 72h reperfusion.3. Under transmission electronic microscope, dot and patch high electronic density protein aggregation was observed after 4h and 24h reperfusion following ischemia. However, none was observed in the samples from 0.5h recovery group and sham group.4. Compared with the gray density value 20.0 + 4.1 labeled with ubiquitin monoclonal antibody in the sham group, the value of the abnormal protein labeled with ubiquitin in the protein aggregation isolated from 0.5h recovery group was 120.4 + 42.7 (p<0.01) . After 4h, 24h, 72h reperfusion, the values were 185.7±51.1 (p<0.01) , 188.0±50.7(p<0.01 )and 29.7±8.7(p<0.05)respectively. With the elongation of the reperfusion time, the quantity of the protein aggregation increase continuously in the cell.5. The distribution of 19S proteasome in the neurons after transient ischemia. In the sham group, the 19S proteasome distributed homogeneously in the nucleus and plasma, but the quantity in the nucleus is higher than in the plasma. Also, there was no significant alteration of 19S proteasome distribution in and between nucleus and plasma after 0.5h and 4h reperfusion. However, after 24h reperfusion, 19S decreased both in the nucleus and plasma and decreased much more in the nucleus. After 72h reperfusion, 19S proteasome disappeared totally in nucleus, and only litter rescued in the plasma. In the sham group, the 20S proteasome distributed homogeneously in the nucleus and plasma, but the quantity in the nucleus is higher than in the plasma. Compared with sham group, 20S proteasome decreased significantly after 0.5h and 4h reperfusion, and decreased much more in the nucleus that in the plasma. However, after 24h reperfusion the 20S proteasome in both the nucleus and plasma recovered. After 72h reperfusion, 20S proteasome disappeared from the nucleus, and increased in the plasma.6. The alterations of 20S proteasome in the protein aggregation. Compared with sham group, 20S proteasome increased significantly after 0.5h reperfusion (p<0. 01) , after that it presented constant decreasing tendency. After 24h reperfusion, the proteasome in the protein aggregates was still higher than that of sham group (p<0. 01) . There was no significant difference between 72h recovery group and sham group.The alteration of 19S in the protein aggregates. Compared with sham group, the 19S proteasome in the protein aggregates increased significantly after 0.5h reperfusion (p<o. 01 ) , after 4h reperfusion the 19S proteasome in the protein aggregates decreased little but still higher than that in sham group (p<o. 01) . After 24h reperfusion, 19S proteasome increased again and reached the peak value (p<0.01) . after 72h reperfusion the 19S proteasome in the protein aggregates decreased little but still higher than that in sham group (p<0. 01) .7. Protein aggregates from 0.5h group and 24h group was furthermore centrifuged by sucrose gradient and analyzed with westernblot. After reperfusion 0.5h, the protein aggregates labeled by monoclonal ubiquitin antibody distributed from No.20 to No.24. After reperfusion 24h, ubiquitin labeled protein aggregated distributed from No.17 to No.21. With 20S antibody, 20S proteasome in the protein aggregates distributed from No.20 to No.23 after 0.5h reperfusion and from No.17 to No.21 after 24h reperfusion. With 19S antibody, 19S proteasome in the protein aggregates distributed from No.20 to No.23 after 0.5h reperfusion and from No.17 to No.21 after 24h reperfusion.8. Proteasome in the supernatant samples from 0.5h and 24h group was separated furthermore with gel filtration, then activity assay and westernblot analysis were performed. Activity assay results showed three peaks, the 1st one is 26S proteasome, the 2nd one is 19S+20S and the 3rd one is 20S. Conclusion: 1. Proteasome activity decrease after transient cerebral ischemia. 2. Proteasome accumulated with other protein to form protein aggregates.3.26S proteasome dissembled after transient cerebral ischemia.

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CLC: > Medicine, health > Neurology and psychiatry > Neurology > Cerebrovascular disease > Acute cerebrovascular disease ( stroke) > Transient ischemic
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