泛素蛋白酶体介导的蛋白降解在海马CA1区突触可塑性中的作用研究
发布时间：2018-09-12 16:21
【摘要】：突触可塑性与使用依赖性的蛋白更新包括蛋白合成,降解,转运和定位的调控紧密联系。联合应用病毒表达载体系统,荧光成像技术和电生理技术研究泛素蛋白酶体系统(UPS)介导的蛋白降解在活性依赖性的突触可塑性中的作用。我们的主要目的是(1)明确与突触可塑性相关的几种主要的突触后蛋白的降解的时空顺序；(2)了解UPS激活过程所包含的信号级联反应；(3)评价单个树突棘水平蛋白降解的输入特异性程度；(4)明确这些重要的突触后蛋白降解对活性依赖性的突触可塑性的影响。 本研究获得以下主要的研究结果： 1、UPS抑制剂MG132通过抑制蛋白酶体活性从而破坏海马CA1区晚期LTP的诱导,但不影响晚期LTP的维持。 2、晚期LTP诱导后10分钟,蛋白酶体活性增加约50%；1小时后蛋白酶体活性恢复到对照水平。在"Strong before Weak" LTP诱导模式中低强度的刺激也足以增加蛋白酶体的活性,只是增加的程度低于高强度刺激,约20%,与未给刺激的脑片相比具有统计学差异。 3、无论是采用‘'Strong before Weak"还是"Weak before Strong" LTP诱导模式,在给予诱导突触标识形成的低强度刺激(weak)时抑制蛋白酶体活性,使接受低强度刺激的突触产生的早期LTP无法延续为晚期LTP。在“Weak before Strong" LTP诱导模式,给予高强度刺激时应用UPS抑制剂可分别抑制两条输入通路的LTP。 4、一个重要的发现是：在"Strong before Weak" LTP诱导模式中,给予S2低强度刺激的同时应用MG132抑制蛋白酶体活性,可使已经表达LTP的突触(S1)发生突触异源去长时程增强现象；NMDA受体抑制剂AP5或者钙调磷酸酶PP2B抑制剂cyclosporin A与MG132同时灌流均可逆转MG132所介导的突触异源去长时程增加。另外蛋白合成抑制剂anisomycin也能抑制MG132介导的突触异源去长时程增强。 5、L-LTP的诱导可以增加PP2B, PP1的活性；在"Strong before Weak" LTP诱导模式中低强度刺激也足以增加PP2B,PP1的活性,PP2B的活性增加可以被MG132所抑制,但PPl的活性增加不能被应用MG132阻断；磷酸酶PP2B抑制剂可以完全取消PP1活性的增加；PP1抑制剂与MG132联合应用不能取消突触异源去长时程增强。 6、成功构建含有eGFP和SPAR融合基因的西门利克森林病毒载体并定向转染成年大鼠海马脑区；与表达eGFP (1.26 spines/μm)的锥体细胞相比,表达eGFP-SPAR (2.67 spines/μm)的CA1区锥体细胞树突棘含量明显增加。 7、诱导LTP的强直刺激可以诱导Plk2 mRNA的转录。 8、诱导LTP的强直刺激可以使SPAR的荧光强度降低,SPAR荧光强度的降低可以被蛋白酶体抑制剂所阻断,因此SPAR降解是通过泛素蛋白酶体系统进行的；蛋白合成抑制剂anisomycin和CDK5抑制剂roscovitine均能抑制SPAR蛋白荧光强度的降低,我们推测蛋白合成抑制剂可能通过抑制Plk2的合成使SPAR不能磷酸化而抑制其经UPS的降解;而CDK5抑制剂可能通过抑制SPAR蛋白特殊位点磷酸化,使得Plk2无法与SPAR结合而抑制SPAR降解。 9、蛋白合成被抑制后,排除激光淬灭的的作用,SPAR蛋白荧光强度仍然有降低,这种荧光强度的降低可被联合应用MG132和NMDA受体阻断剂APV所阻断,而不能被联合应用MG132和roscovitine所阻断,推测在蛋白合成被抑制时,强直刺激可能通过激活NMDA受体参与SPAR蛋白经UPS的降解,但是这种降解是Plk-2非依赖性的。 10、蛋白合成被抑制后,过表达eGFP-SPAR的脑片仍然可以诱导出LTP,而过表达eGFP的脑片无法诱导出LTP,可能是由于蛋白合成抑制剂抑制了Plk2合成,SPAR无法被磷酸化,因而不能经UPS降解,提示SPAR在LTP诱导过程中发挥重要作用。
[Abstract]:Synaptic plasticity is closely related to use-dependent protein renewal, including regulation of protein synthesis, degradation, transport and localization. The role of ubiquitin proteasome system (UPS)-mediated protein degradation in activity-dependent synaptic plasticity was investigated by combining viral expression vector systems, fluorescence imaging and electrophysiological techniques. The main objectives are: (1) to determine the temporal and spatial sequence of degradation of several major postsynaptic proteins related to synaptic plasticity; (2) to understand the signal cascade involved in UPS activation; (3) to evaluate the input specificity of protein degradation at the level of a single dendritic spine; (4) to determine the activity dependence of these important postsynaptic proteins degradation. The influence of synaptic plasticity.
The main findings of this study are as follows:
1. UPS inhibitor MG132 destroys the induction of late LTP in hippocampal CA1 region by inhibiting proteasome activity, but does not affect the maintenance of late LTP.
2. The proteasome activity increased by about 50% 10 minutes after LTP induction, and restored to the control level 1 hour after LTP induction. Differences in study.
3. Whether the''Strong before Weak'or''Weak before Strong'' LTP induction model inhibits proteasome activity when low-intensity stimulation (weak) is given to induce synaptic marker formation, so that the early LTP produced by synapses receiving low-intensity stimulation can not be extended to late LTP. UPS inhibitors can inhibit LTP. of two input pathways respectively.
4. An important finding is that in the "Strong before Weak" LTP induction model, low-intensity stimuli of S2 combined with inhibition of proteasome activity by MG132 can induce synaptic allogeneic long-term potentiation in synapses (S1) that have already expressed LTP. The NMDA receptor inhibitor AP5 or calmodulin phosphatase PP2B inhibitor cyclosporin A is similar to MG132. In addition, anisomycin, an inhibitor of protein synthesis, also inhibited MG132-mediated synaptic allogeneic delaying.
5. L-LTP could increase the activity of PP2B and PP1, and low-intensity stimulation could also increase the activity of PP2B and PP1 in the "Strong before Weak" LTP induction model. The increase of PP2B activity could be inhibited by MG132, but the increase of PPl activity could not be blocked by MG132; PP2B inhibitor could completely cancel the increase of PP1 activity; The combination of MG132 and preparations can not abolish the long term potentiation of synapses.
6. Simulick Forest virus vector containing eGFP and SPAR fusion gene was successfully constructed and transfected into the hippocampus of adult rats. Compared with the pyramidal cells expressing eGFP (1.26 spines/micron), the dendritic spines of CA1 pyramidal cells expressing eGFP-SPAR (2.67 spines/micron) were significantly increased.
7, induction of LTP tetanic stimulation can induce the transcription of Plk2 mRNA.
8. Inducing LTP can decrease the fluorescence intensity of SPAR, and the decrease of SPAR fluorescence intensity can be blocked by proteasome inhibitors, so the degradation of SPAR is carried out through the ubiquitin proteasome system. Protein synthesis inhibitors anisomycin and CDK5 inhibitor roscovitine can inhibit the decrease of SPAR protein fluorescence intensity, we conclude Inhibitors of protein synthesis may inhibit the degradation of SPAR by UPS by inhibiting the synthesis of Plk2, whereas CDK5 inhibitors may inhibit the degradation of SPAR by inhibiting the phosphorylation of SPAR specific sites.
9. When protein synthesis was inhibited and laser quenching was excluded, the fluorescence intensity of SPAR protein still decreased, which could be blocked by the combination of MG132 and NMDA receptor blocker APV, but could not be blocked by the combination of MG132 and roscovitine. It was speculated that when protein synthesis was inhibited, the stimulus might activate N. The MDA receptor is involved in the degradation of SPAR protein through UPS, but this degradation is Plk-2 independent.
10. When protein synthesis is inhibited, LTP can still be induced in brain slices overexpressing eGFP-SPAR, but LTP can not be induced in brain slices overexpressing eGFP. This may be because protein synthesis inhibitors inhibit the synthesis of Plk2 and SPAR can not be phosphorylated, so SPAR can not be degraded by UPS, suggesting that SPAR plays an important role in LTP induction.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R338.8
本文编号：2239562
[Abstract]:Synaptic plasticity is closely related to use-dependent protein renewal, including regulation of protein synthesis, degradation, transport and localization. The role of ubiquitin proteasome system (UPS)-mediated protein degradation in activity-dependent synaptic plasticity was investigated by combining viral expression vector systems, fluorescence imaging and electrophysiological techniques. The main objectives are: (1) to determine the temporal and spatial sequence of degradation of several major postsynaptic proteins related to synaptic plasticity; (2) to understand the signal cascade involved in UPS activation; (3) to evaluate the input specificity of protein degradation at the level of a single dendritic spine; (4) to determine the activity dependence of these important postsynaptic proteins degradation. The influence of synaptic plasticity.
The main findings of this study are as follows:
1. UPS inhibitor MG132 destroys the induction of late LTP in hippocampal CA1 region by inhibiting proteasome activity, but does not affect the maintenance of late LTP.
2. The proteasome activity increased by about 50% 10 minutes after LTP induction, and restored to the control level 1 hour after LTP induction. Differences in study.
3. Whether the''Strong before Weak'or''Weak before Strong'' LTP induction model inhibits proteasome activity when low-intensity stimulation (weak) is given to induce synaptic marker formation, so that the early LTP produced by synapses receiving low-intensity stimulation can not be extended to late LTP. UPS inhibitors can inhibit LTP. of two input pathways respectively.
4. An important finding is that in the "Strong before Weak" LTP induction model, low-intensity stimuli of S2 combined with inhibition of proteasome activity by MG132 can induce synaptic allogeneic long-term potentiation in synapses (S1) that have already expressed LTP. The NMDA receptor inhibitor AP5 or calmodulin phosphatase PP2B inhibitor cyclosporin A is similar to MG132. In addition, anisomycin, an inhibitor of protein synthesis, also inhibited MG132-mediated synaptic allogeneic delaying.
5. L-LTP could increase the activity of PP2B and PP1, and low-intensity stimulation could also increase the activity of PP2B and PP1 in the "Strong before Weak" LTP induction model. The increase of PP2B activity could be inhibited by MG132, but the increase of PPl activity could not be blocked by MG132; PP2B inhibitor could completely cancel the increase of PP1 activity; The combination of MG132 and preparations can not abolish the long term potentiation of synapses.
6. Simulick Forest virus vector containing eGFP and SPAR fusion gene was successfully constructed and transfected into the hippocampus of adult rats. Compared with the pyramidal cells expressing eGFP (1.26 spines/micron), the dendritic spines of CA1 pyramidal cells expressing eGFP-SPAR (2.67 spines/micron) were significantly increased.
7, induction of LTP tetanic stimulation can induce the transcription of Plk2 mRNA.
8. Inducing LTP can decrease the fluorescence intensity of SPAR, and the decrease of SPAR fluorescence intensity can be blocked by proteasome inhibitors, so the degradation of SPAR is carried out through the ubiquitin proteasome system. Protein synthesis inhibitors anisomycin and CDK5 inhibitor roscovitine can inhibit the decrease of SPAR protein fluorescence intensity, we conclude Inhibitors of protein synthesis may inhibit the degradation of SPAR by UPS by inhibiting the synthesis of Plk2, whereas CDK5 inhibitors may inhibit the degradation of SPAR by inhibiting the phosphorylation of SPAR specific sites.
9. When protein synthesis was inhibited and laser quenching was excluded, the fluorescence intensity of SPAR protein still decreased, which could be blocked by the combination of MG132 and NMDA receptor blocker APV, but could not be blocked by the combination of MG132 and roscovitine. It was speculated that when protein synthesis was inhibited, the stimulus might activate N. The MDA receptor is involved in the degradation of SPAR protein through UPS, but this degradation is Plk-2 independent.
10. When protein synthesis is inhibited, LTP can still be induced in brain slices overexpressing eGFP-SPAR, but LTP can not be induced in brain slices overexpressing eGFP. This may be because protein synthesis inhibitors inhibit the synthesis of Plk2 and SPAR can not be phosphorylated, so SPAR can not be degraded by UPS, suggesting that SPAR plays an important role in LTP induction.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R338.8
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