泛素蛋白酶體介導(dǎo)的蛋白降解在海馬CA1區(qū)突觸可塑性中的作用研究
發(fā)布時(shí)間：2018-09-12 16:21
【摘要】：突觸可塑性與使用依賴性的蛋白更新包括蛋白合成,降解,轉(zhuǎn)運(yùn)和定位的調(diào)控緊密聯(lián)系。聯(lián)合應(yīng)用病毒表達(dá)載體系統(tǒng),熒光成像技術(shù)和電生理技術(shù)研究泛素蛋白酶體系統(tǒng)(UPS)介導(dǎo)的蛋白降解在活性依賴性的突觸可塑性中的作用。我們的主要目的是(1)明確與突觸可塑性相關(guān)的幾種主要的突觸后蛋白的降解的時(shí)空順序；(2)了解UPS激活過程所包含的信號級聯(lián)反應(yīng)；(3)評價(jià)單個(gè)樹突棘水平蛋白降解的輸入特異性程度；(4)明確這些重要的突觸后蛋白降解對活性依賴性的突觸可塑性的影響。 本研究獲得以下主要的研究結(jié)果： 1、UPS抑制劑MG132通過抑制蛋白酶體活性從而破壞海馬CA1區(qū)晚期LTP的誘導(dǎo),但不影響晚期LTP的維持。 2、晚期LTP誘導(dǎo)后10分鐘,蛋白酶體活性增加約50%；1小時(shí)后蛋白酶體活性恢復(fù)到對照水平。在"Strong before Weak" LTP誘導(dǎo)模式中低強(qiáng)度的刺激也足以增加蛋白酶體的活性,只是增加的程度低于高強(qiáng)度刺激,約20%,與未給刺激的腦片相比具有統(tǒng)計(jì)學(xué)差異。 3、無論是采用‘'Strong before Weak"還是"Weak before Strong" LTP誘導(dǎo)模式,在給予誘導(dǎo)突觸標(biāo)識形成的低強(qiáng)度刺激(weak)時(shí)抑制蛋白酶體活性,使接受低強(qiáng)度刺激的突觸產(chǎn)生的早期LTP無法延續(xù)為晚期LTP。在“Weak before Strong" LTP誘導(dǎo)模式,給予高強(qiáng)度刺激時(shí)應(yīng)用UPS抑制劑可分別抑制兩條輸入通路的LTP。 4、一個(gè)重要的發(fā)現(xiàn)是：在"Strong before Weak" LTP誘導(dǎo)模式中,給予S2低強(qiáng)度刺激的同時(shí)應(yīng)用MG132抑制蛋白酶體活性,可使已經(jīng)表達(dá)LTP的突觸(S1)發(fā)生突觸異源去長時(shí)程增強(qiáng)現(xiàn)象；NMDA受體抑制劑AP5或者鈣調(diào)磷酸酶PP2B抑制劑cyclosporin A與MG132同時(shí)灌流均可逆轉(zhuǎn)MG132所介導(dǎo)的突觸異源去長時(shí)程增加。另外蛋白合成抑制劑anisomycin也能抑制MG132介導(dǎo)的突觸異源去長時(shí)程增強(qiáng)。 5、L-LTP的誘導(dǎo)可以增加PP2B, PP1的活性；在"Strong before Weak" LTP誘導(dǎo)模式中低強(qiáng)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的活性增加不能被應(yīng)用MG132阻斷；磷酸酶PP2B抑制劑可以完全取消PP1活性的增加；PP1抑制劑與MG132聯(lián)合應(yīng)用不能取消突觸異源去長時(shí)程增強(qiáng)。 6、成功構(gòu)建含有eGFP和SPAR融合基因的西門利克森林病毒載體并定向轉(zhuǎn)染成年大鼠海馬腦區(qū)；與表達(dá)eGFP (1.26 spines/μm)的錐體細(xì)胞相比,表達(dá)eGFP-SPAR (2.67 spines/μm)的CA1區(qū)錐體細(xì)胞樹突棘含量明顯增加。 7、誘導(dǎo)LTP的強(qiáng)直刺激可以誘導(dǎo)Plk2 mRNA的轉(zhuǎn)錄。 8、誘導(dǎo)LTP的強(qiáng)直刺激可以使SPAR的熒光強(qiáng)度降低,SPAR熒光強(qiáng)度的降低可以被蛋白酶體抑制劑所阻斷,因此SPAR降解是通過泛素蛋白酶體系統(tǒng)進(jìn)行的；蛋白合成抑制劑anisomycin和CDK5抑制劑roscovitine均能抑制SPAR蛋白熒光強(qiáng)度的降低,我們推測蛋白合成抑制劑可能通過抑制Plk2的合成使SPAR不能磷酸化而抑制其經(jīng)UPS的降解;而CDK5抑制劑可能通過抑制SPAR蛋白特殊位點(diǎn)磷酸化,使得Plk2無法與SPAR結(jié)合而抑制SPAR降解。 9、蛋白合成被抑制后,排除激光淬滅的的作用,SPAR蛋白熒光強(qiáng)度仍然有降低,這種熒光強(qiáng)度的降低可被聯(lián)合應(yīng)用MG132和NMDA受體阻斷劑APV所阻斷,而不能被聯(lián)合應(yīng)用MG132和roscovitine所阻斷,推測在蛋白合成被抑制時(shí),強(qiáng)直刺激可能通過激活NMDA受體參與SPAR蛋白經(jīng)UPS的降解,但是這種降解是Plk-2非依賴性的。 10、蛋白合成被抑制后,過表達(dá)eGFP-SPAR的腦片仍然可以誘導(dǎo)出LTP,而過表達(dá)eGFP的腦片無法誘導(dǎo)出LTP,可能是由于蛋白合成抑制劑抑制了Plk2合成,SPAR無法被磷酸化,因而不能經(jīng)UPS降解,提示SPAR在LTP誘導(dǎo)過程中發(fā)揮重要作用。
[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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R338.8
本文編號：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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R338.8
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