缺血性卒中后DAPK1死亡信号介导突触损伤的机制研究
发布时间：2018-08-15 16:04
【摘要】：[背景] 卒中是脑损伤的首要原因,缺血性脑卒中是由于脑血流中断,导致大脑局部缺血的一类严重的神经疾病,患者出现语言功能损伤,视力丧失,瘫痪甚至死亡。脑卒中发病率、死亡率、致残率居高不下,伴随着人口老龄化的加剧,脑卒中的发生率还在逐渐上升,被公认为目前严重危害人类健康和生命安全的常见难治性疾病。目前,针对脑缺血造成的损害所采取的治疗手段非常有限,目前唯一有效的治疗是运用组织纤溶酶原激活物(tPA)的溶栓疗法。然而,出于安全考虑和溶栓治疗的时间窗较窄(4.5小时),多数患者只能得到对症支持治疗。因此,针对缺血性卒中脑损伤,探讨新的干预方法和治疗手段来对抗脑损伤并保护神经细胞极其重要。 缺血后神经元突触损伤(包括突触前和突触后损伤)是引起突触传递障碍与神经元死亡的早期病理生理学基础。我们课题组前期研究发现：死亡相关蛋白激酶(Death Associated Protein Kinase.DAPKl),作为一种Ca2+/钙调蛋白依赖的丝氨酸/苏氨酸激酶,在缺血性脑卒中的缺血脑区被激活,并介导神经元死亡。神经元重要的骨架蛋白-微管相关蛋-Tau,在神经元胞体和突起均丰富表达,主要通过促进微管组装以维持轴突转运、神经元形态以及神经元之间的信号交流。Tau的氨基酸序列中富含丝氨酸/苏氨酸残基,许多丝氨酸/苏氨酸激酶(包括DAPK1)参与神经元退行性变性中Tau蛋白异常过度磷酸化,但在缺血性脑损伤过程中,DAPKl是否影响Tau的磷酸化、Tau介导了DAPK1对神经元的哪些损伤作用,这些问题至今尚无报道。Caytaxin作为一种在突触前丰富表达的蛋白,能够调节谷氨酸神经传递,参与神经元突触的凋亡,能引起神经退行性改变与神经元死亡。激活的DAPK1是否与Caytaxin作用并引起Caytaxin的磷酸化、介导缺血性卒中后突触前损伤尚不清楚。 [目的] (1)阐明DAPK1与Tau、DAPKl与Caytaxin相互作用介导缺血性卒中后神经元死亡的细胞与分子机制,为揭示Tau和Caytaxin成为DAPK1下游特异作用底物参与缺血性卒中后神经元死亡提供证据,即缺血性卒中后,激活的DAPK1引起Tau蛋白的异常磷酸化并在树突棘异常聚集,导致树突棘丢失；而在突触前激活的DAPK1磷酸化Caytaxin,促进Cayatxin的表达,引起突触传递功能障碍。 (2)通过合成小分子多肽阻断DAPK1与Tau相互作用探讨缺血性卒中的治疗策略,为研发缺血性卒中的治疗药物奠定理论基础。[方法] 运用大脑中动脉栓塞(MCAO)手术以及光照缺血(PT)构建4月龄小鼠脑缺血模型或相应的Sham假手术作为对照,分别采用C57BL/6J小鼠、CaMK Ⅱα-Cre小鼠、DAPK1-KDloxp/loxp、DAPK1-KD-/-小鼠为研究对象;CaMK II a-Cre小鼠与DAPK1-KDloxp/loxp小鼠杂交,PCR鉴定阳性小鼠通过他莫西芬诱导得到DAPK1-KD-/-小鼠；通过多普勒脑血流仪和激光散斑脑血管成像仪监测MCAO手术中脑血流的情况;通过磁共振(MRI)和TTC染色检测小鼠卒中后大脑缺血面积；Fluoro-Jade C (FJ)染色和TUNEL染色鉴定缺血后分别发生退行性变和凋亡的神经元数目；采用腺相关病毒(AAV-EGFP)感染标记树突棘；运用Western blot(WB)方法检测缺血后DAPK1、磷酸化肌球蛋白轻链(Phospho-myosin light chain, pMLC)、Tau、Caytaxin、剪切型Caspase3(Cleaved-Caspase3),突触相关蛋白PSD95、GluR1、Synapsin I等蛋白的表达水平；通过免疫荧光双标和免疫共沉淀的方法在体内和体外研究DAPK1与Tau以及DAPK1与Caytaxin的相互作用情况；在HEK293T细胞构建表达系统,共转DAPK1不同的突变体(DAPK1△KD、DAPK1△CaM、DAPK1△DD、 DAPK1K42A)与Tau,鉴定DAPK1与Tau相互作用的结构域；质谱检测DAPK1免疫沉淀下来的磷酸化蛋白；AAV-Tau-WT以及rAAV-Tau-S262A病毒感染DAPK1-KD+/+原代神经元,1AAV-Tau-WT感染DAPK1-KD-/-原代神经元,观察神经元树突棘损伤情况,同时记录上述神经元AMPA受体介导的微型兴奋性突触后电位(mEPSC)的幅度和频率；合成多肽阻断DAPK1与Tau的相互作用,静脉注射TAT-R1D小分子多肽观察其是否逆转中风损伤；运用水迷宫、旷场等行为学手段检测脑缺血小鼠以及TAT-R1D多肽治疗后的小鼠的学习记忆和活动情况。 [结果] 1.脑缺血时树突棘损伤早于凋亡的发生 MCAO后再灌注2h到24h,树突棘密度较假手术组明显下降;同时,突触相关蛋白PSD95、GluR1和Synapsin I也明显减少；再灌注6h开始到24h,TUNEL阳性细胞数目明显上升,剪切型的Caspase3再灌注后12h明显上升。 2.DAPK1通过激酶域与Tau相互作用,DAPK1与Caytaxin在突触前相互作用 免疫荧光双标和免疫共沉淀的结果均证实在缺血缺氧状态下DAPK1和Tau以及DAP K1和Caytaxin相互作用形成复合物,而DAPK1和Tau在缺血后表达量均未发生改变,而Caytaxin的表达水平在缺血侧较对侧有明显的增加;运用GST-pull-down实验和构建细胞系表达体系发现DAPK1通过氨基端的激酶域与Tau特异性结合；免疫荧光双标显示DAPK1与Caytaxin在神经元中共定位;免疫荧光及免疫印迹均显示DAPK1与Caytaxin在突触前表达；DAPK1与Caytaxin相互作用在缺血性卒中以及神经元双氧水处理后明显增强。 3.脑缺血时DAPK1被激活,磷酸化Tau Ser262位点,磷酸化Caytaxin Ser46位点 MCAO后,DAPK1的活性则较假手术组明显增加,表现为pMLC的水平明显增加；GPS21软件预测结果显示,DAPK1可能磷酸化Tau Ser262位；质谱结果显示,中风后DAPK1磷酸化Tau Ser262位,磷酸化Caytaxin Ser46HEK293T细胞共转DAPK1和Tau-WT组,Tau pS262以及剪切型的Caspase3水平均明显升高,而在共转DAPK1和Tau-S262A组则未发现此现象；C57BL/6J小鼠MCAO2h和24h后Tau pS262水平升高,而pS202, pS422,以及GSK3P水平未观察到明显改变。 4.磷酸化Tau Ser262位导致树突棘损伤 培养9天的原代神经元感染rAAV-Tau-WT病毒,12天后观察出现了树突棘的丢失,Tau pS262水平上升,突触相关蛋白例如PSD95, GluRl和Synapsin I下降,同时AMPA受体介导的微型兴奋性突触后电位(mEPSC)的幅度和频率均明显下降,相反的,感染\AV-Tau-S262A以及在DAPK1-KD-/-的原代神元感染Tau-WT未观察到Tau pS262上调和上述提及的树突棘损伤；氧糖剥夺(OGD)处理原代神经元60分钟,感染了Tau-S262A原代神经元中TUNEL+/pS262+细胞数目明显少于感染Tau-WT。 5.敲除DAPK1的激酶域减轻缺血损伤 DAPK1-KD-/-小鼠的体重、大脑血管、血流、大脑的结构以及情绪和同窝野生型小鼠相比均未发生明显改变,提示条件性敲除DAPK1激酶域没有影响小鼠的表型;MCAO1h缺血处理,再灌注24小时,磁共振和TTC染色结果显示,DAPK1-KD小鼠缺血面积较DAPK1-KDloxp/loxp小鼠明显下降,Tau pS262水平也明显下降;再灌注3d和7d后,FJ染色和TUNEL显示DAPK1-KD-/-小鼠发生退行性变和凋亡的细胞减少,同时树突棘丢失的现象也减轻,而神经功能评分以及运动运动协调能力有所改善。 6.TAT-R1D对中风损伤产生治疗效应 根据DAPK1与Tau相互作用位点合成一段多肽TAT-R1D,荧光显微镜观察脑片,肽段TAT-R1D被神经元,而不是被小神经胶质或者星形胶质细胞吸收；免疫共沉淀结果显示静脉注射TAT-R1D2mg/kg的剂量,再灌注6小时以内应用TAT-R1D,在脑组织中可以有效的干扰DAPK1-Tau的结合；MCAO再灌注1小时应用TAT-R1D,和应用TAT-s-R1D以及生理盐水比较,Tau pS262下降,突触相关蛋白PSD-95、GluR1、Synapsin1较对照组均有所上调；3d后进行TTC染色,缺血面积减少；而且,TAT-R1D显著地提高TMCAO7天后小鼠的神经功能评分和行为学的表现例如水迷宫和旷场。 [结论] 我们首次发现在缺血性卒中小鼠DAPK1与Tau的相互作用介导了卒中后神经元树突棘丢失以及随后的神经元死亡。缺血后激活的DAPK1磷酸化Tau的Ser262位点,引发Tau在树突棘聚集。利用转基因技术敲除DAPK1的激酶域或者运用小分子多肽TAT-R1D阻断DAPK1与Tau的相互作用保护了树突棘丢失,逆转神经功能损伤。而且,我们还初步探讨了缺血性卒中DAPK1与Caytaxin的相互作用很可能介导卒中后突触前功能障碍。因此,针对DAPK1及其下游底物Tau以及Caytaxin的相互作用设计小分子多肽进行干预,很可能为缺血性卒中的治疗提供新的治疗靶点与策略。
[Abstract]:[background]
Stroke is the primary cause of brain injury. Ischemic stroke is a serious neurological disease caused by the interruption of cerebral blood flow, resulting in local cerebral ischemia. The patients suffer from language impairment, visual loss, paralysis and even death. The incidence, mortality and disability rate of stroke remain high. With the aging of the population, the incidence of stroke is increasing. The rate is rising and is recognized as a common refractory disease that seriously endangers human health and life safety. Currently, the treatment for cerebral ischemia damage is very limited, and the only effective treatment is thrombolytic therapy with tissue plasminogen activator (tPA). However, safety considerations and thrombolytic therapy are considered. The treatment window is narrow (4.5 hours), and most patients can only get symptomatic support treatment. Therefore, it is very important to explore new intervention methods and treatment methods to combat brain injury and protect nerve cells against ischemic stroke.
Neuronal synaptic damage after ischemia (including presynaptic and postsynaptic damage) is the early pathophysiological basis of synaptic transmission disorders and neuronal death. Our previous study found that death-associated protein kinase (DAPKl) is a Ca2+/calmodulin-dependent serine/threonine. Kinases, which are activated in ischemic brain regions and mediate neuronal death, are abundantly expressed in the somas and processes of neurons, mainly by promoting microtubule assembly to maintain axonal transport, neuronal morphology and signal exchange between neurons. Many serine/threonine kinases (including DAPK1) are involved in abnormal hyperphosphorylation of Tau protein in neurodegenerative disorders. However, whether DAPKl affects Tau phosphorylation during ischemic brain injury and which damage effects of DAPK1 on neurons are mediated by Tau have not been reported so far. As a protein abundantly expressed in presynaptic, n can regulate glutamate transmission, participate in neuronal synaptic apoptosis, and cause neurodegenerative changes and neuronal death. Whether activated DAPK1 acts with Caytaxin and induces Caytaxin phosphorylation, which mediates presynaptic injury after ischemic stroke, remains unclear.
[Objective]
(1) To elucidate the cellular and molecular mechanisms of DAPK1 interacting with Tau, DAPKl and Caytaxin mediating neuronal death after ischemic stroke, and to provide evidence that Tau and Caytaxin are downstream specific substrates of DAPK1 and participate in neuronal death after ischemic stroke. Activated DAPK1 causes abnormal phosphorylation of Tau protein after ischemic stroke Abnormal dendritic spine aggregation leads to the loss of dendritic spine, and the pre-synaptic activation of DAPK1 phosphorylated Caytaxin promotes the expression of Cayatxin, causing synaptic transmission dysfunction.
(2) To explore the therapeutic strategy of ischemic stroke by blocking the interaction between DAPK1 and Tau by synthesizing small molecular polypeptides, so as to lay a theoretical foundation for the development of therapeutic drugs for ischemic stroke.
Middle cerebral artery embolization (MCAO) and light ischemia (PT) were used to construct cerebral ischemia model in 4-month-old mice or sham-operated mice as control. C57BL/6J mice, CaMK II alpha-Cre mice, DAPK1-KD loxp/loxp, DAPK1-KD-/-mice were used as study objects. CaMK II a-Cre mice were hybridized with DAPK1-KD loxp/loxp mice and PCR was used to identify positive. DAPK1-KD-/- mice were induced by tamoxifen; cerebral blood flow during MCAO was monitored by Doppler Cerebral Blood Flowmeter and Laser Speckle Cerebrovascular Imaging; cerebral ischemic area was detected by magnetic resonance (MRI) and TTC staining; degeneration was identified by Fluoro-Jade C (FJ) staining and TUNEL staining, respectively. Number of degenerative and apoptotic neurons; Adeno-associated virus (AAV-EGFP) was used to infect dendritic spines; Western blot (WB) was used to detect DAPK1, Phospho-myosin light chain (pMLC), Tau, Caytaxin, sheared-caspase 3, synapse-related protein PSD95, GluR1, SynapIsin and other eggs after ischemia. The interaction between DAPK1 and Tau, DAPK1 and Caytaxin was studied in vivo and in vitro by immunofluorescence double labeling and immunoprecipitation. The expression system was constructed in HEK293T cells to co-transfect different mutants of DAPK1 (DAPK1 KD, DAPK1 CaM, DAPK1 DD, DAPK1K42A) with Tau, and the interaction between DAPK1 and Tau was identified. Structural domains of DAPK1 immunoprecipitated phosphorylated proteins were detected by mass spectrometry; DAPK1-KD+/+ primary neurons were infected by AAV-Tau-WT and rAAV-Tau-S262A viruses, and DAPK1-KD-/-primary neurons were infected by 1AAV-Tau-WT. The damage of dendritic spines was observed and the AMPA receptor-mediated miniature excitatory postsynaptic potentials (mEPs) were recorded. The amplitude and frequency of EPSC, synthetic peptides blocked the interaction between DAPK1 and Tau, and intravenously injected TAT-R1D small molecule peptides to observe whether they reversed stroke injury. Behavioral methods such as water maze and open field were used to detect the learning, memory and activity of cerebral ischemic mice and those after TAT-R1D polypeptide treatment.
[results]
1. the damage of dendritic spine was earlier than that of apoptosis during cerebral ischemia.
The dendritic spine density decreased significantly from 2 h to 24 h after reperfusion in MCAO group, and the synaptic related proteins PSD95, GluR1 and Synapsin I also decreased significantly. The number of TUNEL positive cells increased significantly from 6 h to 24 h after reperfusion, and the number of shear Caspase 3 increased significantly at 12 h after reperfusion.
2.DAPK1 interacts with Tau through kinase domain, and DAPK1 and Caytaxin interact at presynaptic level.
The results of immunofluorescence double labeling and immunoprecipitation confirmed that DAPK1 and Tau and DAP K1 and Caytaxin interacted to form a complex under hypoxic-ischemic condition, but the expression of DAPK1 and Tau did not change after ischemia, while the expression of Caytaxin increased significantly on the ischemic side compared with the contralateral side. DAPK1 specifically binds to Tau via the amino-terminal kinase domain in the cell line expression system; DAPK1 and Caytaxin co-localize in neurons by immunofluorescence double labeling; both immunofluorescence and immunoblotting show that DAPK1 and Caytaxin are pre-synaptic; interaction between DAPK1 and Caytaxin after ischemic stroke and hydrogen peroxide treatment in neurons Obviously enhanced.
3. DAPK1 was activated during cerebral ischemia, phosphorylation of Tau Ser262 site and phosphorylation of Caytaxin Ser46 site.
After MCAO, the activity of DAPK1 was significantly higher than that of sham-operated group, which showed that the level of pMLC was significantly increased; GPS21 software predicted that DAPK1 might phosphorylate Tau Ser262 site; MS results showed that DAPK1 phosphorylated Tau Ser262 site, phosphorylated Caytaxin Ser46HEK293T cells co-transfected DAPK1 and Tau-WT group, Tau pS262 and shear type. Caspase 3 levels were significantly elevated, but not in DAPK1 and Tau-S262A co-transfected mice; Tau pS262 levels were elevated in C57BL/6J mice after 2 and 24 hours of MCAO, while pS202, pS422, and GSK3P levels were not significantly changed.
4. phosphorylation of Tau Ser262 site results in dendritic spine injury.
After 9 days of culture, primary neurons infected with rAAV-Tau-WT virus showed loss of dendritic spines, elevated levels of Tau pS262, decreased levels of synaptic-related proteins such as PSD95, GluRl and Synapsin I, and decreased amplitudes and frequencies of AMPA receptor-mediated miniature excitatory postsynaptic potentials (mEPSC). On the contrary, AV-Tau-S262A infection resulted in a decrease in the formation of dendritic spines. Tau-WT was infected with DAPK1-KD-/-primitive primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial pri
5. knocking out DAPK1 kinase domain to alleviate ischemic injury
The body weight, cerebral blood vessels, blood flow, brain structure and emotions of DAPK1-KD-/- mice did not change significantly compared with wild-type mice in the same nest, suggesting that conditioned knockout of DAPK1 kinase domain did not affect the phenotype of mice; MCAO1-hour ischemia, reperfusion 24 hours, magnetic resonance imaging and TTC staining showed that the ischemic area of DAPK1-KD mice was larger than that of DAPK1-K mice. In Dloxp/loxp mice, the levels of Tau pS262 and degeneration and apoptosis of DAPK1-KD-/- mice were observed by FJ staining and TUNEL at 3 and 7 days after reperfusion. The loss of dendritic spines was also alleviated, and the neurological function score and motor coordination were improved.
Therapeutic effect of 6.TAT-R1D on stroke injury
A peptide TAT-R1D was synthesized according to the site of interaction between DAPK1 and Tau. Fluorescence microscopy showed that TAT-R1D was absorbed by neurons instead of microglia or astrocytes. Immunocoprecipitation results showed that TAT-R1D could be used in brain tissue at the dose of intravenous TAT-R1D2mg/kg within 6 hours of reperfusion. Tau pS262 decreased, synapse-related proteins PSD-95, GluR1 and Synapsin-1 were up-regulated compared with the control group, TTC staining was performed 3 days later, and the ischemic area was reduced; moreover, TAT-R1D significantly increased the neural function of the mice 7 days after TMCAO. Able to score and behave in behavior such as water maze and open field.
[Conclusion]
We found for the first time that the interaction between DAPK1 and Tau in ischemic stroke mice mediated the loss of dendritic spines and subsequent neuronal death after stroke. The activation of DAPK1 phosphorylated Tau at the site of Ser262 triggered Tau aggregation in the dendritic spines. Blocking the interaction between DAPK1 and Tau protects dendritic spines from loss and reverses neurological impairment. Furthermore, we preliminarily investigated that the interaction between DAPK1 and Caytaxin in ischemic stroke may mediate poststroke presynaptic dysfunction. Row intervention is likely to provide new therapeutic targets and strategies for the treatment of ischemic stroke.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R743.3
本文编号：2184705
[Abstract]:[background]
Stroke is the primary cause of brain injury. Ischemic stroke is a serious neurological disease caused by the interruption of cerebral blood flow, resulting in local cerebral ischemia. The patients suffer from language impairment, visual loss, paralysis and even death. The incidence, mortality and disability rate of stroke remain high. With the aging of the population, the incidence of stroke is increasing. The rate is rising and is recognized as a common refractory disease that seriously endangers human health and life safety. Currently, the treatment for cerebral ischemia damage is very limited, and the only effective treatment is thrombolytic therapy with tissue plasminogen activator (tPA). However, safety considerations and thrombolytic therapy are considered. The treatment window is narrow (4.5 hours), and most patients can only get symptomatic support treatment. Therefore, it is very important to explore new intervention methods and treatment methods to combat brain injury and protect nerve cells against ischemic stroke.
Neuronal synaptic damage after ischemia (including presynaptic and postsynaptic damage) is the early pathophysiological basis of synaptic transmission disorders and neuronal death. Our previous study found that death-associated protein kinase (DAPKl) is a Ca2+/calmodulin-dependent serine/threonine. Kinases, which are activated in ischemic brain regions and mediate neuronal death, are abundantly expressed in the somas and processes of neurons, mainly by promoting microtubule assembly to maintain axonal transport, neuronal morphology and signal exchange between neurons. Many serine/threonine kinases (including DAPK1) are involved in abnormal hyperphosphorylation of Tau protein in neurodegenerative disorders. However, whether DAPKl affects Tau phosphorylation during ischemic brain injury and which damage effects of DAPK1 on neurons are mediated by Tau have not been reported so far. As a protein abundantly expressed in presynaptic, n can regulate glutamate transmission, participate in neuronal synaptic apoptosis, and cause neurodegenerative changes and neuronal death. Whether activated DAPK1 acts with Caytaxin and induces Caytaxin phosphorylation, which mediates presynaptic injury after ischemic stroke, remains unclear.
[Objective]
(1) To elucidate the cellular and molecular mechanisms of DAPK1 interacting with Tau, DAPKl and Caytaxin mediating neuronal death after ischemic stroke, and to provide evidence that Tau and Caytaxin are downstream specific substrates of DAPK1 and participate in neuronal death after ischemic stroke. Activated DAPK1 causes abnormal phosphorylation of Tau protein after ischemic stroke Abnormal dendritic spine aggregation leads to the loss of dendritic spine, and the pre-synaptic activation of DAPK1 phosphorylated Caytaxin promotes the expression of Cayatxin, causing synaptic transmission dysfunction.
(2) To explore the therapeutic strategy of ischemic stroke by blocking the interaction between DAPK1 and Tau by synthesizing small molecular polypeptides, so as to lay a theoretical foundation for the development of therapeutic drugs for ischemic stroke.
Middle cerebral artery embolization (MCAO) and light ischemia (PT) were used to construct cerebral ischemia model in 4-month-old mice or sham-operated mice as control. C57BL/6J mice, CaMK II alpha-Cre mice, DAPK1-KD loxp/loxp, DAPK1-KD-/-mice were used as study objects. CaMK II a-Cre mice were hybridized with DAPK1-KD loxp/loxp mice and PCR was used to identify positive. DAPK1-KD-/- mice were induced by tamoxifen; cerebral blood flow during MCAO was monitored by Doppler Cerebral Blood Flowmeter and Laser Speckle Cerebrovascular Imaging; cerebral ischemic area was detected by magnetic resonance (MRI) and TTC staining; degeneration was identified by Fluoro-Jade C (FJ) staining and TUNEL staining, respectively. Number of degenerative and apoptotic neurons; Adeno-associated virus (AAV-EGFP) was used to infect dendritic spines; Western blot (WB) was used to detect DAPK1, Phospho-myosin light chain (pMLC), Tau, Caytaxin, sheared-caspase 3, synapse-related protein PSD95, GluR1, SynapIsin and other eggs after ischemia. The interaction between DAPK1 and Tau, DAPK1 and Caytaxin was studied in vivo and in vitro by immunofluorescence double labeling and immunoprecipitation. The expression system was constructed in HEK293T cells to co-transfect different mutants of DAPK1 (DAPK1 KD, DAPK1 CaM, DAPK1 DD, DAPK1K42A) with Tau, and the interaction between DAPK1 and Tau was identified. Structural domains of DAPK1 immunoprecipitated phosphorylated proteins were detected by mass spectrometry; DAPK1-KD+/+ primary neurons were infected by AAV-Tau-WT and rAAV-Tau-S262A viruses, and DAPK1-KD-/-primary neurons were infected by 1AAV-Tau-WT. The damage of dendritic spines was observed and the AMPA receptor-mediated miniature excitatory postsynaptic potentials (mEPs) were recorded. The amplitude and frequency of EPSC, synthetic peptides blocked the interaction between DAPK1 and Tau, and intravenously injected TAT-R1D small molecule peptides to observe whether they reversed stroke injury. Behavioral methods such as water maze and open field were used to detect the learning, memory and activity of cerebral ischemic mice and those after TAT-R1D polypeptide treatment.
[results]
1. the damage of dendritic spine was earlier than that of apoptosis during cerebral ischemia.
The dendritic spine density decreased significantly from 2 h to 24 h after reperfusion in MCAO group, and the synaptic related proteins PSD95, GluR1 and Synapsin I also decreased significantly. The number of TUNEL positive cells increased significantly from 6 h to 24 h after reperfusion, and the number of shear Caspase 3 increased significantly at 12 h after reperfusion.
2.DAPK1 interacts with Tau through kinase domain, and DAPK1 and Caytaxin interact at presynaptic level.
The results of immunofluorescence double labeling and immunoprecipitation confirmed that DAPK1 and Tau and DAP K1 and Caytaxin interacted to form a complex under hypoxic-ischemic condition, but the expression of DAPK1 and Tau did not change after ischemia, while the expression of Caytaxin increased significantly on the ischemic side compared with the contralateral side. DAPK1 specifically binds to Tau via the amino-terminal kinase domain in the cell line expression system; DAPK1 and Caytaxin co-localize in neurons by immunofluorescence double labeling; both immunofluorescence and immunoblotting show that DAPK1 and Caytaxin are pre-synaptic; interaction between DAPK1 and Caytaxin after ischemic stroke and hydrogen peroxide treatment in neurons Obviously enhanced.
3. DAPK1 was activated during cerebral ischemia, phosphorylation of Tau Ser262 site and phosphorylation of Caytaxin Ser46 site.
After MCAO, the activity of DAPK1 was significantly higher than that of sham-operated group, which showed that the level of pMLC was significantly increased; GPS21 software predicted that DAPK1 might phosphorylate Tau Ser262 site; MS results showed that DAPK1 phosphorylated Tau Ser262 site, phosphorylated Caytaxin Ser46HEK293T cells co-transfected DAPK1 and Tau-WT group, Tau pS262 and shear type. Caspase 3 levels were significantly elevated, but not in DAPK1 and Tau-S262A co-transfected mice; Tau pS262 levels were elevated in C57BL/6J mice after 2 and 24 hours of MCAO, while pS202, pS422, and GSK3P levels were not significantly changed.
4. phosphorylation of Tau Ser262 site results in dendritic spine injury.
After 9 days of culture, primary neurons infected with rAAV-Tau-WT virus showed loss of dendritic spines, elevated levels of Tau pS262, decreased levels of synaptic-related proteins such as PSD95, GluRl and Synapsin I, and decreased amplitudes and frequencies of AMPA receptor-mediated miniature excitatory postsynaptic potentials (mEPSC). On the contrary, AV-Tau-S262A infection resulted in a decrease in the formation of dendritic spines. Tau-WT was infected with DAPK1-KD-/-primitive primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial primordial pri
5. knocking out DAPK1 kinase domain to alleviate ischemic injury
The body weight, cerebral blood vessels, blood flow, brain structure and emotions of DAPK1-KD-/- mice did not change significantly compared with wild-type mice in the same nest, suggesting that conditioned knockout of DAPK1 kinase domain did not affect the phenotype of mice; MCAO1-hour ischemia, reperfusion 24 hours, magnetic resonance imaging and TTC staining showed that the ischemic area of DAPK1-KD mice was larger than that of DAPK1-K mice. In Dloxp/loxp mice, the levels of Tau pS262 and degeneration and apoptosis of DAPK1-KD-/- mice were observed by FJ staining and TUNEL at 3 and 7 days after reperfusion. The loss of dendritic spines was also alleviated, and the neurological function score and motor coordination were improved.
Therapeutic effect of 6.TAT-R1D on stroke injury
A peptide TAT-R1D was synthesized according to the site of interaction between DAPK1 and Tau. Fluorescence microscopy showed that TAT-R1D was absorbed by neurons instead of microglia or astrocytes. Immunocoprecipitation results showed that TAT-R1D could be used in brain tissue at the dose of intravenous TAT-R1D2mg/kg within 6 hours of reperfusion. Tau pS262 decreased, synapse-related proteins PSD-95, GluR1 and Synapsin-1 were up-regulated compared with the control group, TTC staining was performed 3 days later, and the ischemic area was reduced; moreover, TAT-R1D significantly increased the neural function of the mice 7 days after TMCAO. Able to score and behave in behavior such as water maze and open field.
[Conclusion]
We found for the first time that the interaction between DAPK1 and Tau in ischemic stroke mice mediated the loss of dendritic spines and subsequent neuronal death after stroke. The activation of DAPK1 phosphorylated Tau at the site of Ser262 triggered Tau aggregation in the dendritic spines. Blocking the interaction between DAPK1 and Tau protects dendritic spines from loss and reverses neurological impairment. Furthermore, we preliminarily investigated that the interaction between DAPK1 and Caytaxin in ischemic stroke may mediate poststroke presynaptic dysfunction. Row intervention is likely to provide new therapeutic targets and strategies for the treatment of ischemic stroke.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R743.3
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