PSD-95与脑缺血的关系及其调控机制研究

PSD-95（突触后密度蛋白-95）在突触后密度区含量丰富,具有复杂的结构域,与膜受体、离子通道、细胞粘附因子和信号分子等相互作用聚集成大分子复合物,在突触的可塑性、学习记忆、大脑的病理生理紊乱等起重要作用。PSD-95与脑缺血神经元损伤和凋亡的分子机制有密切联系。脑缺血再灌注后PSD-95在缺血侧皮层的变化表现为PSD．95阳性细胞数的减少和细胞形态的受损改变。抑制NMDA受体活性的治疗策略包括破坏受体本身、钙离子通道阻滞剂、破坏PSD-95／NMDAR相互作用、破坏PSD-95／nNOS相互作用、nNOS抑制剂药物干预。已有研究发现在大鼠大脑中动脉栓塞模型中抑制PSD-95复合体之间的相互作用可以改善脑缺血。实验性的PSD-95抑制剂减少了短时间和长时间局部脑缺血大鼠的梗死面积、并恢复相应的运动功能治疗脑缺血。本文重点研究PSD-95与脑缺血的关系及其调控机制。     

永久性局灶性中动脉阻断脑缺血（pMCAO）模型大鼠脑内突触相关蛋白表达的免疫组织化学观察

目的观察永久性局灶性中动脉阻断脑缺血（pMCAO）模型大鼠脑缺血发作后2 d、7 d脑内突触相关蛋白表达的变化。方法制作大鼠永久性局灶性中动脉阻断脑缺血（pMCAO）模型。缺血动物术后随机分为缺血2 d组、缺血7 d组,另设假手术组。在术后2 d、7 d 2个时间点采用HE染色观察动物神经病理学改变,同时采用免疫组织化学法观察动物的缺血侧脑组织突触素-I（synapsin-I）、突触后致密蛋白95（PSD-95）、α-突触核蛋白（α-synuclein）表达情况。结果与假手术组相比,缺血后,模型动物神经元大量变性坏死,数目减少,排列散乱。缺血后2 d,synapsin-I在CA1区、CA3区、皮层表达显著减少（P〈0.05或P〈0.01）,PSD-95在CA1区、皮层表达显著减少（P〈0.05或P〈0.01）,α-synuclein在CA1区神经元产生显著积聚（P〈0.01）;缺血后7 d,synapsin-I在CA1区、皮层表达仍显著降低（P〈0.01）,PSD-95在CA1区、皮层表达显著减少（P〈0.05或P〈0.01）,α-synuclein在CA1、CA3、皮层表达显著增加（P〈0.05或P〈0.01）。结论 pMCAO模型大鼠在脑缺血发生后,神经突触有关蛋白的表达显著改变,并随缺血后不同时间点表达情况不同,这可能与神经元突触重塑有关。突触相关蛋白的表达与缺血损伤程度密切相关。     

大鼠局灶性脑缺血再灌注中nNOS来源的NO对细胞凋亡的影响

目的探讨大鼠局灶性脑缺血再灌注早期nNOS来源的NO对细胞凋亡的影响.方法闭塞大鼠左侧大脑中动脉造成局灶性脑缺血模型,给予选择性nNOS抑制剂-7硝基吲唑,应用原位末端标记法及流式细胞术检测缺血2h再灌注6h细胞凋亡的变化.结果 50mg/kg、25mg/kg剂量的7硝基吲唑可使1、NO含量显著降低.2、NT阳性细胞荧光强度及阳性细胞百分比显著减少.3、TUNEL阳性细胞明显减少.4、细胞凋亡百分率降低,AP峰降低.结论 nNOS来源的NO参与介导脑缺血再灌注早期的细胞凋亡.     

大鼠脑缺血再灌注后海马神经细胞NOS表达与细胞凋亡及复方丹参的保护作用

目的探讨大鼠局灶性脑缺血再灌注后海马神经细胞一氧化氮合酶(NOS)的表达与神经细胞凋亡的关系及中药复方丹参的保护作用。方法采用大脑中动脉内栓线阻断法(MCAO)造成局灶性脑缺血再灌注模型。用原位细胞凋亡检测方法观察海马神经细胞凋亡;用免疫组织化学方法检测大鼠海马神经细胞(nNOS、iNOS)的表达并做图像分析。结果与假手术对照组比较,脑缺血再灌注2h后缺血侧海马CA1、CA3区神经细胞nNOS、iNOS表达升高,并出现神经细胞凋亡,随着再灌注时间的延长,神经细胞iNOS的表达明显增强,凋亡神经细胞数逐渐增多,至24h达高峰,但神经细胞nNOS的表达并未见明显增强。复方丹参保护组神经细胞nNOS、iNOS的表达和凋亡神经细胞数明显低于缺血再灌组(P<0.01)。结论脑缺血再灌注后缺血侧海马CA1、CA3区神经细胞nNOS的表达增强,iNOS的表达显著升高,使NO的形成增加,这可能是介导脑缺血再灌注后神经细胞凋亡的机制之一。复方丹参具有下调神经细胞nNOS、iNOS的表达,减少NO的生成,抑制细胞凋亡,减轻缺血再灌注对大鼠海马损伤的作用。     

GST-pull down方法鉴定Src激酶与PSD-95的直接结合

脚手架蛋白PSD-95通过募集多种蛋白质在包括缺血性脑中风在内的多种神经系统疾病中具有重要的作用。Src蛋白激酶家族是膜相关非受体酪氨酸蛋白激酶中最大的家族,该家族激酶含有与突触后致密蛋白PSD-95相结合的结构域。Src激酶是其家族中主要成员之一,在脑组织中表达丰富。脑缺血/再灌注引起缺血敏感区Src激酶活性的显著增强。之前的研究表明,Src激酶参与调节PSD-95酪氨酸磷酸化。本实验主要通过GST-pull down实验体外鉴定Src与PSD-95之间的直接结合。     

戊四氮点燃癫痫大鼠空间学习记忆改变及海马突触素、突触后致密物PSD-95表达的变化

目的探讨戊四氮点燃癫痫对大鼠空间学习记忆的影响及可能的分子机制。方法戊四氮(pentylenetet-razol,PTZ)点燃建立慢性癫痫(chronic epileptic,CEP)模型,Morris水迷宫进行行为学检测,免疫组织化学方法观察大鼠海马CA1、CA3区突触素(synaptophysin,P38)和突触后致密物95(postsynaptic density 95,PSD-95)的表达,并用计算机图像分析系统对免疫反应结果进行处理。结果水迷宫试验检测癫痫组大鼠空间学习记忆能力受损;免疫组化结果表明其海马CA1、CA3区P38和PSD-95免疫反应产物较对照组明显减少(P<0.01,P<0.05)。结论戊四氮点燃癫痫大鼠伴有学习记忆功能减退,其海马神经元P38和PSD-95的表达减少可能参与了空间学习记忆受损。     

电针对脑缺血神经元凋亡影响的形态学研究

为了探讨针刺治疗“脑卒中”的机制,本研究以大鼠一侧大脑中动脉栓塞后再灌注为动物模型,分别以ＴＵＮＥＬ法和ＰＩ染色法观察电针改善脑缺血性神经元凋亡的情况。结果显示：①局灶性脑缺血能诱导神经元凋亡：缺血侧凋亡神经元数目明显多于对照侧,差异非常显著;②电针能明显抑制神经元凋亡：电针治疗组缺血侧梗塞区凋亡神经元数目明显减少。本文表明电针能抑制脑缺血性神经元凋亡。     

电针对局灶脑缺血/再灌注模型大鼠大脑缺血皮质区P2X7R与NLRP3炎性小体表达的影响

目的观察电针治疗对局灶脑缺血/再灌注模型大鼠大脑缺血皮质区嘌呤受体配体门控性离子通道7(purinergic2X7 receptor,P2X7R)和Nod样受体蛋白3(NOD-like receptor pyrin 3,NLRP3)炎性小体表达的影响,探讨电针治疗减轻局灶脑缺血/再灌注炎性损伤的可能机制。方法雄性SD大鼠随机分为假手术组、模型组、电针组,每组16只。采用改良线栓法制备局灶脑缺血/再灌注模型。以大鼠"百会"、"合谷"和"太冲"为电针穴位。采用Bederson行为学评分评价各组大鼠神经功能缺损程度,Western blot和RT-q PCR检测大脑缺血皮区P2X7R、NLRP3蛋白及m RNA表达情况,ELISA检测脑内IL-1β和IL-18含量,荧光共聚焦显微镜检测脑内Iba-1阳性小胶质细胞数量。结果脑缺血再灌注后24h,电针治疗可明显改善神经功能缺损症状。RT-q PCR和Western blot检测显示,模型组大脑皮质缺血区P2X7R和NLRP3 m RNA和蛋白表达较假手术组明显升高,电针治疗可明显减少脑缺血再灌注后P2X7R和NLRP3 m RNA和蛋白表达的升高。ELISA测定表明,与模型组相比,电针组大脑皮质缺血区IL-1β和IL-18含量明显降低。激光扫描共聚焦显微镜观察发现,脑缺血再灌注后24h,电针治疗可明显减少大脑皮质缺血区Iba-1阳性小胶质细胞数量。结论电针可抑制局灶脑缺血/再灌注模型大鼠脑内P2X7R、NLRP3表达的上调,削弱小胶质细胞激活,减轻炎症因子分泌,从而减轻脑缺血/再灌注炎性损伤。     

氟西汀抑制PTSD大鼠海马神经元PSD-95和突触素Ⅰ水平的下调与学习记忆力的损害

目的探讨氟西汀(fluoxetine,FLXT)对创伤后应激障碍(post-traumatic-stress-disorder,PTSD)大鼠记忆及大鼠海马神经元突触后致密物蛋白95(postsynaptic density 95,PSD-95)和突触素Ⅰ(synapsinⅠ)表达的影响。方法采用国际认定的SPS方法刺激大鼠建立PTSD大鼠模型,应用水迷宫实验观察氟西汀对PTSD大鼠学习记忆的影响,采用免疫荧光染色和免疫印迹法检测海马神经元PSD-95和突触素Ⅰ水平。结果 Morris水迷宫前5天的定位航行实验结果显示,SPS刺激后大鼠(PTSD大鼠)找到水下平台的游泳距离和潜伏期比正常组大鼠长,给予氟西汀的PTSD大鼠找到水下平台的距离和潜伏期较未用氟西汀处理的PTSD大鼠缩短。Morris水迷宫第6天空间探索实验结果显示,PTSD大鼠穿越平台的次数和在靶象限花费时间的百分比明显低于正常对照大鼠,而氟西汀可显著增加PTSD大鼠在水迷宫实验中的穿台次数和在靶象限停留的时间百分比。免疫荧光染色和Western Blot检测显示,PTSD大鼠PSD-95和突触素Ⅰ的水平降低,氟西汀干预可抑制PTSD大鼠PSD-95和突触素Ⅰ水平的降低。结论氟西汀可通过抑制PTSD大鼠海马神经元细胞PSD-95和SynapsinⅠ水平的下调,减轻PTSD大鼠空间记忆和学习能力的损伤。     

A型流感病毒NS1与突触后密度蛋白-95的相互作用

目的 A型流感病毒NS1蛋白是一种多功能的致病因子,能够与被感染细胞中的多种蛋白相互结合,影响并干扰宿主细胞内的信号转导、蛋白质合成及抗病毒反应。突触后密度蛋白(Postsynaptic density protein95,PSD-95)主要存在于神经元及SH-SY-5Y等神经来源的细胞株中。假设NS1能够与PSD-95结合,则更有利于了解A型流感病毒对神经元及相关细胞的作用机制。方法通过酵母双杂交,GST-pull down及免疫荧光技术分别从体外和体内两方面检测NS1与PSD-95的相互作用。结果酵母双杂交表明,仅转染PGAD-NS51/PGBK-PSD-95的QDO有菌落生长,且α-半乳糖苷酶活性显著高于阳性对照;而转染PGAD-NS32/PGBK-PSD-95的QDO无菌落生长;GST-pull down表明仅NS51与PSD-95孵育后,能够被Western-blot检测到;免疫荧光表明NS51与PSD-95可能存在共定位,而NS32与PSD-95则不存在共定位。结论 H5N1(A/chicken/Guangdong/1/2005)的NS1能够与PSD-95结合;反之,H3N2(A/Shantou/602/06)的NS1则不能。     

Treatment of cerebral ischemia by disrupting ischemia-induced interaction of nNOS with PSD-95

Stroke is a major public health problem leading to high rates of death and disability in adults. Excessive stimulation of N-methyl-D-aspartate receptors (NMDARs) and the resulting neuronal nitric oxide synthase (nNOS) activation are crucial for neuronal injury after stroke insult. However, directly inhibiting NMDARs or nNOS can cause severe side effects because they have key physiological functions in the CNS. Here we show that cerebral ischemia induces the interaction of nNOS with postsynaptic density protein-95 (PSD-95). Disrupting nNOS-PSD-95 interaction via overexpressing the N-terminal amino acid residues 1-133 of nNOS (nNOS-N(1-133)) prevented glutamate-induced excitotoxicity and cerebral ischemic damage. Given the mechanism of nNOS-PSD-95 interaction, we developed a series of compounds and discovered a small-molecular inhibitor of the nNOS-PSD-95 interaction, ZL006. This drug blocked the ischemia-induced nNOS-PSD-95 association selectively, had potent neuroprotective activity in vitro and ameliorated focal cerebral ischemic damage in mice and rats subjected to middle cerebral artery occlusion (MCAO) and reperfusion. Moreover, it readily crossed the blood-brain barrier, did not inhibit NMDAR function, catalytic activity of nNOS or spatial memory, and had no effect on aggressive behaviors. Thus, this new drug may serve as a treatment for stroke, perhaps without major side effects.     

Altered interaction between PSD-95 and the NMDA receptor following transient global ischemia

The postsynaptic density (PSD) is a cytoskeletal specialization involved in the anchoring of neurotransmitter receptors and in regulating the response of postsynaptic neurons to synaptic stimulation. The postsynaptic protein PSD-95 binds to NMDA receptor subunits NR2A and NR2B and to signaling molecules such as neuronal nitric oxide synthase and p135synGAP. We investigated the effects of transient cerebral ischemia on protein interactions involving PSD-95 and the NMDA receptor in the rat hippocampus. Ischemia followed by reperfusion resulted in a decrease in the solubility of the NMDA receptor and PSD-95 in 1% sodium deoxycholate, the decrease being greater in the vulnerable CA1 hippocampal subfield than in the less sensitive CA3/dentate gyrus regions. Solubilization of the kainic acid receptor GluR6/7 and the PSD-95 binding proteins, neuronal nitric oxide synthase and p135synGAP, also decreased following ischemia. The association between PSD-95 and NR2A and NR2B, as indicated by coimmunoprecipitation, was less in postischemic samples than in sham-operated controls. Ischemia also resulted in a decrease in the size of protein complexes containing PSD-95, but had only a small effect on the size distribution of complexes containing the NMDA receptor. The results indicate that molecular interactions involving PSD-95 and the NMDA receptor are modified by an ischemic challenge.     

PSD-95 assembles a ternary complex with the N-methyl-D-aspartic acid receptor and a bivalent neuronal NO synthase PDZ domain.

Nitric oxide (NO) biosynthesis in cerebellum is preferentially activated by calcium influx through N-methyl-D-aspartate (NMDA)-type glutamate receptors, suggesting that there is a specific link between these receptors and neuronal NO synthase (nNOS). Here, we find that PSD-95 assembles a postsynaptic protein complex containing nNOS and NMDA receptors. Formation of this complex is mediated by the PDZ domains of PSD-95, which bind to the COOH termini of specific NMDA receptor subunits. In contrast, nNOS is recruited to this complex by a novel PDZ-PDZ interaction in which PSD-95 recognizes an internal motif adjacent to the consensus nNOS PDZ domain. This internal motif is a structured "pseudo-peptide" extension of the nNOS PDZ that interacts with the peptide-binding pocket of PSD-95 PDZ2. This asymmetric interaction leaves the peptide-binding pocket of the nNOS PDZ domain available to interact with additional COOH-terminal PDZ ligands. Accordingly, we find that the nNOS PDZ domain can bind PSD-95 PDZ2 and a COOH-terminal peptide simultaneously. This bivalent nature of the nNOS PDZ domain further expands the scope for assembly of protein networks by PDZ domains.     

Isolation of 2000-kDa complexes of N-methyl-D-aspartate receptor and postsynaptic density 95 from mouse brain

Neurotransmitter receptors in vivo are linked to intracellular adaptor proteins and signalling molecules driving downstream pathways. Methods for physical isolation are essential to answer fundamental questions about the size, structure and composition of in vivo complexes and complement the widely used yeast 2-hybrid method. The N-methyl-D-aspartate receptor (NMDAR) binds postsynaptic density 95 (PSD-95) protein; both are required for synaptic plasticity and learning and participate in other important pathophysiological functions. Here we describe the development and optimization of novel methods for large-scale isolation of NMDAR--PSD-95 complexes from mouse brain including immunoaffinity, immunoprecipitation, ligand-affinity and immobilized PSD-95 binding peptides. Short PDZ binding peptides modelled on NMDAR subunits were shown to isolate NMDAR complexes. Gel filtration indicated the native NMDAR--PSD-95 complexes were 2000 kDa, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed a complexity suggesting a huge network of both structural components and signalling enzymes. These methods can be used to define the structure of the complexes at different synapses and in mice carrying gene mutations as well as new tools for drug discovery.     

Flavonoids from Radix Scutellariae as potential stroke therapeutic agents by targeting the second postsynaptic density 95 (PSD-95)/disc large/zonula occludens-1 (PDZ) domain of PSD-95.

Excessive activation of N-methyl-D-aspartate receptors (NMDARs) and subsequent production of nitric oxide by neuronal nitric oxide synthase (nNOS) contribute to neuronal damage resulting from hypoxic and ischemic insults. NMDARs and nNOS are coupled together at the postsynaptic membrane through their interaction with postsynaptic density protein (PSD) 95 via PSD-95/disc large/zonula occludens-1 (PDZ) domains. We used NMR (nuclear magnetic resonance) spectroscopy to screen medicinal herbs used in traditional Chinese medicine (TCM) stroke therapy for compounds binding to the second PDZ domain (PDZ2) of PSD-95, the domain linking nNOS and PSD-95. Aqueous extract of Huangqin, the root of Scutellaria baicalensis Georgi (Labiatae), showed significant binding to PDZ2 of PSD-95. The binding site of the active components in the extract overlapped with the nNOS/NR2B-binding pocket of PDZ2 of PSD-95. Four flavones, baicalin, norwogonoside, oroxylin A-glucuronide (oroxyloside), and wogonoside were isolated and found to account for the PDZ-binding activity of the extract. NMR titration experiments showed that baicalin and norwogonoside displayed the highest PDZ2 binding affinity, while oroxylin A-glucuronide and wogonoside showed 4-5 fold less potency in binding to the PDZ domain. Identification of the PDZ binding activity of these compounds will allow investigating whether or not it contributes to the observed clinical effects of Radix Scutellariae. Furthermore, these molecules might provide leads for the development of drugs targeting the signaling pathways mediated by PDZ domains.     

Developmental regulation of PSD-95 and nNOS expression in lumbar spinal cord of rats

Postsynaptic density (PSD)-95 is originally isolated from glutamatergic synapse where it serves as a physical tether to allow neuronal nitric oxide synthase (nNOS) signaling by N-methyl-D-aspartate receptor (NMDAR) activity. Considering the physiological importance of glutamate receptor and nitric oxide (NO) during development, we examined the spatiotemporal expression of PSD-95 and nNOS in the lumbar spinal cord at a postnatal stage. Temporally, both gene and protein levels of them gradually increased with age after birth, peaked at the postnatal day 14 (P14), and then decreased to an adult level. In addition, the enhanced coimmunoprecipitations between PSD-95 and nNOS were detected in developing spinal cord. Spatially, PSD-95 staining codistributed with nNOS in NeuN-positive motor neurons and sensory neurons at P14. These findings indicate that PSD-95 and nNOS might collectively participate in spinal cord development.     

Spatiotemporal Expression of PSD-95 and nNOS After Rat Sciatic Nerve Injury

Neuronal nitric oxide synthase (nNOS) has been implicated to influence peripheral nerve lesion and regeneration. Post-synaptic density-95 (PSD-95) is one of nNOS-anchoring proteins and plays an important role in specifying the sites of reaction of NO in nervous system. Here we established a rat sciatic nerve crush (SNC) model to examine the spatiotemporal expression of PSD-95 and nNOS. At gene levels, PSD-95 mRNA diminished shortly after crush, and significantly elevated from 2 days to 2 weeks, whereas nNOS decreased progressively post-operation, reached the valley at 1 day, and markedly up-regulated from 1 to 2 weeks after SNC. The expression of both molecules returned to the control level at 4 weeks post-injury. At protein levels, PSD-95 and nNOS underwent the similar changes as their gene expression except for a time lag during up-regulating. At their peak expression, PSD-95 co-labeled with nNOS in Schwann cells (SCs) of sciatic nerve within 0.5 mm from the lesion site, but had few colocalization in axons. In addition, the interaction between PSD-95 and nNOS enhanced significantly at 2 weeks after SNC. These results suggest a correlation of PSD-95 up-regulation with nNOS in reactive SCs of crushed sciatic nerve, which may lead to understanding the function of PSD-95 during peripheral nerve regeneration. Shangfeng Gao and Min Fei contributed equally to this work.     

The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein.

In CNS synapses, the synaptic junctional complex with associated postsynaptic density is presumed to contain proteins responsible for adhesion between pre- and postsynaptic membranes and for postsynaptic signal transduction. We have found that a prominent, brain-specific protein (PSD-95) enriched in the postsynaptic density fraction from rat brain is highly similar to the Drosophila lethal(1)discs-large-1 (dlg) tumor suppressor protein. The dlg protein is associated with septate junctions in developing flies and contains a guanylate kinase domain that is required for normal control of cell division. The sequence similarity between dlg and PSD-95 suggests that molecular mechanisms critical for growth control in developing organisms may also regulate synapse formation, stabilization, or function in the adult brain.     

Formation of nNOS/PSD-95 PDZ dimer requires a preformed beta-finger structure from the nNOS PDZ domain

PDZ domains are modular protein units that play important roles in organizing signal transduction complexes. PDZ domains mediate interactions with both C-terminal peptide ligands and other PDZ domains. Here, we used PDZ domains from neuronal nitric oxide synthase (nNOS) and postsynaptic density protein-95 (PSD-95) to explore the mechanism for PDZ-dimer formation. The nNOS PDZ domain terminates with a approximately 30 residue amino acid beta-finger peptide that is shown to be required for nNOS/PSD-95 PDZ dimer formation. In addition, formation of the PDZ dimer requires this beta-finger peptide to be physically anchored to the main body of the canonical nNOS PDZ domain. A buried salt bridge between the beta-finger and the PDZ domain induces and stabilizes the beta-hairpin structure of the nNOS PDZ domain. In apo-nNOS, the beta-finger peptide is partially flexible and adopts a transient beta-strand like structure that is stabilized upon PDZ dimer formation. The flexibility of the NOS PDZ beta-finger is likely to play a critical role in supporting the formation of nNOS/PSD-95 complex. The experimental data also suggest that nNOS PDZ and the second PDZ domain of PSD-95 form a "head-to-tail" dimer similar to the nNOS/syntrophin complex characterized by X-ray crystallography.