突触后致密物-95研究新进展

突触后致密物-95(PSD-95)是膜相关的鸟苷酸激酶(MAGUK)家族的一员[1],是突触的支架蛋白与复杂的蛋白一蛋白作用区.PSD-95的结构中包含了3个PDZ区,一个SH3区或WW基序(两个保守的色氨酸残基)和一个同源的鸟苷酸(gGK)区,其中PDZ结构域在不同膜蛋白中具有不同结构.PSD-95在突触部位发挥较多功能,其中最重要的功能是与膜蛋白相互作用,调节它们在突触中的定位.本文仅对PSD-95在生理和病理条件下作用的某些新进展综述如下.     

突触后致密区蛋白-95与突触可塑性研究进展

<正>突触是神经元之间的连接部位,生物信号通过突触前膜经突触传递到突触后膜。突触数量和功效的改变可引起突触可塑性的改变~(〔1〕)。突触可塑性是学习和记忆的神经基础。突触后致密区(PSD)是突触后信号转导和整合的结构基础,而PSD-95是新近在谷氨酸能突触的PSD中发现的一种特殊蛋白质~(〔2〕),能够整合N-甲基-D-天冬氨酸(NMDA)受体信号。该蛋白与突触可塑性密切相关。本文就PSD-95与突触可塑性研究     

APP5肽类似物P165对糖尿病小鼠海马突触蛋白的影响

目的探讨β-淀粉样前体蛋白（APP）5肽类似物P165对糖尿病小鼠海马神经元α-突触核蛋白（α-Syn）、Shank1和突触后致密物质95（PSD-95）的影响。方法链脲佐菌素腹腔注射诱发DM小鼠模型，P165灌胃治疗。5周后，行灌注固定，海马CA1区α-Syn、Shank1和PSD-95免疫组织化学染色。结果与对照组相比，糖尿病（DM）组α-Syn阳性细胞增多（P〈0.01），胞浆突起深染，Shank1和PSD-95阳性细胞减少，染色浅（P〈0.01）；与DM组相比，P165治疗组α-Syn阳性细胞减少（P〈0.05），胞浆突起染色变浅；Shank1和PSD-95阳性细胞增多（P〈0.05），胞浆突起深染。结论糖尿病小鼠海马神经元存在突触蛋白表达的异常，P165可改善DM小鼠海马神经元突触蛋白的表达。     

CaMKⅡ参与内脏痛觉调节的机制

Ca^2＋/钙调蛋白依赖的蛋白激酶Ⅱ（CaMKⅡ）广泛分布于神经系统．是突触后致密物（PSD）的主要构成蛋白之一。CaMKⅡ因其特殊的自身磷酸化机制，可形成突触后膜长时程增强效应（LTP）．在中枢神经系统突触可塑性变化中发挥重要作用。近年研究发现CaMKII尚参与痛觉过敏的形成，且在功能性胃肠病的发病中亦发挥重要作用。本文就CaMKⅡ的独特结构、自身磷酸化机制及其与内脏高敏感的关系作一综述。     

红景天对阿尔茨海默病模型大鼠行为学及海马区突触相关蛋白的影响

目的探讨红景天对阿尔茨海默病(AD)模型大鼠行为学能力及海马组织突触后致密蛋白(PSD-95)、shank-1蛋白表达的影响及其治疗AD的可能作用机制。方法采取腹腔注射D-半乳糖+灌胃AlCl3+一次性注射东莨菪碱法制备AD大鼠模型,用红景天采取灌胃方式对实验大鼠进行干预,并以喜得镇作为对照,观察其干预效果。用Morris水迷宫法检测大鼠学习记忆能力,采用免疫组化法检测大鼠海马区PSD-95、shank-1蛋白水平的变化。结果模型组与空白组相比,学习记忆能力明显减退(P<0.01),PSD-95、shank-1蛋白水平下降(P<0.01);中药组与模型组相比,学习记忆能力显著改善(P<0.01),PSD-95、shank-1蛋白水平提高(P<0.01);中药组和西药组相比,差异无统计学意义(P>0.05)。结论红景天明显增高AD大鼠海马组织中PSD-95、shank-1蛋白水平,从而影响突触可塑性,这可能是其改善AD大鼠学习记忆功能的作用机制之一。     

突触可塑性在肠道感染大鼠模型内脏高敏感形成中的意义

目的 研究旋毛虫感染SD大鼠后内脏敏感性变化和突触可塑性在内脏高敏感中的作用.方法 30只雄性SD大鼠均分为正常对照组、急性感染组(旋毛虫感染2周)和慢性感染组(旋毛虫感染8周).用不同结直肠压力[20、40、60和80 mmHg(1 mmHg=0.133 kPa)]扩张10 S内诱发的腹壁肌电活动形成的曲线下面积(AUC)评估内脏敏感性.观察感染后不同时期结肠组织病理学变化.采用透射电镜观察结肠突触超微结构,RT-PCR和Western印迹法检测回盲部、近端结肠和远端结肠突触素和突触后致密物质(PSD)-95 mRNA和蛋白质水平的表达.结果 ①在40和60 mmHg的扩张压力下,慢性感染组AUC较正常对照组显著升高(P值分别=0.012和0.005);而急性感染组AUC较正常对照组显著降低(P值分别=0.018和0.012).②组织病理学评分结果 显示,急性感染组的炎症积分为23.45±4.10,较正常对照组(9.10±2.42,P=0.027)显著升高;而慢性感染组为13.95±7.96,与正常对照组差异无统计学意义(P=0.78).③急性感染组可见突触前膜线粒体嵴消失,线粒体肿胀或空泡化,与正常对照组相比囊泡数量显著减少,PSD长度显著减少.慢性感染组中,囊泡性质无明显变化,但数量较正常对照组大鼠明显增加,突触后膜电子致密物增厚,颜色加深,长度延长.三组间突触间隙无明显变化.④与正常对照组相比,慢性感染组大鼠回盲部、近端结肠和远端结肠突触素mRNA和蛋白的表达均显著升高(P值均＜0.05);而急性感染组突触素在mRNA和蛋白质水平的表达降低,但差异无统计学意义(P＞0.05).⑤与正常对照组相比,慢性感染组大鼠回盲部、近端结肠和远端结肠PSD-95mRNA和蛋白的表达均显著升高(P值均＜0.05);而急性感染组各部位PSD-95 mRNA和蛋白的表达显著降低(P值均＜0.05).结论 肠道感染大鼠模型内脏高敏感性的形成与突触可塑性有关.     

雷公藤内酯醇对慢性脑缺血模型大鼠海马突触素和突触后致密物95的影响

目的探讨雷公藤内酯醇对慢性脑缺血(CCI)模型大鼠海马神经元突触素(SYN)和突触后致密物95(PSD-95)表达的影响。方法取30只雄性SD大鼠随机分成对照组、模型组、治疗组,每组10只。模型组和治疗组采用双侧颈总动脉永久性结扎法(2-VO)建立CCI模型。对照组大鼠只暴露双侧颈总动脉。治疗组大鼠每日腹腔注射雷公藤内酯醇0.4 mg/kg,连续注射28 d。采用免疫组织化学法和RT-PCR法检测各组大鼠海马SYN和PSD-95蛋白及mRNA的表达。结果免疫组织化学染色结果显示,治疗组大鼠海马CA1区分子层SYN阳性产物数量和平均光密度明显高于模型组;治疗组大鼠海马CA1区锥体层PSD-95阳性细胞数和平均光密度值明显高于模型组。RT-PCR结果显示,治疗组大鼠海马SYN和PSD-95 mRNA含量较模型组增加。结论雷公藤内酯醇能促进大鼠海马SYN和PSD-95蛋白及mRNA的表达。     

补阳还五汤和星蒌承气汤对脑缺血大鼠神经元突触重塑及胶质源性神经营养因子和神经生长因子表达的影响

目的观察补阳还五汤和星蒌承气汤对脑缺血大鼠神经元突触重塑的作用及其对胶质源性神经营养因子(GDNF)、神经生长因子(NGF)和神经生长相关蛋白-43(GAP-43)、突触后致密物-95(PSD-95)表达变化的影响。方法 120只大鼠随机分为假手术组、模型组、尼莫地平组、星蒌承气汤和补阳还五汤组;线栓法制备大脑中动脉阻塞模型;灌胃用药并分别于14 d、28 d取材;电子透射电镜观察神经元突触变化,免疫组织化学法检测GAP-43、PSD-95、NGF、GDNF表达变化。结果与假手术组比较,14 d模型组GDNF表达明显减弱(P0.01),28 d模型组GAP-43表达明显减弱;与模型组比较,补阳还五汤14 d和28 d组GAP-43、PSD-95、NGF、GDNF表达均明显增强(P均0.05),14 d星蒌承气汤组NGF表达显著增强(P0.01),28 d星蒌承气汤组GDNF表达显著增强(P0.01);与尼莫地平比较,14 d补阳还五汤组PSD-95、NGF、GDNF表达均明显增强(P均0.05),28 d补阳还五汤组PSD-95表达显著增强(P0.05);与14 d比较,28 d补阳还五汤组PSD-95表达显著增强(P0.01)。结论补阳还五汤可明显促进缺血后神经元突触重塑,其机制可能是通过上调缺血脑组织中NGF、GDNF的表达从而使GAP-43、PSD-95表达增加而实现。星蒌承气汤可以上调缺血脑组织早期NGF表达而其远期作用不明显。     

α7神经型尼古丁受体激动剂PNU282987对小鼠海马组织突触相关蛋白的影响

目的探讨特异性激动小鼠α7神经型尼古丁受体(α7n AChR)水平对海马组织中突触相关蛋白表达的影响。方法选取6月龄非转基因C57雄性小鼠32只,随机分为对照组(Control),腹腔注射0.5、1.0 mg/kg、3.0 mg/kg PNU282987组各8只。采用Real-time PCR法和蛋白免疫印迹(Western印迹)法分别测定小鼠海马组织中囊泡相关蛋白突触素(Syn)、突触小体相关蛋白(SNAP)-25和突触后致密物(PSD)-95 mRNA及蛋白表达水平的变化。结果与对照组相比,1.0 mg/kg PNU282987组、3.0 mg/kg PNU282987组中突触相关蛋白Syn、PSD-95、SNAP-25的mRNA和蛋白表达水平均显著增高。结论特异性激动小鼠α7n AChR能够使海马组织中突触相关蛋白水平表达增加。这可能提示了α7n AChR与突触密切相关,这可能与其神经保护作用有关。     

Tau蛋白异常修饰及其相关调控机制的研究进展

Tau蛋白由位于17号染色体上的MAPT基因编码,最初于1975年与微管蛋白共纯化出来,作为一种微管结合蛋白,在体外有促进微管组装的作用[1].后续研究发现tau蛋白在中枢神经系统神经元内高度表达,调节微管的稳定和组装[2].突触前、后位点的运输对突触功能来说是很关键的,包含了线粒体、突触囊泡的组分和质膜、离子通道、受体和支架蛋白.     

The neuregulin receptor ErbB-4 interacts with PDZ-containing proteins at neuronal synapses

Neuregulins regulate the expression of ligand- and voltage-gated channels in neurons and skeletal muscle by the activation of their cognate tyrosine kinase receptors, ErbB 1-4. The subcellular distribution and mechanisms that regulate the localization of ErbB receptors are unknown. We have found that ErbB receptors are present in brain subcellular fractions enriched for postsynaptic densities (PSD). The ErbB-4 receptor is unique among the ErbB proteins because its C-terminal tail (T-V-V) conforms to a sequence that binds to a protein motif known as the PDZ domain. Using the yeast two-hybrid system, we found that the C-terminal region of ErbB-4 interacts with the three related membrane-associated guanylate kinases (MAGUKs) PSD-95/SAP90, PSD-93/chapsyn-110, and SAP 102, which harbor three PDZ domains, as well as with beta(2)-syntrophin, which has a single PDZ domain. As with N-methyl-D-aspartate (NMDA) receptors, ErbB4 interacts with the first two PDZ domains of PSD-95. Using coimmunoprecipitation assays, we confirmed the direct interactions between ErbB-4 and PSD-95 in transfected heterologous cells, as well as in vivo, where both proteins are coimmunoprecipitated from brain lysates. Moreover, evidence for colocalization of these proteins was also observed by immunofluorescence in cultured hippocampal neurons. ErbB-4 colocalizes with PSD-95 and NMDA receptors at a subset of excitatory synapses apposed to synaptophysin-positive presynaptic terminals. The capacity of ErbB receptors to interact with PDZ-domain proteins at cell junctions is conserved from invertebrates to mammals. As discussed, the interactions found between receptor tyrosine kinases and MAGUKs at neuronal synapses may have important implications for activity-dependent plasticity.     

PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density

The postsynaptic density (PSD)-95 family of membrane-associated guanylate kinases (MAGUKs) are major scaffolding proteins at the PSD in glutamatergic excitatory synapses, where they maintain and modulate synaptic strength. How MAGUKs underlie synaptic strength at the molecular level is still not well understood. Here, we explore the structural and functional roles of MAGUKs at hippocampal excitatory synapses by simultaneous knocking down PSD-95, PSD-93, and synapse-associated protein (SAP)102 and combining electrophysiology and transmission electron microscopic (TEM) tomography imaging to analyze the resulting changes. Acute MAGUK knockdown greatly reduces synaptic transmission mediated by α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) and N-methyl-d-aspartate receptors (NMDARs). This knockdown leads to a significant rise in the number of silent synapses, diminishes the size of PSDs without changes in pre- or postsynaptic membrane, and depletes the number of membrane-associated PSD-95–like vertical filaments and transmembrane structures, identified as AMPARs and NMDARs by EM tomography. The differential distribution of these receptor-like structures and dependence of their abundance on PSD size matches that of AMPARs and NMDARs in the hippocampal synapses. The loss of these structures following MAGUK knockdown tracks the reduction in postsynaptic AMPAR and NMDAR transmission, confirming the structural identities of these two types of receptors. These results demonstrate that MAGUKs are required for anchoring both types of glutamate receptors at the PSD and are consistent with a structural model where MAGUKs, corresponding to membrane-associated vertical filaments, are the essential structural proteins that anchor and organize both types of glutamate receptors and govern the overall molecular organization of the PSD.The postsynaptic density (PSD) at excitatory glutamatergic synapses, appearing in electron micrographs as a prominent electron-dense thickening lining the postsynaptic membrane (1) is a complex macromolecular machine positioned across from synaptic vesicle release sites at the presynaptic active zone. The PSD clusters and organizes neurotransmitter receptors and signaling molecules at the postsynaptic membrane, transmits and processes synaptic signals, and can undergo structural changes to encode and store information (2–5). Two types of ionotropic glutamate receptors, AMPA receptors (AMPARs) and NMDA receptors (NMDARs), present at PSDs of excitatory synapses (6–10) mediate almost all synaptic transmission in the brain (11). Biochemistry and mass spectrometry of the detergent-extracted cellular fraction of PSDs have additionally identified many proteins associated with AMPAR and NMDAR complexes (12, 13).The membrane-associated guanylate kinases (MAGUKs), a class of abundant scaffold proteins consisting of PSD-95, PSD-93, synapse-associated protein (SAP)102, and SAP97, interact directly with NMDARs (14–18). These MAGUK proteins share conserved modular structures consisting of three PDZ domains (19, 20) and one SH3-GK supermodule (21). PDZ domains of MAGUKs bind to a conserved motif at the extreme C-terminal region of GluN2 subunits of NMDARs (16, 22). PSD-95 controls the number of AMPARs at the PSD through interactions with auxiliary proteins, such as Stargazin/TARPs in complex with AMPARs (23–25). Single-particle tracking of AMPARs provides evidence that AMPAR/Stargazin complexes are stabilized by PSD-95 at the membrane (26), where PSD-95 is thought to provide hot spots for accumulating AMPARs at synapses (27, 28). Germ-line knockout of PSD-95 reduces AMPAR transmission with little effects on NMDARs (29), whereas acute loss of single members of the MAGUK family decreases primarily AMPAR-mediated synaptic transmission (30–32), and removal of multiple MAGUKs results in greater losses of transmission mediated by both AMPARs and NMDARs (30).PSD-95 and PSD-93 include N-terminal palmitoylation sites that enable PSD-95 and PSD-93 to associate with membrane lipids. N-terminal palmitoylation of PSD-95 is necessary for its synaptic localization, clustering of receptors (33–35), and stability at the PSD (36). PSD-95 palmitoylation regulates synaptic strength by controlling the accumulation of AMPARs at the PSD (35). Consistent with these results, a recent immunogold electron microscopy (immuno-EM) mapping of the positions of the two ends of the PSD-95 molecule at the PSD shows that its N terminus is located at the membrane, whereas its C terminus is farther away from the membrane in a relatively extended configuration, where it is vertically oriented with respect to the membrane (3, 4, 37). In contrast, neither SAP102 nor SAP97 has palmitoylation sites. SAP97 contains a L27 domain at the N terminus (31, 38), which might be involved in self-association, and has a role in sorting and trafficking of AMPARs and NMDARs (39) but is not required for basal synaptic transmission (40).The MAGUK family proteins interact with a host of other proteins in the PSD, such as GKAP (41, 42), which binds to the GK domain of the MAGUKs, whereas GKAPs in turn bind Shank and Homer (43–45). Both Shank and Homer can interact with actin-associated proteins, thus indirectly linking the core PSD structure to the actin system prevalent in the cytoplasm of dendritic spines (45). MAGUKs interact with signaling complexes such as AKAPs (46, 47), K channels (48), and postsynaptic adhesion molecules such as neuroligin (49, 50). With an average density of 300–400 molecules per PSD (51, 52), the MAGUKs outnumber glutamate receptors by a significant margin. With so many potential binding partners, the MAGUKs are positioned to play an important role in organizing glutamate receptors as well as other scaffolding and signaling molecules at the PSD (53).We have examined the consequences of knocking down three key MAGUKs on excitatory synaptic transmission and found an ∼80% reduction in both AMPAR and NMDAR synaptic responses (54). Interestingly, despite the rather ubiquitous distribution of MAGUKs at excitatory synapses, the reduction in synaptic AMPAR-mediated transmission appeared to be attributable primarily to an all-or-none loss of functional synapses. We present evidence that after the knockdown, there is an initial uniform decrease in AMPARs across all synapses, but over a 4-d period, a consolidation process in which a “winner-take-all” phenomenon occurs (54).Here, we have used EM tomography (3, 4) to study the structural effects of knocking down the three key MAGUKs at the PSD to develop a molecular model of the organization of the core PSD structure in intact hippocampal spine synapses. PSDs in intact synapses show numerous regularly spaced and membrane-associated vertical filaments containing PSD-95 in extended conformation connecting with NMDAR and AMPAR-type complexes. These vertical structures in turn contact horizontal elements, resulting in a molecular scaffold supporting a core PSD structure (3, 4, 37). Thus, vertical filaments appear to be of crucial importance in sustaining the core PSD structure. Here, we show that knocking down three key synaptic MAGUKs results in a profound loss of vertical filaments and the electron-dense materials manifested by the PSD. The loss of MAGUKs is accompanied by a dramatic loss of both NMDAR- and AMPAR-type structures at the PSD.     

MAGUKs are essential,but redundant,in long-term potentiation

This study presents evidence that the MAGUK family of synaptic scaffolding proteins plays an essential, but redundant, role in long-term potentiation (LTP). The action of PSD-95, but not that of SAP102, requires the binding to the transsynaptic adhesion protein ADAM22, which is required for nanocolumn stabilization. Based on these and previous results, we propose a two-step process in the recruitment of AMPARs during LTP. First, AMPARs, via TARPs, bind to exposed PSD-95 in the PSD. This alone is not adequate to enhance synaptic transmission. Second, the AMPAR/TARP/PSD-95 complex is stabilized in the nanocolumn by binding to ADAM22. A second, ADAM22-independent pathway is proposed for SAP102.

Discovered 50 y ago, long-term potentiation (LTP) remains the most compelling cellular model for learning and memory. It is generally agreed that NMDA receptor (NMDAR)-dependent LTP is mediated primarily by a postsynaptic modification involving the trafficking of the AMPA-type glutamate receptor (AMPAR) (1–4). During the past decade, effort has been focused on the molecular mechanisms underlying both the constitutive and activity-dependent trafficking of these receptors. A family of synaptic scaffolding proteins, referred to as membrane-associated guanylate kinases (MAGUKs) has featured prominently in these studies (5, 6). These proteins contain three PDZ domains, which are involved in protein–protein interactions. The PSD-95 family of synaptic MAGUKs include PSD-95, PSD-93, and SAP102 and are highly expressed at excitatory synapses (5, 7). In terms of AMPAR basal synaptic trafficking, all MAGUKs appear to play overlapping roles (8). Most research has focused on PSD-95. Overexpressing PSD-95 causes a roughly threefold enhancement in AMPAR excitatory postsynaptic currents (EPSCs) with no change in the NMDAR EPSC (9–13). The enhancement mimics LTP, especially with its selective effect on AMPAR EPSCs (as reviewed in ref. 3). Furthermore, PSD-95 occludes LTP, suggesting a common mechanism (9, 12). This suggests that PSD-95 is an essential step in LTP. However, LTP remains intact in cells lacking PSD-95 (14–16), raising the possibility that PSD-93 or SAP102 may play redundant roles.PSD-95 binds to many synaptic proteins (17, 18) including transsynaptic cell-adhesion proteins (e.g., neuroligins, LRRTMs) (19–24). Of particular interest is ADAM22, a member of a large family of catalytically inactive metalloproteases (25, 26), which, via its binding to the secreted protein LGI1, governs transsynaptic nanoalignment. Deleting either ADAM22 (27) or LGI1 (28) reduces AMPAR synaptic transmission. Critical for ADAM22’s function is the presence of a PDZ binding motif (PBM) at the cytoplasmic C terminus. Thus, expressing a mutated form of ADAM22, which lacks the PBM (ADAM22ΔC5) fails to rescue the defect, resulting from the deletion of ADAM22 (27). Furthermore, AMPAR responses are depressed in ADAM22^ΔC5/ΔC5 knockin (KI) mice (29). Previous results found that the typical enhancement in AMPAR responses seen with the overexpression of PSD-95 or the depression observed with the knockdown (KD) of PSD-95 is absent in LGI1 knockout (KO) mice (27). Interestingly, the depression observed with the KD of SAP102 remained intact (27). Complimentary results are seen with ADAM22^ΔC5/ΔC5 KI mice (29) where overexpression of PSD-95 failed to enhance synaptic transmission. Surprisingly, LTP was intact in these mice. What could account for the dissociation of the enhancing action of PSD-95 and LTP?Here we show an essential role for MAGUKs in both basal synaptic transmission and in LTP. Any one of the MAGUKs can substitute for each other. However, coexpression of the MAGUKs suggests differences in their action. The synaptic enhancement seen with the coexpression of PSD-95 and PSD-93 is no greater than the enhancement observed when singly expressed. In contrast the enhancement seen with the coexpression of PSD-95 and SAP102 is additive. In addition, when MAGUK binding to ADAM22 is eliminated in ADAM22^ΔC5/ΔC5 KI mice, PSD-95 is no longer functional, but the action of SAP102 remains intact. Finally, KD of SAP102 in ADAM22^ΔC5/ΔC5 KI mice abolishes LTP. Based on these results we propose a model in which the AMPAR/TARP/PSD-95 complex binds to ADAM22, a transsynaptic adhesion protein essential for nanocolumn stability, which tethers AMPAR receptors in the nanocolumn. An additional pathway involving SAP102 would hold the AMPAR/TARP/SAP102 complex in the nanocolumn by an ADAM22-independent mechanism.     

Intrinsic disorder in the C-terminal domain of the Shaker voltage-activated K+ channel modulates its interaction with scaffold proteins

The interaction of membrane-embedded voltage-activated potassium channels (Kv) with intracellular scaffold proteins, such as the postsynaptic density 95 (PSD-95) protein, is mediated by the channel C-terminal segment. This interaction underlies Kv channel clustering at unique membrane sites and is important for the proper assembly and functioning of the synapse. In the current study, we address the molecular mechanism underlying Kv/PSD-95 interaction. We provide experimental evidence, based on hydrodynamic and spectroscopic analyses, indicating that the isolated C-terminal segment of the archetypical Shaker Kv channel (ShB-C) is a random coil, suggesting that ShB-C belongs to the recently defined class of intrinsically disordered proteins. We show that isolated ShB-C is still able to bind its scaffold protein partner and support protein clustering in vivo, indicating that unfoldedness is compatible with ShB-C activity. Pulldown experiments involving C-terminal chains differing in flexibility or length further demonstrate that intrinsic disorder in the C-terminal segment of the Shaker channel modulates its interaction with the PSD-95 protein. Our results thus suggest that the C-terminal domain of the Shaker Kv channel behaves as an entropic chain and support a "fishing rod" molecular mechanism for Kv channel binding to scaffold proteins. The importance of intrinsically disordered protein segments to the complex processes of synapse assembly, maintenance, and function is discussed.     

Developmental loss of miniature N-methyl-D-aspartate receptor currents in NR2A knockout mice

The N-methyl-d-aspartate (NMDA) glutamate receptor (NMDAR), long implicated in developmental plasticity, shows decay time kinetics that shorten postnatally as NR2A subunits are added to the receptor. Neither the mechanism nor immediate effect of this change is known. We studied developing NMDAR currents by using visual neurons in slices from NR2A knockout (NR2AKO) and WT mice. Both strains show increased dendritic levels of synaptic density scaffolding protein PSD-95 with age. Dendritic levels of NR2A increased at the same time in WT and immunoprecipitated with PSD-95. PSD-95NMDAR binding was significantly decreased in the NR2AKO. Moreover, NMDAR miniature currents (minis) were lost and rise times of NMDAR evoked currents increased in mutant mice. Age-matched WT cells showed NR2A-rich receptors predominating in minis, yet slow NR2B mediated currents persisted in evoked currents. Disrupting photoreceptor activation of retinal ganglion cells eliminated increases in PSD-95 and NR2A in superior collicular dendrites of WT mice and slowed the loss of miniature NMDAR currents in NR2AKOs. These data demonstrate that NMDARs that respond to single quantal events mature faster during development by expressing the NR2A subunit earlier than NMDARs that respond to evoked release. We hypothesize that NR2A-rich NMDARs may be localized to the center of developing synapses by an activity-dependent process that involves the targeting of PSD-95 to the postsynaptic density. Neonatal receptors become restricted to perisynpatic or extrasynaptic sites, where they participate primarily in evoked currents.     

The LGI1–ADAM22 protein complex directs synapse maturation through regulation of PSD-95 function

Synapse development is coordinated by a number of transmembrane and secreted proteins that come together to form synaptic organizing complexes. Whereas a variety of synaptogenic proteins have been characterized, much less is understood about the molecular networks that support the maintenance and functional maturation of nascent synapses. Here, we demonstrate that leucine-rich, glioma-inactivated protein 1 (LGI1), a secreted protein previously shown to modulate synaptic AMPA receptors, is a paracrine signal released from pre- and postsynaptic neurons that acts specifically through a disintegrin and metalloproteinase protein 22 (ADAM22) to set postsynaptic strength. We go on to describe a novel role for ADAM22 in maintaining excitatory synapses through PSD-95/Dlg1/zo-1 (PDZ) domain interactions. Finally, we show that in the absence of LGI1, the mature synapse scaffolding protein PSD-95, but not the immature synapse scaffolding protein SAP102, is unable to modulate synaptic transmission. These results indicate that LGI1 and ADAM22 form an essential synaptic organizing complex that coordinates the maturation of excitatory synapses by regulating the functional incorporation of PSD-95.Proper development of synapses involves recruitment of proteins that establish presynaptic release sites and postsynaptic densities (PSDs), and later coordinate maturation, maintenance, and plasticity of the synapse. In the last decade, a number of transmembrane synaptic adhesion proteins and secreted proteins that initiate and modulate excitatory synapses—termed synaptic organizing proteins—have been identified (1, 2). Whereas the synaptogenic properties of many synaptic organizing proteins have been described in detail (2–11), much less is known about how synaptic organizing proteins regulate synapse maintenance and maturation (1).A key component to the functional maturation of excitatory synapses is the recruitment of AMPA-type glutamate receptors (AMPARs) to the PSD, which is coordinated by PSD-95/Dlg1/zo-1 (PDZ) domain-containing scaffolding proteins (12). Specifically, one family of PDZ proteins, the membrane-associated guanylyl kinases (MAGUKs), is known to determine basal synaptic AMPAR content (13–16). Like synaptic AMPAR content, the expression of different MAGUKs is developmentally regulated; whereas synapse-associated protein 102 (SAP102) is expressed in the early postnatal period and is critical to the function of immature synapses, postsynaptic density proteins 93 and 95 (PSD-93 and PSD-95) are first expressed around postnatal day 10 (P10) and are required for proper function of mature synapses (14, 17). Mice lacking PSD-93 and PSD-95 have synapses with significantly reduced AMPAR content (14), indicating the shift in MAGUK expression is critical to synapse maturation. However, what guides the incorporation of PSD-93 and PSD-95 into developing synapses remains unknown.We recently identified an instructive role for leucine-rich, glioma-inactivated protein 1 (LGI1) in regulating synaptic AMPAR content—application of LGI1 increases and loss of LGI1 decreases synaptic AMPAR localization (18, 19). LGI1 is a secreted protein that is localized to synapses, where it binds to the extracellular domain of the transmembrane a disintegrin and metalloproteinase proteins 11, 22, and 23 (ADAM11, ADAM22, and ADAM23) (19). Notably, LGI1 and ADAM22 are found in complex with the mature MAGUKs, PSD-93 and PSD-95, in vivo (18, 19), and their expression levels follow a similar timeline (17, 18, 20). However, little is known about the neuronal function of ADAM11, ADAM22, and ADAM23, and it is not clear which mediates the function of LGI1 at the synapse. Moreover, the source and destination of secreted LGI1 remains unstudied.Here, we show that LGI1 released from pre- and postsynaptic cells acts in a paracrine fashion to regulate synaptic AMPAR content. We find that the function of LGI1 at the synapse is fully dependent on its interaction with ADAM22, the only ADAM in the LGI1 complex that contains a PDZ-binding motif. ADAM22, in turn, maintains excitatory synapses through PDZ domain interactions. Finally, we demonstrate that PSD-95, but not SAP102, requires LGI1 to function at synapses, indicating that the LGI1–ADAM22 complex directs synapse maturation by controlling the incorporation of the mature MAGUK PSD-95.     

颞叶癫痫大鼠学习记忆障碍与海马区PSD-95表达的关系

目的研究颞叶癫痫大鼠在Morris水迷宫中学习、记忆能力与大鼠海马区PSD-95表达变化的关系。方法随机将40只W istar大鼠分为海人酸（KA）组（28只）和对照组（12只）。KA组采用KA腹腔注射制作颞叶癫痫大鼠模型,根据是否出现自发性再发作（SRS）分SRS组（A组）、无SRS组（B组）;对照组（C组）注射生理盐水。通过Morris水迷宫测验观察各组大鼠注射KA或生理盐水2、6周时的空间学习、记忆能力,采用HE染色观察大鼠海马的组织病理学变化,免疫组化法检测大鼠海马CA1、CA3区PSD-95的表达。结果A组大鼠注射KA或生理盐水6周时海马区未见广泛神经元丢失及胶质增生,偶见局部神经元丢失及胶质增生;B、C组大鼠未见神经元丢失及胶质增生。与A组2周时及B、C组在2、6周时相比,A组6周时的学习、记忆能力明显下降（P〈0.01）,相应海马CA1、CA3区PSD-95表达均明显降低（P〈0.05）。结论颞叶癫痫长期反复发作时海马区PSD-95表达的减少,可能是导致颞叶癫痫大鼠学习、记忆障碍的机制之一。     

PSD-95 and PKC converge in regulating NMDA receptor trafficking and gating

Neuronal NMDA receptors (NMDARs) colocalize with postsynaptic density protein-95 (PSD-95), a putative NMDAR anchoring protein and core component of the PSD, at excitatory synapses. PKC activation and PSD-95 expression each enhance NMDAR channel opening rate and number of functional channels at the cell surface. Here we show in Xenopus oocytes that PSD-95 and PKC potentiate NMDA gating and trafficking in a nonadditive manner. PSD-95 and PKC each enhance NMDA channel activity, with no change in single-channel conductance, reversal potential or mean open time. PSD-95 and PKC each potentiate NMDA channel opening rate (k(beta)) and number of functional channels at the cell surface (N), as indicated by more rapid current decay and enhanced charge transfer in the presence of the open channel blocker MK-801. PSD-95 and PKC each increase NMDAR surface expression, as indicated by immunofluorescence. PKC potentiates NMDA channel function and NMDAR surface expression to the same final absolute values in the absence or presence of PSD-95. Thus, PSD-95 partially occludes PKC potentiation. We further show that Ser-1462, a putative phosphorylation target within the PDZ-binding motif of the NR2A subunit, is required for PSD-95-induced potentiation and partial occlusion of PKC potentiation. Coimmunoprecipitation experiments with cortical neurons in culture indicate that PKC activation promotes assembly of NR2 with NR1, and that the newly assembled NMDARs are not associated with PSD-95. These findings predict that synaptic scaffolding proteins and protein kinases convergently modulate NMDAR gating and trafficking at synaptic sites.