【作者】 顾军；

【导师】 陈建国；

【作者基本信息】 华中科技大学 ， 药理学， 2009， 博士

【摘要】 缺血性神经损伤的机制研究中，兴奋性氨基酸细胞毒性扮演了重要角色并且是重要的治疗中风的研究方向之一。以往研究往往更加关注NMDA受体在其中的重要作用，而另一种谷氨酸受体AMPA受体(α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor)在缺血中的作用如今已经受到日益广泛的关注。AMPA受体在中枢神经系统中广泛分布，由GluR1-4四个亚基组成。大量研究表明，缺乏GluR2的AMPA受体能通透包括钙离子在内的多种二价阳离子，参与了多种病理过程的神经损伤。酸敏感离子通道(acid-sensing ion channel，ASIC)是一类由胞外酸化所激活的阳离子通道，属于Degenerin／epithelial Na+channel(DEG／ENaC)家族成员。ASIC广泛存在于哺乳动物中枢神经系统和外周神经系统。ASIC在神经可塑性和疾病发生中表现出重要的作用。现已发现四种基因编码的6种ASIC亚基。其中，ASIC1a主要通透Na+，对Ca2+也有一定的通透性，它可以被乳酸积累和组织酸化所激活，参与了缺血性脑损伤的病理进程。近年来分子生物学的部分研究表明，一种含有PDZ和BAR结构域的膜转运相关蛋白PICK1(protein that interacts with C kinasel)在AMPA受体的上膜、转运和ASIC的膜定位分布过程中扮演了极为重要的关键角色。研究表明，PICK1蛋白和AMPA受体的相互作用介导了AMPA受体GluR2亚基的内化和循环，从而影响到钙信号相关的突触功能如LTP和LTD。PICK1蛋白和ASICs分布有高度的一致性，并可影响ASICs膜定位。考虑到缺血缺氧后钙通透性的AMPA受体在突触后膜定位的增加以及PICK1对于AMPA受体及ASIC膜分布的调节作用，我们推测PICK1可能在缺血性脑损伤过程中扮演了重要角色。最新研究报道认为PICK1介导了OGD-R引起的海马神经细胞兴奋性氨基酸损伤。本研究探索了PICK1在缺血性脑损伤中的作用。第一部分PICK1对局灶性脑缺血中钙通透性AMPA受体表达及分布的影响目的：研究PICK1对局灶性脑缺血中钙通透性AMPA受体表达及分布的影响。方法：用大脑中动脉栓塞(MCAO)的方法建立局灶性脑缺血模型，采用免疫印迹的方法检测AMPA受体的变化和膜表达的变化。用PICK1基因敲除研究PICK1对缺血引起AMPA受体表达及分布的变化的影响。结果：缺血再灌注损伤后3小时，与假手术组相比，皮质和海马GluR2亚基的表达均尚未发生改变。膜蛋白提取和Western Blotting检测表明，在缺血再灌注损伤后3小时，与假手术组相比，模型组皮质和海马GluR2的膜表达减少。通过免疫共沉淀的方法检测缺血对PICK1与GluR2之间的蛋白质相互作用的影响。与假手术组相比，缺血导致了皮质和海马的PICK1和GluR2的结合增加。免疫荧光的结果类似。进一步用PICK1敲除小鼠研究缺血对AMPA受体GluR2亚基表达、膜表达的影响。研究表明，在PICK1敲除的小鼠上GluR2的膜表达与同窝生的野生型小鼠相比无显著的统计学差异，这表明PICK1敲除对GluR2的膜表达并没有明显的影响。而对野生型和敲除鼠同时给予MCAO模型处理后可以观察到和大鼠类似的变化，即GluR2的膜表达降低。而敲除鼠GluR2下降的程度和野生型相比，明显减轻。结论：缺血引起AMPA受体GluR2亚基膜表达的减少；PICK1参与这种膜转运的过程，PICK1敲除能减弱缺血引起的AMPA受体GluR2亚基膜表达的减少。第二部分PICK1对局灶性脑缺血中ASIC1表达和分布的影响目的：研究局灶性脑缺血中ASIC1表达和分布的影响以及PICK1的影响。方法：用大脑中动脉栓塞(MCAO)的方法建立局灶性脑缺血模型，观察ASIC1a在局灶性脑缺血损伤中的表达变化。用PICK1基因敲除研究PICK1在其中的作用。结果：与假手术组动物同侧相比，模型组大鼠右侧(缺血侧)的皮质ASIC1a表达在缺血再灌注损伤后开始上升，在3小时达到顶峰，随后开始下降，24小时仍略高于假手术组，72小时恢复到正常的水平。通过免疫共沉淀的方法检测缺血对PICK1与ASICs之间的蛋白质相互作用的影响。在缺血再灌注的大鼠皮质组织上，与假手术组相比，等量PICK1结合的ASIC1a量有所增加。为进一步研究PICK1在其中的作用，我们利用PICK1基因敲除鼠研究ASIC1a在缺血后的膜表达变化。可以看到，在普通野生型小鼠上，缺血会导致ASIC1a的膜表达升高。而同窝生PICK1敲除鼠同时给与缺血再灌注损伤模型处理后，与野生型小鼠相比，ASIC的膜表达也发生了升高，但其上升的幅度较小。结论：缺血能够引起ASIC1a的膜表达的上调，PICK1参与这种膜表达的变化，PICK1敲除能够减轻缺血引起的ASIC1a的膜表达的增加。第三部分PICK1对OGD-R所致神经元损伤的作用目的：细胞水平研究PICK1对OGD-R所致神经元损伤的作用方法：体外建立氧糖剥夺再灌注模型。敲除PICK1基因和过表达PICK1基因观察OGD-R所致神经元损伤的变化结果：ODR-R引起细胞活性的下降过程中，PICK1敲除的杂合子(PICK1+／-mice)神经元和PICK1敲除纯合子(PICK1-／-mice)神经元的活性下降都显著小于同窝生野生型小鼠的活性下降。在ODR-Rep对细胞LDH释放的影响实验中，PICK1敲除杂合子和纯合子神经元的LDH释放都显著小于野生型小鼠的LDH释放。PICK1敲除可以显著降低OGD-R引起的凋亡蛋白表达。过表达PICK1蛋白则会增加OGD-R引起的神经细胞株损伤。转染空质粒的SH-SY5Y细胞株OGD-R处理后，经IPP软件分析神经细胞死亡比例约为40％；转染PICK1质粒的SH-SY5Y细胞株OGD-R处理后，经IPP软件分析神经细胞死亡比例约为60％，明显加重OGD-R引起的神经细胞死亡。结论：PICK1在OGD-R所致神经元损伤其重要作用，PICK1敲除可以显著降低OGD-R引起的细胞凋亡。过表达PICK1蛋白则会增加OGD-R引起的神经细胞株损伤。第四部分PICK1敲除对局灶性脑缺血损伤的保护作用目的：在整体水平研究PICK1敲除是否对局灶性脑缺血损伤有保护作用方法：取模型组和假手术组的大鼠右侧(缺血侧)的皮质和海马，提取蛋白，通过免疫印迹法观察PICK1在局灶性脑缺血再灌注损伤后的不同时间点表达变化的情况。结果：与假手术组动物同侧相比，模型组大鼠右侧(缺血侧)的皮质PICK1表达在缺血再灌注损伤后开始上升，在3小时达到顶峰，随后开始下降，72小时恢复到正常的水平(n=3)。而在海马观察到类似的结果。与假手术组动物同侧相比，模型组大鼠右侧(缺血侧)的海马PICK1表达在缺血再灌注损伤后开始上升，但在0小时无统计学差异。在3小时上升达到顶峰，随后开始下降，72小时恢复到正常的水平。免疫组化的结果也显示一致的结果：在缺血再灌注后的3小时，PICK1的表达在海马和皮质显著上升，在72小时恢复到正常水平。无论是对缺血24h损伤还是缺血(1 h)再灌注(24 h)损伤，PICK1敲除小鼠都表现出较好的耐受性，和野生型小鼠相比，梗死面积明显减少。其中，缺血(24h)后PICK1敲除杂合子小鼠的梗死面积(64．28±4．30 mm~3)和PICK1敲除纯合子小鼠的梗死面积(42．14±4．13 mm3)都显著小于同窝生野生型小鼠的梗死面积(79．92±4．15 mm~3)。而在缺血(1h)再灌注(24 h)后，PICK1敲除纯合子小鼠的梗死面积(42．14±4．13 mm~3)小于同窝生野生型小鼠的梗死面积(79．92±4．15 mm~3)。~结论：缺血再灌注损伤的过程中，PICK1表达上升；敲除PICK1基因的对缺血再灌注损伤有显著的保护作用。更多还原

【Abstract】 PICK1:Novel Therapeutic Target for Ischemic Brain InjuryExcitotoxic cell injury plays a critical role in cerebral ischemic neuronal death and itrepresents potential treatment strategy in developing effective therapies to rescue braindamages after stroke.Initial studies often focused on NMDA-type glutamate receptors ascritical mediators and the most intensely investigated drug targets for the focal ischemicinjury treatment.Recent studies support a more central role for another major subtype ofglutamate receptors,α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid(AMPA)-type glutamate receptors in global ischemic brain injury.AMPA receptors areassembled in the CNS from GluR1,-2,-3,and-4 subunits and many emerging new studieshave suggested that the reduction of GluR2 subunit expression,which dictates Ca2+permeability of AMPA receptor channels,plays a crucial role in the pathogenesis ofischemic neuronal cell death.ASICs are one of the members of the degenerin/epithelial Na~+channel superfamily (DEG/ENaC),which was activated by a drop of extracellular pH.There are four genes (ASIC1-ASIC4) encoding six subunits in ASICs family have beenidentified to date.Among them,ASIC1a,which was observed to be activated after lacticacid accumulation and tissue pH reduction during ischemic stroke,contributed to ischemicbrain injury.Studies on molecular aspects have revealed that protein interacting with C-kinase1(PICK1),a PDZ and BAR domain-containing trafficking regulatory protein,regulatedGluR2 subunit membrane trafficking and ASICs localization.Interactions between PICK1and AMPARs have regulated the anchoring,insertion and internalization of synapticAMPARs and played an important role in synaptic functions such as LTP and LTD.PICK1interacts specifically with the C-termini of ASICs through its PDZ domain and contirbutedto their localization.Considering the targeting of GluR2-lacking AMPARs to synapses ofhippocAMPA1 neurons was promoted after ischemic insults and the key regulatary functionof PICK1 in the modulation of Ca2+-permeable AMPA receptor and ASICs,PICK1 mayalso play an important role in ischemic cell injury after stroke.A recent study hasdemonstrated that PICK1-mediated GluR2 trafficking contributed to oxygen/glucose deprived induced hippcampus neuron cell death.Here we have investigated theinvolvement of PICK1 after transient cerebral ischemia.PART1 PICK1 contributed to the trafficking ofAMPAR GluR2subunit induced by focal cerebral ischemiaObjective:To investiage the role of PICK1 in the traffficking of AMPAR GluR2subunit induced by focal cerebral ischemia.Methods:We employed MCAO models to test the protein level and the membraneprotein level of GluR2 after ischemia.Then,knock out the PICK1 gene to test the changeof the protein level and the membrane protein level of GluR2 after ischemia.Results:After ischemia lh and following 3h perfusion,the total protein level ofGluR2 subunit in model group was 1.22±0.18(cortex) and 1.42±0.23(hippocampus),which was 1.25±0.12 (cortex) and 1.38±0.23 (hippocampus) in sham group.But themembrane protein level of GluR2 was decreased significantly (P＜0.05) in the model groupversus the sham group.To examine the role of PICK1 in plasma membrane AMPARsfunction after cerebral ischemia,we first investigated the interactions between GluR2 andPICK1 by coimmunoprecipitation after brain ischemia.In our study,we found thatimmunoprecipitation with an antibody to PICK1 resulted in coprecipitation of GluR2protein.The level of coprecipitated GluR2 was significantly increased,assessedimmediately at 3h after brain ischemia both in hippocampus and cortex.Then we alsoexamined the subcellular co-localization of GluR2 subunit and PICK1 alterations aftercerebral ischemia with immunofluorescence.In our experiment,red fluorescence(590 nm)intensity ratio standed for GluR2 expression and green fluorescence(530 nm) intensity ratiostanded for PICK1 expression.After concomitant,we observed a significant incresement inyellow fluorescence at 3 h after cerebral ischemia both in cortex and hippocampus,suggesting that subcellular co-localization of PICK1 and GluR2 subunit increasedsignificantly in these experimental settings and the functional associations between PICK1and AMPA receptors may also be upregulated.In order to further identify the role of PICK1 played in modulation of GluR2 membrane trafficking after ischemia,we tested theplasma membrane GluR2 level after MCAO treatment in wide type and PICK1 knockoutmice.In our current experiment,PICK1 knockout did not affect the plasma membraneGluR2 level in shamed mice.Brain ischemia reduced the plasma membrane level of GluR2both in PICK1+/+ and PICK1-/-mice,indicating that there exsited other molecularmechanisms for GluR2 membrane traffic modulation after ischemia excepted PICK1.Conclusion:PICK1 knockout attenuated the loss of GluR2 plasma membrane levelafter ischemia in mice.PART2 PICK1 contributed to theASIC1 membrane traffickinginduced by focal cerebral ischemiaObjective:To test whether the PICK1 contributed to the ASIC 1 membrane traffickinginduced by focal cerebral ischemia.Methods:We employed MCAO models to test the protein level and the membraneprotein level of ASICs after ischemia.We evaluated ASIC1 a expression in rats at 0h,3h,6h,12h,24h and 72h after MCAO by western blot analysis.Results:The results demonstrated that ASICla expression was significantlyupregulated at 0 and 3h after ischemia with peaks at 3 h after ischemia than sham group incortex.After ischemia 1h and following 3h perfusion,the membrane protein level ofASIC1a protein was increased significantly (P＜0.05) in the model group versus the shamgroup.To examine the role of PICK1 in plasma membrane ASIC1a function after cerebralischemia,we first investigated the interactions between ASIC1a and PICK1 bycoimmunoprecipitation after brain ischemia.In our study,we found thatimmunoprecipitation with an antibody to PICK1 resulted in coprecipitation of ASIC1aprotein.The level of coprecipitated ASIC1a was significantly increased,assessedimmediately at 3h after brain ischemia both in hippocampus and cortex.We also usedPICK1 knockout mice to determine the effects of PICK1 on the alterations of subcellular distribution of ASIC1a after ischemia.In the KO mice,the level of ASIC1a membraneprotein was significantly less than WT mice both in shame group and in model group.Conclusion:PICK1 knockout attenuated the enhance of ASIC1a plasma membranelevel after ischemia in mice.PART3 Effect of PICK1 on OGD-Reperfucion induced neuronalcell injuryObjective:To investigated the effect of PICK1 on OGD-Reperfucion inducedneuronal cell injury.Methods:We employed an in vitro ischemic model of oxygen-glucose deprivationfollowed by reperfusion 24 h (OGD-Rep).We cultured PICK1 knockout cells andgenerated PICK1 gene overexpressing cells.Results:Cortical neurons subjected to OGD-Rep manifested a remarkable decrease ofcell viabilities and cells of PICK1 KO mice exhibited a much more resistant property toischemic damages in the MTT assay and the LDH leakages analyze.The expression of Baxunder OGD 4 h and reperfusion 24 h was also decreased by depleting PICK1 gene afterOGD-Rep.Although PICK1 knocking out protected primary cultured mouse corticalneurons from OGD-Rep insults,we further investigated whether overexpression of PICK1aggravated the cell injury induced by OGD-Reperfucion.We generated PICK1 geneoverexpressing SH-SY5Y cells by plasmid transfection and G418 screen.We used PI die toindicate the cell death after OGD-R and we found SH-SY5Y cells transfection with PICK1were more fragile after OGD-R treatment than SH-SY5Y cells transfection with EGFP-N 1vehicle plasmid.Conclusion:Knockout PICK1 protected neurons from OGD-Rep insults whileoverexpression PICK1 aggravated the cell injury induced by OGD-Reperfucion. PART4 Protective effect of PICK1 knockout on cerebral focalbrain ischemiaObjective:To investigate the protective effect of PICK1 knockout on cerebral focalbrain ischemiaMethods:We employed MCAO models to test the protein level of PICK1 afterischemia and to test.the protective effect of PICK1 knockout on cerebral focal brainischemia.Results:To determine whether PICK1 was involved in ischemic brain injury,weevaluated PICK1 expression in rats at 0h,3h,6h,12h,24h and 72h after MCAO by westernblot analysis.The results demonstrated that PICK1 expression was significantlyupregulated at 0,3 and 6h after ischemia with peaks at 3 h after ischemia than sham groupin cortex.In hippcampus,PICK1 expression was also higher than sham group.Interestingly,PICK1 protein level was significantly downregulated and returned to basal level observedin sham group at 24h after ischemia.In order to confirm PICK1 expression alterations indifferent brain areas after ischemia,we also performed an immunohistochemistryexperiment which showed a similar result.Although some study have demonstrated thatcaspase-dependent and caspase-independent signalling of apoptosis in the penumbra wasobserved only after 4h post-ischemia,the upregulation of PICK1 gene expression at earlytime after ischemia may be qualified as novel candidate mechanism mediating ischemicneuronal injury.To investigate that whether PICK1 gene was involved in ischemic brain injury in vivoaccordingly,we employed PICK1-/-mice to test in the model of focal ischemia.Cerebralischemia was induced by middle cerebral artery occlusion (MCAO) and infarct volume wasdetermined by TTC staining at 2 hr following ischemia or 24h following reperfusion.Aftersubjected to 2 hr MCAO,PICK1+/+ mice displayed significant infarct volumes and thevalue was about 79.92±4.15 mm~3 (n=6).In PICK1+/-mice,it was shown that smallerinfarct volumes with the value of 64.28±4.30 mm~3 (n=6,P＜0.01 vsWT).The smallest infarct volumes were observed in PICK1-/-mice,which was only 42.14±4.13 mm~3 (n=6,P＜0.01 vs wildtype and vs heterozygote).Compared with PICK1+/+ mice,after 24hreperfusion the infarct volumes in PICK1-/-mice were reduced almost 45%.Conclusion:These results have suggested that PICK1 gene participated in ischemicbrain injury and disrupting PICK1 gene expression exhibited significant neuroprotectiveeffects against brain ischemia.更多还原