PICK1: Novel Therapeutic Target For Ischemic Brain Injury

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.