The Roles Of Homer1a In Regulating Endoplasmic Reticulum And Mitochondria Functions After Neural Ischemic Reperfusion Injury

[Background]As the most common subtype of cerebral stroke,ischemic stroke(IS)has substantially high morbidity and mortality,thus makes it one of the top three health-killers worldwide.Neuroprotection after IS ictus prevails that researches now focus on many potential targets including excitatory neurotransimitter,calcium overload,free radical injury,apoptosis and inflammation.Yet the prognosis of IS patients still showed little improvement.Recently,the endoplasmic reticulum(ER)and mitochondria are found to contribute to cell apoptosis via regulating certain pathways in ischemia core.The ER-mitochondria pathways also can regulate neuron death via unfolded protein response(UPR)and calcium overload in the ischemic penumbra.Hence,the ER-mitochondria signal dysfunction may plays key roles in post-IS neural injury and reserches focus on it may shed new light on post-ischemia neuroprotection research.Nevertheless,the key molecules involved in ERmitochondria functions are still unclear.Homer protein is a scaffold protein that usually expresses on Postsynaptic density(PSD).As the most common subtype,Homer1 expresses largely on excitatory synapses and involved in signal conduction,synaptogenesis and receptor aggregation.There are two subtypes of Homer1:Homerla and Homer1b/c.Homerla lacks C-terminal amino-domain,thus plays as a negative regulator of Homer1b/c.Our previous researches showed that,in different models,Homerla participates in mitochondria function and mitochondrial autophage as well as influences the ER-related-signal CHOP expression,indicate that Homerla may plays important roles in regulating ER-mitochondria function after IS.[Objectives]1.To testify the roles of Homerla after neural ischemic reperfusion injury.2.To elucidate the changes of ER and mitochondrial functions as well as the regulatory roles of Homerla on ER and mitochondrial after neural ischemic reperfusion injury.3.To further explore the molecular mechanisms of Homer1a’s effects on ER-mitochondria after neural ischemic reperfusion injury.[Methods]1.C57BL/6 mice were used to induce in vivo ischemic reperfusion injury via middle cerebral artery(MCA)occlusion&reperfusion model.Primary cortical neurons were cultured to induce in vitro ischemic reperfusion injury via Oxy-glucose deprivation&reperfusion(OGDr)model.2.Lactate dehydrogenase(LDH)release assay and cell viability test were used to assess the cell injury.3.ProteoExtract Cytosol/Mitochondria Fractionation kit was used to extract cytosol and mito-protein respectively.4.Western blot(WB)and immunofluorescence were wused to detect the expression of target molecules.5.Cortical neuronal apoptosis was evaluated by Terminal dUTP Nick end labeling(TUNEL)and Caspase-9 activation level.6.The level of reactive oxygen species(ROS)in cortial neurons was detected by 2,7dichlorodihydrofluorescein diacetate(H2DCFDA)assay.7.The calcium load level in cortial neurons was detected using Rhod-2AM probe.8.Mitochondrial membrane potential was evaluated by rhodamine 123 staining and mitochondrial energy metabolism was measured by adenosine triphosphate(ATP)assay.9.Specific lentivirus transfection was used to up-regulate the Homer la expression in neurons.10.Pathway-specific drugs were used to inhibit/activate target pathways.[Results]Section one:After the ischemic reperfusion injury,the Homer la exhibited expression upregulation mainly in cortical neurons.In primary cortical neurons after OGDr injry,the Homer la expression upregulation occured in hours and lasted at least 48 hours.Overexpression of Homerla via lentivirus alleviated the morphological changes,damage and death in neurons induced by OGDr injury.Section two:After the ischemic reperfusion injury in mice,neurons showed endoplasmic reticulum stress(ERS)activation,mitochondrial dysfunction and malfunctional ERmitochondrial interaction.The experiment in vitro showed similar results and Homerla overexpression attenuated ERS activity and partially reversed the mitochondria dysfunction.Section three:In primary cortical neurons after OGDr injury,Homerla relieved mitochondria dysfuntion by inhibiting ERS activity,and the neuroprotective effects were achieved mainly via inhibiting PERK pathway in ERS.[Conclusion]These studies find that Homerla upregulates in neurons after ischemic reperfusion injury and plays endogenous protective roles.In addition,our studies showed that ERS activation plays pivotal roles in neuron mitochondria dysfunction after ischemic reperfusion injury,and Homer la overexpression can inhibits ERS thus protects mitochondria functions.These results indicated a novel intervention focus on ER-mitochondria function that may be promising targets of treatment for neural ischemic reperfusion injury.