Study Of GOLPH3Mediated Golgi Apparatus Stress In Oxygen-Glucose Deprivation/Reoxygenation Model

Objective:It is widely accepted that the oxidative stress is the major cause of neuronal death in brain ischemia. Increasing evidence implicate organelle-dependent initiation of Oxidative stress related cell death is a research area worthy of pursuing. Recently, researchers suggest that the Golgi apparatus like ER and Mitochondria also presents an exciting role in stress sensing of cell death pathways and acts as a common downstream-effecter, which undergoes disassembly and fragmentation during apoptosis in several neurological disorders, and described it by the term of "GA stress" for the first time.Mammalian GOLPH3, also known as GPP34/GMx33/MIDAS, is an exciting new class of Golgi outer membrane protein involved in vesicular trafficking, Golgi architecture maintaining, receptor sorting, protein glycosylation and cell signaling pathways that localize to the cytoplasmic face of the trans-Golgi. We predict the possibility that GOLPH3for its diverse functions closely related with Golgi may exist as a stress-related Golgi-related protein that is mobilized during GA stress response.We have successfully used a cell culture model of oxygen and glucose deprivation following reoxygen (OGD/R) to mimic the ischemia/reperfusion injury in vitro. Our studies will focus on the role of GOLPH3in Golgi stress-sensing and the functional significance of GOLPH3in oxidative stress response of GA during ischemia/reperfusion injury caused by OGD/R model.Experimental Methods:1. We employed a stable OGD/R model to mimic ischemic-like conditions in vitro. The neuronal injury was measured by the morphologic changes studied under Light Microscopes and detection of cell apoptosis;2. Experimental grouping was designed as follows:control group; toxic drug intervention group (Thapsigargin/TG or NMDA); common OGD/R group; OGD/R group with drug intervention (N-acetylcysteine/NAC or4-phenylbutyrate acid/4-PBA). The latter two groups were divided into four different subsets for various reoxygen time points following4hours’OGD (OGD/R4H,12H,24H and48H);3. The protein level of GOLPH3, GRP78, LC3Ⅰ/Ⅱ, p75NTR was determined by western blotting;4. GOLPH3ShRNA were designed and transfected into N2A cells by Lipofectamine2000, GOLPH3RNA level were analyzed by RT-PCR;5. The rates of apoptosis were measured by flow cytometric analysis; 6. The intracellular ROS production was detected by fluorescence method;7. The concentration of BDNF in cell supernatant was analyzed by ELISAkit;8. Confocal Immuno-fluorographs of GOLPH3and DAPI were taken to determine the sub-cellular location of GOLPH3.Result:1. The ROS detection and GRP78expression as well as the morphology change of N2A cells showed that the stress reaction of ER and Mitochondria were strongly induced by OGD/R model and direct injury were brought by severe ischemia/reperfusion;2. The intervention of anti-oxidant drugs (NAC or4-PBA) before OGD/R resulted in significant decrease in ROS production of24H group (P<0.01). While, GRP78expression in4H (P<0.01) or24H (P<0.05) groups was decreased after4-PBA intervention, down-regulation of GRP78was only detected in4H group after NAC intervention;3. The intervention of cytotoxic drugs by Thapsigargin/TG or NMDA significantly increased the ROS production and GRP78expression in N2A cells (P<0.01);4. GOLPH3expression were induced by OGD/R model in a time-dependent manner, increased remarkably in OGD/R groups than in control (P<0.01). The intervention of NAC or4-PBA both decreased the expression of GOLPH3in OGD/R4H group (P<0.05). Correlation analysis indicated that GOLPH3was highly co-related with ROS and GRP78in OGD/R model; Cytotoxic drugs intervention of TG or NMDA induced the highest expression level of GOLPH3(P<0.01)5. Confocal Immuno-fluorographs showed that the GOLPH3labeled Golgi apparatus localized to a compact peri-nuclear ribbon, consisting of multiple vesicle-like structures arranged in close opposition to each other to form stacks. Meanwhile, a little amount of G3staining diffused in the cytoplasm. In contrast, after24-hour reoxygenation following24-hour OGD exposure, the compact vesicle-like structures by G3staining fragmented into punctuate-like structures, dispersed throughout the cytoplasm with increased intensity; TG or NMDA intervention which can induce Golgi Fragmentation here also induced notable sub-cellular apposition change of GOLPH3into intensive fragments;6. RNAi-Mediated Silencing research of GOLPH3by mus-505ShRNA could effectively inhibit the RNA as well as its protein level of GOLPH3in N2A cells;7. RNAi-Mediated Silencing research found out that GOLPH3promote the degradation of p75NTR and decrease the secretion of BDNF by N2A cells in OGD/R24H group (P<0.01);8. A time-dependent elevation of autophagy level after OGD/R injury were detected by LC3II/I ratio, GOLPH3suppression could induce the LC3II/I ratio of OGD/R4H group (P<0.05), but inhibit that of24H group (P<0.01).9. RNAi-Mediated Silencing of GOLPH3research demonstrated that in OGD/R24H group GOLPH3promote ROS production and increase the apoptosis rates of N2A cells, mediate a specific apoptotic pathway of Golgi apparatus Stress (GAS) during oxidative stress (P<0.01);Conclusion:1. GOLPH3is a Golgi-originated stress-related protein; Its expression is closely related with the level of oxidative stress;2. By inhibiting the secretion of BDNF, promoting the degradation of p75NTR, and affecting the formation of stress-related primary autophagosome, GOLPH3mediates Golgi apparatus Stress (GAS) to participate in OGD/R model induced oxidative stress.