Effect Of Mitochondrial Calcium Uniporter On Autophagy And Mitophagy In SH-SY5Y Cells

Cerebral ischemia/reperfusion(I/R)injury can cause neurons damage,leading to apoptosis or necrosis,causing ischemic brain tissue neurological dysfunction.Autophagy is a process for the degradation of cytosolic components and organelles through their delivery to the lysosomes.It is a fundamental cellular process promoting cells survival under various environmental stress conditions.As the crucial organelles for energy production and the major source of reactive oxygen species(ROS)in living cells,mitochondria are also the principal targets of ischemia/reperfusion(I/R)injury.I/R injury causes ROS overproduction,cellular components damage and triggers mitochondrial dysfunction.The damaged mitochondria are selectively eliminated by the process of mitophagy.Thus,mitophagy plays an important role in mitochondrial quality control and cell survival,and it is also essential for the cells to against I/R injury by the timely elimination of dysfunctional mitochondria.Many studies indicated that both defective and excessive mitophagy are linked to cell death.The clearance of damaged mitochondria via mitophagy is beneficial for neuron survival,while excessive autophagy can result in removal of too many essential organelles,such as mitochondria,and contribute to the autophagic cell death.Mitochondrial calcium uniporter(MCU)is the most important channel responsible for Ca2+ influx into mitochondria.Ca2+ signal plays a potential role in modulating and/or triggering mitophagy.It has been proved that MCU can regulate the mitochondrial permeability transition pore(MPTP)in isolated cortical mitochondria.MPTP also plays an important role in the induction of mitophagy.Furthermore,mitochondria are dynamic organelles and form a dynamic reticulum that is continuously remodeled by fusion and fission events.Fission can produce metabolically different daughter units.The daughter mitochondria with healthier membrane potential will continue to participate in fusion and fission cycles,whereas the depolarized daughter mitochondria are unlikely to undergo fusion and degraded through mitophagy.Hence,mitochondrial fission may be the upstream event and essential for mitophagy.Interestingly,MCU can regulate the process of mitochondrial fission and fusion.Inhibition of mitochondrial fission or induction of fusion can abolish mitophagy.Mdivi-1 can attenuate cell apoptosis,upregulated Bcl-2expression and down-regulated Drp1,Bax as well as Cytochrome C expression and protected brain from I/R by suppressing the ROS initiated mitochondrial pathway.As there is a complex association between apoptosis and autophagy,even though the exact role of MCU in modulating mitophagy remains unclear,we assume that MCU may have a tight relationship with mitophagy and contribute to the final fate of neurons.Objectives: The present study is aimed to explore the effect of I/R injury on neurons autophagy and mitophagy,and to explore the effect of MCU on I/R induced autophagy and mitophagy.Methods: This study constructed an in vitro I/R model by subjecting oxygen and glucose deprivation/reperfusion(OGD/RP)model to SH-SY5 Y cells to mimic the cerebral I/R injury.Cells were randomly divided into 6 groups: Control group: cells were cultured in normal condition without any treatment;OGD/RP group: cells were treated with OGD for 6 h and then returned to normoxic conditions for 24 h;OGD/RP + RU360group: cells were pretreated with RU360(10 μM)for 30 min before OGD and then returned to normoxic conditions for 24 h;OGD/RP + Sper group: cells were pretreated with spermine(10 μM)for 30 min before OGD and then returned to normoxic conditions for 24 h;RU360 group: cells were pretreated with RU360(10 μM)for 30 min and then cultured without drug for 24 h;Sper group: cells were pretreated with spermine(10 μM)for 30 min and then cultured without drug for 24 h.Cell viability was assayed by using cell counting kit-8(cck-8).MMP assay kit with JC-1 was used to detect MMP.Transmission electron microscopy(TEM)examination was used to observe OGD/RP induced formation of autophagosomes and ultrastructural changes of cell organelles.Western blot was used to detect the expression of Beclin-1,P62 and Tom20 in different groups.Statistical analysis was performed of SPSS 18.0 statistics package.Data were expressed as means ± SD(x±s).One-way ANOVA was applied for multiple comparisons.p <0.05 was considered statistically significant.Results: Cell viability assay: Cell viability was significantly lower in OGD/RP group,OGD/RP + RU360 group and OGD/RP + Sper group compared with control group(p﹤0.001),no significant difference was observed in RU360 and Sper group(p﹥0.05);Cell viability in OGD/RP + RU360 group was higher,compared with OGD/RP group(p﹤0.001).TEM examination: After OGD/RP treatment,numerous of autophagic vacuoles(AVs)with characteristic morphological features of autophagosomes weredetected;In addition,some partially degraded mitochondria surrounding by double membranes of typical autophagosomes were observed;The number of mitochondria was also reduced compared with control group;Clear cristae could still be observed and the amount of AVs was decreased obviously in OGD/RP + RU360 group,which showed similar mitochondrial morphology compared with control group.MMP assay: The MMP was significantly lower in OGD/RP group,OGD/RP + RU360 group and OGD/RP + Sper group compared with control group(p﹤0.001),no significant difference was observed in RU360 and Sper group(p﹥0.05);The MMP of OGD/RP + RU360 group was higher than that of OGD/RP group.Western blot showed that the expression of Beclin-1 was increased and the expressions of P62 and Tom20 were decreased in OGD/RP group,OGD/RP + RU360 group and OGD/RP + Sper group compared with control group;the expressions of these proteins in OGD/RP + Sper group were similar to those in OGD/RP group;the expression of P62 and Tom20 were increased and the expression of Beclin-1was decreased in OGD/RP + RU360 group compared with OGD/RP group.Conclusion: 1.OGD/RP injury induced autophagy and mitophagy in SH-SY5 Y cells.2.MCU can modulate OGD/RP induced autophagy and mitophagy in SH-SY5 Y cells.3.Inhibition of MCU can inhibit excessive autophagy and mitophagy by maintaining mitochondrial morphology,MMP and function,thus protecting SH-SY5 Y cells from OGD/RP injury.