Mechanism Of DDAH-1 Alleviating Cerebral Ischemia-reperfusion Injury By Increasing Endoplasmic Reticulum-mitochondrial Tethering

Ischemic stroke（IS）is one of the major diseases worldwide characterized by high morbidity,high mortality,and high disability.Although intravenous thrombolysis or endovascular treatment restore blood perfusion in the ischemic area,the secondary ischemia-reperfusion injury is the main cause of neurological deficits in patients.Therefore,it is vital to explore the target that effectively reduces the neuronal injury induced by cerebral ischemia-reperfusion,which is meaningful for the prevention and treatment of IS.Dimethylarginine dimethylaminohydrolase（DDAH）is a highly conserved protein responsible for methylated arginine metabolism.Two isoforms of DDAH exist,DDAH-1 and DDAH-2.DDAH-1 is mainly expressed in the central nervous system,and it has been confirmed that mouse DDAH-1 is mainly distributed in the cerebral cortex,hippocampus and amygdala.DDAH degrades asymmetric dimethylarginine（ADMA）,which competitively inhibits its activity due to its similar structure to nitric oxide synthase（NOS）thereby blocking the synthesis process of nitric oxide（NO）.As the main hydrolytic enzyme of ADMA degradation,DDAH-1 plays a dominant role in regulating ADMA and NO levels.Elevated plasma ADMA levels have been associated with stroke occurrence.Studies have shown that loss of DDAH-1 function due to DDAH-1 gene polymorphisms in the population is closely related to increased risk of thrombosis and stroke.However,the regulatory role and specific mechanism of DDAH-1 in IS are not clear.Neuronal death is the core issue of IS pathological changes,and mitochondrial dysfunction is an early event of neuronal ischemia-reperfusion injury,including mitochondrial calcium overload,mitochondrial membrane potential（MMP）depolarization and mitochondrial respiratory chain complex（MRCC）activity reduction.They lead to the accumulation of reactive oxygen species（ROS）,which further causes the opening of mitochondrial permeability transition pores and the release of pro-apoptotic factors,and finally triggers neuronal apoptosis or necrosis.The maintenance of normal mitochondrial function plays a very important role in neuronal energy metabolism,and the positive correlation between ADMA and mitochondrial dysfunction and oxidative stress status has been demonstrated in multiple pathological changes.Whether DDAH-1 regulates mitochondrial damage via ADMA in IS neurons requires further exploration.Mitochondria tether with the endoplasmic reticulum（ER）through the mitochondria-associated endoplasmic reticulum membrane（MAM）,which is essential for maintaining the normal function and homeostasis of mitochondria.Changes in ER-mitochondrial tethering can mediate the development of stroke,but the advantages and disadvantages of MAM formation are still controversial in the pathological study of cerebral ischemia-reperfusion injury.In this study,we first determined whether ER-mitochondrial tethering protects against neuronal injury induced by ischemia-reperfusion.A variety of proteins are recognized to be involved in the interaction between mitochondria and ER.Mitochondrial fusion protein 2（MFN2）has also been reported to be enriched in MAM and is one of the most important proteins involved in tethering mitochondria and ER.One of the major functions of MAM is regulating the Ca²⁺transport from ER to mitochondria,which is mainly dependent on the activation of the inositol trisphosphate receptor（IP3R）on ER.Little is known about the association of DDAH-1 with ER-mitochondrial tethering within IS neurons,thus we explore whether DDAH-1 regulates Mitochondrial homeostasis via ER-mitochondrial tethering in IS neurons.This study was divided into three parts to explore the protective effect of DDAH-1 on neuronal injury induced by ischemia-reperfusion and its specific mechanism.In the first part,the middle cerebral artery occlusion/reperfusion（MCAO/R）model was established in DDAH-1 global knockout(DDAH-1^-/-)rats to preliminarily clarify whether DDAH-1 plays a protective role in cerebral ischemia-reperfusion injury in rats.In the second part,primary cortical neurons were cultured to establish an oxygen-glucose deprivation/reperfusion（OGD/R）model,and the protective effect of DDAH-1on mitochondrial injury induced by OGD/R was explored by transmission electron microscopy（TEM）,plasmid transfection,and a series of molecular biological means.In the third part,the protective effect and molecular mechanism of DDAH-1 on neuronal OGD/R injury via increasing ER-mitochondrial tethering were also explored in vitro by TEM,blue-native polyacrylamide gel electrophoresis（BN-PAGE）,and co-immunoprecipitation（CO-IP）.This study provides a new direction for the mechanistic study of cerebral ischemia-reperfusion injury.Part 1 DDAH-1 attenuates cerebral ischemia-reperfusion injury in ratsObjective:To investigate the protective effect of DDAH-1 on neurological injury induced by MCAO/R in rats.Methods:Firstly,the distribution and expression of DDAH-1 in different brain regions and in neurons and endothelial cells were detected by immunofluorescence staining and western blotting,The gene knockout efficiency of DDAH-1^-/-rats and its effect on anatomical indicators were verified.After that,we established MCAO/R models to compare the behavioral changes of DDAH-1^-/-and DDAH-1^+/+rats using modified neurological severity scores（m NSS）.The volume of cerebral infarction was tested by2,3,5-triphenyltetrazolium chloride（TTC）staining.The brain edema was reflected via brain water content.The apoptosis of cells in the infarcted area was detected by TUNEL staining.The leakage of Evan’s blue dye（EBD）and the expression levels of tight junction（TJ）proteins were recorded as reflection of blood-brain barrier damage.The concentrations of ADMA in plasma and infarcted area and NO levels in different brain regions were also detected.Meanwhile,L-arginine（ARG）（substrate for NO）was supplemented to DDAH-1^-/-rats and the above indicators were observed.Results:We observed that DDAH-1 was mainly expressed in neurons of the cerebral cortex.Following DDAH-1 depletion,there was no DDAH-1 expression in the brain,and ADMA levels were increased significantly in various brain regions,especially in the cerebral cortex.No significant change was seen in body weight,brain tissue weight or other anatomical structure.After MCAO/R modeling,compared with DDAH-1^+/+rats,DDAH-1^-/-rats showed more severe neurological deficits and brain edema,increased infarct volume and number of apoptosis,and aggravated blood-brain barrier injury（increased EBD leakage and TJ protein degradation）.After ARG was supplemented in DDAH-1^-/-rats,the m NSS score,infarct volume,brain water content and number of apoptotic cells were significantly reduced,and blood-brain barrier damage was alleviated.Finally,compared with DDAH-1^+/+rats,DDAH-1^-/-rats had significantly higher ADMA levels in plasma and infarct area.DDAH-1^-/-rats also showed significantly lower NO levels in infarct area,but there was no significant difference in non-infarct area between DDAH-1^-/-and DDAH-1^+/+rats.Conclusion:DDAH-1 attenuates cerebral ischemia-reperfusion injury in rats,which may be related to the regulation of ADMA and NO level in infarct area.Part 2 DDAH-1 attenuates mitochondrial damage induced by OGD/R in neurons Objective:To clarify the protective effect of DDAH-1 on mitochondrial damage induced by OGD/R in cultured cortical neurons.Methods:Rat primary cortical neurons were cultured in vitro to establish OGD/R models.We constructed DDAH-1 small interfering RNA（si RNA）and DDAH-1overexpression plasmids and transfected cells with them,after that ARG or ADMA were supplemented respectively.Cell viability was determined by cell counting kit-8（CCK-8）.The intracellular ROS accumulation was detected by ROS kit and DCFH-DA probe.Intracellular ADMA concentration was detected by enzyme linked immunosorbent assay（ELISA）and NO concentration was measured by cellular NO kit.The DDAH-1 expression on mitochondria was detected by immunofluorescence staining.MMP in neurons was measured with mitochondrial membrane potential（JC-1）kit.The activities of MRCC-Ⅰ,-Ⅱ,-Ⅲ,-Ⅳwere also determined by MRCC kit.Rhod-2AM staining were carried out to show the level of Ca²⁺in mitochondria,and TEM was used to observe mitochondrial ultrastructure.Results:DDAH-1 was highly expressed in cortical neuron mitochondria.After DDAH-1 knockdown in OGD/R cortical neurons,their cell death and ROS accumulation were exacerbated,accompanied by up-regulated intracellular ADMA levels.Although the ARG supplementation increased the NO levels,there was no improvement for the neuronal viability and ROS levels.Meanwhile,DDAH-1 knockdown resulted in more severe mitochondrial damage,as manifested by the reduction of MMP and MRCC activity,and aggravated calcium overload in mitochondria and damage of mitochondrial integrity.Conversely,overexpression of DDAH-1 significantly attenuated OGD/R-induced neuronal death and ROS accumulation.It also decreased intracellular ADMA levels and attenuated the damage of OGD/R on MMP,MRCC activity,mitochondrial calcium balance,and mitochondrial integrity,while ADMA intervention attenuated the protective effect of DDAH-1 overexpression.Conclusion:DDAH-1 attenuates damages of mitochondrial function and integrity in primary cortical neurons induced by OGD/R.Part 3 DDAH-1 attenuates neuronal OGD/R injury by phosphorylatingMARCH5 and increasing ER-mitochondrial tetheringObjective:To explore the molecular mechanism of DDAH-1 attenuating neuronal OGD/R injury by increasing ER-mitochondrial tethering.Methods:Rat primary cortical neurons were cultured in vitro to establish OGD/R models.We constructed MFN2 si RNA,DDAH-1 si RNA and DDAH-1 overexpression plasmids and transfected cells with them.The ultrastructure of MAM was observed by TEM.Cell viability was determined by CCK-8.The steady-state expression level of MFN2 was detected by immunoblotting,and the oligomerization state of MFN2 was assessed by BN-PAGE.The expression and phosphorylation level of membrane-associated RING-CH5（MARCH5）,a MFN2-activating protein,were detected by CO-IP and immunoblotting.The MARCH5 double-phosphorylation site mutant(MARCH5^S38/59A)plasmid was further constructed and transfected,and its effects on MARCH5 phosphorylation,MFN2 oligomerization,MAM formation,mitochondrial function and integrity,and cell viability were detected.Finally,the expression and phosphorylation level of IP3R,a main protein regulating Ca²⁺transport on MAM,were detected by CO-IP and immunoblotting.Results:OGD/R resulted in the reduction of neuronal MAM formation.Knockdown of MFN2 expression further inhibited the MAM formation and aggravated the death of OGD/R neurons.OGD/R also led to decreased MFN2 expression,MFN2oligomerization and MARCH5 phosphorylation levels,but had no significant effect on total expression levels of MARCH5.Knockdown of DDAH-1 expression aggravated the down-regulated ER-mitochondrial tethering,MFN2 oligomerization and MARCH5phosphorylation levels caused by OGD/R,but had no effect on MFN2 expression.However,overexpression of DDAH-1 significantly reduced the MAM damage caused by OGD/R,as manifested by up-regulated MARCH5 phosphorylation level,MFN2oligomerization and MAM formation,while the protective effect of DDAH-1overexpression was weakened after ADMA treatment.MARCH5^S38/59A plasmid transfection did not significantly affect the down-regulated MARCH5 phosphorylation level caused by OGD/R,but significantly reduced MARCH5 phosphorylation level,MFN2 oligomerization and MAM formation after DDAH-1 overexpression,also attenuated the protective effect of DDAH-1 overexpression on mitochondrial function,integrity,and cell viability.DDAH-1 knockdown aggravated the decrease in IP3R phosphorylation caused by OGD/R,while overexpression of DDAH-1 significantly restored IP3R phosphorylation and subsequent ADMA intervention counteracted the effect of DDAH-1 overexpression.Conclusion:DDAH-1 increases ER-mitochondrial tethering by maintaining MARCH5phosphorylation state and prompting MFN2 oligomerization state,while enhancing the phosphorylation level of IP3R and inhibiting its Ca²⁺transporting activity,protecting the mitochondrial homeostasis and activity of OGD/R neurons.