The Role Of Sirt3 In Oxidative Stress-induced Neuronal Injury And Related Mechanism

Background and purpose:Sirtuins(Sirtl-7)are a family of phylogenetically conserved nicotinamide adenine nucleotide(NAD+)-dependent protein deacetylases,among which Sirt3 resides primarily in the mitochondria and serves as a stress responsive deacetylase playing a role in protecting cells in multiple pathological conditions.Sirt3 regulates mitochondrial function and metabolism in response to caloric restriction and stress,including ATP generation and reactive oxygen species(ROS)production.However,the exact role of Sirt3 in oxidative stress-induced neuronal cell injury has not yet been fully elucidated.Sirtl is another well-characterized member of this family,it has also been shown to play a vital role in resistance to cellular stress.However,the interaction between these two sirtuins has not been fully determined.Autophagy is an evolutionarily conserved process for the bulk degradation and recycling of cytosolic proteins and organelles.Although the existence and activation of autophagy after ischemic stroke is undisputable,whether autophagy is a protective or detrimental mechanism for ischemic neuronal injury is still a topic of debate.Mitophagy is the selective degradation of mitochondria by autophagy.HIF-1/BNIP3 has been proved to be one of the most important signaling pathways involved in mitophagy.Several recent studies have shown that Sirt3 may play a role in regulating mitophagy through downstream proteins of BNIP3 and FOXO3.This study aims to investigate the role of Sirt3 in H2O2 or oxygen and glucose deprivation(OGD)induced oxidative neuronal injury and the effects of Sirtl-Sirt3 axis in blood-brain barrier(BBB)permeability.Methods:An in vitro H2O2 or OGD injury model has been applied to HT22 cell culture and primary cortical neuron.Co-clutured model of human brain microvascular endothelial cells and human astrocytes has been used to mimic BBB.Cell viability assay,flow cytometry and lactate dehydrogenase(LDH)and cytochrome c release assay have been performed to investigate cell injury.Measurements of caspase-3,reactive oxygen species(ROS)generation and antioxidant enzymes activities have been used to study the role of Sirt3 on oxidative stress.Calcium imaging,ATP synthesis measurement and RT-qPCR has been applied to measure mitochondrial dysfunction.We also used transepithelial electrical resistance(TEER)to test BBB permeability and electron microscope to monitor the functions of organelles under ultrastructure level.Several other molecular biological and immunological techniques including Western Blot and ICC have also been used to study protein expression.Results:In this study,we found 1)H2O2 or OGD treatment increased the expression of Sirt3 in a dose-and time-dependent manner at both mRNA and protein levels.2)Knockdown of Sirt3 with a specific small interfering RNA(siRNA)exacerbated H2O2-or OGD-induced neuronal injury.3)Overexpression of Sirt3 by lentivirus transfection attenuated cytochrome c and LDH release,increase of Bax/Bcl-2 ratio as well as caspase-9/caspase-3 activity induced by H2O2 or OGD,and eventually inhibited apoptotic neuronal death.4)Sirt3 significantly reduced ROS generation and lipid peroxidation after injury,increased the activities of endogenous antioxidant enzymes.5)The intra-mitochondrial Ca2+overload,but not increase of cytosolic Ca2+ after H2O2 or OGD treatment,was strongly attenuated after Sirt3 overexpression.6)H2O2-or OGD-induced inhibition of mitochondrial complex activities and ATP synthesis,decrease of mitochondrial Ca2+ buffering capacity,and mitochondrial swelling were all partly reversed by Sirt3.7)Sirt3 also increased the content of mitochondrial DNA(mtDNA)and the expression of mitochondrial biogenesis related transcription factors and preserved MMP.8)Sirt3 increased autophagy in OGD-injured neurons,which was also confirmed by the increased expression of Beclin-1 as well as LC3-? to LC3-? conversion.9)The autophagy inhibitor 3-MA and bafilomycin A1 partially prevented the effects of Sirt3 on LDH release and apoptosis after OGD.10)Overexpression of Sirt3 in cortical neurons markedly increased the phosphorylation of AMPK,whereas the phosphor-mTOR(p-mTOR)levels decreased both in the presence and absence of OGD insult.11)Pre-treatment with the AMPK inhibitor compound C partially reversed the protective effects of Sirt3.12)Suppression of Sirtl via siRNA or salermide significantly decreased BBB permeability and attenuated mitochondrial ROS generation,whereas Sirt3 knockdown increased BBB permeability.13)Application of the AMPK inhibitor compound C partially prevented the effects of Sirtl-Sirt3 axis on BBB permeability after OGD.14)The results of flow cytometry and cytochrome c release demonstrated that Sirtl and Sirt3 exerted opposite effects on OGD-induced apoptosis.Conclusion:Taken together,these results suggest that Sirt3 acts as a prosurvival factor playing an essential role to protect neurons under H2O2 or OGD induced oxidative stress.It protects neurons possibly through inhibiting ROS accumulation and activation of mitochondrial apoptotic pathway via regulating mitochondrial Ca2+ homeostasis and mitochondrial biogenesis.Sirt3 also protects against OGD insult by inducing autophagy through regulation of the AMPK-mTOR pathway.In addition,Sirtl-Sirt3 axis may act as an important modulator in BBB physiology,and can be a therapeutic target for ischemic stroke via regulating mitochondrial ROS generation.