Sphingosine kinase 1 overexpression induces MFN2 fragmentation and alters mitochondrial matrix Ca2+ handling in HeLa cells

Sphingosine kinase 1 (SK1) converts sphingosine to the bioactive lipid sphingosine 1-phosphate (S1P). S1P binds to G-protein-coupled receptors (S1PR_1–5) to regulate cellular events, including Ca²⁺ signaling. The SK1/S1P axis and Ca²⁺ signaling both play important roles in health and disease. In this respect, Ca²⁺ microdomains at the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are of importance in oncogenesis. Mitofusin 2 (MFN2) modulates ER-mitochondria contacts, and dysregulation of MFN2 is associated with malignancies. We show that overexpression of SK1 augments agonist-induced Ca²⁺ release from the ER resulting in increased mitochondrial matrix Ca²⁺. Also, overexpression of SK1 induces MFN2 fragmentation, likely through increased calpain activity. Further, expressing putative calpain-cleaved MFN2 N- and C-terminal fragments increases mitochondrial matrix Ca²⁺ during agonist stimulation, mimicking the SK1 overexpression in cells. Moreover, SK1 overexpression enhances cellular respiration and cell migration. Thus, SK1 regulates MFN2 fragmentation resulting in increased mitochondrial Ca²⁺ and downstream cellular effects.     

Model structures of inactive and peptide agonist bound C5aR: Insights into agonist binding,selectivity and activation

C5a receptor (C5aR) is one of the major chemoattractant receptors of the druggable proteome that binds C5a, the proinflammatory polypeptide of complement cascade, triggering inflammation and SEPSIS. Here, we report the model structures of C5aR in both inactive and peptide agonist (YSFKPMPLaR; a=D-Ala) bound meta-active state. Assembled in CYANA and evolved over molecular dynamics (MD) in POPC bilayer, the inactive C5aR demonstrates a topologically unique compact heptahelical bundle topology harboring a β-hairpin in extracellular loop 2 (ECL2), derived from the atomistic folding simulations. The peptide agonist bound meta-active C5aR deciphers the “site2” at an atomistic resolution in the extracellular surface (ECS), in contrast to the previously hypothesized inter-helical crevice. With estimated Ki≈2.75 μM, the meta-active C5aR excellently rationalizes the IC₅₀ (0.1–13 μM) and EC₅₀ (0.01–6 μM) values, displayed by the peptide agonist in several signaling studies. Moreover, with Ki≈5.3×10⁵ μM, the “site2” also illustrates selectivity, by discriminating the stereochemical mutant peptide (YSFkPMPLaR; k=D-Lys), known to be inert toward C5aR, up to 1 mM concentration. Topologically juxtaposed between the structures of rhodopsin and CXCR1, the C5aR models also display excellent structural correlations with the other G-protein coupled receptors (GPCRs). The models elaborated in the current study unravel many important structural insights previously not known for regulating the agonist binding and activation mechanism of C5aR.     

Helix 8 in chemotactic receptors of the complement system

Host response to infection involves the activation of the complement system leading to the production of anaphylatoxins C3a and C5a. Complement factor C5a exerts its effect through the activation of C5aR1, chemotactic receptor 1, and triggers the G protein-coupled signaling cascade. Orthosteric and allosteric antagonists of C5aR1 are a novel strategy for anti-inflammatory therapies. Here, we discuss recent crystal structures of inactive C5aR1 in terms of an inverted orientation of helix H8, unobserved in other GPCR structures. An analysis of mutual interactions of subunits in the C5aR1—G protein complex has provided new insights into the activation mechanism of this distinct receptor. By comparing two C5aR receptors C5aR1 and C5aR2 we explained differences between their signaling pathways on the molecular level. By means of molecular dynamics we explained why C5aR2 cannot transduce signal through the G protein pathway but instead recruits beta-arrestin. A comparison of microsecond MD trajectories started from active and inactive C5aR1 receptor conformations has provided insights into details of local and global changes in the transmembrane domain induced by interactions with the Gα subunit and explained the impact of inverted H8 on the C5aR1 activation.     

Role of mitofusin 2 in the renal stress response

The role of mitofusin 2 (MFN2), a key regulator of mitochondrial morphology and function in the renal stress response is unknown. To assess its role, the MFN2 floxed gene was conditionally deleted in the kidney of mice (MFN2 cKO) by Pax2 promoter driven Cre expression (Pax2Cre). MFN2 cKO caused severe mitochondrial fragmentation in renal epithelial cells that are critical for normal kidney tubular function. However, despite a small (20%) decrease in nephron number, newborn cKO pups had organ or tubular function that did not differ from littermate Cre-negative pups. MFN2 deficiency in proximal tubule epithelial cells in primary culture induced mitochondrial fragmentation but did not significantly alter ATP turnover, maximal mitochondrial oxidative reserve capacity, or the low level of oxygen consumption during cyanide exposure. MFN2 deficiency also did not increase apoptosis of tubule epithelial cells under non-stress conditions. In contrast, metabolic stress caused by ATP depletion exacerbated mitochondrial outer membrane injury and increased apoptosis by 80% in MFN2 deficient vs. control cells. Despite similar stress-induced Bax 6A7 epitope exposure in MFN2 deficient and control cells, MFN2 deficiency significantly increased mitochondrial Bax accumulation and was associated with greater release of both apoptosis inducing factor and cytochrome c. In conclusion, MFN2 deficiency in the kidney causes mitochondrial fragmentation but does not affect kidney or tubular function during development or under non-stress conditions. However, MFN2 deficiency exacerbates renal epithelial cell injury by promoting Bax-mediated mitochondrial outer membrane injury and apoptosis.     

依达拉奉对MPP+处理的PC12细胞线粒体融合和分裂平衡的保护作用

本文旨在探讨依达拉奉(edaravone, Eda)对帕金森病细胞模型线粒体融合、分裂动态平衡的作用及机制。用500μmol/L1-甲基-4-苯基吡啶离子(1-methyl-4-phenylpyridinium, MPP^+)处理PC12细胞建立帕金森病细胞模型,采用噻唑蓝(MTT)比色法检测不同浓度Eda对MPP^+处理的PC12细胞存活率的影响,用激光共聚焦显微镜检测线粒体形态,用Western blot检测线粒体融合与分裂相关蛋白OPA1、MFN2、DRP1和Fis1的表达变化。结果显示,预先加入不同浓度的Eda能减轻MPP^+处理的PC12细胞损伤,作用呈一定的量效关系;经MPP^+处理48 h,PC12细胞线粒体出现碎片化,OPA1和MFN2蛋白表达下调,DRP1和Fis1蛋白表达上调,而Eda预处理能逆转PC12细胞的上述变化,但对Fis1的蛋白表达没有影响。以上结果提示,Eda可上调OPA1和MFN2的蛋白表达,下调DRP1的表达,从而抑制线粒体碎片化,发挥神经细胞线粒体保护作用。     

MARCH-V is a novel mitofusin 2- and Drp1-binding protein able to change mitochondrial morphology

Mitofusins and Drp1 are key components in mitochondrial membrane fusion and division, but the molecular mechanism underlying the regulation of their activities remains to be clarified. Here, we identified human membrane-associated RING-CH (MARCH)-V as a novel transmembrane protein of the mitochondrial outer membrane. Immunoprecipitation studies demonstrated that MARCH-V interacts with mitofusin 2 (MFN2) and ubiquitinated forms of Drp1. Overexpression of MARCH-V promoted the formation of long tubular mitochondria in a manner that depends on MFN2 activity. By contrast, mutations in the RING finger caused fragmentation of mitochondria. We also show that MARCH-V promotes ubiquitination of Drp1. These results indicate that MARCH-V has a crucial role in the control of mitochondrial morphology by regulating MFN2 and Drp1 activities.     

MFN2 Couples Glutamate Excitotoxicity and Mitochondrial Dysfunction in Motor Neurons

Mitochondrial dysfunction plays a central role in glutamate-evoked neuronal excitotoxicity, and mitochondrial fission/fusion dynamics are essential for mitochondrial morphology and function. Here, we establish a novel mechanistic linker among glutamate excitotoxicity, mitochondrial dynamics, and mitochondrial dysfunction in spinal cord motor neurons. Ca²⁺-dependent activation of the cysteine protease calpain in response to glutamate results in the degradation of a key mitochondrial outer membrane fusion regulator, mitofusin 2 (MFN2), and leads to MFN2-mediated mitochondrial fragmentation preceding glutamate-induced neuronal death. MFN2 deficiency impairs mitochondrial function, induces motor neuronal death, and renders motor neurons vulnerable to glutamate excitotoxicity. Conversely, MFN2 overexpression blocks glutamate-induced mitochondrial fragmentation, mitochondrial dysfunction, and/or neuronal death in spinal cord motor neurons both in vitro and in mice. The inhibition of calpain activation also alleviates glutamate-induced excitotoxicity of mitochondria and neurons. Overall, these results suggest that glutamate excitotoxicity causes mitochondrial dysfunction by impairing mitochondrial dynamics via calpain-mediated MFN2 degradation in motor neurons and thus present a molecular mechanism coupling glutamate excitotoxicity and mitochondrial dysfunction.     

Neutraligands of C5a can potentially occlude the interaction of C5a with the complement receptors C5aR1 and C5aR2

The complement system is central to the rapid immune response witnessed in vertebrates and invertebrates, which plays a crucial role in physiology and pathophysiology. Complement activation fuels the proteolytic cascade, which produces several complement fragments that interacts with a distinct set of complement receptors. Among all the complement fragments, C5a is one of the most potent anaphylatoxins, which exerts solid pro-inflammatory responses in a myriad of tissues by binding to the complement receptors such as C5aR1 (CD88, C5aR) and C5aR2 (GPR77, C5L2), which are part of the rhodopsin subfamily of G-protein coupled receptors. In terms of signaling cascade, recruitment of C5aR1 or C5aR2 by C5a triggers the association of either G-proteins or β-arrestins, providing a protective response under normal physiological conditions and a destructive response under pathophysiological conditions. As a result, both deficiency and unregulated activation of the complement lead to clinical conditions that require therapeutic intervention. Indeed, complement therapeutics targeting either the complement fragments or the complement receptors are being actively pursued by both industry and academia. In this context, the model structural complex of C5a–C5aR1 interactions, followed by a biophysical evaluation of the model complex, has been elaborated on earlier. In addition, through the drug repurposing strategy, we have shown that small molecule drugs such as raloxifene and prednisone may act as neutraligands of C5a by effectively binding to C5a and altering its biologically active molecular conformation. Very recently, structural models illustrating the intermolecular interaction of C5a with C5aR2 have also been elaborated by our group. In the current study, we provide the biophysical validation of the C5a-C5aR2 model complex by recruiting major synthetic peptide fragments of C5aR2 against C5a. In addition, the ability of the selected neutraligands to hinder the interaction of C5a with the peptide fragments derived from both C5aR1 and C5aR2 has also been explored. Overall, the computational and experimental data provided in the current study supports the idea that small molecule drugs targeting C5a can potentially neutralize C5a's ability to interact effectively with its cognate complement receptors, which can be beneficial in modulating the destructive signaling response of C5a under pathological conditions.     

Icariin attenuates excessive alcohol consumption-induced susceptibility to atrial fibrillation through SIRT3 signaling

Excessive alcohol consumption has long been identified as a risk factor for adverse atrial remodeling and atrial fibrillation (AF). Icariin is a principal active component from traditional Chinese medicine Herba Epimedii and has been demonstrated to exert potential antiarrhythmic effect. The present study was designed to evaluate the effect of icariin against alcohol-induced atrial remodeling and disruption of mitochondrial dynamics and furthermore, to elucidate the underlying mechanisms. Excessive alcohol-treated C57BL/6 J mice were infected with serotype 9 adeno-associated virus (AAV9) carrying mouse SIRT3 gene or negative control virus. Meanwhile, icariin (50 mg/kg/d) was administered to the animals in the presence or absence of AAV9 carrying SIRT3 shRNA. We noted that 8 weeks of icariin treatment effectively attenuated alcohol consumption-induced atrial structural and electrical remodeling as evidenced by reduced AF inducibility and reversed atrial electrical conduction pattern as well as atrial enlargement. Furthermore, icariin-treated group exhibited significantly enhanced atrial SIRT3-AMPK signaling, decreased atrial mitoSOX fluorescence and mitochondrial fission markers, elevated mitochondrial fusion markers (MFN1, MFN2) as well as NRF-1-Tfam-mediated mitochondrial biogenesis. Importantly, these beneficial effects were mimicked by SIRT3 overexpression while abolished by SIRT3 knockdown. These data revealed that targeting atrial SIRT3-AMPK signaling and preserving mitochondrial dynamics might serve as the novel therapeutic strategy against alcohol-induced AF genesis. Additionally, icariin ameliorated atrial remodeling and mitochondrial dysfunction by activating SIRT3-AMPK signaling, highlighting the use of icariin as a promising antiarrhythmic agent in this circumstance.     

The role of the ribosomal protein S19 C-terminus in Gi protein-dependent alternative activation of p38 MAP kinase via the C5a receptor in HMC-1 cells

We have demonstrated that an alternative C5a receptor (C5aR) ligand, the homodimer of ribosomal protein S19 (RP S19), contains a unique C-terminus (I₁₃₄–H₁₄₅) that is distinct from the moieties involved in the C5a–C5aR interaction. To examine the role of I₁₃₄–H₁₄₅ in the ligand–C5aR interaction, we connected this peptide to the C-terminus of C5a (C5a/RP S19) and found that it endowed the second binding moiety of RP S19 (L₁₃₁DR) with a relatively higher binding affinity to the C5aR on a human mast cell line, HMC-1. In contrast to the C5aR, the second C5aR C5L2 worked as a decoy receptor. As a result, the mitogen-activated protein kinase (MAPK) downstream of the Gi protein exchanged extracellular-signal regulated kinase for p38MAPK. This alternative p38MAPK activation could be pharmacologically suppressed not only by the downregulation of phosphoinositide 3-kinase (PI3K) by LY294002, but also by the over-activation of protein kinase C by phorbol 12-myristate 13-acetate. The activation was reproduced upon C5a–C5aR interaction by a simultaneous suppression of PI3K and phospholipase C with LY294002 and U73122 at low concentrations. Moreover, p38MAPK phosphorylation upstream of the pertussis toxin-dependent extracellular Ca²⁺ entry was also suppressed by high concentrations of MgCl₂, which blocks melastatin-type transient receptor potential Ca²⁺ channels (TRPMs). The active conformation of C5aR upon the ligation by C5a, at least on HMC-1 cells, is changed by the additional interaction of the I₁₃₄–H₁₄₅ peptide, which seems to guide the alternative activation of p38MAPK. This activation is then amplified by a novel positive feedback loop between p38MAPK and TRPM.     

C5L2 is required for C5a-triggered receptor internalization and ERK signaling

C5L2 is a receptor that binds to C5a and belongs to the family of G protein-coupled receptors, but its role in physiological C5a-mediated responses remains under debate. Here we show that, like the canonical C5a receptor C5aR, C5L2 plays a pro-inflammatory role in a murine model of acute experimental colitis. We demonstrate that C5L2 physically interacts with C5aR and is required for optimal C5a-mediated C5aR internalization and associated ERK activation. Abrogation of C5a-induced receptor internalization by treatment with the dynamin inhibitor dynasore^TM impaired C5a-induced MEK and ERK signaling. Although the presence of C5aR alone was sufficient to recruit the scaffold protein β-arrestin1 to the cell membrane in response to C5a stimulation, it was inadequate to mediate AP2 recruitment and subsequent C5aR internalization. Expression of C5L2 allowed normal internalization of C5aR in response to C5a stimulation, followed by normal ERK signaling. Thus, our work reveals an essential role for C5L2 in C5a-triggered, AP2-dependent C5aR internalization and downstream ERK signaling.     

MFN2 silencing promotes neural differentiation of embryonic stem cells via the Akt signaling pathway

Mitofusin 2 (MFN2) is a regulatory protein participating in mitochondria dynamics, cell proliferation, death, differentiation, and so on. This study aims at revealing the functional role of MFN2 in the pluripotency maintenance and primitive differetiation of embryonic stem cell (ESCs). A dox inducible silencing and routine overexpressing approach was used to downregulate and upregulate MFN2 expression, respectively. We have compared the morphology, cell proliferation, and expression level of pluripotent genes in various groups. We also used directed differentiation methods to test the differentiation capacity of various groups. The Akt signaling pathway was explored by the western blot assay. MFN2 upregulation in ESCs exhibited a typical cell morphology and similar cell proliferation, but decreased pluripotent gene markers. In addition, MFN2 overexpression inhibited ESCs differentiation into the mesendoderm, while MFN2 silencing ESCs exhibited a normal cell morphology, slower cell proliferation and elevated pluripotency markers. For differentiation, MFN2 silencing ESCs exhibited enhanced three germs' differentiation ability. Moreover, the protein levels of phosphorylated Akt308 and Akt473 decreased in MFN2 silenced ESCs, and recovered in the neural differentiation process. When treated with the Akt inhibitor, the neural differentiation capacity of the MFN2 silenced ESCs can reverse to a normal level. Taken together, the data indicated that the appropriate level of MFN2 expression is essential for pluripotency and differentiation capacity in ESCs. The increased neural differentiation ability by MFN2 silencing is strongly related to the Akt signaling pathway.     

Regulation of C3a receptor signaling in human mast cells by G protein coupled receptor kinases

Background

The complement component C3a activates human mast cells via its cell surface G protein coupled receptor (GPCR) C3aR. For most GPCRs, agonist-induced receptor phosphorylation leads to receptor desensitization, internalization as well as activation of downstream signaling pathways such as ERK1/2 phosphorylation. Previous studies in transfected COS cells overexpressing G protein coupled receptor kinases (GRKs) demonstrated that GRK2, GRK3, GRK5 and GRK6 participate in agonist-induced C3aR phosphorylation. However, the roles of these GRKs on the regulation of C3aR signaling and mediator release in human mast cells remain unknown.

Methodology/Principal Findings

We utilized lentivirus short hairpin (sh)RNA to stably knockdown the expression of GRK2, GRK3, GRK5 and GRK6 in human mast cell lines, HMC-1 and LAD2, that endogenously express C3aR. Silencing GRK2 or GRK3 expression caused a more sustained Ca²⁺ mobilization, attenuated C3aR desensitization, and enhanced degranulation as well as ERK1/2 phosphorylation when compared to shRNA control cells. By contrast, GRK5 or GRK6 knockdown had no effect on C3aR desensitization, but caused a significant decrease in C3a-induced mast cell degranulation. Interestingly, GRK5 or GRK6 knockdown rendered mast cells more responsive to C3a for ERK1/2 phosphorylation.

Conclusion/Significance

This study demonstrates that GRK2 and GRK3 are involved in C3aR desensitization. Furthermore, it reveals the novel finding that GRK5 and GRK6 promote C3a-induced mast cell degranulation but inhibit ERK1/2 phosphorylation via C3aR desensitization-independent mechanisms. These findings thus reveal a new level of complexity for C3aR regulation by GRKs in human mast cells.     

Loss of sirtuin 1 and mitofusin 2 contributes to enhanced ischemia/reperfusion injury in aged livers

Ischemia/reperfusion (I/R) injury is a causative factor contributing to morbidity and mortality during liver resection and transplantation. Livers from elderly patients have a poorer recovery from these surgeries, indicating reduced reparative capacity with aging. Mechanisms underlying this age‐mediated hypersensitivity to I/R injury remain poorly understood. Here, we investigated how sirtuin 1 (SIRT1) and mitofusin 2 (MFN2) are affected by I/R in aged livers. Young (3 months) and old (23–26 months) male C57/BL6 mice were subjected to hepatic I/R in vivo. Primary hepatocytes isolated from each age group were also exposed to simulated in vitro I/R. Biochemical, genetic, and imaging analyses were performed to assess cell death, autophagy flux, mitophagy, and mitochondrial function. Compared to young mice, old livers showed accelerated liver injury following mild I/R. Reperfusion of old hepatocytes also showed necrosis, accompanied with defective autophagy, onset of the mitochondrial permeability transition, and mitochondrial dysfunction. Biochemical analysis indicated a near‐complete loss of both SIRT1 and MFN2 after I/R in old hepatocytes, which did not occur in young cells. Overexpression of either SIRT1 or MFN2 alone in old hepatocytes failed to mitigate I/R injury, while co‐overexpression of both proteins promoted autophagy and prevented mitochondrial dysfunction and cell death after reperfusion. Genetic approaches with deletion and point mutants revealed that SIRT1 deacetylated K655 and K662 residues in the C‐terminus of MFN2, leading to autophagy activation. The SIRT1‐MFN2 axis is pivotal during I/R recovery and may be a novel therapeutic target to reduce I/R injury in aged livers.     

Apoptotic signaling induced by H2O2-mediated oxidative stress in differentiated C2C12 myotubes

AimsApoptotic signaling proteins were evaluated in postmitotic skeletal myotubes to test the hypothesis that oxidative stress induced by H₂O₂ activates both caspase-dependent and caspase-independent apoptotic proteins in differentiated C2C12 myotubes. We hypothesized that oxidative stress would decrease anti-apoptotic protein levels in C2C12 myotubes.Main methodsApoptotic regulatory factors and apoptosis-associated proteins including Bcl-2, Bax, Apaf-1, XIAP, ARC, cleaved PARP, p53, p21^Cip1/Waf1, c-Myc, HSP70, CuZnSOD, and MnSOD protein content were measured by immunoblots.Key findingsH₂O₂ induced apoptosis in myotubes as shown by DNA laddering and an elevation of apoptotic DNA fragmentation. Cell death ELISA showed increase in the extent of apoptotic DNA fragmentation following treatment with H₂O₂. Treatment with 4 mM of H₂O₂ for 24 or 96 h caused increase in Bax (56%, 227%), cytochrome c (282%, 701%), Smac/DIABLO (155%, 260%), caspase-3 protease activity (51%, 141%), and nuclear and cytosolic p53 (719%, 1581%) levels in the myotubes. As an estimate of the mitochondrial AIF release to the cytosol, AIF protein content measured in the mitochondria-free cytosolic fraction was elevated by 65% after 96 h treatment with 4 mM of H₂O₂. AIF measured in the nuclear protein fraction increased by 74% and 352% following treatment with 4 mM of H₂O₂ for 24 and 96 h, respectively. Bcl-2 declined in myotubes by 61% and 69% after 24 or 96 h of treatment in 4 mM H₂O₂, respectively.SignificanceThese findings indicate that both caspase-dependent and caspase-independent mechanisms are involved in coordinating the activation of apoptosis induced by H₂O₂ in differentiated myotubes.     

Dengue Virus Impairs Mitochondrial Fusion by Cleaving Mitofusins

Mitochondria are highly dynamic subcellular organelles participating in many signaling pathways such as antiviral innate immunity and cell death cascades. Here we found that mitochondrial fusion was impaired in dengue virus (DENV) infected cells. Two mitofusins (MFN1 and MFN2), which mediate mitochondrial fusion and participate in the proper function of mitochondria, were cleaved by DENV protease NS2B3. By knockdown and overexpression approaches, these two MFNs showed diverse functions in DENV infection. MFN1 was required for efficient antiviral retinoic acid-inducible gene I–like receptor signaling to suppress DENV replication, while MFN2 participated in maintaining mitochondrial membrane potential (MMP) to attenuate DENV-induced cell death. Cleaving MFN1 and MFN2 by DENV protease suppressed mitochondrial fusion and deteriorated DENV-induced cytopathic effects through subverting interferon production and facilitating MMP disruption. Thus, MFNs participate in host defense against DENV infection by promoting the antiviral response and cell survival, and DENV regulates mitochondrial morphology by cleaving MFNs to manipulate the outcome of infection.     

Paraoxonase 2 protects against the CML mediated mitochondrial dysfunction through modulating JNK pathway in human retinal cells

BackgroundParaoxonase 2 (PON2) a known anti-apoptotic protein, has not been explored against N^ε-(carboxymethyl)lysine (CML), induced mitochondrial dysfunction and apoptosis in human retinal cells. Hence this present study aims to investigate the potential role of PON2 in mitigating CML-induced mitochondrial dysfunction in these cells.MethodsPON2 protein was quantified in HRECs (Human retinal endothelial cells), ARPE-19 (Retinal pigment epithelial cells) cells upon CML treatment and also in cadaveric diabetic retina vs respective controls. ROS production, mitochondrial membrane potential (MMP), mitochondrial permeability transition pore (mPTP) opening, the release of Cyt-c, Bax, Caspase-3, Fis1, Mfn1, Mfn2, mitochondrial morphology, and the signaling pathway was assessed using DCFDA, JC-1, CoCl₂, immunofluorescence or western blotting analysis in both loss-of-function or gain-of-function experiments.ResultsPON2 protein was downregulated in HREC and ARPE-19 cells upon CML treatment as well as in the diabetic retina (p = 0.035). Decrease in PON2 augments Fis1 expression resulting in fragmentation of mitochondria and enhances the ROS production, decreases MMP, facilitates mPTP opening, and induces the release of Cyt-c, which activates the pro-apoptotic pathway. Whereas PON2 overexpression similar to SP600125 (a specific JNK inhibitor) was able to decrease Fis1 (p = 0.036) and reverse the Bcl-2 and Bax ratio, and inhibit the JNK1/2 signaling pathway.ConclusionOur results confirm that PON2 has an anti-apoptotic role against the CML mediated mitochondrial dysfunction and inhibits apoptosis through the JNK-Fis1 axis.General significanceWe hypothesis that enhancing PON2 may provide a better therapeutic potential against diabetic vascular disease.