Cyclophilin Research Articles

Chrysophanol localizes in mitochondria to promote cell death through upregulation of mitochondrial cyclophilin D in HepG2 cells

ObjectiveChrysophanol (Chry) displays potent anticancer activity in human cancer cells and animal models, but the cellular targets of Chry have not been fully defined. Herein, we speculated whether mitochondria were a target involved in Chry-induced cytotoxicity. MethodsHuman liver cancer cell line HepG2 was incubated. The cytotoxicity was evaluated by MTT assay. Mitochondria localization was evaluated by a confocal microscopy. Mitochondrial membrane potential ΔΨm was detected by TMRE staining and determined by the flow cytometer. The levels of ATP, mitochondrial superoxide anions, and GSH/GSSG were determined according to the assay kits. The apoptosis were evaluated through Hoechst33342/PI and Annexin V/PI staining, respectively. The expression of cyclophilin D (CyPD) was determined by immunoblot method, and the interaction between CyPD and Chry was analyzed by molecule docking procedure. ResultsChry itself mainly localized in mitochondria to cause mitochondrial dysfunction and cell death in HepG2 cells. As regard to the mechanism, cyclosporin A as the inhibitor for the formation of mitochondrial permeability transition pore (mPTP) moderately suppressed cell death, indicating mPTP involved in the process of cell death. Further, Chry enhanced the protein expression of Cyclophilin D (CyPD) which is a molecular componentry and a modulator of mPTP, while antioxidant N-acetyl-L-cysteine inhibited the expression of CyPD. Molecule docking procedure disclosed two hydrogen-bonds existed in CyPD-Chry complex with −11.94 kal/mol of the binding affinity value. Besides, the mtDNA-deficient HepG2-ρ0 cells were much resistant to Chry-induced cell death, indicating mtDNA at least partly participated in cell death. A combination of Chry and VP-16 produced the synergism effect toward cell viability and ΔΨm, while Chry combined with Cis-Pt elicited the antagonism effect. ConclusionTaken together, enrichment in mitochondria and actions on mPTP, CyPD and mtDNA provides an insight into the anticancer mechanism of Chry. The combination therapy for Chry with clinical drugs may deserve to further explore.

Selection and Validation of Reference Genes for RT-qPCR Analysis in Spinacia oleracea under Abiotic Stress

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is an accurate and convenient method for mRNA quantification. Selection of optimal reference gene(s) is an important step in RT-qPCR experiments. However, the stability of housekeeping genes in spinach (Spinacia oleracea) under various abiotic stresses is unclear. Evaluating the stability of candidate genes and determining the optimal gene(s) for normalization of gene expression in spinach are necessary to investigate the gene expression patterns during development and stress response. In this study, ten housekeeping genes, 18S ribosomal RNA (18S rRNA), actin, ADP ribosylation factor (ARF), cytochrome c oxidase subunit 5C (COX), cyclophilin (CYP), elongation factor 1-alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H3 (H3), 50S ribosomal protein L2 (RPL2), and tubulin alpha chain (TUBα) from spinach, were selected as candidates in roots, stems, leaves, flowers, and seedlings in response to high temperature, CdCl2, NaCl, NaHCO3, and Na2CO3 stresses. The expression of these genes was quantified by RT-qPCR and evaluated by NormFinder, BestKeeper, and geNorm. 18S rRNA, actin, ARF, COX, CYP, EF1α, GAPDH, H3, and RPL2 were detected as optimal reference genes for gene expression analysis of different organs and stress responses. The results were further confirmed by the expression pattern normalized with different reference genes of two heat-responsive genes. Here, we optimized the detection method of the gene expression pattern in spinach. Our results provide the optimal candidate reference genes which were crucial for RT-qPCR analysis.

Cyclophilin A is a mitochondrial factor that forms complexes with p23 - correlative evidence for an anti-apoptotic action.

Cyclophilin A (CyPA, also known as PPIA) is an abundant and ubiquitously expressed protein belonging to the immunophilin family, which has intrinsic peptidyl-prolyl-(cis/trans)-isomerase enzymatic activity. CyPA mediates immunosuppressive action of the cyclic undecapeptide cyclosporine A and is also involved in multiple cellular processes, such as protein folding, intracellular trafficking, signal transduction and transcriptional regulation. CyPA is abundantly expressed in cancer cells, and, owing to its chaperone nature, its expression is induced upon the onset of stress. In this study, we demonstrated that a significant pool of this immunophilin is primarily an intramitochondrial factor that migrates to the nucleus when cells are stimulated with stressors. CyPA shows anti-apoptotic action per se and the capability of forming ternary complexes with cytochrome c and the small acidic co-chaperone p23, the latter interaction being independent of the usual association of p23 with the heat-shock protein of 90 kDa, Hsp90. These CyPA•p23 complexes enhance the anti-apoptotic response of the cell, suggesting that both proteins form a functional unit, the high level of expression of which plays a significant role in cell survival.

Identification of Celastrol as a Novel Therapeutic Agent for Pulmonary Arterial Hypertension and Right Ventricular Failure Through Suppression of Bsg (Basigin)/CyPA (Cyclophilin A).

Pulmonary arterial hypertension is characterized by abnormal proliferation of pulmonary artery smooth muscle cells and vascular remodeling, which leads to right ventricular (RV) failure. Bsg (Basigin) is a transmembrane glycoprotein that promotes myofibroblast differentiation, cell proliferation, and matrix metalloproteinase activation. CyPA (cyclophilin A) binds to its receptor Bsg and promotes pulmonary artery smooth muscle cell proliferation and inflammatory cell recruitment. We previously reported that Bsg promotes cardiac fibrosis and failure in the left ventricle in response to pressure-overload in mice. However, the roles of Bsg and CyPA in RV failure remain to be elucidated. Approach and Results: First, we found that protein levels of Bsg and CyPA were upregulated in the heart of hypoxia-induced pulmonary hypertension (PH) in mice and monocrotaline-induced PH in rats. Furthermore, cardiomyocyte-specific Bsg-overexpressing mice showed exacerbated RV hypertrophy, fibrosis, and dysfunction compared with their littermates under chronic hypoxia and pulmonary artery banding. Treatment with celastrol, which we identified as a suppressor of Bsg and CyPA by drug screening, decreased proliferation, reactive oxygen species, and inflammatory cytokines in pulmonary artery smooth muscle cells. Furthermore, celastrol treatment ameliorated RV systolic pressure, hypertrophy, fibrosis, and dysfunction in hypoxia-induced PH in mice and SU5416/hypoxia-induced PH in rats with reduced Bsg, CyPA, and inflammatory cytokines in the hearts and lungs. These results indicate that elevated Bsg in pressure-overloaded RV exacerbates RV dysfunction and that celastrol ameliorates RV dysfunction in PH model animals by suppressing Bsg and its ligand CyPA. Thus, celastrol can be a novel drug for PH and RV failure that targets Bsg and CyPA. Graphic Abstract: A graphic abstract is available for this article.

The effect of mitochondrial calcium uniporter and cyclophilin D knockout on resistance of brain mitochondria to Ca2+-induced damage

The mitochondrial calcium uniporter (MCU) and cyclophilin D (CyD) are key players in induction of the permeability transition pore (PTP), which leads to mitochondrial depolarization and swelling, the major signs of Ca2+-induced mitochondrial damage. Mitochondrial depolarization inhibits ATP production, whereas swelling results in the release of mitochondrial pro-apoptotic proteins. The extent to which simultaneous deletion of MCU and CyD inhibits PTP induction and prevents damage of brain mitochondria is not clear. Here, we investigated the effects of MCU and CyD deletion on the propensity for PTP induction using mitochondria isolated from the brains of MCU-KO, CyD-KO, and newly created MCU/CyD-double knockout (DKO) mice. Neither deletion of MCU nor of CyD affected respiration or membrane potential in mitochondria isolated from the brains of these mice. Mitochondria from MCU-KO and MCU/CyD-DKO mice displayed reduced Ca2+ uptake and diminished extent of PTP induction. The Ca2+ uptake by mitochondria from CyD-KO mice was increased compared with mitochondria from WT mice. Deletion of CyD prevented mitochondrial swelling and resulted in transient depolarization in response to Ca2+, but it did not prevent Ca2+-induced delayed mitochondrial depolarization. Mitochondria from MCU/CyD-DKO mice did not swell in response to Ca2+, but they did exhibit mild sustained depolarization. Dibucaine, an inhibitor of the Ca2+-activated mitochondrial phospholipase A2, attenuated and bovine serum albumin completely eliminated the sustained depolarization. This suggests the involvement of phospholipase A2 and free fatty acids. Thus, in addition to induction of the classical PTP, alternative deleterious mechanisms may contribute to mitochondrial damage following exposure to elevated Ca2+.

Trypanosoma cruzi infection in Cyclophilin D deficient mice

Trypanosoma cruzi is the causative agent of Chagas disease, which is endemic in Latin America and around the world through mother to child transmission. The heart is the organ most frequently affected in the chronic stage of the human infection and depends on mitochondria for the required energy for its activity. Cyclophilins are involved in protein folding and the mitochondrial isoform, Cyclophilin D (CyPD), has a crucial role in the opening of the mitochondrial permeability transition pore. In the present study, we infected CyPD deficient mice, with ablation of the Ppif gene, with T. cruzi parasites and the course of the infection was analyzed. Parasite load, quantified by PCR, was significantly lower in skeletal and cardiac tissues of Ppif-/- mice compared to wild type mice. In vitro cultured cardiomyocytes and macrophages from mice lacking CyPD exhibited lower percentage of infected cells and number of intracellular parasites than those observed for wild type mice. Although histopathological analysis of heart and mRNA of heart cytokines showed differences between T. cruzi-infected mice compared to the uninfected animals, no significant differences were found mice due to the ablation of the Ppif gene. Our results suggest that cells deficient for mitochondrial CyPD, inhibited for the mitochondrial membrane potential collapse, reduces the severity of parasite aggression and spread of cellular infection.

Dexmedetomidine Ameliorates Hippocampus Injury and Cognitive Dysfunction Induced by Hepatic Ischemia/Reperfusion by Activating SIRT3-Mediated Mitophagy and Inhibiting Activation of the NLRP3 Inflammasome in Young Rats.

Hepatic ischemia-reperfusion (HIR) has been proven to trigger oxidative stress and pyroptosis in the hippocampus. Sirtuin 3 (SIRT3) is an essential mitochondrial protein deacetylase regulating oxidative stress and mitophagy. Dexmedetomidine (Dex) has been demonstrated to confer neuroprotection in different brain injury models. However, whether the protective effects of Dex following HIR are orchestrated by activation of SIRT3-mediated mitophagy and inhibition of NOD-like receptor protein 3 (NLRP3) inflammasome activation remains unknown. Herein, two-week-old rats were treated with Dex or a selective SIRT3 inhibitor (3-TYP)/autophagy inhibitor (3-MA) and then subjected to HIR. The results revealed that Dex treatment effectively attenuated neuroinflammation and cognitive deficits via upregulating SIRT3 expression and activity. Furthermore, Dex treatment inhibited the activation of NLRP3 inflammasome, while 3-TYP and 3-MA eliminated the protective effects of Dex, suggesting that SIRT3-mediated mitophagy executes the protective effects of Dex. Moreover, 3-TYP treatment downregulated the expression level of SIRT3 downstream proteins: forkhead-box-protein 3α (FOXO3α), superoxide dismutase 2 (SOD2), peroxiredoxin 3 (PRDX3), and cyclophilin D (CYP-D), which were barely influenced by 3-MA treatment. Notably, both 3-TYP and 3-MA were able to offset the antioxidative and antiapoptosis effects of Dex, indicating that SIRT3-mediated mitophagy may be the last step and the major pathway executing the neuroprotective effects of Dex. In conclusion, Dex inhibits HIR-induced NLRP3 inflammasome activation mainly by triggering SIRT3-mediated mitophagy.

Expression of Cyclophilin A in Coronary Artery Plaque with Intraplaque Hemorrhage Is More Frequent in Deceased Patients Who Had Impaired Kidney Function.

Patients with impaired kidney function have a high frequency of intraplaque hemorrhage (IPH) in their coronary arteries. Levels of cyclophilin A (CyPA), an indirect matrix metalloproteinase inducer, are increased in deceased patients who had impaired kidney function. In this study, we have examined the relationship between IPH and CyPA.We examined 47 samples of coronary plaque from 27 cadavers with coronary stenosis. These sections, all with > 50% coronary stenosis, were stained with an antibody against CyPA and the expression of CyPA was semi-quantified. Cadavers and plaques were classified into one of two groups depending on the presence or absence of IPH. IPH was defined as the presence of red blood cells stained with hematoxylin and eosin (HE) indicative of overt acute hemorrhage.In an individual analysis, estimation of glomerular filtration rate (eGFR) in the IPH group was significantly lower than that in the non-IPH group (P = 0.002). In a histological analysis, the percentage of stained area of CyPA in the IPH group was significantly higher than that in the non-IPH group (P < 0.0001).IPH was associated with a significantly higher expression of CyPA in this study. In addition, patients with IPH in their coronary arteries had significantly impaired kidney function.

Structural and functional analyses of the PPIase domain of Arabidopsisthaliana CYP71 reveal its catalytic activity toward histone H3.

Arabidopsisthaliana CYP71 (AtCYP71) is a chromatin-remodeling protein that promotes shoot apical meristem (SAM) differentiation. The N terminus of AtCYP71 contains a noncanonical WD domain, and the C terminus contains an enzymatic peptidyl-prolyl isomerase (PPIase) cyclophilin (CYP) domain. To date, there has been no characterization of CYP71, and its mode of action remains unknown. Here, we report the crystal structure of the CYP domain of AtCYP71 at 1.9Å resolution. The structure shows key differences when compared to the canonical CYP fold of human CypA. To the best our knowledge, this is the first A.thaliana CYP structure with a conserved active site loop. Using nuclear magnetic resonance spectroscopy, we demonstrate that the CYP domain is active toward histone H3. Our findings suggest that the PPIase activity of the CYP domain is important for the function of AtCYP71 in chromatin remodeling during organogenesis.

Selection of Reference Genes for qRT-PCR Analysis in Medicinal Plant Glycyrrhiza under Abiotic Stresses and Hormonal Treatments.

Best known as licorice, Glycyrrhiza Linn., a genus of herbaceous perennial legume, has been used as a traditional herbal medicine in Asia and a flavoring agent for tobacco and food industry in Europe and America. Abiotic stresses and hormonal treatments can significantly impact the development and metabolism of secondary metabolites in Glycyrrhiza. To better understand the biosynthesis of the trace-amount bioactive compounds, we first screened for the suitable reference genes for quantitative real-time reverse transcription PCR (qRT-PCR) analysis in Glycyrrhiza. The expression profiles of 14 candidate reference genes, including Actin1 (ACT), Clathrin complex AP1 (CAC), Cyclophilin (CYP), Heat-shock protein 40 (DNAJ), Dehydration responsive element binding gene (DREB), Translation elongation factor1 (EF1), Ras related protein (RAN), Translation initiation factor (TIF1), β-Tubulin (TUB), Ubiquitin-conjugating enzyme E2 (UBC2), ATP binding-box transpoter 2 (ABCC2), COP9 signal compex subunit 3 (COPS3), Citrate synthase (CS), and R3H domain protein 2 (R3HDM2) from two congeneric species, Glycyrrhiza uralensis F. and Glycyrrhiza inflata B., were examined under abiotic stresses (osmotic and salinity) and hormonal treatments (Abscisic acid (ABA) and methyl jasmonic acid (MeJA)) using a panel of software, including geNorm, NormFinder, BestKeeper, and Delta CT. The overall stability, however, was provided by RefFinder, a comprehensive ranking system integrating inputs from all four algorithms. In G. uralensis, the most stable reference genes under osmotic stress, salt stress, ABA treatment, and MeJA treatment were TIF1, DNAJ, CS, and ABCC2 for leaves and DNAJ, DREB, CAC, and CAC for roots, respectively. In comparison, the top ranked genes were TUB, CAC, UBC2, and RAN for leaves and TIF1, ABCC2, CAC, and UBC2 for roots, respectively, under stress and hormonal treatments in G. inflata. ACT and TIF1, on the other hand, were the least stable genes under the most experimental conditions in the two congeneric species. Finally, our survey of the reference genes in legume shows that EF, ACT, UBC2, and TUB were the top choices for the abiotic stresses while EF, UBC2, CAC, and ABCC2 were recommended for the hormonal treatments in Leguminosae. Our combined results provide reliable normalizers for accurate gene quantifications in Glycyrrhiza species, which will allow us to exploit its medicinal potential in general and antiviral activities in particular.

Detachment Activated CyPA/CD147 Induces Cancer Stem Cell Potential in Non-stem Breast Cancer Cells.

BackgroundCancer stem cells (CSCs), responsible for cancer metastasis and recurrence, are generated from non-CSCs after chemo-radiation therapy. This study investigated the induction of CSC potential in non-stem breast cancer cells and the underlying molecular mechanisms in detachment culture.MethodsBulk breast cancer cells, or sorted non-CSCs and CSCs were cultured under an attached or detached condition to assess CSC numbers, ability to form tumor spheres, expression of stemness markers, and chemoresistance. Lentivirus carrying CD147 shRNA or cDNA was used to manipulate CD147 expression, while CD147 ligand recombinant cyclophilin A (CyPA) or its inhibitor was used to activate or inhibit CD147 signaling.ResultsDetachment promoted anoikis resistance, chemoresistance, sphere formation, self-renewal, and expression of stemness markers in breast cancer cells. Detachment increased functional ALDH+ or CD44highCD24–/low CSCs, and induced CSC potential in ALDH– or CD44lowCD24high non-CSCs. Upon detachment, both CD147 expression and CyPA secretion were enhanced, and CyPA-CD147 activation mediated detachment induced CSC potential in non-CSCs via STAT3 signaling. Clinically, CD147 and pSTAT3 were highly co-expressed and correlated with poor overall survival and tumor recurrence in breast cancer patients.ConclusionThis study demonstrates that detachment induces the generation of CSCs from non-stem breast cancer cells via CyPA-CD147 signaling, indicating that targeting CD147 may serve as a potential novel therapeutic strategy for lethal metastatic breast cancer by eliminating induced CSCs.

Cyclophilin A (CyPA) as a Novel Biomarker for Early Detection of Diabetic Nephropathy in an Animal Model.

Background and AimType 2 diabetes mellitus (DM) is the most common single cause of the end-stage renal disease (ESRD). Cyclophilin A (CyPA) is an 18-kD protein. The connection between diabetic nephropathy (DN) and the secreted form of CyPA (sCyPA) has been elucidated in this study that aims to investigate sCyPA correlation with renal dysfunction.Materials and MethodsThirty-four male adult Wistar rats weighing 180–220 g were used. Animals were divided into a study group and a control group, 17 rats in each. Streptozotocin (STZ) and nicotine amide were used to damage some pancreatic cells for induction of type 2 DM. Comparison was made between the study and the control groups. Moreover, a comparison was made between the members of the study group before and after induction of DN.ResultsThe rat model that exhibited a higher concentration of urinary sCyPA was detected early in the eighth week. There was a significantly higher level of 24-h urinary CyPA in the study group compared to the control group (p-value=0.004) and there was a significant elevation in the 24-h urinary Cyp-A in the study group after injection of STZ compared to the values before injection (p-value <0.001). Immunohistochemical analysis of renal tissue revealed that the mean expression of CyPA was higher in the study group than in the control group. For the urinary 24-h CYP-A, using a cutoff of 1.15 ng/mL, the accuracy was 72.4%, sensitivity was (77.8%) and specificity was (67%).ConclusionAccording to this animal study, we proved that CyPA is a valuable marker for DN. It is a more sensitive, noninvasive and rapid biomarker for early detection of any renal affection in human diabetic patients.

Cyclophilin A plays an important role in embryo implantation through activating Stat3.

Embryo implantation is a crucial step for the successful establishment of mammalian pregnancy. Cyclophilin A (CYPA) is a ubiquitously expressed intracellular protein and is secreted in response to inflammatory stimuli to regulate diverse cellular functions. However, there are currently no reports about the role of CYPA in embryo implantation. Here, we examine the expression pattern of CYPA during mouse early pregnancy and explore the potential role of CYPA during implantation. CYPA is expressed in the subluminal stroma surrounding the implanting blastocyst on day 5 of pregnancy, but not at inter-implantation sites. In ovariectomized mice, estrogen and progesterone significantly stimulate CYPA expression. When pregnant mice are injected intraperitoneally with CYPA inhibitor, the numbers of implantation sites are significantly reduced. Using an in vitro stromal cell culture system, Ppia siRNA knockdown of CYPA and CYPA-specific inhibitor treatment partially inhibits levels of CD147, MMP3 and MMP9. Decreased CYPA expression also significantly inhibits Stat3 activity and expands estrogen responsiveness. Taken together, CYPA may play an important role during mouse embryo implantation.

The Unique Cysteine of F-ATP Synthase OSCP Subunit Participates in Modulation of the Permeability Transition Pore

The mitochondrial permeability transition pore (PTP) is a Ca2+-activated channel that plays a key role in cell death. Thiol oxidation facilitates PTP opening, yet the targets and molecular mechanisms still await a definition. Here, we investigate the role of C141 of F-ATP synthase oligomycin sensitivity conferral protein (OSCP) subunit in PTP modulation by oxidation. We find that the OSCP C141S mutation confers resistance to PTP opening and cell death by diamide and MitoParaquat only when cyclophilin D (CyPD) has been ablated, a protective role that can be explained by CyPD shielding C141 from oxidants. The mutation decreases apoptosis in zebrafish embryos, indicating that this OSCP residue is involved in development. Site-directed mutagenesis in yeast suggests that other conserved cysteines in the α, γ, and c subunits of F-ATP synthase are not involved in PTP modulation. Thus, OSCP provides a strategic site that regulates PTP opening by the interplay between CyPD (un)binding and thiol oxidation-reduction.

Extracellular cyclophilin A induces cardiac hypertrophy via the ERK/p47phox pathway

Excessive reactive oxygen species (ROS) are a critical driver of cardiac hypertrophy developing into heart failure. Cyclophilin A (CyPA), a member of the cyclophilin family, has been highlighted as a main secreted ROS-induced factor. The mechanism by which extracellular CyPA interacts with cardiomyocytes is unclear. We showed that extracellular CyPA is upregulated in cardiac hypertrophy rats and expressed around hypertrophic cardiomyocytes. Cell experiments further confirmed that extracellular CyPA induces H9c2 cardiomyocytes hypertrophy via ROS generation. Extracellular CyPA-induced ROS is derived from nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, and extracellular CyPA activates p47phox membrane translocation through ERK1/2 pathway. When blocking extracellular matrix metalloproteinase inducer (EMMPRIN), most of the extracellular CyPA effects were significantly inhibited. The current study shows that extracellular CyPA is one of the key factors linking oxidative stress and cardiac hypertrophy, and may be a potential target for cardiac hypertrophy therapy.

Downregulation of CyclophilinA/CD147 Axis Induces Cell Apoptosis and Inhibits Glioma Aggressiveness.

Gliomas are the most common primary tumors in the brain with poor prognosis. Previous studies have detected high expression of Cyclophilin A (CyPA) and CD147, respectively, in glioma. However, the correlation between their expressions and glioma prognosis remains unclear. Here, we investigated the expression of CyPA and CD147 in different types of glioma and characterized their relationships with clinical features, prognosis, and cell proliferation. Results showed that CyPA and CD147 expressions were elevated in higher grade gliomas. Moreover, the knockdown of CyPA and CD147 by RNA interference significantly induced cell express apoptosis biomarkers such as Annexin V and inhibited proliferation biomarkers like EdU in glioma cells. In summary, our findings revealed that high expression of CyPA and CD147 correlated with glioma grades. Moreover, downregulation of the Cyclophilin A/CD147 axis induces cell apoptosis and inhibits glioma aggressiveness. Those indicating CyPA and CD147 could be used as both potential predictive biomarkers and a potential therapeutic target.

The aged hematopoietic system promotes hippocampal-dependent cognitive decline.

The aged systemic milieu promotes cellular and cognitive impairments in the hippocampus. Here, we report that aging of the hematopoietic system directly contributes to the pro‐aging effects of old blood on cognition. Using a heterochronic hematopoietic stem cell (HSC) transplantation model (in which the blood of young mice is reconstituted with old HSCs), we find that exposure to an old hematopoietic system inhibits hippocampal neurogenesis, decreases synaptic marker expression, and impairs cognition. We identify a number of factors elevated in the blood of young mice reconstituted with old HSCs, of which cyclophilin A (CyPA) acts as a pro‐aging factor. Increased systemic levels of CyPA impair cognition in young mice, while inhibition of CyPA in aged mice improves cognition. Together, these data identify age‐related changes in the hematopoietic system as drivers of hippocampal aging.

The Release of Cyclophilin A from Rapamycin-Stimulated Vascular Smooth Muscle Cells Mediated by Myosin II Activation: Involvement of Apoptosis but Not Autophagy

Introduction: The exocytosis of cyclophilin A (CyPA) by a vesicular pathway in response to reactive oxygen species has been determined. However, other sources of extracellular CyPA remain obscure. Objective: The aim of this study was to determine the role of autophagy in the secretion of CyPA. Methods and Results: Rapamycin induced the activation of autophagy and release of CyPA from primary cultured rat aortic smooth muscle cells (RASMCs). However, inhibition of autophagy by knockdown of Atg7 or chloroquine did not affect the rapamycin-induced release of CyPA. With the exception of myosin II activity, rho-associated coiled-coil kinase (ROCK), actin remodelling, and synaptic vesicles were not implicated in the release of rapamycin-induced CyPA. Finally, we confirmed that rapamycin-induced extracellular CyPA originated from apoptotic RASMCs. Furthermore, the decreased activation of myosin II by blebbistatin blocked the release of CyPA from apoptotic RASMCs induced by rapamycin. Conclusions: Rapamycin induced the release of CyPA from apoptotic RASMCs but did not affect exocytosis through autophagosomes. ROCK, actin remodelling, and synaptic vesicles were not involved in the apoptosis-related release of CyPA. Myosin II activation modulated the apoptosis of vascular smooth muscle cells and the release of CyPA from rapamycin-induced apoptotic cell death.

Comparative Effects of Alpha- and Gamma-Tocopherol on Mitochondrial Functions in Alzheimer\u2019s Disease In Vitro Model

Vitamin E acts as an antioxidant and reduces the level of reactive oxygen species (ROS) in Alzheimer’s disease (AD). Alpha-tocopherol (ATF) is the most widely studied form of vitamin E besides gamma-tocopherol (GTF) which also shows beneficial effects in AD. The levels of amyloid-beta (Aβ) and amyloid precursor protein (APP) increased in the brains of AD patients, and mutations in the APP gene are known to enhance the production of Aβ. Mitochondrial function was shown to be affected by the increased level of Aβ and may induce cell death. Here, we aimed to compare the effects of ATF and GTF on their ability to reduce Aβ level, modulate mitochondrial function and reduce the apoptosis marker in SH-SY5Y cells stably transfected with the wild-type or mutant form of the APP gene. The Aβ level was measured by ELISA, the mitochondrial ROS and ATP level were quantified by fluorescence and luciferase assay respectively whereas the complex V enzyme activity was measured by spectrophotometry. The expressions of genes involved in the regulation of mitochondrial membrane permeability such as voltage dependent anion channel (VDAC1), adenine nucleotide translocase (ANT), and cyclophilin D (CYPD) were determined by quantitative real-time polymerase chain reaction (qRT-PCR), while the expressions of cyclophilin D (CypD), cytochrome c, Bcl2 associated X (BAX), B cell lymphoma-2 (Bcl-2), and pro-caspase-3 were determined by western blot. Our results showed that mitochondrial ROS level was elevated accompanied by decreased ATP level and complex V enzyme activity in SH-SY5Y cells expressing the mutant APP gene (p < 0.05). Treatment with both ATF and GTF reduced the mitochondrial ROS level with maximum reduction was observed in the cells treated with high concentrations of ATF and GTF (p < 0.05). However, only GTF at 80 µM significantly increase the ATP level and complex V enzyme activity (p < 0.05). VDAC1 and CYPD were downregulated and CypD protein was significantly overexpressed in cells transfected with the wild-type (WT) and mutant APP gene (p < 0.05). Cytochrome c release, the ratio of BAX/Bcl-2, and pro-caspase-3 expression increased in cells expressing mutated APP gene (p < 0.05). The expression of CypD and pro-caspase 3 protein, and the ratio of BAX/Bcl-2 were increased in the following order; SH-SY5Y-APP-WT < SH-SY5Y-APP Swe <SH-SY5Y-APP Swe/Ind. Treatment with both ATF and GTF reduced the release of cytochrome c and the ratio of BAX/Bcl-2. However, only GTF significantly reduced the expression of CypD and pro-caspase-3, suggestive of its unique role in AD. In conclusion, GTF has an effect that was not shown by ATF and thus suggest its potential role in the development of therapeutic agents for AD.

Concentrations of Serum Cyclophilin A in Patients With Bell Palsy.

Cyclophilin A (CyPA) is the responder protein to stimuli that cause inflammation. To date, no association among CyPA and Bell palsy has been reported. The concentrations of Serum CyPA were measured in 90 healthy participants and 92 patients with Bell palsy. Serum samples of patients and the control group were compared on the basis of CyPA levels. Facial latency and amplitude values on electromyography were evaluated and compared with serum CyPA concentrations. A total of 28, 37, 19, and 8 patients had grade 3, 4, 5, and 6 facial palsy cases, respectively. Comparing the control group and the patient group showed significant differences in CyPA values (P < 0.001). Cyclophilin A value can be evaluated as a marker with high disease discrimination capability. The results also showed that at low CyPA, the average recovery time was shorter than that of high CyPA (41.6 ± 5.7 days vs 62.8 ± 10.2 days, P = 0.036). We found no statistically significant relationship between electromyography parameters and CyPA level. (Facial latency: r: -0.014, P: 0.948; facial amplitude r: -0.081, P: 0.713). Serum CyPA concentrations increased in response to inflammation in Bell palsy patients. However, CyPA could not be used as an early prognostic marker in Bell palsy, low CyPA indicates the shorter average recovery time than that of high CyPA.