Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease. At present, no definite ALS biomarkers are available. In this study, exosomes from the plasma of patients with ALS and healthy controls were extracted, and differentially expressed exosomal proteins were compared. Among them, the expression of exosomal coronin-1a (CORO1A) was 5.3-fold higher than that in the controls. CORO1A increased with disease progression at a certain proportion in the plasma of patients with ALS and in the spinal cord of ALS mice. CORO1A was also overexpressed in NSC-34 motor neuron-like cells, and apoptosis, oxidative stress, and autophagic protein expression were evaluated. CORO1A overexpression resulted in increased apoptosis and oxidative stress, overactivated autophagy, and hindered the formation of autolysosomes. Moreover, CORO1A activated Ca²⁺-dependent phosphatase calcineurin, thereby blocking the fusion of autophagosomes and lysosomes. The inhibition of calcineurin activation by cyclosporin A reversed the damaged autolysosomes. In conclusion, the role of CORO1A in ALS pathogenesis was discovered, potentially affecting the disease onset and progression by blocking autophagic flux. Therefore, CORO1A might be a potential biomarker and therapeutic target for ALS.
Mice ; Animals ; Amyotrophic Lateral Sclerosis/pathology* ; Calcineurin/metabolism* ; Motor Neurons/pathology* ; Microfilament Proteins/metabolism* ; Cytoskeletal Proteins/metabolism*

OBJECTIVETo investigate the effects of evodiamine (Evo), a component of Evodiaminedia rutaecarpa (Juss.) Benth, on cardiomyocyte hypertrophy induced by angiotensin II (Ang II) and further explore the potential mechanisms.

METHODSCardiomyocytes from neonatal Sprague Dawley rats were isolated and characterized, and then the cadiomyocyte cultures were randomly divided into control, model (Ang II 0.1 μmol/L), and Evo (0.03, 0.3, 3 μmol/L) groups. The cardiomyocyte surface area, protein level, intracellular free calcium ([Ca]) concentration, activity of nitric oxide synthase (NOS) and content of nitric oxide (NO) were measured, respectively. The mRNA expressions of atrial natriuretic factor (ANF), calcineurin (CaN), extracellular signal-regulated kinase-2 (ERK-2), and endothelial nitric oxide synthase (eNOS) of cardiomyocytes were analyzed by real-time reverse transcriptionpolymerase chain reaction. The protein expressions of calcineurin catalytic subunit (CnA) and mitogen-activated protein kinase phosphatase-1 (MKP-1) were detected by Western blot analysis.

RESULTSCompared with the control group, Ang II induced cardiomyocytes hypertrophy, as evidenced by increased cardiomyocyte surface area, protein content, and ANF mRNA expression; increased intracellular free calcium ([Ca]) concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but decreased MKP-1 protein expression (P<0.05 or P<0.01). Compared with Ang II, Evo (0.3, 3 μmol/L) significantly attenuated Ang II-induced cardiomyocyte hypertrophy, decreased the [Ca] concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but increased MKP-1 protein expression (P<0.05 or P<0.01). Most interestingly, Evo increased the NOS activity and NO production, and upregulated the eNOS mRNA expression (P<0.05).

CONCLUSIONEvo signifificantly attenuated Ang II-induced cardiomyocyte hypertrophy, and this effect was partly due to promotion of NO production, reduction of [Ca]i concentration, and inhibition of CaN and ERK-2 signal transduction pathways.

Angiotensin II ; Animals ; Atrial Natriuretic Factor ; metabolism ; Calcineurin ; genetics ; metabolism ; Calcium ; metabolism ; Dual Specificity Phosphatase 1 ; genetics ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; genetics ; metabolism ; Hypertrophy ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Quinazolines ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley

OBJECTIVE: To investigate the effects of hydrogen sulfide (HS) on the negatively regulation of cardiomyocyte hypertrophy and the relationship between the effect of HS with miRNA-133a-mediated Ca/calcineurin/NFATc4 signal pathway. METHODS: Cardiomyocyte hypertrophy was induced by isoproterenol (ISO). The cell surface area was measured by image analysis system (Leica). The expression of brain natriuretic peptide(BNP), β-myosin heavy chain(β-MHC), cystathionase (CSE), miRNA-133a, calcineurin (CaN) were detected by qRT-PCR. The protein expressions of CaN、nuclear factors of activated T cells (NFATc4) were detected by Western blot. The concentration of HS in the cardiomyocyte was detected by Elisa. The concentration of intracellular calcium was measured by calcium imaging using confocal microscope. The nuclear translocation of NFATc4 was checked by immuno-fluorescence cell staining technique. RESULTS: ①The level of system of CSE/HS and expression of miRNA-133a were significantly reduced in cardiomyocyte hypertrophy. Pretreatment with NaHS increased the concentration of HS and the expression of miRNA-133a mRNA in cardiomyocytes, and suppressed cardiomyocyte hypertrophy. ②The concentration of intracellular calcium, the expression of CaN and nulear protein NFATc4 were significantly increased, and the nuclear translocation of NFATc4 were obviously enhanced in cardiomyocyte hypertrophy. NaHS pretreatment markedly inhibited these effects of ISO induced cardiomyocyte hypertrophy. ③Application of antagomir-133a reversed the inhibitory effects of NaHS on cardiomyocyte hypertrophy, and increased the influx of intracellular calcium, and elevated the expression of CaN and nuclear protein NFATc4, and enhanced the nuclear translocation of NFATc4. CONCLUSIONS HS can negatively regulate cardiomyocyte hypertrophy. The effects might be associated with HS increasing expression of miRNA-133a and inhibiting inactivation of Ca/calcineurin/NFATc4 signal pathway.
Animals ; Calcineurin ; metabolism ; Cardiomegaly ; chemically induced ; metabolism ; Cells, Cultured ; Cystathionine gamma-Lyase ; metabolism ; Hydrogen Sulfide ; metabolism ; MicroRNAs ; metabolism ; Myocytes, Cardiac ; metabolism ; Myosin Heavy Chains ; metabolism ; NFATC Transcription Factors ; metabolism ; Natriuretic Peptide, Brain ; metabolism ; Nerve Tissue Proteins ; metabolism ; Rats ; Signal Transduction

OBJECTIVETo observe the effect of total ginsenosides (TG) on monocrotaline (MCT) induced right ventricular hypertrophy rats, and to explore its correlation with calcineurin (CaN) pathway.

METHODSFifty male Sprague Dawley rats were randomly divided into the normal control group, the MCT model group, and the low, middle, high dose TG treatment groups, 10 in each group. All medication was performed by peritoneal injection for 18 days. Right ventricular peak systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and right ventricular weight/body weight (RVW/BW) were measured. Intracellular free calcium concentrations were measured by Ca2+ fluorescence indicator Fura2/AM. The atrial natriuretic factor (ANF) and CaN mRNA expression of the myocardial tissue were quantitatively analyzed by Real-time PCR. The protein expression of CaN was detected by Western blot.

RESULTSCompared with the MCT model group, preventive treatment of TG at the 3 doses could significantly reduce RVSP, RVHI, RVW/BW, and ANF mRNA expression, and decrease Ca2+ concentration in myocardial cells, CaN mRNA and protein expression in the myocardial tissue.

CONCLUSIONTG could obviously improve MCT-induced right ventricular hypertrophy, which was possibly achieved through suppressing MCT-activated CaN signal transduction.

Animals ; Atrial Natriuretic Factor ; Calcineurin ; metabolism ; Calcineurin Inhibitors ; therapeutic use ; Ginsenosides ; therapeutic use ; Heart Ventricles ; Hypertrophy, Right Ventricular ; drug therapy ; metabolism ; Male ; Monocrotaline ; Myocardium ; Myocytes, Cardiac ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Signal Transduction

OBJECTIVEThis study investigated the role and mechanism of calcineurin (CaN)-nuclear factor of activated T cells (NFAT) pathway in the myoblast apoptosis induced by cyclic tensile strain.

METHODSMyoblasts were cultured using an in vitro-mechanical stimulation model and imposed with tension for different hours with a multi-channel cell stress loading system. Cyclosporine (CsA) was used as CaN inhibitor to clarify the role of CaN in the apoptosis induced by cyclic stress. Hochest 33258 staining and flow cytometry detection were performed to detect the apoptotic cells. Real-time polymerase chain reaction was conducted to detect the mRNA expression of CaN and NFAT. Protein levels of NFAT3 were evaluated by Western blot.

RESULTSThe apoptosis rate increased with the extension of loading time. The mRNA expression of the CaN subunits, CnA and CnB, and the protein levels of NFAT3 also increased. When the myoblasts were incubated with CsA, the apoptosis rate decreased, the mRNA expression of CnA and NFAT3 significantly decreased, and the NFAT3 protein expression levels became significantly lower than those of the groups without CsA.

CONCLUSIONContinuous cyclic tensile stress can induce myoblast apoptosis. The CaN-NFAT signaling pathway may be involved in the cyclic stretch-induced apoptosis of myoblasts.

Apoptosis ; Calcineurin ; genetics ; Cyclosporine ; Flow Cytometry ; Myoblasts ; physiology ; NFATC Transcription Factors ; metabolism ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; T-Lymphocytes

This study was aimed to compare the differential expressions of calcineurin (PP2B, PP3) in the mouse Pre-B cell lines (S9) and the tumor cell lines (S4C2) derived from pre-B lymphocytes, and to clarify its possible mechanism involving in the leukemia cell apoptosis. The quantitative real-time PCR was used to detect the differential expressions of H2AX-associated phosphakinase ATM, ATR, DNA-PKs, JNK1, P38 and the γ-H2AX-related phosphatase PP1, PP2A, calcineurin, PP4, PP6, PP5 between S9 and S4C2 cell lines. CCK-8 assay and flow cytometry were used to detect the effect of imatinib (IM) and cyclosporine A (CsA) on cytotoxicity and apoptosis of 2 cell lines. The Western blot was used to detect the effects of 2 drugs on apoptosis of S9 and S4C2 cell lines. The results showed that the expression level of calcineurin gene in the leukemia cell S4C2 was about 3.5 times of that in S9 cells, while the expression of other genes in these 2 kinds of cells was not significantly different. The apoptosis and toxicity of IM and CsA on S4C2 cells was significantly stronger than that on S9 cells. The expression level of calcineurin in S4C2 cells was higher than that in S9 cells.When CsA inhibited the calcineurin activity, the expression of DNA damage marker γ-H2AX in S9 cells was significantly lower than that in S4C2 cells,while the expression level of γ-H2AX between the two cell lines was no significantly different after treatment with imatinib, the expression level of γ-H2AX in S9 cells was lower than that in S4C2 cells when the two drugs were combined. It is concluded that the calcineurin plays a role of anti-apoptosis in B leukemic cells, cyclosporine A can promote the leukemia cell apoptosis.
Animals ; Apoptosis ; Calcineurin ; metabolism ; Cell Line, Tumor ; Cyclosporine ; DNA Damage ; Flow Cytometry ; Leukemia ; metabolism ; Mice ; Precursor Cells, B-Lymphoid ; metabolism ; Real-Time Polymerase Chain Reaction

OBJECTIVETo investigate the effects of rituximab (RTX) in children with steroid-dependent nephrotic syndrome.

METHODFive cases of children with steroid-dependent nephrotic syndrome seen from May 2012 to February 2013 in whom only steroid plus calcineurin inhibitor was effective and the disease recurred on reduction of dose were enrolled into this study, including 3 males and 2 females. Calcineurin inhibitors were stopped and steroids was changed to full dose. After the general condition improved, RTX was given at a dose of 375 mg/m(2), once a week for a total of three times for one course. After urine protein became negative for five days, the dose of steroid was changed to 2 mg/kg every other day, thereafter the dose was reduced by 5 mg per every 2 weeks, until discontinuation. After regular monitoring, when peripheral blood B cells were ≥ 3%, a second RTX was added.

RESULTUrine protein was negative in 2-7 days in 5 patients after the first RTX treatment. Before treatment B lymphocytes in peripheral blood was 7.8% to 13.0% and after the first course of RTX treatment decreased to 0 in the first 6 to 8 months at the beginning of recovery, while in the first 7 to 10 months to 3.3%-6.1%, after a second RTX was given, B lymphocytes were reduced to 0, but in two cases (cases 1 and 3) B lymphocytes rose again at 16 and 17 months, in the first 17 and 18 months rose to 4.16% and 4.17%, RTX was given once again respectively. B lymphocytes were reduced to 0 again. Currently the 5 patients continued to be negative for urine protein, maintaining remission for 12 to 20 months.RTX infusion had no significant side effects, and side effects of steroid and calcineurin inhibitor disappeared.

CONCLUSIONIn children with steroid-dependent and only calcineurin inhibitor effective nephritic syndrome, relapse may still occur after improvement of nephrotic syndrome, after the first RTX treatment, regular monitoring of B lymphocytes, RTX supplementary treatment in advance can help discontinuation of steroids and immunosuppressive agents and maintain remission.

Anti-Inflammatory Agents ; administration & dosage ; therapeutic use ; Antibodies, Monoclonal, Murine-Derived ; administration & dosage ; therapeutic use ; Antigens, CD19 ; metabolism ; B-Lymphocytes ; drug effects ; metabolism ; Calcineurin Inhibitors ; administration & dosage ; therapeutic use ; Child ; Child, Preschool ; Drug Administration Schedule ; Female ; Follow-Up Studies ; Humans ; Immunosuppressive Agents ; administration & dosage ; therapeutic use ; Lymphocyte Count ; Male ; Nephrotic Syndrome ; drug therapy ; metabolism ; Proteinuria ; urine ; Recurrence ; Remission Induction ; Rituximab ; Treatment Outcome

OBJECTIVEThe purpose of the present study was to observe the changes in CD4+CD25+Nrp1+Treg cells after irradiation with different doses and explore the possible molecular mechanisms involved.

METHODSICR mice and mouse lymphoma cell line (EL-4 cells) was used. The expressions of CD4, CD25, Nrp1, calcineurin and PKC-α were detected by flow cytometry. The expressions of TGF-β1, IL-10, PKA and cAMP were estimated with ELISA.

RESULTSAt 12 h after irradiation, the expression of Nrp1 increased significantly in 4.0 Gy group, compared with sham-irradiation group (P<0.05) in the spleen and thymus, respectively, when ICR mice received whole-body irradiation (WBI). Meanwhile the synthesis of Interleukin 10 (IL-10) and transforming growth factor-β1 (TGF-β1) increased significantly after high dose irradiation (HDR) (> or = 1.0 Gy). In addition, the expression of cAMP and PKA protein increased, while PKC-α, calcineurin decreased at 12h in thymus cells after 4.0 Gy X-irradiation. While TGF-β1 was clearly inhibited when the PLC-PIP2 signal pathway was stimulated or the cAMP-PKA signal pathway was blocked after 4.0 Gy X-irradiation, this did not limit the up-regulation of CD4+CD25+Nrp1+Treg cells after ionizing radiation.

CONCLUSIONThese results indicated that HDR might induce CD4+CD25+Nrp1+Treg cells production and stimulate TGF-β1 secretion by regulating signal molecules in mice.

Animals ; Calcineurin ; genetics ; metabolism ; Cyclic AMP ; metabolism ; Dose-Response Relationship, Radiation ; Female ; Gene Expression Regulation ; radiation effects ; Immunosuppression ; Interleukin-10 ; genetics ; metabolism ; Lymphocyte Subsets ; physiology ; Male ; Mice ; Neuropilin-1 ; genetics ; metabolism ; Phosphoinositide Phospholipase C ; genetics ; metabolism ; Protein Kinases ; genetics ; metabolism ; Signal Transduction ; Transforming Growth Factor beta ; genetics ; metabolism ; Whole-Body Irradiation ; adverse effects

OBJECTIVETo investigate the regulation of calcineurin (CaN) by endoplasmic reticulum stress (ERS) in podocytes in vitro and in vivo at the stage microalbuminuria in diabetic nehropathy (DN).

METHODSThe urinary albumin excretions of C57BLKS/J (Lepr) db/db and db/m mice at the ages of 6, 9, and 12 weeks were measured. The expressions of CaN and synaptopodin of these mice were observed. In immortalized mouse podocytes, the expression of podocyte CaN incubated with different concentrations of paltimate was quantitatively determined by real-time PCR. The changes of CaN incubated with paltimate with or without ursodeoxy-cholic acid (UDCA) were analyzed by confocal microscopy and Western blotting.

RESULTSAs urine protein increased, the expression of CaN was enhanced and the expression of synaptopodin was reduced in early stage DN db/db mice potocytes. In immortalized mouse podocytes, as the concentrations of palmitate increased, CaN mRNA increased. By confocal microscopy, the fluorescence intensity of CaN increased in palmitate treatment group. After co-incubation with palmitate and UDCA, the fluorescence intensity decreased. The similar results were shown by Western blotting.

CONCLUSIONAt the stage of microalbuminuria in DN, ERS in podocytes up-regulates the expression of CaN.

Animals ; Calcineurin ; metabolism ; Cells, Cultured ; Diabetes Mellitus, Experimental ; metabolism ; Diabetic Nephropathies ; metabolism ; Endoplasmic Reticulum Stress ; Male ; Mice ; Mice, Inbred C57BL ; Microfilament Proteins ; metabolism ; Podocytes ; metabolism

Apoptosis ; Calcineurin ; metabolism ; Chymotrypsin ; metabolism ; Humans ; Lysosomes ; enzymology ; Mitochondria ; metabolism ; pathology ; Mitochondrial Dynamics ; Neuroblastoma ; metabolism ; pathology