Decreased Calcineurin Activity Research Articles

Calcineurin inhibition causes persistent hypertension through hypothalamic NMDA receptor‐dependent sympathetic outflow

Calcineurin is a calcium/calmodulin‐dependent serine/threonine protein phosphatase, and hypertension is a major side effect of clinically used calcineurin inhibitors. Although increased sympathetic nerve discharges are associated with calcineurin inhibitor‐induced hypertension, the sources of sympathetic outflow are unknown. In this study, we determined the role of the paraventricular nuclear (PVN) of the hypothalamus in calcineurin inhibitor‐induced hypertension. Immunocytochemistry labeling showed that calcineurin was expressed on spinally projecting PVN neurons. Radiotelemetry recordings showed that systemic administration of tacrolimus (FK506), a specific calcineurin inhibitor, for 14 days caused a gradual and significant increase in arterial blood pressure in rats, which lasted at least another 7 days after discontinuing FK506 treatment. FK506 significantly decreased calcineurin activity in the PVN and increased the protein level of GluN1, the NMDA receptor obligatory subunit, and serine/threonine phosphorylation levels of GluN1 in the PVN synaptosomes. Electrophysiological recordings revealed that the spontaneous firing rate, frequency of mEPSCs, and amplitude of puff NMDA‐elicited currents in spinally projecting PVN neurons were significant greater in FK506‐treated rats than in vehicle‐treated rats. Remarkably, treatment with the NMDA receptor antagonist AP5 normalized the stimulatory effects of FK506 on PVN neurons of FK506‐treated rats. Furthermore, microinjection of AP5 into the PVN decreased renal sympathetic nerve discharges and blood pressure increased in FK506‐treated rats. Systemic administration of memantine, a clinically used NMDA receptor antagonist, also significantly lowered the high blood pressure in FK506‐treated rats. Collectively, our findings indicate that calcineurin inhibitor‐induced persistent hypertension results from augmented sympathetic output through increased NMDA receptor phosphorylation and activity in the PVN.

Regulation of dual leucine zipper kinase activity through its interaction with calcineurin

Hyperglycemia enhancing the intracellular levels of reactive oxygen species (ROS) contributes to dysfunction and progressive loss of beta cells and thereby to diabetes mellitus. The oxidation sensitive calcium/calmodulin dependent phosphatase calcineurin promotes pancreatic beta cell function and survival whereas the dual leucine zipper kinase (DLK) induces apoptosis. Therefore, it was studied whether calcineurin interferes with DLK action. In a beta cell line similar concentrations of H2O2 decreased calcineurin activity and activated DLK. DLK interacted via its φLxVP motif (aa 362–365) with the interface of the calcineurin subunits A and B. Mutation of the Val prevented this protein protein interaction, hinting at a distinct φLxVP motif. Indeed, mutational analysis revealed an ordered structure of DLK's φLxVP motif whereby Val mediates the interaction with calcineurin and Leu maintains an enzymatically active conformation. Overexpression of DLK wild-type but not the DLK mutant unable to bind calcineurin diminished calcineurin-induced nuclear localisation of the nuclear factor of activated T-cells (NFAT), suggesting that both, DLK and NFAT compete for the substrate binding site of calcineurin. The calcineurin binding-deficient DLK mutant exhibited increased DLK activity measured as phosphorylation of the downstream c-Jun N-terminal kinase, inhibition of CRE-dependent gene transcription and induction of apoptosis. These findings show that calcineurin interacts with DLK; and inhibition of calcineurin increases DLK activity. Hence, this study demonstrates a novel mechanism regulating DLK action. These findings suggest that ROS through inhibition of calcineurin enhance DLK activity and thereby lead to beta cell dysfunction and loss and ultimately diabetes mellitus.

The CnB1 p.D102A variant is linked to dilated cardiomyopathy via impaired Calcineurin activity

Background:The role of calcineurin (protein phosphatase 2B (PP2B)) in the pathogenesis of human dilated cardiomyopathy (DCM) has not been fully elucidated. We determined the potential involvement of calcineurin in the pathogenesis of DCM caused by mutations in CnB1, a subunit of calcineurin.Methods: By whole-exome sequencing, we identified a new CnB1 variant in a Han Chinese proband with cardiomyopathy from a 3-generation family with 2 normal individuals and 3 individuals with familial dilated cardiomyopathy. The potential pathogenic variant was validated by Sanger sequencing. We performed functional and mechanistic experiments in a CnB1-knockin (KI) mouse model and at the cellular level.Results: We detected a rare heterozygous CnB1 variant (p.D102A)in a proband with dilated cardiomyopathy. This variant was localized to the EF hand 3 region of CnB1, where no variants have been previously reported. KI mice harboring the p.D102A variant exhibited decreased cardiac function and cardiac dilatation. Immunoblotting, RT-PCR and immunofluorescence results showed decreased cardiomyocyte size and heart failure-related protein expression. A calcineurin activity assay demonstrated decreased calcineurin activity in the KI mice, accompanied by the decreased ability of CnB1 to bind CnA.Conclusions: CnB1 p.D102A is a disease-associated variantthat confers susceptibility to cardiac dilatation. This variant is associated with impaired calcineurin activity and a subsequent decrease in the ability of CnB1 to bind CnA.

Dimethyl fumarate exerts neuroprotection by modulating calcineurin/NFAT1 and NFκB dependent BACE1 activity in Aβ1-42 treated neuroblastoma SH-SY5Y cells

Ample studies indicate that calcineurin, a Ca2+/calmodulin-sensitive phosphatase, plays a key role in the initiation and/or clinical progression of Alzheimer's disease, and alteration of calcineurin has been confirmed in Alzheimer’s disease brain, impacting tau proteins and amyloid beta (Aβ) levels and resulting in neuronal cell death. As, it is sensible to deliberate the likelihood of calcineurin inhibition as a pharmacological target in the progress of novel Alzheimer’s disease therapies, we investigated the neuroprotective efficacy of dimethyl fumarate (DMF) via calcineurin dependent downstream targets in oligomeric Aβ1-42 treated neuroblastoma SH-SY5Y cells. DMF pre-treatment reduced LDH release, increased cell survival and decreased calcineurin activity in Aβ1-42-incubated cells. DMF was found to block calcineurin dependent apoptosis induced by Aβ1-42 through Bcl-2 linked death protein (BAD) and cAMP response element binding (CREB) dephosphorylation; moreover, rescued the opposing effect of Aβ1-42 on CREB-driven transcription in cells transfected with the CRE-SEAP reporter gene indicating its efficacy on long term potentiation and synaptic plasticity. DMF reversed Aβ1-42-induced enhancement in the active form of nuclear factor of activated T-cells (NFAT1) and further associated beta-site amyloid precursor protein cleaving enzyme-1 (BACE1) expression, BACE1 promoter activity and BACE1 enzymatic activity. DMF exhibited another neuroprotective mechanism against Aβ1-42 treatment by reducing nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) dependent BACE1 gene transcription. DMF does not alter either beta amyloid precursor protein (βAPP) or its mRNA levels in SH-SY5Y cells, confirming that it does not possess amyloidogenic effect. Inhibiting molecular events related to Aβ1-42-induced calcineurin activity by DMF, may be an approach towards finding suitable treatment for Alzheimer’s disease.

Role of PI3Kα and sarcolemmal ATP-sensitive potassium channels in epoxyeicosatrienoic acid mediated cardioprotection

Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid that have known cardioprotective properties. While the mechanism(s) remains unknown, evidence suggests that phosphoinositide 3-kinase (PI3K) and sarcolemmal ATP-sensitive potassium channels (pmK(ATP)) are important. However the role of specific PI3K isoforms and corresponding intracellular mechanisms remains unknown. To study this, mice hearts were perfused in Langendorff mode for 40 min of baseline and subjected to 20 or 30 min of global no-flow ischemia followed by 40 min of reperfusion. C57BL6 mice perfused with 11,12-EET (1 μM) had improved postischemic recovery, whereas co-perfusion with PI3Kα inhibitor, PI-103 (0.1 μM), abolished the EET-mediated effect. In contrast, blocking of PI3Kβ or PI3Kγ isoforms failed to inhibit EET-mediated cardioprotection. In addition to the improved post-ischemic recovery, increased levels of p-Akt, decreased calcineurin activity and decreased translocation of proapoptotic protein BAD to mitochondria were noted in EET-treated hearts. Perfusion of 11,12-EET to Kir6.2 deficient mice (pmK(ATP)) failed to improve postischemic recovery, decrease calcineurin activity and translocation of proapoptotic protein BAD, however increased levels of p-Akt were still observed. Patch-clamp experiments demonstrated that 11,12-EET could not activate pmK(ATP) currents in myocytes pre-treated with PI-103. Mechanistic studies in H9c2 cells demonstrate that 11,12-EET limits anoxia-reoxygenation triggered Ca(2+) accumulation and maintains mitochondrial ΔΨm compared to controls. Both PI-103 and glibenclamide (10 μM, pmK(ATP) inhibitor) abolished EET cytoprotection. Together our data suggest that EET-mediated cardioprotection involves activation of PI3Kα, upstream of pmK(ATP), which prevents Ca(2+) overload and maintains mitochondrial function.

Decreased Calcineurin Activity Suppresses Protein Synthesis and Activates AMP Dependent Kinase (AMPK)

CAβ knock out mice (CAβ−/−) kidneys have normal development and function but do not hypertrophy to diabetes. We examined calcineurinˈs role in protein metabolism and in Akt, mitogen associated kinase (MAPK) and AMPK signaling in proximal tubule cells in epidermal growth factor (EGF) and/or cyclosporin A (CYA) treated NRK‐52E renal epithelial cells and SV40 immortalized cell lines created from CAβ−/− and wildtype litter mate proximal tubules. CYA (4μM) decreased protein content/well (48h) by 14±2% and protein synthesis (18h) by 51±2% of control in NRK‐52E cells. In contrast, protein degradation(0h–28h) was decreased by 10±1%. Inhibitors upstream of MAPK or Akt reduced protein synthesis by 48±3% or 56±2%. However, CYA increased both basal and EGF‐induced MAPK and AKT phosphorylation (1h) by ~3‐fold. AMPK, which acts to inhibit the mammalian target of rapamycin, phosphorylation was 6 fold higher with CYA compared to control. CAβ −/− tubular cells had lower protein per well (48h, 20±2%) and EGF‐induced protein synthesis than CAβ +/+ cells. In H2O2 treated CAβ −/− cells, AMPK phosphorylation was >10 fold higher in CAβ −/− compared with CAβ +/+ cells. Decreased calcineurin activity reduces protein accumulation in renal tubular cells via reduced protein synthesis possibly through activation of AMPK.

Role of calcineurin in inhibiting disadvantageous associations

Calcineurin is an important calcium-dependent phosphatase that is evolutionarily conserved in all studied species, and has been implicated in the consolidation and maintenance of new memories. However, recent evidence has extended the role of calcineurin. In contrast to learning tasks that require behavioral acquisition, extinction tasks that require behavioral inhibition have been shown to be reliant on calcineurin. In the present study, using a Morris water maze, we have demonstrated that pharmacological inhibition of calcineurin causes augmentation of spatial learning and perseveration of spatial reversal-learning in a dose-dependent manner. Direct infusions of a specific calcineurin inhibitor, cyclosporine A, into the dorsal hippocampi bilaterally, prior to spatial learning, led to increased learning, whereas similar injections of cyclosporine A following a spatial learning task and prior to a spatial reversal-learning task resulted in perseveration of reversal-learning. Our results indicate that injections of cyclosporin A resulted in decreased calcineurin activity in the dorsal hippocampus and increased difficulty in switching to new task demands, in a dose-dependent manner, despite evidence indicating no deficit in ability to learn new information. Therefore, calcineurin activity contributes to the inhibition of previously learned but unwanted behavioral responses during competitive spatial learning. Involvement of calcineurin in extinction of fear memory has recently been demonstrated. Our results also indicate that calcineurin activity plays a role in memory extinction in spatial memory tasks, and therefore, suggest that calcineurin might be an important molecule in mediating behavioral flexibility in general.

Striatal-Enriched Protein Tyrosine Phosphatase Expression and Activity in Huntington's Disease: A STEP in the Resistance to Excitotoxicity

Striatal-enriched protein tyrosine phosphatase (STEP) is highly expressed in striatal projection neurons, the neuronal population most affected in Huntington's disease. Here, we examined STEP expression and phosphorylation, which regulates its activity, in N-terminal exon-1 and full-length mutant huntingtin mouse models. R6/1 mice displayed reduced STEP protein levels in the striatum and cortex, whereas its phosphorylation was increased in the striatum, cortex, and hippocampus. The early increase in striatal STEP phosphorylation levels correlated with a deregulation of the protein kinase A pathway, and decreased calcineurin activity at later stages further contributes to an enhancement of STEP phosphorylation and inactivation. Accordingly, we detected an accumulation of phosphorylated ERK2 and p38, two targets of STEP, in R6/1 mice striatum at advanced stages of the disease. Activation of STEP participates in excitotoxic-induced cell death. Because Huntington's disease mouse models develop resistance to excitotoxicity, we analyzed whether decreased STEP activity was involved in this process. After intrastriatal quinolinic acid (QUIN) injection, we detected higher phosphorylated STEP levels in R6/1 than in wild-type mice, suggesting that STEP inactivation could mediate neuroprotection in R6/1 striatum. In agreement, intrastriatal injection of TAT-STEP increased QUIN-induced cell death. R6/2, Tet/HD94, and Hdh(Q7/Q111) mice striatum also displayed decreased STEP protein and increased phosphorylation levels. In Tet/HD94 mice striatum, mutant huntingtin transgene shutdown reestablished STEP expression. In conclusion, the STEP pathway is severely downregulated in the presence of mutant huntingtin and may participate in compensatory mechanisms activated by striatal neurons that lead to resistance to excitotoxicity.

Hippocampal CA3 calcineurin activity participates in depressive‐like behavior in rats

Calcineurin is a serine/threonine protein phosphatase that regulates neurotransmission, neuronal structure and plasticity, and neuronal excitability in mood disorders, including depression. Increasing evidence has suggested that calcineurin is involved in the regulation of depressive-like behavior. However, little is known about the neurobiological mechanisms that underlie the mood-regulating effects of calcineurin. We investigated the potential mechanism by which calcineurin mediates the development of depressive-like behavior and the involvement of calcineurin in the action of antidepressant medication in the chronic mild stress (CMS) model. The results showed that rats exposed to CMS had decreased calcineurin activity, measured by increased phospho-synapsin I S62/67 (pSynapsin) and decreased calcineurin-Aα levels, specifically in the CA3 but not CA1 or dentate gyrus (DG) subfields of the hippocampus. Calcineurin inhibition in the CA3 but not DG by microinfusion of cyclosporine-A (2 μg) induced depressive-like behavior in normal rats and exacerbated depressive-like performance in CMS-treated rats. Additionally, calcineurin inhibition in the CA3 but not DG reversed the antidepressant-like activity of venlafaxine. Calcineurin inhibition in the CA3 also reduced metabotropic glutamate 2/3 receptor (mGluR2/3) expression levels. mGluR2/3 activation by its agonist LY354740 (100 ng) in the CA3 reversed the depressive-like behavior induced by cyclosporine-A administration. Finally, chronic venlafaxine (40 mg/kg) treatment increased calcineurin activity, reflected by decreased pSynapsin and increased calcineurin-Aα protein levels in the CA3 but not CA1 or DG. These findings indicate that CA3 calcineurin signaling probably mediated through mGluR2/3 participates in the development of depression and the behavioral responses to antidepressant treatment.

Decreased calcineurin activity in circulation of Duchenne muscular dystrophy

Objectives: In an analysis of enzymes in easily accessible tissues like blood cells, serum can provide a valuable information and a simple tool for disease and carrier detection. In the study presented we have analyzed calcineurin activity in Duchenne muscular dystrophy (DMD) and carrier sera and lymphocytes for its diagnostic value and its status in DMD pathology. Design and methods: We have monitored calcineurin activity in sera and lymphocytes of DMD, in carriers and in controls using colorimetric method by following the p-nitrophenol released in the presence and absence of Trifluoperazine (TFP), an inhibitor of calcineurin. Results: Results showed a significant decrease in serum and lymphocyte calcineurin activity in DMD ( p < 0.001) without alteration in carriers compared to normal. Conclusion: Further studies are required to understand possible alterations mediated by calcineurin with reference to DMD lymphocytes as any alteration in phosphorylation/dephosphorylation pathway can disturb the normal functioning of these cells. The decreased calcineurin activity observed in DMD serum compared with controls could be further examined for its diagnostic utility.

Combined effects of unloading and Cyclosporin A on myosin heavy chain in slow muscles of rats

In skeletal muscle, calcineurin has been associated both in the molecular regulation of fiber-type expression, and in phenotypic responses to altered neuromuscular activity. A decreased calcineurin activity could be involved in the unweighting-induced slow-to-fast myosin transition. This study examines both specific and combined effects of unloading and cyclosporin A (CsA) treatment, a well-known inhibitor of calcineurin, on the distribution of myosin heavy chain (MHC) isoforms in soleus (SOL) and adductor digitorum longus (ADL) muscles of rats. Female rats were randomly assigned to 4 groups (n=8 in each group): weight-bearing or hindlimb-unloaded, treated either by CsA (CsA-WB, CsA-HU) or placebo (WB, HU) daily for 3 weeks. The MHC profile of SOL and ADL was analysed by SDS-PAGE electrophoresis and calcineurin activity estimated by mRNA levels of modulatory calcineurin interacting protein 1 (MCIP-1). A similar slow-to-fast transition was observed in both SOL and ADL of HU, with a de novo expression of MHC-IIX and MHC-IIB. By contrast, in CsA-WB rats a transition limited to MHC-I toward MHC-IIA was observed only in SOL. Unloading and CsA have additional effects on the MHC profile of SOL, whereas in ADL the distribution of MHC was similar in HU and CsA-HU groups. Unloading and CsA decreased MCIP-1 levels in SOL, but have no effect in ADL. These results suggest that a decrease in calcineurin activity cannot fully explain the slow-to-fast transition observed in response to unloading. Support: Service de Santé des Armées

Serum calcineurin activity in relation to oxidative stress and glycemic control in type II diabetes mellitus

Objectives: In view of the well-recognized prevalence of oxidative stress in diabetes mellitus and the susceptibility of calcineurin (Ca 2+-calmodulin dependent protein phosphatase 2 B) to free radicals, calcineurin was assayed in the sera of type II diabetic patients. Design and methods: Serum contents of thiobarbituric acid reactive substances, calcineurin and calmodulin, as well as activities of calcineurin and superoxide dismutase were measured in 81 diabetic patients and compared with age-matched controls. Results: Oxidative stress in diabetic subjects was evidenced by increased thiobarbituric acid reactive substances, decreased superoxide dismutase activity concomitant with decreased calcineurin activity in sera. The observed decrease in calcineurin activity had a reciprocal correlation with fasting blood sugar, thiobarbituric acid reactive substances, and glycosylated hemoglobin. Conclusion: The inverse correlation observed between serum calcineurin activity and glycosylated hemoglobin levels suggests that an assay of serum calcineurin activity may be useful in simultaneous assessment of oxidative stress and glycemic control in type II diabetes mellitus.

Alterations in slow-twitch muscle phenotype in transgenic mice overexpressing the Ca2+ buffering protein parvalbumin.

The purpose of this study was to determine whether induced expression of the Ca2+ buffering protein parvalbumin (PV) in slow-twitch fibres would lead to alterations in physiological, biochemical and molecular properties reflective of a fast fibre phenotype. Transgenic (TG) mice were generated that overexpressed PV in slow (type I) muscle fibres. In soleus muscle (SOL; 58 % type I fibres) total PV expression was 2- to 6-fold higher in TG compared to wild-type (WT) mice. Maximum twitch and tetanic tensions were similar in WT and TG but force at subtetanic frequencies (30 and 50 Hz) was reduced in TG SOL. Twitch time-to-peak tension and half-relaxation time were significantly decreased in TG SOL (time-to-peak tension: 39.3 +/- 2.6 vs. 55.1 +/- 4.7 ms; half-relaxation time: 42.1 +/- 3.5 vs. 68.1 +/- 9.6 ms, P < 0.05 for TG vs. WT, respectively; n = 8-10). There was a significant increase in expression of type IIa myosin heavy chain (MHC) and ryanodine receptor at the mRNA level in TG SOL but there were no differences in MHC expression at the protein level and thus no difference in fibre type. Whole muscle succinate dehydrogenase activity was reduced by 12 +/- 0.4 % in TG SOL and single fibre glycerol-3-phosphate dehydrogenase activity was decreased in a subset of type IIa fibres. These differences were associated with a 64 % reduction in calcineurin activity in TG SOL. These data show that overexpression of PV, resulting in decreased calcineurin activity, can alter the functional and metabolic profile of muscle and influence the expression of key marker genes in a predominantly slow-twitch muscle with minimal effects on the expression of muscle contractile proteins.

Calcineurin as a potential contributor in estradiol regulation of hippocampal synaptic function

Estradiol influences Ca 2+ regulation and Ca 2+-dependent synaptic plasticity, suggesting estrogenic effects on Ca 2+-dependent enzymes that regulate synaptic plasticity may mediate hormonal influences on cognition. In ovariectomized female rats, injections of estradiol benzoate (EB, 10 μg) reduced hippocampal cytosolic activity of serine/threonine protein phosphatases, calcineurin and protein phosphatase 1 (PP1). The decreased activity was rapid and recovered substantially over a 24-h period. Decreased calcineurin activity was associated with a decreased level of calcineurin in the cytosol. In contrast, expression of PP1 was not altered suggesting that the level of calcineurin activity regulated PP1 activity. EB application to hippocampal slices rapidly decreased cytosolic phosphatase activity, which was not blocked by the estrogen receptor antagonist, ICI 182780. Decreased phosphatase activity was associated with an increase in CA3–CA1 synaptic transmission. In addition, EB application shifted synaptic plasticity, blocking the induction of long-term depression and facilitating the establishment of long-term potentiation. The reduction in calcineurin activity and shift in synaptic plasticity were mimicked to a lesser extent by 17-α-estradiol. From these results we suggest that EB can act to rapidly influence Ca 2+ signaling pathways including the activity of Ca 2+-regulated phosphatases involved in synaptic plasticity.

Clinical significance of serum calcineurin in acute leukemia

Background: Calcineurin is involved in T-lymphocyte activation as well as in the maturation of hematopoietic cells. Identification of this predominantly intracellular phosphatase and of calmodulin (CaM) in human sera warranted their assessment in different types of acute leukemias. Methods: Phosphatase activity of calcineurin (CaN) was assayed, involving the measurement of trifluoperazine-sensitive neutral phosphatase, in sera of leukemic patients before and after treatment. Calcineurin and calmodulin contents were also determined by ELISA employing monoclonal antibodies specific to the proteins. Results: The activity of calcineurin was decreased by 75% and 85% in sera of patients diagnosed either for acute lymphoid leukemia and acute myeloid leukemia, respectively, without apparent changes in calmodulin or calcineurin contents under both these conditions. In addition, the decreased calcineurin activity in acute myeloid leukemia was restored to levels comparable to non-leukemic individuals upon treatment. This was not observed in cases of acute lymphoid leukemia. Conclusions: These results suggest diagnostic utility in the measurement of serum calcineurin activity in acute leukemia. Restoration of normal calcineurin activity in patients undergoing treatment for acute myeloid leukemia may provide a means to monitor patient response to the prescribed therapeutic regimen.