Calcineurin Research Articles

Mechanism of tacrolimus in the treatment of lupus nephritis.

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder, with more than half of the patients developing lupus nephritis (LN), which significantly contributes to chronic kidney disease (CKD) and end-stage renal disease (ESRD). The treatment of lupus nephritis has always been challenging. Tacrolimus (TAC), an effective immunosuppressant, has been increasingly used in the treatment of LN in recent years. This review aims to explore the mechanisms of action of tacrolimus in treating LN. Firstly, we briefly introduce the pharmacological properties of tacrolimus, including its role as a calcineurin (CaN) inhibitor, exerting immunosuppressive effects by inhibiting T cell activation and cytokine production. Subsequently, we focus on various other immunomodulatory mechanisms of tacrolimus in LN therapy, including its effects on T cells, B cells, and immune cells in kidney. Particularly, we emphasize tacrolimus' regulatory effect on inflammatory mediators and its importance in modulating the Th1/Th2 and Th17/Treg balance. Additionally, we review its effects on actin cytoskeleton, angiotensin II (Ang II)-specific vascular contraction, and P-glycoprotein activity, summarizing its impacts on non-immune mechanisms. Finally, we summarize the efficacy and safety of tacrolimus in clinical studies and trials. Although some studies have shown significant efficacy of tacrolimus in treating LN, its safety remains a challenge. We outline the potential adverse reactions of long-term tacrolimus use and provide suggestions on effectively monitoring and managing these adverse reactions in clinical practice. In general, tacrolimus, as a novel immunosuppressant, holds promising prospects for treating LN. Of course, further research is needed to better understand its therapeutic mechanisms and ensure its safety and efficacy in clinical practice.

Inhibition of Cdc42-Interacting Protein 4-calcineurin complexes attenuate pathological cardiac remodeling after myocardial infarction

Myocardial infarction (MI) is a major cause of mortality and morbidity worldwide. Heart failure due to MI results from both acute ischemia/reperfusion (I/R) injury and subsequent post-MI pathological cardiac remodeling. The Ca2+/calmodulin-dependent protein phosphatase calcineurin (CaN) is a critical mediator of pathological cardiac remodeling. Along with its role in the immune system, the pro-survival function of CaN complicates the targeting of the phosphatase in heart disease. CaNAβ is a CaN isoform uniquely associated with pathological cardiac hypertrophy. We have discovered that pools of cardiomyocyte CaNAβ are anchored via the unique CaNAβ N-terminal polyproline domain (PP) to scaffold proteins such as CIP4 (Cdc42 interacting protein 4, TRIP10), creating discrete Ca2+-CaN signaling compartments. Pathological cardiac remodeling induced by pressure overload was inhibited by CIP4 gene knock-out and by inhibition of CaNAβ binding to CIP4 using an adeno-associated virus (AAV) vector that expresses the CaNAβ PP peptide. It remains unknown whether PP-anchored CaNAβ also participates in CaN-dependent cardioprotective signaling in I/R injury. We now consider whether CIP4-CaNAβ signalosomes are required for myocyte survival in ischemic cardiomyopathy. To test the relevance of CIP4 to ischemic reperfusion (IR) injury, tamoxifen-inducible, cardiac-specific CIP4 knock-out (CIP4 CKO), control Tg(Myh6-cre/Esr1*), and control CIP4f/f mice were subjected to 30 minutes of ischemia by transient left anterior descending coronary artery (LAD) ligation. Cardiac function and structure were evaluated 24 hours after I/R by echocardiography and histology. Left ventricular ejection fraction was significantly preserved, while infarct size was significantly decreased by CIP4 CKO when compared to the two control cohorts. Separate mice subjected to permanent LAD ligation showed that CIP4 CKO resulted in long-term preservation of cardiac function. To disrupt CaNAβ-CIP4 complexes in vivo, the CaNAβ PP domain peptide was expressed again using AAV. In contrast to the expression of the VIVIT pan-CaN inhibitory peptide, CaNAβ PP peptide expression did not exacerbate I/R injury and in fact preserved cardiac function immediately after infarction. Moreover, VIVIT peptide expression worsened, while CaNAβ PP peptide expression improved cardiac function long term after induction of MI by permanent LAD ligation. Together, these results suggest that CIP4-CaNAβ signalosomes do not contribute to cardioprotection, while inducing pathological cardiac remodeling. These results suggest that targeting of CIP4-CaNAβ signalosomes constitute a novel strategy for inhibiting I/R injury and preventing the development of heart failure in response to diverse forms of cardiovascular disease. This work was supported by National Institutes of Health R01 grants HL126950 and HL158052. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Calcineurin mediates the inhibitory effects of TNF on NKCC2 phosphorylation

We previously showed that TNF inhibits NKCC2 activity and phosphorylation along the TAL, however, the mechanism has not yet been defined. As calcineurin (CN) activity in synoviocytes was increased by proinflammatory cytokines such as IL-1 and TNF, we tested the hypothesis that induction of CN activity and expression of CN isoforms is part of a pathway by which TNF inhibits NKCC2 phosphorylation. In this study, we found that CN activity increased by approximately 2-fold in primary cultures of medullary thick ascending limb (mTAL) cells that were maintained at 37oC/5%CO2 then challenged with mouse recombinant TNF (1 nM) for 60 min. Similarly, TNF induced a 3-fold increase in CN activity when mTAL cells were exposed to hypertonic media (400 mOsm/Kg H2O), an effect that was prevented by cyclosporine A (CsA). In contrast, silencing TNF production in mTAL cells using lentivirus (U6-TNF-ex4) significantly reduced CN activity by approximately 56% in cells exposed to 400 mOsm/Kg H2O for 1 h. CN activity also was inhibited in cells pretreated with CsA for 30 min followed by incubation in 400 mOsm/Kg H2O. Our data indicate that NKCC2 phosphorylation was significantly decreased in cultured mTAL cells challenged with TNF (1 nM) for 4 hr while inhibition of CN activity with CsA increased pNKCC2 expression. Analysis of qRT-PCR experiments showed that mTAL cells express the calcineurin A subunit (CNA) isoforms a (Sequence ID: NM_001293622.1) and b (sequence ID: NM_001310426.1). However, only the mRNA abundance of the CNAb isoform increased when mTAL cells were incubated with TNF (1 nM) for 4 hr under NS or HS (400 mOsm/Kg H2O) conditions as TNF had no effect on the a isoform of CNA. In vivo, both TNF and CNAb expression increased in outer medulla (OM) from mice given 1% NaCl in the drinking water (HS) for 7 days while intrarenal silencing of TNF with the U6-TNF-ex4 lentivirus reduced expression of CNAb under NS or HS conditions. Similarly, intrarenal injection of a lentivirus construct that specifically silenced CNAb in the kidney (U6-CNAb-ex6) reduced expression of CNAb in mice ingesting HS for 7 days. Interestingly, co-administration of U6-CNAb-ex6 with U6-TNF-ex4 increased pNKCC2 expression by approximately 63% in the OM from mice compared with silencing of TNF alone. Collectively, these data are the first to demonstrate that TNF increases CN activity as well as specific isoform expression in the kidney. Since NKCC2 has been identified as a target of the CNAb isoform, which is increased by TNF, these data demonstrate that a calcineurin-dependent signaling pathway is part of the mechanism by which TNF inhibits the phosphorylation of NKCC2. NIH grants R01 HL133077 and HL153525. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Ca2+-dependent phosphatase calcineurin dephosphorylates TBK1 to suppress antiviral innate immunity.

Tumor necrosis factor receptor-associated factor family member-associated NF-κB activator-binding kinase 1 (TBK1) plays a key role in the induction of the type 1 interferon (IFN-I) response, which is an important component of innate antiviral defense. Viruses target calcium (Ca2+) signaling networks, which participate in the regulation of the viral life cycle, as well as mediate the host antiviral response. Although many studies have focused on the role of Ca2+ signaling in the regulation of IFN-I, the relationship between Ca2+ and TBK1 in different infection models requires further elucidation. Here, we examined the effects of the Newcastle disease virus (NDV)-induced increase in intracellular Ca2+ levels on the suppression of host antiviral responses. We demonstrated that intracellular Ca2+ increased significantly during NDV infection, leading to impaired IFN-I production and antiviral immunity through the activation of calcineurin (CaN). Depletion of Ca²+ was found to lead to a significant increase in virus-induced IFN-I production resulting in the inhibition of viral replication. Mechanistically, the accumulation of Ca2+ in response to viral infection increases the phosphatase activity of CaN, which in turn dephosphorylates and inactivates TBK1 in a Ca2+-dependent manner. Furthermore, the inhibition of CaN on viral replication was counteracted in TBK1 knockout cells. Together, our data demonstrate that NDV hijacks Ca2+ signaling networks to negatively regulate innate immunity via the CaN-TBK1 signaling axis. Thus, our findings not only identify the mechanism by which viruses exploit Ca2+ signaling to evade the host antiviral response but also, more importantly, highlight the potential role of Ca2+ homeostasis in the viral innate immune response.IMPORTANCEViral infections disrupt intracellular Ca2+ homeostasis, which affects the regulation of various host processes to create conditions that are conducive for their own proliferation, including the host immune response. The mechanism by which viruses trigger TBK1 activation and IFN-I induction through viral pathogen-associated molecular patterns has been well defined. However, the effects of virus-mediated Ca2+ imbalance on the IFN-I pathway requires further elucidation, especially with respect to TBK1 activation. Herein, we report that NDV infection causes an increase in intracellular free Ca2+ that leads to activation of the serine/threonine phosphatase CaN, which subsequently dephosphorylates TBK1 and negatively regulates IFN-I production. Furthermore, depletion of Ca2+ or inhibition of CaN activity exerts antiviral effects by promoting the production of IFN-I and inhibiting viral replication. Thus, our results reveal the potential role of Ca2+ in the innate immune response to viruses and provide a theoretical reference for the treatment of viral infectious diseases.

Amyloid-β-induced dendritic spine elimination requires Ca2+-permeable AMPA receptors, AKAP-Calcineurin-NFAT signaling, and the NFAT target gene Mdm2.

Alzheimer's Disease (AD) is associated with brain accumulation of synaptotoxic amyloid-β (Aβ) peptides produced by the proteolytic processing of amyloid precursor protein (APP). Cognitive impairments associated with AD correlate with dendritic spine and excitatory synapse loss, particularly within the hippocampus. In rodents, soluble Aβ oligomers impair hippocampus-dependent learning and memory, promote dendritic spine loss, inhibit NMDA-type glutamate receptor (NMDAR)-dependent long-term potentiation (LTP), and promote synaptic depression (LTD), at least in part through activation of the Ca2+-CaM-dependent phosphatase calcineurin (CaN). Yet, questions remain regarding Aβ-dependent postsynaptic CaN signaling specifically at the synapse to mediate its synaptotoxicity. Here, we use pharmacologic and genetic approaches to demonstrate a role for postsynaptic signaling via A kinase-anchoring protein 150 (AKAP150)-scaffolded CaN in mediating Aβ-induced dendritic spine loss in hippocampal neurons from rats and mice of both sexes. In particular, we found that Ca2+-permeable AMPA-type glutamate receptors (CP-AMPARs), which were previously shown to signal through AKAP-anchored CaN to promote both LTD and Aβ-dependent inhibition of LTP, are also required upstream of AKAP-CaN signaling to mediate spine loss via overexpression of APP containing multiple mutations linked to familial, early-onset AD and increased Aβ production. In addition, we found that the CaN-dependent nuclear factor of activated T-cells (NFAT) transcription factors are required downstream to promote Aβ-mediated dendritic spine loss. Finally, we identified the E3-ubiquitin ligase Mdm2, which was previously linked to LTD and developmental synapse elimination, as a downstream NFAT target gene upregulated by Aβ whose enzymatic activity is required for Aβ-mediated spine loss.Significance Statement Impaired hippocampal function and synapse loss are hallmarks of AD linked to Aβ oligomers. Aβ exposure acutely blocks hippocampal LTP and enhances LTD and chronically leads to dendritic spine synapse loss. In particular, Aβ hijacks normal plasticity mechanisms, biasing them toward synapse weakening/elimination, with previous studies broadly linking CaN phosphatase signaling to this synaptic dysfunction. However, we do not understand how Aβ engages signaling specifically at synapses. Here we elucidate a synapse-to-nucleus signaling pathway coordinated by the postsynaptic scaffold protein AKAP150 that is activated by Ca2+ influx through CP-AMPARs and transduced to nucleus by CaN-NFAT signaling to transcriptionally upregulate the E3-ubiquitin ligase Mdm2 that is required for Aβ-mediated spine loss. These findings identify Mdm2 as potential therapeutic target for AD.

TRPV4 Channels Promote Pathological, but Not Physiological, Cardiac Remodeling through the Activation of Calcineurin/NFAT and TRPC6.

TRPV4 channels, which respond to mechanical activation by permeating Ca2+ into the cell, may play a pivotal role in cardiac remodeling during cardiac overload. Our study aimed to investigate TRPV4 involvement in pathological and physiological remodeling through Ca2+-dependent signaling. TRPV4 expression was assessed in heart failure (HF) models, induced by isoproterenol infusion or transverse aortic constriction, and in exercise-induced adaptive remodeling models. The impact of genetic TRPV4 inhibition on HF was studied by echocardiography, histology, gene and protein analysis, arrhythmia inducibility, Ca2+ dynamics, calcineurin (CN) activity, and NFAT nuclear translocation. TRPV4 expression exclusively increased in HF models, strongly correlating with fibrosis. Isoproterenol-administered transgenic TRPV4-/- mice did not exhibit HF features. Cardiac fibroblasts (CFb) from TRPV4+/+ animals, compared to TRPV4-/-, displayed significant TRPV4 overexpression, elevated Ca2+ influx, and enhanced CN/NFATc3 pathway activation. TRPC6 expression paralleled that of TRPV4 in all models, with no increase in TRPV4-/- mice. In cultured CFb, the activation of TRPV4 by GSK1016790A increased TRPC6 expression, which led to enhanced CN/NFATc3 activation through synergistic action of both channels. In conclusion, TRPV4 channels contribute to pathological remodeling by promoting fibrosis and inducing TRPC6 upregulation through the activation of Ca2+-dependent CN/NFATc3 signaling. These results pose TRPV4 as a primary mediator of the pathological response.

MITOCHONDRIAL DYSFUNCTION CAUSES CECAL PATCH (CP) INFLAMMATION IN ULCERATIVE COLITIS

Abstract BACKGROUND Up to 17% of distal ulcerative colitis (UC) patients have discontinuous (proximal) inflammation referred to as Cecal Patch (CP), associated with greater abdominal pain, bleeding and diarrhea. Despite these known associations, the pathogenesis of CP lesions is poorly understood. Recent data suggest that reduced mitochondrial (Mito) function predicts unfavorable clinical outcomes in active UC (Haberman et al; Nat Comm 2019). We posit that inflammation and tissue injury induced in UC-associated CP involves Mito dysfunction. METHOD UC patients with/without cecal disease and normal controls were biopsied. Tissue from CP lesions or normal caecum were collected in Allprotect® (AP) and formalin. Total RNA from AP-preserved biopsies were used for cDNA synthesis and RT-qPCR for Mito complexes, cytokines, anti-oxidant and calcineurin (CaN) signaling genes and tissue processed for IHC for Mito complexes. RESULT Transcriptomic analysis showed significant downregulation in genes associated with Mito biogenesis (PGC1α, TFAM, NRF2) and Mito complexes (Ndufa1, Ndufa4, Ndufb2, Ndufb6, mtCO1, mtCO2, Cox5B, Cox6A1, ATP5A1, ATP5B, ATP5E) in CP lesions compared to normal caecum from active patients. Major regulators of Mito biogenesis (PGC1α and TFAM) revealed ~39% and ~24% reductions of mRNA respectively, while NRF2 (anti-oxidant regulator) showed ~62% reduced mRNA in CP lesions compared to normal cecum from distal UC patients. Electron transport chain complex mRNAs showed 31%, 47%, and 48% reduction in complex I (Ndufa1, Ndufa4, Ndufb2, Ndufb6), complex IV (mtCO1, mtCO2, Cox5B, Cox6A1), and complex V (ATP5A1, ATP5B, ATP5E) levels, respectively in CP lesions compared to normal controls. Concomitantly, the IHC staining also showed significant reductions in Ndufb6, MTCO1 and Cox5A protein levels in CP lesions compared to normal cecum sections. The CP-related decreased markers of Mito biogenesis and function correlates with increased proinflammatory cytokine mRNA (IL-6: 2-fold; TGFβ1: 1.6-fold; IL-2: 2.6-fold) and decreased anti-inflammatory (IL10: 3.2-fold) gene expression in CP lesions. Further investigation demonstrated that repressed Mito function in CP lesions is associated with downregulation of anti-oxidant genes (SOD1, SOD2, Prdx3, Gpx4, Trx2) compared to normal cecum. Inflammation-induced Mito dysfunction in CP lesions also stimulates CaN signaling as demonstrated by significant increases in mRNA levels of CaN and downstream regulated genes (Cathepsin-L, Rel-A Glut4, CREB-BP and Igf-1r). CONCLUSION These data represent the first indication that Mito dysfunction occurs in CP associated with distal UC. Furthermore, we speculate that Mito dysfunction-induced CaN signaling promotes regional (CP) inflammatory responses. These important findings indicate that targeting Mito metabolism may be an important therapeutic goal of treating UC patients with CP.

Tacrolimus Induces Pain by Upregulating the Synaptic Expression of AMPA Receptors in the Spinal Cord Dorsal Horn

To investigate the effects of long-term administration of tacrolimus (also known as FK506) on the pain-related behaviors in mice and to study the underlying mechanism of pain induced by FK506 via measuring the effect of FK506 on the synaptic expression and phosphorylation of alpha-amino-3-hyroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor in the spinal cord dorsal horn of mice. 1) A total of 24 mice were evenly and randomly assigned to two groups, a FK506 group and a Saline group. The FK506 group was given daily intraperitoneal injection of FK506 and the Saline group received normal saline. Both groups received injection once a day for 7 days in a row. Some of the mice ( n=6 in each group) were monitored for the changes in the paw withdrawal threshold (PWT), the paw withdrawal latency (PWL), and the spontaneous pain behaviors to establish the pain model. The other mice ( n=6 in each group) of each group underwent isolation of the dorsal horn when obvious pain symptoms were induced on day 7 of injection. Then, immunoblotting was performed to determine the synaptic expression and phosphorylation levels of GluA1 and GluA2 subunits of AMPA receptors. 2) The mice were randomly divided into two groups, FK506+calcineurin (CaN) group and FK506+Saline group ( n=6 in each group). After the pain model was constructed, the mice were given intrathecal injection of recombinant CaN (also know as 33 U) or normal saline. Then, 60 minutes later, the PWT and the PWL of the mice were measured to investigate the role of CaN in FK506-induced pain. 3) Another18 mice were selected. The mice were randomly and evenly assigned to three groups, a control group (receiving intraperitoneal injection of normal saline followed by intrathecal injection of normal saline), FK506+Saline group (receiving intraperitoneal injection of FK506 followed by intrathecal injection of normal saline) and FK506+CaN group (receiving intraperitoneal injection of FK506 followed by intrathecal injection of CaN). Then, 60 minutes later, the spinal cords were isolated and subjected to immunoblotting assay to determine the role of CaN in FK506-induced AMPA receptor modification. 1) After 7 consecutive days of intraperitoneal injection of FK506, the PWT and PWL of mice dropped significantly, reaching on day 7 as low as 22.3%±0.05% and 66.6%±0.05% of the control group, respectively ( P<0.01). The FK506-treated mice displayed evident spontaneous pain behavior, presenting significantly increased licking activities ( P<0.01). These results indicated that FK506-induced pain model was successfully established. Immunoblotting assay showed that the total expressions of GluA1 and GluA2 subunits in the spinal dorsal horn of the FK506 group remained unchanged in comparison with those of the Saline group. However, FK506 specifically induced an increase in the synaptic expression of GluA1. In addition, the phosphorylation levels of GluA1 at Ser845 and Ser831 in FK506-treated mice were significantly increased in comparison with those of the control group ( P<0.05). 2) Compared with those of the mice in the FK506+Saline group, the PWT and the PWL of mice in the FK506+CaN group were significantly increased ( P<0.05). 3) Compared with those of the FK506+Saline group, the synaptic expression of GluA1 were decreased in FK506+CaN group ( P<0.01) and the phosphorylation levels of GluA1 at Ser845 and Ser831 were significantly downregulated ( P<0.001). The hyper-expression and hyperphosphorylation of GluA1 subunit in the spinal cord dorsal horn resulting from CaN inhibition contributes to the FK506-induced pain syndrome. FK506 induces the synaptic hyper-expression and hyperphosphorylation of GluA1 in the dorsal horn of the spinal cord through CaN inhibition, thereby inducing pain.

Arginine vasopressin induces ferroptosis to promote heart failure via activation of the V1aR/CaN/NFATC3 pathway.

Arginine vasopressin (AVP) is a key contributor to heart failure (HF), but the underlying mechanisms remain unclear. In the present study, a mouse model of HF and human cardiomyocyte (HCM) cells treated with dDAVP are generated in vivo and in vitro, respectively. Hematoxylin and eosin (HE) staining is used to evaluate the morphological changes in the myocardial tissues. A colorimetric method is used to measure the iron concentration, Fe 2+ concentration and malondialdehyde (MDA) level. Western blot analysis is used to examine the protein levels of the V1a receptor (V1aR), calcineurin (CaN), nuclear factor of activated T cells isoform C3 (NFATC3), glutathione peroxidase 4 (GPX4) and acyl-CoA synthase long chain family member 4 (ACSL4). Immunoprecipitation (IP) and luciferase reporter assays are performed to determine the interaction between NFATC3 and ACSL4. Both in vivo and in vitro experiments reveal that the V1aR-CaN-NFATC3 signaling pathway and ferroptosis are upregulated in HFs, which are verified by the elevated protein levels of V1aR, CaN, NFATC3 and ACSL4; reduced GPX4 protein level; and enhanced Fe 2+ and MDA levels. We further find that inhibiting NFATC3 by suppressing the V1aR/CaN/NFATC3 pathway via V1aR/CaN inhibitors or sh-NFATC3 not only alleviates HF but also inhibits AVP-induced ferroptosis. Mechanistically, sh-NFATC3 significantly reverses the increase in AVP-induced ACSL4 protein level, Fe 2+ concentration, and MDA level by directly interacting with ACSL4. Our results demonstrate that AVP enhances ACSL4 expression by activating the V1aR/CaN/NFATC3 pathway to induce ferroptosis, thus contributing to HF. This study may lead to the proposal of a novel therapeutic strategy for HF.

Targeting Reactive Astrocytes in Vascular Dementia: Investigation of Neuronal-Astrocyte-Vascular Interactions.

Historically known as neuronal support cells, astrocytes are now widely studied for their close structural and functional interactions with multiple neural cell types and cerebral vessels where they maintain an ideal environment for optimized brain function. Under pathological conditions, astrocytes become reactive and lose key protective functions. In this commentary, we discuss our recent work in The Journal of Neuroscience (Sompol et al., 2023) that showed Ca2+ dysregulation in reactive astrocytes, as well as hyperactivation of the Ca2+-dependent protein phosphatase calcineurin (CN) and the Nuclear Factor of Activated T Cells (NFATs), in a diet-induced hyperhomocystienemia (HHcy) mouse model of Vascular Contributions to Cognitive Impairment and Dementia (VCID). Intravital multiphoton imaging coupled with whisker stimulation was used to explore astrocyte Ca2+ signaling and neurovascular function under active phase, fully awake conditions. Interestingly, evoked Ca2+ transients in individual astrocytes were greater, even though intercorrelated Ca2+ signaling across networks of astrocytes was impaired in HHcy mice. Blockade of astrocytic CN/NFAT reduced signs of astrocyte reactivity, normalized cerebrovascular function, and improved hippocampal synaptic strength and hippocampal dependent cognition in HHcy mice, revealing a previously unrecognized deficit regarding neuron-astrocyte-vascular interactions. These findings strongly support the use of astrocyte targeting strategies to mitigate pathophysiological changes associated with VCID and other Alzheimer's-related dementias.

A new metal ion chelator attenuates human tau accumulation-induced neurodegeneration and memory deficits in mice

Neuronal neurofibrillary tangles containing Tau hyperphosphorylation proteins are a typical pathological marker of Alzheimer's disease (AD). The level of tangles in neurons correlates positively with severe dementia. However, how Tau induces cognitive dysfunction is still unknown, which leads to a lack of effective treatments for AD. Metal ions deposition occurs with tangles in AD brain autopsy. Reduced metal ion can improve the pathology of AD. To explore whether abnormally phosphorylated Tau causes metal ion deposition, we overexpressed human full-length Tau (hTau) in the hippocampal CA3 area of mice and primary cultured hippocampal neurons (CPHN) and found that Tau accumulation induced iron deposition and activated calcineurin (CaN), which dephosphorylates glycogen synthase kinase 3 beta (GSK3β), mediating Tau hyperphosphorylation. Simultaneous activation of CaN dephosphorylates cyclic-AMP response binding protein (CREB), leading to synaptic deficits and memory impairment, as shown in our previous study; this seems to be a vicious cycle exacerbating tauopathy. In the current study, we developed a new metal ion chelator that displayed a significant inhibitory effect on Tau phosphorylation and memory impairment by chelating iron ions in vivo and in vitro. These findings provide new insight into the mechanism of memory impairment induced by Tau accumulation and develop a novel potential treatment for tauopathy in AD.

Macromolecular Complex Including MLL3, Carabin and Calcineurin Regulates Cardiac Remodeling.

Cardiac hypertrophy is an intermediate stage in the development of heart failure. The structural and functional processes occurring in cardiac hypertrophy include extensive gene reprogramming, which is dependent on epigenetic regulation and chromatin remodeling. However, the chromatin remodelers and their regulatory functions involved in the pathogenesis of cardiac hypertrophy are not well characterized. Protein interaction was determined by immunoprecipitation assay in primary cardiomyocytes and mouse cardiac samples subjected or not to transverse aortic constriction for 1 week. Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) experiments were performed on the chromatin of adult mouse cardiomyocytes. We report that the calcium-activated protein phosphatase CaN (calcineurin), its endogenous inhibitory protein carabin, the STK24 (STE20-like protein kinase 3), and the histone monomethyltransferase, MLL3 (mixed lineage leukemia 3) form altogether a macromolecular complex at the chromatin of cardiomyocytes. Under basal conditions, carabin prevents CaN activation while the serine/threonine kinase STK24 maintains MLL3 inactive via phosphorylation. After 1 week of transverse aortic constriction, both carabin and STK24 are released from the CaN-MLL3 complex leading to the activation of CaN, dephosphorylation of MLL3, and in turn, histone H3 lysine 4 monomethylation. Selective cardiac MLL3 knockdown mitigates hypertrophy, and chromatin immunoprecipitation and DNA sequencing analysis demonstrates that MLL3 is de novo recruited at the transcriptional start site of genes implicated in cardiomyopathy in stress conditions. We also show that CaN and MLL3 colocalize at chromatin and that CaN activates MLL3 histone methyl transferase activity at distal intergenic regions under hypertrophic conditions. Our study reveals an unsuspected epigenetic mechanism of CaN that directly regulates MLL3 histone methyl transferase activity to promote cardiac remodeling.

Trpc6 knockout protects against renal fibrosis by restraining the CN‑NFAT2 signaling pathway in T2DM mice.

Diabetic kidney disease (DKD), one of the common complications of type‑2 diabetes mellitus (T2DM), has become the principal cause of end‑stage kidney disease. Transient receptor potential channel 6 (TRPC6), one of non‑selective cation channels with significant calcium‑permeability, is associated with renal fibrosis. However, the mechanism of TRPC6 in T2DM‑induced renal fibrosis is still not entirely understood. The present study explored the potential mechanism of Trpc6 knockout in T2DM‑induced renal fibrosis in Trpc6‑/‑ mice. The results showed that Trpc6 knockout inhibited the loss of body weight and the increase of fasting blood glucose (FBG) and significantly improved renal dysfunction and glomerular fibrosis in T2DM mice. The present study also indicated that Trpc6 knockout significantly lowered the expression of phosphorylated (p‑)SMAD2/3, TGF‑β, calcineurin (CN), nuclear factor of activated T‑cell (NFAT)2 and Nod‑like receptor (NLR) 3 inflammasome‑associated proteins. Calcium imaging results revealed that Trpc6 knockdown could decrease the levels of [Ca2+]i and inhibited calcium homeostasis imbalance. Moreover, it was found that knockout of Trpc6 had no significant influence on lipid disposition and reactive oxygen species generation in the kidney cortex. The present study suggested that knockout of Trpc6 may alleviate glomerular fibrosis and delay DKD progression by reducing [Ca2+]i overload and inhibiting the CN‑NFAT2 pathway in T2DM mice.

Targeting CaN/NFAT in Alzheimer's brain degeneration.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive loss of cognitive functions. While the exact causes of this debilitating disorder remain elusive, numerous investigations have characterized its two core pathologies: the presence of β-amyloid plaques and tau tangles. Additionally, multiple studies of postmortem brain tissue, as well as results from AD preclinical models, have consistently demonstrated the presence of a sustained inflammatory response. As the persistent immune response is associated with neurodegeneration, it became clear that it may also exacerbate other AD pathologies, providing a link between the initial deposition of β-amyloid plaques and the later development of neurofibrillary tangles. Initially discovered in T cells, the nuclear factor of activated T-cells (NFAT) is one of the main transcription factors driving the expression of inflammatory genes and thus regulating immune responses. NFAT-dependent production of inflammatory mediators is controlled by Ca2+-dependent protein phosphatase calcineurin (CaN), which dephosphorylates NFAT and promotes its transcriptional activity. A substantial body of evidence has demonstrated that aberrant CaN/NFAT signaling is linked to several pathologies observed in AD, including neuronal apoptosis, synaptic deficits, and glia activation. In view of this, the role of NFAT isoforms in AD has been linked to disease progression at different stages, some of which are paralleled to diminished cognitive status. The use of classical inhibitors of CaN/NFAT signaling, such as tacrolimus or cyclosporine, or adeno-associated viruses to specifically inhibit astrocytic NFAT activation, has alleviated some symptoms of AD by diminishing β-amyloid neurotoxicity and neuroinflammation. In this article, we discuss the recent findings related to the contribution of CaN/NFAT signaling to the progression of AD and highlight the possible benefits of targeting this pathway in AD treatment.

Low-frequency electromagnetic fields influence the expression of calcium metabolism related proteins in leukocytic cell lines

Our study aimed to verify the hypothesis concerning low-frequency magnetic fields (LF-MFs)-related changes in cell viability through the biomechanism(s) based on calcineurin (CaN)-mediated signaling pathways triggered via ROS-like molecules. For experiments, Mono Mac 6 and U937 leukocytic cell lines were chosen and exposed to various LF-MFs and/or puromycin (PMC). The protein expression level of key regulatory proteins of calcium metabolism was examined by Western Blot analysis. In turn, the reactive oxygen species (ROS) and cell viability parameters were evaluated by cytochrome C reduction assay and flow cytometry, respectively. The simultaneous action of applied MF and PMC influenced cell viability in a MF-dependent manner. The changes in cell viability were correlated with protein expression and ROS levels. It was verified experimentally that applied stress stimuli influence cell susceptibility to undergo cell death. Moreover, the evoked bioeffects might be recognized as specific to both types of leukocyte populations.

CDN1163 alleviates SERCA2 dysfunction-induced pulmonary vascular remodeling by inhibiting the phenotypic transition of pulmonary artery smooth muscle cells

ABSTRACT Background and Purpose Substitution of Cys674 (C674) in the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) causes SERCA2 dysfunction which leads to activated inositol requiring enzyme 1 alpha (IRE1α) and spliced X-box binding protein 1 (XBP1s) pathway accelerating cell proliferation of pulmonary artery smooth muscle cells (PASMCs) followed by significant pulmonary vascular remodeling resembling human pulmonary hypertension. Based on this knowledge, we intend to investigate other potential mechanisms involved in SERCA2 dysfunction-induced pulmonary vascular remodeling. Experimental Approach Heterozygous SERCA2 C674S knock-in (SKI) mice of which half of cysteine in 674 was substituted by serine to mimic the partial irreversible oxidation of C674 were used. The lungs of SKI mice and their littermate wild-type mice were collected for PASMC culture, protein expression, and pulmonary vascular remodeling analysis. Results SERCA2 dysfunction increased intracellular Ca2+ levels, which activated Ca2+-dependent calcineurin (CaN) and promoted the nuclear translocation and protein expression of the nuclear factor of activated T-lymphocytes 4 (NFAT4) in an IRE1α/XBP1s pathway-independent manner. In SKI PASMCs, the scavenge of intracellular Ca2+ by BAPTA-AM or inhibition of CaN by cyclosporin A can prevent PASMC phenotypic transition. CDN1163, a SERCA2 agonist, suppressed the activation of CaN/NFAT4 and IRE1α/XBP1s pathways, reversed the protein expression of PASMC phenotypic transition markers and cell cycle-related proteins, and inhibited cell proliferation and migration when given to SKI PASMCs. Furthermore, CDN1163 ameliorated pulmonary vascular remodeling in SKI mice. Conclusions and Implications SERCA2 dysfunction promotes PASMC phenotypic transition and pulmonary vascular remodeling by multiple mechanisms, which could be improved by SERCA2 agonist CDN1163.

Oleanolic acid promotes skeletal muscle fiber type transformation by activating TGR5-mediated CaN signaling pathway

In recent years, the impact of bile acids and their representative G protein-coupled bile acid receptor 1 Takeda-G-protein-receptor-5 (TGR5) signaling pathway on muscle function and metabolic health has gained considerable interest. Increasing the content of slow muscle fibers has been recognized as an effective strategy to improve metabolic health. Oleanolic acid (OA) is a naturally occurring triterpenoid compound derived from plants, which can activate TGR5. The aim of this study was to investigate the effect of OA and TGR5 on muscle fiber types and further explore the underlying TGR5-dependent mechanisms. In this study, mice were divided into three groups and dietary supplementation with 0, 50, or 100 mg/kg OA. In addition, C2C12 cells were treated with OA at concentrations of 0, 5, 10, and 20 µM. Our studies revealed that OA promoted the conversion of fast to slow muscle fibers. In addition, it was found that OA activated the TGR5-mediated calcineurin (CaN)/nuclear factor of activated T cells cytoplasmic 1 (NFATc1) signaling pathway. Further mechanistic investigations demonstrated that inhibiting TGR5 and CaN abolished the effects of OA on muscle fiber types transformation. In conclusion, this study found that OA promotes the transformation of fast muscle fibers to slow muscle fibers through the TGR5-mediated CaN/NFATc1 signaling pathway.

The Ca2+/Calmodulin-dependent Calcineurin/NFAT Signaling Pathway in the Pathogenesis of Insulin Resistance in Skeletal Muscle.

Skeletal muscle is the tissue directly involved in insulin-stimulated glucose uptake. Glucose is the primary energy substrate for contracting muscles, and proper metabolism of glucose is essential for health. Contractile activity and the associated Ca2+signaling regulate functional capacity and muscle mass. A high concentration of Ca2+and the presence of calmodulin (CaM) leads to the activation of calcineurin (CaN), a protein with serine-threonine phosphatase activity. The signaling pathway linked with CaN and transcription factors like the nuclear factor of activated T cells (NFAT) is essential for skeletal muscle development and reprogramming of fast-twitch to slow-twitch fibers. CaN activation may promote metabolic adaptations in muscle cells, resulting in better insulin-stimulated glucose transport. The molecular mechanisms underlying the altered insulin response remain unclear. The role of the CaN/NFAT pathway in regulating skeletal muscle hypertrophy is better described than its involvement in the pathogenesis of insulin resistance. Thus, there are opportunities for future research in that field. This review presents the role of CaN/NFAT signaling and suggests the relationship with insulin-resistant muscles.

Long-term normalization of calcineurin activity in model mice rescues Pin1 and attenuates Alzheimer’s phenotypes without blocking peripheral T cell IL-2 response

BackgroundCurrent treatments for Alzheimer’s disease (AD) have largely failed to yield significant therapeutic benefits. Novel approaches are desperately needed to help address this immense public health issue. Data suggests that early intervention at the first stages of mild cognitive impairment may have a greater chance for success. The calcineurin (CN)-Pin1 signaling cascade can be selectively targeted with tacrolimus (FK506), a highly specific, FDA-approved CN inhibitor used safely for > 20 years in solid organ transplant recipients. AD prevalence was significantly reduced in solid organ recipients treated with FK506.MethodsTime release pellets were used to deliver constant FK506 dosage to APP/PS1 mice without deleterious manipulation or handling. Immunofluorescence, histology, molecular biology, and behavior were used to evaluate changes in AD pathology.ResultsFK506 can be safely and consistently delivered into juvenile APP/PS1 mice via time-release pellets to levels roughly seen in transplant patients, leading to the normalization of CN activity and reduction or elimination of AD pathologies including synapse loss, neuroinflammation, and cognitive impairment. Pin1 activity and function were rescued despite the continuing presence of high levels of transgenic Aβ42. Indicators of neuroinflammation including Iba1 positivity and IL-6 production were also reduced to normal levels. Peripheral blood mononuclear cells (PBMC) obtained during treatment or splenocytes isolated at euthanasia activated normally after mitogens.ConclusionsLow-dose, constant FK506 can normalize CNS CN and Pin1 activity, suppress neuroinflammation, and attenuate AD-associated pathology without blocking peripheral IL-2 responses making repurposed FK506 a viable option for early, therapeutic intervention in AD.

Taurine promotes muscle fiber type transformation through CaN/NFATc1 signaling in porcine myoblasts.

This study investigated the effect of taurine (TAU) on the muscle fiber type transformation in porcine myoblasts and its molecular mechanisms. The findings revealed that TAU augmented the protein expression of slow MyHC and the enzyme activities of oxidative metabolism markers like malate dehydrogenase and succinic dehydrogenase. Conversely, it curtailed the expression of fast MyHC and glycolytic metabolism enzyme activity of lactate dehydrogenase. Moreover, TAU elevated the expression of genes associated with oxidative fiber while diminishing the expression of those linked to glycolytic fibers, suggesting that TAU promoted the muscle fiber type transformation from glycolytic fiber to oxidative fiber. Additionally, TAU notably enhanced the expression of key molecules of calcineurin (CaN)/nuclear factor of activated T cells c1 (NFATc1) signaling and the CaN activity in porcine myoblasts. However, CaN inhibitor cyclosporine A abolished these effects induced by TAU. Our results indicated that TAU regulated the muscle fiber type transformation from glycolytic to oxidative fiber by activation of CaN/NFATc1 signaling.