Calcineurin Pathway Research Articles

Verapamil enhances the activity of Caspofungin against Cryptococcus neoformans， coinciding with inhibited Ca2+/CN pathway and damage to cell wall integrity

Given the challenges posed by toxicity and drug resistance in the treatment of cryptococcal infections, we sought to explore the antifungal potential of verapamil (VER), a calcium channel blocker, against Cryptococcus neoformans (C. neoformans), and its potential synergy with antifungals, specifically caspofungin (CAS). In vitro and in vivo (Galleria mellonella) models were employed to assess VER's antifungal activity and its interaction with CAS. Mechanisms underlying the synergism were explored through analysis of cell wall integrity, membrane permeability, and gene expression related to the calcineurin pathway. Additionally, the influence of Ca2+ on chitin deacetylase activity was investigated. VER exhibited a pronounced antifungal effect on C. neoformans and synergized with CAS, enhancing antifungal efficacy in Galleria mellonella. VER reduced chitosan content and disrupted cell wall integrity, evidenced by melanin leakage and fluorescence staining. VER+CAS modified membrane permeability, triggering intracellular ROS accumulation and mitochondrial membrane potential alterations. VER mitigated CAS-induced calcium fluctuations and downregulated calcineurin pathway genes. Furthermore, it was found that the enzyme activity of chitin deacetylase of C. neoformans is significantly influenced by the presence of Ca2+, suggesting that the use of VER may affect this activity. The synergistic antifungal effect of VER and CAS represents a promising therapeutic strategy for cryptococcal infections. The multifaceted mechanisms, including disruption of cell wall integrity and modulation of membrane permeability, and regulation of intracellular calcium signaling pathways, offer new insights into antifungal drug development.

Phosphate availability conditions caspofungin tolerance, capsule attachment and titan cell formation in Cryptococcus neoformans.

There is an urgent need for new antifungal drugs to treat invasive fungal diseases. Unfortunately, the echinocandin drugs that are fungicidal against other important fungal pathogens are ineffective against Cryptococcus neoformans, the causative agent of life-threatening meningoencephalitis in immunocompromised people. Contributing mechanisms for echinocandin tolerance are emerging with connections to calcineurin signaling, the cell wall, and membrane composition. In this context, we discovered that a defect in phosphate uptake impairs the tolerance of C. neoformans to the echinocandin caspofungin. Our previous analysis of mutants lacking three high affinity phosphate transporters revealed reduced elaboration of the polysaccharide capsule and attenuated virulence in mice. We investigated the underlying mechanisms and found that loss of the transporters and altered phosphate availability influences the cell wall and membrane composition. These changes contribute to the shedding of capsule polysaccharide thus explaining the reduced size of capsules on mutants lacking the phosphate transporters. We also found an influence of the calcineurin pathway including calcium sensitivity and an involvement of the endoplasmic reticulum in the response to phosphate limitation. Furthermore, we identified membrane and lipid composition changes consistent with the role of phosphate in phospholipid biosynthesis and with previous studies implicating membrane integrity in caspofungin tolerance. Finally, we discovered a contribution of phosphate to titan cell formation, a cell type that displays modified cell wall and capsule composition. Overall, our analysis reinforces the importance of phosphate as a regulator of cell wall and membrane composition with implications for capsule attachment and antifungal drug susceptibility.

Depression proteomic profiling in adolescents with transcriptome analyses in independent cohorts.

Depression is a major global burden with unclear pathophysiology and poor treatment outcomes. Diagnosis of depression continues to rely primarily on behavioral rather than biological methods. Investigating tools that might aid in diagnosing and treating early-onset depression is essential for improving the prognosis of the disease course. While there is increasing evidence of possible biomarkers in adult depression, studies investigating this subject in adolescents are lacking. In the current study, we analyzed protein levels in 461 adolescents assessed for depression using the Development and Well-Being Assessment (DAWBA) questionnaire as part of the domestic Psychiatric Health in Adolescent Study conducted in Uppsala, Sweden. We used the Proseek Multiplex Neuro Exploratory panel with Proximity Extension Assay technology provided by Olink Bioscience, followed by transcriptome analyses for the genes corresponding to the significant proteins, using four publicly available cohorts. We identified a total of seven proteins showing different levels between DAWBA risk groups at nominal significance, including RBKS, CRADD, ASGR1, HMOX2, PPP3R1, CD63, and PMVK. Transcriptomic analyses for these genes showed nominally significant replication of PPP3R1 in two of four cohorts including whole blood and prefrontal cortex, while ASGR1 and CD63 were replicated in only one cohort. Our study on adolescent depression revealed protein-level and transcriptomic differences, particularly in PPP3R1, pointing to the involvement of the calcineurin pathway in depression. Our findings regarding PPP3R1 also support the role of the prefrontal cortex in depression and reinforce the significance of investigating prefrontal cortex-related mechanisms in depression.

Heat stress-induced NO enhanced perylenequinone biosynthesis of Shiraia sp. via calcium signaling pathway

Perylenequinones (PQs) are natural photosensitizing compounds used as photodynamic therapy, and heat stress (HS) is the main limiting factor of mycelial growth and secondary metabolism of fungi. This study aimed to unravel the impact of HS-induced Ca2+ and the calcium signaling pathway on PQ biosynthesis of Shiraia sp. Slf14(w). Meanwhile, the intricate interplay between HS-induced NO and Ca2+ and the calcium signaling pathway was investigated. The outcomes disclosed that Ca2+ and the calcium signaling pathway activated by HS could effectively enhance the production of PQs in Shiraia sp. Slf14(w). Further investigations elucidated the specific mechanism through which NO signaling molecules induced by HS act upon the Ca2+/CaM (calmodulin) signaling pathway, thus propelling PQ biosynthesis in Shiraia sp. Slf14(w). This was substantiated by decoding the downstream positioning of the CaM/CaN (calcineurin) pathway in relation to NO through comprehensive analyses encompassing transcript levels, enzyme assays, and the introduction of chemical agents. Concurrently, the engagement of Ca2+ and the calcium signaling pathway in heat shock signaling was also evidenced. The implications of our study underscore the pivotal role of HS-induced Ca2+ and the calcium signaling pathway, which not only participate in heat shock signal transduction but also play an instrumental role in promoting PQ biosynthesis. Consequently, our study not only enriches our comprehension of the mechanisms driving HS signaling transduction in fungi but also offers novel insights into the PQ synthesis paradigm within Shiraia sp. Slf14(w).Key points• The calcium signaling pathway was proposed to participate in PQ biosynthesis under HS.• HS-induced NO was revealed to act upon the calcium signaling pathway for the first time.

Beneficial effects of resistance training on both mild and severe mouse dystrophic muscle function as a preclinical option for Duchenne muscular dystrophy.

Mechanical overloading (OVL) resulting from the ablation of muscle agonists, a supra-physiological model of resistance training, reduces skeletal muscle fragility, i.e. the immediate maximal force drop following lengthening contractions, and increases maximal force production, in mdx mice, a murine model of Duchene muscular dystrophy (DMD). Here, we further analyzed these beneficial effects of OVL by determining whether they were blocked by cyclosporin, an inhibitor of the calcineurin pathway, and whether there were also observed in the D2-mdx mice, a more severe murine DMD model. We found that cyclosporin did not block the beneficial effect of 1-month OVL on plantaris muscle fragility in mdx mice, nor did it limit the increases in maximal force and muscle weight (an index of hypertrophy). Fragility and maximal force were also ameliorated by OVL in the plantaris muscle of D2-mdx mice. In addition, OVL increased the expression of utrophin, cytoplamic γ-actin, MyoD, and p-Akt in the D2-mdx mice, proteins playing an important role in fragility, maximal force gain and muscle growth. In conclusion, OVL reduced fragility and increased maximal force in the more frequently used mild mdx model but also in D2-mdx mice, a severe model of DMD, closer to human physiopathology. Moreover, these beneficial effects of OVL did not seem to be related to the activation of the calcineurin pathway. Thus, this preclinical study suggests that resistance training could have a potential benefit in the improvement of the quality of life of DMD patients.

Inhibition of Myocardial Remodeling and Heart Failure by Traditional Herbal Medications: Evidence from Ginseng and ginkgo biloba.

Herbal-based medications have been used as therapeutic agents for thousands of years, particularly in Asian cultures. It is now well established that these herbal medications contain potent bioactive phytochemicals which exert a plethora of beneficial effects such as those seen on the cardiovascular system. Among the most widely studied of these herbal agents is ginseng, a member of the genus Panax, which has been shown to produce beneficial effects in terms of reducing cardiac pathology, at least in experimental studies. The beneficial effects of ginseng observed in such studies are likely attributable to their constituent ginsenosides, which are steroid-like saponins of which there are at least 100 and which vary according to ginseng species. Many ginseng species such as Panax ginseng (also known as Asian ginseng) and P quinquefolius (North American ginseng) as well as specific ginsenosides have been shown to attenuate hypertrophy as well as other indices of myocardial remodeling in a wide variety of experimental models. Ginkgo biloba on the other hand has been much less studied although the leaf extract of the ancient ginkgo tree has similarly consistently been shown to produce anti-remodeling effects. Ginkgo's primary bioactive constituents are thought to be terpene trilactones called ginkgolides, of which there are currently seven known types. Ginkgo and ginkgolides have also been shown to produce anti-remodeling effects as have been shown for ginseng in a variety of experimental models, in some cases via similar mechanisms. Although a common single mechanism for the salutary effects of these compounds is unlikely, there are a number of examples of shared effects including antioxidant and antiapoptotic effects as well as inhibition of pro-hypertrophic intracellular signaling such as that involving the calcineurin pathway which results in the upregulation of pro-hypertrophic genes. Robust clinical evidence represented by large scale phase 3 trials is lacking although there is limited supporting evidence from small trials at least with respect to ginseng. Taken together, both ginseng and ginkgo as well as their bioactive components offer potential as adjuvant therapy for the treatment of myocardial remodeling and heart failure.

MAP Kinase FgHog1 and Importin β FgNmd5 Regulate Calcium Homeostasis in Fusarium graminearum.

Maintaining cellular calcium (Ca2+) homeostasis is essential for many aspects of cellular life. The high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway responsible for signal integration and transduction plays crucial roles in environmental adaptation, especially in the response to osmotic stress. Hog1 is activated by transient Ca2+ increase in yeast, but the functions of the HOG pathway in Ca2+ homeostasis are largely unknown. We found that the HOG pathway was involved in the regulation of Ca2+ homeostasis in Fusarium graminearum, a devastating fungal pathogen of cereal crops. The deletion mutants of HOG pathway displayed increased sensitivity to Ca2+ and FK506, and elevated intracellular Ca2+ content. Ca2+ treatment induced the phosphorylation of FgHog1, and the phosphorylated FgHog1 was transported into the nucleus by importin β FgNmd5. Moreover, the increased phosphorylation and nuclear accumulation of FgHog1 upon Ca2+ treatment is independent of the calcineurin pathway that is conserved and downstream of the Ca2+ signal. Taken together, this study reported the novel function of FgHog1 in the regulation of Ca2+ homeostasis in F. graminearum, which advance the understanding of the HOG pathway and the association between the HOG and calcineurin pathways in fungi.

Caspofungin-induced β(1,3)-glucan exposure in Candida albicans is driven by increased chitin levels.

To successfully induce disease, Candida albicans must effectively evade the host immune system. One mechanism used by C. albicans to achieve this is to mask immunogenic β(1,3)-glucan epitopes within its cell wall under an outer layer of mannosylated glycoproteins. Consequently, induction of β(1,3)-glucan exposure (unmasking) via genetic or chemical manipulation increases fungal recognition by host immune cells in vitro and attenuates disease during systemic infection in mice. Treatment with the echinocandin caspofungin is one of the most potent drivers of β(1,3)-glucan exposure. Several reports using murine infection models suggest a role for the immune system, and specifically host β(1,3)-glucan receptors, in mediating the efficacy of echinocandin treatment in vivo. However, the mechanism by which caspofungin-induced unmasking occurs is not well understood. In this report, we show that foci of unmasking co-localize with areas of increased chitin within the yeast cell wall in response to caspofungin, and that inhibition of chitin synthesis via nikkomycin Z attenuates caspofungin-induced β(1,3)-glucan exposure. Furthermore, we find that both the calcineurin and Mkc1 mitogen-activated protein kinase pathways work synergistically to regulate β(1,3)-glucan exposure and chitin synthesis in response to drug treatment. When either of these pathways are interrupted, it results in a bimodal population of cells containing either high or low chitin content. Importantly, increased unmasking correlates with increased chitin content within these cells. Microscopy further indicates that caspofungin-induced unmasking correlates with actively growing cells. Collectively, our work presents a model in which chitin synthesis induces unmasking within the cell wall in response to caspofungin in growing cells. IMPORTANCE Systemic candidiasis has reported mortality rates ranging from 20% to 40%. The echinocandins, including caspofungin, are first-line antifungals used to treat systemic candidiasis. However, studies in mice have shown that echinocandin efficacy relies on both its cidal impacts on Candida albicans, as well as a functional immune system to successfully clear invading fungi. In addition to direct C. albicans killing, caspofungin increases exposure (unmasking) of immunogenic β(1,3)-glucan moieties. To evade immune detection, β(1,3)-glucan is normally masked within the C. albicans cell wall. Consequently, unmasked β(1,3)-glucan renders these cells more visible to the host immune system and attenuates disease progression. Therefore, discovery of how caspofungin-induced unmasking occurs is needed to elucidate how the drug facilitates host immune system-mediated clearance in vivo. We report a strong and consistent correlation between chitin deposition and unmasking in response to caspofungin and propose a model in which altered chitin synthesis drives increased unmasking during drug exposure.

Targeting Ca2+-dependent pathways to promote corneal epithelial wound healing induced by CISD2 deficiency

Chronic epithelial defects of the cornea, which are usually associated with severe dry eye disease, diabetes mellitus, chemical injuries or neurotrophic keratitis, as well as aging, are an unmet clinical need. CDGSH Iron Sulfur Domain 2 (CISD2) is the causative gene for Wolfram syndrome 2 (WFS2; MIM 604928). CISD2 protein is significantly decreased in the corneal epithelium of patients with various corneal epithelial diseases. Here we summarize the most updated publications and discuss the central role of CISD2 in corneal repair, as well as providing new results describing how targeting Ca2+-dependent pathways can improve corneal epithelial regeneration. This review mainly focuses on the following topics. Firstly, an overview of the cornea and of corneal epithelial wound healing. The key players involved in this process, such as Ca2+, various growth factors/cytokines, extracellular matrix remodeling, focal adhesions and proteinases, are briefly discussed. Secondly, it is well known that CISD2 plays an essential role in corneal epithelial regeneration via the maintenance of intracellular Ca2+ homeostasis. CISD2 deficiency dysregulates cytosolic Ca2+, impairs cell proliferation and migration, decreases mitochondrial function and increases oxidative stress. As a consequence, these abnormalities bring about poor epithelial wound healing and this, in turn, will lead to persistent corneal regeneration and limbal progenitor cell exhaustion. Thirdly, CISD2 deficiency induces three distinct Ca2+-dependent pathways, namely the calcineurin, CaMKII and PKCα signaling pathways. Intriguingly, inhibition of each of the Ca2+-dependent pathways seems to reverse cytosolic Ca2+ dysregulation and restore cell migration during corneal wound healing. Notably, cyclosporin, an inhibitor of calcineurin, appears to have a dual effect on both inflammatory and corneal epithelial cells. Finally, corneal transcriptomic analyses have revealed that there are six major functional groupings of differential expression genes when CISD2 deficiency is present: (1) inflammation and cell death; (2) cell proliferation, migration and differentiation; (3) cell adhesion, junction and interaction; (4) Ca2+ homeostasis; (5) wound healing and extracellular matrix; and (6) oxidative stress and aging. This review highlights the importance of CISD2 in corneal epithelial regeneration and identifies the potential of repurposing venerable FDA-approved drugs that target Ca2+-dependent pathways for new uses, namely treating chronic epithelial defects of the cornea.

Newly synthesized glycoprotein profiling to identify molecular signatures of warm ischemic injury in donor lungs.

Despite recent technological advances such as ex vivo lung perfusion (EVLP), the outcome of lung transplantation remains unsatisfactory with ischemic injury being a common cause for primary graft dysfunction. New therapeutic developments are hampered by limited understanding of pathogenic mediators of ischemic injury to donor lung grafts. Here, to identify novel proteomic effectors underlying the development of lung graft dysfunction, using bioorthogonal protein engineering, we selectively captured and identified newly synthesized glycoproteins (NewS-glycoproteins) produced during EVLP with unprecedented temporal resolution of 4 h. Comparing the NewS-glycoproteomes in lungs with and without warm ischemic injury, we discovered highly specific proteomic signatures with altered synthesis in ischemic lungs, which exhibited close association to hypoxia response pathways. Inspired by the discovered protein signatures, pharmacological modulation of the calcineurin pathway during EVLP of ischemic lungs offered graft protection and improved posttransplantation outcome. In summary, the described EVLP-NewS-glycoproteomics strategy delivers an effective new means to reveal molecular mediators of donor lung pathophysiology and offers the potential to guide future therapeutic development.NEW & NOTEWORTHY This study developed and implemented a bioorthogonal strategy to chemoselectively label, enrich, and characterize newly synthesized (NewS-)glycoproteins during 4-h ex vivo lung perfusion (EVLP). Through this approach, the investigators uncovered specific proteomic signatures associated with warm ischemic injury in donor lung grafts. These signatures exhibit high biological relevance to ischemia-reperfusion injury, validating the robustness of the presented approach.

Loss of A Metalloproteinase and Disintegrin 15 leads to exacerbated myocardial hypertrophy and dilation through activation of the calcineurin pathway in male but not female micev

Cardiac dysfunction associated with cardiomyopathies is characterized by myocardial hypertrophy and dilation involving many cellular and molecular signalling events. A disintegrin and metalloproteinases (ADAMs) are membrane-bound proteinases with diverse functions, whose role in heart disease remains underexplored. ADAM15 is expressed in the heart and is downregulated in the failing human heart. In left ventricular (LV) specimens, ADAM15 levels did not change in patients with concentric hypertrophy (preserved ejection fraction) but was markedly decreased in hypertrophic and dilated failing cardiomyopathy. We investigated the role of ADAM15 in pressure overloaded cardiomyopathy in both male and female C57BL/6 mice. Loss of ADAM15 alone did not cause cardiomyopathy in both male and female mice up to 1 year of age. Male and female wildtype (WT) and ADAM15 knockout ( Adam15 -/- ) mice were subjected to cardiac pressure overload by transverse aortic constriction (TAC) at 8 weeks of age. Adam15 -/- female mice had comparable cardiac function and hypertrophy to their WT controls while, Adam15 -/- male mice exhibited worsened decompensated hypertrophy and dilation with increased activation of mitogen activated protein kinases (pJNK, pERK1/2) compared to WT. Expression of integrin-α7 (but not integrin β1) increased significantly more in male Adam15 -/- -TAC hearts, while the interaction of integrins with basement membrane (laminin) decreased consistent with increased LV dilation. Activity of calcineurin was higher in male Adam15 -/- -TAC hearts along with greater de-phosphorylation of its target transcription factor, NFAT1. Calcineurin inhibition (cyclosporin A) blocked the excess hypertrophy and dilation in male Adam15 -/- mice post-TAC. Calcineurin signalling has been shown to be inhibited by 17β-estradiol (E2), the circulating form of estrogen. Thus, the sex-dependent phenotypic differences in male and female Adam15 -/- mice following pressure overload could be attributed to the inherent increased inhibition of calcineurin-mediated signalling due to the increased expression of estrogen in female mice. Research supported by Canadian Institutes of Health Research (CIHR) grant This is the full abstract presented at the American Physiology Summit 2023 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 Ycx1 protein encoded by the yeast YDL206W gene plays a role in calcium and calcineurin signaling

Calcium is ubiquitously present in all living cells and plays important regulatory roles in a wide variety of biological processes. In yeast, many effects of calcium are mediated via the action of calcineurin, a calcium/calmodulin-dependent protein phosphatase. Proper signaling of calcium and calcineurin is important in yeast, and the calcineurin pathway has emerged as a valuable target for developing novel antifungal drugs. Here, we report a role of YDL206W in calcium and calcineurin signaling in yeast. YDL206W is an uncharacterized gene in yeast, encoding a protein with two sodium/calcium exchange domains. Disrupting the YDL206W gene leads to a diminished level of calcium-induced activation of calcineurin and a reduced accumulation of cytosolic calcium. Consistent with a role of calcineurin in regulating pheromone and cell wall integrity signaling, the ydl206wΔ mutants display an enhanced growth arrest induced by pheromone treatment and poor growth at elevated temperature. Subcellular localization studies indicate that YDL206W is localized in endoplasmic reticulum and Golgi. Together, our results reveal YDL206W as a new regulator for calcineurin signaling in yeast and suggest a role of the endoplasmic reticulum and Golgi in regulating cytosolic calcium in yeast.

The Conserved Yeast Protein Knr4 Involved in Cell Wall Integrity Is a Multi-domain Intrinsically Disordered Protein

Knr4/Smi1 proteins are specific to the fungal kingdom and their deletion in the model yeast Saccharomyces cerevisiae and the human pathogen Candida albicans results in hypersensitivity to specific antifungal agents and a wide range of parietal stresses. In S. cerevisiae, Knr4 is located at the crossroads of several signalling pathways, including the conserved cell wall integrity and calcineurin pathways. Knr4 interacts genetically and physically with several protein members of those pathways. Its sequence suggests that it contains large intrinsically disordered regions. Here, a combination of small-angle X-ray scattering (SAXS) and crystallographic analysis led to a comprehensive structural view of Knr4. This experimental work unambiguously showed that Knr4 comprises two large intrinsically disordered regions flanking a central globular domain whose structure has been established. The structured domain is itself interrupted by a disordered loop. Using the CRISPR/Cas9 genome editing technique, strains expressing KNR4 genes deleted from different domains were constructed. The N-terminal domain and the loop are essential for optimal resistance to cell wall-binding stressors. The C-terminal disordered domain, on the other hand, acts as a negative regulator of this function of Knr4. The identification of molecular recognition features, the possible presence of secondary structure in these disordered domains and the functional importance of the disordered domains revealed here designate these domains as putative interacting spots with partners in either pathway. Targeting these interacting regions is a promising route to the discovery of inhibitory molecules that could increase the susceptibility of pathogens to the antifungals currently in clinical use.

HIV-1 gp120 protein promotes HAND through the calcineurin pathway activation

For over two decades, highly active antiretroviral therapy (HAART) was able to help prolong the life expectancy of people living with HIV-1 (PLWH) and eliminate the virus to an undetectable level. However, an increased prevalence of HIV- associated neurocognitive disorders (HAND) was observed. These symptoms range from neuronal dysfunction to cell death. Among the markers of neuronal deregulation, we cite the alteration of synaptic plasticity and neuronal communications. Clinically, these dysfunctions led to neurocognitive disorders such as learning alteration and loss of spatial memory, which promote premature brain aging even in HAART-treated patients. In support of these observations, we showed that the gp120 protein deregulates miR-499-5p and its downstream target, the calcineurin (CaN) protein. The gp120 protein also promotes the accumulation of calcium (Ca2+) and reactive oxygen species (ROS) inside the neurons leading to the activation of CaN and the inhibition of miR-499-5p. gp120 protein also caused mitochondrial fragmentation and changes in shape and size. The use of mimic miR-499 restored mitochondrial functions, appearance, and size. These results demonstrated the additional effect of the gp120 protein on neurons through the miR-499-5p/calcineurin pathway.

Calcineurin-dependent regulation of gap junction conductance and connexin phosphorylation in guinea pig left atrium

Atrial fibrillation (AF) occurs from disordered atrial action potential conduction and is associated with reduced gap junction electrical conductance (Gj). The Ca2+ and calmodulin-dependent phosphatase, calcineurin, reduces Gj in ventricular myocardium via a protein phosphatase-1 (PP1)-dependent pathway culminating in phosphorylation of serine368 on connexin43 (pSer368-Cx43). However, characterisation of corresponding pathways in left atrial myocardium, which have a more complex connexin subtype profile, is undefined and was the aim of this study. Gj was measured in guinea-pig left atrium from the frequency-dependent variation of intracellular impedance; intracellular [Ca2+], ([Ca2+]i) in low-Na solution was measured by Fura-2 fluorescence. Phosphorylation of guinea-pig Ser368-Cx43 residues was measured by Western blot; Cx40 was immunoprecipitated and probed for serine/threonine residue phosphorylation. Low-Na solution reversibly reduced Gj, in turn attenuated or prevented by calcineurin inhibitors cyclosporin-A or CAIP, respectively. Moreover, Ser368-Cx43 phosphorylation in low-Na solution was also prevented by CAIP. Changes were partially prevented by fostreicin (FST), a protein phosphatase-2A (PP2A) inhibitor; but not by tautomycin, a PP1 inhibitor. Serine/threonine residues on Cx40 were also phosphorylated in low-Na solution; prevented by CAIP and attenuated by FST. Reduced Gj with raised [Ca2+]i is paralleled by a changed Cx43/Cx40 phosphorylation status; changes mediated by calcineurin and PP2A-dependent pathways, but not PP1. The pharmacological profile underlying changes to guinea-pig atrial gap junction electrical conductance with raised intracellular [Ca2+]i is fundamentally different from that in ventricular myocardium. This provides a targeted drug model whereby atrial and ventricular myocardium can be selectively targeted to correct conduction defects.

TRPML1-Induced Lysosomal Ca2+ Signals Activate AQP2 Translocation and Water Flux in Renal Collecting Duct Cells.

Lysosomes are acidic Ca2+ storage organelles that actively generate local Ca2+ signaling events to regulate a plethora of cell functions. Here, we characterized lysosomal Ca2+ signals in mouse renal collecting duct (CD) cells and we assessed their putative role in aquaporin 2 (AQP2)-dependent water reabsorption. Bafilomycin A1 and ML-SA1 triggered similar Ca2+ oscillations, in the absence of extracellular Ca2+, by alkalizing the acidic lysosomal pH or activating the lysosomal cation channel mucolipin 1 (TRPML1), respectively. TRPML1-dependent Ca2+ signals were blocked either pharmacologically or by lysosomes' osmotic permeabilization, thus indicating these organelles as primary sources of Ca2+ release. Lysosome-induced Ca2+ oscillations were sustained by endoplasmic reticulum (ER) Ca2+ content, while bafilomycin A1 and ML-SA1 did not directly interfere with ER Ca2+ homeostasis per se. TRPML1 activation strongly increased AQP2 apical expression and depolymerized the actin cytoskeleton, thereby boosting water flux in response to an hypoosmotic stimulus. These effects were strictly dependent on the activation of the Ca2+/calcineurin pathway. Conversely, bafilomycin A1 led to perinuclear accumulation of AQP2 vesicles without affecting water permeability. Overall, lysosomal Ca2+ signaling events can be differently decoded to modulate Ca2+-dependent cellular functions related to the dock/fusion of AQP2-transporting vesicles in principal cells of the CD.

Calcineurin activation and scaffolding in Aß‐induced synaptic dysfunction

AbstractBackgroundCalcineurin (CaN) is a protein phosphatase that is essential for synaptic long‐term depression (LTD) through its regulation of AMPA‐type glutamate receptors (AMPAR). AMPAR regulation by CaN is critically mediated by the postsynaptic scaffold protein A‐kinase anchoring protein 150 (AKAP150), which localizes a pool of CaN to the postsynaptic membrane. Our lab has generated knockin mice carrying a mutant version of AKAP150 lacking its CaN‐binding site, the PxIxIT motif; AKAP150ΔPIX (PIX). LTD does not occur in these mice, underscoring the importance of the synaptic pool of CaN for normal synaptic function and plasticity. Pharmacological inhibition of calcineurin prevents diverse manifestations of Aβ‐mediated synaptotoxicity, including long‐term potentiation (LTP) blockade, synapse loss, and impaired learning and memory in AD mouse models. However, the importance of synaptically‐localized calcineurin in Aβ synaptotoxicity is unknown.MethodTo test our novel hypothesis that Aβ synaptotoxicity requires spatial positioning of CaN at the synapse via AKAP150 anchoring, we used our innovative AKAP150ΔPIX (PIX) knock‐in mouse model. Primary hippocampal neuronal cultures and acute hippocampal slices from WT and PIX mice were treated with various doses of Aβ oligomers and assayed for Aβ’s effect on dendritic spine density, quantal calcium transients through NMDA receptors (both in culture), and LTP through extracellular field recordings (in slices). Additionally, utilizing the 5xFAD AD mouse model, we generated PIX/5xFAD mice to test long‐term behavioral (via radial arm water maze and contextual fear conditioning tasks) and electrophysiological effects (through field recordings) of the PIX mutation on the 5xFAD phenotype in 6‐month‐old animals.ResultThe PIX mutation protects against acute Aβ‐mediated synaptic loss, decreased Ca2+ entry through NMDA receptors, and LTP deficits. Long‐term, the PIX mutation rescues contextual fear conditioning memory, but not radial arm water maze performance or chronic LTP deficits of 5xFAD mice.ConclusionSynaptically‐localized calcineurin is necessary for early Aβ‐mediated synaptic deficits. Long‐term Aβ exposure engages mechanisms beyond its AKAP‐anchored calcineurin pathway.

Loss of ADAM15 Exacerbates Transition to Decompensated Myocardial Hypertrophy and Dilation Through Activation of the Calcineurin Pathway.

Myocardial hypertrophy and dilation are key features of cardiomyopathies and involve several cellular and molecular events. ADAMs (a disintegrin and metalloproteinases) are membrane-bound proteinases with diverse functions whose role in heart disease remains underexplored. ADAM15 is expressed in the heart and is downregulated in the failing human heart. We investigated the role ADAM15 in pressure overload cardiomyopathy. We assessed ADAM15 levels in myocardial specimens from patients. Its direct role in pressure overload was investigated by subjecting wildtype and Adam15-deficient mice to transverse aortic constriction (TAC). ADAM15 levels did not change in patients with concentric hypertrophy, but markedly decreased in eccentric hypertrophy and heart failure. Loss of ADAM15 alone did not cause cardiomyopathy in mice (1 year old). After TAC, Adam15-/- mice exhibited worsened eccentric hypertrophy and dilation with greater increase in hypertrophy markers (pJNK, pERK1/2; Nppb, Nppa, Myh7, Acta1) compared with wildtype-TAC. Expression of integrin-α7 (but not integrin β1) increased significantly more in Adam15-/--TAC hearts, while the interaction of these integrins with basement membrane (laminin), decreased consistent with worsened left ventricle dilation. In vitro, ADAM15 knockdown increased cardiomyocyte hypertrophy in response to mechanical stretch. Adam15-/--TAC hearts exhibited increased calcineurin activity and de-phosphorylation of nuclear factor of activated T cells. Calcineurin inhibition (cyclosporin-A) blocked the excess hypertrophy and dilation in Adam15-/--TAC mice. Proteome profiling demonstrated the increased abundance of the key proteins linked to worsened DCM in Adam15-/--TAC. This is the first report demonstrating that ADAM15 can suppress hypertrophy through regulating the integrin-laminin interaction and the calcineurin pathway.

Potential Antifungal Targets for Aspergillus sp. from the Calcineurin and Heat Shock Protein Pathways.

Aspergillus species, especially A. fumigatus, and to a lesser extent others (A. flavus, A. niger, A. terreus), although rarely pathogenic to healthy humans, can be very aggressive to immunocompromised patients (they are opportunistic pathogens). Although survival rates for such infections have improved in recent decades following the introduction of azole derivatives, they remain a clinical challenge. The fact that current antifungals act as fungistatic rather than fungicide, that they have limited safety, and that resistance is becoming increasingly common make the need for new, more effective, and safer therapies to become more acute. Over the last decades, knowledge about the molecular biology of A. fumigatus and other Aspergillus species, and particularly of calcineurin, Hsp90, and their signaling pathway proteins, has progressed remarkably. Although calcineurin has attracted much interest, its adverse effects, particularly its immunosuppressive effects, make it less attractive than it might at first appear. The situation is not very different for Hsp90. Other proteins from their signaling pathways, such as protein kinases phosphorylating the four SPRR serine residues, CrzA, rcnA, pmcA-pmcC (particularly pmcC), rfeF, BAR adapter protein(s), the phkB histidine kinase, sskB MAP kinase kinase, zfpA, htfA, ctfA, SwoH (nucleoside diphosphate kinase), CchA, MidA, FKBP12, the K27 lysine position from Hsp90, PkcA, MpkA, RlmA, brlA, abaA, wetA, other heat shock proteins (Hsp70, Hsp40, Hsp12) currently appear promising and deserve further investigation as potential targets for antifungal drug development.

Modulation of calcineurin signaling during development.

Calcineurin signaling pathways are suppressed in Down syndrome (trisomy 21), by overexpression of genes that are located on chromosome 21. Two key genes are the regulator of calcineurin 1 (RCAN1), also called the Down syndrome critical region 1 (DSCR1), and the dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A). The suppressed calcineurin pathway may potentially be restored using small-molecule DYRK inhibitors, which have been proposed as therapeutics in Down syndrome. However, little is known about the benefits and risks of such treatments during various stages of embryonic development, fetal development, and childhood. We examined the modulation of calcineurin signaling during development, using zebrafish as a model system. To mimic suppressed calcineurin signaling in Down syndrome, zebrafish were exposed to the calcineurin inhibitors cyclosporine and tacrolimus during development. We found that suppression of calcineurin signaling changed specific larval behaviors, including activity and responses to acoustic and visual stimuli, depending on the period of exposure. Cotreatment with the DYRK inhibitor proINDY restored a few of these behaviors but also induced a range of adverse side effects including decreased activity and reduced optomotor responses to visual stimuli. Based on these results, we conclude that proINDY has limited benefits and substantial risks when used during development. We propose that zebrafish is an efficient model system for preliminary safety and efficacy tests of other DYRK inhibitors that aim to restore calcineurin signaling during neural development.