Protein Phosphatase Calcineurin Research Articles

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.

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.

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.

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.

Calcineurin is required for Candida glabrata Pdr1 transcriptional activation.

Fluconazole is one of the most commonly used antifungals today. A result of this has been the inevitable selection of fluconazole-resistant organisms. This is an especially acute problem in the pathogenic yeast Candida glabrata. Elevated minimal inhibitory concentrations for fluconazole in C. glabrata are frequently associated with substitution mutations within the Zn2Cys6 zinc cluster-containing transcription factor-encoding gene PDR1. These mutant Pdr1 regulators drive constitutively high expression of target genes like CDR1 that encodes an ATP-binding cassette transporter thought to act as a drug efflux pump. Exposure of C. glabrata to fluconazole induced expression of both Pdr1 and CDR1, although little is known of the molecular basis underlying the upstream signals that trigger Pdr1 activation. Here, we show that the protein phosphatase calcineurin is required for fluconazole-dependent induction of Pdr1 transcriptional regulation. Calcineurin catalytic activity is required for normal Pdr1 regulation, and a hyperactive form of this phosphatase can decrease susceptibility to the echinocandin caspofungin but does not show a similar change for fluconazole susceptibility. Loss of calcineurin from strains expressing two different gain-of-function forms of Pdr1 also caused a decrease in CDR1 expression and increased fluconazole susceptibility, demonstrating that even these hyperactive Pdr1 regulatory mutants cannot bypass the requirement for calcineurin. Our data implicate calcineurin activity as a link tying azole and echinocandin susceptibility together via the control of transcription factor activity.IMPORTANCEDrug-resistant microorganisms are a problem in the treatment of all infectious diseases; this is an especially acute problem with fungi due to the existence of only three major classes of antifungal drugs, including the azole drug fluconazole. In the pathogenic yeast Candida glabrata, mutant forms of a transcription factor called Pdr1 are commonly associated with decreased fluconazole susceptibility and poor clinical outcomes. Here, we identify a protein phosphatase called calcineurin that is required for fluconazole-dependent induction of Pdr1 transcriptional activation and associated drug susceptibility. Gain-of-function mutant forms of Pdr1 still required the presence of calcineurin to confer normally decreased fluconazole susceptibility. Previous studies showed that calcineurin controls susceptibility to the echinocandin class of antifungal drugs, and our data demonstrate that this protein phosphatase is also required for normal azole drug susceptibility. Calcineurin plays a central role in susceptibility to two of the three major classes of antifungal drugs in C. glabrata.

Melatonin inhibits Japanese encephalitis virus replication and neurotoxicity via calcineurin-autophagy pathways

BackgroundJapanese encephalitis virus (JEV) is a mosquito-borne flavivirus that has no specific treatment except for supportive medical care. JEV is a neurotropic virus that affects the nervous system and triggers inflammation in the brain.MethodsMelatonin is used as a sleep-inducing agent in neurophysiology and may serve as a protective agent against neurological and neurodegenerative diseases. Herein, we investigated the effects of melatonin and the critical roles of the serine/threonine protein phosphatase calcineurin during JEV infection in SK-N-SH neuroblastoma cells.ResultsMelatonin treatment decreased JEV replication and JEV-mediated neurotoxicity. Calcineurin activity was increased by JEV infection and inhibited by melatonin treatment. Through calcineurin regulation, melatonin decreased the JEV-mediated neuroinflammatory response and attenuated JEV-induced autophagy.ConclusionsCalcineurin inactivation has a protective effect in JEV-infected neuronal cells, and melatonin is a novel resource for the development of anti-JEV agents.

Is It Time to Repurpose Calcineurin Inhibitors for the Treatment of Cognitive Impairment and Dementia?

Numerous preclinical and human tissue studies implicate the protein phosphatase calcineurin (CN) as a pathophysiologic mechanism in Alzheimer's disease (AD) and other neurodegenerative conditions. Using public electronic records of tens of thousands of individuals across the United States, Silva et al. (2023) show that use of the FDA-approved CN inhibitor, tacrolimus (for purposes of immunosuppression) is also associated with reduced prevalence of dementia-related symptoms. Notably, the study controls for age, sex, and race as well as multiple risk factors for AD. The results suggest that tacrolimus, and possibly other immunosuppressants could be repurposed for the treatment of AD-related dementia.

Abstract P2021: Targeting Of Cdc42-interacting Protein 4 -calcineurin Signaling Complexes Is Cardioprotective During Ischemia/Reperfusion Injury

Myocardial infarction (MI) is a major cause of mortality and morbidity worldwide. Heart failure due to MI results from both acute ischemic/reperfusion (I/R) injury and subsequent post-MI pathological cardiac remodeling. The Ca 2+ /calmodulin-dependent protein phosphatase calcineurin (CaN), which promotes pathological cardiac remodeling, has been problematic as a therapeutic target due to its role in cardioprotection, including during I/R injury. We have shown that a select pool of CaNAβ holoenzyme is localized within the cardiomyocyte by scaffold protein Cdc42-Interacting Protein 4 (CIP4/TRIP10), such that the CaNAβ N-terminal polyproline (PP) domain binds directly to the CIP4 C-terminal SH3 domain. 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. We now consider whether targeting CIP4-CaNAβ signalosomes is beneficial in ischemic cardiomyopathy. To test the relevance of CIP4 in vivo to ischemic reperfusion (IR) injury, tamoxifen-inducible, cardiac-specific CIP4 knock-out (CIP4 CKO), control Tg(Myh6-cre/Esr1*), and control CIP4 f/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 reperfusion 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, AAV has been used to express the CaNAβ PP domain peptide. In contrast to the expression of the VIVIT pan-CaN inhibitory peptide, CaNAβ PP peptide expression did not worsen I/R injury. Together, these results suggest that PP-anchored CaNAβ does not contribute to cardioprotection. Targeting CIP4 signalosomes may constitute a novel strategy for inhibiting I/R injury and preventing the development of heart failure in response to diverse forms of cardiovascular disease.

Calmodulin Regulates Transient Receptor Potential TRPM3 and TRPM8-Induced Gene Transcription.

Calmodulin is a small protein that binds Ca2+ ions via four EF-hand motifs. The Ca2+/calmodulin complex as well as Ca2+-free calmodulin regulate the activities of numerous enzymes and ion channels. Here, we used genetic and pharmacological tools to study the functional role of calmodulin in regulating signal transduction of TRPM3 and TRPM8 channels. Both TRPM3 and TRPM8 are important regulators of thermosensation. Gene transcription triggered by stimulation of TRPM3 or TRPM8 channels was significantly impaired in cells expressing a calmodulin mutant with mutations in all four EF-hand Ca2+ binding motifs. Similarly, incubation of cells with the calmodulin inhibitor ophiobolin A reduced TRPM3 and TRPM8-induced signaling. The Ca2+/calmodulin-dependent protein phosphatase calcineurin was shown to negatively regulate TRPM3-induced gene transcription. Here, we show that TRPM8-induced transcription is also regulated by calcineurin. We propose that calmodulin plays a dual role in regulating TRPM3 and TRPM8 functions. Calmodulin is required for the activation of TRPM3 and TRPM8-induced intracellular signaling, most likely through a direct interaction with the channels. Ca2+ influx through TRPM3 and TRPM8 feeds back to TRPM3 and TRPM8-induced signaling by activation of the calmodulin-regulated enzyme calcineurin, which acts as a negative feedback loop for both TRPM3 and TRPM8 channel signaling.

The serine-threonine protein phosphatases that regulate the thiazide-sensitive NaCl cotransporter.

The activity of the Na+-Cl- cotransporter (NCC) in the distal convoluted tubule (DCT) is finely tuned by phosphorylation networks involving serine/threonine kinases and phosphatases. While much attention has been paid to the With-No-lysine (K) kinase (WNK)- STE20-related Proline Alanine rich Kinase (SPAK)/Oxidative Stress Responsive kinase 1 (OSR1) signaling pathway, there remain many unanswered questions regarding phosphatase-mediated modulation of NCC and its interactors. The phosphatases shown to regulate NCC's activity, directly or indirectly, are protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), calcineurin (CN), and protein phosphatase 4 (PP4). PP1 has been suggested to directly dephosphorylate WNK4, SPAK, and NCC. This phosphatase increases its abundance and activity when extracellular K+ is increased, which leads to distinct inhibitory mechanisms towards NCC. Inhibitor-1 (I1), oppositely, inhibits PP1 when phosphorylated by protein kinase A (PKA). CN inhibitors, like tacrolimus and cyclosporin A, increase NCC phosphorylation, giving an explanation to the Familial Hyperkalemic Hypertension-like syndrome that affects some patients treated with these drugs. CN inhibitors can prevent high K+-induced dephosphorylation of NCC. CN can also dephosphorylate and activate Kelch-like protein 3 (KLHL3), thus decreasing WNK abundance. PP2A and PP4 have been shown in in vitro models to regulate NCC or its upstream activators. However, no studies in native kidneys or tubules have been performed to test their physiological role in NCC regulation. This review focuses on these dephosphorylation mediators and the transduction mechanisms possibly involved in physiological states that require of the modulation of the dephosphorylation rate of NCC.

The calcineurin regulator Sarah enables distinct forms of homeostatic plasticity at the Drosophila neuromuscular junction.

Introduction: The ability of synapses to maintain physiological levels of evoked neurotransmission is essential for neuronal stability. A variety of perturbations can disrupt neurotransmission, but synapses often compensate for disruptions and work to stabilize activity levels, using forms of homeostatic synaptic plasticity. Presynaptic homeostatic potentiation (PHP) is one such mechanism. PHP is expressed at the Drosophila melanogaster larval neuromuscular junction (NMJ) synapse, as well as other NMJs. In PHP, presynaptic neurotransmitter release increases to offset the effects of impairing muscle transmitter receptors. Prior Drosophila work has studied PHP using different ways to perturb muscle receptor function-either acutely (using pharmacology) or chronically (using genetics). Some of our prior data suggested that cytoplasmic calcium signaling was important for expression of PHP after genetic impairment of glutamate receptors. Here we followed up on that observation. Methods: We used a combination of transgenic Drosophila RNA interference and overexpression lines, along with NMJ electrophysiology, synapse imaging, and pharmacology to test if regulators of the calcium/calmodulin-dependent protein phosphatase calcineurin are necessary for the normal expression of PHP. Results: We found that either pre- or postsynaptic dysregulation of a Drosophila gene regulating calcineurin, sarah (sra), blocks PHP. Tissue-specific manipulations showed that either increases or decreases in sra expression are detrimental to PHP. Additionally, pharmacologically and genetically induced forms of expression of PHP are functionally separable depending entirely upon which sra genetic manipulation is used. Surprisingly, dual-tissue pre- and postsynaptic sra knockdown or overexpression can ameliorate PHP blocks revealed in single-tissue experiments. Pharmacological and genetic inhibition of calcineurin corroborated this latter finding. Discussion: Our results suggest tight calcineurin regulation is needed across multiple tissue types to stabilize peripheral synaptic outputs.

Glucose Homeostasis and Pancreatic Islet Size Are Regulated by the Transcription Factors Elk-1 and Egr-1 and the Protein Phosphatase Calcineurin.

Pancreatic β-cells synthesize and secrete insulin. A key feature of diabetes mellitus is the loss of these cells. A decrease in the number of β-cells results in decreased biosynthesis of insulin. Increasing the number of β-cells should restore adequate insulin biosynthesis leading to adequate insulin secretion. Therefore, identifying proteins that regulate the number of β-cells is a high priority in diabetes research. In this review article, we summerize the results of three sophisticated transgenic mouse models showing that the transcription factors Elk-1 and Egr-1 and the Ca2+/calmodulin-regulated protein phosphatase calcineurin control the formation of sufficiently large pancreatic islets. Impairment of the biological activity of Egr-1 and Elk-1 in pancreatic β-cells leads to glucose intolerance and dysregulation of glucose homeostasis, the process that maintains glucose concentration in the blood within a narrow range. Transgenic mice expressing an activated calcineurin mutant also had smaller islets and showed hyperglycemia. Calcineurin induces dephosphorylation of Elk-1 which subsequently impairs Egr-1 biosynthesis and the biological functions of Elk-1 and Egr-1 to regulate islet size and glucose homeostasis.

Nuanced Interactions between AKAP79 and STIM1 with Orai1 Ca2+ Channels at Endoplasmic Reticulum-Plasma Membrane Junctions Sustain NFAT Activation.

A-kinase anchoring protein 79 (AKAP79) is a human scaffolding protein that organizes Ca2+/calmodulin-dependent protein phosphatase calcineurin, calmodulin, cAMP-dependent protein kinase, protein kinase C, and the transcription factor nuclear factor of activated T cells (NFAT1) into a signalosome at the plasma membrane. Upon Ca2+ store depletion, AKAP79 interacts with the N-terminus of STIM1-gated Orai1 Ca2+ channels, enabling Ca2+ nanodomains to stimulate calcineurin. Calcineurin then dephosphorylates and activates NFAT1, which then translocates to the nucleus. A fundamental question is how signalosomes maintain long-term signaling when key effectors are released and therefore removed beyond the reach of the activating signal. Here, we show that the AKAP79-Orai1 interaction is considerably more transient than that of STIM1-Orai1. Free AKAP79, with calcineurin and NFAT1 in tow, is able to replace rapidly AKAP79 devoid of NFAT1 on Orai1, in the presence of continuous Ca2+ entry. We also show that Ca2+ nanodomains near Orai1 channels activate almost the entire cytosolic pool of NFAT1. Recycling of inactive NFAT1 from the cytoplasm to AKAP79 in the plasma membrane, coupled with the relatively weak interaction between AKAP79 and Orai1, maintain excitation-transcription coupling. By measuring rates for AKAP79-NFAT interaction, we formulate a mathematical model that simulates NFAT dynamics at the plasma membrane.

Calcineurin Inhibitors Synergize with Manogepix to Kill Diverse Human Fungal Pathogens.

Invasive fungal infections have mortality rates of 30–90%, depending on patient co-morbidities and the causative pathogen. The frequent emergence of drug resistance reduces the efficacy of currently approved treatment options, highlighting an urgent need for antifungals with new modes of action. Addressing this need, fosmanogepix (N-phosphonooxymethylene prodrug of manogepix; MGX) is the first in a new class of gepix drugs, and acts as a broad-spectrum, orally bioavailable inhibitor of the essential fungal glycosylphosphatidylinositol (GPI) acyltransferase Gwt1. MGX inhibits the growth of diverse fungal pathogens and causes accumulation of immature GPI-anchored proteins in the fungal endoplasmic reticulum. Relevant to the ongoing clinical development of fosmanogepix, we report a synergistic, fungicidal interaction between MGX and inhibitors of the protein phosphatase calcineurin against important human fungal pathogens. To investigate this synergy further, we evaluated a library of 124 conditional expression mutants covering 95% of the genes encoding proteins involved in GPI-anchor biosynthesis or proteins predicted to be GPI-anchored. Strong negative chemical-genetic interactions between the calcineurin inhibitor FK506 and eleven GPI-anchor biosynthesis genes were identified, indicating that calcineurin signalling is required for fungal tolerance to not only MGX, but to inhibition of the GPI-anchor biosynthesis pathway more broadly. Depletion of these GPI-anchor biosynthesis genes, like MGX treatment, also exposed fungal cell wall (1→3)-β-D-glucans. Taken together, these findings suggest the increased risk of invasive fungal infections associated with use of calcineurin inhibitors as immunosuppressants may be mitigated by their synergistic fungicidal interaction with (fos)manogepix and its ability to enhance exposure of immunostimulatory glucans.

Development of a novel pharmacophore model to screen specific inhibitors for the serine-threonine protein phosphatase calcineurin

Calcineurin (CaN) is a calcium/calmodulin-dependent serine/threonine phosphatase with a crucial role in cellular homeostasis. It is also the target of the Food and Drug Administration (FDA) approved immunosuppressant drugs FK506 and cyclosporine A. Recent work from our group and others indicated that an uncontrolled increase in CaN activity causes synaptic dysfunction and neuronal death in various models of neurodegenerative diseases associated with calcium dysregulation. Furthermore, pharmacological normalization of CaN activity can prevent disease progression in animal models. However, none of the FDA-approved CaN inhibitors bind CaN directly, leading to adverse side effects. The development of direct CaN inhibitors is required to reduce off-target effects, but its highly conserved active site and similar mechanism of action with other protein serine/threonine phosphatases impose a significant challenge. In this work, we developed a novel pharmacophore model to screen for CaN-specific inhibitors. Then, we performed a virtual screen for molecules having the pharmacophore model. We also show that the molecules identified in this screen can inhibit CaN with a low micromolar IC50. Interestingly, the inhibitors identified from the screen do not inhibit phosphoprotein phosphatase 2A, a member of the serine/threonine phosphatase family that shares 43% sequence identity with the CaN active site. The pharmacophore model that we developed and validated in this work may help to accelerate the development of specific CaN inhibitors.

Calcineurin requirement for in vivo insular cortex LTD and CTA-extinction

Currently, it is widely accepted that memory extinction involves the formation of a new associative memory rather than unlearning of the information previously acquired. Nonetheless, the cellular and molecular mechanisms underlying this process are still unclear. In this regard, it has been suggested that while kinases modulate conditioning and LTP, phosphatases are relevant for extinction and LTD. In particular, the protein phosphatase calcineurin (CaN) has been involved in the extinction of some behavioral tasks along with LTD. Indeed, studies of our research group have demonstrated that induction of LTD in the basolateral nucleus of the amygdala (Bla) to the insular cortex (IC) pathway facilitates the extinction of conditioned taste aversion (CTA), while the induction of LTP in this pathway slows it down. In addition, we have shown that the extinction of CTA elicits an increase of CaN. The aim of the present study was to evaluate the participation of calcineurin in the extinction of CTA and in the expression of in vivo LTD in the Bla-IC pathway. For this purpose, we chemically inhibited calcineurin in the IC of adult male Wistar rats, either during CTA-extinction or thirty minutes after LTD induction in the Bla-IC pathway. Our results show that calcineurin inhibition slows down the CTA-extinction and blocks the maintenance of LTD. Furthermore, we show that CaN levels increase after LTD induction. These findings support the idea that calcineurin is a key molecular actor for both CTA extinction and LTD expression in the IC, a highly relevant neocortical area for the processing of aversively motivated learning tasks, suggesting that both processes are associated at a molecular level.

Ca2+/Calcineurin‐mediated release of exosomal miR‐23a regulates astrocyte‐neuron cross‐talk and protects against neuroinflammation and neurodegeneration

AbstractBackgroundAstrocyte dysfunction and associated neuroinflammation is well implicated in various neurodegenerative disorders like Alzheimer’s (AD) and Parkinson’s diseases (PD). Importantly, Ca2+/Calcineurin signaling is reported to induce astrocyte dysfunction in AD (1) but very little is known regarding PD. So, our objective was to study the role of Ca2+/Calcineurin signaling in astrocyte‐neuron cross‐talk during PD‐related neuroinflammation which is unreported till date.Method1.Mammalian cell culture 2.Immunoblotting. 3.qRT‐PCR. 4.Exosome isolation. 5.Intracellular Ca2+ measurement. 6.Transfection (miRNA mimics). 7. 3’UTR cloning. 8.Luciferase assay.ResultWe treated human astrocytic 1321N1 cells with Rotenone, a neuro‐toxin known to induce PD‐associated neuroinflammation (2). At the dose of Rotenone which caused about 40% death in 24 hrs, simultaneous activation of the Ca2+‐dependent protein phosphatase Calcineurin and intracellular surge in the Ca2+ levels were also observed. Now, miR‐23a is an anti‐apoptotic miRNA and Calcineurin/NFAT‐mediated upregulation of miR‐23a has been reported in cardiac hypertrophy (3). Surprisingly, in our study, Rotenone caused a Calcineurin‐mediated decrease of intracellular miR‐23a levels in astrocytes. Now this decrease, we found, was due to a concomitant release of miR‐23a via exosomes. Can this anti‐apoptotic miR‐23a released by astrocytes protect the neurons? To answer this, we treated neuronal SH‐SY5Y cells with Rotenone and found that the pro‐apoptotic protein PUMA was upregulated although the miR‐23a levels remained unchanged. Interestingly, over‐expression of miR‐23a caused attenuation of the Rotenone‐mediated death in neurons. PUMA being a predicted target of miR‐23a, we cloned the 3’UTR of PUMA and subsequent luciferase assay revealed that indeed PUMA is a direct target of miR‐23a under our experimental conditions.ConclusionOur results suggest a novel mechanism by which Ca2+/Calcineurin‐mediated release of miR‐23a through exosomes regulates astrocyte‐neuron cross‐talk and plays a probable role in protection against neuroinflammation and neurodegeneration.

Critical Protein-Protein Interactions Determine the Biological Activity of Elk-1, a Master Regulator of Stimulus-Induced Gene Transcription.

Elk-1 is a transcription factor that binds together with a dimer of the serum response factor (SRF) to the serum-response element (SRE), a genetic element that connects cellular stimulation with gene transcription. Elk-1 plays an important role in the regulation of cellular proliferation and apoptosis, thymocyte development, glucose homeostasis and brain function. The biological function of Elk-1 relies essentially on the interaction with other proteins. Elk-1 binds to SRF and generates a functional ternary complex that is required to activate SRE-mediated gene transcription. Elk-1 is kept in an inactive state under basal conditions via binding of a SUMO-histone deacetylase complex. Phosphorylation by extracellular signal-regulated protein kinase, c-Jun N-terminal protein kinase or p38 upregulates the transcriptional activity of Elk-1, mediated by binding to the mediator of RNA polymerase II transcription (Mediator) and the transcriptional coactivator p300. Strong and extended phosphorylation of Elk-1 attenuates Mediator and p300 recruitment and allows the binding of the mSin3A-histone deacetylase corepressor complex. The subsequent dephosphorylation of Elk-1, catalyzed by the protein phosphatase calcineurin, facilitates the re-SUMOylation of Elk-1, transforming Elk-1 back to a transcriptionally inactive state. Thus, numerous protein–protein interactions control the activation cycle of Elk-1 and are essential for its biological function.

Alpha-arrestins Aly1/Art6 and Aly2/Art3 regulate trafficking of the glycerophosphoinositol transporter Git1 and impact phospholipid homeostasis.

Phosphatidylinositol (PI) is an essential phospholipid, critical to membrane bilayers. The complete deacylation of PI by B-type phospholipases produces intracellular and extracellular glycerophosphoinositol (GPI). Extracellular GPI is transported into the cell via Git1, a member of the Major Facilitator Superfamily of transporters at the yeast plasma membrane. Internalized GPI is degraded to produce inositol, phosphate and glycerol, thereby contributing to these pools. GIT1 gene expression is controlled by nutrient balance, with phosphate or inositol starvation increasing GIT1 expression to stimulate GPI uptake. However, less is known about control of Git1 protein levels or localization. We find that the α-arrestins, an important class of protein trafficking adaptor, regulate Git1 localization and this is dependent upon their interaction with the ubiquitin ligase Rsp5. Specifically, α-arrestin Aly2 stimulates Git1 trafficking to the vacuole under basal conditions, but in response to GPI-treatment, either Aly1 or Aly2 promote Git1 vacuole trafficking. Cell surface retention of Git1, as occurs in aly1∆aly2∆ cells, is linked to impaired growth in the presence of exogenous GPI and results in increased uptake of radiolabeled GPI, suggesting that accumulation of GPI somehow causes cellular toxicity. Regulation of α-arrestin Aly1 by the protein phosphatase calcineurin improves steady-state and substrate-induced trafficking of Git1, however, calcineurin plays a larger role in Git1 trafficking beyond regulation of α-arrestins. Interestingly, loss of Aly1 and Aly2 increased phosphatidylinositol-3-phosphate on the limiting membrane of the vacuole, and this was further exacerbated by GPI addition, suggesting that the effect is partially linked to Git1. Loss of Aly1 and Aly2 leads to increased incorporation of inositol label from [3 H]-inositol-labelled GPI into PI, confirming that internalized GPI influences PI balance and indicating a role for the a-arrestins in this regulation. The α-arrestins Aly1 and Aly2 are novel regulators of Git1 trafficking with previously unanticipated roles in controlling phospholipid distribution and balance. To our knowledge, this is the first example of α-arrestin regulation of phosphatidyliniositol-3-phosphate levels. In future studies it will be exciting to determine if other α-arrestins similarly alter PI and PIPs to change the cellular landscape.

Abstract P515: Dynamics Of Cardiomyocyte Differentiation In Isogenic Induced Pluripotent Stem Cells Derived From Trisomy21 Patients Hints Molecular Pathology Of Congenital Heart Defects In Down Syndrome

Down syndrome (DS) is the most frequently occurring human chromosomal disorder and is responsible for a range of both congenital defects and progressive, degenerative conditions. For instance, an estimated 50% DS neonates are born with congenital heart defects (CHD) and more than 50% of DS adults develop early onset Alzheimer’s. Using induced pluripotent stem cells (iPSCs) derived from DS patients and isogenic controls we previously demonstrated the presence of a hyper-metabolic, hyper-fused mitochondrial network in trisomic iPSCs (3S-iPSCs) compared to disomic (2S-iPSCs) controls. Furthermore, mitochondrial function was normalized by siRNA depletion of RCAN1, an inhibitor of the protein phosphatase calcineurin (CN). Both CN signaling and mitochondrial metabolism have been implicated in a variety of steps during the progression from embryonic stem cells to cardiac progenitors, including self-renewal, exit from pluripotency, and commitment to cardiac verses hematopoietic lineages. Based on this, we hypothesized that the dynamics of many of these processes will be altered over the course of differentiation of 3S-iPSCs to cardiomyocytes when compared to 2S-iPSCs. Here, we investigate the temporal expression of pluripotency associated genes and lineage associated genes as well as cardiac mesoderm and mature cardiomyocyte specific genes. We also define and compare changes in CN activity, expression of specific CN isoforms, mitochondrial expansion, ROS generation, and activation of stress responses. Our study identifies early developmental and metabolic sequelae capable of contributing to CHD in DS that may result from a disruption in the normal balance in crosstalk between CN and RCAN1.