MICU1 is an essential gatekeeper for MCU-mediated mitochondrial Ca(2+) uptake that regulates cell survival.

Mitochondrial Ca(2+) (Ca(2+)(m)) uptake is mediated by an inner membrane Ca(2+) channel called the uniporter. Ca(2+) uptake is driven by the considerable voltage present across the inner membrane (ΔΨ(m)) generated by proton pumping by the respiratory chain. Mitochondrial matrix Ca(2+) concentration is maintained five to six orders of magnitude lower than its equilibrium level, but the molecular mechanisms for how this is achieved are not clear. Here, we demonstrate that the mitochondrial protein MICU1 is required to preserve normal [Ca(2+)](m) under basal conditions. In its absence, mitochondria become constitutively loaded with Ca(2+), triggering excessive reactive oxygen species generation and sensitivity to apoptotic stress. MICU1 interacts with the uniporter pore-forming subunit MCU and sets a Ca(2+) threshold for Ca(2+)(m) uptake without affecting the kinetic properties of MCU-mediated Ca(2+) uptake. Thus, MICU1 is a gatekeeper of MCU-mediated Ca(2+)(m) uptake that is essential to prevent [Ca(2+)](m) overload and associated stress.

Epstein-Barr virus-induced ectopic CD137 expression helps nasopharyngeal carcinoma to escape immune surveillance and enables targeting by chimeric antigen receptors.

Non-keratinizing nasopharyngeal carcinoma (NPC) is a malignancy with a poor prognosis for relapsing patients and those with metastatic disease. Here, we identify a novel disease mechanism of NPC which may be its Achilles' heel that makes it susceptible to immunotherapy. CD137 is a potent costimulatory receptor on activated T cells, and CD137 agonists strongly enhance anti-tumor immune responses. A negative feedback mechanism prevents overstimulation by transferring CD137 from T cells to CD137 ligand (CD137L)-expressing antigen presenting cells (APC) during cognate interaction, upon which the CD137-CD137L complex is internalized and degraded. We found ectopic expression of CD137 on 42 of 122 (34.4%) NPC cases, and that CD137 is induced by the Epstein-Barr virus latent membrane protein (LMP) 1. CD137 expression enables NPC to hijack the inbuilt negative feedback mechanism to downregulate the costimulatory CD137L on APC, facilitating its escape from immune surveillance. Further, the ectopically expressed CD137 signals into NPC cells via the p38-MAPK pathway, and induces the expression of IL-6, IL-8 and Laminin γ2. As much as ectopic CD137 expression may support the growth and spread of NPC, it may be a target for its immunotherapeutic elimination. Natural killer cells that express a CD137-specific chimeric antigen receptor induce death in CD137+ NPC cells, in vitro, and in vivo in a murine xenograft model. These data identify a novel immune escape mechanism of NPC, and lay the foundation for an urgently needed immunotherapeutic approach for NPC.

Mitochondrial Dysfunction in Age-Related Metabolic Disorders.

Aging is a natural biological process in living organisms characterized by receding bioenergetics. Mitochondria are crucial for cellular bioenergetics and thus an important contributor to age-related energetics deterioration. In addition, mitochondria play a major role in calcium signaling, redox homeostasis, and thermogenesis making this organelle a major cellular component that dictates the fate of a cell. To maintain its quantity and quality, mitochondria undergo multiple processes such as fission, fusion, and mitophagy to eliminate or replace damaged mitochondria. While this bioenergetics machinery is properly protected, the functional decline associated with age and age-related metabolic diseases is mostly a result of failure in such protective mechanisms. In addition, metabolic by-products like reactive oxygen species also aid in this destructive pathway. Mitochondrial dysfunction has always been thought to be associated with diseases. Moreover, studies in recent years have pointed out that aging contributes to the decay of mitochondrial health by promoting imbalances in key mitochondrial-regulated pathways. Hence, it is crucial to understand the nexus of mitochondrial dysfunction in age-related diseases. This review focuses on various aspects of basic mitochondrial biology and its status in aging and age-related metabolic diseases.

Epigenetic regulation of microglial phosphatidylinositol 3-kinase pathway involved in long-term potentiation and synaptic plasticity in rats.

Microglia are the main form of immune defense in the central nervous system. Microglia express phosphatidylinositol 3-kinase (PI3K), which has been shown to play a significant role in synaptic plasticity in neurons and inflammation via microglia. This study shows that microglial PI3K is regulated epigenetically through histone modifications and posttranslationally through sumoylation and is involved in long-term potentiation (LTP) by modulating the expression of brain-derived neurotrophic factor (BDNF), which has been shown to be involved in neuronal synaptic plasticity. Sodium butyrate, a histone deacetylase inhibitor, upregulates PI3K expression, the phosphorylation of its downstream effectors, AKT and cAMP response element-binding protein (CREB), and the expression of BDNF in microglia, suggesting that BDNF secretion is regulated in microglia via epigenetic regulation of PI3K. Further, knockdown of SUMO1 in BV2 microglia results in a decrease in the expression of PI3K, the phosphorylation of AKT and CREB, as well as the expression of BDNF. These results suggest that microglial PI3K is epigenetically regulated by histone modifications and posttranslationally modified by sumoylation, leading to altered expression of BDNF. Whole-cell voltage-clamp showed the involvement of microglia in neuronal LTP, as selective ablation or disruption of microglia with clodronate in rat hippocampal slices abolished LTP. However, LTP was rescued when the same hippocampal slices were treated with active PI3K or BDNF, indicating that microglial PI3K/AKT signaling contributes to LTP and synaptic plasticity. Understanding the mechanisms by which microglial PI3K influences synapses provides insights into the ways it can modulate synaptic transmission and plasticity in learning and memory.

Transcriptome analysis reveals intermittent fasting-induced genetic changes in ischemic stroke.

Genetic changes due to dietary intervention in the form of either calorie restriction (CR) or intermittent fasting (IF) are not reported in detail until now. However, it is well established that both CR and IF extend the lifespan and protect against neurodegenerative diseases and stroke. The current research aims were first to describe the transcriptomic changes in brains of IF mice and, second, to determine whether IF induces extensive transcriptomic changes following ischemic stroke to protect the brain from injury. Mice were randomly assigned to ad libitum feeding (AL), 12 (IF12) or 16 (IF16) h daily fasting. Each diet group was then subjected to sham surgery or middle cerebral artery occlusion and consecutive reperfusion. Mid-coronal sections of ipsilateral cerebral tissue were harvested at the end of the 1 h ischemic period or at 3, 12, 24 or 72 h of reperfusion, and genome-wide mRNA expression was quantified by RNA sequencing. The cerebral transcriptome of mice in AL group exhibited robust, sustained up-regulation of detrimental genetic pathways under ischemic stroke, but activation of these pathways was suppressed in IF16 group. Interestingly, the cerebral transcriptome of AL mice was largely unchanged during the 1 h of ischemia, whereas mice in IF16 group exhibited extensive up-regulation of genetic pathways involved in neuroplasticity and down-regulation of protein synthesis. Our data provide a genetic molecular framework for understanding how IF protects brain cells against damage caused by ischemic stroke, and reveal cellular signaling and bioenergetic pathways to target in the development of clinical interventions.

Dynamic contrast-enhanced MRI of brown and beige adipose tissues.

PURPOSE: The vascular blood flow in brown adipose tissue (BAT) is important for handling triglyceride clearance, increased blood flow and oxygenation. We used dynamic contrast-enhanced (DCE)-MRI and fat fraction (FF) imaging for investigating vascular perfusion kinetics in brown and beige adipose tissues with cold exposure or treatment with ß3-adrenergic agonist. METHODS: FF imaging and DCE-MRI using gadolinium-diethylenetriaminepentaacetic acid were performed in interscapular BAT (iBAT) and beige tissues using male Wister rats (n = 38). Imaging was performed at thermoneutral condition and with either cold exposure, treatment with pharmacological agent CL-316,243, or saline. DCE-MRI and FF data were co-registered to enhance the understanding of metabolic activity. RESULTS: Uptake of contrast agent in activated iBAT and beige tissues were significantly (P < .05) higher than nonactivated iBAT. The Ktrans and kep increased significantly in iBAT and beige tissues after treatment with either cold exposure or ß3-adrenergic agonist. The FF decreased in activated iBAT and beige tissues. The Ktrans and FF from iBAT and beige tissues were inversely correlated (r = 0.97; r = 0.94). Significant increase in vascular endothelial growth factor expression and Ktrans in activated iBAT and beige tissues were in agreement with the increased vasculature and vascular perfusion kinetics. The iBAT and beige tissues were validated by measuring molecular markers. CONCLUSION: Increased Ktrans and decreased FF in iBAT and beige tissues were in agreement with the vascular perfusion kinetics facilitating the clearance of free fatty acids. The methodology can be extended for the screening of browning agents.

Tissue-selective restriction of RNA editing of CaV1.3 by splicing factor SRSF9.

Adenosine DeAminases acting on RNA (ADAR) catalyzes adenosine-to-inosine (A-to-I) conversion within RNA duplex structures. While A-to-I editing is often dynamically regulated in a spatial-temporal manner, the mechanisms underlying its tissue-selective restriction remain elusive. We have previously reported that transcripts of voltage-gated calcium channel CaV1.3 are subject to brain-selective A-to-I RNA editing by ADAR2. Here, we show that editing of CaV1.3 mRNA is dependent on a 40 bp RNA duplex formed between exon 41 and an evolutionarily conserved editing site complementary sequence (ECS) located within the preceding intron. Heterologous expression of a mouse minigene that contained the ECS, intermediate intronic sequence and exon 41 with ADAR2 yielded robust editing. Interestingly, editing of CaV1.3 was potently inhibited by serine/arginine-rich splicing factor 9 (SRSF9). Mechanistically, the inhibitory effect of SRSF9 required direct RNA interaction. Selective down-regulation of SRSF9 in neurons provides a basis for the neuron-specific editing of CaV1.3 transcripts.

TRPM2 channels protect against cardiac ischemia-reperfusion injury: role of mitochondria.

Cardiac TRPM2 channels were activated by intracellular adenosine diphosphate-ribose and blocked by flufenamic acid. In adult cardiac myocytes the ratio of GCa to GNa of TRPM2 channels was 0.56 ± 0.02. To explore the cellular mechanisms by which TRPM2 channels protect against cardiac ischemia/reperfusion (I/R) injury, we analyzed proteomes from WT and TRPM2 KO hearts subjected to I/R. The canonical pathways that exhibited the largest difference between WT-I/R and KO-I/R hearts were mitochondrial dysfunction and the tricarboxylic acid cycle. Complexes I, III, and IV were down-regulated, whereas complexes II and V were up-regulated in KO-I/R compared with WT-I/R hearts. Western blots confirmed reduced expression of the Complex I subunit and other mitochondria-associated proteins in KO-I/R hearts. Bioenergetic analyses revealed that KO myocytes had a lower mitochondrial membrane potential, mitochondrial Ca(2+) uptake, ATP levels, and O2 consumption but higher mitochondrial superoxide levels. Additionally, mitochondrial Ca(2+) uniporter (MCU) currents were lower in KO myocytes, indicating reduced mitochondrial Ca(2+) uptake was likely due to both lower ψm and MCU activity. Similar to isolated myocytes, O2 consumption and ATP levels were also reduced in KO hearts. Under a simulated I/R model, aberrant mitochondrial bioenergetics was exacerbated in KO myocytes. Reactive oxygen species levels were also significantly higher in KO-I/R compared with WT-I/R heart slices, consistent with mitochondrial dysfunction in KO-I/R hearts. We conclude that TRPM2 channels protect the heart from I/R injury by ameliorating mitochondrial dysfunction and reducing reactive oxygen species levels.

Histone 3.3 participates in a self-sustaining cascade of apoptosis that contributes to the progression of chronic obstructive pulmonary disease.

RATIONALE: Shifts in the gene expression of nuclear protein in chronic obstructive pulmonary disease (COPD), a progressive disease that is characterized by extensive lung inflammation and apoptosis, are common; however, the extent of the elevation of the core histones, which are the major components of nuclear proteins and their consequences in COPD, has not been characterized, which is important because extracellular histones are cytotoxic to endothelial and airway epithelial cells. OBJECTIVES: To investigate the role of extracellular histones in COPD disease progression. METHODS: We analyzed the nuclear lung proteomes of ex-smokers with and without the disease. Further studies on the consequences of H3.3 were also performed. MEASUREMENTS AND MAIN RESULTS: A striking finding was a COPD-specific eightfold increase of hyperacetylated histone H3.3. The hyperacetylation renders H3.3 resistant to proteasomal degradation despite ubiquitination; when combined with the reduction in proteasome activity that is known for COPD, this resistance helps account for the increased levels of H3.3. Using anti-H3 antibodies, we found H3.3 in the airway lumen, alveolar fluid, and plasma of COPD samples. H3.3 was cytotoxic to lung structural cells via a mechanism that involves the perturbation of Ca(2+) homeostasis and mitochondrial toxicity. We used the primary human airway epithelial cells and found that the antibodies to either the C or N terminus of H3 could partially reverse H3.3 toxicity. CONCLUSIONS: Our data indicate that there is an uncontrolled positive feedback loop in which the damaged cells release acetylated H3.3, which causes more damage, adds H3.3 release, and contributes toward the disease progression.

Role of Mitogen-Activated Protein (MAP) Kinase Pathways in Metabolic Diseases.

Physiological processes that govern the normal functioning of mammalian cells are regulated by a myriad of signalling pathways. Mammalian mitogen-activated protein (MAP) kinases constitute one of the major signalling arms and have been broadly classified into four groups that include extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), p38, and ERK5. Each signalling cascade is governed by a wide array of external and cellular stimuli, which play a critical part in mammalian cells in the regulation of various key responses, such as mitogenic growth, differentiation, stress responses, as well as inflammation. This evolutionarily conserved MAP kinase signalling arm is also important for metabolic maintenance, which is tightly coordinated via complicated mechanisms that include the intricate interaction of scaffold proteins, recognition through cognate motifs, action of phosphatases, distinct subcellular localisation, and even post-translational modifications. Aberration in the signalling pathway itself or their regulation has been implicated in the disruption of metabolic homeostasis, which provides a pathophysiological foundation in the development of metabolic syndrome. Metabolic syndrome is an umbrella term that usually includes a group of closely associated metabolic diseases such as hyperglycaemia, hyperlipidaemia, and hypertension. These risk factors exacerbate the development of obesity, diabetes, atherosclerosis, cardiovascular diseases, and hepatic diseases, which have accounted for an increase in the worldwide morbidity and mortality rate. This review aims to summarise recent findings that have implicated MAP kinase signalling in the development of metabolic diseases, highlighting the potential therapeutic targets of this pathway to be investigated further for the attenuation of these diseases.

Intracellular endothelin type B receptor-driven Ca2+ signal elicits nitric oxide production in endothelial cells.

Endothelin-1 exerts its actions via activation of ET(A) and ET(B) G(q/11) protein-coupled receptors, located in the plasmalemma, cytoplasm, and nucleus. Although the autocrine/paracrine nature of endothelin-1 signaling has been extensively studied, its intracrine role has been largely attributed to interaction with receptors located on nuclear membranes and the nucleoplasm. Because ET(B) receptors have been shown to be targeted to endolysosomes, we used intracellular microinjection and concurrent imaging methods to test their involvement in Ca(2+) signaling and subsequential NO production. We provide evidence that microinjected endothelin-1 produces a dose-dependent elevation in cytosolic calcium concentration in ET(B)-transfected cells and endothelial cells; this response is sensitive to ET(B) but not ET(A) receptor blockade. In endothelial cells, the endothelin-1-induced Ca(2+) response is abolished upon endolysosomal but not Golgi disruption. Moreover, the effect is prevented by inhibition of microautophagy and is sensitive to inhibitors of the phospholipase C and inositol 1,4,5-trisphosphate receptor. Furthermore, intracellular endothelin-1 increases nitric oxide via an ET(B)-dependent mechanism. Our results indicate for the first time that intracellular endothelin-1 activates endolysosomal ET(B) receptors and increase cytosolic Ca(2+) and nitric oxide production. Endothelin-1 acts in an intracrine fashion on endolysosomal ET(B) to induce nitric oxide formation, thus modulating endothelial function.

The second member of transient receptor potential-melastatin channel family protects hearts from ischemia-reperfusion injury.

The second member of the transient receptor potential-melastatin channel family (TRPM2) is expressed in the heart and vasculature. TRPM2 channels were expressed in the sarcolemma and transverse tubules of adult left ventricular (LV) myocytes. Cardiac TRPM2 channels were functional since activation with H2O2 resulted in Ca(2+) influx that was dependent on extracellular Ca(2+), was significantly higher in wild-type (WT) myocytes compared with TRPM2 knockout (KO) myocytes, and inhibited by clotrimazole in WT myocytes. At rest, there were no differences in LV mass, heart rate, fractional shortening, and +dP/dt between WT and KO hearts. At 2-3 days after ischemia-reperfusion (I/R), despite similar areas at risk and infarct sizes, KO hearts had lower fractional shortening and +dP/dt compared with WT hearts. Compared with WT I/R myocytes, expression of the Na(+)/Ca(2+) exchanger (NCX1) and NCX1 current were increased, expression of the α1-subunit of Na(+)-K(+)-ATPase and Na(+) pump current were decreased, and action potential duration was prolonged in KO I/R myocytes. Post-I/R, intracellular Ca(2+) concentration transients and contraction amplitudes were equally depressed in WT and KO myocytes. After 2 h of hypoxia followed by 30 min of reoxygenation, levels of ROS were significantly higher in KO compared with WT LV myocytes. Compared with WT I/R hearts, oxygen radical scavenging enzymes (SODs) and their upstream regulators (forkhead box transcription factors and hypoxia-inducible factor) were lower, whereas NADPH oxidase was higher, in KO I/R hearts. We conclude that TRPM2 channels protected hearts from I/R injury by decreasing generation and enhancing scavenging of ROS, thereby reducing I/R-induced oxidative stress.

Hyperhomocysteinemia impairs endothelium-derived hyperpolarizing factor-mediated vasorelaxation in transgenic cystathionine beta synthase-deficient mice.

Hyperhomocysteinemia (HHcy) is associated with endothelial dysfunction (ED), but the mechanism is largely unknown. In this study, we investigated the role and mechanism of HHcy-induced ED in microvasculature in our newly established mouse model of severe HHcy (plasma total homocysteine, 169.5 µM). We found that severe HHcy impaired nitric oxide (NO)- and endothelium-derived hyperpolarizing factor (EDHF)-mediated, endothelium-dependent relaxations of small mesenteric arteries (SMAs). Endothelium-independent and prostacyclin-mediated endothelium-dependent relaxations were not changed. A nonselective Ca(2+)-activated potassium channel (K(Ca)) inhibitor completely blocked EDHF-mediated relaxation. Selective blockers for small-conductance K(Ca) (SK) or intermediate-conductance K(Ca) (IK) failed to inhibit EDHF-mediated relaxation in HHcy mice. HHcy increased the levels of SK3 and IK1 protein, superoxide (O(2)(-)), and 3-nitrotyrosine in the endothelium of SMAs. Preincubation with antioxidants and peroxynitrite (ONOO(-)) inhibitors improved endothelium-dependent and EDHF-mediated relaxations and decreased O(2)(-) production in SMAs from HHcy mice. Further, EDHF-mediated relaxation was inhibited by ONOO(-) and prevented by catalase in the control mice. Finally, L-homocysteine stimulated O(2)(-) production, which was reversed by antioxidants, and increased SK/IK protein levels and tyrosine nitration in cultured human cardiac microvascular endothelial cells. Our results suggest that HHcy impairs EDHF relaxation in SMAs by inhibiting SK/IK activities via oxidation- and tyrosine nitration-related mechanisms.

Multiomics analyses reveal dynamic bioenergetic pathways and functional remodeling of the heart during intermittent fasting.

Intermittent fasting (IF) has been shown to reduce cardiovascular risk factors in both animals and humans, and can protect the heart against ischemic injury in models of myocardial infarction. However, the underlying molecular mechanisms behind these effects remain unclear. To shed light on the molecular and cellular adaptations of the heart to IF, we conducted comprehensive system-wide analyses of the proteome, phosphoproteome, and transcriptome, followed by functional analysis. Using advanced mass spectrometry, we profiled the proteome and phosphoproteome of heart tissues obtained from mice that were maintained on daily 12- or 16 hr fasting, every-other-day fasting, or ad libitum control feeding regimens for 6 months. We also performed RNA sequencing to evaluate whether the observed molecular responses to IF occur at the transcriptional or post-transcriptional levels. Our analyses revealed that IF significantly affected pathways that regulate cyclic GMP signaling, lipid and amino acid metabolism, cell adhesion, cell death, and inflammation. Furthermore, we found that the impact of IF on different metabolic processes varied depending on the length of the fasting regimen. Short IF regimens showed a higher correlation of pathway alteration, while longer IF regimens had an inverse correlation of metabolic processes such as fatty acid oxidation and immune processes. Additionally, functional echocardiographic analyses demonstrated that IF enhances stress-induced cardiac performance. Our systematic multi-omics study provides a molecular framework for understanding how IF impacts the heart's function and its vulnerability to injury and disease.

Hypoxia-induced acidosis uncouples the STIM-Orai calcium signaling complex.

The endoplasmic reticulum Ca(2+)-sensing STIM proteins mediate Ca(2+) entry signals by coupling to activate plasma membrane Orai channels. We reveal that STIM-Orai coupling is rapidly blocked by hypoxia and the ensuing decrease in cytosolic pH. In smooth muscle cells or HEK293 cells coexpressing STIM1 and Orai1, acute hypoxic conditions rapidly blocked store-operated Ca(2+) entry and the Orai1-mediated Ca(2+) release-activated Ca(2+) current (I(CRAC)). Hypoxia-induced blockade of Ca(2+) entry and I(CRAC) was reversed by NH(4)(+)-induced cytosolic alkalinization. Hypoxia and acidification both blocked I(CRAC) induced by the short STIM1 Orai-activating region. Although hypoxia induced STIM1 translocation into junctions, it did not dissociate the STIM1-Orai1 complex. However, both hypoxia and cytosolic acidosis rapidly decreased Förster resonance energy transfer (FRET) between STIM1-YFP and Orai1-CFP. Thus, although hypoxia promotes STIM1 junctional accumulation, the ensuing acidification functionally uncouples the STIM1-Orai1 complex providing an important mechanism protecting cells from Ca(2+) overload under hypoxic stress conditions.

Integrative epigenomic and transcriptomic analyses reveal metabolic switching by intermittent fasting in brain.

Intermittent fasting (IF) remains the most effective intervention to achieve robust anti-aging effects and attenuation of age-related diseases in various species. Epigenetic modifications mediate the biological effects of several environmental factors on gene expression; however, no information is available on the effects of IF on the epigenome. Here, we first found that IF for 3 months caused modulation of H3K9 trimethylation (H3K9me3) in the cerebellum, which in turn orchestrated a plethora of transcriptomic changes involved in robust metabolic switching processes commonly observed during IF. Second, a portion of both the epigenomic and transcriptomic modulations induced by IF was remarkably preserved for at least 3 months post-IF refeeding, indicating that memory of IF-induced epigenetic changes was maintained. Notably, though, we found that termination of IF resulted in a loss of H3K9me3 regulation of the transcriptome. Collectively, our study characterizes the novel effects of IF on the epigenetic-transcriptomic axis, which controls myriad metabolic processes. The comprehensive analyses undertaken in this study reveal a molecular framework for understanding how IF impacts the metabolo-epigenetic axis of the brain and will serve as a valuable resource for future research.

miR-142-3p Regulates BDNF Expression in Activated Rodent Microglia Through Its Target CAMK2A.

Microglia, the innate immune effector cells of the mammalian central nervous system (CNS), are involved in the development, homeostasis, and pathology of CNS. Microglia become activated in response to various insults and injuries and protect the CNS by phagocytosing the invading pathogens, dead neurons, and other cellular debris. Recent studies have demonstrated that the epigenetic mechanisms ensure the coordinated regulation of genes involved in microglial activation. In this study, we performed a microRNA (miRNA) microarray in activated primary microglia derived from rat pup's brain and identified differentially expressed miRNAs targeting key genes involved in cell survival, apoptosis, and inflammatory responses. Interestingly, miR-142-3p, one of the highly up-regulated miRNAs in microglia upon lipopolysaccharide (LPS)-mediated activation, compared to untreated primary microglia cells was predicted to target Ca2+/calmodulin dependent kinase 2a (CAMK2A). Further, luciferase reporter assay confirmed that miR-142-3p targets the 3'UTR of Camk2a. CAMK2A has been implicated in regulating the expression of brain-derived neurotrophic factor (BDNF) and long-term potentiation (LTP), a cellular mechanism underlying memory and learning. Given this, this study further focused on understanding the miR-142-3p mediated regulation of the CAMK2A-BDNF pathway via Cyclic AMP-responsive element-binding protein (CREB) in activated microglia. The results revealed that CAMK2A was downregulated in activated microglia, suggesting an inverse relationship between miR-142-3p and Camk2a in activated microglia. Overexpression of miR-142-3p in microglia was found to decrease the expression of CAMK2A and subsequently BDNF through regulation of CREB phosphorylation. Functional analysis through shRNA-mediated stable knockdown of CAMK2A in microglia confirmed that the regulation of BDNF by miR-142-3p is via CAMK2A. Overall, this study provides a database of differentially expressed miRNAs in activated primary microglia and reveals that microglial miR-142-3p regulates the CAMK2A-CREB-BDNF pathway which is involved in synaptic plasticity.

Oxygen Glucose Deprivation Induced Prosurvival Autophagy Is Insufficient to Rescue Endothelial Function.

Endothelial dysfunction, referring to a disturbance in the vascular homeostasis, has been implicated in many disease conditions including ischemic/reperfusion injury and atherosclerosis. Endothelial mitochondria have been increasingly recognized as a regulator of calcium homeostasis which has implications in the execution of diverse cellular events and energy production. The mitochondrial calcium uniporter complex through which calcium enters the mitochondria is composed of several proteins, including the pore-forming subunit MCU and its regulators MCUR1, MICU1, and MICU2. Mitochondrial calcium overload leads to opening of MPTP (mitochondrial permeability transition pore) and results in apoptotic cell death. Whereas, blockage of calcium entry into the mitochondria results in reduced ATP production thereby activates AMPK-mediated pro-survival autophagy. Here, we investigated the expression of mitochondrial calcium uniporter complex components (MCU, MCUR1, MICU1, and MICU2), induction of autophagy and apoptotic cell death in endothelial cells in response to oxygen-glucose deprivation. Human pulmonary microvascular endothelial cells (HPMVECs) were subjected to oxygen-glucose deprivation (OGD) at 3-h timepoints up to 12 h. Interestingly, except MCUR1 which was significantly downregulated, all other components of the uniporter (MCU, MICU1, and MICU2) remained unchanged. MCUR1 downregulation has been shown to activate AMPK mediated pro-survival autophagy. Similarly, MCUR1 downregulation in response to OGD resulted in AMPK phosphorylation and LC3 processing indicating the activation of pro-survival autophagy. Despite the activation of autophagy, OGD induced Caspase-mediated apoptotic cell death. Blockade of autophagy did not reduce OGD-induced apoptotic cell death whereas serum starvation conferred enough cellular and functional protection. In conclusion, the autophagic flux induced by MCUR1 downregulation in response to OGD is insufficient in protecting endothelial cells from undergoing apoptotic cell death and requires enhancement of autophagic flux by additional means such as serum starvation.

ER-luminal [Ca2+] regulation of InsP3 receptor gating mediated by an ER-luminal peripheral Ca2+-binding protein.

Modulating cytoplasmic Ca2+ concentration ([Ca2+]i) by endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate receptor (InsP3R) Ca2+-release channels is a universal signaling pathway that regulates numerous cell-physiological processes. Whereas much is known regarding regulation of InsP3R activity by cytoplasmic ligands and processes, its regulation by ER-luminal Ca2+ concentration ([Ca2+]ER) is poorly understood and controversial. We discovered that the InsP3R is regulated by a peripheral membrane-associated ER-luminal protein that strongly inhibits the channel in the presence of high, physiological [Ca2+]ER. The widely-expressed Ca2+-binding protein annexin A1 (ANXA1) is present in the nuclear envelope lumen and, through interaction with a luminal region of the channel, can modify high-[Ca2+]ER inhibition of InsP3R activity. Genetic knockdown of ANXA1 expression enhanced global and local elementary InsP3-mediated Ca2+ signaling events. Thus, [Ca2+]ER is a major regulator of InsP3R channel activity and InsP3R-mediated [Ca2+]i signaling in cells by controlling an interaction of the channel with a peripheral membrane-associated Ca2+-binding protein, likely ANXA1.