磷脂酶D1信号对神经干细胞向神经分化的重要影响

磷脂酶D1(PLD1)在细胞生长、存活、分化、膜转运和细胞骨架组织等多种功能的调控中发挥重要作用。近年来研究发现,PLD1在神经干细胞（NSCs）向神经元的分化中也起关键作用。PLD1参与多种信号通路如Rho家族GTP酶和Ca²⁺信号通路的调节,影响轴突生长、突触发育及其可塑性。因此,PLD1作为神经系统中一种重要的信号分子引起了广泛的关注。本文综述了PLD1的结构、功能、作用机制及其在NSCs向神经分化中的调控作用,对深入研究NSCs的分化和神经元的再生有重要的指导意义。     

Chemical signaling under abiotic stress environment in plants

Many chemicals are critical for plant growth and development and play an important role in integrating various stress signals and controlling downstream stress responses by modulating gene expression machinery and regulating various transporters/pumps and biochemical reactions. These chemicals include calcium (Ca²⁺), cyclic nucleotides, polyphosphoinositides, nitric oxide (NO), sugars, abscisic acid (ABA), jasmonates (JA), salicylic acid (SA) and polyamines. Ca²⁺ is one of the very important ubiquitous second messengers in signal transduction pathways and usually its concentration increases in response to the stimuli including stress signals. Many Ca²⁺ sensors detect the Ca²⁺ signals and direct them to downstream signaling pathways by binding and activating diverse targets. cAMP or cGMP protects the cell with ion toxicity. Phosphoinositides are known to be involved both in transmission of signal across the plasma membrane and in intracellular signaling. NO activates various defense genes and acts as a developmental regulator in plants. Sugars affect the expression of many genes involved in photosynthesis, glycolysis, nitrogen metabolism, sucrose and starch metabolism, defense mechanisms and cell cycle regulation. ABA, JA, SA and polyamines are also involved in many stress responses. Cross-talk between these chemical signaling pathways is very common in plant responses to abiotic and bitotic factors. In this article we have described the role of these chemicals in initiating signaling under stress conditions mainly the abiotic stress.Key words: ABA, abiotic stress, Ca²⁺ binding proteins, calcium signaling, cyclic nucleotides, nitric oxide, phosphoinositides signaling, signal transduction, sugar signaling     

The Diversity of Calcium Sensor Proteins in the Regulation of Neuronal Function

Calcium signaling in neurons as in other cell types mediates changes in gene expression, cell growth, development, survival, and cell death. However, neuronal Ca²⁺ signaling processes have become adapted to modulate the function of other important pathways including axon outgrowth and changes in synaptic strength. Ca²⁺ plays a key role as the trigger for fast neurotransmitter release. The ubiquitous Ca²⁺ sensor calmodulin is involved in various aspects of neuronal regulation. The mechanisms by which changes in intracellular Ca²⁺ concentration in neurons can bring about such diverse responses has, however, become a topic of widespread interest that has recently focused on the roles of specialized neuronal Ca²⁺ sensors. In this article, we summarize synaptotagmins in neurotransmitter release, the neuronal roles of calmodulin, and the functional significance of the NCS and the CaBP/calneuron protein families of neuronal Ca²⁺ sensors.Calcium signaling in many cell types can mediate changes in gene expression, cell growth, development, survival, and cell death. However, neuronal calcium signaling processes have become adapted to modulate the function of important pathways in the brain, including neuronal survival, axon outgrowth, and changes in synaptic strength. Changes in the concentration of intracellular free Ca²⁺ ([Ca²⁺]i) are essential for the transmission of information through the nervous system as the trigger for neurotransmitter release at synapses. In addition, alterations in [Ca²⁺]i can lead to a wide range of different physiological changes that can modify neuronal functions over time scales of milliseconds through tens of minutes to days or longer (Berridge 1998). Many of these processes have been shown to be dependent upon the particular route of Ca²⁺ entry into the cell. It has long been known that the physiological outcome from a change in [Ca²⁺]i depends on its location, amplitude, and duration. The importance of location becomes even more pronounced in neurons because of their complex and extended morphologies. [Ca²⁺]i also regulates neuronal development and neuronal survival (Spitzer 2006). In addition, modifications to Ca²⁺ signaling pathways have been suggested to underlie various neuropathological disorders (Braunewell 2005; Berridge 2010).Highly localized Ca²⁺ elevations (Augustine et al. 2003) formed following Ca²⁺ entry though voltage-gated Ca²⁺ channels (VGCCs) lead to synaptic vesicle fusion with the presynaptic membrane and thereby allow neurotransmitter release within less than a millisecond. Differently localized and timed Ca²⁺ signals can, for example, result in changes to the properties of the VGCCs (Catterall and Few 2008) or lead to changes in gene expression (Bito et al. 1997). Postsynaptic Ca²⁺ signals arising from activation of NMDA receptors give rise to two important processes in synaptic plasticity, long term potentiation (LTP) and long term depression (LTD). LTP and LTD are examples of the way synaptic transmission can change synaptic efficacy and are thought to be important in modulating learning and memory. Importantly, the Ca²⁺ signals that bring about either LTP or LTD differ only in their timing and duration. LTP is triggered by Ca²⁺ signals on the micromolar scale for shorter durations, whereas LTD is triggered by changes in [Ca²⁺]i on the nanomolar scale for longer durations (Yang et al. 1999). Specific Ca²⁺ signals are likely to be decoded by different Ca²⁺ sensor proteins. These are proteins that undergo a conformational change on Ca²⁺ binding and then interact with and regulate various target proteins. Among those Ca²⁺ sensors that are important for neuronal function are the synaptotagmins that control neurotransmitter release (Chapman 2008), the ubiquitous EF-hand containing sensor calmodulin that has many neuronal roles, and the more recently discovered neuronal EF-hand containing proteins, including the neuronal calcium sensor (NCS) protein (Burgoyne 2007) and the calcium-binding protein (CaBP)/calneuron (Haeseleer et al. 2002) families. We will briefly review synaptotagmins and the neuronal functions of calmodulin but concentrate on the NCS and CaBP families of Ca²⁺ sensors.     

Ca2+-mediated Mitochondrial Reactive Oxygen Species Metabolism Augments Wnt/β-Catenin Pathway Activation to Facilitate Cell Differentiation

Emerging evidence suggests that reactive oxygen species (ROS) can stimulate the Wnt/β-catenin pathway in a number of cellular processes. However, potential sources of endogenous ROS have not been thoroughly explored. Here, we show that growth factor depletion in human neural progenitor cells induces ROS production in mitochondria. Elevated ROS levels augment activation of Wnt/β-catenin signaling that regulates neural differentiation. We find that growth factor depletion stimulates the release of Ca²⁺ from the endoplasmic reticulum stores. Ca²⁺ subsequently accumulates in the mitochondria and triggers ROS production. The inhibition of mitochondrial Ca²⁺ uptake with simultaneous growth factor depletion prevents the rise in ROS metabolism. Moreover, low ROS levels block the dissociation of the Wnt effector Dishevelled from nucleoredoxin. Attenuation of the response amplitudes of pathway effectors delays the onset of the Wnt/β-catenin pathway activation and results in markedly impaired neuronal differentiation. Our findings reveal Ca²⁺-mediated ROS metabolic cues that fine-tune the efficiency of cell differentiation by modulating the extent of the Wnt/β-catenin signaling output.     

SK2 channel regulation of neuronal excitability,synaptic transmission,and brain rhythmic activity in health and diseases

Small conductance calcium-activated potassium channels (SKs) are solely activated by intracellular Ca²⁺ and their activation leads to potassium efflux, thereby repolarizing/hyperpolarizing membrane potential. Thus, these channels play a critical role in synaptic transmission, and consequently in information transmission along the neuronal circuits expressing them. SKs are widely but not homogeneously distributed in the central nervous system (CNS). Activation of SKs requires submicromolar cytoplasmic Ca²⁺ concentrations, which are reached following either Ca²⁺ release from intracellular Ca²⁺ stores or influx through Ca²⁺ permeable membrane channels. Both Ca²⁺ sensitivity and synaptic levels of SKs are regulated by protein kinases and phosphatases, and degradation pathways. SKs in turn control the activity of multiple Ca²⁺ channels. They are therefore critically involved in coordinating diverse Ca²⁺ signaling pathways and controlling Ca²⁺ signal amplitude and duration. This review highlights recent advances in our understanding of the regulation of SK2 channels and of their roles in normal brain functions, including synaptic plasticity, learning and memory, and rhythmic activities. It will also discuss how alterations in their expression and regulation might contribute to various brain disorders such as Angelman Syndrome, Alzheimer's disease and Parkinson's disease.     

The SERCA2: A Gatekeeper of Neuronal Calcium Homeostasis in the Brain

Calcium (Ca²⁺) ions are prominent cell signaling regulators that carry information for a variety of cellular processes and are critical for neuronal survival and function. Furthermore, Ca²⁺ acts as a prominent second messenger that modulates divergent intracellular cascades in the nerve cells. Therefore, nerve cells have developed intricate Ca²⁺ signaling pathways to couple the Ca²⁺ signal to their biochemical machinery. Notably, intracellular Ca²⁺ homeostasis greatly relies on the rapid redistribution of Ca²⁺ ions into the diverse subcellular organelles which serve as Ca²⁺ stores, including the endoplasmic reticulum (ER). It is well established that Ca²⁺ released into the neuronal cytoplasm is pumped back into the ER by the sarco-/ER Ca²⁺ ATPase 2 (SERCA2), a P-type ion-motive ATPase that resides on the ER membrane. Even though the SERCA2 is constitutively expressed in nerve cells, its precise role in brain physiology and pathophysiology is not well-characterized. Intriguingly, SERCA2-dependent Ca²⁺ dysregulation has been implicated in several disorders that affect cognitive function, including Darier’s disease, schizophrenia, Alzheimer’s disease, and cerebral ischemia. The current review summarizes knowledge on the expression pattern of the different SERCA2 isoforms in the nervous system, and further discusses evidence of SERCA2 dysregulation in various neuropsychiatric disorders. To the best of our knowledge, this is the first literature review that specifically highlights the critical role of the SERCA2 in the brain. Advancing knowledge on the role of SERCA2 in maintaining neuronal Ca²⁺ homeostasis may ultimately lead to the development of safer and more effective pharmacotherapies to combat debilitating neuropsychiatric disorders.     

Quinolinate-induced Rat Striatal Excitotoxicity Impairs Endoplasmic Reticulum Ca2+-ATPase Function

Excessive activation of NMDA glutamate receptors and the resulting loss of intracellular Ca²⁺ homeostasis may be lethal (excitotoxic) to neurons. Such excitotoxicity can be induced in vivo by intrastriatal infusion of quinolinate, as this substance selectively activates NMDA receptors. The aim of the present research was to investigate whether the in vivo treatment of striatal tissue with quinolinate would lead to an early impairment of sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) activity or mitochondrial Ca²⁺ sequestration, two intracellular mechanisms involved in Ca²⁺ homeostasis and signaling. Sodium quinolinate was infused intrastriatally into adult rats, and 6 h later the brains were removed and the corpora striata dissected. At this time point, striatal sections stained with Fluoro-Jade, a cellular marker of cell death, showed initial signs of neuronal degeneration. In addition, SERCA activity decreased 39% in relation to the activity observed in the control striata. A corresponding decrease of the same magnitude in ⁴⁵Ca²⁺ uptake by striatal microsomes was also found in the treated striata. Western blot analysis did not indicate any decrease in SERCA levels in striatal tissue after quinolinate infusion. Mitochondrial Ca²⁺ sequestration was still preserved in quinolinate-treated striatal tissue when the assay was carried out in the presence of physiological concentrations of ATP and Mg²⁺. These results suggest that impairment of the SERCA function may be an early event in excitotoxicity.     

Cell adhesion and intracellular calcium signaling in neurons

Cell adhesion molecules (CAMs) play indispensable roles in the developing and mature brain by regulating neuronal migration and differentiation, neurite outgrowth, axonal fasciculation, synapse formation and synaptic plasticity. CAM-mediated changes in neuronal behavior depend on a number of intracellular signaling cascades including changes in various second messengers, among which CAM-dependent changes in intracellular Ca²⁺ levels play a prominent role. Ca²⁺ is an essential secondary intracellular signaling molecule that regulates fundamental cellular functions in various cell types, including neurons. We present a systematic review of the studies reporting changes in intracellular Ca²⁺ levels in response to activation of the immunoglobulin superfamily CAMs, cadherins and integrins in neurons. We also analyze current experimental evidence on the Ca²⁺ sources and channels involved in intracellular Ca²⁺ increases mediated by CAMs of these families, and systematically review the role of the voltage-dependent Ca²⁺ channels (VDCCs) in neurite outgrowth induced by activation of these CAMs. Molecular mechanisms linking CAMs to VDCCs and intracellular Ca²⁺ stores in neurons are discussed.     

The renaissance of Ca2+-binding proteins in the nervous system: secretagogin takes center stage

Effective control of the Ca²⁺ homeostasis in any living cell is paramount to coordinate some of the most essential physiological processes, including cell division, morphological differentiation, and intercellular communication. Therefore, effective homeostatic mechanisms have evolved to maintain the intracellular Ca²⁺ concentration at physiologically adequate levels, as well as to regulate the spatial and temporal dynamics of Ca²⁺signaling at subcellular resolution. Members of the superfamily of EF-hand Ca²⁺-binding proteins are effective to either attenuate intracellular Ca²⁺ transients as stochiometric buffers or function as Ca²⁺ sensors whose conformational change upon Ca²⁺ binding triggers protein-protein interactions, leading to cell state-specific intracellular signaling events. In the central nervous system, some EF-hand Ca²⁺-binding proteins are restricted to specific subtypes of neurons or glia, with their expression under developmental and/or metabolic control. Therefore, Ca²⁺-binding proteins are widely used as molecular markers of cell identity whilst also predicting excitability and neurotransmitter release profiles in response to electrical stimuli. Secretagogin is a novel member of the group of EF-hand Ca²⁺-binding proteins whose expression precedes that of many other Ca²⁺-binding proteins in postmitotic, migratory neurons in the embryonic nervous system. Secretagogin expression persists during neurogenesis in the adult brain, yet becomes confined to regionalized subsets of differentiated neurons in the adult central and peripheral nervous and neuroendocrine systems. Secretagogin may be implicated in the control of neuronal turnover and differentiation, particularly since it is re-expressed in neoplastic brain and endocrine tumors and modulates cell proliferation in vitro. Alternatively, and since secretagogin can bind to SNARE proteins, it might function as a Ca²⁺ sensor/coincidence detector modulating vesicular exocytosis of neurotransmitters, neuropeptides or hormones. Thus, secretagogin emerges as a functionally multifaceted Ca²⁺-binding protein whose molecular characterization can unravel a new and fundamental dimension of Ca²⁺signaling under physiological and disease conditions in the nervous system and beyond.     

Stress responses mediated by the CBL calcium sensors in plants

Calcium ions (Ca²⁺) are involved as second messenger in plant responses to a broad array of environmental stimuli, including biotic and abiotic stresses. Therefore, understanding Ca²⁺-signaling mechanisms may lead to the development of transgenic crops with enhanced tolerance to adverse environmental conditions. In order to initiate the signaling cascades and give rise to relevant cellular and physiological responses, changes in the parameters of Ca²⁺ transients should be first detected by appropriate Ca²⁺ sensors in plant cells. In this regard, elucidations of plant Ca²⁺ sensors and their target molecules are critical steps for unraveling the Ca²⁺ signal transduction pathways. Recent studies have revealed that plants possess many Ca²⁺-binding proteins with different properties, which can serve as distinct Ca²⁺ sensors. This present review mainly focuses on a family of calcineurin B-like Ca²⁺ sensors which has been most recently identified from higher plants including Arabidopsis, rice, maize and pea.     

Calcineurin Mediates Synaptic Scaling Via Synaptic Trafficking of Ca2+-Permeable AMPA Receptors

Homeostatic synaptic plasticity is a negative-feedback mechanism for compensating excessive excitation or inhibition of neuronal activity. When neuronal activity is chronically suppressed, neurons increase synaptic strength across all affected synapses via synaptic scaling. One mechanism for this change is alteration of synaptic AMPA receptor (AMPAR) accumulation. Although decreased intracellular Ca²⁺ levels caused by chronic inhibition of neuronal activity are believed to be an important trigger of synaptic scaling, the mechanism of Ca²⁺-mediated AMPAR-dependent synaptic scaling is not yet understood. Here, we use dissociated mouse cortical neurons and employ Ca²⁺ imaging, electrophysiological, cell biological, and biochemical approaches to describe a novel mechanism in which homeostasis of Ca²⁺ signaling modulates activity deprivation-induced synaptic scaling by three steps: (1) suppression of neuronal activity decreases somatic Ca²⁺ signals; (2) reduced activity of calcineurin, a Ca²⁺-dependent serine/threonine phosphatase, increases synaptic expression of Ca²⁺-permeable AMPARs (CPARs) by stabilizing GluA1 phosphorylation; and (3) Ca²⁺ influx via CPARs restores CREB phosphorylation as a homeostatic response by Ca²⁺-induced Ca²⁺ release from the ER. Therefore, we suggest that synaptic scaling not only maintains neuronal stability by increasing postsynaptic strength but also maintains nuclear Ca²⁺ signaling by synaptic expression of CPARs and ER Ca²⁺ propagation.     

Calcium and secondary CPK signaling in plants in response to herbivore attack

Plant Ca²⁺ signals are involved in a sizable array of intracellular signaling pathways after pest invasion. Upon herbivore feeding there is a dramatic Ca²⁺ influx, followed by the activation of Ca²⁺-dependent signal transduction pathways that include interacting downstream networks of kinases for defense responses. Notably, Ca²⁺-binding sensory proteins such as Ca²⁺-dependent protein kinases (CPKs) have recently been documented to mediate the signaling following Ca²⁺ influx after herbivory, in phytohormone-independent manners. Here, we review the sequence of signal transductions triggered by herbivory-evoked Ca²⁺ signaling leading to CPK actions for defense responses, and discuss in a comparative way the involvement of CPKs in the signal transduction of a variety of other biotic and abiotic stresses.     

Dysregulation of neural calcium signaling in Alzheimer disease,bipolar disorder and schizophrenia

Neurons have highly developed Ca²⁺ signaling systems responsible for regulating a large number of neural functions such as the control of brain rhythms, information processing and the changes in synaptic plasticity that underpin learning and memory. The tonic excitatory drive, which is activated by the ascending arousal system, is particularly important for processes such as sensory perception, cognition and consciousness. The Ca²⁺ signaling pathway is a key component of this arousal system that regulates the neuronal excitability responsible for controlling the neural brain rhythms required for information processing and cognition. Dysregulation of the Ca²⁺ signaling pathway responsible for many of these neuronal processes has been implicated in the development of some of the major neural diseases in man such as Alzheimer disease, bipolar disorder and schizophrenia. Various treatments, which are known to act by reducing the activity of Ca²⁺ signaling, have proved successful in alleviating the symptoms of some of these neural diseases.     

Spontaneous calcium transients in the immature adult-born neurons of the olfactory bulb

Spontaneous neuronal activity and concomitant intracellular Ca²⁺ signaling are abundant during early perinatal development and are well known for their key role in neuronal proliferation, migration, differentiation and wiring. However, much less is known about the in vivo patterns of spontaneous Ca²⁺ signaling in immature adult-born cells. Here, by using two-photon Ca²⁺ imaging, we analyzed spontaneous in vivo Ca²⁺ signaling in adult-born juxtaglomerular cells of the mouse olfactory bulb over the time period of 5 weeks, from the day of their arrival in the glomerular layer till their stable integration into the preexisting neural network. We show that spontaneous Ca²⁺ transients are ubiquitously present in adult-born cells right after their arrival, require activation of voltage-gated Na⁺ channels and are little sensitive to isoflurane anesthesia. Interestingly, several parameters of this spontaneous activity, such as the area under the curve, the time spent in the active state as well as the fraction of continuously active cells show a bell-shaped dependence on cell’s age, all peaking in 3–4 weeks old cells. This data firmly document the in vivo presence of spontaneous Ca²⁺ signaling during the layer-specific maturation of adult-born neurons in the olfactory bulb and motivate further analyses of the functional role(s) of this activity.     

The Local Control of Cytosolic Ca2+ as a Propagator of CNS Communication—Integration of Mitochondrial Transport Mechanisms and Cellular Responses

Ca²⁺ signals propagate in wave form along individual cells of the central nervous system(CNS) and through networks of connected cells of neuronal and multiple glial cell types. Inorder for wave fronts to convey information, signaling mechanisms are required that allowwaves to propagate reproducibly and without decrement in signal strength over long distances.CNS Ca²⁺ waves are under specific integrated local control, made possible by interactions atlocal subcellular microdomains between endoplasmic reticulum and mitochondria. Activemitochondria located near the mouth of inositol trisphosphate receptor (InsP₃R) channel clustersin glia take up Ca²⁺, which may prevent a buildup of Ca²⁺ around the InsP₃R channel, therebydecreasing the rate of Ca²⁺-induced receptor inactivation, and prolonging channel open time.Mitochondria may amplify InsP;i3-dependent Ca2;pl signals by a transient permeability transitionin response to Ca²⁺ uptake into the mitochondrion. Other evidence suggests privileged accessinto mitochondria for Ca²⁺ entering neurons by glutamatergic receptor channels. This enablesspecific signal modulation as the Ca²⁺ wave is propagated into neurons, such that mitochondrialocated close to glutamate channels can prolong the neuronal cytosolic response time bysuccessive uptake and release of Ca²⁺. Disruption of mitochondrial function deregulates theability of CNS-derived cells to undergo normal Ca²⁺ signaling and wave propagation.     

Two Pore Channel 2 Differentially Modulates Neural Differentiation of Mouse Embryonic Stem Cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) is an endogenous Ca²⁺ mobilizing nucleotide presented in various species. NAADP mobilizes Ca²⁺ from acidic organelles through two pore channel 2 (TPC2) in many cell types and it has been previously shown that NAADP can potently induce neuronal differentiation in PC12 cells. Here we examined the role of TPC2 signaling in the neural differentiation of mouse embryonic stem (ES) cells. We found that the expression of TPC2 was markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebounded during the late stages of neurogenesis. Correspondingly, TPC2 knockdown accelerated mouse ES cell differentiation into neural progenitors but inhibited these neural progenitors from committing to neurons. Overexpression of TPC2, on the other hand, inhibited mouse ES cell from entering the early neural lineage. Interestingly, TPC2 knockdown had no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Taken together, our data indicate that TPC2 signaling plays a temporal and differential role in modulating the neural lineage entry of mouse ES cells, in that TPC2 signaling inhibits ES cell entry to early neural progenitors, but is required for late neuronal differentiation.     

Glutamate Regulates the Frequency of Spontaneous Synchronized Ca<Superscript>2+</Superscript> Spikes Through Group II Metabotropic Glutamate Receptor in Cultured Mouse Cortical Networks

1. Synchronized spontaneous intracellular Ca²⁺ spikes in networked neurons are believed to play a major role in the development and plasticity of neural circuits. Glutamate-induced signals through the ionotropic glutamate receptors (iGluRs) are profoundly involved in the generation of synchronized Ca²⁺ spikes.1 2. In this study, we examined the involvement of metabotropic glutamate receptors (mGluRs) in cultured mouse cortical neurons. We pharmacologically revealed that glutamate-induced signals through inclusive mGluRs decreased the frequency of Ca²⁺ spikes. Further experiments indicated that this suppressive effect on the spike frequency was mainly due to the signal through group II mGluR, inactivation of adenylate cyclase-cAMP-PKA signaling pathway. Group I mGluR had little involvement in the spike frequency.3. Taken together, glutamate generates the synchronized Ca²⁺ spikes through iGluRs and modulates simultaneously their frequency through group II mGluR–adenylate cyclase–cAMP–PKA signaling pathway in the present in vitro neural network. These results provide the evidence of the profound role of group II mGluR in the spontaneous and synchronous neural activities.     

Characterization of calcium signals in human embryonic stem cells and in their differentiated offspring by a stably integrated calcium indicator protein

Intracellular calcium signaling pathways play a major role in cellular responses such as proliferation, differentiation and apoptosis. Human embryonic stem cells (hESC) provide new possibilities to explore the development and differentiation of various cell types of the human body. Intracellular calcium responses to various ligands and the calcium signaling pathways, however, have not been thoroughly studied in embryonic stem cells and in their differentiated progenies. In our previous work we demonstrated that the use of the fluorescent calcium indicator Fluo-4 with confocal microscopy allows sensitive and reliable measurements of calcium modulation in human embryonic stem cells and stem-cell derived cardiomyocytes. Here we developed a human embryonic stem cell line stably expressing a genetically encoded Ca^2 + indicator (GCaMP2) using a transposon-based gene delivery system. We found that the differentiation properties were fully preserved in the GCaMP2-expressing hESC lines and Ca imaging could be performed without the need of toxic dye-loading of the cells. In undifferentiated hES cells the calcium signals induced by various ligands, ATP, LPA, trypsin or angiotensin II were comparable to those in Fluo-4 loaded cells. In accordance with previous findings, no calcium signal was evoked by thrombin, histamine or GABA. Cardiomyocyte colonies differentiated from hES-GCaMP2 cells could be recognized by spontaneous contractions and Ca^2 + oscillations. GCaMP2-expressing neural cells were identified based on their morphological and immuno-staining properties and Ca signals were characterized on those cells. Characteristics of both the spontaneous and ligand-induced Ca^2 + signals, as well as their pharmacological modification could be successfully examined in these model cells by fluorescence imaging.     

The mitochondrial aspartate/glutamate carrier isoform 1 gene expression is regulated by CREB in neuronal cells

The aspartate/glutamate carrier isoform 1 is an essential mitochondrial transporter that exchanges intramitochondrial aspartate and cytosolic glutamate across the inner mitochondrial membrane. It is expressed in brain, heart and muscle and is involved in important biological processes, including myelination. However, the signals that regulate the expression of this transporter are still largely unknown. In this study we first identify a CREB binding site within the aspartate/glutamate carrier gene promoter that acts as a strong enhancer element in neuronal SH-SY5Y cells. This element is regulated by active, phosphorylated CREB protein and by signal pathways that modify the activity of CREB itself and, most noticeably, by intracellular Ca²⁺ levels. Specifically, aspartate/glutamate carrier gene expression is induced via CREB by forskolin while it is inhibited by the PKA inhibitor, H89. Furthermore, the CREB-induced activation of gene expression is increased by thapsigargin, which enhances cytosolic Ca²⁺, while it is inhibited by BAPTA-AM that reduces cytosolic Ca²⁺ or by STO-609, which inhibits CaMK-IV phosphorylation. We further show that CREB-dependent regulation of aspartate/glutamate carrier gene expression occurs in neuronal cells in response to pathological (inflammation) and physiological (differentiation) conditions. Since this carrier is necessary for neuronal functions and is involved in myelinogenesis, our results highlight that targeting of CREB activity and Ca²⁺ might be therapeutically exploited to increase aspartate/glutamate carrier gene expression in neurodegenerative diseases.     

Calcium signaling in mammalian egg activation and embryo development: The influence of subcellular localization

Calcium (Ca²⁺) signals drive the fundamental events surrounding fertilization and the activation of development in all species examined to date. Initial studies of Ca²⁺ signaling at fertilization in marine animals were tightly linked to new discoveries of bioluminescent proteins and their use as fluorescent Ca²⁺ sensors. Since that time, there has been rapid progress in our understanding of the key functions for Ca²⁺ in many cell types and of the impact of cellular localization on Ca²⁺ signaling pathways. In this review, which focuses on mammalian egg activation, we consider how Ca²⁺ is regulated and stored at different stages of oocyte development and examine the functions of molecules that serve as both regulators of Ca²⁺ release and effectors of Ca²⁺ signals. We then summarize studies exploring how Ca²⁺ directs downstream effectors mediating both egg activation and later signaling events required for successful preimplantation embryo development. Throughout this review, we focus attention on how localization of Ca²⁺ signals influences downstream signaling events, and attempt to highlight gaps in our knowledge that are ripe for future research. Mol. Reprod. Dev. 79: 742–756, 2012. Published 2012. This article is a US government work and, as such, is in the public domain in the United States of America.