Engineered protein systems have been created to impart new functions, or “re-program” mammalian cells for applications including cancer and HIV/AIDS therapies. The successful development of mammalian cells for re-programming will depend on having well-defined, modular systems. Migration is a particularly important cell function that will determine the efficiency and efficacy of many re-programming applications in vivo, and Rho proteins are responsible for regulation of cell migration natively. While there have been several reports of photo-activated Rho proteins, no strategy has been developed such that Rho proteins and cell migration can be controlled by a variety of extracellular stimuli that may be compatible with signaling in large organisms. Here, several methods are described for engineering Ca2+-sensitive Rho proteins so that the large, natural toolbox of Ca2+-mobilizing proteins can use the Ca2+ intermediate to activate Rho proteins in response to a variety of exogenous stimuli, including chemicals, growth factors, and light. First, an unreported calmodulin binding site was identified in RhoA. This knowledge was used to create a tandem fusion of RhoA and calmodulin that mediated Ca2+-sensitive bleb retraction in response to a variety of Ca2+-elevating chemicals. Ca2+-mobilizing modules including channelrhodopsin-2 and nicotinic acetylcholine receptor α4 were used for light- and acetylcholine-dependent bleb retraction. Second, a more robust morphology switch was created by embedding a calmodulin binding site into RhoA to enable Ca2+-responsive bleb formation. A wider range of Ca2+-mobilizing modules were also used here including LOVS1K/Orai1 and vascular endothelial growth factor 2. Combining Ca2+-mobilizing and Ca2+-responsive modules increased amoeboid-like cell migration in wound closure and transwell assays. Finally, the embedded peptide design was applied to Rac1 and Cdc42 to enable control of new morphologies and migration modes. The modular Ca2+ control over Rho proteins developed here is an important contribution to cell re-programming because it shows that control over cell migration can be rewired in a way that is flexible and tunable.

Engineered protein systems have been created to impart new functions, or “re-program” mammalian cells for applications including cancer and HIV/AIDS therapies. The successful development of mammalian cells for re-programming will depend on having well-defined, modular systems. Migration is a particularly important cell function that will determine the efficiency and efficacy of many re-programming applications in vivo, and Rho proteins are responsible for regulation of cell migration natively. While there have been several reports of photo-activated Rho proteins, no strategy has been developed such that Rho proteins and cell migration can be controlled by a variety of extracellular stimuli that may be compatible with signaling in large organisms. Here, several methods are described for engineering Ca2+-sensitive Rho proteins so that the large, natural toolbox of Ca2+-mobilizing proteins can use the Ca2+ intermediate to activate Rho proteins in response to a variety of exogenous stimuli, including chemicals, growth factors, and light. First, an unreported calmodulin binding site was identified in RhoA. This knowledge was used to create a tandem fusion of RhoA and calmodulin that mediated Ca2+-sensitive bleb retraction in response to a variety of Ca2+-elevating chemicals. Ca2+-mobilizing modules including channelrhodopsin-2 and nicotinic acetylcholine receptor α4 were used for light- and acetylcholine-dependent bleb retraction. Second, a more robust morphology switch was created by embedding a calmodulin binding site into RhoA to enable Ca2+-responsive bleb formation. A wider range of Ca2+-mobilizing modules were also used here including LOVS1K/Orai1 and vascular endothelial growth factor 2. Combining Ca2+-mobilizing and Ca2+-responsive modules increased amoeboid-like cell migration in wound closure and transwell assays. Finally, the embedded peptide design was applied to Rac1 and Cdc42 to enable control of new morphologies and migration modes. The modular Ca2+ control over Rho proteins developed here is an important contribution to cell re-programming because it shows that control over cell migration can be rewired in a way that is flexible and tunable.

As sessile organisms, coordination of development and reproduction in a dynamic, and often stressful, environment presents a particular challenge for plants. Rapid processing of internal and external cues by complex signal transduction pathways leads to stimulus-appropriate physiological responses on an organismal scale. In plants, myriad signaling pathways are mediated by calcium (Ca2+) signals, and it is thought that different stimuli elicit unique patterns of Ca2+ influx into cells (termed Ca2+ ‘signatures’) that encode information important for proper physiological responses. Encoding of information in the form of Ca2+ signatures requires that decoding elements be present in cells to direct downstream cellular processes. This role is filled by Ca2+-binding proteins that serve as Ca2+ sensors. Interestingly, plant genomes encode multiple expanded families of Ca2+ sensors not found in animal genomes. Among these, the calmodulin (CaM)-like proteins (CMLs) are represented by a 50 member family in Arabidopsis. On the basis of structural homology, CMLs are predicted to function like conserved CaM, however, little work has been done to address this question. Biochemical characterization of CML39 indicates that it possesses structural properties consistent with function as a Ca2+ sensor. Analysis of transgenic CML39 loss-of-function (cml39) mutants revealed that CML39 is important for proper seedling establishment in the absence of exogenous metabolisable carbon as cml39 seedlings entered a state of developmental arrest shortly after germination. cml39 mutants also exhibited a conditional ‘de-etiolated’ phenotype when grown in complete darkness and exaggerated hypocotyl elongation under a short-day light regime. Genetic data suggest that CML39 functions in signaling pathways downstream of light perception, and this idea is supported by the observation that CML39 iii is expressed in light-sensing tissues, and that subunit 5 of the COP9 signalosome, a protein critical for photomorphogenesis, was identified as a putative target of CML39. Collectively, results show that CML39 is Ca2+ sensor that serves a critical regulatory role during seedling establishment when sucrose is limited, and importantly, further underscore the pervasiveness of Ca2+ signaling in plant growth and development. / Thesis (Ph.D, Biology) -- Queen's University, 2013-05-29 18:16:25.769

Mitochondria plays a crucial role in cells by maintaining energy metabolism and directing cell death mechanisms by buffering calcium (Ca2+ )from cytosol. Therefore, the Ca2+ overload of mitochondria due to the upregulated cytosolic Ca2+ , observed in many neurological disorders is hypothesized to be a key pathway leading to mitochondrial dysfunction and cell death. In particular, Ca2+ homeostasis disruptions due to Alzheimer’ s disease (AD)-causing presenilins (PS1/PS2) and oligomeric forms of β-amyloid peptides Aβ commonly found in AD patients are presumed to cause detrimental effects on the mitochondria and its ability to function properly. We begin by showing that Familial Alzheimer’s disease (FAD)-causing PS mutants affect intracellular Ca2+ ([Ca2+]i) homeostasis by enhancing the gating of inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) Ca2+ channels on the endoplasmic reticulum (ER), leading to exaggerated Ca2+ release into the cytoplasm. Using experimental IP3R-mediated Ca2+ release data in conjunction with a computational model of mitochondrial bioenergetics, we explore how the differences in mitochondrial Ca2+ uptake in control cells and cells expressing FAD-causing PS mutants affect key variables such as ATP, reactive oxygen species (ROS), NADH, and mitochondrial Ca2+ ([Ca2+ ]m). We find that as a result of exaggerated [Ca2+]i in FAD-causing mutant PS-expressing cells, the rate of oxygen consumption increases dramatically and overcomes the Ca2+ dependent enzymes that stimulate NADH production. This leads to decreased rates of proton pumping due to diminished membrane potential (Ψm) along with less ATP and enhanced ROS production. These results show that through Ca2+ signaling disruption, mutant PS leads to mitochondrial dysfunction and potentially cell death. Next, the model for the mitochondria is expanded to include the mitochondrial uniporter (MCU) that senses Ca2+ in the microdomain formed by the close proximity of mitochondria and ER. Ca2+ concentration in the microdomain ([Ca2+] mic) depends on the distance between the cluster of IP3R channels (r) on ER and mitochondria, the number of IP3R in the cluster (nIP3R), and open-probability (Po) of IP3R. Using the same experimental results for Ca2+ release though IP3R due to FAD-causing PS mutants, in conjunction with a computational model of mitochondrial bioenergetics, a data-driven Markov chain model for IP3R gating, and a model for the dynamics of the mitochondrial permeability transition pore (PTP), we explore the difference in mitochondrial Ca2+ uptake in cells expressing wild type (PS1-WT) and FAD-causing mutant (PS1-M146L) PS. We find that increased mitochondrial [Ca2+]m due to the gain-of-function enhancement of IP3R channels in the cell expressing PS1-M146L leads to the opening of PTP in high conductance state (PTPh), where the latency of opening is inversely correlated with r and proportional to nIP3R. Furthermore, we observe diminished inner mitochondrial Ψm, [NADH], [Ca2+]m, and [ATP] when PTP opens. Additionally, we explore how parameters such as the pH gradient, inorganic phosphate concentration, and the rate of the Na+/ Ca2+ -exchanger affect the latency of PTP to open in PTPh. Intracellular accumulation of oligomeric forms of Aβ are now believed to play a key role in the early phase of AD as their rise correlates well with the early symptoms of the disease. Extensive evidence points to impaired neuronal Ca2+ homeostasis as a direct consequence of the intracellular Aβ oligomers. To study the effect of intracellular Aβ on Ca2+ signaling and the resulting mitochondrial dysfunction, we employed data-driven modeling in conjunction with total internal reflection fluorescence (TIRF) microscopy (TIRFM). High resolution fluorescence TIRFM together with detailed computational modeling provides a powerful approach towards the understanding of a wide range of Ca2+ signals mediated by the IP3R. Achieving this requires a close agreement between Ca2+ signals from computational models and TIRFM experiments. However, we found that elementary Ca2+ release events, puffs, imaged through TIRFM do not show the rapid single-channel opening and closing during x and between puffs using data-driven single channel models. TIRFM also shows a rapid equilibration of 10 ms after a channel opens or closes which is not achievable in simulation using standard Ca2+ diffusion coefficients and reaction rates between indicator dye and Ca2+. Using the widely used Ca2+ diffusion coefficients and reaction rates, our simulations show equilibration rates that are eight times slower than TIRFM imaging. We show that to get equilibrium rates consistent with observed values, the diffusion coefficients and reaction rates have to be significantly higher than the values reported in the literature. Once a close agreement between experiment and model is achieved, we use multiscale modeling in conjunction with patch-clamp electrophysiology of IP3R and fluorescence imaging of whole-cell Ca2+ response, induced by intracellular Aβ42 oligomers to show that Aβ42 inflicts cytotoxicity by impairing mitochondrial function. Driven by patch-clamp experiments, we first model the kinetics of IP3R, which is then extended to build a model for the whole-cell Ca2+ signals. The whole-cell model is then fitted to fluorescence signals to quantify the overall Ca2+ release from the ER by intracellular Aβ42 oligomers through G-protein-mediated stimulation of IP3 production. The estimated IP3 concentration as a function of intracellular Aβ42 content together with the whole-cell model allows us to show that Aβ42 oligomers impair mitochondrial function through pathological Ca2+ uptake and the resulting reduced mitochondrial inner membrane potential, leading to an overall lower ATP and increased production of reactive oxygen species and [H2O2]. We further show that mitochondrial function can be restored by the addition of Ca2+ buffer EGTA, in accordance with the observed abrogation of Aβ42 cytotoxicity by EGTA in our live cells experiments. Finally, our modeling study was extended to other pathological phenomena such as epileptic seizures and spreading depolarizations (SD) and their effects on mitochondria by incorporating conservation of particles and charge, and accounting for the energy required to restore ionic gradients to the neuron. By examining the dynamics as a function of potassium and oxygen we can account for a wide range of neuronal hyperactivity from seizures, normoxic SD, and hypoxic SD (HSD) in the model. Together with a detailed model of mitochondria xi and Ca2+ -release through the ER, we determine mitochondrial dysfunction and potential recovery mechanisms from HSD. Our results demonstrate that HSD causes detrimental mitochondrial dysfunction that can only be recovered by restoration of oxygen. Once oxygen is replenished to the neuron, organic phosphate and pH gradients along the mitochondria determine how rapid the neuron recovers from HSD.

Chronic exposure to arsenic-contaminated drinking water is correlated with obstructive lung disease (i.e. chronic obstructive pulmonary disease (COPD), bronchiectasis), reduced lung function and other respiratory effects (e.g. chronic cough, chest sounds). Researchers have associated arsenic exposure with reduced airway immunity. The airway epithelial innate immune system protects underlying tissue from inhaled particulates and pathogens through a variety of mechanisms. Such defects in innate immunity are associated with chronic bacterial infections and development of obstructive airway diseases, including COPD and bronchiectasis. We hypothesize that arsenic exposure may lead to recurrent lung infection and eventual obstructive lung disease by compromising mechanisms essential in airway innate immunity. In the work presented herein we evaluated the effects of arsenic on airway epithelial barrier properties, wound repair capacity, and signaling pathways essential in innate immunity. We previously published that acute (24 hr) arsenic (0.4-3.9 μM as Naarsenite) slowed wound repair in a human bronchial epithelial cell line (16HBE14o-). In the first study we hypothesized arsenic may be affecting wound repair by altering Ca²⁺ signaling that is important in multiple aspects of wound repair, including cell migration. We found wound-induced Ca²⁺ signaling was largely mediated by paracrine ATP in 16HBE14o- cells, and acute (24 hr) arsenic (0.8, 3.9 μM) exposure reduced ATPmediated Ca²⁺ signaling. We identified functional reductions in the ATP receptors P2Y₂ and P2X₄ following arsenic exposure. Both of these receptors are essential in airway innate immunity (e.g. mucociliary clearance). In the second study we found similar reductions in wound repair capacity and ATP-mediated Ca²⁺ signaling in 16HBE14o cells using a chronic (4-5 week) low-dose (0.13, 0.33 μM) arsenic exposure representative of U.S. drinking water standards. Further, wound-induced Ca²⁺ signaling was reduced in primary cultured tracheal cells derived from mice fed arsenic-free or arsenic-supplemented (50 ppb; 1μM=75 ppb) water for four weeks prior to experimentation. In the last study we demonstrated that the structure and function of the airway epithelial barrier was altered by a five-day exposure of arsenic (0.8, 3.9 μM). We conclude that arsenic at environmentally relevant levels compromises key functions in airway epithelial innate immunity that may underlie development of lung disease.

Ion signaling plays fundamental role in cell survival. Na+ and Ca2+ are critical players in ion signaling. Cells spend the major amount of energy to maintain and regulate Na+ and Ca2+ gradients across the cell membrane. Any disruption in cellular energy consumption by plasma membrane ATPases affects ion signaling and vice versa. This thesis is a combination of four separate research studies. In the first study, We measured ATP consumption dynamics of Na+/K+-ATPase using a genetically encoded fluorescent indicator called Perceval HR. we demonstrate that PercevalHR is an excellent tool to visualize ATP:ADP in mammalian cells. In the second study, We studied the role of calcium signaling and TRP channels in angiotensin II type 1 receptor  signaling cascade. We prove that low inhibition of CaV1.2 with physiological and therapeutically relevant concentration of Angiotensin II up regulate AT1R signaling. In the third study, We studied the role of the TRPM5 channel in regulating insulin secretion, and cytoplasmic free calcium concentration in the rat β-cells by usingtriphenyl phosphine oxide, a selective inhibitor of the channel. In the fourth study, We tested whether, the genetically engineered human β-cell line (EndoC-BH1) could be used as models for studying Ca2+signaling in the context of Type II Diabetes. We found that the EndoC-BH1 cells could be a relevant model to study stimulus-secretion coupling and Ca2+ signaling in the human β-cells. / <p>QC 20170328</p>

A atividade nervosa simpática está aumentada na insuficiência cardíaca (IC) e relacionada com a gravidade e o prognóstico da doença. Camundongos com deleção dos receptores adrenérgicos &#945;2A e &#945;2C (&#945;2A/&#945;2CARKO) desenvolvem IC induzida pela hiperestimulação simpática e apresentam 50% de mortalidade aos sete meses de idade. A diminuição na contratilidade cardíaca, a perda de cardiomiócitos e a intolerância à realização de esforço físico sugerem esse modelo genético para o estudo de possíveis terapias farmacológicas e não-farmacológicas para o tratamento da IC. O presente estudo avalia o possível efeito terapêutico do treinamento físico (TF) na cardiomiopatia induzida pela hiperatividade simpática. Os benefícios sobre a tolerância ao esforço e a função sistólica observados nos camundongos &#945;2A/&#945;2CARKO com o TF foram acompanhados pelo aumento do pico do transiente de Ca2+ intracelular e da expressão de proteínas cardíacas que regulam o transiente de Ca2+ SERCA2 (20 %), fosfo-PLB-Ser16 (92 %), fosfo-PLB-Tre17 (285 %), bem como pela redução na expressão do NCX e da PP1. Portanto, esse estudo fornece evidências de alterações na sinalização intracelular de Ca2+ nos camundongos &#945;2A/&#945;2CARKO que contribuem para o agravamento da IC nesse modelo, e que o TF melhora a função ventricular associada ao aumento no transiente de Ca2+ intracelular no cardiomiócito. / The sympathetic nervous activity is increased in heart failure (HF) and is associated with the severity and prognosis of disease. Mice lacking both &#945;2A and &#945;2C adrenergic receptors (&#945;2A/&#945;2CARKO) develop sympathetic hyperactivity- induced HF and present 50% mortality rate by seven mo of age. The decreased cardiac contractility, cardiomyocytes degradation and exercise intolerance suggest that these mice are a good genetic model to unravel molecular mechanisms involved in the improvements of ventricular function by different pharmacological and non-pharmacological therapies for HF. The present study was underlined to test the possible therapeutic effect of exercise training in sympathetic hyperactivity- induced HF. The improved exercise tolerance and systolic function after exercise training in &#945;2A/&#945;2CARKO was accompanied by increased intracellular Ca2+ transient and the expression of cardiac proteins which regulate Ca2+ transients, such as expression SERCA2 (20%), phospho-PLB-Ser16 (92%), phospho-PLB-Tre17 (285%), paralleled by reduction in NCX and PP1 expression. Therefore, this study provide direct evidence for the altered intracellular Ca2+ signaling in &#945;2A/&#945;2CARKO mice and that exercise training improves the ventricular function associated with an increase in intracellular Ca2+ transient in cardiomyocyte.

A atividade nervosa simpática está aumentada na insuficiência cardíaca (IC) e relacionada com a gravidade e o prognóstico da doença. Camundongos com deleção dos receptores adrenérgicos &#945;2A e &#945;2C (&#945;2A/&#945;2CARKO) desenvolvem IC induzida pela hiperestimulação simpática e apresentam 50% de mortalidade aos sete meses de idade. A diminuição na contratilidade cardíaca, a perda de cardiomiócitos e a intolerância à realização de esforço físico sugerem esse modelo genético para o estudo de possíveis terapias farmacológicas e não-farmacológicas para o tratamento da IC. O presente estudo avalia o possível efeito terapêutico do treinamento físico (TF) na cardiomiopatia induzida pela hiperatividade simpática. Os benefícios sobre a tolerância ao esforço e a função sistólica observados nos camundongos &#945;2A/&#945;2CARKO com o TF foram acompanhados pelo aumento do pico do transiente de Ca2+ intracelular e da expressão de proteínas cardíacas que regulam o transiente de Ca2+ SERCA2 (20 %), fosfo-PLB-Ser16 (92 %), fosfo-PLB-Tre17 (285 %), bem como pela redução na expressão do NCX e da PP1. Portanto, esse estudo fornece evidências de alterações na sinalização intracelular de Ca2+ nos camundongos &#945;2A/&#945;2CARKO que contribuem para o agravamento da IC nesse modelo, e que o TF melhora a função ventricular associada ao aumento no transiente de Ca2+ intracelular no cardiomiócito. / The sympathetic nervous activity is increased in heart failure (HF) and is associated with the severity and prognosis of disease. Mice lacking both &#945;2A and &#945;2C adrenergic receptors (&#945;2A/&#945;2CARKO) develop sympathetic hyperactivity- induced HF and present 50% mortality rate by seven mo of age. The decreased cardiac contractility, cardiomyocytes degradation and exercise intolerance suggest that these mice are a good genetic model to unravel molecular mechanisms involved in the improvements of ventricular function by different pharmacological and non-pharmacological therapies for HF. The present study was underlined to test the possible therapeutic effect of exercise training in sympathetic hyperactivity- induced HF. The improved exercise tolerance and systolic function after exercise training in &#945;2A/&#945;2CARKO was accompanied by increased intracellular Ca2+ transient and the expression of cardiac proteins which regulate Ca2+ transients, such as expression SERCA2 (20%), phospho-PLB-Ser16 (92%), phospho-PLB-Tre17 (285%), paralleled by reduction in NCX and PP1 expression. Therefore, this study provide direct evidence for the altered intracellular Ca2+ signaling in &#945;2A/&#945;2CARKO mice and that exercise training improves the ventricular function associated with an increase in intracellular Ca2+ transient in cardiomyocyte.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19753号 / 工博第4208号 / 新制||工||1649(附属図書館) / 32789 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 濵地 格, 教授 梅田 眞郷 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Receptores purinérgicos e canais de cálcio voltagem-dependentes estão envolvidos em diversos processos biológicos como na gastrulação, durante o desenvolvimento embrionário, e na diferenciação neural. Quando ativados, canais de cálcio voltagem-dependentes e receptores purinérgicos do tipo P2, ativados por nucleotídeos, desencadeiam transientes de cálcio intracelulares controlando diversos processos biológicos. Neste trabalho, nós estudamos a participação de canais de cálcio voltagem-dependentes e receptores do tipo P2 na geração de transientes de cálcio espontâneos e sua regulação na expressão de fatores de transcrição relacionados com a neurogênese utilizando como modelo células tronco (CTE) induzidas à diferenciação em células tronco neurais (NSC) com ácido retinóico. Descrevemos que CTE indiferenciadas podem ter a proliferação acelerada pela ativação de receptores P2X7, enquanto que a expressão e a atividade desse receptor precisam ser inibidas para o progresso da diferenciação em neuroblasto. Além disso, ao longo da diferenciação neural, por análise em tempo real dos níveis de cálcio intracelular livre identificamos 3 padrões de oscilações espontâneas de cálcio (onda, pico e unique), e mostramos que ondas e picos tiveram a frequência e amplitude aumentadas conforme o andamento da diferenciação. Células tratadas com o inibidor do receptor de inositol 1,4,5-trifosfato (IP3R), Xestospongin C, apresentaram picos mas não ondas, indicando que ondas dependem exclusivamente de cálcio oriundo do retículo endoplasmático pela ativação de IP3R. NSC de telencéfalo de embrião de camundongos transgênicos ou pré-diferenciadas de CTE tratadas com Bz-ATP, o agonista do receptor P2X7, e com 2SUTP, agonista de P2Y2 e P2Y4, aumentaram a frequência e a amplitude das oscilações espontâneas de cálcio do tipo pico. Dados, obtidos por microscopia de luminescência, da expressão em tempo real de gene repórter luciferase fusionado à Mash1 e Ngn2 revelou que a ativação dos receptores P2Y2/P2Y4 aumentou a expressão estável de Mash1 enquanto que ativação do receptor P2X7 levou ao aumento de Ngn2. Além disso, células na presença do quelante de cálcio extracelular (EGTA) ou do depletor dos estoques intracelulares de cálcio do retículo endoplasmático (thapsigargin) apresentaram redução na expressão de Mash1 e Ngn2, indicando que ambos são regulados pela sinalização de cálcio. A investigação dos canais de cálcio voltagem-dependentes demonstrou que o influxo de cálcio gerado por despolarização da membrana de NSC diferenciadas de CTE é decorrente da ativação de canais de cálcio voltagem-dependentes do tipo L. Além disso, esse influxo pode controlar o destino celular por estabilizar expressão de Mash1 e induzir a diferenciação neuronal por fosforilação e translocação do fator de transcrição CREB. Esses dados sugerem que os receptores P2X7, P2Y2, P2Y4 e canais de cálcio voltagem-dependentes do tipo L podem modular as oscilações espontâneas de cálcio durante a diferenciação neural e consequentemente alteram o padrão de expressão de Mash1 e Ngn2 favorecendo a decisão do destino celular neuronal. / Purinergic receptors and voltage gated Ca2+ channels have been attributed with developmental functions including gastrulation and neural differentiation. Upon activation, nucleotide-activated P2 purinergic receptor and voltage-gated Ca2+ channel subtypes trigger intracellular calcium transients controlling cellular processes. Here, we studied the participation of voltage-gated calcium channels and P2 receptor activity in spontaneous calcium transients and consequent regulation expression of transcription factors related to retinoic acid-induced neurogenesis of mouse neural stem and embryonic stem cells (ESC). In embryonic pluripotent stem cells, proliferation is accelerated by P2X7 receptor activation, while receptor expression / activity needs to be down-regulated for the progress of neuroblast differentiation. Moreover, along neural differentiation time lapse imaging with means of a cytosolic calcium-sensitive fluorescent probe provided different patterns of spontaneous calcium transients (waves and spikes) showing that both, frequency and amplitude increased along differentiation. Cells treated with the inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor Xestospongin C showed spikes but not waves, indicating that waves exclusively depended on calcium release from endoplasmic reticulum by IP3R activation. Cells treated with the P2X7 receptor subtype agonist Bz-ATP and the P2Y2 and P2Y4 receptor 2-S-UTP increased frequency and amplitudes of calcium transients, mainly spikes, in embryonic telencephalon neural stem cells (NSC) and NSC pre-differentiated from ESC. Data obtained by luminescence time lapse imaging of stable transfected cells with Mash1 or Ngn2 promoter-protein fusion to luciferase reporter construct revealed increased Mash1 expression due to activation of P2Y2/P2Y4 receptor subtypes, while increased expression of Ngn2 was observed following P2X7 receptor activation. In addition, cells imaged in presence of the extracellular calcium chelator EGTA or following endoplasmic reticulum calcium store depletion by thapsigargin showed a decrease in Mash1 and Ngn2 expression, indicating that both are regulated by calcium signaling. Investigation of the roles of voltage gated Ca2+ channels in neural differentiation showed that Ca2+ influx in NSC pre-differentiated from ESC is due to membrane depolarization and L-type voltage gated Ca2+ channel activation, thereby controlling cell fate decision, by stabilizing the expression of MASH1 and inducing differentiation, by phosphorylation of the transcription factor CREB. Altogether these data suggest that P2X7, P2Y2, P2Y4 receptors and L-type voltage gated Ca2+ channels can modulate spontaneous calcium oscillations during neural differentiation and consequently change the Mash1 and Ngn2 expression patterns, thus favoring the cell fate decision to the neuronal phenotype.