Application of hydrogen peroxide (H2O2) caused an increase in cytosolic Ca2+ levels ([Ca2+]i) in both cell types. The [Ca2+]i rises diminished in the presence of U73122, a phospholipase C inhibitor, or Xestospongin C (XeC), an inhibitor for inositol-1,4,5-trisphosphate (IP3) receptors. Removal of Ca2+ from the bath also decreased the [Ca2+]i rises in response to H2O2. In addition, treatment of endothelial cells with H2O2 reduced the [Ca2+]i responses to subsequent challenge of ATP. The decreased [Ca2+]i responses to ATP were resulted from a pre-depletion of intracellular Ca2+ stores by H2O2. Interestingly, we also found that Ca2+ store depletion was more sensitive to H2O2 treatment in endothelial cells of mesenteric arteries than those of aortas. Hypoxanthine-xanthine oxidase (HX-XO) was also found to induce [Ca2+]i rises in both types of endothelial cells, the effect of which was mediated by superoxide anions and H2O2 but not by hydroxyl radical. H2O2 contribution in HX-XO-induced [Ca2+]i rises were more significant in endothelial cells from mesenteric arteries than those from aortas. In summary, H2O2 could induce store Ca2+ release via phospholipase C-IP3 pathway in endothelial cells. Resultant emptying of intracellular Ca2+ stores contributed to the reduced [Ca2+]i responses to subsequent ATP challenge. The [Ca2+]i responses were more sensitive to H2O2 in endothelial cells of small-sized arteries than those of large-sized arteries.We compared the Ca Functionally, ROS play a key role in physiological and pathological processes in endothelial cells. For example, H2O2 at physiological concentration serves as an endothelium-derived hyperpolarizing factor (EDHF), mediating vascular relaxation 2+]iVascular endothelial cells 2O22+]i rises in vascular endothelial cells. The [Ca2+]i rises could result from ROS actions on the plasma membrane ion channels 3 production 3 receptors 2+-ATPase 2+]i, ROS treatment may alter the [Ca2+]i responses of endothelial cells to a variety of physiological agonists including ATP and bradykinin 2+]i rises 2+]i responses.Reports showed that superoxide anions 2+]i in endothelial cells, most of these reports only investigated the endothelial cells derived from large-sized arteries 2+]i in endothelial cells of small-sized arteries has received little attention 2+]i responses in endothelial cells from different-sized arteries. Large-sized arteries and small-sized arteries differ in their function. Small-sized arteries such as mesenteric arteries are resistance arteries that play a key role in blood pressure control. Vasoactive factors in small-sized arteries are often different from that in large-sized arteries. For example, while nitric oxide is the major vasodilator in large arteries, EDHFs often play a more important role as vasodilators in small-sized arteries Although there have been a great number of studies investigating the ROS effect on [Ca2O2 on [Ca2+]i in endothelial cells from large-sized arteries, aortas (aortic ECs), and small-sized arteries, mesenteric arteries (MAECs). We found that H2O2 stimulated IP3 production to induce store Ca2+ release in both cell types. H2O2 treatment depleted intracellular [Ca2+]i stores, resulted in a decreased [Ca2+]i response to subsequent ATP challenge. The Ca2+ store depletion was more sensitive to H2O2 in endothelial cells of small-sized arteries than those of large-sized arteries.In the present study, we compared the effect of H2O2 on [Ca2+]i was investigated in aortic ECs and MAECs. H2O2 at 5 mM caused marked [Ca2+]i rises in both types of cells that were bathed in normal physiological saline solution (N-PSS) containing 1 mM Ca2+ , suggesting a contribution of Ca2+ entry to the H2O2-induced [Ca2+]i rises. Significant [Ca2+]i rises to H2O2 could still be observed even when bath was Ca2+-free, suggesting that store Ca2+ release also contributed to the H2O2-induced [Ca2+]i rises.The effect of H mM Ca2+ . The amp3-sensitive Ca2+ stores are the major intracellular Ca2+ stores, and that the Ca2+ release from the stores hinges on the production on IP3, which is generated through activity of phospholipase C (PLC) 3 receptor inhibitor, at 10 µM for 20 min almost abolished the H2O2-induced [Ca2+]i rises in both aortic ECs and MAECs. Furthermore, a PLC inhibitor U73122 (10 µM) markedly reduced the H2O2-induced [Ca2+]i rises, whereas its inactive analog U73343 (10 µM) had no effect -induced [Ca2+]i rises. The cells were first pre-incubated with H2O2 (500 µM or 1 mM) for 30 min, followed by 30 µM ATP application to evoke [Ca2+]i responses. 2+]i rises in response to ATP in aortic ECs and MAECs that were pre-incubated with different concentrations of H2O2. A marked difference was found between aortic ECs and MAECs. While both cells lost the [Ca2+]i responses to ATP after 1 mM H2O2 treatment, a relatively low concentration of 500 µM H2O2 could abolish the ATP responses in MAECs but had no effect in aortic ECs -induced [Ca2+]i rises in cells bathed in a nominal Ca2+-free solution  in MAECs but had no significant effect in aortic ECs. These data suggest that MAECs were more sensitive to H2O2 treatment than aortic ECs with regard to their responses in Ca2+ store depletion and ATP-induced [Ca2+]i rises. The controls in 2O2. In time control, Mag-fluo4 fluorescence only decreased by 8±6% (n = 3) in MAECs and by 3±7% (n = 3) in aortic ECs. The small reduction in Mag-fluo4 fluorescence in the control experiments could be due to light-sensitive quenching of Mag-fluo4 as described elsewhere To further confirm the findings, Mag-fluo4/AM, a dye that stains Ca2+ store release in aortic ECs and MAECs in the absence of 2O2H pretreatment. Cells bathed in a nominal Ca2+-free solution were challenged with different concentrations of ATP. In both cell types, ATP evoked [Ca2+]i rises in a concentration dependent manner , an enzyme that causes superoxide dismutation, reduced the [Ca2+]i rises  than in aortic ECs   . Combineal cells .2+]i change is an important early signal for ROS-induced endothelial function and dysfunction. However, only a few studies have investigated ROS-induced Ca2+ signaling in the endothelial cells derived from small-sized arteries 2+]i responses in endothelial cells from different-sized arteries. In the present study, we compared the effect of H2O2 on [Ca2+]i in endothelial cells from large-sized arteries and small-sized arteries. The results show that H2O2 stimulated [Ca2+]i rises in both cell types. The H2O2-induced [Ca2+]i rises could be blocked by U73122 and XeC, suggesting that the signaling cascade involves phospholiase C activity, IP3 production, and Ca2+ release through IP3 receptors. The increased IP3 production following H2O2 treatment was confirmed by IP3 measurement. It is well documented that Ca2+ release from IP3-sensitive Ca2+ stores would stimulate Ca2+ influx through store-operated Ca2+ entry mechanism 2O2 treatment could enhance Ca2+ entry when the bath solution contained Ca2+.[Ca2+]i responses to subsequent agonist challenge in endothelial cells 2O2 and superoxide anions were found to reduce the agonist-induced [Ca2+]i rises 2+]i responses 2O2 treatment reduced the [Ca2+]i responses to ATP in H2O2 concentration-dependent and H2O2 incubation time-dependent manners in mouse aortic ECs and MAECs. The reduced [Ca2+]i responses to ATP were due to a pre-depletion of intracellular Ca2+ stores during H2O2 treatment. Two lines of evidence support this: 1) After H2O2 treatment, the store Ca2+ release in response to ATP became much smaller. 2) Direct measurement of store Ca2+ content by Mag-fluo4 demonstrated a reduction in store Ca2+ content after H2O2 treatment. Interestingly, our data clearly indicate that endothelial cells from small-sized arteries (MAECs) were more sensitive to H2O2 treatment than those of large-sized arteries (aortic ECs) with regard to their store Ca2+ release and subsequent [Ca2+]i responses to ATP. This type of differential sensitivity/response of store Ca2+ release to ROS treatment could explain some data conflicts in the literature. For example, Volk et al., reported that, in rat liver artery endothelial cells, ROS treatment had no effect on the [Ca2+]i responses to subsequent ATP or histamine challenge 2+ depletion. As a result, no change in [Ca2+]i responses to agonists would be expected.There are conflicts in reports as to how ROS treatment would affect the [Ca2+]i responses to H2O2 in MAECs than in aortic ECs? H2O2-induced IP3 production was similar in MAECs and aortic ECs, therefore IP3 production was not the reason. Alternatively, this could be due to more abundant IP3 receptor expression and/or a higher IP3 receptor sensitivity to IP3 in MAECs than in aortic ECs. If this is true, [Ca2+]i responses to other agonists is also expected to be higher in MAECs than in aortic ECs. Indeed, we found that similar high sensitivity of intracellular store Ca2+ release to ATP in MAECs than in aortic ECs  than in those of large-sized arteries (aortic ECs). Previously, different reports have claimed different ROS, including H2O22+]i rises in endothelial cells. The discrepancy in results could be due to a variety of factors including endothelial cell sources and/or culture conditions.H2+ entry and store Ca2+ release contributed to the H2O2-induced [Ca2+]i rises in endothelial cells. H2O2 treatment depleted the intracellular Ca2+ stores, resulting in reduced [Ca2+]i responses to subsequent agonist challenge. The store Ca2+ release and subsequent [Ca2+]i responses to ATP were more sensitive to H2O2 treatment in endothelial cells of small-sized arteries than those of large-sized arteries. This study highlights the similarity and difference of ROS-induced [Ca2+]i responses in endothelial cells from large-sized arteries and small-sized arteries.In conclusion, we found both CaWe followed Guide for Animal Care and Use of Laboratory Animals published by the US National Institute of Health. The protocols for animal experiments were approved by Animal Experimentation Ethics Committee, The Chinese University of Hong Kong .2. Primary cultured aortic endothelial cells (aortic ECs) and mesenteric artery endothelial cells (MAECs) were dissociated from mouse aorta and mesenteric arteries of the first to tertiary branches , respectively, using the methods described elsewhere Animals were supplied by the Laboratory Animal Service Center of the Chinese University of Hong Kong . We followed Guide for Animal Care and Use of Laboratory Animals published by the US National Institute of Health. The protocols for animal experiments were approved by Animal Experimentation Ethics Committee, The Chinese University of Hong Kong . Male C57 mice (8–12 weeks) were sacrificed by inhalation of CO2+]i responses to H2O2 or HX-XO or ATP. Briefly, the cells were seeded on circular glass discs at 37°C overnight supplemented with the culture medium. For the fluorescence dye loading, cells were incubated for 1 hr in dark at room temperature with 10 µM Fluo4/AM and 0.02% Pluronic acid F-127 in normal physiological saline solution (N-PSS), which contained in mM: 1 CaCl2, 140 NaCl, 1 KCl, 1 MgCl2, 10 glucose, and 5 Hepes at pH 7.4. The circular discs containing the endothelial cells were then pinned in a specially designed chamber. The chamber was placed on the stage of an inverted microscope (Nikon Diaphot 200). During experiments, cells were bathed in N-PSS or 0.5Ca2+-PSS or 0Ca2+-PSS. The composition of 0.5Ca2+-PSS and 0Ca2+-PSS was similar to N-PSS except for Ca2+ concentration . All agents were applied directly to the bath along the side of the chamber. Solutions were then mixed by pipetting gently up and down for a few times. Experiments were performed at room temperature. Fluorescence signals were recorded by MRC-1000 Laser Scanning Confocal Imaging System with MRC-1000 software (Bio-Rad) with the excitation wavelength of 488 nm and a 515 nm-long pass emission filter. The Ca2+ responses were displayed as the ratio of fluorescence relative to the intensity before H2O2 or ATP or HX-XO (F1/F0). Due to variation in [Ca2+]i responses between different batches of cells, each series of experiments had its own control.Cells were prepared and loaded with a membrane permeant fluorescence dye Fluo4/AM  for observing their [Ca2+ level in intracellular Ca2+ stores. Briefly, cells were seeded on circular glass plates at 37°C overnight supplemented with the culture medium. As for the fluorescence dye loading, cells were incubated with 5 µM Mag-fluo4/AM in dark at 37°C for 45 min, and 0.02% Pluronic acid F-127 in N-PSS. Cells were then washed with the indicator-free N-PSS and incubated at 37°C for 45 min to unload the Mag-fluo4 from cytoplasm. The circular discs containing the endothelial cells were then pinned down in a specially designed chamber. The chamber was placed on the stage of an inverted microscope (Nikon Diaphot 200). Mag-fluo4 fluorescence was recorded by MRC-1000 Laser Scanning Confocal Imaging System with MRC-1000 software (Bio-Rad) with the excitation wavelength of 488 nm and a 515 nm-long pass emission filter. The cells were then treated with or without H2O2 for 30 minutes. Because Mag-fluo4 fluorescence was reported to be light-sensitive and could be quenched by light exposure, laser emission to samples was cut off during the period of H2O2 treatment. Fluorescence signals were then collected before and after 30-minute H2O2 treatment. The change in store Ca2+ content is displayed as Mag-fluo4 intensity change in percentage.Cells were loaded with fluorescence dye Mag-fluo4/AM  for observing the Ca3 was measured using HitHunter™ IP3 Assay Fluorescence Polarization Detection-Green Kits , a reliable and convenient methodology based on competitive binding between an IP3 fluorescence tracer and unlabeled IP3 from the cell lysates or standards. Free IP3 competes at the IP3 binding protein and allows the IP3 tracer to rotate freely upon excitation with plane polarized light. The polarized signal is inversely proportional to the amount of the free unlabelled IP3. Thus, polarization signal is decreased when the concentration of free IP3 is increased 2O2  for 5 min in black 96-well plates. The cellular reactions were terminated by placing cells on ice followed by addition of 0.2 N perchloric acid to lyse the cells. The plate was then shaken at 650 rpm for 5 min. The IP3 tracer was subsequently added to each well, followed by IP3 binding protein. The polarized fluorescence from the IP3 tracer (fluorescein) was read using a Wallac EnVision™ Microplate Reader  with a polarization mirror, a 485 nm excitation filter and a 530 nm emission filter. IP3 concentration was calculated from the IP3 standard curve and expressed as pmole/1×106 cells.The amount of IPData Analysis was performed with Software Confocal Assistant and Metafluor. All representative traces were plotted by using Prism 3.0 . Summarized data were expressed as the mean±SEM and analyzed with two-tailed Student's t test at a p<0.05 level of significance.


These changes occur primarily as a result of ER Ca2+ overload. We argue that normalization of intracellular Ca2+ homeostasis could be a strategy for development of effective disease-modifying therapies. The current review summarizes recent data about changes in ER Ca2+ signaling in AD. Ca2+ channels that are discussed in the current review include: inositol trisphosphate receptors, ryanodine receptors, presenilins as ER Ca2+ leak channels, and neuronal SOC channels. We discuss how function of these channels is altered in AD and how important are resulting Ca2+ signaling changes for AD pathogenesis.Alzheimer disease (AD) is a major threat of twenty-first century that is responsible for the majority of dementia in the elderly. Development of effective AD-preventing therapies are the top priority tasks for neuroscience research. Amyloid hypothesis of AD is a dominant idea in the field, but so far all amyloid-targeting therapies have failed in clinical trials. In addition to amyloid accumulation, there are consistent reports of abnormal calcium signaling in AD neurons. AD neurons exhibit enhanced intracellular calcium (Ca Ca2+-mediated signal transduction connects membrane excitability and biological functions of neurons ranging from proliferation, secretion, gene expression, ATP production, cell death to memory formation and its loss. Acting at the border of electrical and signaling “worlds” of the cell, Ca2+-permeable channels play a major role in many key aspects of neuronal functions. Due to the huge importance of the calcium as the second messenger neurons utilize many approaches to regulate intracellular Ca2+ content, mainly via local signal transduction pathways. Neuronal Ca2+ influx can be maintained by different Ca2+-permeable channels, such as voltage-gated Ca2+ channels of plasma membrane, N-methyl-D-aspartate (NMDA) receptors, α-amino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid (AMPA) receptors, nicotinic receptors, store-operated Ca2+ channels (SOC). Ca2+ can also be released from intracellular stores of endoplasmic reticulum (ER) via inositol-1,4,5-trisphosphate receptors (InsP3R) and ryanodine receptors (RyanRs). Mitochondria also play an important role in intracellular Ca2+ handling. Neurons are highly susceptible to any changes in intracellular Ca2+ concentrations: insufficient intracellular Ca2+ content lead to abnormal functioning of neurons, whereas excessive Ca2+ levels cause cell death  is the threat of twenty-first century that is responsible for the majority of senile dementia. AD progresses slowly and affects neurons in the brain. Currently there are two main proteins whose dysfunctions and accumulation in the brain are correlated with the disease progress. The first is 40–42 long beta-amyloid (Aβ) peptides that constitute a major part of neuritic plaques and cause excessive neurotoxicity. These peptides are cleaved from the amyloid precursor protein (APP) by β- and γ-secretases . The sec in vivo . It has ntration . Recent hosphate  and by shosphate .2+ accumulation in the ER in the absence of Aβ pathology  has been linked to late-onset AD by genetic evidence  mutant constructs showed an upregulation of InsP3R-mediated Ca2+ release . Also, it was reported that Aβ oligomers stimulate synaptic mGluR5 receptors linked with InsP3 production  in 3xTg-AD mice is exaggerated in response to synaptic stimulation, including NMDAR-mediated Ca2+ influx , presenilin 2 (PSEN2), and APP genes that are linked to early onset FAD. The majority, nearly 200, of these mutations are within PSEN1. To date many known PSEN1 mutations contribute to Ca2+ disruptions in ER Ca2+ signaling . Interestingly, changes in expression of STIM1 and STIM2 proteins were found in PS knockout and FAD mutant cells  proteins. STIM 1 and STIM2 protein reside in ER, and reduction in ER Cachannels . The molnt cells , suggestnt cells . These int cells , 2010a.2+ dysregulation in AD . FAD linked mutations in PS cause disruption of PS Ca2+ leak function. As a result Ca2+ is accumulating inside of the ER. Similar increase in ER Ca2+ levels occur as a result of brain aging. In order to compensate for ER Ca2+ overload neurons mount two physiological responses: (1) upregulate gating of InsP3R and expression/activity of RyanR, and (2) downregulate activity of neuronal SOC . We hypothesize that these initially protective responses with time become toxic and eventually lead to synaptic dysfunction, synaptic loss, impaired plasticity, and learning, loss of memories and neurodegeneration. The role of RyanR in these processes is likely to be more significant than the role of InsP3R, as InsP3R predominantly localized in the soma, whereas RyanR are abundant in the postsynaptic and presynaptic terminals. Dantrolene provides a possible way to suppress RyanR-mediated Ca2+ release pharmacologically, but there are significant issues with specificity of dantrolene effects and its delivery to the brain. Neuronal SOC pathway provides a novel potential target for AD treatment that should be explored further.In the summary we would like to conclude with our working hypothesis for ER CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


GnRH binding to G-protein-coupled receptors triggers Ca2+ mobilization from InsP3-sensitive intracellular pools, generating the global Ca2+ elevations necessary for secretion. Ca2+ signaling responses to increasing (GnRH) vary in stereotyped fashion from subthreshold to baseline spiking (oscillatory), to biphasic (spike-oscillatory or spike-plateau). This progression varies somewhat in gonadotrophs from different species and biological preparations. Both baseline spiking and biphasic GnRH-induced Ca2+ signals control LH/FSH synthesis and exocytosis. Estradiol and testosterone regulate gonadotropin secretion through feedback mechanisms, while FSH synthesis and release are influenced by activin, inhibin, and follistatin. Adaptation to physiological events like the estrous cycle, involves changes in GnRH sensitivity and LH/FSH synthesis: in proestrus, estradiol feedback regulation abruptly changes from negative to positive, causing the pre-ovulatory LH surge. Similarly, when testosterone levels drop after orquiectomy the lack of negative feedback on pituitary and hypothalamus boosts both GnRH and LH secretion, gonadotrophs GnRH sensitivity increases, and Ca2+ signaling patterns change. In addition, gonadotrophs proliferate and grow. These plastic changes denote a more vigorous functional adaptation in response to an extraordinary functional demand.Pituitary gonadotrophs are a small fraction of the anterior pituitary population, yet they synthesize gonadotropins: luteinizing (LH) and follicle-stimulating (FSH), essential for gametogenesis and steroidogenesis. LH is secreted via a regulated pathway while FSH release is mostly constitutive and controlled by synthesis. Although gonadotrophs fire action potentials spontaneously, the intracellular Ca The reproductive function and sexual maturation is under the control of the hypothalamic-pituitary-gonadal axis. Pituitary gonadotrophs, which constitute 7–15% of the anterior pituitary gland secrete two dimeric glycoproteins, gonadotropins, luteinizing (LH) and follicle-stimulating (FSH) hormones that play an essential role in the control of steroidogenesis, gametogenesis, and ovulation  and monohormonal (15%) gonadotrophs whose percentage shifts under different physiological conditions, such as castration or estrous cycle . LH and 2+]i) rise, gonadotrophs as other pituitary endocrine cells display spontaneous intracellular Ca2+ transients in dependence of changes in the membrane electrical activity. However, this membrane potential oscillations are small and do not produce the necessary [Ca2+]i increase to generate hormonal secretion (2+ (in situ) present robust and stereotyped dose-dependent intracellular Ca2+ signals in response to suprathreshold concentrations of GnRH , resulting in the rapid hydrolysis of phosphatidylinositol 4, 5-biphosphate (PIP2) and the production of two second messengers: diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (InsP3); long lasting GnRH stimulation (∼5–10 min) could also activate phospholipase D (PLD) and phospholipase A2 (PLA2)  activation which in turn reduces depolarization-mediated Ca2+ influx, while increasing gonadotropin secretion , which is a member of the rhodopsin-like G-protein-coupled receptors (GPCR) superfamily . Upon Gn2 (PLA2) . InsP3 gecretion   than in the cytosol (50–250 nM) by the pumping activity of the sarco-ER Ca2+ ATPase (SERCA) located in the ER membrane , a ligand-gated Ca2+ channel that opens after InsP3 binding  sites on the cytoplasmic side of the InsP3R is essential for channel opening. In fact, Ca2+ and InsP3 operate as co-agonists. Ca2+ signal amplification and spreading phenomena, involving assemblies of InsP3Rs originate from this synergistic role of Ca2+  site of the receptor, which closes the InsP3R channel. This occurs when cytosolic Ca2+ close to the InsP3Rs is high, i.e., after an episode of fast release. As in most pituitary cells, agonist stimulation in gonadotrophs produce a [Ca2+]i peak which decays to sustained Ca2+ level (plateau phase). At intermediate GnRH concentration the initial Ca2+ spike is often followed by large [Ca2+]i oscillations resulting from opening and closing cycles of the InsP3R channels as a consequence of [Ca2+]i fluctuations near its cytoplasmic side (2+ oscillations is determined by the dose of GnRH applied and the intracellular (InsP3) reached   activity, with a consequent diminution on gonadotropin secretion  , 29, pitlin (ET) , and sublin (ET) . Converseceptors  under thdotrophs , being t release , also inoduction . The actecretion –36; toni release ; its rec release .2+ signaling: i.e., subthreshold GnRH concentrations produce either small monophasic Ca2+ transients or irregular, small Ca2+ spikes. With higher GnRH concentrations (0.1–10 nM) regular, oscillatory, frequency-modulated, large Ca2+ transients (baseline Ca2+ spiking) are produced. Eventually (∼50–100 nM GnRH), these Ca2+ spikes fuse into an amplitude-modulated biphasic Ca2+ response  and hormones that alter their synthesis, release, and/or storage can dynamically regulate their output.Dissociated pituitary gonadotrophs respond to increasing doses of GnRH with a stereotyped progression of intracellular Caresponse , 10, 39 response . It is rocytosis  and that release . Further release . Convers release . It rema2+]i is the key signal to trigger regulated exocytosis in neuronal and endocrine tissues. Endocrine cell models used to study the role of Ca2+ in exocytosis include adrenal chromaffin and PC12 cells  proteins as effectors. Syntaxin, SNAP25 (synaptosome-associated protein of 25 kDa in molecular weight), and synaptobrevin  constitute SNARE proteins. Syntaxin and SNAP25  are the plasma membrane proteins to which VAMP couples , before its association to the trans-SNAREs. Finally, synaptotagmin detects the [Ca2+] elevation and provides the extra drive needed to overcome the energy barrier of lipid-to-lipid interaction, allowing membrane fusion (Secretory granules must undergo a well-defined series of events: 1) recruitment, (2) tethering at the plasma membrane, (3) priming, and (4) vesicle fusion with the plasma membrane. Regulated hormone secretion is a Ca recruitms Figure –58. Vesie fusion , this dae fusion , in addie fusion .Along the follicular phase of the estrus cycle, LH secretion is maximal while FSH secretion is reduced; even though gonadotrophs secrete both hormones, the mechanisms underlying this differential release are unclear. FSH appears to be released mostly through the constitutive pathway in accordance to its rate of synthesis. Conversely, LH-containing granules are released through the regulated pathway in response to GnRH, with no effect on LHβ mRNA production . Moreove2+ levels and PKC activation, which as a consequence activated mitogen-activated protein kinase (MAPK) cascade, culminating in an activation of extracellular-signal-regulated kinase (ERK) 1/2, cJun NH2-terminale kinase (JNK), p38 MAPK, and ERK 5 , whose autophosphorylation could be important in transmitting Ca2+ pulse frequency and amplitude signals, as fast and high-amplitude Ca2+ influxes, which results in greater and/or sustained Ca2+/CALM1 levels  mRNAs levels, which reflect changes in gene transcription in pituitary gonadotrophs, are GnRH pulse frequency modulated –67. GnRHnd ERK 5 –80, it i1 levels , 81  exerts a direct action at the pituitary level through its α-receptor –97, incr spiking , 106. Ne spiking , 107–109 spiking , 104 and spiking –112. How spiking –117, alt2+ mobilization and Ca2+ entry to gonadotrophs. In E-primed cells P alters the intracellular Ca2+ signaling patterns produced by GnRH. In the short-term P treatment shifts subthreshold [Ca2+]i responses to oscillatory, and oscillatory to biphasic responses; in contrast, long-term P exposure led to decreased GnRH sensitivity, changing oscillatory response into subthreshold [Ca2+]i response profiles (Progesterone (P) exerts some of its effects at hypothalamic level, decreasing GnRH secretion and pulse frequency , 103 conprofiles , 106.Androgens [testosterone (T) and 5α-dihydrotestosterone (DHT)] are important component of the male gonadal feedback and they act either at the hypothalamic level by regulating the secretion of GnRH into the hypophyseal portal circulation –122, dirAt hypothalamic level, T reduce GnRH synthesis , 124–1272+]i is essential for the transduction of GnRH signal; T but specially DHT regulate GnRH-induced [Ca2+]i variations  show oscillatory responses regardless of the GnRH concentration. Correlated with this results it has been demonstrated that in T or DHT treated cells, there is an inhibition of the GnRH increase in [Ca2+]i; at low GnRH doses (0.1 nM) 30% of gonadotrophs were unable to initiated threshold spiking and in the residual cells the frequency of oscillations decreased, as in controls, androgen treated cells, respond with a spike-plateau type of signal to 1 nM GnRH, but the frequency of spiking was also reduced  bind to activins with high-affinity modulating its actions , 163. AcOne mechanism that contributes to differential FSH and LH production may be related to the observation that different patterns of GnRH pulses produce differential effects on inhibin/activin and follistatin mRNA levels . Estrogein vivo organization. However, as it has been described in this and other works, most of the studies have been done in individual cell activity where this networks and relations are disrupted, due to methodological difficulties, just few recently approaches has been done in the understanding of the endocrine activity in a tissue context.Endocrine cells are organized in three-dimensional networks, which facilitate the coordination of the activity of thousands of individual cells to respond to different regulation factors and achieve hormone output , 165. ThIn this regard, the distribution of gonadotrophs in fixed and live slices at different female reproductive stages has been analyzed . Across 2+ imaging in male mouse acute pituitary slices  under increasing doses of GnRH stimulation exhibited a progression of Ca2+ signaling patterns termed “non-canonical” [i.e., oscillatory responses at a given (GnRH) and transient responses at both lower and higher concentrations as described before in this review; Figure in situ gonadotroph population.The possibility of changes in gonadotrophs activity within its tissue context and physiological conditions was recently addressed using Cay slices , 145. Cecultures , 145. Ho2+ signaling has been reported in dissociated cells  to increasing GnRH were significantly augmented (80% of GnRH responding gonadotrophs) and “canonical” sequences were significantly reduced  for GnRH decreased from 0.17 nM (control) to 0.07 nM after GnX, suggesting an increased GnRH responsiveness of the gonadotroph population  was seen, no matter the size of the cell, they generated intracellular Ca2+ signals smaller than 40 fluorescence arbitrary units (a.u.) poorly correlated with the cell size   increase in gonadotropin secretion that characterizes this condition , 168 somed cells  and thered cells . Neverth reduced    (Figure Functional adaptation of the gonadotrophs in the pituitary gland to different external and internal conditions may involucrate not just alterations in cell number, size, and morphology, as it has been considered for many years, recent methodological techniques allowed us to understand that it is a more complicated process that involucrates different aspects at the cellular physiology level but in coordination with the whole tissue environment.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, specific mechanisms of this regulation and their relative contribution to pacemaker function remain mainly unknown. Our review summarizes available data and existing numerical modeling approaches to understand Ca2+ and CaMKII effects on the SAN. Data interpretation and future directions to address the problem are given within the coupled-clock theory, i.e., a modern view on the cardiac pacemaker cell function generated by a system of sarcolemmal and intracellular proteins.Sinoatrial node (SAN) is the primary heart pacemaker which initiates each heartbeat under normal conditions. Numerous experimental data have demonstrated that Ca Interactions within the system are extremely complex and their detailed investigation requires numerical model simulations. The CaMKII function in pacemaker cells has not been systematically studied using numerical simulations. Our review summarizes major principles of the coupled-clock theory, available data, and existing numerical modeling approaches that are important to delineate future numerical integration and exploration of CaMKII within the pacemaker cell system.Under normal conditions, SAN cells (SANC) generate spontaneous rhythmic action potentials (AP) that initiate the heartbeat. The evolution of thought regarding the cardiac pacemaker cell operation paradigm switched back and forth between intracellular origin  and cel2+-clock ]. A morefindings . The key m) to a cell excitation threshold of ~-40 mV. The coupled-clock theory postulates that the DD is generated by the two coupled oscillators, Ca2+-clock and M-clock, rather than just by M-clock alone .Operation of the coupled-clock system has been explored in recent numerical model studies , d, and + current  and release . Ventricular muscle cells can spontaneously cycle Ca2+ (under conditions of high Ca2+ loading) via global Ca2+ waves via regenerative Ca2+-induced Ca2+ release (CICR) propagating by Ca2+ diffusion . The rhythmic LCRs are generated in the absence of M-clock, e.g., under voltage clamp or in membrane-permeabilized SANC [when [Ca2+] is normal, ~100 nM, review , whose function is enhanced by phosphorylation via basal activity of PKA (2+-activated adenylyl cyclases (ACs) which are highly expressed in SANC  in rabbit SANC . In this special issue Wu and Anderson discuss in detail experimental evidence for contribution of CaMKII activity to SAN function during health and heart disease  and its future numerical modeling.CaMKII indirectly senses [Caactivity .  and RyR. Studies in isolated rabbit SANC suggested that CaMKII regulates the pacemaker activity via modulating I CaL inactivation and reactivation  depresses the basal rate and amplitude of spontaneous APs in SANC of rabbit  and guintivation  and LCR tivation . I f is hibition .2+ transients, CaMKII is ideally suited to respond to changes in SAN rhythm. For example, electrical stimulation alone increases CaMKII-dependent phosphorylation of phospholamban at CaMKII phosphorylation site in a frequency-dependent manner in ventricular myocytes  has been obtained in studies of specific I f inhibitor ivabradine  by testing effects of specific perturbations of either clock in rabbit SANC. The M-clock was specifically perturbed by ivabradine that at low concentrations (<3 μM) specifically inhibits I f, i.e., it does not suppress I CaL . Later activation of diastolic I NCX by the LCRs (and I NCX-linked DD acceleration) leads to a delayed activation of I CaL, i.e., M-clock slowing. Thus, inhibition of the M-clock inhibits (indirectly) Ca2+-clock that further suppresses the M-clock, and so on, until the coupled-clock system attains a new steady-state.Numerical simulations  using a 2+ influx produced by ivabradine not only decreases Ca2+ available for SR pumping, but also likely decreases protein phosphorylation signaling via Ca2+-activated-CaMKII and Ca2+-activated-ACs-cAMP/PKA pathways. This leads to further reductions in the average Ca2+ influx and, therefore, SR Ca2+ loading and AP firing rate. Simultaneously, reduction in cAMP shifts the I f activation curve (effecting further M-clock slowing). If the “biochemical” crosstalk is lacking, model simulations , with the LCR period reporting the resultant complex effect , rate-dependent effects on both clocks, ultimately resulting in mutual entrainment of the clocks (Heart rate reductions produced by ivabradine or HCN4 mutations have been interpreted as a pure result of insufficient e clocks .2+ control theory (2+ release units (CRUs) to fire. While partially periodic LCRs (comprising Ca2+ clocks) in cardiac pacemaker cells are generated by the CRUs, they are, in fact, a product of complex local interactions of proteins residing in both cell membrane and the SR, i.e., RyR, SERCA, LCCh, and NCX. These interactions, in turn, are regulated by PKA and CaMKII signaling .The local Cal theory  remains 2+, CaM, and phosphatase activity , without assuming either a discrete sub-membrane compartment or an inactivated state of the RyR. The model succeeded in reproducing observed propagating local Ca2+ releases and realistic pacemaker rates only when RyR locations were assigned taking into account irregular, hierarchical distribution of RyR clusters  observed in 3D confocal scan sections of immunofluorescence staining. When the RyR sensitivity is very high or the NCX density is low, synchronization is lost, causing sympathetic stimulation to reduce (rather than increase) beating rate, often exhibiting arrhythmias . Thus, the “local” models, predicting the realistic scale of Ca2+ signals within the inhomogeneous signaling network of SANC, seem to be a better choice to explore CaMKII effects in future studies of pacemaker cells.Compared to previous models, lacking local Ca2+ and CaMKII roles in cardiac pacemaker cells. Taking into account emerging importance of local Ca2+ control in cardiac pacemaker cells and also importance of local CaMKII signaling (reported in ventricular myocytes), accurate interpretation of experimental data on CaMKII effects in pacemaker cells will likely require integration of local  with key regulatory molecules , effector molecules , and energy production of the system. This knowledge will contribute greatly to our understanding of cardiac impulse initiation and specific role of CaMKII signaling in the pacemaker regulation.In this review we have summarized the present state of experimental and numerical modeling studies on Caof local  and moleof local  mechanisof local . The newBoth authors contributed to the conception of the work, drafted the paper, approved the version to be published, and are accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N-glycan processing enzymes are arranged along the early secretory pathway, forming an assembly line to facilitate the step-by-step modification of oligosaccharides on glycoproteins. Thus, these enzymes provide excellent tools to study signals and mechanisms, promoting their localization and retention in the endoplasmic reticulum (ER) and Golgi apparatus. Herein, we focused on a detailed investigation of amino acid sequence motifs present in their short cytoplasmic tails in respect to ER export. Using site-directed mutagenesis, we determined that single arginine/lysine residues within the cytoplasmic tail are sufficient to promote rapid Golgi targeting of Golgi-resident N-acetylglucosaminyltransferase I (GnTI) and α-mannosidase II (GMII). Furthermore, we reveal that an intact ER export motif is essential for proper in vivofunction of GnTI. Coexpression studies with Sar1p provided evidence for COPII-dependent transport of GnTI to the Golgi. Our data provide evidence that efficient ER export of Golgi-resident plant N-glycan processing enzymes occurs through a selective mechanism based on recognition of single basic amino acids present in their cytoplasmic tails.Plant  The plant Golgi apparatus consists of numerous separate stacks of cisternae, which are distributed throughout the cytoplasm and often associated with the endoplasmic reticulum (ER) N-glycosylation is an abundant covalent protein modification in all eukaryotic cells. The core oligosaccharide, which is transferred to nascent proteins from a lipid-linked precursor, is extensively modified by removal and addition of sugar residues in the ER and subsequently in the Golgi apparatus  Sar1p, indicating that the export of these glycosyltransferases occurs through the formation of COPII vesicles at ER export sites (ERES) N-glycan processing enzymes is influenced by amino acid sequence motifs present in the cytoplasmic tail. Using a series of deletion mutants, we determined the protein domains required for efficient Golgi retention. Furthermore, using site-directed mutagenesis, we identified amino acid residues required for efficient ER export. For one enzyme, Golgi-resident N-acetylglucosaminyltransferase I (GnTI), the functional significance of proper Golgi targeting was tested by complementation studies in GnTI-deficient Arabidopsis thalianaplants. Coexpression of GnTI with Sar1p forms indicated the presence of COPII-dependent transport to the Golgi. Our data show that despite the unique structural characteristics of the plant ER–Golgi interface, the ER-to-Golgi transport mechanisms of glycosyltransferases seem to be similar in mammals and plants.In this work, we investigated whether the ER export of different Golgi-resident N. tabacumGnTI consisting of a short cytoplasmic tail (11 amino acids), a single transmembrane domain (18 amino acids) and a lumenal stem region (48 amino acids) are sufficient for targeting of a reporter protein to the Golgi apparatus in plants in vivolocalization in more detail, we generated constructs, where the GnTI CTS region was fused to the N-terminal part of monomeric red fluorescent protein (mRFP) and made deletions thereof  of  thereof . These cpparatus . The retpparatus ,4,27  To analyse the effect of complete removal of the lumenal stem region on targeting of GnTI, the cytoplasmic tail and transmembrane domain were fused to mRFP (GnTI-CT-mRFP) and transiently expressed in ructures , which cructures . These dRGYKFCCDFR, basic amino acid residues are shown in bold) contains three basic amino acids, which could promote ER exit, as demonstrated for mammalian glycosylation enzymes RAATS, CAARTS and CAAATS; N. benthamiana. The chimeric protein lacking all three basic amino acids in the cytoplasmic tail (CAAATS) displayed a predominant ER steady-state location with little Golgi labelling (AARTS) was targeted to the Golgi apparatus quite similar to the wild-type form (GnTI-CTS-mRFP) (RAATS) was detected both in the ER and in the Golgi apparatus  , thus repparatus , with a pparatus . ColocalAAATS) was caused by overexpression, we compared the expression levels in extracts 1–3 days after infiltration by immunoblotting using anti-red fluorescent protein (RFP) antibodies. A band of the expected size was clearly visible after 2 and 3 days for GnTI-CTS-mRFP and GnTI-CAAATS-mRFP  of GMII containing only 10 amino acids fused to its transmembrane domain is sufficient for Golgi targeting and retention 10 tail of GMII play a similar role as observed for the positively charged amino acids of GnTI, chimeric proteins consisting of the C10 tail , the basic amino acids were replaced by alanine residues and the proteins transiently expressed in N. benthamianaleaf epidermal cells. GMII-C10AKATS-GFP with one remaining basic amino acid displayed a Golgi distribution like GMII-C10TS-GFP (10AAATS-GFP) resulted in a predominant ER steady-state localization . Near perfect colocalization of GMII-C10T-cyan fluorescent protein (CFP) with ST–mRFP , with alanine residues led to accumulation of a GFP fusion protein in the ER  heavy chain between the GnTI-CTS region and the fluorescent protein , respectively, and different localization when a wild-type form was coexpressed with a mutated one (GnTI-CTS-mRFP/GnTI-CAAATS-GFPglyc or GnTI-CAAATS-mRFP/GnTI-CTS-GFPglyc) regardless of the used fluorescent protein tag (data not shown).To provide further evidence for correct topology of the tail-mutated GnTI protein, we generated a glycoprotein reporter by exchanging mRFP with GFP and inserting a protein fragment with a single  protein . The resal cells . CoexpreN-glycosylation site facing the ER lumen. Thus, transfer of the oligosaccharide precursor can occur, while the opposite orientation with the chimeric glycoprotein reporter in the cytosol would not result in any N-glycosylated protein. Western blot detection of proteins extracted from infiltrated leaves showed a band of expected size  and the mannose-binding lectin concanavalin A (Con A), indicating that both proteins are N-glycosylated and thus have proper orientation. Hence, we exclude that the observed difference in localization of the tail-mutated chimeric GnTI protein is triggered by a failure to acquire a type II membrane topology.Correct topology of the chimeric proteins, which differ only in their cytoplasmic tail, will orient the chimeric protein with the tibodies . ImportaN-glycan profile. The presence of oligo-mannosidic structures (like Man8) attached to the glycoprotein is a hallmark of retention in the ER, while forward movement to the Golgi will lead to processing of N-glycans and the formation of complex N-glycans carrying β1,2-xylose and core α1,3-fucose residues (main glycoform: GnGnXF). The analysis of the purified chimeric GnTI-CTS-GFPglyc proteins by western blot with anti-HRP antibodies and Con A already indicated differences in their content of complex and oligo-mannosidic N-glycans. The signal with anti-HRP antibody was stronger for the wild-type form, while Con A gave the opposite result when equal amounts of protein were analysed  was performed on tryptic peptides of both purified proteins. In the case of GnTI-CTS-GFPglyc, the major peak was found to be the complex N-glycan structure GnGnXF  and the mutated form (GnTI-CAAATS-GFPglyc). The complex-type content of the wild-type form amounted to 88%, whereas the mutant form contained much less (32%)  . The disss (32%) . These dA. thalianaα-glucosidase I (GCSI) N-glycans of this ER-retained glycoreporter (GCSI-CTS-GFPglyc) were exclusively of the oligo-mannosidic type  with GFP fused to the C-terminus  resulted in a weak staining signal in most of the lines, and some lines did not show any staining. These findings were confirmed by matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of total N-glycans isolated from transformed cgl1plants, which showed the presence of complex-type N-glycans for wild-type GnTI, whereas the tail-mutated form produced only oligo-mannosidic structures  is clearly in favour of the first hypothesis. In line with this, the observed failure for ER export could be the result of a loss of interaction between the cytoplasmic tail of the cargo protein and the COPII components. For mammalian glycosyltransferases, direct in vitrointeraction with the small GTPase Sar1p, which initiates COPII coat assembly, has been demonstrated in vitrobinding of the cytoplasmic tails of mammalian glycosylation enzymes to another COPII protein has been reported Accumulation of Golgi-resident glycosylation enzymes with altered cytoplasmic tails in the ER could be caused by exclusion from anterograde transport or increased recruitment from the Golgi by retrograde transport. Our finding of increased amounts of unprocessed oligo-mannosidic N. tabacumGnTI complementary DNA (cDNA) HindIII/SalI-linearized cloning vector puc19 to produce vector puc19Nt1. The mRFP coding sequence was amplified with Turbo Pfu polymerase (Stratagene) from pRSETB-mRFP  using primers mRFP1/2 and cloned into the SalI/BamHI site of puc19Nt1 (puc19Nt1-mRFP). The GnTI-CTS-mRFP coding sequence was excised with XbaI/BamHI and ligated into the binary plant expression vector pPT2 Spromoter. The CT-mRFP and T-mRFP constructs were generated by ligation of overlapping primers NtGnTI-11F/-11R and NtGnTI-12F/-12R, respectively, into XbaI/KpnI-digested vector pPFH3, which is a derivative of pPT2 XbaI/KpnI, and overlapping primers NtC3F/R (GnTI-CRAATS-mRFP), NtC4F/R (GnTI-CAARTS-mRFP) and NtC8F/R (GnTI-CAAATS-mRFP) were inserted.The GnTI CTS region was amplified with primers NtGnTI-1/-2 (for primer sequences see 10TS region was amplified by polymerase chain reaction (PCR) from the A. thalianaGMII cDNA 10AAATS-region was amplified with Ath-MII-41F/-44R and the C10AKATS-region was amplified with Ath-MII-48F/-44R. The C10T region was amplified using primers Ath-MII-40F/-42R and C10AAAT with Ath-MII-41F/-42R. All PCR products were XbaI/BamHI digested and inserted into p20F 10TS, -GMII-C10AAATS, -GMII-C10AKATS and -GMII-C10AAAT) or into p23 10T).To generate the GMII constructs, the CBamHI/BglII digested and ligated into BamHI-linearized vector p20F to create vector p20F-Fc. GnTI-CTS was amplified using primers NtGnTI-13F/-16R and cloned into the XbaI/BamHI site of p20F-Fc. The cytoplasmic-domain-swap vectors were generated by XbaI/KpnI excision of the GnTI-C-tail and insertion of NtC8F/R (GnTI-CAAATS) and XT-C1F/R overlapping primers (XylT-C-GnTI-TS) or the PCR product from A. thalianagenomic DNA using primers GCSI-1/-2 (GCSI-C-GnTI-TS). GCSI-CTS-GFPglyc was generated by PCR amplification from A. thalianagenomic DNA using oligos GCSI-1/-3 and cloned into the XbaI/BamHI site of p20F-Fc. GnTI-C-GCSI-TS-GFPglyc was generated by replacing the GnTI-TS region with a PCR fragment containing the coding sequence for the GCSI TS region obtained by using primers GCSI-4/-3.The glycoprotein–GFP vectors were generated by PCR amplification of a human IgG1 heavy chain fragment from vector pTRAp-2G12-Ds XbaI/BamHI site of p20F. Vector pNT-GnTI-Mut was generated by site-directed mutagenesis of the KpnI site at position 1272 of the tobacco GnTI coding sequence in vector p5/2 AAA-GFP.The full-length GnTI-GFP expression vector was generated by PCR using primers NtGnTI-13F/-15R and vector pNT-GnTI-Mut as template and inserted into the BamHI/SalI-digested vector p20ChisSP (unpublished data), which is derived from pPT2 and contains a chitinase signal peptide.To generate the mRFP-HDEL vector (pmRFP-HDEL), mRFP was amplified using primers mRFP5-F/6-R and ligated into N. benthamianaplants were used for Agrobacterium tumefaciens(strain UIA143)-mediated transient expression of indicated constructs using the agroinfiltration technique described previously (600) of the bacterial suspensions used for plant transformations was 0.03 for all constructs except for ST–GFP/mRFP 0.05, ERD2-GFP 0.08, full-length GnTI-GFP and GnTIAAA-GFP 0.05. Constructs expressed in Sar1p experiments were infiltrated at the following OD600: ST–GFP 0.2, ERD2-GFP 0.2, Sar1-YFP 0.05, untagged Sar1[H74L]p 0.03 and GnTI-CTS-GFP 0.2.Four-week-old to 5-week-old eviously ,51. The min dimethyl sulphoxide) used at a concentration of 50 mmfor 10 min before confocal analysis mageJ and Adobe Photoshop CS.Sections of transformed leaves were analysed 2–4 days postinfiltration (dpi) on a Leica TCS SP2 or Zeiss LSM 510 confocal microscope both equipped with ×63 and ×100 oil immersion objectives using appropriate filters or spectral selections. Images presented in this manuscript were taken at 2 dpi, unless stated otherwise. For ease of imaging motile structures, leaf segments were incubated with n-ethylmaleimide , circularity ‘0–1.0’ default, and show ‘outline’.For quantifying the number of Golgi stacks, 4-μm thick three-dimensional z-stack images (no cropping) were captured with a ×40 oil immersion objective of a Zeiss LSM 510 using identical settings throughout the experiment. Per experiment, eight z-stacks were analysed . In IN. benthamianaleaves as described previously mTris/HCl (pH 7.2) and 0.5 mmdithioerythritol (DTE), and 8.5 μL was incubated in the presence or absence of 1% Triton-X-100 with 0.5 μg trypsin (Sigma) for 60 min at 37°C. To stop the reaction, 20 μg soybean trypsin inhibitor (Sigma) was added and samples were subjected to SDS–PAGE and immunoblotting using rabbit anti-RFP  or mouse anti-GFP (Roche) antibodies for detection.Microsomal fractions were prepared from 500 mg infiltrated N. benthamianaleaves 600 of 0.2. For purification, 800 mg infiltrated leaf material was homogenized in liquid nitrogen, resuspended in 8 mL of precooled extraction buffer  and incubated for 15 min at 4°C. Insoluble material was removed by several centrifugation steps at 5000 × gfor 10 min at 4°C. The clear supernatant was incubated with 40 μL rProtein A–Sepharose , and the fusion proteins were eluted by boiling in Laemmli sample buffer. Protein gel blot analysis of crude extracts or purified proteins was performed using anti-HRP antibodies as described previously Purification of the CTS-GFP fusion proteins  was separated by SDS–PAGE (10%) under reducing conditions, and polypeptides were detected by Coomassie Brilliant Blue staining. The corresponding band was excised from the gel, destained, carbamidomethylated, in-gel trypsin digested and analysed by LC-ESI-MS as described recently To analyse the


Endothelial mPGES-1 exacerbates KA-induced neuronal damage in in vivo experiments. In in vitro experiments, mPGES-1 produces PGE2, which enhances astrocytic Ca2+ levels via the EP3 receptor and increases Ca2+-dependent glutamate release, thus aggravating neuronal injury. This novel endothelium-astrocyte-neuron signaling pathway may be crucial for driving neuronal damage after repetitive seizures and could be a new therapeutic target for epilepsy and other brain disorders.Neurons interact closely with astrocytes via glutamate; this neuron-glia circuit may play a pivotal role in synaptic transmission. On the other hand, astrocytes contact vascular endothelial cells with their end-feet. It is becoming obvious that non-neuronal cells play a critical role in regulating the neuronal activity in the brain. We find that kainic acid (KA) administration induces the expression of microsomal prostaglandin E synthase-1 (mPGES-1) in venous endothelial cells and the prostaglandin E In the brain, PGE2 is also involved in pathological processes, such as fever, seizure and cerebral ischemia i) levels directly through the PGE2 receptor. Moreover, astrocytes can modulate synaptic transmission through the release of glutamate i levels and, subsequently, evoking sustained glutamate release, ultimately leading to neuronal damage. To investigate the mechanisms of the neuronal damage while maintaining the intercellular associations among endothelial cells, astrocytes and neurons, we used hippocampal slice culture prepared from wild-type (WT) and mPGES-1−/− mice.Brain endothelial cells are surrounded by astrocytic end-feet , suggesttrocytes ; therefolutamate –30, whiclutamate . Taken t2 concentration in the slices. This increase in PGE2 was not observed in the mPGES-1−/− slices (2+]i and astrocytes in the slices with Fluo-4 (Ca2+ indicator) and sulforhodamine101 , respectively  i levels, particularly around the CA3 stratum radiatum, and also increased the number of Fluo-4 labeled astrocytes i levels. Moreover, we confirmed whether exogenous PGE2 increases the [Ca2+]i levels in cultured neurons, because a [Ca2+]i increase in neurons might cause neuronal damage. We found that PGE2 could raise the [Ca2+]i levels in neurons co-cultured with astrocytes, but not without astrocytes (unpublished data). In addition, the [Ca2+]i increase in neurons was found to follow the [Ca2+]i increase in astrocytes (unpublished data). These results suggest that PGE2 indirectly increases the neuronal [Ca2+]i levels via the astrocytic [Ca2+]i increase and subsequent glutamate release. Finally, we tested whether this PGE2-evoked glutamate release from astrocytes occurs in a Ca2+-dependent manner.To validate the above findings on the endogenous PGEtrocytes . Moreove1 region . These r2+ chelator, BAPTA-AM, was applied to the slices. BAPTA-AM diminished the increase in the [Ca2+]i levels in the astrocytes in the WT slice and abolished the increase in glutamate concentration [2+-dependent glutamate release from neighboring astrocytes [A membrane-permeable Cantration . Moreovetrocytes .2 produced by endothelial mPGES-1 activates the astrocytic EP3 receptor to elevate the [Ca2+]i levels in astrocytes, causing Ca2+-dependent glutamate release and leading to neuronal injury , PGE2 may require another factor, such as a concomitant activation of astrocytic EP3, to elevate [Ca2+]i levels in astrocytes after KA treatment. Furthermore, it has been shown that PGE2 barely increases [Ca2+]i levels in neurons (unpublished data) and cannot evoke neuronal current directly i levels in astrocytes, which may in turn release glutamate and feedback to extrasynaptic NMDAR NR2B , , N-methyDAR NR2B . Thus, t2 to astrocytes, which would in turn affect neurons. The interaction among neurons, astrocytes and endothelial cells may be a key for investigating the processes of neuropathological disorders.The results described here indicate that brain endothelial cells are not merely a physiological barrier between the blood and brain, but may also act as a signal transducer or amplifier. In particular, endothelial cells may be active under pathological conditions, such as in epileptic seizure. In response to such insults, endothelial cells would continuously supply a large amount of PGE2 is synthesized by inducible mPGES-1 in cooperation with COX-2 in vascular endothelial cells in response to KA treatment. Moreover, endothelial PGE2 activates astrocytic the EP3 receptor to elevate [Ca2+]i levels in astrocytes, causing Ca2+-dependent glutamate release and, subsequently, stimulating neuronal damage. This is a first clarification of a mechanism for neuronal damage regulated by endothelial cells; therefore, this review emphasizes that brain endothelial cells act as a signal transducer or amplifier, especially, under pathological conditions, such as epileptic seizure. The analysis of the interactions among neurons, astrocytes and endothelial cells provides a better understanding of the processes of neuropathological disorders and will facilitate the development of new treatments.We find that PGE


Saccharomyces cerevisiae to reveal that meiotic DSB formation is modulated by the Mec1(ATR) branch of the DNA damage signalling cascade, promoting DSB formation when Spo11-mediated catalysis is compromised. Activation of the positive feedback pathway correlates with the formation of single-stranded DNA (ssDNA) recombination intermediates and activation of the downstream kinase, Mek1. We show that the requirement for checkpoint activation can be rescued by prolonging meiotic prophase by deleting the NDT80 transcription factor, and that even transient prophase arrest caused by Ndt80 depletion is sufficient to restore meiotic spore viability in checkpoint mutants. Our observations are unexpected given recent reports that the complementary kinase pathway Tel1(ATM) acts to inhibit DSB formation. We propose that such antagonistic regulation of DSB formation by Mec1 and Tel1 creates a regulatory mechanism, where the absolute frequency of DSBs is maintained at a level optimal for genetic exchange and efficient chromosome segregation.During meiosis, formation and repair of programmed DNA double-strand breaks (DSBs) create genetic exchange between homologous chromosomes—a process that is critical for reductional meiotic chromosome segregation and the production of genetically diverse sexually reproducing populations. Meiotic DSB formation is a complex process, requiring numerous proteins, of which Spo11 is the evolutionarily conserved catalytic subunit. Precisely how Spo11 and its accessory proteins function or are regulated is unclear. Here, we use  Halving the chromosome copy number occurs during meiosis—a specialized pair of sequential nuclear divisions that first segregates homologous chromosomes, then sister chromatids. In many organisms, accurate segregation of homologous chromosomes during meiosis I requires the formation and repair of numerous DNA double-strand breaks (DSBs) distributed across the genome. DSB repair uses homologous recombination to drive interaction and genetic exchange between homologous chromosomes, which facilitates accurate chromosome disjunction at anaphase I. Much of our detailed understanding of the mechanisms of meiotic recombination comes from studies performed in the budding yeast revisiae .S. cerevisiae, meiotic DSB formation requires the function of 10 proteins, of which Spo11 is the catalytic subunit. The precise function of the nine accessory proteins is unclear, as are the detailed mechanisms that govern the frequency and distribution of Spo11–DSB formation across the genome. Spo11 shares similarity to a type-II topoisomerase (Top6A) from archaea and initiates DSB formation via a transesterification reaction that coordinately severs the DNA backbone on both strands, leaving Spo11 monomers covalently attached to the 5′ ends of the DSB (reviewed in [In iewed in ).Spo11–DSBs are processed nucleolytically by the Mre11, Sae2 and Exo1 proteins, creating DSBs with extended 3′-ending single-stranded termini , which aIn wild-type cells, the ssDNA ends are used in a homology search by the eukaryotic RecA orthologues, Rad51 and Dmc1 ,23, creaRemoving the function of checkpoint proteins causes entry into the meiotic divisions of mutants that accumulate unrepaired DSBs, leading to cell death . InteresRecently, it has been shown that mutation of another PIKK kinase, ATM, results in increased DSB formation in mammals and in flies ,46, and 3.3.1.a [dmc1Δ [dmc1Δ strains .In each budding yeast meiotic cell, accurate reductional segregation of homologous chromosomes at the first nuclear division involves the formation and repair of approximately 160 Spo11–DSBs ,49. The figure 1a ,48). Spoa . The absa )—a statea [dmc1Δ . During cumulate ), such aSPO11-HA3-His6 allele (henceforth referred to as spo11-HA) to enrich for Spo11-oligo complexes. This C-terminal epitope tag is reported to moderately reduce the catalytic activity of Spo11 . BecausHIS4::LEU2 reporter locus on chromosome III [HIS4::LEU2, there are two prominent DSB sites flanking the LEU2 insertion (a). In the untagged strain (SPO11+), DSB signals peaked at approximately 6% of the total lane signal (c), with more of the DSBs arising at site 1 (d). Contrary to our expectation, total DSB signals were not reduced by the Spo11-HA tag (c). However, the distribution was altered such that now a similar proportion of DSBs was detected at site 1 and site 2 .Figuree). In both the tagged and untagged Spo11 strains, DSBs accumulated to a similar extent, reaching approximately 15% and approximately 18% of total lane signal, respectively (f). As before, the distribution of events was altered by the HA-tag, such that the fractions of DSBs forming at site 1 and site 2 were now similar (d). A second strong hotspot region, ARE1, showed the same trend of relatively unchanged DSB frequency when the spo11-HA allele was present .Accurately measuring DSB abundance from a culture of wild-type cells is complicated by overlapping kinetics of DSB formation and repair. To work around this issue, we remeasured the DSB frequency in strains similar to those we had used for the Spo11-oligo analysis, where the Dmc1 repair protein was mutated, causing DSBs to accumulate e. In botectively f. As bef similar d. A secorad24Δ strain (b–f). Reduced MEC1 checkpoint activity (as caused by mutations such as rad24Δ) results in rapid exonucleolytic hyper-resection of the DSB ends creating extensive 3′-ending ssDNA tails that cause DSB molecules to migrate much more rapidly and heterogeneously when separated by agarose electrophoresis [HIS4::LEU2, despite the signal arising in rad24Δ being more diffuse, DSBs remained readily detectable above lane background signals, peaking at 9% (c), slightly greater than wild-type cells, possibly due to a slight delay in DSB repair causing DSB signals to transiently accumulate to higher steady-state levels. In rad24Δdmc1Δ cells, despite extensive hyper-resection, DSBs accumulated and persisted similar to RAD24dmc1Δ, reaching a maximum level of approximately 16–20% of the total DNA . Notably, in the rad24Δ and rad24Δdmc1Δ backgrounds, barely any signal was observed at DSB site 2, resulting in a significantly altered ratio compared with RAD24+ cells (d). In addition, we observed increases in the frequency of ectopic recombination products in rad24Δ strains , as has been reported by others [Next, we assessed DSB formation in the checkpoint-defective Δ strain b–f. Reduphoresis . At HIS4ng at 9% c, slightotal DNA e,f. Nota4+ cells d. In addy others .RAD24+ cells, the combination of spo11-HA and rad24Δ resulted in a substantial reduction in DSB signal (b–f). DSBs were reduced to 50% of the rad24Δ level in rad24Δspo11-HA cells , and to less than 25% of the rad24Δdmc1Δ level in the rad24Δdmc1Δspo11-HA strain, where DSB signals were barely detected despite being expected to accumulate . These reductions in DSB abundance correlated with reduced abundance of ectopic recombination products . Similar effects were observed at the ARE1 locus . In rad24Δdmc1Δ cells, despite extensive hyper-resection, DSBs accumulated to levels similar to both the dmc1Δ and dmc1Δspo11-HA controls . By contrast, DSBs were undetectable in the dmc1Δrad24Δspo11-HA strain . This surprising reduction in DSB abundance was reproduced in two independently generated isolates of the same rad24Δdmc1Δspo11-HA genotype, indicating that the phenotype was not due to an underlying defect in the genotype of the strain nor a culture problem (data not shown).In contrast to the situation in B signal b–f. DSBsHA cells b,c, and cumulate e,f. Thesrad24Δspo11-HA strains was a general phenomenon affecting global DSB levels, we assessed total DSB signals in the dmc1Δ background across four chromosomes using pulsed-field gel electrophoresis . We detected an approximately 25–30% reduction in DSBs in the dmc1Δspo11-HA strain relative to dmc1Δ . In rad24Δdmc1Δ, DSBs were reduced by 10–20% relative to dmc1Δ . Finally, in the dmc1Δrad24Δspo11-HA strain, DSB frequency fell to just a third of the dmc1Δ level . Representative comparisons of lane profile plots are presented in the electronic supplementary material (figure S3). Collectively, these results indicate that the reduced abundance of Spo11-oligo complexes we had observed in rad24Δdmc1Δspo11-HA strains  is correlated with a reduced ability to detect DSBs by southern blotting.To determine whether the synergistic reduction in DSB abundance observed in  strains b,c is coS. cerevisiae, as in mammals, meiotic recombination is necessary for accurate chromosome segregation—with errors in this process causing aneuploidy. To determine whether the molecular defects in DSBs and Spo11-oligo abundance correlate with defects in meiotic chromosome segregation, we measured the viability of the haploid spores formed upon completion of meiosis. By itself, the Spo11-HA tag is reported to have very little effect on spore viability [spo11-HA alone causes no major reduction in spore viability (i). By contrast, in the rad24Δ strain, the spo11-HA allele caused spore viability to drop from 30% down to less than 5% (i).In iability ,53. We ciability i. By con than 5% i.3.2.spo11-HA allele, on the other hand, is reported to be a DSB formation hypomorph, variably reducing DSBs by 11–50% [DMC1 and dmc1Δ backgrounds, DSB frequency at two recombination hotspots (HIS4::LEU2 and ARE1), and across three other chromosomes, is reduced less than we expected by spo11-HA; and (ii) DSB frequency, however, is more severely reduced when Rad24 checkpoint activity is abolished in spo11-HA; and spo11-HA dmc1Δ strains.Mutation of the Rad24-dependent DDR was not expected to affect meiotic DSB formation. The y 11–50% . Contrarspo11-HA made use of the rad50S mutant background [rad50S , DSB signals accumulate due to a failure to remove Spo11 from the DSB end [sae2Δ background at HIS4::LEU2 , ARE1  and across the length of four chromosomes . In contrast to our previous results in SAE2+ cells  and now more consistent with the published work [spo11-HA sae2Δ strain compared with sae2Δ . Moreover, removal of checkpoint activity (rad24Δ) in sae2Δ strains had little impact on DSB formation in either SPO11+ or spo11-HA .Previous analysis of DSB abundance in ckground . In rad5 DSB end ,52. We rS4::LEU2 j,k, ARE1omosomes g,h. In ched work , we obsespo11-HA is deficient in DSB formation (as observed in sae2Δ), such a defect is substantially corrected in wild-type and dmc1Δ cells by a process that is dependent on the activity of the Rad24 (Mec1/ATR) checkpoint. We propose that these observations reveal the presence of a DDR-dependent positive feedback mechanism that is activated by ssDNA and promotes DSB formation under conditions of suboptimal catalysis (i.e. the spo11-HA hypomorph). Consistent with this idea, we note that the final frequency of DSBs that accumulates in rad24Δdmc1Δspo11-HA cells (as assessed by PFGE) is very similar to that observed in sae2Δspo11-HA , which, because of the absence of ssDNA intermediates, we expect will also lack efficient Rad24/Mec1 activation [Taken together, these observations suggest that while spo11-HA g,h, whitivation .mec1 cultures, we used an allele of MEC1 where expression is specifically repressed during meiosis . DSBs accumulated to high levels in both rad17Δdmc1Δ and pCLB2-MEC1 dmc1Δ . By contrast, in spo11-HA derivatives, DSB levels were reduced to approximately 30% of the SPO11+ levels, as were ectopic recombination products , largely mimicking the synergistic DSB defects we observed with rad24Δ (rad24Δspo11-HA strains (i), loss of the MEC1 or RAD17 activity in the spo11-HA background also caused a defect in spore viability . Overall, the effects conferred by the loss of Rad17 were less severe than Rad24 or Mec1, suggesting that there may be previously uncharacterized differences between the roles of these DDR components. Nevertheless, we tentatively conclude that the proposed positive feedback mechanism uses all major components of the canonical ssDNA-dependent DDR pathway.To determine whether the proposed feedback mechanism was a general feature of the DDR, we repeated experiments in strains mutated for components of either the checkpoint clamp (Rad17) or the Mec1(ATR) kinase itself . Owing th rad24Δ . Further strains i, loss o3.3.spo11-HA allele), we investigated whether other Spo11 hypomorphs also benefit from Rad24/Mec1 checkpoint activation. We selected a weak hypomorphic allele of SPO11, spo11-D290A, which contains a point mutation in the putative Toprim domain thought to aid coordination of a metal ion necessary for DSB catalysis [spo11-D290A (untagged) allele to be a good candidate for testing the requirement for checkpoint activity.The precise reason for the hypomorphic behaviour of the Spo11-HA protein is unknown. To determine whether the positive feedback mechanism we have uncovered is a general feature of meiotic recombination regulation . In the dmc1Δ background, the spo11-D290A allele resulted in a 30% reduction in DSB formation, and a dramatic redistribution of events away from DSB 1 and towards DSB 2 . These effects were slightly more pronounced than the defects caused by spo11-HA (b–f), but nevertheless, in the DMC1+ background, spo11-D290A supported almost wild-type levels of spore viability (e). In the sae2Δ background, spo11-D290A caused a more severe 50% reduction in DSB signal at HIS4::LEU2 compared with SPO11sae2Δ , and the same redistribution of events towards DSB 2 (d).We first measured DSB formation at the U2 locus a–d. In tds DSB 2 a,b,d. Thspo11-HA b–f, but iability e. In the11sae2Δ a,c, and ds DSB 2 d.Figuref,g). We detected no reduction in DSB formation in the dmc1Δspo11-D290A background compared with dmc1Δ . By contrast, the spo11-D290A allele reduced DSB formation in the sae2Δ background by approximately 40% , similar to what we had observed for spo11-HA (h). Loss of rad24Δ checkpoint activity caused DSB levels at HIS4::LEU2 to drop below our detection limit in the spo11-D290A dmc1Δ background , and a 60–70% reduction in DSBs relative to the dmc1Δ control, as measured by PFGE , down to the same frequency of DSBs observed in sae2Δspo11-D290A. Finally, we asked whether spore survival in the spo11-D290A strain required Rad24/Mec1 activity (figure 4e). Consistent with our observations made with the spo11-HA allele, spore viability in rad24Δspo11-D290A fell to just 2.7%.We next estimated DSB formation globally using PFGE and three chromosome probes f,g. We dspo11-HA h. Loss oCollectively, these results support the view that activation of the Rad24/Mec1 checkpoint by ssDNA intermediates acts as a general mechanism to promote DSB formation when the efficiency of Spo11–DSB formation is compromised.3.4.a,b). In wild-type and spo11-HA cultures, slower migrating phosphorylated forms of Mek1 appear coincidently with the time of DSB recombination intermediates ). Phosphorylated Mek1 was slightly delayed in rad24Δ cells, suggesting a partial requirement for Rad24 to achieve Mek1 activation (figure 5a(ii)). Strikingly, phosphorylation of Mek1 was completely abolished in the rad24Δspo11-HA strain (figure 5a(ii)).Activation of the DDR kinases (Mec1 and Tel1 in budding yeast) causes phosphorylation of Hop1 and subsequent activation of Mek1 , a meiotfigure 5DMC1+ cells, we repeated the analysis in the dmc1Δ background, where DSB recombination intermediates and Mek1 signals would usually be expected to persist (b). In dmc1Δ and dmc1Δspo11-HA, there was a steady accumulation in the abundance of multiple-shifted species of Mek1, indicating varying levels of phosphorylation (b). In the rad24Δdmc1Δ strain, much of this hyper-phosphorylation was lost, confirming that hyper-activation of Mek1 is Rad24-dependent (b). In the rad24Δspo11-HA dmc1Δ strain, all residual Mek1 phosphorylation was abolished (b), and not visible even when overexposing the film (figure 5b(ii)). Similar loss of Mek1 phosphorylation was observed in pCLB2-MEC1 dmc1Δspo11-HA strains (data not shown). Thus, activated Mek1 correlates strongly with the ability to accumulate appreciable levels of meiotic DSBs.Because Mek1 phosphorylation signals are transient in  persist b. In dmcrylation b. In theependent b. In thebolished b, and no3.5.spo11-HA and rad24Δ, could be explained by the observed failure to fully activate Mek1, and thus to appropriately control repair partner choice, resulting in increased occurrence of Rad51-dependent inter-sister recombination. To test this idea, we repeated our DSB analyses in rad51Δdmc1Δ strains, thereby preventing all recombination-mediated pathways of DSB repair . In the rad24Δrad51Δdmc1Δspo11-HA strain, despite having removed the potential for both Rad51 and Dmc1-dependent repair, accumulated DSB signals were barely increased relative to rad24Δdmc1Δspo11-HA, remaining approximately threefold lower than the SPO11+  control. Analysis of DSB formation across four chromosomes revealed a similar relationship . Specifically, we detected no increase in DSB abundance in rad24Δdmc1Δrad51Δspo11-HA relative to rad24Δdmc1Δspo11-HA . We conclude that the loss of DSB signal observed in rad24Δspo11-HA strains is not primarily due to changes in repair partner choice.During meiosis, DSB repair reactions take place preferentially between homologous chromosomes rather than between sister chromatids ,55. The B repair . As expee signal a,b. In te SPO11+ a,b contrtionship c,d. Spec3.6.e). This delay is thought to be caused by errors in recombination. We noted that the spo11-HA allele alleviated the meiotic delay observed in the rad24Δ strain (e), consistent with the observed synergistic reduction in DSB signal alleviating recombination problems. However, it was possible that the reduced DSB signal in rad24Δspo11-HA and rad24Δdmc1Δspo11-HA cells compared with rad24ΔSPO11+ cells was itself due to the more rapid cell cycle progression beyond prophase I that arises in the rad24Δspo11-HA double mutant compared with rad24ΔSPO11+ (e).DDR mutants initiate the first meiotic nuclear division about 1 h later than wild-type cells  figure e. This dΔ strain e, consisΔSPO11+ e.ndt80Δrad24Δrad51Δdmc1Δspo11-HA background, where all DSB signals should accumulate, and where the cells will be forced to arrest at late prophase I in a checkpoint-independent manner owing to deletion of the NDT80 gene. To our surprise, the ndt80Δ-enforced prophase arrest was sufficient to restore DSB accumulation at HIS4::LEU2 to the otherwise DSB-deficient rad24Δrad51Δdmc1Δspo11-HA strain (figure 6a(ii)).Exit from prophase I into anaphase I is controlled by expression of the Ndt80 transcription factor ,57. To dndt80Δ-enforced arrest was not unique to HIS4::LEU2, we measured DSB formation across four chromosomes using PFGE . Consistent with our results at HIS4::LEU2, the ndt80Δ arrest caused DSB signals to return to levels indistinguishable from the rad24Δdmc1Δrad51Δ control . Taken together, our results suggest that the apparent positive feedback on meiotic DSB formation is mediated by Mec1-dependent inhibition of Ndt80.To confirm that the restoration of DSB formation by ing PFGE d,f. Cons control d,f. Take3.7.rad24Δspo11-HA and rad24Δspo11-D290A strains is due to precocious Ndt80 expression and/or activation causing more rapid passage through meiotic prophase, which we suppose prevents efficient DSB formation by the hypomorphic Spo11-HA and Spo11-D290A proteins.Our results suggest that the synergistic reduction in DSB signal observed in NDT80 transcription factor under the control of the GAL1-10 promoter, where expression can be induced by addition of oestradiol to the growth media [NDT80 gene (data not shown). By contrast, samples induced with oestradiol after 8 h in meiosis sporulated efficiently and permitted us to assess the viability of the haploid spores  to checkpoint-defective Spo11 hypomorphs. To test this idea, we placed the th media . As exped spores . Dramatiprophase . IntriguΔ strain . This su3.8.rad24Δ strains is directly due to Ndt80 expression or instead to the onset of anaphase I, which ensues as a result of the Ndt80-dependent transcriptional cascade, we asked whether arresting cells at the metaphase–anaphase transition is sufficient to restore DSB formation in NDT80+rad24Δdmc1Δspo11-HA cells. The budding yeast Polo-like kinase, Cdc5, regulates late prophase events including Holliday junction resolution and the dissociation of Rec8 cohesin from meiotic chromosomes to enable their segregation [CDC5 is an essential gene, we took advantage of a pCLB2-CDC5 allele developed previously, which, similar to our pCLB2-MEC1 allele, is not expressed during meiosis [To determine whether the DSB defects in regation ,60. Beca meiosis ,60.HIS4::LEU2 . In rad24Δdmc1ΔpCLB2-CDC5 cells, DSB frequencies rose to 25% of total DNA at late time points , a modest increase relative to CDC5+ that may be due to prolonged prophase I arrest. Cdc5 depletion also had almost no effect on DSB accumulation in the rad24Δdmc1Δspo11-HA background, with measured signals barely rising above general lane background . Our observation that DSB formation is restored by NDT80 deletion (Depletion of Cdc5 caused cells to arrest in prophase (data not shown), yet had little effect on measured DSB frequency at deletion , but not4.4.1.Meiotic DSB formation is regulated at numerous levels by relatively poorly understood mechanisms. Although Spo11 is the evolutionarily conserved catalytic component, its activity depends on interactions made between itself and the accessory DSB complex proteins, and with the chromosomal substrate. Spo11 activity is modulated temporally by the local timing of DNA replication  and by ca).Here, by using an allele of Spo11, which is hypomorphic for DSB catalysis, we additionally reveal that DSB formation is modulated in a positive manner by activation of the DNA damage checkpoint machinery Mec1(ATR). Recent work in mice, flies and yeast –47, and figure 8a,b): the rate of Spo11–DSB formation is initially increased concomitantly with CDK/DDK-dependent phosphorylation of Mer2 [spo11-HA and spo11-D290A strains, DSB accumulation is initially slower, but that activation of the Rad24/Mec1(ATR) kinase by ssDNA formation at emergent DSBs causes transient inhibition of Ndt80, which enables the rate of DSB formation to increase until it reaches a plateau similar to that observed in wild-type cells. Therefore, activation of this positive feedback results in the similar efficiency of DSB formation that we observe in SPO11+ relative to spo11-HA or spo11-D290A strains. By contrast, in the absence of the Mec1 checkpoint pathway, precocious Ndt80 activity results in a residual low rate of DSB formation in the hypomorphic spo11 strains. Our model is consistent with the increased Ndt80 transcription observed in the dmc1Δrad17Δ checkpoint mutant relative to dmc1Δ [sae2Δspo11-HA and sae2Δspo11-D290A strains, which, owing to an inability to produce ssDNA, may also exhibit precocious Ndt80 activation.Our model contains a number of features a,b: the  of Mer2 –64, but to dmc1Δ . We suggHIS4::LEU2 hotspot for Tel1 in S. cerevisiae [TEL1 increases the rate and/or prolongs the activity of the Spo11–DSB machinery .Overall, our results are surprising given the proposed negative role of ATM in DSB formation in mouse and flies ,46, and revisiae . We assuIt is worth noting that we have revealed the requirement of the Mec1 checkpoint response in situations where Spo11 activity is downregulated due to defined genetic modification. Although we are unable to exclude that the mechanism we describe reflects a unique consequence of this mutant situation, we interpret our results to indicate that the transient prophase delay caused by Mec1 activation will also promote meiotic DSB formation in other suboptimal circumstances. For example, mutation in a gene other than Spo11, defects in chromosome pairing and/or synapsis or perhaps slower Spo11–DSB formation for an environmental reason (see below).4.2.Mec1 and Tel1 are members of the PIKK-family, and preferentially phosphorylate substrate serine and threonine residues that precede a glutamine (SQ/TQ sites ). One ofcis- and/or trans-inhibition, is similar to that discussed by Zhang et al. [The spatial and temporal distributions of meiotic DSB formation may also be modulated by meiotic chromosome morphogenesis. For example, chromosome axis-associated proteins involved in Spo11–DSB formation are excluded from synapsed chromosomes ,72, suggg et al. . We wish4.3.HIS4::LEU2 DSB site 1 hotspot arose fortuitously via the ectopic insertion of a short bacterial DNA sequence during integration of the LEU2 marker downstream of the HIS4 gene [spo11-HA and spo11-D290A, have such a severe effect on DSB site 1, but not at site 2 nor at DSB formation generally across the genome.The IS4 gene . DSB sitIS4 gene . The preIS4 gene . Given ta), yet is in the top 3% of sites ranked according to the frequency of mapped Spo11-oligonucleotides —with Spo11-oligo hits about 10 times greater than at other similarly narrow sites. Together, this makes DSB site 1 have the second highest density of mapped Spo11-oligonucleotides per bp , and a significant outlier relative to hotspots of similar width . We speculate that these unique characteristics render DSB site 1 particularly sensitive to reductions in Spo11's catalytic activity. By contrast, DSB site 2 maps to the promoter of the inserted LEU2 gene, and probably behaves in a way more similar to canonical DSB sites and provides a buffer for defects in DSB formation at site 1.DSB site 1 is particularly narrow relative to its heat . When compared to all other mapped DSB hotspots , DSB sitrad24Δ caused an increase in the frequency of DSBs at site 1 . Changes in either parameter can alter measured gene conversion frequencies, and chromosomal estimates of DSB formation were made in a rad50S background, where, like sae2Δ, Spo11–DSBs accumulate without ssDNA resection [During meiosis, only a portion of DSBs repair as interhomologue crossover events—the prerequisites for chiasma formation and the accurate segregation of homologous chromosomes. In budding yeast, about half of total DSB events resolve as a crossover ,75, whereostasis . Howeveresection .rad50S/sae2Δ when compared with resection-proficient wild-type or dmc1Δ strains due to the activation of Mec1 substantially improving DSB formation. Assuming our observations are an accurate reflection of DSB formation in repair-proficient (DMC1+) meiosis, then the strength of crossover homeostasis may need to be reviewed. Alternatively, it is possible that the substantial increases in DSB formation we report for dmc1Δspo11-HA and dmc1Δspo11-D290A compared with sae2Δspo11-HA and sae2Δspo11-D290A are themselves overestimates due to checkpoint hyper-activation stimulating DSB formation in a manner that would not occur in repair-proficient cells. Developing accurate and quantitative measures of genome-wide recombination is needed to resolve such conundrums.Our work here suggests that the measured frequency of DSB formation obtained in such hypomorphic Spo11 strains may be substantially different in 4.5.The initiation of meiotic recombination by DSB formation is a potentially catastrophic event for the maintenance of genome stability: any unrepaired DSB can result in the loss of genetic information upon nuclear division. In this context, it makes sense for the DSB programme to be tightly temporally regulated: DSBs should form only in meiosis, and only after local DNA replication has occurred. DSB formation and repair must also occur efficiently across the genome prior to the onset of anaphase. We suggest that the positive and negative action of the meiotic checkpoint helps to make this system efficient and robust under what may often be suboptimal conditions. At this point, we note that budding yeast did not evolve to enter meiosis under the controlled laboratory conditions in which most experiments are performed. In the wild, variation in genotype and in the local environment may cause substantial deficiencies in these processes. Indeed, effects on recombination caused by polymorphism, temperature and nutritional deficiencies have all been reported ,77–79. I5.5.1.Saccharomyces cerevisiae strains  are isogenic to the SK1 subtype and were generated using standard genetic techniques. The base strain genotype is ho::LYS2, lys2, ura3, arg4-nsp, leu2::hisG, his4X::LEU2, nuc1::LEU2. Spo11 protein is tagged with the HA3-His6::kanMX4 epitope [spo11-D290A::kanMX4 allele is a single copy point mutation integrated without epitope tag at the natural Spo11 locus [dmc1Δ::LEU2, dmc1Δ::hphMX, sae2Δ::kanMX6, mek1Δ::URA3, rad51Δ::hisG-URA3-hisG, rad17Δ::natMX, rad24Δ::hphMX and ndt80Δ::hphMX are all full replacements of the ORF with the selection marker. In the pCLB2-MEC1 and pCLB2-CDC5 alleles, the natural MEC1 or CDC5 promoter is replaced with the CLB2 promoter, whose activity is downregulated upon meiotic entry  were diluted 100-fold into YPA (1% yeast extract/2% peptone/1% K-acetate) and grown vigorously for 14 h at 30°C. Cells were collected by centrifugation, washed once in water, resuspended in an equal volume of prewarmed 2% K-acetate containing diluted amino acid supplements and shaken vigorously at 30°C.  epitope . The spo11 locus . dmc1Δ::igure S4 ,60,80,81pression .5.2.HIS4:LEU2, genomic DNA was digested with PstI, fractionated on 0.7% agarose in 1× tris-acetate-EDTA (TAE) for approximately 18 h at room temperature, transferred to nylon membrane under denaturing conditions then hybridized with a probe that recognizes the MXR2 locus. For DSBs at ARE1, genomic DNA was digested with BglII, fractionated and transferred as above, and then hybridized with a probe that recognizes the RSC6 locus. To measure the total DSB formation along whole chromosomes, chromosomal-length DNA was prepared after first immobilizing cells in agarose plugs as described [–1; switch angle 120°; figures 2g and 6c, ramped switch time of 15–22 s over 25 h, then constant switch time of 45 s for 4 h; figures 4f and 6f, ramped switch time of 20–50 s over 28 h. After transfer to nylon membrane under denaturing conditions, genomic DNA was sequentially hybridized with DNA probes that localize close to the left telomere of four chromosomes. Probes used were CHA1 (Chr III), RMD6 (Chr V), CBP2 (Chr VIII) and JEN1 (Chr XI). Between each hybridization, radiolabelled probes were removed by sequential washes in 0.4 M NaOH (2 × 5 min), 2× SSC (2 × 5 min) and six rinses in distilled water, and then air-dried on Whatman paper for 1 h and stored at 4°C. Radioactive signals were collected on phosphor screens, scanned with a Fuji FLA5100 and quantified using ImageGauge software (FujiFilm). DSBs at HIS4::LEU2 are reported as a percentage of the total lane signal after background subtraction. For analysis of PFGE experiments, DSBs occurring far from the probed chromosome end will be underrepresented whenever they arise on a chromosome with additional DSBs closer to the probe. To correct for this, the measured frequency of DSBs  was estimated using the following standard formula: corrected DSB fraction = –ln(1 – measured DSB fraction). This correction assumes that DSBs at different loci on the same chromosome occur independently of one another.DSB signals were detected using standard techniques by indirect end-labelling of specific genomic loci after fractionation and transfer to nylon membranes . For DSBescribed . Chromosrad24Δdmc1Δ (two experiments). Where the exact same sample has been analysed on different gels, the average measure for that sample is used before calculating the average and standard deviation of the experimental repeats. For PFGE data, DSB frequencies for all strains are expressed relative to the average frequency of DSBs forming in dmc1Δ strain.Values plotted with standard deviation bars are the mean of at least two independent time-course experiments. For PFGE experiments, all data are the average of at least three independent time courses of each strain except for 5.3.−1 DAPI. Cells were scored for the presence of one, two or four nuclei and percentage at each time point calculated.Cells were fixed in 100% methanol and aliquots mixed with 1 µg ml5.4.−1 in a 150 mM sodium phosphate buffer at 37°C for 10 min. Dissected spores were incubated for 2 days at 30°C on YPD and scored for percentage viability per strain and viable spores per tetrad. For the pGAL-NDT80 experiments, synchronized cultures were split after 8 h in 2% K-acetate, and one fraction induced to sporulate by addition of beta-oestradiol to a final concentration of 2 µM. Cultures were then incubated for a further 40 h at 30°C prior to dissection. Where shown, error bars are 95% confidence limits.Fresh diploid colonies were incubated in 2% K-acetate liquid for 48 h at 30°C, and then incubated with zymolyase 100 T at a final concentration of 10 µg ml5.5.32P dCTP using Terminal deoxynucleotidyl transferase (Fermentas) and fractionated on a 7.5% SDS-PAGE gel. Following transfer to polyvinylidene difluoride (PVDF) membrane, radioactive signals were collected on phosphor screens, scanned with a Fuji FLA5100 and quantified using ImageGauge software.Spo11-oligonucleotide complexes were detected by immunoprecipitation and end-labelling following established methods ,50. Brie5.6.2 for 3 h at 100 V. Gels were soaked in 1× CAPS transfer buffer (10 mM CAPS/10% methanol) containing 1 mM EDTA for 10 min and then transferred to PVDF membrane in 1 × CAPS for 1 h at constant 0.6 A. Membrane was probed with anti-Mek1  and 20 µM MnCl-Segundo ), follow