Recent evidence indicates that long-term cultures of rat hippocampal neurons, resembling aged neurons, undergo cell death after treatment with Aβo, whereas short-term cultures, resembling young neurons, do not. These in vitro changes are associated with the remodeling of intracellular Ca2+ homeostasis with aging, thus providing a simplistic model for investigating Ca2+ remodeling in aging. In vitro aged neurons show increased resting cytosolic Ca2+ concentration, enhanced Ca2+ store content, and Ca2+ release from the endoplasmic reticulum (ER). Ca2+ transfer from the endoplasmic reticulum (ER) to mitochondria is also enhanced. Aged neurons also show decreased store-operated Ca2+ entry (SOCE), a Ca2+ entry pathway related to memory storage. At the molecular level, in vitro remodeling is associated with changes in the expression of Ca2+ channels resembling in vivo aging, including changes in N-methyl-D-aspartate NMDA receptor and inositol 1,4,5-trisphosphate (IP3) receptor isoforms, increased expression of the mitochondrial calcium uniporter (MCU), and decreased expression of Orai1/Stim1, the molecular players involved in SOCE. Additionally, Aβo treatment exacerbates most of the changes observed in aged neurons and enhances susceptibility to cell death. Conversely, the solely effect of Aβo in young neurons is to increase ER–mitochondria colocalization and enhance Ca2+ transfer from ER to mitochondria without inducing neuronal damage. We propose that cultured rat hippocampal neurons may be a useful model to investigate Ca2+ remodeling in aging and in age-related neurodegenerative disorders.Aging is often associated with a cognitive decline and a susceptibility to neuronal damage. It is also the most important risk factor for neurodegenerative disorders, particularly Alzheimer’s disease (AD). AD is related to an excess of neurotoxic oligomers of amyloid β peptide (Aβo); however, the molecular mechanisms are still highly controversial. Intracellular Ca Consistently, NMDA receptor (NMDARs) antagonists as, for instance, Memantine, partially inhibit NMDARs and the massive Ca2+ influx linked to NMDA receptor activation, thus protecting neurons against damage. In fact, Memantine is one of the few drugs approved for neurodegenerative diseases treatment, such as AD. Neuronal damage linked to intracellular Ca2+ homeostasis has been also related to neuroinflammation due to pathogen-associated molecular patterns and endogenous ligands known as damage-associated molecular patterns released upon cell damage and necrosis. Consistently, several non-steroidal anti-inflammatory drugs (NSAIDs) may help preventing the cognitive decline associated with aging mit). Consistently, inhibiting the driving force for mitochondrial Ca2+ uptake (loss of ∆Ψm) with low concentrations of mitochondrial uncouplers, such as FCCP, or several NSAIDs acting as mild mitochondrial uncouplers, prevented NMDA-induced mitochondrial Ca2+ uptake and neuron cell death [2+ channel remodeling, at least in vitro. This remodeling depends on changes in the expression of NMDAR subunits, enhanced Ca2+ entry and mitochondrial Ca2+ overload. In addition, inhibiting this mitochondrial Ca2+ overload protects from neuronal cell death. Interestingly, this mechanism may explain the anti-inflammatory-independent mechanism of neuroprotection afforded by low concentrations of NSAIDs [Accordingly, enhanced Ca neurons . Howeverll death . Taking f NSAIDs . 2+ entry into the cytosol and mitochondrial Ca2+ uptake in rat cerebellar granule cells and hippocampal neurons leading to mitochondrial Ca2+ overload and apoptosis [2+ entry and mitochondrial Ca2+ overload in primary cultures of both rat cerebellar neurons and hippocampal neurons [AD is the neurodegenerative disorder most commonly associated with age, which implies neuronal cell death and progressive cognitive impairment. Even though it is one of the most prevalent dementias, the direct cause of AD is still unknown. It has been proposed that soluble low molecular weight aggregates (oligomers) of the amyloid β peptide (Aβo) are the main neurotoxin in AD. Aβo promote Capoptosis . These epoptosis . Howeverpoptosis ,27. Neve neurons ,27. 2+-permeable pores or non-specific ion channels in the plasma membrane [2+-leak channel [2+ release through the leak channels, leading to an increase of Ca2+ in the ER and increasing the vulnerability to neurodegeneration [2+ overload in genetic cases of AD. Aβ itself has been hypothesized to form Camembrane ,29. On t channel , indepenneration . Therefo2+ concentration in young neurons, but started to increase cytosolic Ca2+ at 8 DIV, and the effects increased further in aged neurons at >13 DIV. These effects were observed only in neurons but not in glial cells extracted from rat hippocampi, as demonstrated by simultaneous Ca2+ imaging and immunofluorescence in the same individual cells. Moreover, Aβo promoted no rise in mitochondrial Ca2+ concentration in short-term cultures of rat hippocampal neurons. However, Aβo induced a small rise in mitochondrial Ca2+ at 8 DIV, and the effects increased rather dramatically in aged neurons at >13 DIV [Interestingly, Aβo had no effect on apoptosis in short-term cultures of rat hippocampal neurons, but started to promote cell death at 8 DIV and, after longer culture periods (>13 DIV), oligomers caused apoptosis in more than 40% of the neurons as assessed by fluorescence imaging . Consist >13 DIV . In addi2+ uptake using low concentrations of mitochondrial uncouplers or several NSAIDs prevented apoptosis induced by Aβo without having any effect on cytosolic Ca2+ [2+ overload rather than to their anti-inflammatory activity. However, it must be emphasized that neuroprotection is only achieved at low NSAID concentrations that depolarize mitochondria only partially. At large concentrations, NSAIDs collapse mitochondrial potential and induce apoptosis even in the absence of Aβo [2+ as a key player in the Aβo-induced neurotoxicity. In addition, they provide strong evidence that NSAIDs may protect against Aβo toxicity and perhaps AD by preventing the associated mitochondrial Ca2+ overload. Interestingly, inhibition of mitochondrial Calic Ca2+ . The effe of Aβo . These dUnfortunately, latest trials with NSAIDs aimed at preventing or treating AD have given rather pessimistic results. In the last trial carried out in cognitively intact individuals at risk (INTREPAD), the sustained treatment with naproxen sodium increased the frequency of adverse health effects but did not reduce apparent progression of pre-symptomatic AD . Further2+ signaling is also involved in this regard. Specifically, toll-like receptors (TLRs) are transmembrane pattern-recognition receptors of the innate immune system that recognize several pathogen-derived and tissue damage-related ligands. It has been proposed that TLR signaling may contribute to age-related neurodegenerative diseases, including AD, suggesting a possible interplay between inflammation and Aβo in AD [2+ and promotes apoptosis in long-term cultures of rat hippocampal neurons but not in short-term cultured neurons [2+ and neuronal apoptosis induced by LPS were prevented by the TLR4 antagonist CAY10614. Interestingly, hippocampal neurons express TLR4 receptors and the expression level increases with time in culture consistently with the rise in TLR4 in aging brains [2+ rises and apoptosis induced by LPS. These results suggest that in vitro aging increases the susceptibility of rat hippocampal neurons to LPS-induced damage, and these effects seem to be related to changes in the TLR4 expression [AD has also been related to neuroinflammation. The AD brain shows increased levels of pro-inflammatory factors, such as pro-inflammatory cytokines, complement components, and proteases, which are recognized by different receptors in glial cells and neurons. Neuroinflammation comprises a set of cellular and molecular responses, and recent evidence suggests that Caβo in AD . Consist neurons . Both, tg brains . Furtherpression . Altogether, these data suggest that rat hippocampal neurons in long-term culture may be a good model to investigate age-related changes in susceptibility to neurotoxins related to excitotoxicity, AD, and neuroinflammation. 2+ signaling. Therefore, changes in intracellular Ca2+ homeostasis (Ca2+ remodeling) could be involved in the aging process. These changes have recently been explored in the model of a long-term culture of rat hippocampal neurons. Data suggest that long-term cultured neurons resume many of the characteristics of aged neurons, at least regarding susceptibility to neuron damage and changes in expression of receptors targeted by neurotoxins related to age-dependent neurodegenerative processes and Ca2+ oscillations after a few days in vitro, when neurons express mature NMDARs [2+ concentration relative to the short-term cultures instead [2+ oscillations. Intriguingly, in aged neurons, Aβo treatment resumes synchronic oscillations [2+ hyperactivity has also been shown in in vivo mouse models of cerebral amyloidosis [One of the characteristics of cultured hippocampal neurons is that they connect each other forming neural networks. This is evidenced by their ability to display spontaneous and synchronous intracellular Cae NMDARs ,37. Thes instead . Chronicllations . Ca2+ hyloidosis . The fun2+ entry pathway activated after Ca2+ release from the ER induced by physiological agonists such as acetylcholine or glutamate activating metabotropic receptors. SOCE is involved in many different physiological functions in most cell types [2+ sensor, the stromal interacting molecular 1 (STIM1), with a plasma membrane channel, Orai1. Other isoforms of these proteins, including STIM2, Orai2, and Orai3, can also be involved. Rat hippocampal neurons display SOCE and express both Orai1 and STIM1, the classic molecular players involved in SOCE. Interestingly, the amplitude of SOCE in rat hippocampal neurons decreases significantly with culture time [2+ induced by extracellular Ca2+ addition to cultured neurons pre-treated with thapsigargin (commonly used to deplete intracellular Ca2+ stores) is easily recorded in short-term cultures of rat hippocampal neurons, and decreases significantly in long-term cultures [2+ homeostasis which is harmful for neurons. If this is confirmed, then the reversal of Ca2+ remodeling could be an efficient novel approach for AD prevention. SOCE is an important Call types . In neurll types . At the ure time . Specificultures . Loss ofcultures . Accordi2+ uptake is critically involved in neuronal apoptosis. Due to the low Ca2+-affinity of the MCU, mitochondria take up Ca2+ from microdomains, which exhibit high Ca2+ concentrations and are typically formed at the inner mouth of plasma membrane Ca2+ channels [2+ domains formed nearby IP3 receptor channels in the ER, particularly those located at the interface between the ER and mitochondria, at the mitochondria-associated membranes, the so-called MAMs [2+ content of intracellular Ca2+ stores, the extent of Ca2+ release though the IP3 receptors, and the resulting Ca2+ uptake by mitochondria are key parameters of intracellular Ca2+ homeostasis in neurons. Recent data indicate that in vitro aging is associated with changes in several parameters consistently with an age-related remodeling of intracellular Ca2+ homeostasis [2+ store content increases with time in culture. This has been shown by the size of the rise in cytosolic Ca2+ induced by low concentrations of the Ca2+ ionophore ionomycin that release Ca2+ from intracellular stores [3 synthesis and the ensuing activation of IP3 receptors, induce a Ca2+ release from the ER that is significantly enhanced in aged cultures compared to young cultures [2+ release is enhanced in aging neurons. This may be a logic consequence of enhanced resting intracellular Ca2+ levels in aged cells as mentioned above, but this may be also mediated by changes in the expression of IP3 receptors. In fact, immunofluorescence imaging using specific antibodies against different IP3 receptor subunits suggests that the expression of all three IP3 receptor subunits  is enhanced in aged neurons relative to young neurons [2+ release in aged neurons. Interestingly, it has also been reported that Aβo treatment enhances further Ca2+ store content and Ca2+ release induced by acetylcholine or caffeine. Again, these effects are only observed in aged cultures of rat hippocampal neurons but not in young cultures [2+ store content and Ca2+ release from intracellular stores, and these effects are further potentiated by Aβo treatment . The above evidence indicates that, in contrast to young neurons, most of the Ca2+ released by the ER is taken by the surrounding mitochondria in the aged neurons [2+ uptake using bioluminescence imaging of individual neurons transfected with mitochondria-targeted aequorin [2+ release after acetylcholine stimulation induced no or very little mitochondrial Ca2+ uptake in young cultures. On the contrary, it induced large mitochondrial Ca2+ uptake in the aged cultures [2+ transfer from the ER to mitochondria [Specifically, Calvo-Rodriguez et al.  showed n+ uptake . However neurons . This viaequorin . In thiscultures . Therefochondria . 2+ from ER to mitochondria in the aged neurons. The first one has been mentioned above: increased ER Ca2+ content together with enhanced ER Ca2+ release. Changes in expression of acetylcholine, IP3, and ryanodine receptors could contribute to these changes. As stated above, immunofluorescence of cultured neurons suggests upregulation of all three IP3 receptor subunits in long-term cultures [2+ transfer between these two organelles. Several mechanisms could contribute to the enhanced transfer of Cacultures . Second,cultures , which c2+ transfer ER–mitochondria. As stated above, mitochondrial Ca2+ uptake depends on MCU and ∆Ψm. Recent evidence indicates that MCU expression increases with time in culture, suggesting that MCU is upregulated in aged hippocampal neurons [2+ uptake in aged cultures described above. However, since Ca2+ release from the ER is also enhanced in aged neurons, the specific contribution of each mechanism cannot be distinguished. This was analyzed in detail using permeabilized neurons subjected to a clamped cytosolic Ca2+ concentration. In this experimental setting, mitochondrial Ca2+ uptake was paradoxically lower in aged neurons compared to young neurons. This cannot be attributed to a decreased MCU expression. Instead, it could be explained by the partial loss of ∆Ψm reported in aged neurons [2+ transfer mediated by several mechanisms mostly related to enhanced ER Ca2+ release and increased ER–mitochondria colocalization in aged neurons in culture [Mitochondria may also contribute to the aging-induced enhanced Ca neurons . These d neurons . Therefo culture . 2+ transfer from ER to mitochondria in aged neurons? It may favor the activation of Ca2+-dependent dehydrogenases involved in the Krebs cycle, thus supporting ATP synthesis in metabolically compromised aged neurons. However, this improvement of energy production increases the risk of mitochondrial Ca2+ overload and apoptosis. In consequence, the remodeling of intracellular Ca2+ homeostasis observed in aged neurons in vitro may contribute to explain some of the characteristics of brain aging, including cognitive decline and enhanced susceptibility to neuron damage. What are the functional consequences of enhanced Ca2+ transfer from ER to mitochondria has also been investigated in the context of AD, in cultures of rat hippocampal neurons treated with Aβo. Evidence indicates that a 24 h treatment with Aβo increases the ER–mitochondria Ca2+ transfer. This effect is observed in short-term cultures of rat hippocampal neurons [2+, SOCE, Ca2+ store content or ER Ca2+ release in young cultures. Therefore, Aβo-enhanced Ca2+ transfer from ER to mitochondria cannot be attributed to possible effects on Ca2+ store content, Ca2+ release, or changes in expression of MCU since Aβo treatment in young neurons does not influence any of the above mentioned parameters. However, Aβo treatment also increases ER–mitochondria colocalization in young cultures. Therefore, these data imply that Aβo increase ER–mitochondria Ca2+ transfer most likely by promoting the colocalization of these two organelles. In fact, it has been proposed that modulation of the ER–mitochondria interface could be “the” physiological role of Aβo [2+ overload is limited in this scenario by the low Ca2+ store content, the small Ca2+ release, and the low expression levels of IP3 receptors and MCU. Ca neurons . Remarkae of Aβo . This fue of Aβo . NeverthIt is not entirely clear how a peptide such as Aβ could mechanistically affect the intracellular localization of ER–mitochondria and increase their interaction. Area-Gomez et al. first found increased ER–mitochondrial contacts in AD patient fibroblasts and PS-mutant cells . Additio2+ store content, large Ca2+ release after stimulation, and increased expression of IP3 receptors and MCU. Surprisingly, in aged neurons treated with Aβo, Ca2+ transfer from ERto mitochondria is rather abolished [2+ overload, and enhanced ER–mitochondrial coupling may favor failure of mitochondrial Ca2+ homeostasis and mPTP opening, leading to apoptosis [2+ remodeling with the physiological effect of Aβo increasing ER–mitochondria coupling may be critically involved in the catastrophic consequences of neuronal damage in AD. Some of the evidences reviewed here should be confirmed in in vivo aging. However, the preparation of long-term cultures of rat hippocampal neurons as a model of aging [2+ fluxes, particularly at the ER–mitochondria interface, should be considered further for fighting cognitive decline and neurodegenerative disorders in the elderly. Aβo effects on apoptosis may be quite different in the aging scenario. Aged neurons do display enhanced Cabolished . This efbolished . Howeverpoptosis . Furtherof aging ,38 is wo2+ homeostasis that prevent spine stability and increase the susceptibility to cell death induced by different insults. These changes include loss of store-operated Ca2+ channels involved in spine stability and enhanced Ca2+ functional coupling between the ER and mitochondria. At the functional level, Ca2+ remodeling favors energy production at the expense of increased susceptibility to apoptosis induced by mitochondrial Ca2+ overload. Treatment of cultured cells with Aβo enhances this coupling in young neurons. However, in aged neurons, enhanced coupling induced by oligomers disrupts mitochondrial Ca2+ homeostasis and mitochondrial Ca2+ buffer capacity. Further research is required to ascertain whether these changes may contribute to a cognitive decline and enhanced susceptibility to cell death in aged neurons in vivo. Aging is a highly complex, physiological process in which cell, organ, and whole organism dysfunctions are prone to accumulate. In spite of its complexity, some of the characteristics of healthy aging are observed in cells cultured for long periods of time. Evidence indicates that this may happen in long-term isolated rat hippocampal neurons in primary culture. First, cells acquire characteristics of differentiated neurons, express high levels of neurotransmitter receptors, and connect to other neurons to form neural networks with spontaneous synchronic activity. After more than two weeks in culture, neurons start displaying many characteristics of senescent or aged cells, and the expression of aging markers increase. These phenotypic changes are associated with changes in intracellular Ca


Ca2+ release from the ER mainly occurs through two classes of Ca2+ channels, inositol 1,4,5-trisphosphate receptors (InsP3Rs) and ryanodine receptors (RyRs). Calsequestrin (CASQ) and calreticulin (CR) are the most abundant Ca2+-binding proteins allowing ER Ca2+ storage. The hippocampus is one of the brain regions expressing CASQ, but its role in neuronal activity, plasticity, and the learning processes is poorly investigated. Here, we used knockout mice lacking both CASQ type-1 and type-2 isoforms (double (d)CASQ-null mice) to: a) evaluate in adulthood the neuronal electrophysiological properties and synaptic plasticity in the hippocampal Cornu Ammonis 1 (CA1) field and b) study the performance of knockout mice in spatial learning tasks. The ablation of CASQ increased the CA1 neuron excitability and improved the long-term potentiation (LTP) maintenance. Consistently, (d)CASQ-null mice performed significantly better than controls in the Morris Water Maze task, needing a shorter time to develop a spatial preference for the goal. The Ca2+ handling analysis in CA1 pyramidal cells showed a decrement of Ca2+ transient amplitude in (d)CASQ-null mouse neurons, which is consistent with a decrease in afterhyperpolarization improving LTP. Altogether, our findings suggest that CASQ deletion affects activity-dependent ER Ca2+ release, thus facilitating synaptic plasticity and spatial learning in post-natal development.Experimental evidence highlights the involvement of the endoplasmic reticulum (ER)-mediated Ca Throughout the training sessions, the (d)CASQ-null mice did not show any motor impairment; in particular, the ability to swim and the resistance in swimming seemed not to be affected by the ablation of CASQ.Considering the enhancement in synaptic plasticity and its role in the memory process, spatial learning was evaluated by using the paradigm of Morris Water Maze, a hippocampus-dependent learning test commonly adopted to assess long-term memory . Before In both groups of mice, as an effect of the learning process, the escape latency became progressively shorter, showing an asymptotic trend A. This f2+ dynamics are affected by a lack of CASQ; (iii) LTP in the Schaffer collateral-CA1 pathway is facilitated in mice lacking CASQ; and, finally, (iv) hippocampus-dependent spatial learning is improved in mutant mice.The present work aimed to gain insight into the role of CASQ in hippocampal activity. In this regard, although it was demonstrated that CASQ is expressed in the hippocampus , an inve2+ signals at the pre- and postsynaptic levels; in particular, a transient postsynaptic elevation of intracellular Ca2+ concentration is required 0 and during a single action potential Δ[Ca2+]AP were calculated according to Maravall and collegues [In each recorded neuron, the endogenous buffer capacity ; Sigma-Aldrich, Italy), biocytin was detected as previously described [Throughout the whole-cell recordings, except those for Caescribed ,91. A coescribed , whereasescribed .n = 5 for each group) were tested for spatial learning using the paradigm of Morris water maze, as previously described [Adult CTRL and (d)CASQ-null male mice (escribed . A pre-tp ≤ 0.05 and ** p ≤ 0.01.Before the application of parametric statistics, each data distribution was explored using the Shapiro–Wilk test to assess the normality of the distributions. The data were expressed as mean ± SEM. Differences between the experimental groups were statistically evaluated using GraphPad Prism 8 Software. A series of unpaired Student’s t-tests and two-way ANOVA tests followed by Sidak post-hoc tests were appropriately applied to interpret the results. The significant difference was established at *


Evidence shows stretch-dependent Ca2+ influx during the SFR occurs through some mechanism that is continuously active for several minutes following stretch. Many of the candidate ion channels are located primarily in the t-tubules, which are consequently lost in heart disease. Our aim, therefore, was to investigate the impact of t-tubule loss on the SFR in non-failing cardiac trabeculae in which expression of the different Ca2+ handling proteins was not altered by any disease process. For comparison, we also investigated the effect of formamide detubulation of trabeculae on β-adrenergic activation (1 μM isoproterenol), since this is another key regulator of cardiac force. Measurement of intracellular calcium ([Ca2+]i) and isometric stress were made in RV trabeculae from rat hearts before, during and after formamide treatment (1.5 M for 5 min), which on washout seals the surface sarcolemmal t-tubule openings. Results showed detubulation slowed the time course of Ca2+ transients and twitch force, with time-to-peak, maximum rate-of-rise, and relaxation prolonged in trabeculae at optimal length (Lo). Formamide treatment also prevented development of the SFR following a step change in length from 90 to 100% Lo, and blunted the response to β-adrenergic activation (1 μM isoproterenol).Ventricular muscle has a biphasic response to stretch. There is an immediate increase in force that coincides with the stretch which is followed by a second phase that takes several minutes for force to develop to a new steady state. The initial increase in force is due to changes in myofilament properties, whereas the second, slower component of the stretch response (known as the “slow force response” or SFR) is accompanied by a steady increase in Ca This causes a ∼10-fold increase in cytosolic [Ca2+]i, during the “Ca2+ transient,” which initiates cross-bridge cycling and force production. Many of the ion channels essential to EC coupling and Ca2+ transport are present in greater density in the t-tubule SL, which makes up 64% of the total sarcolemmal area of rat ventricular myocytes  that occur at the Z disc. These invaginations form a complex tubular system that is continuous with the extracellular environment, commonly referred to as the “transverse tubules” (or “t-tubules”). Linkages between t-tubules are also present in the longitudinal axis of myocytes , providimyocytes . These iional SR . This sp2+]i returning to resting (diastolic) levels. Although the main protein responsible for removal of cytosolic Ca2+ is the sarco-endoplasmic reticulum ATPase (SERCA), the Na+-Ca2+ exchanger (NCX) also contributes. NCXs are also concentrated in the myocyte t-tubule SL  to investigate the impact of detubulation on the slow force response to stretch which relies predominantly on transport of ions across the t-tubule SL. We also investigated the effects of detubulation on the β-adrenergic response which, along with stretch, is a key regulator of cardiac contraction. We hypothesized that pharmacological detubulation of trabeculae from healthy hearts would have an impact on both the magnitude of the SFR and on the response to β-adrenergic stimulation since both mechanisms augment force by receptor activation and/or ion transport across the t-tubule SL. However, β-adrenergic receptors are distributed throughout both the surface and t-tubule sarcolemma  to examine the impact of acutely disrupting t-tubules on o) was set by adding CaCl2 from a 1 M stock solution. The dissection and initial mounting solution contained 0.25 mM [Ca2+]o, and 20 mM 2,3-butanedione monoxime (BDM) for cardio-protection . All chemicals were purchased from Sigma  unless stated otherwise.Trabeculae were continuously superfused with a HCOotection . Once mo2+ transients were fitted by a five parameter function as described previously  followed by Sidak’s multiple comparisons test using Prism 7.0c  depending on the experimental protocol. A statistically significant difference was assumed at P < 0.05.Data were acquired using LabVIEW  and analyzed off-line using a custom-written program . Caeviously . All res2+]i and force throughout the whole detubulation process we show a continuous record of simultaneous Ca2+ transients (fura-2 340/380 fluorescence) and their corresponding twitch force from a representative trabecula before, during and after exposure to 1.5 M formamide. 2+]i was increased during formamide exposure (ii), returning to pre-formamide control levels after ∼5 min of formamide washout (iv). Peak force also increased during formamide, with no discernible change in the diastolic force. Comparison of Ca2+ transient and twitch force kinetics before and after formamide treatment are shown in 2+ transients and twitch force in comparison to pre-formamide control (i), with a ∼20 ms delay in reaching peak force post-formamide treatment, as is illustrated by the gray lines on the force-time plot which are superimposed in time for comparison.To demonstrate the effects of formamide on [Ca2+]i was longer during and after formamide treatment, with the time constant of Ca2+ transient decay prolonged during the formamide exposure (ii), but not different to the pre-formamide control after washout. Active stress was decreased during and after formamide, with slower relaxation, although the maximum rate-of-rise of stress increased. An additional line (v) was added to n = 4 trabeculae showing data 30 min after formamide washout. Although the Ca2+ transients in these trabeculae were not different to pre-formamide controls, the delay in time to peak [Ca2+]i and time to 50% relaxation of force continued, suggesting the surface SL t-tubule openings remained sealed for this period of time.2+ transients that result from stretch-dependent Ca2+ influx via transport proteins thought to be located predominantly in the t-tubules. We therefore investigated whether detubulation would eliminate the SFR. Following trabeculae stretch there was an immediate increase in active stress, which was not affected by formamide treatment  suggesting detubulation had no impact on the Frank-Starling mechanism. Active stress increased in Control trabeculae stretched from 90 to 100% Lo showing a SFR after 3 min of 114% . An example SFR from a control trabecula is shown in Stretching cardiac muscle results in a biphasic increase in the force of contraction. There is an immediate increase in the force of contraction that coincides with the stretch, which is followed by a slower developing force increase that takes several minutes to reach a maximum, known as the “slow force response” or SFR. The SFR is accompanied by increasing Ca2+ transients, twitches and phase plots from the same representative trabecula are shown after formamide treatment in 2+ sensitivity in intact trabeculae since the crossbridge cycling is in equilibrium at this point. 2+ transient amplitude data from control and formamide treated trabeculae. There was a small but significant increase in Ca2+ transient amplitude during the SFR in control trabeculae that was not observed following formamide treatment. Consequently for formamide treated trabeculae, the SFR was reduced to 104%  investigate the impact of detubulation on the slow force response to mechanical stretch; and (iii) determine whether detubulation modified the response to ß-adrenergic activation, since the ß1 receptors are not restricted to the t-tubule SL  measure the changes in the time course of Caubule SL .o). Simultaneous recordings of Ca2+ transients and isometric force were made before, during and after 5 min of formamide exposure. During formamide, we observed increased diastolic [Ca2+]i and larger Ca2+ transients in trabeculae, with reduced stress. We also observed a prolonged time to Ca2+ transient peak and a slower time constant of decay .Formamide has been used in the past to detubulate isolated ventricular myocytes e.g., , and to of decay . This is2+]i was decreased following formamide washout, and the time course of both the Ca2+ transients and twitch force were slower. This is consistent with impaired excitation-contraction coupling which has previously been reported after detubulation of isolated myocytes due to reduced L-type Ca2+ current  as contributing to Na+ influx during the SFR , rather than via NHE1. TRPC channels are mechanosensitive, and have been implicated in the Ca2+ and/or Na+ influx during the SFR .M-LW contributed to the conceptualization. AP, SK, and CD contributed to the experimental investigations. M-LW, SK, AP, and CD contributed to the data analysis. M-LW, SK, and AP contributed to the manuscript preparation. M-LW contributed to the funding acquisition and administration.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.


Ca2+ plays an essential role in lens homeostasis, and its dysregulation causes cataracts. Ca2+ associated CaM is a well-established inhibitor of gap junction coupling. Recent studies suggest that elevated intracellular Ca2+ activates Cx hemichannels in lens fiber cells and Cx46 directly interacts with CaM. A Cx46 site mutation (Cx46-G143R), which is associated with congenital Coppock cataracts, shows an increased Cx46-CaM interaction and this interaction is insensitive to Ca2+, given that depletion of Ca2+ reduces the interaction between CaM and wild-type Cx46. Moreover, inhibition of CaM function greatly reduces the hemichannel activity in the Cx46 G143R mutant. These research findings suggest a new regulatory mechanism by which enhanced association of Cx46 with CaM leads to the increase in hemichannel activity and dysregulation may lead to cataract development. In this review, we will first discuss the involvement of Ca2+/CaM in lens homeostasis and pathology, and follow by providing a general overview of Ca2+/CaM in the regulation of Cx46 gap junctions. We discuss the most recent studies concerning the molecular mechanism of Ca2+/CaM in regulating Cx46 hemichannels. Finally, we will offer perspectives of the impacts of Ca2+/CaM and dysregulation on Cx46 channels and vice versa.Connexins are the structural components of gap junctions and hemichannels that mediate the communication and exchange of small molecules between cells, and between the intracellular and extracellular environment, respectively. Connexin (Cx) 46 is predominately expressed in lens fiber cells, where they function in maintaining the homeostasis and transparency of the lens. Cx46 mutations are associated with impairment of channel function, which results in the development of congenital cataracts. Cx46 gap junctions and hemichannels are closely regulated by multiple mechanisms. Key regulators of Cx46 channel function include Ca The lens is an avascular transparent organ suspended between the anterior chamber and the back of the eye. It focuses and transmits light to the retina. Due to the lack of direct blood supply, the lens obtains oxygen and nutrients primarily from the aqueous humor, and vasculature in the iris and retina . Lens anGap junction channels are the pathways through which lens fiber cells communicate with each other and to cells at the lens surface . These c+ and Ca2+ (rich in the extracellular environment) enter the lens circulation from the anterior and posterior poles, then move to the center through relatively high resistance extracellular spaces i) is strictly regulated to avoid the adverse effects of Ca2+ overload. The Ca2+ concentration in human aqueous humor is estimated at 1.34 mM. Human lens fibers [Ca2+]i during resting condition is relatively high, around 9 μM, several times higher compared with lens epithelial cells that are around 80 nM [2+]i across different regions of the lens. Mature lens fibers have higher [Ca2+]i close to 500 nM, and this concentration progressively decreases to roughly half in outer fibers. Ca2+ pumps that are enriched in the equatorial region of the lens drive the flow of water and ions. This microcirculation allows the distribution of nutrients and the removal of metabolic waste in the lens i ranging between 0.7 to 1.5 μM, which is high enough to activate proteases and induce protein aggregation i rise at junctional membranes [2+]i, in the range of ~100 nM to low µM, can effectively regulate gap junction channel gating in salivary gland cells [2+]i to ~110 to ~400 nM. Lowering the [Ca2+]i to ~260 nM by saline containing EGTA results in cells ability to regain dye transfer capacity  is mediated by pH and affects gap junction gating i induces the activation of CaM-dependent kinases (CamK) [2+]i increase activates Cx45 gap junction communication. However, there is no evidence that CaMKII directly phosphorylates Cx45 [2+]i, cytoplasmic acidification, or Ca2+ ionophores are all shown to activate CaM, which interacts with the cytoplasmic loop domain of the Cxs, and leads to the reduction of Cx gap junction channel activity . CaM mutants lacking one or more of the four high-affinity Ca2+-binding sites strongly inhibit the formation of functional Cx32 channels . In the absence of Ca2+, no interaction between the WT IL domain and CaM is observed, whereas the interaction between CaM and the G143R mutant is still detectable. This lack of adaptability to the in situ physiological environment by the mutation might partially be responsible for malfunction of channel activities  and Na+. This change provokes depolarization and increase of [Ca2+]i, leading to CaM activation, which stabilizes Cx46 hemichannels in a leaky conformation [2+ and the activation of multiple kinases [2+ has been not been fully explored. Moreover, the proteins and the chain of events in this opposite regulation remain unknown. The role of CaM as common regulator of gap junction communication and hemichannels activity has not been investigated in detail. The evidence suggests that inhibitors of CaM may provide advantages that could keep the beneficial role of gap junction communication during injury, such as ion buffering [Changes in membrane potential appear to play an essential role in hemichannel activity ,16. Undetathione ,83. Thistathione . The lens fibers . Lens fiTP, NAD+ . Permeabormation . Concomiormation  and Cx46ormation . These eormation . Aberranormation . The stuormation ,88. In b kinases ,87. Howeuffering ,90. Furtuffering ,91.


CEBPA, FLT3, IDH1, and IDH2 have been associated with AML initiation and prognosis, the molecular mechanisms underlying this disease remain unclear. In this study, we used a systemic co-expression analysis method, namely weighted gene co-expression network analysis (WGCNA), to identify new candidate genes associated with adult AML progression and prognosis. We identified around 5,138 differentially expressed genes (DEGs) between AML samples (from The Cancer Genome Atlas database) and normal control samples (from the Genotype-Tissue Expression database). WGCNA identified nine co-expression modules with significant differences based on the DEGs. Among modules, the turquoise and blue ones were the most relevant to AML . GO term and KEGG pathway analyses revealed that pathways that are commonly dysregulated in AML were all enriched in the blue and turquoise modules. A total of 15 hub genes were identified to be crucial for AML progression. PIVOT analysis revealed non-coding RNAs, transcriptional factors, and drugs associated with the hub genes. Finally, survival analysis revealed that one of the hub genes, CEACAM5, was significantly associated with AML prognosis and could serve as a potential target for AML treatment.Acute myeloid leukemia (AML) is a malignancy of hematopoietic stem cells. Although many candidate genes such as  DNMT3A, ASXL1, IDH1, and IDH2 are frequently mutated in patients with AML. These gene mutations are considered to be acquired early in AML. In contrast, mutations in FLT3, RAS, and NPM1 are regarded as secondary events in leukemogenesis. Nevertheless, these findings cannot fully explain why and how AML occurs. Hence, bioinformatic analysis is also used to decipher the molecular mechanisms of leukemogenesis and AML progression, which could provide new therapeutic targets for AML treatment.Acute myeloid leukemia (AML) is a hematopoietic malignancy with high heterogeneity. The incidence of AML is approximately 4.3 per 100,000 per year in the United States according to the Surveillance, Epidemiology, and End Results database. The mortality of patients with AML is still relatively high, despite the application of advanced therapy methods such as intensive chemotherapy, bone marrow transplantation, and targeted therapy. In the past decades, through novel high-throughput sequencing techniques, multiple somatic mutations have been identified as associated with AML initiation or prognosis. For example, Various bio-informatic methods have been used to uncover the mechanisms underlying AML progression or to construct prognosis signatures for patient risk stratification –3. PreviAfter screening and filtration of RNA-seq data in TCGA  and GTEx2 reached 0.9 at this moment of time  and module membership (MM) analyses for genes in the blue and turquoise modules to examine whether they were well correlated with the AML phenotype. As shown in BDP1, RFX7, LARP4, TCEGR1, MPHOSPH9, CCDC18, PDS5A, FANCL, and ICE2 in the turquoise module; and SERPINB7, CEACAM5, MUC2, RHOV, ALDH3A1, and CBLC in the blue module. Visualization of the co-expression network for the turquoise and blue modules are depicted in Hub genes were considered as genes with connectivity > 5 in both blue and turquoise modules. The 15 hub genes that were identified as associated with AML progression in the present study were: In order to obtain a comprehensive understanding of the biological functions of genes in the turquoise and blue modules, we carried out GO and KEGG pathway enrichment analyses. According to GO analysis, genes in the turquoise module were most enriched in DNA replication, chromosome segregation, microtubule cytoskeleton organization, and nuclear division . In contin vitro and in vivo [Transcription factors are a variety of proteins participating in the initiation of transcription. Mutations or functional dysregulation of transcription factors may result in the transformation of hematopoietic precursors into leukemic stem cells. For example, CCAAT/enhancer binding protein alpha (CEBPA) deficiency inhibits the differentiation of myeloid cells both  in vivo . A totalNon-coding RNAs are important post-transcriptional regulators. Their dysregulation has been tightly correlated to AML progression in a post-transcriptional level. For example, transcriptional activation of some long non-coding RNAs, such as GAS6-AS2, has been reported to lead to chemotherapy resistance in AML . In the CEACAM5 was significantly associated with worse OS for patients with AML  as positively correlated with the triple-negative breast cancer subtype using the WGCNA method [WGCNA is a bio-informatic method used to identify clusters of biologically relevant genes associated with a particular disease . It has A method . AccordiA method , chromosA method , and micA method , have beA method , and neuA method , DNA repA method , chemokiBDP1, RFX7, LARP4, TCEGR1, MPHOSPH9, CCDC18, ICE2, SERPINB7, RHOV, and CBLC are understudied in leukemia. It was reported that the deletion of RFX7, encoding for a transcriptional factor, decreases natural killer (NK) cell maintenance and immunity [MPHOSPH9 was reported as a susceptible locus for multiple sclerosis [CDCC18 was reported as a susceptibility gene for familial colorectal cancer. PDS5A is a cell cycle-related gene and precocious dissociation of PDS5A is a translocation partner of MLL in AML [FANCL promoter region was also suggested to be associated with sporadic acute leukemia [MUC2 belongs to the mucin family, is located on 11p15.5, and is associated with childhood AML [CEACAM5 has been widely studied in various kinds of cancers, including breast cancer [Regarding the identified hub genes, the functions of immunity . The dysimmunity . MPHOSPHclerosis . CDCC18 L in AML . FANCL iL in AML . Moreoveleukemia . MUC2 behood AML . CEACAM5t cancer , colorect cancer , 29, pant cancer , gastrict cancer , and cart cancer . It is at cancer .Further, we investigated transcription factors and non-coding RNAs associated with AML. CEBPA mutations , 35 and https://portal.gdc.cancer.gov/) database. The age of the patients ranged from 18 to 88 years, and 45.0% of the patients were females. Control data from whole-blood samples of healthy subjects were obtained from GTEx  database. A total of 755 control samples were included in the present study. For each sample, probe data less than 25% were excluded from the study. After annotating a probe name to each gene symbol according to the annotation files, RNA-seq data from different sources were unified by realigning raw reads, removing degraded samples, and performing batch effect correction to correct non-biological variation. Batch effect correction was performed using the SVAseq R package. AML-related genes in the NCBI GENE and OMIM databases were also downloaded for co-expression network construction.RNA-seq data of 151 samples from adult patients with AML were downloaded from TCGA . GO and KEGG analyses were accordingly applied. Results with P-value < 0.05 were considered as significant terms and pathways, respectively.To get a comprehensive understanding of the function of genes associated with AML, a functional annotation was carried out for the two most relevant modules using Database for Annotation, Visualization, and Integrated Discovery (DAVID)  time and the living status were downloaded from TCGA database. Survival analysis was performed using the Kaplan-Meier method using R Bioconductor. Adjusted P values <0.05 were considered statistically significant.Supplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4


In the immune system, calcium signals play a central role in a variety of cellular functions such as proliferation, differentiation, apoptosis, and numerous gene transcriptions. During an immune response, the engagement of T-cell and B-cell antigen receptors induces a decrease in the intracellular Ca2+ store and then activates store-operated Ca2+ entry (SOCE) to raise the intracellular Ca2+ concentration, which is mediated by the Ca2+ release-activated Ca2+ (CRAC) channels. Recently, identification of the two critical regulators of the CRAC channel, stromal interaction molecule (STIM) and Orai1, has broadened our understanding of the regulatory mechanisms of Ca2+ signaling in lymphocytes. Repetitive or prolonged increase in intracellular Ca2+ is required for the calcineurin-mediated dephosphorylation of the nuclear factor of an activated T cell (NFAT). Recent data indicate that Ca2+-calcineurin-NFAT1 to 4 pathways are dysregulated in autoimmune diseases. Therefore, calcineurin inhibitors, cyclosporine and tacrolimus, have been used for the treatment of such autoimmune diseases as systemic lupus erythematosus and rheumatoid arthritis. Here, we review the role of the Ca2+-calcineurin–NFAT signaling pathway in health and diseases, focusing on the STIM and Orai1, and discuss the deregulated calcium-mediated calcineurin-NFAT pathway in autoimmune diseases.Calcium (Ca However, the cytosolic Ca2+ levels are approximately in the range of 100 nM, which is 10,000 times lower than ECF Ca2+ concentrations .Calcium (Cany cells  Table 12+ is a s2+-permeable channels are involved in the influx of extracellular Ca2+: voltage-operated Ca2+ channels (VOCCs), receptor-operated channels (ROCs), store-operated channels (SOCs) like Ca2+ release-activated Ca2+ (CRAC) channels, and second messenger-operated channels (SMOCs)  is mediated by activated CRAC channels (Various Ca (SMOCs) . VOCCs a to them . SOCs ar to them . SOCs archannels .2+ signaling generated by SOCE and CRAC channels play different roles in immune cells. Short-duration functions occur within minutes. Short-duration types of Ca2+ signal transduction are not related to new gene expression, but to motility and the degranulation of T cells  receptor activation , a Ca2+ sensor protein presented in the ER, which is responsible for CRAC channel activation. The second regulator is Orai1, one subunit of CRAC channels. This review focuses on the roles of Ca2+ and the signal pathway upstream and downstream of Ca2+ flow in the immune cells and its involvement in autoimmunity and immunologic diseases.Signaling between the plasma membrane and ER, or between ER and mitochondria, is required for the regulation of a direct Catransfer . It has 2+ influx to the cells. However, their physiological importance or function in immune cells remains unclear.VOCCs are located in electrically excitable cells, and they are activated by membrane depolarization. Usually, VOCCs function as the major passages of Ca2+ channels are mostly non-selective ion channels. Among them, TRP channels consist of six subfamilies according to the amino acid sequence: the TRPC , TRPV (vanilloid), TRPA (ankyrin), TRPM (melastatin), TRPML (mucolipin), and TRPP (polycystin) groups  groups . The TRP) groups . Since tmembrane . Second,ntration . Third, channels .2+ store depletion  screen , there wx factor , and vesx factor . Howeveran cells . Unlike nd STIM2 . Both STER lumen . Mutatio+ stores , 24, sug2+ stores in ER and lead to the activation of SOCE via Orai1. However, RNAi-mediated knockdown of STIM2 selectively decreases baseline levels of Ca2+ cytosolic and ER Ca2+ concentrations. Oh-Hora et al.  in the ER membrane  abrogates Ca2+ influx  , aspartate (D), or glutamine (Q). The mutant proteins were transduced into SCID T cells, and then SOCE was analyzed . IP3 opens IP3 receptors (IP3R) and allows Ca2+ efflux from the ER Ca2+ reservoir  complexes bind to TCR, protein tyrosine kinases are activated. This binding induces the tyrosine phosphorylation and the activation of phospholipase C, gamma 1 (PLC-γ1) . PLC-γ1 2+ influx increases intracellular Ca2+ concentration, intracellular Ca2+ activates other signaling pathway and transcription factors. The pathway known to date is , and , which phosphorylates several effector proteins by transferring phosphates from ATP to serine and threonine residues on the proteins , and  nuclear  complex . This coproteins . Calcineproteins .2+ concentration increases the calmodulin binding to calcineurin, NFAT translocates to the nucleus  and two isoforms of calcineurin B  , 41. Whe nucleus –42 and t nucleus . Periphe nucleus , 45, sugtivation .2+ signal is crucial to a variety of functions of neutrophils as well as lymphocytes. In neutrophils, Ca2+ signaling starts with the binding of various cell surface receptors, including Fc-gamma receptors (FcγRs), G-protein-coupled receptors (GPCRs), and integrins  critically regulates the differentiation of monocytes/macrophages to osteoclasts, the key immune cells involved in bone resorption and destruction. An increasing body of evidence suggests that the Caructures . The Ca2poptosis . Interesg and Li  also havStim1 and Orai1 silencing inhibits RANKL-induced Ca2+ oscillation in RAW264.7 macrophages , systemic lupus erythematosus (SLE), Sjögren's syndrome (SS), and psoriasis, focusing on dysregulated Ca2+ signaling in autoreactive T cells  DR1 and 4 subtypes, smoking, and infectious agents, are thought to contribute to RA etiology and pathogenesis . Most RA+ T cells are thought to be the principal mediators of RA progression, how CD4+ T cells are induced and activated needs clarification. Sakaguchi et al.  within Orai1 were selected for genotyping. As a result, the SNP rs7135617 has a significant correlation with the risk of RA, indicating that genetic polymorphism of Orai1 contributes to the susceptibility to RA.Since RA has a strong genetic background, there is research into whether it is associated with Can et al.  investig2+ is a key regulator of a variety of transcription factors, control of Ca2+ influx is essential for the activation and function of the adaptive immune response. Liu et al.  expressions, suggesting that calcineurin regulates the catabolic and anabolic activities of chondrocytes  , which is caused by a generalized immune cell dysfunction that promotes B cell hyperactivity. Several studies have demonstrated abnormal signaling through BCR, and this modified signaling results in increased Ca signals –94. It hcontrols . Such in(SHIP-1) .2+ signals via the activation of SHIP and hydrolysis of phosphatidyl-inositol-3,4,5-trisphosphate and also by inhibiting CD19-mediated Ca2+ responses (phospholipase Cγ2 (PLCγ2) can lead to severe spontaneous inflammation and systemic autoimmunity  T cells. These double-negative T cells produce more inflammatory cytokines, including IL-17 and IFN-γ  is an animal model of brain inflammation, where autoreactive T and B cells to neuro-antigens, such as myelin basic protein and myelin oligodendrocyte glycoprotein (MOG), play a major role. EAE is a representative Th1 and Th17 cell-mediated central nervous system (CNS) disease  and has 2+ signal pathway is closely related to the development of EAE. For example, decreased levels of STIM1 protein greatly impair the production of neuro-antigen-specific T cell responses (reduced Th1/Th17 responses), and this results in complete protection from EAE  is controlled (2+-calcineurin–NFAT pathway with cyclosporine A and tacrolimus has been the established treatment options of RA, lupus nephritis, and psoriasis patients who show an insufficient response to methotrexate. Therefore, CRAC channels in addition to the calcineurin–NFAT pathway are crucial to the lymphocyte function and development of autoimmunity, perhaps providing a new therapeutic target to treat human autoimmune diseases.In this review, we have discussed recent advances in understanding the role played by the Cantrolled . MoreoveY-JP, S-AY, MK, and W-UK conceptualized the article, reviewed the literature, and wrote the manuscript.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, the role of EGFR-mediated Ca2+ signaling in acquired drug resistance is not fully understood. Here, we analyzed alterations of intracellular Ca2+ ([Ca2+]i) responses between gefitinib-sensitive NSCLC PC-9 cells and gefitinib-resistant NSCLC PC-9/GR cells, and we found that acute EGF treatment elicited intracellular Ca2+ ([Ca2+]i) oscillations in PC-9 cells but not in PC-9/GR cells. PC-9/GR cells presented a more sustained basal [Ca2+]i level, lower endoplasmic reticulum Ca2+ level, and higher spontaneous extracellular Ca2+ ([Ca2+]e) influx than PC-9 cells. Notably, restricting [Ca2+]e in both cell types induced identical [Ca2+]i oscillations, dependent on phospholipase C and EGFR activation. Consequently, restricting [Ca2+]e in PC-9/GR cells upregulated gefitinib-mediated poly (ADP-ribose) polymerase cleavage, an increase in Bax/Bcl-2 ratio, cytotoxicity, and apoptosis. In addition, nuclear factor of activated T cell (NFAT1) induction in response to EGF was inhibited by gefitinib in PC-9 cells, whereas EGF-mediated NFAT1 induction in PC-9/GR cells was sustained regardless of gefitinib treatment. Restricting [Ca2+]e in PC-9/GR cells significantly reduced EGF-mediated NFAT1 induction. These findings indicate that spontaneous [Ca2+]e influx in NSCLC cells plays a pivotal role in developing acquired drug resistance and suggest that restricting [Ca2+]e may be a potential strategy for modulating drug-sensitivity.Non-small cell lung cancer (NSCLC), one of the leading causes of cancer-related death, has a low 5-year survival rate owing to the inevitable acquired resistance toward antitumor drugs, platinum-based chemotherapy, and targeted therapy. Epidermal growth factor (EGF)-EGF receptor (EGFR) signaling activates downstream events leading to phospholipase C/inositol trisphosphate (IP The incidence of non-small cell lung cancer (NSCLC) is steadily increasing and accounts for 85% of lung cancer subtypes. Owing to its recurrence, NSCLC has a low 5-year survival rate of <15% . MoreoveEGFR, ALK, BRAF, KRAS, HER2, ROS1, RET, and MET, are therapeutic targets for treating NSCLC. TKIs targeting these fundamentally inhibit signaling cascades related to cell proliferation, by which cancer cells frequently show acquired drug resistance owing to gene mutations, including rearrangement, amplification, and point mutation [2+ ([Ca2+]i) signaling pathway in NSCLC, in which EGFR activation sequentially elicits phospholipase Cγ (PLCγ) phosphorylation, inositol trisphosphate (IP3) production, Ca2+ release from the endoplasmic reticulum (ER), and protein kinase C activation. Acute human glioma cell stimulation with EGF evokes intracellular Ca2+ responses as oscillations, which are blocked by EGFR inhibitors e) levels increase the sensitivity of PC-9 cells to afatinib e restriction on gefitinib-sensitive and gefitinib-resistant cells elicited identical [Ca2+]i oscillations, which were associated with the modulation of gefitinib efficacy.In this study, we demonstrated that the EGF-mediated Ca2. To determine the effects of [Ca2+]e restriction, the culture medium was exchanged with RPMI-1640 without CaCl2  and 1 mM CaCl2 was added. Cyclopiazonic acid was purchased from Enzo Life Sciences . The mouse monoclonal antibodies against nuclear factor of activated T cell , Orai1 (cat # sc-377281), Bcl-2 (cat # sc-509), and Bax (cat # sc-20067) were obtained from Santa Cruz Biotechnology . U-compounds (U73122 and U73343) were obtained from Sigma Aldrich . Human EGF, gefitinib, and rabbit polyclonal antibodies against poly (ADP-ribose) polymerase-1 , stromal interaction molecule 1 , sarco/endoplasmic reticulum Ca2+-ATPase 2 , and β-actin (cat #4967) were obtained from Cell Signaling Technology, Inc. .The lung adenocarcinoma cell line, PC-9, and its gefitinib-resistant sub-cell line, PC-9/GR possessing the T790M mutation, were gifted by Dr. Jin Kyung, Rho . Cells w2+]i was measured using a Ca2+-sensitive fluorescence dye, Fura-2/AM (Sigma Aldrich) as described previously i responses between PC-9 and PC-9/GR cells, we performed a ratiometric assay using Fura-2/AM. Acute treatment with 200 ng/mL of EGF induced [Ca2+]i oscillations in PC-9 cells but not in PC-9/GR cells . Cells were treated with cyclopiazonic acid to deplete ER Ca2+. The spontaneous Ca2+ influx (indicated as F1) was greater in PC-9/GR cells than in PC-9 cells, and ER Ca2+ content in PC-9/GR cells was 15% lower than that in PC-9 cells (2+ entry (SOCE). Expression of dimeric Orai1 and STIM1 showed no significant difference between PC-9 and PC-9/GR cells, whereas SERCA2 in PC-9/GR cells was significantly reduced by about 35% compared to PC-9 , an inhibitor of PLC, and gefitinib (0.1 μM) in PC-9 and PC-9/GR cells  and incubated in a culture medium supplemented with or without Ca2+. After 24- and 48-h incubation periods, cytotoxicity and cell viability, respectively, were evaluated using enzymatic assays. We first showed that gefitinib-treated PC-9 cells exhibited a significant increase in cytotoxicity and decrease in cell viability in a dose-dependent manner regardless of [Ca2+]e , followed by treatment with gefitinib, either in the absence or presence of [Ca2+]e. PLC inhibition by U73122 and [Ca2+]e restriction synergistically exacerbated cytotoxicity and cell viability  compared to cells treated with the same dose of gefitinib in the presence of [Ca2+]e  cells were incubated. EGF-mediated NFAT1 induction was significantly reduced by gefitinib (5 μM) in PC-9 cells Click here for additional data file.S1 Raw images(PPTX)Click here for additional data file.


The online version of this article (10.1140/epjp/s13360-020-00613-3) contains supplementary material, which is available to authorized users. Firstke bands . This iske bands . The gapmentclasspt{minimake bands  and in pke bands , 18. WhiU is further increased by confinement, resulting in an enhancement of spin fluctuations [The proximity of SIO to a metal–insulator phase boundary gives rise to anomalous properties such as non-Fermi liquid behavior and enhanced paramagnetism due to a ferromagnetic instability , 20. Sigtuations . This bric order , 24. Theic state –27. HereSIO films were grown by pulsed laser deposition on ous work , 21. X-rous work . MagnetoT) characteristics of three SIO films of different thicknesses are shown in Fig. Resistivity  and the film normal. Figure B applied along z and y  and in the yz-plane (bottom). Interestingly, 4 peaks are observed at yz-plane displays two peaks of larger magnitude, while the peaks heights are equal in the xy-plane.We study the MR of the 6 u.c. film further by varying the angle between the magnetic field (entclass1pt{minimaentclass1pt{minimaM) and current (I), reflecting the difference between transport scattering matrix elements for the M. Owing to the anisotropy of the electronic structure with respect to the magnetization angle, these matrix elements may change when M is rotated [AMR consists of a noncrystalline and a crystalline component, which have very different microscopic origins. The noncrystalline component depends on the angle between the magnetization , the angle between M and I remains constant and the AMR is determined by the varying angle between magnetization and crystal axes. In this way, the crystalline component of the AMR can be isolated.The two contributions to the AMR can be identified by measuring the magnitude of the MR while rotating B in the xy-, yz-, and xz-planes. The measurements are performed at the base temperature of the system (R) is large and its variation with magnetic field is small, we use a Wheatstone bridge circuit to accurately measure the change in resistance  is likely due to a slight misalignment in angle. This indicates that this component [proportional to I and M varies in the yz-plane, whereas it is always xz-plane. Instead, this component depends on the relative angle between B and the film normal and is also present at low fields. It cannot be attributed to classical MR, as this would provide a positive contribution to the MR when B is perpendicular to the plane. Therefore, this MR most likely originates from the anisotropy of quantum corrections, as the negative MR associated with weak localization is largest when B is along z.Figure xy-plane. In the xz- and yz-planes, the peaks at xy-plane is fourfold symmetric, while it shows two large and two small lobes in the xz and yz-planes. The magnitude of the fourfold symmetric component [proportional to B and the crystal axes. We note that this AMR component cannot be measured in polycrystalline samples as the contributions from different crystalline domains average out. The sign and symmetry of this component is not compatible with classical MR or weak (anti)localization. The negative MR is largest when the field is oriented at At higher fields localization. A fourfold symmetric component appears as B is increased, and the critical field corresponds well to the quantum critical point previously reported by Cao et al. [In conclusion, we showed that the low-temperature, negative MR component in SIO thin films is anisotropic with respect to the magnetic field orientation. The twofold symmetric component, present only when the angle between o et al. . We attro et al. –31.Supplementary material 1 (pdf 628 KB)Below is the link to the electronic supplementary material.


SETD2 mutation, which led to its loss of function, was identified in AML. However, the distribution and function of SETD2 mutation in AML remained largely unknown. Herein, we integrated SETD2-mutated AML cases from our center and literature reports, and found that NPM1 mutation was the most common concomitant genetic alteration with SETD2 mutation in AML, with its frequency even higher than MLL rearrangement and AML1-ETO. Though this result indicated the cooperation of SETD2 and NPM1 mutations in leukemogenesis, our functional study showed that SETD2 was required for the proliferation of NPM1-mutated AML cell line OCI-AML3, but not MLL-rearranged AML cell line THP-1, via maintaining its direct target NPM1 expression, which was just opposite to its role of tumor suppressor. Therefore, we speculated that SETD2 possibly had two different faces in distinct subtypes and stages of AML.SETD2 is the only methyltransferase for H3K36me3, and our previous study has firstly demonstrated that it functioned as one tumor suppressor in hematopoiesis. Consistent with it, The online version contains supplementary material available at 10.1186/s40364-020-00243-y. To the editorSETD2 mutation affected AML, in which its distribution remained not fully understood [SETD2 mutation in NPM1-mutated AML.SETD2 has been demonstrated as one tumor suppresser in hematopoiesis , and SET9/L, Hb: 98 g/L, PLT: 48 × 109/L, circulated blast: 80%. BM examination exhibited 67.5% myeblasts with the immunophenotype of CD11b-CD13dim + CD14-CD15dim + CD33 + CD34partial + CD35-CD38dim + CD45 + CD64-CD65dim + CD71 + CD117 + CD123dim + HLA-DR-. Though karyotype was normal and CBF or MLL rearrangements were negative, NPM1, SETD2, NRAS and ETV6 mutations were identified. Therefore, AML with mutated NPM1 was diagnosed. After receiving the operation for co-existed acute appendicitis, she accepted IA regimen as induction therapy, and CR1 was achieved. Subsequently, she received three cycle of medium-dose cytarabine regimen. However, AML relapsed at the 3 months after cessation of chemotherapy, and 72% myeloblasts re-emerged in BM. Due to the early recurrence, she accepted HAA and CLAG regimen successively, and achieved CR2. However, the leukemic clones were not eradicated reflected by persistent above mutations. Therefore, allogeneic semi-compatible HSCT was immediately conducted. As follow-up, CR was still maintained at the 15 months after HSCT , so SETD2R2109X was one driver in cancer. However, SETD2 deficiency was not sufficient to generate AML, so additional hits were required [SETD2 mutation [NPM1 mutation rather than MLL rearrangement or AML1-ETO was the most common co-existed genetic alteration of SETD2 mutation in AML  . Therefore, our results indicated that SETD2 knockdown-mediated OCI-AML3 proliferation inhibition was possibly attributed to NPM1 down-regulation.Loss of SETD2 function accelerated the progression of mutation , while NSETD2 mutation in NPM1-mutated AML remained mysterious. Theoretically, SETD2 and NPM1 mutations probably cooperated in leukemogenesis. However, our results showed that SETD2 was required for the maintenance of OCI-AML3. To our knowledge, two possibilities existed: firstly, SETD2 mutation played different roles in the initiation and maintenance of NPM1-mutated AML; secondly, additional genetic alteration influenced SETD2 function in NPM1-mutated AML. Therefore, further investigations were needed in the furture.The detailed role of Additional file 1: Figure S1. The OS of SETD2- wild type and mutated AML patients from the summary of TCGA, TARGET, and OHSU studies.


MT), which corresponds to the transcriptional activation of mitochondrial chaperones, proteases, and antioxidant enzymes to repair defective mitochondria. Transcription of target UPRMT genes is epigenetically regulated by Histone 3-specific methylation. Age-dependency of this regulation could explain a differential UPRMT activity in early developmental stages or aged organisms. At the same time, precise tuning of mitochondrial stress responses is crucial for maintaining neuronal homeostasis. However, compared to other mitochondrial and stress response programs, the role of UPRMT in neurodegenerative disease is barely understood and studies in this topic are just emerging. In this review, we document the reported evidence characterizing the evolutionarily conserved regulation of the UPRMT and summarize the recent advances in understanding the role of the pathway in neurodegenerative diseases and aging.Aging is the time-dependent functional decline that increases the vulnerability to different forms of stress, constituting the major risk factor for the development of neurodegenerative diseases. Dysfunctional mitochondria significantly contribute to aging phenotypes, accumulating particularly in post-mitotic cells, including neurons. To cope with deleterious effects, mitochondria feature different mechanisms for quality control. One such mechanism is the mitochondrial unfolded protein response (UPR Thus, UPRMT is the transcriptional program that stabilizes mitochondrial homeostasis and reduces misfolded protein amount in the organelle, increasing the mitochondrial response capability to stress stimuli , alteration of mitochondrial dynamics, accumulation of unfolded proteins, deletion of mitochondrial DNA (mtDNA), inhibition of mitochondrial chaperones or proteases, and the increase of reactive oxygen species (ROS) levels , was described as associated with the accumulation of proteins in the mitochondrial intermembrane space . Activation of UPRMT requires the dimethylation of lysine 3 of histone 3 (H3K9), which translates into a compacted and overall silenced chromatin state. At the same time although, other chromatin portions remain loose, favoring the binding of UPRMT regulators such as DVE-1  exhibit a hormetic phenotype as they show increased longevity despite their mild mitochondrial dysfunction  activates UPRMT as a mitigation mechanism. Without atfs-1 dependent UPRMT activation, lifespan decreases, and dopamine neurons degenerate  is caused by decreased dopamine secretion from damaged dopaminergic neurons  is the most common motor neuron disease and its complex etiology is explained by the almost 30 causative genes that have been linked to familial cases  is characterized by key neuropathological hallmarks such as the abnormal accumulation of the amyloid-β (Aβ) peptide (reviewed in Masters et al., MT activation extends mitochondrial function, further characterization of the pathway will provide stronger hints to understand neuronal homeostasis and healthspan extension. So far, it seems that UPRMT activation is partially modulated by the age-dependent methylation levels of Histone 3. As H3K9 is differentially methylated in specific brain regions (Snigdha et al., MT could differ in distinct neuronal types. This fact raises concerns when thinking about therapeutic approaches since systemic inhibition of UPRMT could be beneficial for cell types with a dysregulated activation of UPRMT, but detrimental for another that requires its activation. Therefore, the fine-tuning of UPRMT in different pathogenic contexts will be a crucial consideration for future studies. In the case of PD, AD and ALS, incipient evidence has emerged in the last years highlighting also an over-activation of UPRMT as contributors of the ND pathomechanisms. Future studies on this topic should focus on determining whether known ND causative genes are associated to UPRMT components on an early neurodegenerative stage, or whether UPRMT is only activated on a late, non-reversible stage as a consequence of an overall neuronal decay. Precise pharmacological modulation of the mitochondrial stress response could bring new alternatives to restore compromised neuronal functions, with a prospective increase in the life quality of ND patients and the elderly population.Mitochondrial dysfunction is a hallmark of aging and age-related neurodegenerative diseases (Kennedy et al., FM-C and MS planned, researched, and wrote the manuscript.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.


NPM1 and FLT3 mutational status, our gene expression-based DNA repair score might be used as a biomarker to predict outcomes for patients with CN-AML. DNA repair score has the potential to identify CN-AML patients whose tumor cells are dependent on specific DNA repair pathways to design new targeted therapies.Acute myeloid leukemia (AML) is the second most frequent type of adult leukemias. Cytogenetically normal acute myeloid leukemias represent about 50% of total adult AML, exhibit no chromosomal abnormalities, and present high heterogeneity regarding the clinical outcome. Deregulation of DNA repair mechanisms is involved in the adaptation of cancer cells to replicative stress and resistance to genotoxic agents. We investigate the prognostic value of genes related to the major DNA repair pathways. The data reveals specific patterns of gene expression in CN-AML that have prognostic value. Combined with NPM1 mutations of FLT3 internal tandem duplication (FLT3-ITD), clinical outcomes remain heterogeneous in this subset of AML. Given the role of genomic instability in leukemogenesis, expression analysis of DNA repair genes might be relevant to sharpen prognosis evaluation in CN-AML. A publicly available gene expression profile dataset from two independent cohorts of patients with CN-AML were analyzed (GSE12417). We investigated the prognostic value of 175 genes involved in DNA repair. Among these genes, 23 were associated with a prognostic value. The prognostic information provided by these genes was summed in a DNA repair score, allowing to define a group of patients  with poor median overall survival (OS) of 233 days (95% CI: 184–260). These results were confirmed in two validation cohorts. In multivariate Cox analysis, the DNA repair score, NPM1, and FLT3-ITD mutational status remained independent prognosis factors in CN-AML. Combining these parameters allowed the identification of three risk groups with different clinical outcomes in both training and validation cohorts. Combined with NPM1 and FLT3 mutational status, our GE-based DNA repair score might be used as a biomarker to predict outcomes for patients with CN-AML. DNA repair score has the potential to identify CN-AML patients whose tumor cells are dependent on specific DNA repair pathways to design new therapeutic avenues.Cytogenetically normal acute myeloid leukemias (CN-AML) represent about 50% of total adult AML. Despite the well-known prognosis role of gene mutations such as  NPM1, signal transduction genes (FLT3), or myeloid transcription factor genes  [Acute myeloid leukemia (AML) is the second most frequent type of adult leukemias. When analyzed with conventional cytogenetics, about 40–50% of AML exhibits no chromosomal abnormalities and are defined as “cytogenetically normal AML” (CN-AML) . Recurre, RUNX1) . Based ogenes FLT, or myel, RUNX1) .Even if the study of the mutational landscape by new DNA sequencing technologies demonstrated a low mutation frequency in AML compared to other cancers , genomicFLT3-ITD mutations, occurring in about 20–25% of CN-AML, leads to constitutive activation of FLT3 and therefore confers a growth advantage to leukemic cells. Several studies showed that the level of reactive oxygen species (ROS) was increased in FLT3-ITD mutated AML cells and correlated with high levels of double-strand break (DSB) and lower efficiency of non-homologous end joining (NHEJ) repair pathway [FLT3-ITD in DNA damages and acquired drug resistance of AML cells, such as telomere-related genome instability [NPM1 is the most commonly mutated gene in CN-AML, with more than 50 described mutations. The prognostic significance of these mutations and co-mutations in other genes has been widely studied [NPM1 mutations in AML result in APEX1 abnormal cytoplasmic accumulation, and impaired base excision repair (BER) activity [NPM1-mutated AML.FLT3 and NPM1 have also been shown to play a role in the DNA damage response in AML.  pathway . Other mtability , or paratability . NPM1 is studied . The rol studied . NPM1 is studied ,28. Theractivity , potentiIn the current study, we investigate the prognostic value of genes related to the major DNA repair pathways. The data reveals specific patterns of gene expression in CN-AML that have prognostic value. Therefore, the expression analysis of DNA repair genes might be relevant in the context of CN-AML to sharpen prognosis evaluation of this heterogeneous AML subset.http://repairtoire.genesilico.pl) and review of the literature (https://cran.r-project.org/web/packages/maxstat/index.html), we identified 23 out of the 175 genes whose level of expression had a prognostic value in the two independent cohorts. This approach allowed the selection of DNA repair genes that could provide a selective advantage to CN-AML cells through adaptation to replicative stress and chemoresistance. Among them, a high expression of 19 genes was associated with a poor outcome , homologous recombination repair (HRR), NHEJ, and Fanconi (FANC) pathways—defined using the REPAIRtoire database (terature . Using t outcome . No statwww.depmap.org) [APEX1 (BER), RTEL1 (HRR), and COPS6 (NER) were identified as significant essential AML genes   ,31. Intectively) .Then, we searched to combine the prognostic information of these genes in a GE-based DNA repair risk score. The 23 DNA repair genes associated with a prognostic value included 4 coding genes for the BER pathway, 6 genes for the FANC pathway, 6 genes for the HRR pathway, 3 genes for the MMR pathway, and 8 genes for the NER pathway . Four oun = 20), group III included patients with high NER and HRR risk scores (n = 87), and group II included patients with NER or HRR high-risk score value (n = 55).In Cox multivariate analysis, only HRR and NER scores remained associated with overall survival in the training cohort . Therefop = 0.016 between group I and II; p < 0.001 between group II and III).After a median follow-up of 1176 days (95% CI: 916-NR), the median overall survival (OS) was 293 days (95% CI: 252–461) for the whole training cohort . One-yeap = 0.287), OS was still statistically different between risk groups II and III .We searched to validate these results in an independent cohort of 78 patients. HRR and NER scores computed with training cohort parameters were also prognostic in this validation cohort . The glop = 0.03) for overall survival  with CN-AML ,37,38 ussurvival . Furthersurvival  suggestiThere was no significant difference between subgroups according to age in these three cohorts . AltogetNPM1 mutations and FLT3-ITD  are well-described prognosis factors in CN-AML, we conducted another Cox analysis to determine whether our DNA repair score provides additional prognostic information. Prognostic classification according to NPM1 and FLT3 mutational status was established in both cohort according to actual recommendations [NPM1 mutation were classified as “better outcome”, patients with only FLT3-ITD were classified as “adverse prognosis” and patients with both or none of these mutations were classified as “intermediate prognosis”. Kaplan–Meier survival curves according to NPM1 and FLT3 mutational status are presented in Because NPM1/FLT3 mutation classification remained independently associated with survival , and NPM1 / FLT3 mutational status . The sum of the prognostic information was computed for all patients, splitting the training cohort in five groups. When the Kaplan–Meier analysis did not show a significant OS difference between consecutive groups, we merged the two groups. This approach resulted in three prognostic groups with significant different OS values: group A for patients with 0 or 1 point, group B for patients with 2 points, and group C for patients with 3 or 4 points. , 326 days (95% CI: 127-NR) and 236 days (95% CI: 190–263) respectively for patients in groups A, B, and C. One-year OS was 90.3% (95% CI: 80.5–100) in group A, 49.3% (95% CI: 37.1–65.7) in group B and 24.2% (95% CI: 16.2–36.2) in group C. These results were confirmed in the validation cohort where median OS was not reached (95% CI: 1278-NR), 516 days (95% CI: 308-NR) and 253 days (95% CI: 52–403) for patients respectively in groups A, B, and C. One-year OS was 92.6% (95% CI: 83.2–100) in group A, 54.9% (95% CI: 39.8–75.7) in group B and 26.5% (95% CI: 12.4–55.8) in group C. OS was statistically different between groups A, B, and C in both training and validation cohorts . AltogetNPM1, FLT3, or CEBPA, outcomes in CN-AML remain heterogeneous, underlying the wide diversity of this AML subset. In this study, we developed a GE-based score using data from genes involved in the DNA damage response. Our model succeeded to predict poor outcomes in three independent cohorts of adult patients with CN-AML treated with intensive chemotherapy. Combining DNA repair score with NPM1 and FLT3-ITD mutational status allows us to distinguish three prognostic groups including a low-risk group with a not reached median OS after a median follow-up of more than 3 years in both cohorts, a high-risk group with a median OS of about 8 months in both cohorts, and an intermediate risk-group. Furthermore, the DNA repair score is also a prognostic factor for EFS underlining a potential role of DNA repair pathway deregulation in disease relapse. This model may therefore be used for risk stratification in CN-AML since DNA repair scores could be calculated for new patients using Affymetrix microarray data if the same methodology of normalization is applied. Prospective validation of DNA repair score prognostic value using RNA-seq data will be of interest in future investigations.Despite an improvement in prognosis classification, mostly based on the identification of gene mutations such as RAD51-G135C has been suggested to be correlated with the onset of therapy-related AML by several case-control studies, even if two meta-analyses seem to dismiss the role of this polymorphism in de novo AML onset [Among the GEP-based defined DNA-repair scores built into our study, HRR and NER scores remained independent prognostic factors in CN-AML. HRR pathway is a process involved in DNA double-strand break (DSB) repair, in which complementary sister chromatid is used as a template for an error-free repair of DNA sequence ,40. AmonML onset ,53,54. XML onset ,51,52. OML onset . These dNPM1/FLT3 mutational status remained statistically associated with the outcome in CN-AML. The poor prognosis of FLT3-ITD mutated CN-AML has been demonstrated for a high ITD-to-wild-type allelic ratio (>0.5) [FLT3-ITD would have a worse prognosis compared to the others. Moreover, other prognostic mutations defined in ELN 2017 classification  were not available. A combination of DNA repair scores with the mutational status for these genes may provide a more precise risk classification of patients according to recommendations. Studying the correlation between our DNA repair score and others frequently mutated genes in AML may also be of interest, especially for genes involved in chromatin organization and stability . Therefore, it will be compelling to investigate the link between FLT3-ITD allelic ratio, recurrent CN-AML mutations, and the described GEP-based DNA-repair risk score in a new prospective cohort.Interestingly, when compared using multivariate analysis, the DNA repair score and o (>0.5) . UnfortuAPEX1 (BER), RTEL1 (HRR), and COPS6 (NER) are essential AML genes. Among these genes, COPS6 overexpression is associated with poor outcomes in many solid tumors. Interestingly, COPS6 depletion showed in vivo efficacy against glioblastoma [Intensive chemotherapy for CN-AML patients usually includes cytarabine and anthracyclines (daunorubicine or idarubicine) . Cytarabblastoma , cervicablastoma , or papiblastoma , throughTherefore, inhibiting DNA repair might be a promising strategy to improve the efficacy of genotoxic drugs and overcome drug resistance, according to the principle of “synthetic lethality” ,64. PolyOur data support the potential of DNA repair scores to identify CN-AML patients whose malignant cells are dependent on specific DNA repair pathways to design targeted therapy with ATM, CHEK-1/2, RAD51, or CDK7 inhibitors exploiting the addiction to deregulated DNA repair mechanisms.NPM1 and FLT3 mutational status were kindly provided for each patient by Metzeler et al. [FLT3-ITD allelic ratio and mutational status for other prognostic genes  were not available in these cohorts. All patients were treated with intensive chemotherapy. Another cohort (Verhaak cohort) comprising 181 patients with de novo CN-AML was used for validation. The median age at diagnosis was 46 years in this cohort All the patient’s characteristics have been previously reported [Gene expression microarray data from two independent cohorts of adult patients diagnosed with CN-AML were used. One patient was excluded from each cohort due to a diagnosis of myelodysplastic syndrome. Thus, the first cohort (training set) included 162 patients and the second one  78 patients. At least 20 metaphases were analyzed for each patient to confirm the normal karyotype. At the beginning of treatment, the median age was 57.5 years in the training cohort and 62 years in the validation cohort. Pretreatment clinical characteristics of patients have been described previously . At diagr et al. . FLT3-ITreported ,37,38.http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE12417 and GSE14468. They were performed using Affymetrix HG-U133 A&B arrays for training cohort and Affymetrix HG-U133 Plus 2.0 arrays for the validation and Verhaak sets. Normalization of microarray data was performed using the variance stabilizing normalization algorithm, and probe set signals calculated by the median polish method [Affymetrix gene expression data are publicly available via the online Gene Expression Omnibus  and review of the literature .DNA repair gene list was defined using the REPAIRtoire database  and SPSS Statistics version 23.0.0.0 for Mac .The DNA repair score may be useful to identify high-risk CN-AML patients and define the best DNA repair inhibitor to use in combination with conventional treatment to improve patients’ outcomes. The DNA repair score could also be valuable for adapting targeted treatment according to the drug resistance mechanisms selected during the clonal evolution of relapsing AML. These advances may improve the survival of CN-AML patients, and limit the side effects of treatment, improving compliance with dosing regimens and overall quality of life.


These properties make ASX a promising therapeutic agent against pathological conditions that entail oxidative and Ca2+ dysregulation. Here, we studied whether ASX protects neurons from N-methyl-D-aspartate (NMDA)-induced excitotoxicity, a noxious process which decreases cellular viability, alters gene expression and promotes excessive mROS production. Incubation of the neuronal cell line SH-SY5Y with NMDA decreased cellular viability and increased mitochondrial superoxide production; pre-incubation with ASX prevented these effects. Additionally, incubation of SH-SY5Y cells with ASX effectively reduced the basal mROS production and prevented hydrogen peroxide-induced cell death. In primary hippocampal neurons, transfected with a genetically encoded cytoplasmic Ca2+ sensor, ASX also prevented the increase in intracellular Ca2+ concentration induced by NMDA. We suggest that, by preventing the noxious mROS and Ca2+ increases that occur under excitotoxic conditions, ASX could be useful as a therapeutic agent in neurodegenerative pathologies that involve alterations in Ca2+ homeostasis and ROS generation.Astaxanthin (ASX) is a carotenoid pigment with strong antioxidant properties. We have reported previously that ASX protects neurons from the noxious effects of amyloid-β peptide oligomers, which promote excessive mitochondrial reactive oxygen species (mROS) production and induce a sustained increase in cytoplasmic Ca Due to 2+ influx through NMDAR is amplified and propagated by a mechanism known as Ca2+-induced Ca2+ release (CICR), a process in which the intracellular Ca2+ channels resident in the endoplasmic reticulum, Ryanodine Receptors (RyR) and Inositol 1,4,5-trisphosphate receptors (IP3R), have a central role ) ) . Thus, C([Ca2+]) , and as ng aging  or in pang aging .2+ levels in cells treated with excitotoxic NMDA concentrations. We found that ASX decreased mitochondrial ROS levels and alleviated the mitochondrial dysfunction induced in SH-SY5Y cells by NMDA and also prevented H2O2-induced cell death. In addition, ASX attenuated the [Ca2+] increase displayed by primary rat hippocampal neurons treated with NMDA.Currently, intensive research efforts are dedicated to find compounds with biological activity against oxidative stress and excitotoxicity in neurons. In this work, we studied the effects of ASX in NMDAR-mediated excitotoxicity. To this aim, we evaluated the effects of ASX on mitochondrial ROS production, cell viability and intracellular CaSH-SY5Y cells, a human neuroblastoma cell line derived from the catecholaminergic line SK-N-SH , have beTo detect the presence of NMDAR in undifferentiated SH-SY5Y cells, we performed an immunofluorescence analysis using a specific antibody against the NR1 subunit of the NMDAR. A representative image, illustrated in Cellular metabolic activity was evaluated after incubation of SH-SY5Y cells with tetrazolium (MTT) salt, which becomes reduced in cells with metabolically active mitochondria. Although the reduction process is not exclusively mitochondrial; it occurs necessarily in living cells, so the MTT assay has been widely used as a marker of cell viability . MTT red2+] [2+] levels after NMDA addition to SH-SY5Y cells previously loaded with the Ca2+ probe Fluo 4-AM. 2+] increase induced by NMDA (NMDA = 0.314 ± 0.024 v/s NMDA + APV = −0.037 ± 0.011), an indication that NMDAR activation mediates the intracellular [Ca2+] elevation induced by NMDA.Activation of NMDAR in the primary hippocampal neurons generates a measurable increase in cytoplasmic [Ca2+] . Hence, ASX has been used to improve mitochondrial integrity and combat oxidative stress  due to t2+] and mitochondrial superoxide levels but did not affect cellular metabolic activity . Of note, as illustrated in We found that incubation of SH-SY5Y for up to 2 h with a low concentration of NMDA (16 μM), closer to the physiological range, also increased both  has an important role in the mechanisms underlying memory processes and synaptic dysfunctions [2+] levels in cultured hippocampal neurons treated with NMDA. For this purpose, hippocampal neurons were transfected with the genetically encoded Ca2+ sensor GCamp3, and subsequently treated for 24 h either with vehicle  treated with 200 µM NMDA. Preincubation with ASX reduced the fluorescence increase by 40% and induced NMDA by 200 µM, which reflects the increase in cytoplasmic [Ca2+], induced by 200 µM NMDA , compared to control neurons incubated only with NMDA.The regulation of intracellular  and ROS levels induced by the subsequent application of a cytotoxic stimulus (200 µM NMDA), thus normalizing the aberrant crosstalk between Ca2+and ROS signaling that is characteristic of cytotoxic conditions. First, we used a chemical Ca2+-sensitive probe to show that NMDAR activation in SH-SY5Y cells was dose-dependent. The present results showing NMDAR-mediated [Ca2+] increases in SH-SY5Y cells are consistent with our previously published study, in which we demonstrated that the addition of NMDA to primary hippocampal cultures elicits persistent neuronal Ca2+ signals that require functional NMDA receptors  increase, such as that evoked by a high concentration of NMDA (200 µM), leads to sizable mitochondrial depolarization and excessive mitochondrial ROS generation [2+] and mitochondrial superoxide levels but did not affect cell metabolic activity  reported that cells pretreated with ASX exhibited a lower increase in cellular oxidative state following hydrogen peroxide addition compared to untreated cells . AnotherEven though ASX exerts its antioxidant activity by absorbing the energy of excited singlet oxygen or other radical species via the polyene chain present in its chemical structure , its act2+ signaling in different cellular systems  increase caused by NMDAR activation promotes nNOS and NOX2 activities; the ensuing ROS generation together with the cytoplasmic [Ca2+] increase lead to activation of Ca2+ release mediated by the type-2 RyR isoform  elicited by NMDAR activation, SH-SY5Y cells were incubated with 1.5 µM Fluo-4 AM for 30 min at 37 °C in extracellular magnesium-free buffer that contained in mM: 4.8 KCl, 118 NaCl, 3 CaCl2, 10 Glucose, 0.01 Glycine, 20 Hepes/Tris, pH 7.4. The cells were washed with an extracellular buffer for NMDA experiments to remove probe excess. The basal fluorescence intensity was continuously monitored (λ Ex/Em: 496/506 nm) during 4 min before and 20 min after NMDA addition, using the multimode microplate reader Synergy, HT (Biotek). Fluo-4 fluorescence levels were expressed as ∆F/F0, where F0 corresponds to the average basal fluorescence acquired during 4 min prior to NMDA addition, while ΔF corresponds to the difference between each fluorescence value measured after NMDA incubation (F) and F0.To detect changes in intracellular ) induced by 200 μM NMDA did not saturate the signal of the sensor. All experiments were done at room temperature (20–22 °C).Primary hippocampal neurons  transfected with the GCamp-actin EGFP vector were treated by 24 h with 10 μM ASX. Next, control cells (without treatment) and ASX-treated cells were transferred to extracellular solution for NMDA experiments (in mM: 4.8 KCl, 118 NaCl, 3 CaClp < 0.05.Data are expressed as means ± standard error. One-way ANOVA or Bonferroni’s Multiple Comparisons test were used for data analysis. Differences were considered significant at p < 0.05.The results were analyzed using a reading test provided by the GraphPad Prism 5 software. Statistical significance was considered at