RUNX1, NPM1, or SRSF2 transcriptionally modulate cell state in a manner that primes leukemic cells for glucocorticoid sensitivity. In clinical practice, inflammatory markers such as serum ferritin or IL-6 have a strong prognostic impact and may directly affect disease progression, whereas interesting preliminary data suggested that dexamethasone may improve the outcome for AML patients with a high white blood cell count, which paves the way to develop prospective clinical trials that evaluate the role of glucocorticoids in AML.Recent advances in the description of the tumor microenvironment of acute myeloid leukemia, including the comprehensive analysis of the leukemic stem cell niche and clonal evolution, indicate that inflammation may play a major role in many aspects of acute myeloid leukemia (AML) such as disease progression, chemoresistance, and myelosuppression. Studies on the mechanisms of resistance to chemotherapy or tyrosine kinase inhibitors along with high-throughput drug screening have underpinned the potential role of glucocorticoids in this disease classically described as steroid-resistant in contrast to acute lymphoblastic leukemia. Moreover, some mutated oncogenes such as  Acute myeloid leukemia (AML) is a myeloid malignancy induced by the oncogenic transformation of hematopoietic progenitors in the bone marrow leading to the destruction of blood tissue responsible for acute pancytopenia, severe bleeding, and infection . For oveIn the era of next-generation sequencing, considerable progress has been made in understanding leukemogenesis, the genetic diversity of AML, gene-gene interactions, clonal evolution, and the assessment of treatment responses –11. SeveHerpesviridae infections, was documented in 75% of the cases. In this study, hemophagocytic lymphohistiocytosis was associated with poor overall survival, which suggests that inflammation may impact prognosis in AML patients  and FTL (ferritin light chain) genes  genes . Ferritin) genes . Inflammn) genes , 40, 41.n) genes . Therefon) genes , 44. AMLe tumors , 35, 46.e tumors . Overalle tumors , 49.Another point for discussion is the cellular origin of ferritin in AML, although it is thought to reside in the mononuclear phagocyte system and in hepatocytes in non-malignant conditions –52. MacrAccumulating evidence suggest that in AML increased serum ferritin levels are due to both a dysregulated inflammatory response and disease burden, and may have a direct impact on disease progression and response to therapy. Therefore, pharmacological modulation of this pathway could be a new therapeutic target in AML (Glucocorticoids such as dexamethasone are anti-inflammatory drugs widely used in acute lymphoblastic leukemia (ALL) and other lymphoid malignancies . There aIn adult AML, dexamethasone has long been used to prevent or treat a severe inflammatory condition known as differentiation syndrome in patients with acute promyelocytic leukemia (APL) treated with all trans-retinoic acid (ATRA) and/or arsenic trioxide, or more recently in AML patients treated with IDH or FLT3 inhibitors , 64. In via microvascular invasion, hyperviscosity, leukocytic microthrombi, and oxygen consumption. Mediators of inflammation induced by leukemic blasts and endothelial cells play a central role in the pathogenesis of leukostasis (Approximately 20% of AML patients present with a high WBC count (>50 G/L) at diagnosis . This iskostasis . Studieskostasis , 69. Beckostasis . A subsekostasis . Moreovekostasis .Glucocorticoid drugs act through different targets and mechanisms to control inflammation . After cUnlike ALL, AML blasts are not usually sensitive to pharmacologic doses of glucocorticoids . In vitrIn clinic, glucocorticoids may increase the risk of bacterial or invasive fungal infections by worsening the immunosuppression of AML patients during intensive chemotherapy. Therefore, these drugs have received little attention in the field. However, we discuss below recent emerging preclinical data suggesting that glucocorticoids may have a positive therapeutic impact in certain settings in AML.DCK gene that encodes deoxycytidine kinase, the rate-limiting enzyme in the metabolic activation of cytarabine. Moreover, upregulation of GR protein expression was also observed in cytarabine-resistant AML cells and contribute to the acquisition of glucocorticoid sensitivity. However, it should be noted that in a rat model of AML, dexamethasone decreased the activity of deoxycytidine kinase  are a reliable functional readout to monitor the activity of leukemia-initiating/stem cells (LICs), an AML subpopulation thought to be at the origin of relapse , 92. Usintiation . Furthermic bulk . As descmic bulk , 94, 95.mic bulk . Therefomic bulk , 96.RUNX1-RUNX1T1 fusion gene generated by chromosomal translocation and represents approximatively 10% of AML. The RUNX1-RUNX1T1 oncoprotein acts as a dominant negative regulator of RUNX1 transcriptional activity thereby repressing the expression of RUNX1-dependant genes involved in myeloid differentiation  increased RUNX1 binding to its target genes and decreased RUNX1-RUNX1T1 binding. The unbound RUNX1-RUNX1T1 might be subjected to proteasome-mediated degradation, resulting in a reduced level of oncoproteins in the nucleus. This leads to a significant change in gene expression with RUNX1 dominance over RUNX1-RUNX1T1 promoting RUNX1-mediated hematopoietic differentiation and inhibition of RUNX1-RUNX1T1-mediated stem cell maintenance. In addition, a combination of dexamethasone with cytarabine or doxorubicin was synergistic in this AML cell line, which suggests that combining dexamethasone with “3+7” induction and then with high-dose cytarabine during the consolidation phase could be a treatment option to be explored in patients with t/RUNX1-RUNX1T1 AML  translocation is induced by the oncogenic activity of the ntiation , 98. RUNntiation . In a chignature . Methylpid cells . A more ML cells . MoreoveX1T1 AML , 102. ItX1T1 AML .RUNX1 (RUNX1mut AML) is a provisional entity that accounts for 10% of the newly diagnosed patients and is associated with a poor prognosis , normal karyotype or noncomplex karyotype with frequent association with trisomy 13. RUNX1 mutations are mutually exclusive of recurrent translocations in AML and co-occur with ASXL1 mutations and other gene mutations including epigenetic modifiers , spliceosome complex  and STAG2, PHF6, and BCOR  were much more responsive to glucocorticoids than AML sample wild type RUNX1 allele or with missense RUNX1 mutations, which are known to have no impact on RUNX1 function. Furthermore, down-regulation of RUNX1 expression was associated with acquired dexamethasone sensitivity. Glucocorticoid sensitivity was reverted by the GR antagonist RU486 or NR3C1 knock-down, which confirms that glucocorticoid activity in AML cells is dependent on their interaction with the GR. It is important to note that RUNX1mut AML samples expressed increased levels of NR3C1 compared to most AML subgroups. Another study showed a similar acquisition of glucocorticoid sensitivity in blastic-phase CML cells with RUNX1 mutations  gene are among the most frequent molecular alterations in AML (~35%) and represent a distinct entity in AML according to the 2016 WHO classification , 113. Whc impact . Unmutattivation .NPM1 mutated cell line, demonstrated in vitro and in vivo sensitivity to dexamethasone compared to wild type cell lines and primary AML samples with NPM1 mutation were also more sensitive to dexamethasone-induced apoptosis than wild type samples  in the juxtamembrane domain and point mutations in the tyrosine kinase domain (TKD). FLT3 mutations are associated with an aggressive disease course especially FLT3-ITD which predicts early relapse and a poor prognosis. Through clonal selection under chemotherapy, a higher mutant allelic burden is frequently observed at relapse, which indicates that AML cells have become more addicted to FLT3 signaling. In the preclinical setting, FLT3-mutant allelic burden and clinical status (diagnosis versus relapse samples) are predictive of a response to FLT3 inhibitors in AML  up-regulated both the inflammatory response gene and GR expression. This resulted in synergistic activity of the combination of FLT3 inhibitors and glucocorticoids through apoptosis induction both in vitro and in xenotransplantation mice models  or myeloproliferative disorders  or therapeutic (cytarabine and FLT3 inhibitors) stress, AML displays up-regulation of the inflammation gene response and GR expression that induces sensitization to glucocorticoid therapeutic action partially through the modulation of key apoptotic protein . Younger patients (18–60 years) with favorable or intermediate risk AML, in their first complete remission following standard induction chemotherapy, receive either high-dose cytarabine alone (standard of care) or high-dose cytarabine plus dexamethasone  . The DEXAML patients who fail to achieve a complete response with standard induction therapy (refractory AML) or who relapse after achieving remission have a very poor prognosis . TreatmeAs indicated above, AML samples collected at relapse are much more sensitive to glucocorticoids than diagnosis samples , 84. MorRepositioning approved drugs for other indications is an interesting subject for clinical trials by cooperating study groups . Even thThe author confirms being the sole contributor of this work and has approved it for publication.Funding from the Fondation Toulouse Cancer Santé, Force Hémato (AO 2017), the French Ministry of Health  and the Ligue Régionale Contre le Cancer (AO 2018).The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cilia are present on nearly every cell type in either a motile or non-motile form; motile cilia generate fluid flow needed for a variety of biological processes, such as left–right body patterning during development, while non-motile cilia serve as the signaling powerhouses of the cell, with vital singling receptors localized to their ciliary membranes. Much of the research currently available on Ca2+-dependent cellular actions and primary cilia are tissue-specific processes. However, basic stimuli-sensing pathways, such as mechanosensation, chemosensation, and electrical sensation (electrosensation), are complex processes entangled in many intersecting pathways; an overview of proposed functions involving cilia and Ca2+ interplay will be briefly summarized here. Next, we will focus on summarizing the evidence for their interactions in basic cellular activities, including the cell cycle, cell polarity and migration, neuronal pattering, glucose-mediated insulin secretion, biliary regulation, and bone formation. Literature investigating the role of cilia and Ca2+-dependent processes at a single-cellular level appears to be scarce, though overlapping signaling pathways imply that cilia and Ca2+ interact with each other on this level in widespread and varied ways on a perpetual basis. Vastly different cellular functions across many different cell types depend on context-specific Ca2+ and cilia interactions to trigger the correct physiological responses, and abnormalities in these interactions, whether at the tissue or the single-cell level, can result in diseases known as ciliopathies; due to their clinical relevance, pathological alterations of cilia function and Ca2+ signaling will also be briefly touched upon throughout this review.The calcium ion (Ca The success of this complex role may, in part, be due to the heterogenic distribution of Ca2+ in areas called “Ca2+ microdomains” found in resting and stimulated cells [Adaptation has long been thought to be the key evolutionary development required for life to exist. We owe our longevity as a species to our ability to adapt to changes in our external environment. Before large-scale adaptations can occur, cells must first possess the ability to adapt and respond to external stimuli in the environment. Because cells face a barrage of minute chemical and physical stimuli on a perpetual basis, cells must precisely signal in order to correctly respond to stress. These signals require messengers whose concentrations can change quickly depending on time and location. Calcium cations  and transient receptor potential cation channel subfamily V member 4 (TRPV4), are present on both the ciliary membrane and basal body; the intracellular anchor point the cilium originates from [2+ into the primary cilia precedes a rise in cytosolic Ca2+; however, the exact mechanism by which this happens remains unknown [2+ signaling but one that is instead cell-type specific, as the type of Ca2+ channels and ciliary activation pathways vary by cell type. For example, PC-2 and TRPV4 have been localized to the primary cilia in both the kidney and bone cells, but research suggests a polycystin-mediated Ca2+ influx in the kidney and a TRPV4-mediated influx in bone [2+-dependent biologic processes that may be mediated through ciliary signaling.Although primary cilia appear to be a continuous extension of the cellular membrane, they have a distinctly unique membrane composition compared to the rest of the cell. Ciliary membranes have a vast array of specifically localized receptors and channels making the detection, transmission, and translation of external mechanical or chemical stimuli their principal function within the cell ,9. High tes from . It is g unknown ,14,15,16 in bone ,18,19,20The sensory role of the primary cilium is highly specialized depending upon the tissue or organ system it is localized in. Well-known examples of their specialized roles include mechanosensitive cilia found in blood vessels, kidney, and bone; chemosensitive cilia in the nose; and recently, new evidence has suggested an electrosensitive role of primary cilia in the nervous system ,22,23. T2+ levels that initiate signaling pathways within the cell. Generally, non-motile primary cilia are said to have a 9 + 0 structure, or 9 doublet microtubules arranged in a circle and emanating from an intracellular anchor point called the basal body  to activate endothelial nitric oxide synthase (eNOS) . Polycystin 1 (PC-1), a mechanosensitive transmembrane protein, and PC-2, a Caion rate ,32. In rion rate ,35,36,37ntenance . Similarntenance . This ree (eNOS)  [19,40,4ow rates . When te2+ involved in secondary messaging within the cilium is still under debate. A study by Delling et al. showed that in cells bearing primary cilia, the Ca2+ wave originated out in the cytoplasm before propagating in the primary cilium [2+ mobilization [2+ flux, and therefore more studies are needed to follow up on this hypothesis [2+-responsive mechanosensors, the origin of the mechanically induced Ca2+ waves is currently a mystery [2+ increases, an increase in ciliary Ca2+ follows, which indirectly potentiates the activity of the ciliary PC-2 channel; if TRP channels are not involved in this process, other possible mechanosensitive proteins that do not elicit changes in ciliary Ca2+, such as G-protein-coupled receptor (GPCR) proteins, may be responsible [However, despite many studies supporting the model in y cilium ,43, sugglization . While ppothesis . If primponsible ,45.2+, depolarizing the neuron and transmitting the signal to the brain where it is interpreted accordingly [2+-activated chloride channels, or in the cilia structure itself can lead to an impaired sense of smell (anosmia) [All primary cilia have a very diverse chemosensory role, housing a wide array of receptors and proteins for ligands, ranging from hormones to neurotransmitters, endogenous molecules, and even exogenous molecules ,46,47,48ordingly ,51. Pertanosmia) ,50,51. Wanosmia) .2+ channels [2+ indicators which report extracellular-dependent changes in intracellular Ca2+ levels suggestive of an electrical current being carried across the ciliary membrane [2+ oscillations. More interestingly, this study reported that knocking out IFT88 , or PKD1 (encoding for PC-1), the stem cells lost their ability to respond to electrical field stimulation, as in differentiation and osteogenic markers, but an increase in cytoplasmic Ca2+ was still observed. This could be explained by either cytoplasmic Ca2+ being needed to precede cilia sensory function, enabling the cilia to sense the electrical stimuli, or a separate Ca2+ microdomain forming in the cilia that is distinct from cytosolic Ca2+. Together, this suggests that primary cilia have a critical role in translating Ca2+-induced cellular responses into electrical stimulation [Whether or not primary cilia detect and transduce electrical stimuli is difficult to identify, as studies show results that are highly variable and dependent on the frequency of stimuli, the strength of the electrical field, and cell type ,54. To mchannels ,56, and channels ,58 may ichannels . More dimembrane . While imembrane . This ismulation .2+ concentrations. The ability of Ca2+ to bind and initiate changes in protein shape and charge, which are important parameters in protein function, make it an ideal secondary messenger. Ca2+ carries out a vast array of complex cellular processes, and many different factors cause fluxes in intracellular Ca2+; hormones, growth factors, cytokines, and neurotransmitters all increase intracellular Ca2+, but due to the temporal and spatial nature of Ca2+, a cell is able to tailor its response to any specific hormone or stimuli [2+ cannot be chemically altered. Thus, to exert control over Ca2+, cells must chelate, compartmentalize, or extrude it [2+-dependent cellular actions and cilia are tissue-specific processes , the concentrations of which are also tightly regulated during cell division. Several studies have reported that overexpression of CaM shortens the G1 phase and accelerates proliferation; interestingly, cilia length has been shown to directly impact the time spent in this phase, with more evidence suggesting cells with shortened cilia enter S-phase more rapidly [2+/CaM exerts its proliferative effect through the use of Ca2+/CaM-dependent kinases I, II, and IV. One way Ca2+/CaM helps coordinate entry into the cell cycle is through the centrosome cycle. For division to occur, the centrioles disengage and migrate to opposite poles in the cell, where they then duplicate, creating the two mitotic spindle poles. Studies show Ca2+/CaM and CaM-dependent protein kinase II (CaMKII) regulate the expression of centriolar coiled-coil protein of 110 kDa (CP110), in addition to several other transcription factors that are necessary for centrosomal duplication, correct spindle formation, and cytokinesis regulation [2+ channels: TRPV2 , PC-2, and TRPC1  [2+ permeable channels allows for an isolated rise in intraciliary Ca2+, creating a localized signaling pathway directly from the cilia, to the basal body, and then to the nucleus. Further evidence shows CaM and Ca2+/CaMKII localize to cilia and are downstream of the ciliary mechanosensory PC-1/PC-2 complex. As cilia bend, the activation of PC-1 triggers two signaling cascades. The first is through the activation of PC-2, enabling an extracellular Ca2+ influx and leading to the activation of Ca2+/CaM-dependent pathways. Secondly, the intracellular tail of PC-1 gets cleaved and translocated to the nucleus, where it regulates DNA transcription via genes related to proliferation [Although several connections to the cell cycle can be made through ciliary-localized proliferative pathways, the connection between primary cilia and the cell cycle lies in the dependence on the centrosome ,77. Howeesponses . In mamm rapidly ,79,80. Agulation ,81. Studember 1) . The higferation ,66,82,832+. In cell migration, Ca2+ is a player in sensing direction, cytoskeletal rearrangements, traction force, and the localization of focal adhesion molecules. Doyle et al. reported, in polarized cells, Ca2+ transients increased starting at the “front” of the cell and gradually moved towards the “rear.” In contrast, the observations in non-polarized cells showed an even distribution of Ca2+ throughout the cell [2+ in not yet fully understood, there is evidence to suggest that cilia, potentially through ciliary-dependent Ca2+ cascades, may stand as a point of reference for the coordinated movement of cells, interpreting and aiding in creating an environment with opposing and synergistic signals [The directional movement of cells to specific locations is fundamental for immune responses, tissue homeostasis, and many developmental processes ,85. The the cell . These rthe cell . While t signals .2+ and activating “left-sided” genes [2+ gradient, but only the two-cilia hypothesis provides an explanation for how this signal is generated and transduced [2+ distribution in the embryo, as well as a disruption in left-right patterning. This suggests that asymmetric Ca2+ is important for proper patterning and is mediated by PC-2 [Neurons originate from proliferating cell progenitors in the neuroepithelium. The neuroepithelium forms the wall of the neural tube, the embryonic precursor to the central nervous system. Neuronal patterning is the process by which cells in the developing nervous system differentiate into distinct identities and is heavily controlled by several extracellular factors and signaling gradients across the center axis of the nervous system . Thus, cell polarity is essential for proper asymmetric divisions, leading to neurogenesis, neuronal positioning, and cellular differentiation ,94. Manyd” genes . Interesansduced . The two by PC-2 ,69.2+ fluctuations have been shown to be important for neural migration along with other regulated developmental events, such as axonal outgrowth and maturation of signaling properties [2+ activity that stimulated proliferation [2+ signals may be due, in part, to standard cytoskeletal or cell-cell based communication, with the evidence presented above, as well as the mounting experimental and literary data not addressed here, primary cilia could play a significant part in mediating these essential Ca2+ fluctuations. While there is little investigation into the link between early neuronal Ca2+ signaling and primary cilia, there is also no evidence implicating any other structure or pathway in causing these oscillating Ca2+ levels. On studies performed on ventral spinal neurons during development in vitro, many of the proposed mechanisms for the oscillating Ca2+ influxes were ruled implausible [−-mediated transmission did not control these individual Ca2+ transients [2+ and require the ion’s entry through Ca2+ channels in the plasma membrane, a function that primary cilia are proposed to moderate in other cell types. L- and T-type channels were also not singularly responsible for these oscillations either, as their pharmacological block with Ni2+ or nifedipine only slowed down these events [2+ to re-supply drained intracellular stores was proposed, and then was quickly proved unlikely when the use of thapsigargin, a potent blocker of this transport system, had no effect on the Ca2+ oscillations [2+ activated SK  channels, also had no apparent effect on these oscillations [2+ signaling roles in their respective cell types, these neuronal primary cilia could play a significant role in mediating these Ca2+ oscillations.In addition to the flow of CSF fluid generated by motile cilia, spontaneous Caoperties ,110,111.feration . While slausible . The posansients . These se events . A mystellations ,115. Thellations . Through2+ signaling mainly occurs in the autocrine insulin secretion pathway and will therefore be the focus of this section [Pancreatic islets regulate glucose homeostasis through the secretion of various peptide hormones and are comprised of β-cells, which interact with each other through both paracrine and autocrine mechanisms in order to serve this function . β-cells section ,118. Bec2+ increase, insulin receptors are recruited to the primary cilia [2+ triggers the first-phase insulin secretion, then a period of continuous Ca2+ oscillations triggers the second-phase insulin secretion [2+ dynamics [2+ currents typically observed after glucose stimulation were abolished in the cilia-less βCKO line, leading to impaired insulin secretion, which established that glucose-mediated Ca2+ oscillations are cilia-dependent [2+ regulation by β-cell primary cilium is unclear, the existence of Ca2+ channels on the ciliary membrane, as well as the Ca2+-fluxes serving as moderators of cytosolic Ca2+ levels are strong evidence of its vital role in glucose-mediated insulin secretion [In normal functioning pancreatic islets, in response to a glucose-mediated cytosolic Cary cilia . The seqecretion ,120. Expdynamics . The casependent . While tecretion ,42,121.2+ which in general has varying effects on bile secretion [When discussing primary cilia, often the sensory roles are looked at in isolation; in the kidneys they are mechanosensors, in the nose they are chemosensors. This, however, is not always an accurate depiction of just how vital it is for primary cilia to have its their unique multi-sensory functions. Much like most lumens, primary cilia can be found extending into the bile duct from cholangiocytes. Here, primary cilia simultaneously use all their sensory properties to tightly regulate luminal tonicity, biliary composition, and bile flow ,74,122. ecretion ,74.2+ and mineral deposition associated with bone formation, a mechanism attributed to intact primary cilium with properly localized polycystin complexes and several TRP channel family proteins [2+, which triggers cellular responses. When researchers pharmacologically inhibited Ca2+ channels, the osteocytes’ ability to respond to mechanical cues was impaired, and in vivo treatment with Ca2+ channel inhibitors was found to reduce skeletal responses to mechanical forces. ATP also increased upon mechanical stimulation, and several in vitro studies demonstrated that intracellular Ca2+ is required for this ATP response. However, the exact mechanism by which Ca2+ controls ATP release is still not completely understood [2+ levels and had impaired intracellular Ca2+ responses to fluid-shear stress, as well as ciliary structural aberrations like a reduction in cilia length and number. In vivo, Kif3a null mice display lower bone density, altered bone volume, reduced bone formation rates, and impaired mechanical properties; all of which supports the idea that osteoblast function may be mediated through ciliary polycystins [2+ influx but instead translate the signal through another, as of yet unidentified pathway [2+-independent pathway in osteocytes [Mechanotransduction, in general, is critical for proper bone and cartilage homeostasis as its ability to adapt to its external environment is partly due to the fluid-saturated and porous nature of the tissue . Reducedproteins ,75. The derstood . Kif3a (derstood . Osteoblycystins . Despite pathway . One proteocytes . 2+ is often involved in cilia-dependent biological processes. Primary cilia have become recognized as being signaling powerhouses of the cell, and their signaling is often dependent on the presence of Ca2+. Depending on the organ system, primary cilia act as mechanosensors, chemosensors, electrosensors, or any combination of these. Working together, Ca2+ and cilia drive a vast array of pathways and processes within the body. The cell cycle, cell polarity and migration, neuronal patterning, glucose-mediated insulin secretion, biliary regulation, and bone formation are just some of the known and proposed ways that Ca2+ and cilia work together to maintain overall homeostasis. While the dysfunction of cilia and wayward Ca2+ signaling causes an ever-growing list of diseases, known as ciliopathies, the true extent of Ca2+ and cilia interactions is still poorly understood, yet likely more intertwined and prevalent than previously thought.Calcium cations are highly diverse secondary messengers present in a vast number of signaling pathways within the body. Because of its versatility, Ca


Calcium channels show selective permeability to Ca2+ and facilitate Ca2+ entry into the cytoplasm, which are normally located in the plasmatic and intracellular membranes. The increase of cytosolic Ca2+ modulates a variety of signalling pathways and results in the transcription of target genes that contribute to adipogenesis, a key cellular event includes proliferation and differentiation of adipocyte. In the past decades, the involvement of some Ca2+-permeable ion channels, such as Ca2+ release-activated Ca2+ channels, transient receptor potential channels, voltage-gated calcium channels and others, in adipogenesis has been extensively explored. In the present review, we provided a summary of the expression and contributions of these Ca2+-permeable channels in mediating Ca2+ influxes that drive adipogenesis. Moreover, we discussed their potentials as future therapeutic targets.As an important second messenger in adipocytes, calcium ions (Ca Obesity is believed to be the result from an imbalance between energy intake and energy expenditure , and it Adipocytes are derived from mesenchymal precursor cells . The pro2+) [2+ functions as an excellent messenger molecule that is widely involved in various cellular events, including generating action potentials, regulating enzyme activity, and bridging the extracellular and intracellular signal transduction [Calcium signalling plays a vital role in maintaining normal cellular functions, including proliferation, differentiation, homoeostasis, and apoptosis. The level of total body calcium is normally regulated. About 98% of total body calcium is stored in the skeleton as calcium phosphate. The calcium concentration of the extracellular fluid (ECF) is about 2.2–2.6 mmol/L in the form of total calcium, and about 1.3–1.5 mmol/L in the form of free calcium ions (Ca2+) . The cal2+) . Therefosduction .2+, which could trigger a sudden increase of the cytosolic Ca2+ level up to 500–1000 nm/L. The cytosolic Ca2+ rises either by entering the cell through Ca2+-permeable ion channels on the plasma membrane, such as voltage-gated Ca2+ channels and transient receptor potential (TRP) channels and/or by release from the intracellular Ca2+ stores, such as the ER and mitochondria [2+ is then quickly removed from cytoplasm by various types of Ca2+ pumps, including the Ca2+-ATPase and Na+/Ca2+ exchanger on the plasma membrane pumping Ca2+ out of cells, and Sarco/endoplasmic reticulum Ca2+ pump (SERCA) on the ER membrane and the mitochondria calcium uniporter (MCU) on the mitochondria membrane, which pump Ca2+ back into the ER and mitochondria, respectively [2+ is precisely regulated by these proteins to balance the intracellular Ca2+ homoeostasis, which is fundamental for maintaining normal cell functions [Calcium channels play fundamental roles in regulating the concentration of cytosolic Cachondria . Once thectively ,20. The unctions .2+ has been implicated in regulating adipocyte differentiation [2+ concentrations are increased in 3T3-L1 pre-adipocytes after Ca2+-ATPase inhibitor treatment, such as thapsigargin, which in turn efficiently inhibits adipocyte differentiation, and impairs specific gene expression of adipocytes and reduces the accumulation of lipid drops [2+-dependent phosphatase [2+ levels has negative effects on 3T3-L1 adipocyte differentiation similar to that seen in other cell types [Calcium signalling is also a vital event in the process of adipogenesis, as it regulates various fundamental processes including proliferation, differentiation, energy metabolism and obesity. Cytosolic Cantiation , which pid drops ,23. Thessphatase  or by acsphatase . By contsphatase . These rsphatase . These rll types .2+ is also involved in the modulation of adipogenesis. It has been reported that low extracellular Ca2+ promotes adipogenesis and high extracellular Ca2+ attenuates adipogenesis [2+ accelerates differentiation and high extracellular Ca2+ suppresses differentiation in mouse brown adipocytes [2+ enhances the proliferation of primary mouse bone marrow mesenchymal stem cells (BMSCs) but not differentiation [2+ promotes the proliferation of BMSCs via a calcium-sensing receptor and ERK signalling pathway [2+ enhances adipogenesis probably through L-type Cav channel [2+ involve membrane receptors, signalling molecules, and a diverse array of Ca2+ channels. In the next session, we summarized the calcium channels involved in regulating the concentration of cytosolic Ca2+ and adipogenesis.Extracellular Caogenesis  in 3T3-Logenesis ,30. Extripocytes . Moreoventiation . And hig pathway . However channel  and calc channel  in porci2+ influx via store-operated Ca2+ channels (SOCs) in the plasma membrane provides increased cytosolic Ca2+ level and sustains the activity of several intracellular enzymes including calcineurin, which is critical for adipogenesis [2+ stores [2+ (CRAC) channels are one type of SOCs. A well-described mechanism of extracellular Ca2+ entering adipocytes is through the CRAC channel, which is found by stromal interaction molecule 1 (STIM1) [2+ entry, and STIM1, the regulatory subunit located in the ER membrane. STIM1 function as a Ca2+ sensing protein monitoring the fluctuation of Ca2+ levels in ER, which normally spread out in the ER membrane and oligomerize during the low level of Ca2+. Orai1 and STIM1 function together to induce Ca2+ influx from the extracellular space into intracellular fluid, which is termed store-operated Ca2+ entry (SOCE) [Caogenesis . SOCs ch+ stores , which i+ stores . Selecti (STIM1)  and Orai (STIM1) ,40. It iy (SOCE) .2+ influx via CRAC channels and inhibits the 3T3-L1 adipocyte differentiation [2+ influx and inhibits 3T3-L1 adipocyte differentiation, without affecting their proliferation and growth arrest. The over-expression of STIM1 also induces the down-regulation of adiponectin and C/EBPα. Besides, the down-regulation of endogenous STIM1 promotes 3T3-L1 adipocyte differentiation, resulting in the up-regulation of C/EBPα and adiponectin [Drosophila [Drosophila. Acute dysfunction of STIM1 in the fat storage tissue triggers hyperphagia in flies [2+ influx in adipogenesis and obesity.The functional presence of STIM1 and ORAI1 has also been confirmed in many non-excitable cell types . It has ntiation . These rponectin . Anotherosophila . Impairmin flies . These s2+ and sodium (Na+) permeation [Another major class of calcium-permeable channels is TRP family. TRP channels are normally non-selective to Carmeation . The strrmeation . The TRPrmeation ,51. TRP rmeation . To datermeation . In the In terms of TRPV family, several TRPV channels have been reported to be expressed in adipocytes and play important roles in proliferation, differentiation, thermogenesis of adipocytes and obesity.2+ influx and impaired differentiation in 3T3-L1 pre-adipocytes [in vivo, but not in TRPV1 knockout mice [2+ level of adipocytes and promotes the browning of WAT. Moreover, activation of TRPV1 increases the expression level of thermogenic genes, such as UCP1, and induces the browning process in 3T3-L1 pre-adipocytes [In the past years, TRPV1 has been studied for its involvement in adipocyte differentiation and energy metabolism, which further related to obesity management. As the first identified TRP channels, TRPV1 is activated by capsaicin, a highly selective agonist of TRPV1, or when the surrounding temperature is higher than 43°C. TRPV1 is expressed in 3T3-L1 preadipocytes and adipose tissue both in animals and humans. TRPV1 has also been reported to play a key role in the regulation of food intake and glucose homoeostasis in WAT during the development of obesity . Activatipocytes , which iipocytes . In matuipocytes . Lackingipocytes . Howeverout mice ,57. Receout mice . It has ipocytes . In BAT,ipocytes . Similaripocytes . Howeveripocytes  and obesipocytes . Furtheripocytes . These cin vivo [2+ influx in adipogenesis has not been fully understood.TRPV3 often form functional heteromeric channels with TRPV1 , which ain vivo . Activat2+ influx plays an important role in BAT differentiation and thermogenesis. And TRPV2 could be a target for preventing human obesity and other metabolic-related diseases [TRPV2 and TRPV4 function as an osmo- and/or mechano-sensor, which could be activated by hypotonic solution or mechanical stimulation . TRPV2 hdiseases . However2+ permeable channels. The amount of Ca2+ influx through one single TRPV4 channel is assessed to be around 100 times more than that of the L-type Ca2+ channel, which in turn to simulate various Ca2+-dependent signalling cascades [Ppargc1a and Ucp1, which further promotes the browning process in 3T3-F442A adipocytes. Besides, pharmacological inhibition or deletion of TRPV4 also activates energy expenditure and protects mice from HFD-induced obesity in vivo [2+ level [2+ influx, adipogenesis and obesity.TRPV4 has been reported to be highly expressed in adipose tissue , such ascascades . Downreg in vivo . These r2+ level . However2+ level . These r2+ signalling pathway. The homomeric TRPC1 alone does not functionally work on the plasma membrane [2+ influx in adipocytes via CRAC channels, such as ORAI1 and STIM1 [2+ influx [2+ and Na+ ions, the exact role of TRPCs mediate signalling in adipogenesis and obesity needs further studies.Several TRPC  channels have been reported function as SOCs, by interacting with the key players of SOCE, such as ORAI1 and STIM1, which we have discussed previously. TRPC1 usually forms a tetrameric complex with TRPC4 or TRPC5 and interacts with each other to stimulate the intracellular Camembrane . It has nd STIM1 . The act+ influx . TRPC1, + influx . Indeed,+ influx . Besides+ influx . Since TOther TRP channels, such as TRPM8 and TRPPs, have also been reported to be involved in adipogenesis and obesity. TRPM8 is known as a cold sensing channel with a temperature threshold lower than 26-28°C , which cTRPP is a type of non-selective ion channel, which has been proved to be associated with autosomal dominant polycystic kidney . TRPP hav channels, namely voltage-gated calcium channels, are one of the major calcium-permeable channels, which are widely expressed in numerous cell types such as neurons and adipocytes, and play important roles in regulating cellular processes, including release of neurotransmitters in neurons, and activation, differentiation and proliferation in adipocytes, respectively [v channels are grouped into three subtypes: L-type high-voltage-activated (HVA) Cav channel is encoded by Cav1; other HVA Cav channels, such as P/Q-type, R-type, and N-type, are encoded by Cav2; and low-voltage-activated (LVA) T-type Cav channel is encoded by Cav3. Some of these Cav channels have been proved to be involved in the regulation of intracellular calcium homoeostasis and adipocyte differentiation. The presence of L-type Cav has been reported to be expressed in adipocytes by experimental and clinical studies. L-type Cav is also involved in high extracellular Ca2+-stimulated adipogenesis in porcine BMSCs [v blocker, Nifedipine. Besides, the distribution of L-type Cav and the basal levels of intracellular calcium have been proved to be regulated by the growth hormone in rat adipocytes [v has been firstly reported to be expressed in 3T3-F442A pre-adipocytes [v3.1 has been proved to be expressed in cultured pre-adipocytes and 3T3-L1 pre-adipocytes, and the protein-expression level of Cav3.1 is significantly decreased in the differentiated adipocytes [v by NNC55-0396, a T-type Cav inhibitor, inhibits the proliferation in pre-adipocytes [v3.1 or treated with TTA-A2, another selective blocker of T-type Cav, are resistant to HFD induced increase of fat mass and body weight gain [v channels, such as Cav3.1, appear to be potential targets for the prevention and treatment of obesity.Caectively ,98. Cav ne BMSCs , becauseipocytes ,100. Theipocytes . Then Caipocytes . It has ipocytes . It has ght gain . These r2+ homoeostasis [2+ homoeostasis [P2 receptors are one type of purinergic receptor, which is in response to the release of ATP. They have been classified into five subclasses, including ionotropic receptors such as P2X, P2Z, and metabotropic receptors such as P2Y, P2 U, and P2 T. P2 receptors have been reported to be involved in several cellular functions, such as vascular reactivity, cytokine secretion, and cell proliferation and migration . Howevereostasis . In BATseostasis , cytosoleostasis , and celeostasis . In 3T3-eostasis . It has eostasis . Althoug2+-permeable channels, such as CRAC channels, TRPs, Cav and other channels in adipogenesis (2+ homoeostasis through manipulating these calcium channels affects the downstream signalling pathways which in turn promotes or inhibits adipocyte differentiation and subsequently affects obesity. However, there are still some open questions that should be carefully considered in future studies. First, how do pre-adipocytes and mature adipocytes balance the Ca2+ fluxes among different components to maintain Ca2+ homoeostasis? Second, is there any other Ca2+ permeable channels involved in adipogenesis and obesity, such as high-voltage-activated Cav channels and NMDA receptors. Third, is the effect of cytosolic Ca2+ on adipocyte differentiation phase-dependent? Further in-depth studies are required to determine the best therapeutic targets of calcium channels for clinical treatment of obesity and related metabolic disorders. Such studies could pave the way for new clinical approaches to treating human obesity and related metabolic diseases.In the present review, we summarized the recent research progress of Caogenesis . Disturb


Ca2+ leak channels are thought to be key regulators of the reticular Ca2+ homeostasis and cell survival. The present study aimed to determine whether a particular reticular Ca2+ leak channel, the translocon, also known as translocation channel, could be a relevant target against ischemia/reperfusion-mediated heart injury. To achieve this objective, we first used an intramyocardial adenoviral strategy to express biosensors in order to assess Ca2+ variations in freshly isolated adult mouse cardiomyocytes to show that translocon is a functional reticular Ca2+ leak channel. Interestingly, translocon activation by puromycin mobilized a ryanodine receptor (RyR)-independent reticular Ca2+ pool and did not affect the excitation–concentration coupling. Second, puromycin pretreatment decreased mitochondrial Ca2+ content and slowed down the mitochondrial permeability transition pore (mPTP) opening and the rate of cytosolic Ca2+ increase during hypoxia. Finally, this translocon pre-activation also protected cardiomyocytes after in vitro hypoxia reoxygenation and reduced infarct size in mice submitted to in vivo ischemia-reperfusion. Altogether, our report emphasizes the role of translocon in cardioprotection and highlights a new paradigm in cardioprotection by functionally uncoupling the RyR-dependent Ca2+ stores and translocon-dependent Ca2+ stores. During myocardial infarction, dysregulation of Ca To this end, we used an in vivo adenoviral delivery strategy based on an intramyocardial injection of an adenovirus encoding a FRET-based reticular Ca2+ sensor, D4ER  using fura-2 loaded cells. Because acute puromycin perfusion failed to trigger a significant increase in [Ca2+]cyto, we submitted CM to a 30 min puromycin pretreatment, combined or not with emetine (as a control in order to inhibit puromycin activity). Steady-state [Ca2+]cyto was unchanged after puromycin-pretreatment (fluorescent ratio: 0.4583 vs. 0.4577 in control (Ctrl)), but was slightly decreased after a pretreatment with emetine prior to puromycin  and the plasma membrane Na+/Ca2+ exchanger (NCX), and absorption by the mitochondria [2+ leakage triggered by puromycin treatment did not change the global [Ca2+]cyto at rest, even if TLC participated in its regulation (as shown by its inhibition by emetine prior to puromycin application). Next, we analyzed whether puromycin treatment could modify the cytosolic Ca emetine A–C. To e emetine B. Moreovchondria . Thus, C2+ store (activated by puromycin) and the RyR-dependent Ca2+ store (sensitive to caffeine) and to evaluate their relative size, we first perfused caffeine to deplete the RyR-dependent stores, and consecutively we applied ionomycin to reveal the residual reticular Ca2+ pool . Third, the increase in the cytosolic Ca2+ concentration over a 30 min hypoxia was lowered by ≈64% in CM pretreated with puromycin compared to control CM . Lastly, caffeine-dependent Ca2+ stores after the 30 min hypoxia were similar between control and puromycin-preconditioned CM . This could have impaired the detection of significant differences in the mitochondrial Ca2+ content in control and puromycin-treated CM. We therefore performed an indirect measurement of the mitochondrial Ca2+ content. FCCP, an ionophore used to decouple ATP synthesis from the mitochondrial respiratory chain, induced the leak of mitochondrial Ca2+ content, which was measured by the cytosolic fura2-AM probe (Kd = 140nM). Puromycin pretreatment significantly decreased mitochondrial Ca2+ stores by ≈31% . Moreover, although [Ca2+]mito rose with the pacing frequency in control CM, interestingly this rise was shifted to higher frequencies of pacing in CM treated with puromycin . Because no difference could be detected in cytosol  in isolated mitochondria of CM and did not find any effect of puromycin pretreatment on either the Ca2+ intake or mPTP opening . We next questioned if the puromycin-mediated decrease in mitochondrial Ca” method E,F. Cycl2+ by decreasing both steady-state mitochondrial Ca2+ content and the average Ca2+ level in mitochondria of beating CM. This may explain how puromycin pretreatment slowed down mPTP opening by decreasing reticular Ca2+ leak during ischemia and augmenting the tolerance to Ca2+ of mitochondria by reducing the average mitochondrial Ca2+ concentration. An enhanced tolerance of mitochondria to Ca2+ load has been revealed to be crucial to evade mPTP opening and in triggering cell death [2+ leak at low doses) in a preconditioning protocol in rats by decreasing infarct size and improving left ventricular function. However, it is obvious that SERCA inhibition will also lead to inhibition of CICR and thus prevent CM contraction [Jointly, our results point out that pharmaceutical activation of TLC enhances mitochondrial tolerance to handle Call death . A similll death , as theytraction .2+ leak via TLC. Second, hypoxia triggers an increase in cytosolic Ca2+ concentration, partly due to a decline of ATP synthesis necessary for SERCA pumps to reuptake Ca2+ into the lumen. An interesting study characterized a calmoduline (CaM)-binding motif in Sec61α [2+-dependent manner. Then, in the context of hypoxia, we may hypothesize that Ca2+ permeability of TLC is reduced.To our knowledge, nothing is known about modulation of TLC activity during hypoxia or ischemia. First, one can assume that translation is reduced during hypoxia and it should be the same for the associated Can Sec61α . Using m2+ homeostasis.In the following, we examined whether puromycin pretreatment  might be an effective cardioprotective strategy. CM death after hypoxia/reoxygenation (H/R) was appraised by a multilabeling flow cytometry analysis. Propidium iodide quantification was performed after 1 h 30 min hypoxia and 1 h reoxygenation with or without puromycin 200 µM pretreatment. Results highlighted a protective effect of puromycin by reducing cell mortality by ≈6% compared to control H/R (56% vs. 62%; Because TLC stimulation by puromycin enhanced cell survival when submitted to H/R, we assessed its putative protective action in an in vivo mouse model of myocardial I/R preconditioned or not with puromycin. In order to optimize the treatment efficiency, we performed dose–effect experiments. Infarct size estimation was figured out after 45 min ischemia and 24 h reperfusion C. Optima2+ depletion via TLC activation, which in turn might decrease Ca2+ toxicity in both cytosol and mitochondria. One can easily imagine that TLC activation, prior to ischemia, increases the reticular Ca2+ leak. In turn, it might cause a mild ER stress, from which there is growing evidence that it contributes to protect CM from cell death [In brief, these last results underlined the fact that CM death can be partly prevented during ischemia by a moderate reticular Call death .2+ store is correlated with the modulation of several processes involved in cell fate—mitochondrial tolerance for cytoplasmic Ca2+ increase, mPTP opening, and tilting the cell balance on the side of cell survival. In fact, most studies on CM protection during ischemia/reperfusion relied on avoidance of mitochondria death and emphasized the concept of direct inhibition of mPTP. We believe that the multiple consequences of the decrease in reticular Ca2+ content could induce a robust cardioprotective effect, less prone to individual fluctuations. Even though our results rely on pre-activation of TLC, that is, I/R preconditioning, and cannot be used during myocardial infarction, this study opens new perspectives on Ca2+ modulation that could be useful for further investigations on postconditioning, thus broadening future therapeutic possibilities.Our results showed for the first time that the modulation of TLC-dependent reticular Ca


Neuronal calcium sensor 1 (NCS1) is a ubiquitously expressed Ca2+‐binding protein that promotes tumor aggressiveness by enhancing cell survival and metastasis. However, the underlying mechanism by which NCS1 contributes to increased tumor aggressiveness has yet to be identified. In this study, we aimed to determine (a) whether NCS1 expression changes in response to external stimuli, (b) the importance of NCS1 for cell survival and migration, and (c) the cellular mechanism(s) through which NSC1 modulates these outcomes. We found that NCS1 abundance increases under conditions of stress, most prominently after stimulation with the pro‐inflammatory cytokine tumor necrosis factor α, in a manner dependent on nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NFκB). We found that NFκB signaling is activated in human breast cancer tissue, which was accompanied by an increase in NCS1 mRNA expression. Further exploration into the relevance of NCS1 in breast cancer progression showed that knockout of NCS1 (NCS1 KO) caused decreased cell survival and motility, increased baseline intracellular Ca2+ levels, and decreased inositol 1,4,5‐trisphosphate‐mediated Ca2+ responses. Protein kinase B (Akt) activity was decreased in NCS1 KO cells, which could be rescued by buffering intracellular Ca2+. Conversely, Akt activity was increased in cells overexpressing NCS1 (NCS1 OE). We therefore conclude that NCS1 acts as cellular stress response protein up‐regulated by stress‐induced NFκB signaling and that NCS1 influences cell survival and motility through effects on Ca2+ signaling and Akt pathway activation.Changes in intracellular calcium (Ca 2+)‐binding protein NCS1 promotes increased tumor aggressiveness. We characterize NCS1 as a stress response protein that is up‐regulated by exogenous stressors, including the cytokine tumor necrosis factor α, through a nuclear factor kappa‐light‐chain‐enhancer of activated B cells‐dependent mechanism. Increased NCS1 expression leads to disrupted intracellular Ca2+ signaling and activation of the protein kinase B pathway, leading to increased cell survival and migration.This study investigates the underlying mechanism by which the calcium (Ca Aktprotein kinase B2+CacalciumERendoplasmic reticulumInsP3inositol 1,4,5‐trisphosphateInsP3Rinositol 1,4,5‐trisphosphate receptorIκBαnuclear factor of kappa‐light polypeptide gene enhancer in B‐cell inhibition, alphaNCS1neuronal calcium sensor 1NFκBnuclear factor kappa‐light‐chain‐enhancer of activated B cellsp‐Aktphosphorylated AktPI3Kphosphoinositide‐3‐kinasePP2Accatalytic subunit of protein phosphatase 2AtBHPtert‐butylhydroperoxideTGthapsigarginTNF‐αtumor necrosis factor alpha12+) signaling leads to a variety of pathological conditions , such that following defined stimuli, Ca2+ levels can be raised and used as an effective second messenger. Ca2+ transients cause a broad variety of outcomes not only due to the tightly regulated spatial and temporal occurrence of Ca2+ signals, but also due to the existence of Ca2+‐binding proteins. Upon binding of Ca2+, these proteins can change conformation and interact with other effector proteins. As such, Ca2+‐binding proteins are essential for regulation of cellular homeostasis.Disrupted intracellular calcium , which shares multiple characteristics with the S100 protein family. Like S100 proteins, NCS1 belongs to the EF‐hand superfamily of Ca2+‐binding proteins   lacking NCS1 expression. Finally, we investigated which cellular mechanisms are used by NSC1 to affect cell survival and migration, focusing on Ca2+ homeostasis, InsP3‐mediated Ca2+ signaling, and phosphatidylinositol 3‐kinase (PI3K)‐protein kinase B/Akt pathway (Akt pathway) activation. Overall, we describe a novel mechanism through which NCS1 functions as a stress response protein to promote cell survival and motility.In this study, we aimed to investigate the mechanisms by which NCS1 enhances tumor progression and aggressiveness. Our primary goal was to determine the specific extracellular conditions and intracellular signaling pathway(s) that lead to increased NCS1 expression in cancer cells. For this purpose, we explored whether NCS1 expression levels change in response to external stimuli 22.12 in Dulbecco's modified essential medium (DMEM) supplemented with 10% FBS, 1% l‐glutamine, and 1% penicillin/streptomycin. MDA‐MB231 cells stably overexpressing NCS1 . ATCC validates all cell lines by short‐tandem repeat analysis. MDA‐MB231 cell lines were maintained at 37 °C with 5% CO2.2−1 TNF‐α  for 24–36 h. For NFκB inhibition, 1 μm Bay 11‐7082 (Sigma‐Aldrich) was used for 24 h. Bay 11‐7082 was either applied alone or together with TNF‐α. To induce oxidative stress, cells were treated with 10 μm tert‐butylhydroperoxide (tBHP) for 20 h. To buffer intracellular Ca2+, MDA‐MB231 cells were treated with 1 μm BAPTA‐AM for 30 min. To induce high extracellular Ca2+ levels, we added 5 mm Ca2+ to the cell culture medium for 24 h. To induce endoplasmic reticulum (ER) stress, cells were treated with 1 μm thapsigargin (TG) for 24 h.To study the effect of cellular stressors on NCS1 expression, SHSY5Y cells were treated with various stimuli. To specifically activate the transcription factor NFκB, cells were treated with 10 ng·mL2.3Ct method , and 18S .RNA was extracted using an RNeasy Mini Kit  according to the manufacturer's protocol. Briefly, cells were lysed and homogenized using QIAshredder spin columns (QIAGEN) and RNA was bound to a RNeasy Mini Spin Column. To eliminate genomic DNA contamination, on‐column DNA digestion was performed using RNAase‐Free DNAse I in buffer RD (QIAGEN). After several washing steps, RNA was eluted. RNA concentration and quality were assessed with spectrophotometry . Using a High‐Capacity cDNA Reverse Transcription Kit , 1 μg RNA was then reverse‐transcribed to cDNA in a total reaction volume of 20 μL. Subsequently, the cDNA reaction was diluted with a dilution factor of 1 : 3. Real‐time quantitative PCR was performed on MicroAmp Fast 96‐well reaction plates  using 1 μL of the diluted cDNA reaction per well and Power SYBR Green PCR Master Mix (Applied Biosystems) in a 7300 Real‐Time PCR System (Thermo Fisher Scientific). Data were analyzed using the ΔΔ2.42.4.1Cells were lysed in ice‐cold mammalian protein extraction reagent (PER) buffer (Thermo Fisher Scientific) supplemented with a protease inhibitor, sodium fluoride, and sodium orthovanadate .2.4.2To obtain cytosolic and nuclear fractions, NE‐PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific) were used according to the manufacturer's protocol. Cells were harvested with TrypLE, centrifuged, and washed in PBS. A reagent ratio of 200 cytoplasmic extraction reagent I (CER I): 11 cytoplasmic extraction reagent II (CER II): 100 nuclear extraction reagent (NER) was used. Ice‐cold CER I containing protease inhibitor was added to the cell pellet and vortexed to fully suspend the cell pellet. The cell suspension was incubated for 10 min on ice. Ice‐cold CER II was added, vortexed, and incubated for 1 min on ice. After an additional vortexing step and following centrifugation, the cytoplasmic extract was obtained from the supernatant. Ice‐cold NER was added to the insoluble fraction (pellet) containing the nuclei. After several vortexing steps, the suspension was centrifuged and the nuclear protein fraction was obtained from the supernatant.All protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). SDS/PAGE was performed with 10–30 μg protein. Protein was transferred to a polyvinylidene difluoride membrane , resulting blots were blocked for 1 h at room temperature (RT) in 5% nonfat dry milk or, for phosphorylated proteins, in 5% BSA in 1X Tris‐buffered saline supplemented with Tween‐20 (TBS‐T), then incubated with primary antibody overnight at 4 °C. The following antibodies were used NCS1 , GAPDH , Akt , phospho‐Akt , NFκB p65 , nuclear factor of kappa‐light polypeptide gene enhancer in B‐cell inhibition, alpha , Lamin . Following primary antibody incubation, membranes were washed with 1X TBS‐T and incubated with a horseradish peroxidase‐labeled goat IgG (Santa Cruz Biotechnology) at RT for 2 h. Membranes were washed, incubated in Pierce ECL western blotting solution (Thermo Fisher Scientific) and developed on X‐ray film in a dark room.2.5et al., NCS1 promoter region. After obtaining a list of transcription factors that potentially bind 200 kB upstream to 200 kB downstream of the predicted transcriptional start site (TSS) of NCS1, we reviewed the literature on their involvement in Ca2+ signaling or cancer progression.The TRANSFAC database (Matys 2.6n = 5) and human breast tumor (n = 8) tissue as described previously  and analyzed using Qlucore Omics Explorer . Gene expression values were log2‐transformed and fold change calculated. A curated list of genes associated with TNF signaling via NFκB (BioCarta Gene Set M5890) was collected from Gene Set Enrichment Analysis Gene Sets (Broad Institute) and used to generate an expression heat map of differentially regulated (P < 0.05) NFκB‐regulated genes in normal versus cancerous breast tissue. Gene expression values of NCS1 mRNA were plotted in normal versus cancerous breast tissue and against expression values of the catalytic subunit of protein phosphatase 2A (PP2Ac).RNA microarray analysis was performed on normal human breast  according to the manufacturer's protocol. The insert was validated by sequencing. Quality and concentration of the DNA were validated with spectrophotometry . For transfection of MDA‐MB231 cells, MDA‐MB231 cells were plated on 100‐cm dishes 24 h prior to transfection. Afterward, cells were transfected for 48 h with either the NCS1‐PX458 vector or the scrambled PX458 vector control. Transfection was performed using FuGene HD transfection reagent  according to the manufacturer's protocol. As the PX458 vector contains GFP, FACS cell sorting was performed to isolate successfully transfected cells by selecting single GFP‐positive cells into 96‐well plates. These cells were grown in complete growth medium until the wells were confluent. Colonies were tested for successful NCS1 KO by western blot analysis and IF. The primary antibody for NCS1 was obtained from Abcam (ab116230) and was used in 1 : 2000 dilution for western blotting and 1 : 500 for IF.To study the cellular effect of high NCS1 expression, MDA‐MB231 cells were stably transfected with a vector encoding for human 2.8For fluorescence microscopy, MDA‐MB231 WT and NCS1 KO cells were seeded on sterile glass coverslips and grown to 80% confluency. Medium was removed, and each coverslip was washed twice with 2 mL of 1× PBS . Fixation was performed for 15 mi at RT with 4% paraformaldehyde. After several washes in 1× PBS supplemented with 0.1% Tween‐20 (PBS‐T), cells were permeabilized in 0.1% Triton X‐100 for 5 min. Following permeabilization, cells were washed and blocked for 1 h at RT in 1X PBS‐T supplemented with 10% normal goat serum . Cells were incubated at 4 °C overnight with a rabbit anti–NCS1 monoclonal antibody  diluted in blocking solution. After washing with PBS, cells were incubated with AlexaFluor‐488 goat anti‐rabbit secondary antibody  and rhodamine‐conjugated phalloidin  for 2 h at RT in the dark. Cells were washed with PBS and mounted on glass slides with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Slides were cured overnight before images were captured with a confocal microscope .2.9Cell proliferation was assessed using a luminescent‐based assay that measures the number of viable cells in culture based on ATP levels (CellTiter‐Glo Luminescence Cell Viability Assay [Promega]). One thousand cells were seeded into each well of a sterile 96‐well plate except for the wells at the edges of the plate. The relative number of viable cells was measured from day 0 to 4 by adding 100 μL of CellTiter‐Glo Reagent (Promega) to 10 wells per timepoint and genotype. The cells were incubated in this reagent for 15 min at RT to stabilize the luminescent signal. Luminescence was measured on a microplate reader . Three independent experiments were performed using MDA‐MB231 WT and NCS1 KO cells with 10 wells per timepoint and genotype.2.10Scratch motility assays were performed by growing MDA‐MB231 WT and NCS1 KO cells to confluency in six‐well plates. After cells were serum‐starved (1% FBS) for 12 h, a T‐shaped wound was induced in the cell layer using a sterile pipette tip. Cells were washed with sterile PBS to remove detached cells. Wound width was assessed at timepoints of 0 and 24 h by capturing images of the scratch under a light microscope. Wound widths at different locations of the wound were measured using ImageJ software and the mean for each wound was calculated. The difference between the width at 0 and 24 h was calculated and depicted as mean wound closure in cm in 24 h. Four independent experiments were performed in triplicate for each genotype.2.112. After 10 days, cells were fixed with 100% ice‐cold methanol and colonies were stained with 2.5% crystal violet. Plates were scanned with a conventional scanner and the area covered by cell colonies was analyzed with ImageJ . To measure the response to UTP, the Ca2+ reporter dye Fluo‐4‐AM (Thermo Fisher Scientific) was used. Baseline Ca2+ and response to TG were measured using the ratiometric dye Fura‐2‐AM (Thermo Fisher Scientific). Ca2+ responses to UTP and TG were measured in Ca2+‐free conditions to abolish effects of extracellular Ca2+ on the measured response. Baseline Ca2+ was assessed in Ca2+‐containing conditions. Ca2+‐containing HEPES‐buffered saline (HBS) contained 1.25 mm CaCl2, 19.7 mm HEPES, 4.7 mm KCl, 1.2 mm KH2PO4, 1 mm MgSO4, 130 mm NaCl, and 5 mm dextrose in dH2O, pH 7.4 at RT. In Ca2+‐free HBS buffer CaCl2 was replaced by 1.25 mm MgCl2 and additionally 0.1 mm EGTA was added. MDA‐MB231 WT or NCS1 KO cells were plated on glass coverslips 1 day prior to imaging at a density of 100 000 cells per coverslip. After 1 day of growth, cells were incubated at 37 °C in 5% CO2 for 40 min with Fura‐2‐AM or Fluo‐4‐AM dissolved in Ca2+‐containing HBS buffer with 0.03% pluronic acid (Thermo Fisher Scientific). Excess dye was washed away with Ca2+‐containing HBS buffer. Cells were incubated for another 10 min in Ca2+‐containing HBS. To measure the effect of the intracellular Ca2+ chelator BAPTA‐AM on baseline Ca2+ levels, WT and NCS1 KO cells were incubated with Fura‐2‐AM supplemented with 1 μm BAPTA‐AM prior to imaging. Once in view on the fluorescence microscope, approximately 20 single cells plus one empty area (background) were identified and defined as regions of interest. For Ca2+ responses to UTP and TG, cells were perfused with Ca2+‐free HBS for 30 s before imaging was started. After 10 s of imaging (F0), cells were stimulated with 10 µm UTP or 5 µm TG in Ca2+‐free HBS. After 30 s of stimulation with UTP or 60 s stimulation with TG, cells were perfused with Ca2+‐free HBS until the fluorescence signal returned to baseline. For baseline Ca2+ measurements, the ratiometric fluorescence signal of unstimulated cells in Ca2+‐containing HBS buffer was recorded over 100 s. To investigate the Ca2+ response to stimulation with UTP measured with Fluo‐4‐AM, data were analyzed by subtracting the background from all measured values followed by normalization of each timepoint to the average of the first 10 s (F1/F0). Maximal amplitude, area under the curve (AUC), and rate of rise were calculated using graphpad prism 7 . Baseline Ca2+ measurements with Fura‐2‐AM were analyzed by calculating the mean ratio of fluorescence at 340 and 380 nm under unstimulated conditions in Ca2+‐containing HBS buffer. The response to TG measured with Fura‐2‐AM was analyzed by normalizing the 340/380 ratio of each timepoint to the mean 340/380 ratio at baseline.Fluorescence‐based Ca2.13graphpad prism 7. Gaussian distribution of datasets was checked with Shapiro–Wilk normality test before applying parametric statistical tests. Significance between two groups was assessed with unpaired Student's t‐tests if the data passed normality test. For comparison of groups of three or more, ordinary one‐way ANOVA followed by Tukey post hoc test was performed. In cases of failed normality test, we applied nonparametric Mann–Whitney U‐test. P values < 0.05 were considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001.Unless otherwise specified, each experiment was performed at least three times. Depicted are bar graphs including individual data points showing mean ± SD or box plots showing the median, interquartile range, and whiskers ranging from the minimum to maximum value. Experiments were blinded for treatments and genotypes where applicable. Statistical analyses were performed using 33.1NCS1 to identify potential transcription factor binding sites using TRANSFAC, a database encompassing information on eukaryotic transcription factors, their genomic binding sites, and DNA binding profiles . This compound inhibits the degradation of IκBα, therefore preventing nuclear translocation of NFκB. After treating SHSY5Y cells with Bay for 24 h and immunoblotting for IκBα, we saw that IκBα protein levels increased Fig. , demonstion Fig. . These dase Fig. , confirm3.3via NFκB activation and NCS1 up‐regulation in human cancer, we analyzed the gene expression patterns of NFκB‐regulated genes and NCS1 expression in a dataset from an RNA microarray performed on human breast cancer and noncancerous breast tissue. A heat map of NFκB‐regulated genes was generated from RNA expression values of noncancerous  and cancerous (tumor) breast cancer. We found that genes transcriptionally regulated by NFκB have a distinct expression pattern in breast cancer tissue compared to normal breast tissue, indicating the differential regulation of NFκB signaling in breast cancer  cells were confirmed through immunofluorescence (IF) staining Fig.  and west3.5et al., et al., et al., et al., Whereas previous publications focused on the effect of NCS1 overexpression (NCS1 OE) on promoting tumor aggressiveness , is a well‐established survival‐promoting protein  reinforce its importance for cell survival and motility, and (c) demonstrate that NCS1 modulates Ca2+ signaling and Akt activity, both of which are important for cancer progression  establish the CaκB Pahl,  and led et al., et al., 2+ signaling and consequent regulation of Akt activation. We found that NCS1 regulates Ca2+ homeostasis and signaling by maintaining low intracellular Ca2+ levels under resting conditions and by enhancing InsP3‐dependent ER Ca2+ release. Additionally, we found that NCS1 increases Akt signaling in a Ca2+‐dependent manner. Both Ca2+ signaling and Akt are well‐established modulators of cell survival and motility , and F31DK118836 (ALB) supported this work. We acknowledge the use of the Yale Center for Cellular and Molecular Imaging (NIH grants 5P30DK034989 and OD020142). BEE is a founder of Osmol Therapeutics, a company that is targeting NCS1 for therapeutic purposes. The other authors declare no conflicts of interest.HKG and BEE designed the study. HKG performed the majority of the experiments, analyzed the results, and drafted the manuscript. TTF, ALB, and JAS performed additional experiments. HKG, TTF, ALB, and BEE edited the manuscript. All authors edited and agreed to the final version of the manuscript.Fig S1. NCS1 mRNA expression does not change with all cell stressors.Click here for additional data file.Fig S2. Wound healing assay.Click here for additional data file.Fig S3. MDA‐MB231 cells stably overexpressing NCS1.Click here for additional data file.Fig S4. Effect of NCS1 expression on endogenous Akt inhibitor PP2Ac.Click here for additional data file.Fig S1‐S4Click here for additional data file.


To assess the safety of biosimilar intravitreal aflibercept  compared to the reference product  in rabbit eyes through functional and histologic studies.  Forty New Zealand albino rabbits were recruited to the study and were divided into four groups to be sacrificed at 48 hours, one, two, and four weeks after injections. In each group, five rabbits received 0.05 mL (2 mg) biosimilar aflibercept in the right eye and 0.05 mL saline in the left eye as the control, and in a similar manner, the remaining five rabbits received the reference drug in the right eye and saline in the left eye. All the rabbits underwent comprehensive ophthalmic examination and electroretinography (ERG) tests at baseline and also just before enucleation at the specific predefined time points. The enucleated eyes were prepared for retinal toxicity histological examination.  No retinal toxicity was observed based on histologic and ERG findings in all groups. Choroidal congestion was revealed after 1 week in an eye that was injected with biosimilar aflibercept, although the similar finding was detected in the contralateral eye which received saline. Also, one subject which received the reference drug showed chronic vitritis and lymphoplasmocytic reaction of the optic disc at week 4. The remaining subjects showed no histologic changes.  The 2 mg intravitreal injection of biosimilar aflibercept  was found to be nontoxic in rabbit eyes in the short-term period. Further studies are required to warrant the efficacy and safety profile of the drug in human subjects. In 1989, the VEGF was introduced as a protein responsible for vascular permeability and endothelial cell proliferation that plays a key role in angiogenesis. Thereafter, it became the core of the treatment of ophthalmic vascular and degenerative disorders .Previously, level of intravitreal VEGF has been revealed to be correlated with the severity of the retinal vascular diseases, and inhibition of this protein in experimental models has been shown to regress the new vessels of the retina and choroid and prevent visual impairment , 3.®  received FDA approval for the treatment of neovascular age-related macular degeneration (nAMD) and diabetic macular edema (DME) [Aflibercept under the brand name of Eyleama (DME) . It consma (DME) .As many of these patients need frequent intravitreal injections, one arising issue, in this regard, is the treatment burdens imposed on the health care systems , 6.Nowadays, many biosimilar agents have been commercially available. They are exactly analogous to the reference drugs in terms of structure, safety, and efficacy but with different production methods and lower cost relative to the reference drugs, which facilitate treatment access particularly in the developing countries , 8.This study was conducted to determine the safety and possible toxicity of the intravitreal injection of the biosimilar aflibercept  in albino rabbits in terms of histopathologic and functional evaluations.Forty New Zealand albino rabbits, between 1.5 and 2.5 kg, were recruited to assess the safety of intravitreal biosimilar aflibercept injection. All procedures were performed following the principal tenets of using animals in ophthalmic and vision research (ARVO). In addition, approval from Tehran University of Medical Science's Institutional Review Board (IRB) and ethics committee was obtained.The rabbits were kept in an air-conditioned room with a 12-hour light-dark cycle and fed with standard processed laboratory food.®) in their right eye and saline in the left.Four groups of the rabbits (10 in each groups) were planned for the injections. Ten rabbits were programmed or assigned to be sacrificed 48 hours after injection, and the remaining were divided into three separate groups to be sacrificed one (10 rabbits), two (10 rabbits), and four weeks (10 rabbits) after injections. Clinical examinations and electroretinogram (ERG) were performed at baseline and just before sacrificing the rabbits. Five rabbits of each group were randomly selected to receive biosimilar aflibercept in their right eye and saline in the left, and the remaining five rabbits in each group received the reference drug  and xylazine hydrochloride (5 mg/kg). Eye examinations were done through the dilated pupil by topical tropicamide (0.5%) eye drop at each session.The rabbits were anesthetized with the aforementioned combination of drugs. They underwent baseline ophthalmic examination and ERG, and then the injection was performed under a sterile condition using a 29-gauge needle after instillation of povidone-iodine 5% into fornixes for 2 minutes.All injections were performed through 1.5 mm posterior to the limbus into the midvitreous. Chloramphenicol and timolol eye drops were applied to the eyes for the first three days after injections.All the eyes underwent thorough ophthalmic examination at baseline, first and second days following injections. Furthermore, the examinations were performed at weeks one, two, and four after injection using the hand-held slit-lamp and indirect ophthalmoscope before considering the eyes for enucleation.Anterior chamber and vitreous cavity were carefully examined with the highest magnification to detect any cell or flare. At each follow-up visit, all the eyes underwent indirect ophthalmoscopy to be assured of sharp and clear imaging of the disc and the retina and to exclude any pathologic finding in these structures.Any conjunctival injection, corneal opacity, or ulcer, crystalline lens damage or abnormality, inflammatory responses in the anterior chamber or the vitreous cavity, and retinal pathology were recorded.a- and b-wave were measured as ERG responses. The a-wave amplitude was defined as the distance from the isoelectric line to the negative spike and the b-wave as the distance from the negative spike to the positive spike. Latencies of the a and b waves were measured form the time of presenting the stimuli.All the rabbits underwent ERG, using the electrophysiological test system , at baseline and days 2, 7, 14, and 28 following injections regarding their groups. Before the test, all dark-adapted rabbits were anesthetized, and ERG was performed by means of a contact lens on the cornea, a negative electrode, and ground electrode that were placed at the orbital rim and ear, respectively. The intensity of the flash light was selected 10 cdsm [At the final visit, all rabbits underwent ophthalmic examination and ERG under deep anaesthesia, and then they were euthanized using 100 mg/kg of sodium pentobarbital intravenous injection, and the eyes were enucleated with cautions to prevent probable globe violation. All eyes were transferred into a neutral formalin medium as soon as possible and then fixed by 10% formalin to prevent tissue degradation.µm thickness was prepared and stained with hematoxylin and eosin (H&E).Tissue processing and specimen preparation were performed by a pathologist well experienced in ophthalmic diseases, and a section of the tissue with 4 We performed the mentioned process for almost all parts of the eye including conjunctiva, cornea, iris, ciliary body, lens, choroid, retina, sclera, optic disc, anterior chamber, and angle vitreous by means of a light microscope.Any abnormal finding including edema, haemorrhage, inflammatory responses, necrosis, and deposition of the unusual materials was considered. Also, different retinal layers' thickness, number of the ganglion cells, and changes of the retinal pigment epithelium (RPE) layers were taken into account.®) groups and also between the biosimilar aflibercept and the saline groups, separately. The changes more than 20% of the baseline in the ERG parameters were considered significant in each group. All data were analyzed using SPSS , and the significance value of P was considered less than 0.05.The Mann–Whitney U-test was used to analyse the effect of the injections on the ERG parameters between the biosimilar aflibercept and the reference drug . These ERG parameters showed no significant difference in each time point after injections between the biosimilar aflibercept-injected eyes and the control groups   .As aforementioned, the changes of ERG parameters more than 20% were considered significant during the study. Tables a-wave amplitude at day 14 in the biosimilar group compared with the baseline, fairly comparable to the Eylea arm that was 10.5% at week 4, no significant changes were found in a-wave amplitude and implicit time after injections in each group.Dark-adapted bright flash ERG was done on all rabbits before the intravitreal injection as a baseline standard and then at each time point. Despite of a 14.6% reduction of b-wave amplitude and latency during the follow-up visits of the subjects. There was a comparable reduction of b-wave amplitude in biosimilar aflibercept and the reference drug-injected eyes  at week 4.Similarly, there were no clinically significant changes in Considering presence of chronic conjunctivitis in some specimens from both control and drug-injected groups, this pathological finding is not necessarily related to drug administration.All the eyes which were enucleated 2 days after biosimilar aflibercept injection had no pathologic finding unless one that showed mild chronic conjunctivitis and iridocyclitis; similar finding was evident in the contralateral eye which received saline.®).Among rabbits that were enucleated one week after injections, mild choroidal congestion was reported in both eyes of a rabbit that received biosimilar aflibercept and normal saline into the right and left eye, respectively. Chronic vitritis and lymphoplasmocytic infiltration of the optic nerve head were detected at 4 weeks after injection in an eye that was injected with the reference drug  was detected  which was partly comparable with the reference drug group at month 1 (10.5%).In agreement with previous studies , we consb-wave amplitude during the study; the maximum change in the biosimilar aflibercept group belonged to week 4 (9.1%) which was similar with the reference drug group (10%). Also, there was no statistically significant difference in ERG parameters between the biosimilar group and the two other groups at different time points  was normal. Intravitreal injection of the drugs can damage the eye by several mechanisms including the direct toxicity of the vehicle, inflammatory response against the immunogenic molecules, and interaction with the VEGF pathway and signalling which play a role in inflammatory responses [Likewise, there was no significant change in the e points . The milesponses .On the basis of the ERG and histologic evaluation, the biosimilar did not show significant toxicity on the ocular tissues, particularly, the retina. However, it should be taken into account that the ERG mainly represents the photoreceptors and bipolar cell function and is not applied to the ganglion cells , 25. FurThis study showed the safety of intravitreal biosimilar aflibercept injection in a short-term evaluation. However, further studies are necessary to validate the safety profile and probable efficacy of this new biosimilar drug in longer follow-up durations.


Little was known about the exact mitochondrial Ca2+ homeostasis in the living brain, particularly in AD. Only now, with the development of intravital imaging techniques and transgenic mouse models of the disease, we are able to directly observe Ca2+ levels in specific regions or particular subcellular compartments of cells, such as mitochondria. Using multiphoton microscopy, a Ca2+ reporter targeted to mitochondria and a mouse model of cerebral β amyloidosis (APP/PS1), our recent study  found elevated mitochondrial Ca2+ concentration in the transgenic mouse after plaque deposition, and after topical application of natural soluble amyloid beta (Aβ) oligomers to the healthy mouse brain at concentrations similar to those found in the human brain. Elevated Ca2+ in mitochondria preceded neuronal death and could be targeted for neuroprotective therapies in AD. Here, we describe our main findings and pose new questions for future studies aimed at better understanding mitochondrial Ca2+ dyshomeostasis in AD.Alzheimer's disease (AD), the most common cause of dementia, affects millions of people worldwide. Suggested mechanisms of neurotoxicity in AD include impaired calcium (Ca However, excessive levels of Ca2+ in mitochondria, i.e. mitochondrial Ca2+ overload, can lead to an increase of reactive oxidative stress (ROS) species, decrease of ATP production and eventually to cell death via apoptosis. Cytosolic Ca2+ homeostasis is impaired in AD, as evidenced by hyperactive neurons close to amyloid plaques or high cytosolic Ca2+ in transgenic mice with plaque deposition. But, is mitochondrial Ca2+ homeostasis also impaired in vivo in AD? Are Ca2+ levels increased or diminished in the neurons? In vitro studies suggested that mitochondrial Ca2+ homeostasis was altered in the presence of Aβ oligomers (Aβo), but these questions remained unresolved in vivo. To address this issue, we used a transgenic mouse model of cerebral amyloidosis, APP/PS1 – a model that develops amyloid plaques comparable to those from human patients starting at around four months of age – and intravital imaging with multiphoton microscopy. To measure Ca2+ concentration in mitochondria, we used a ratiometric Ca2+ reporter  targeted to neuronal mitochondria (hSyn.2mt.YC3.6), that we injected as adenovirus (AAV) in the somatosensory cortex of the mice. This ratiometric reporter, based on Förster resonance energy transfer (FRET), allows for quantitative measurement of absolute Ca2+ concentration and direct comparison between cells and compartments. We observed mitochondrial Ca2+ overload in the neurons of old APP/PS1 transgenic mice, once plaque deposition had started, unlike the young transgenic mice (pre-pathology). Transgenic mice showed twice as many mitochondria with high Ca2+ compared to the wild-type (Wt) mice. This difference was found in both somas and neurites. Other studies have previously reported no changes in cytosolic Ca2+ or neuronal function before plaque deposition, supporting the notion that mitochondrial Ca2+ overload in neurons follows, rather than precedes, amyloid plaque deposition. Figure 1 shows an example of in vivo multiphoton microscopy images of mitochondria in neurons in APP/PS1 mouse and in Wt mouse after transduction of the AAV. We did not find any correlation between mitochondrial Ca2+ overload and the distance to plaques, suggesting that diffusible soluble Aβ species , rather than individual plaques, are responsible for the mitochondrial Ca2+ increase in the neurons of the transgenic mouse.Ca2+ increase in neurons, we topically applied naturally secreted Aβo  at concentrations similar to those found in the human AD brain directly onto the surface of the living brain of a Wt naïve mouse (C57BL/6J) expressing the adenovirus hSyn.2mtYC3.6. Application of TgCM transiently increased mitochondrial Ca2+ concentration in the neurons, whereas Wt conditioned media (obtained from Wt littermates) or Aβ-immunodepleted TgCM did not. Neurites were more susceptible to this increase than somas. These results confirmed our previous observations in the APP/PS1 transgenic mouse, and implicated soluble Aβo in the mitochondrial Ca2+ dyshomeostasis in vivo. After returning to normal mitochondrial Ca2+ levels, we observed that mitochondrial shape and size were altered – mitochondria were smaller and rounded –, suggesting that the toxic effects of Aβo in vivo were not only restricted to mitochondrial Ca2+, but also to mitochondrial morphology. Additionally, this suggests that the change in size and shape is a secondary event after a mitochondrial Ca2+ increase. It is believed that mitochondrial fragmentation precedes neuronal cell death, which establishes a connection with mitochondrial Ca2+. Interestingly, the APP/PS1 transgenic mouse also exhibits fragmented and swollen mitochondria once the pathology is present, and the brains from AD patients show impaired balance of mitochondrial fission and fusion proteins.To confirm that soluble Aβ species drive the mitochondrial Ca2+ uptake? And is it possible to inhibit it in order to prevent or decrease the mitochondrial Ca2+ overload? The mitochondrial Ca2+ uniporter (MCU) complex is the main pathway for mitochondrial Ca2+ influx. However, the mechanism by which Aβ induces a Ca2+ increase in neuronal mitochondria in vivo was not previously known. Various hypotheses proposed that Aβ could create pores in membranes, form a Ca2+-permeable pore itself or act on native channels. In this study, we demonstrated that blocking the MCU complex in vivo with Ru360 – a specific blocker of the channel – prevented the mitochondrial Ca2+ uptake elicited by TgCM. To confirm that Ru360 application did not render neurons insensitive to Aβ or blocked the Ca2+ influx channels of the plasma membrane, we measured the cytosolic Ca2+ influx induced by Aβo with the same Ca2+ reporter (YC) targeted to the cytosol of neurons (CBA.YC3.6). We observed that Ru360 did not interfere with the rise in the cytosolic Ca2+ induced by Aβ. These results suggest that MCU is required for the increase in mitochondrial Ca2+ induced by Aβ in vivo, and points to MCU as a potential target candidate for AD.How does Aβ induce mitochondrial Ca2+ in the in vivo brain? We observed that in the APP/PS1 transgenic mouse, the small fraction of neurons with elevated mitochondrial Ca2+ in the soma died within 24 hours. Their nuclei became condensed (a sign of apoptosis) and the AAV fluorescence in the soma was lost. This effect did not happen in cells with normal mitochondrial Ca2+ levels. Rapid neuronal death was previously observed in the APP/PS1 model following increased oxidative stress in the cytosol. Our in vitro data showed that, in addition, soluble Aβ triggered opening of the mitochondrial permeability transition pore (mPTP), a decrease of the mitochondrial membrane potential and activation of caspases, events that were secondary to the increase in mitochondrial Ca2+. All these findings together link high mitochondrial Ca2+, oxidative stress and neuronal cell death via apoptosis in vivo in AD.What are the implications for increased neuronal mitochondrial Ca2+ homeostasis impaired in the human Alzheimer's brain? Because direct analysis of mitochondrial Ca2+ levels in the human brain is not feasible, could we evaluate the proteins or channels involved in mitochondrial Ca2+ transport in the human brain? And if we can, to what extend are they affected? Using data publicly available at the AMP-AD (Accelerating Medicines Partnership-Alzheimer's disease) knowledge portal, we evaluated the differential expression of genes encoding proteins involved in the mitochondrial Ca2+ transport. Ca2+ influx into mitochondria is mainly led by the MCU complex, which includes the MCU pore, the regulatory subunits MICU1 and MICu2, the essential regulator EMRE, and other proteins such as MCUb and MICU3. Ca2+ efflux from mitochondria is primarily controlled by the Na+/Ca2+ exchanger (NCLX). We compared the expression of the genes encoding these proteins between AD subjects and control individuals using microarray and RNA-seq datasets from the MSBB (Mount Sinai Brain Bank) and the ROSMAP (Religious Orders Study and Memory and Aging Project) cohorts. We observed that the MCU and all subunits of the complex – Ca2+ influx – were downregulated in AD compared with control subjects, whereas NCLX – Ca2+ efflux – was the only one upregulated. These results suggest a dysregulation in the human genes encoding for mitochondrial Ca2+ transport, and imply a counteracting effect to avoid mitochondrial Ca2+ overload in the neurons in the AD human brain.Lastly, is mitochondrial Ca2+ in AD. It suggests that Aβ aggregates (particularly soluble Aβ) increase mitochondrial Ca2+ in AD via the MCU in vivo, an effect that is linked to neuronal cell death via apoptosis . We demonstrated that the extent of mitochondrial Ca2+ alterations are spatially restricted in the living brain and highlight the need for imaging studies that can probe these anatomically specific effects. However, this work highlights other questions that remain unresolved. Does mitochondrial Ca2+ elevation occur concomitantly with plaque formation? Is it possible to observe mitochondrial morphological abnormalities and altered dynamics  induced by Aβ in vivo? Does tau have any effect on mitochondria, both in terms of Ca2+ abnormalities and motility? Additionally, our studies point to MCU as a possible therapeutic target for AD and other neurodegenerative diseases in which mitochondrial Ca2+ is impaired, however, further efforts will be needed to unveil the clinical relevance of our findings.This study helped clarify the understanding of the dynamics of mitochondrial Ca