Nucleophosmin (NPM1) protein regulates several cellular processes and is predominantly located in the nucleolus, owing to the localization signal provided by two tryptophan residues. In acute myeloid leukemia (AML), NPM1 gene is frequently mutated, leading to the aberrant translocation of the protein into cytoplasm. In the present work, we classified NPM1 mutations according to the loss of either one or both tryptophan residues as non-A-like and A-like mutations, respectively, and evaluated their biological features. We found that non-A-like mutations partially delocalize NPM1 protein into the cytoplasm, with a proportion of remaining nucleolar protein preserving p53 protein expression and downstream activity. Different HOXA and HOXB gene expression and cell death pathway activation between A-like and non-A-like NPM1-mutated cells were shown, with an enhanced sensitivity to chemotherapy for AML cells with non-A-like mutations. This study suggests the need for a sub-classification of NPM1-mutated AML, with subsequent implications in the therapeutic management.Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling protein, predominantly located in the nucleolus, that regulates a multiplicity of different biological processes. NPM1 localization in the cell is finely tuned by specific signal motifs, with two tryptophan residues (Trp) being essential for the nucleolar localization. In acute myeloid leukemia (AML), several NPM1 mutations have been reported, all resulting in cytoplasmic delocalization, but the putative biological and clinical significance of different variants are still debated. We explored HOXA and HOXB gene expression profile in AML patients and found a differential expression between NPM1 mutations inducing the loss of two (A-like) Trp residues and those determining the loss of one Trp residue (non-A-like). We thus expressed NPM1 A-like- or non-A-like-mutated vectors in AML cell lines finding that NPM1 partially remained in the nucleolus in the non-A-like NPM1-mutated cells. As a result, only in A-like-mutated cells we detected HOXA5, HOXA10, and HOXB5 hyper-expression and p14ARF/p21/p53 pathway deregulation, leading to reduced sensitivity to the treatment with either chemotherapy or Venetoclax, as compared to non-A-like cells. Overall, we identified that the NPM1 mutational status mediates crucial biological characteristics of AML cells, providing the basis for further sub-classification and, potentially, management of this subgroup of patients. Nucleophosmin NPM1) is a multifunctional, nucleocytoplasmic shuttling protein, that shows a predominant nucleolar localization  is a mul. It perfThe nucleus-cytoplasm shuttling of NPM1 is regulated by specific signal motifs. A bipartite nuclear localization signal (NLS) drives NPM1 from the cytoplasm to the nucleoplasm. NPM1 then localizes in the nucleolus through an aromatic nucleolar localization signal (NoLS), a 3-helix structure located at the C-terminus. NoLS contains two tryptophan residues (W288 and W290) that stabilize the hydrophobic core of triple helix, enabling its proper folding ,11.The human NPM1 gene is located on chromosome 5q35 and contains 12 exons. NPM1 mutations have been identified in about one-third of adults and 6–8% of pediatric patients with acute myeloid leukemia (AML) ,14,15, aNPM1 mutations significantly associate with normal karyotype and often with abnormalities of the FLT3 gene, in particular with the internal tandem duplication (ITD). The favorable prognostic impact in the FLT3ITD mutation for pediatric AML is still controversial ,18.To date, more than 40 NPM1 variants have been reported in adult AML ,21. MostHere, we aimed to investigate the heterogeneity of NPM1 mutations, and found that NPM1 partially remains in the nucleolus when one Trp residue within NoLS is maintained. This diversity translates into distinct HOX gene expression and p14ARF-p53 pro-apoptotic pathway activation, with a different susceptibility to drug treatment.Vector construction and site-specific mutagenesis. pEGFP-C1-NPM1wt vector was previously described ; NPM1 waIn vitro cell culture and treatments. The leukemia cell line HL-60  was maintained in RPMI 1640 , while SHI-1 (DMSZ) in DMEM (Thermo Fisher Scientific). All the media were supplemented with 10% FBS (Thermo Fisher Scientific), 2 mM glutamine , and 100 U/mL streptomycin/penicillin . Primary cells were obtained by bone marrow samples of pediatric patients affected by de novo AML, provided by the pediatric OncoHematology Lab of Padova Hospital, being the Associazione Italiana Emato Oncologia Pediatrica (AIEOP) reference center for the pediatric AML diagnosis. Diagnosis of leukemia was established according to standard criteria based on immune-histochemical staining, immune-phenotyping, cytogenetic studies, and molecular genetics, as detailed in the AIEOP AML 2002/01 treatment protocol . Primary6 HL-60 or SHI-1 were nucleofected with 1.2 μg of pEGFP plasmids, using Sol V (Lonza).Cell transfection. Transient cell transfection was performed using the Nucleofector technology , according to the manufacturer’s instructions. Briefly, 5 × 10Immunofluorescence. EGFP fluorescent cells were analyzed after cytospin, 8 h post transfection, using Zeiss 800 Confocal microscope . Nuclei were stained with DAPI .Western Blot. Western blots were hybridized with anti-GFP , anti-p53 (Pab 1801), anti-HDAC1 , anti-GAPDH , all from Santa Cruz Biotecnology ; anti-p21 , anti-p14ARF . The horseradish peroxidase-conjugated secondary antibody was either anti-rabbit or mouse (Merck Millipore). Immunoblotting was performed as previously described , and sigGene expression (GEP) analysis. Gene expression data were downloaded from GEO Database (GSE75461) . Unsuper−ΔΔCt method.Quantitative real-time PCR. Total RNA was isolated using Trizol (Invitrogen—Thermo Fisher Scientific). One μg of RNA was reverse-transcribed into cDNA using the SuperScript II system (Invitrogen—Thermo Fisher Scientific) according to the manufacturer’s instructions. Expression of mRNA was measured by real time PCR (RQ-PCR) on an ABI 7900HD platform  using the Platinum™ SYBR™ Green qPCR SuperMix (Invitrogen—Thermo Fisher Scientific) and normalized on GUS housekeeping gene, using the 2Apoptosis analysis. Apoptosis was evaluated by double staining with Annexin-V/7-Aminoactinomycin D  and analyzed using Cytomic FC500 . Apoptotic cells were expressed as the percentage of Annexin-V-positive and/or 7AAD-positive cells and were compared to control (DMSO).® assay  following the manufacturer’s guidelines. Briefly, 50 × 103 cells in 100 μL of proper medium were cultured in sterile 96-well plates and incubated at 37 °C. Cell viability-ATP production was evaluated at the indicated time points by adding 100 μL CellTiter-Glo® reagent, shaking plates for 2 min then incubating for 20 min at room temperature and recording the luminescence with the VICTOR3TM multilabel plate reader  with an integration time of 1 s per well.Cell viability assay. Cell viability of AML cells was evaluated using CellTiter-Glot-test was adopted for significance between differences in means when two groups were evaluated; ANOVA was used to compare more than two groups. Graphs and associated statistical analyses were generated using GraphPad Prism 8 . All data are presented as mean ± standard error of the mean (S.E.M.). * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001 were considered statistically significant.Data evaluation and statistical analyses. The In order to evaluate the intracellular localization of mutated NPM1, we generated EGFP-NPM1mut constructs and transfected HL-60 cells. Briefly, by mutagenesis of the NPM1 wild type plasmid  we generp < 0.05, ** p < 0.01, *** p < 0.001), supporting the hypothesis that the activation of crucial HOX genes differ according to the mutation type.Aberrant expression of HOX genes has been frequently associated with specific AML genetic subtypes, including NPM1-mutated. Therefore, we next evaluated HOX gene expression, in order to identify a possible correlation with the different NPM1 mutations. Through the analysis of our previously described pediatric AML cohort gene expression signature . These results are even more evident when drug-induced apoptosis was analyzed clustering together the NPM1 A-like mutations compared with the non-A-like mutations . We confirmed the observed heterogeneity of NPM1 mutations in triggering apoptosis by treating another cell line, the SHI-1 cells with Eto, AraC, or Ven (p < 0.05). Similar results were obtained by evaluating cell viability through ATP measurement 24 h after treatment with Eto: in SHI-1 transfected with NPM1mut7, treatment resulted in a significantly higher reduction of cell viability than in cells transfected with NPM1mutB (p < 0.05). To further validate these findings, we tested AraC in two NPM1-mutated AML primary samples, confirming that the non-A-like NPM1-mutated AML cells were more sensitive to the effect displayed by AraC than A-like-mutated cells (p < 0.01). Moreover, we attempted to treat transfected cells with a combined treatment showing that this combo was more effective in NPM1mut7, but further drug combinations need to be tested in this context of NPM1-mutational status , Cytarabine (AraC), or with the BCL-2-targeting drug Venetoclax (Ven). As shown in l status .In patients affected by AML, NPM1-isolated mutations are markers associated with relatively good prognosis and, therefore, patients carrying these abnormalities are included in the standard risk prognostic group in national and international trials . HoweverWe showed that in non-A-like NPM1 mutations, the residual Trp allowed a partial nucleolar NPM1 protein expression, partially sustaining its physiological function. An important feature of NPM1-mutated AML is the upregulation of HOX family genes , that weAnother important feature associated with the NPM1 localization is represented by p53-dependent apoptosis. Here, we showed that p53 and p21 levels are modulated according to the number of Trp residues substituted at the C-terminus of NPM1. In particular, both p53 and p21 were less expressed in A-like-mutated cells than in non-A-like-mutated blasts, due to p14ARF reduction and cytoplasmic dislocation driven by A-like NPM1 mutation  with theOverall, our results prove that different NPM1 mutations mediate diverse clinical-biological characteristics of AML cells, suggesting that patients with AML harboring a NPM1 mutation may benefit from a refined sub-classification (A-like or non-A-like-mutated) and a subsequent tailored management including the possibility of novel target treatment strategies to improve their outcome.This study suggests that the classification of NPM1-mutated AML in A-like or non-A-like cases can reflect different biological and clinical peculiarities. Further deconvolution of the heterogeneity of NPM1 mutations in pediatric AML may pave the way for more tailored and possibly effective therapeutic strategies.


In vitro maturation of human primary myoblasts using 2D culture remains a challenging process and leads to immature fibers with poor internal organization and function. This would however represent a valuable system to study muscle physiology or pathophysiology from patient myoblasts, at a single-cell level.2+ measurements upon electrical stimulations were performed at various time points during maturation to assess the organization and function of the myotubes.Human primary myoblasts were cultured on 800-nm wide striated surface between two layers of Matrigel, and in a media supplemented with an inhibitor of TGFβ receptor. Gene expression, immunofluorescence, and Ca2+ responses to repetitive electrical stimulations.We show that after 10 days in culture, myotubes display numerous functional acetylcholine receptor clusters and express the adult isoforms of myosin heavy chain and dihydropyridine receptor. In addition, the myotubes are internally well organized with striations of α-actinin and STIM1, and occasionally ryanodine receptor 1. We also demonstrate that the myotubes present robust Ca2+ signals.The present method describes a fast and efficient system to obtain well matured and functional myotubes in 2D culture allowing thorough analysis of single-cell CaThe online version contains supplementary material available at 10.1186/s13395-021-00268-3. The conformational change of DHPR is transmitted to the ryanodine receptor 1 (RyR1), a Ca2+ channel localized at the terminal cisternae of sarcoplasmic reticulum (SR). The opening of RyR1 leads to a strong release of Ca2+ from the SR and eventually muscle contraction, an overall process called excitation–contraction (EC) coupling. Because of the size and organization of muscle fibers, and even if important progresses were accomplished over the years, it remains challenging to obtain well differentiated and matured myofibers in vitro. Several models of myogenesis were designed to generate muscle tissue that could be eventually engrafted in damaged/diseased muscles . One. One2+ coutcomes –11.in vitro maturation system that uses striated surface to culture human primary myoblasts. We report evidence of clear beneficial effects of growing and differentiating muscle cells on a striated surface, both at structural and functional levels. We show that on this surface, myocytes display rapidly a high level of internal organization and numerous AChR clustering. Finally, this culture model allows thorough analysis of single-cell robust and repetitive Ca2+ transients elicited by electrical field stimulations.In the present study, we describe an Human primary myoblasts were isolated from semitendinous muscle samples obtained after orthopedic surgery  on patients without known muscular diseases. All samples were collected anonymously after obtaining a written consent and approval by the University of Geneva (protocol CCER no. PB_2016-01793 12-259) accepted by the Swiss Regulatory Health Authorities and approved by the “Commission Cantonale d'Ethique de la Recherche” from the Geneva Cantonal Authorities, Switzerland). The purification of myoblasts was performed as previously described , 372+ se2+ transients upon electrical field stimulation. Already at 4 days of culture, electrical stimulations resulted in large Ca2+ transients of almost all myotubes, highlighting a functional EC coupling. This time frame is rapid if we compared it to the 8 days needed to obtain 50% of myotubes responding to electrical stimulation with classical 2D culture of human cells [2+ recordings, the amplitude of the first transient, the kinetic of the Ca2+ recovery phase, and the sustained nature of the signals during the 3-min stimulation. None of these parameters significantly changed between 4 and 10 days, showing that even if the cells displayed a better internal organization at 10 days, this did not correlate with an improved Ca2+ signals. This is however in agreement with the expression levels of RYR1, CACNA1S, and ATP2A1/2 that remain stable during the maturation period we analyzed.Finally, we assessed the ability of myofibers to elicit cytosolic Caan cells . We analMYH, increased adult CACNA1S isoform expression, and a high level of internal fiber organization. Further improvements are however required to obtain myofibers that resemble more the adult organization and function. Nevertheless, this rapid and easy-to-perform 2D culture, which does not require sophisticated technology, presents certainly great advantages for further single-cell studies designed to investigate the processes of skeletal muscle maturation.Overall, we proposed a 2D culture system of human myotubes that reach after 10 days a high level of maturation, with the presence of numerous functional AChR clusters, the expression of adult Additional file 1: Movie 1. Spontaneous myotube contraction on 6-day old culture on nanopattern surface.Additional file 2: Supplemental Figure 1. The reserve cell population is not affected by the surface pattern. (A) Immunofluorescence of nuclei of 4-day old myotubes on Flat (upper panel) or nanopattern surface . Cell nuclei were stained using an antibody against Pax7 (in green), MyoD (in red) and all nuclei were counterstained with DAPI (in blue). Scale bar: 50 μm. Images shown are representative of six independent experiments.  Percentage of Pax7 positive nuclei (B) and MyoD positive nuclei (C). Error bars are Mean ± SEM. Supplementary Figure 2. Length of the sarcomere on culture myotubes and on adult tissue. (A-B) Representative images of 10-day old myotubes cultured on nanopattern surface (A) and adult human muscle tissue (B) Scale bar: 10 μm. (C-D) Corresponding line profiles (white dotted line) of α-actinin normalized fluorescence intensity. Supplemental Figure 3. Organization of AChR clusters and Ca2+ response to acetylcholine on flat and striated surface. (A) Representative images of 4-day old myotubes cultured on flat (left panel) and nanopattern surface (right panel) and immunolabeled for α-actinin (red) and α-bungarotoxin  together with a DAPI staining (blue). Scale bar: 50 μm. (B) Myotubes were loaded with 5 μM Cal520-AM to measure changes in cytosolic Ca2+ and stimulated with 10 μM of acetylcholine (ACh). Traces represent the mean ± SEM of Ca2+ responses of myotubes differentiated for 4 day on flat surface (left panel) and striated surface (right panel). (C) Statistical evaluation of the amplitude of the peak following ACh stimulation. Error bars are Mean ± SEM. (D) Bar chart representing the percentage of responding cells on both surfaces. Data were obtained from three independent experiments.


Due to the importance of Ca2+ in inducing the immune response, some viruses have evolved mechanisms to modulate intracellular Ca2+ concentrations and the mobilization of this cation through Ca2+ channels to increase their infectivity and to evade the immune system using different mechanisms. For instance, some viral infections require the influx of Ca2+ through ionic channels as a first step to enter the cell, as well as their replication and budding. Moreover, through the expression of viral proteins on the surface of infected cells, Ca2+ channels function can be altered, enhancing the pathogen evasion of the adaptive immune response. In this article, we review those ion channels and ion transporters that are essential for the function of immune cells. Specifically, cation channels and Ca2+ channels in the context of viral infections and their contribution to the modulation of adaptive immune responses.Most cellular functions require of ion homeostasis and ion movement. Among others, ion channels play a crucial role in controlling the homeostasis of anions and cations concentration between the extracellular and intracellular compartments. Calcium (Ca Na+, K+ganelles . Therefoganelles . Given tganelles . Overallganelles . Usuallyganelles .2+ release-activated Ca2+ (CRAC) channels, voltage-gated such as K+ channel (Kv)1.3 and Ca2+-activated K+ channel (KCa)3.1, transient receptor potential (TRP) channels, P2X receptors family and pannexin hemichannels, voltage-gated Ca2+, Na+, and H+ channels, and Mg2+ and Zn2+ channels, among others  and the subsequent cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol  trisphosphate (IP3) and diacylglycerol (DAG) by PLCγ. After this, the binding of IP3 with to the IP3-receptor (IP3R) on the endoplasmic reticulum (ER) allows the diffusion of Ca2+ into the cytoplasm  and ORAI 1–3 proteins . During immunological synapsis, PMCAs are in close contact with CRAC channels, which modulates the activity of the ATPase pump. However, PMCAs efflux of Ca+2 from the T cell is reduced, to allow mitochondrial control of Ca+2 flux (2+ ATPase (SERCA), which removes cytosolic Ca2+ into the ER and the mitochondria  consists of another group of channels related to immunity and myeloid and lymphoid cell modulation . These c and Na+ . P2X7 rene cells . Activatonocytes . Furtheronocytes . These conocytes . Remarkaonocytes . Becauseonocytes . This moonocytes . Activatonocytes . Gasdermonocytes . Pyroptoonocytes , which donocytes . The infonocytes . Gasdermonocytes . Remarkat manner . TherefoDrosophila melanogaster mutants and constitute a superfamily including 6 subfamilies in humans: TRP canonical (TRCP), TRP vanilloid (TRPV), TRP melastatin (TRPM), TRP ankyrin (TRPA), TRP mulcolipin, and TRP polycystic  is a highly selective Mg2+ channel that is not permeable to other divalent cations such as Ca2+, Zn2+, and Ni2+  in T cells, but rather the primary function of this channel might be related to Ca2+ influx  and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) are ionotropic glutamate receptors that deserve mention, as they are considered receptors linked to ion channels and are relevant during glutamatergic transmission in the central nervous system , and the function of these channels during the immunological synapsis and other processes such as T cell activation, proliferation, and differentiation, will be discussed in this section.One of the most relevant processes that initiate an adaptive immune response is the interaction between antigen-presenting cells (particularly DCs) and T cells , 2011. Tv1.3 results in an impairment in the expression of maturation markers, MHC-II expression, and secretion of cytokines . After activation of T lymphocytes, there is a release of Ca2+ from the ER into the cytoplasm that leads to the activation of CRAC channel  are formed in the immediate vicinity of the T cell receptor-pMHC complex and concentrate most CD28 and T cell receptor molecules . Surroun channel . This wiotential . The prihe cSMAC . The cenhe cSMAC . The meche cSMAC . Becausehe cSMAC .2+, Zn2+, and Ca2+ and the activity of ion channels and transporters  into DAG and IP3, molecules that act as second messengers in these cells .Phospholipase Cγ is composed of three SH domains needed to achieve proper phosphorylation of the phospholipase . The act2+ entry . Signifi2+ entry . In summ2+ into T cells is dependent on the transmembrane electrostatic potential  using a path-clamp measurement to determine membrane potential while stimulating the cell with a femtosecond laser  polarization  pathway  co-activates the Iκ kinase (IKK) and phosphorylates IκB  and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) into the nucleus will induce the transcription of genes encoding for cytokines, such as IL-2 . Ca2+ inpathways . The fir pathway . For the pathway . This ex pathway . To acti+ influx . Certain+ influx . It is a+ influx . Calcineates IκB . Phosphoates IκB . Therefoates IκB . This meates IκB . To actiytoplasm . Using Hnd LITAF .2+ influx is the JNK pathway. Activation of c-Jun modulated by PKCθ regulates MAPK cascade of SEK1/MKK4 and phosphorylates JNK  (2+ channel)   and tranchannel) . Additio and Na+ , creatines to IC . The pro2+ is essential for numerous functions of cells. Interestingly, viruses can take advantage of this by modulating the intracellular concentration of Ca2+ ([Ca2+]i) used in processes such as viral replication  and six accessory proteins  L-type calcium channel expressed in human monocyte-derived DCs (mo-DCs) plays an essential role in Ca2+ mobilization. This function was demonstrated by the use of nifedipine (NFP) which inhibit the DPHR function, affecting Ca2+ mobilization and engulfment of apoptotic bodies by mo-DCs  L-type calcium channel, which when blocked with verapamil  the Caaffected . The intwith VPM . Like otar media . Additioar media . HIV Tatfections .2+]i, which occurs first in astrocytes and then in neurons, and also that NMDARs may mediate this effect in both cells types  and four proteins found inside the viral structure  and decrease IL-2 production, which may explain the absence of a long-lasting protective immunity  and sixteen non-structural proteins nsp1-16; . Patientndicator . Moreovendicator , and in channels . Previouectivity . Further+ and K+ . Indeed,ammasome . The SARll death . Moreovell death .2+ channel for SARS-CoV-2 infection has been observed in Vero E6 and Calu-3 cells, and the use of calcium channels blockers, such as amlodipine, felodipine, and nifedipine, has been studied  . HSVs be2+]i, mediated by the ER and Ca2+ influx from extracellular space (3-sensitive Ca2+ stores, probably available through the opening of Ca2+ channels in the ER membrane, help HSV enter the cell. On the other hand, the chelation of Ca2+ and pharmacological inhibition of IP3R impairs viral infection (v3.2 T-type Ca2+ channel that regulates the excitability of neurons. Significantly, the expression of this channel is reduced by HSV-1 infection, which depends on viral replication and protein synthesis (2+ influx, which can be blocked by the use of Ca2+ channels blockers and, therein, suppresses viral infection. T-type, but not L-type Ca2+ channel, impact HSV-2 infection, as proven by the use of Ca2+ channels blockers (2+ modulation and the role of Ca2+ channels involved in this process. On the other hand, T cell proliferation can be modulated by the HSV-1 gD protein (2+ mobilization that is required for the T cell receptor signaling (2+ channels in immune cells remains poorly understood and requires more studies to explore the potential development of new therapeutic strategies that target these components.Herpes simplex viruses infection of epithelial cell lines induces a transient increase in i and enhance their infectivity in target cells (2+ channels expressed in immune cells allows viruses to impair the immune responses as an evasion mechanism (2+ channels blockers, and their effects on viral infections are still required.Moreover, viruses such as HIV, hRSV, SARS-CoV2, HBV, and HSV, among others, have proteins that can interfere with the normal function of Caet cells . Viral mechanism . DespiteAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The endoplasmic reticulum Ca2+ sensorsSTIM1 and STIM2 are the key components of store-operated Ca2+ entryin mammalian cells. Under physiological conditions, STIM proteins areresponsible for store-operated Ca2+ entry activation. The STIM1 andSTIM2 proteins differ in their potency for activating different store-operatedchannels. At the moment, there are no selective modulators of the STIM proteinactivity. We screened a library of small molecules and found the 4-MPTCcompound, which selectively inhibited STIM2-dependent store-operatedCa2+ entry (IC50 = 1 μM) and had almost noeffect on the STIM1-dependent activation of store-operated channels.Store-operated Ca Under physiological conditions, the intracellularCa2+ response to an agonist includes not only entry of extracellularCa2+ into the cell, but also depletion of the intracellularCa2+ stores located in the endoplasmic reticulum (ER) [2+ entry into the cell in response to the depletion ofintracellular Ca2+ stores or store-operated Ca2+ entry[2+ ion influx into the cell. The entry is induced by STIM proteins(STIM1 and STIM2), which are Ca2+ sensors in the ER lumen. The STIM1protein, which is the main activator of store-operated Ca2+ entry,was the first to be characterized [2+ ions and ability to interact with plasmamembrane channels [2+ andis a weaker activator of store-operated Ca2+ entry than STIM1. STIM1is most likely responsible for the cellular Ca2+ response to anextracellular signal, while STIM2 regulates the basal levels of cytosolic andstored Ca2+ [2+ concentration, which is caused by anenhanced activity of STIM proteins and leads to cell death, is observed inHuntington’s disease [An increase in the concentration of cytoplasmic Calum (ER) . Plasma cterized ,4. The channels . STIM2 ired Ca2+ . In addired Ca2+ . Under pred Ca2+ , which ared Ca2+ , 10and red Ca2+ , 12, 13  disease , 15, Alz disease , 17, cer disease , and tra disease , 20. Cha disease  and colo disease . Thus, c2+entry inhibitors. Most of these inhibitors modulate the activity ofstore-operated Ca2+ channels. However, these compounds are oftenpoorly characterized and have more than one target. One of the most commonlyused compounds, 2-aminoethoxydiphenyl borate (2-APB), was first characterizedas a blocker of IP3-induced Ca2+ release [2+entry inhibitor at concentrations exceeding 50 μM. In addition, 2-APB, ata concentration of 5 μM, can potentiate store-operated entry [2+ leakfrom the ER lumen [Researchers have actively used a wide range of store-operated Ca release . It is ned entry . The meced entry , 26 and ed entry channelsER lumen .2+ stores are filled, STIM proteins are in an inactiveconformation stabilized by the interaction between the CC1 (Coiled-Coil 1) andSOAR (STIMOrai Activating Region) domains. Following Ca2+ storedepletion, STIM proteins undergo multimerization, change their conformation,and expose the SOAR domain for interaction with plasma membrane channels [2+ entry bytransforming STIM2 into its active conformation [When ER Cachannels . The 2-Aormation . On the ormation .2+entry. In this work, we have tested a library of 250 chemical compoundsreceived from InterBioScreen Ltd. possessing a chemical structure similar tothat of 2-APB, in order to identify a selective modulator of STIM2 activity. A4-MPTC compound was found to inhibit STIM2- dependent Ca2+ entry(IC50 = 1 μM) but had almost no effect on theSTIM1-mediated mechanism of storeoperated channel activation. The other 249compounds from the library had a divergent, and non-selective, effect.Thus, 2-APB directly interacts with STIM proteins and provides a good basis forthe search for a more selective modulator of store-operated CaCell lines2.The following HEK293-derived cell lines, kindly provided by Jonathan Soboloffand Mohamed Trebak, were used in the study: STIM1Orai3 (a cell line expressingexogenous STIM1-YFP and Orai3-CFP proteins), STIM2Orai3 (a cell line expressingexogenous STIM2-YFP and Orai3-CFP proteins) , STIM1 KFluorescence analysis2+ concentration were measured usinga Fluo-4 AM calcium indicator . The cells wereplated into 96-well culture plates 48 h prior to the analysis. The cells werefirst incubated in a HBSS solution  containing 5μM Fluo-4 AM for 1 h and then in a HBSS solution supplemented with either4-MPTC  or 1% DMSO  for 30min. Measurements were performed in the presence of 2 mM calcium in theextracellular solution using a FLUOstar Omega microplate reader . Data are presented as Fluo-4 fluorescence intensity values normalizedto the basal fluorescence value.Changes in the intracellular CaElectrophoresis and immunoblottingThe cells were grown in 60-mm Petri dishes and then lysed by adding a proteaseinhibitor cocktail. Proteins were separated by 8% denaturing PAGE. The proteinswere transferred to a nitrocellulose membrane using a semi-dry transfer unit. Primary antibodies to STIM1 , STIM2 , and α-tubulin were diluted at a ratio of 1 : 1000. Next,secondary anti-mouse IgG antibodies  againstα-tubulin and anti-rabbit IgG antibodies against STIM1 and STIM2 were used. Blots were visualized on a BioRad CellImaging System .Low-molecular-weight compounds for screening, including 4-MPTC, were kindlyprovided by InterBio- Screen Ltd. (ibscreen.com) in dry form. The compoundswere dissolved in DMSO to a final concentration of 10 mM.Statistical analysisp < 0.05,“**”–p < 0.01,“***”–p < 0.001; “n.s.”–not statistically significant differences.Statistical analysis was performed using the Origin 8 software. The results offluorescence measurements were checked for normality using the Fisher’stest. Data groups were compared using the Bonferroni test. Statisticallysignificant differences are denoted in figures as follows:“*”–the confidence interval of Fig. 1A).The effect of the test compounds on the amplitudeof a cellular Ca2+ signal in response to the depletion ofintracellular Ca2+ stores was recorded using the Fluo-4 AM calciumindicator. Intracellular Ca2+ stores were depleted by adding 1μM thapsigargin (Tg), a selective inhibitor of the ER Ca2+pump, to the extracellular solution. At the first stage, the effect of thelibrary of 2-APB analogs on the Tg-induced Ca2+ response was tested.For this purpose, the cells were incubated in HBSS solutions containing one ofthe 250 test compounds (at a concentration of 100 μM) for 30 min prior tostarting the experiments. Next, the amplitude of the Ca2+ responseto the addition of 1 μM Tg was assessed. As a result of library screening,we selected 4-MPTC,the compound that most stronglyaffected the Tg-induced Ca2+ response in STIM2Orai3 cells: theCa2+ response was inhibited by 39 Ѓ} 3% compared to that inthe cells incubated in a solution supplemented with 1% DMSO.Since 4-MPTC significantly inhibits the Tg-inducedCa2+ response in cells with increased STIM2 and Orai3 levels, we maysuggest that 4-MPTC modulates the activity of these proteins. The direct actionof 4-MPTC on Orai3 is supported by the fact that 2-APB can activate the Orai3channel [2+ response and decreased its amplitude by 12Ѓ} 3%.Furthermore, incubation of HEK293 cellsexpressing exogenous STIM1 and Orai3 proteins (the STIM1Orai3 cell line) with4-MPTC did not inhibit the amplitude of the Tg-induced Ca2+ responseand, therefore, did not decrease the activity of theOrai3 channels. Hence, the Orai3 protein is not a selective target for 4-MPTC.In order to search for low-molecular-weight compounds that modulate theactivity of STIM2 proteins, we used a model cell line derived from HEK293 cellsstably expressing exogenous STIM2 and Orai3 proteins (STIM2Orai3 cell line).Since 4-MPTC significantly reduced the Ca2+ responseamplitude but did not alter the curve’s shape in STIM2Orai3 cells,we may suggest that this compound affects thepathway of store-operated calcium entry activation through STIM2, but notthrough the STIM1 protein. A change in the curve’s shape for the Orai3 KOand STIM1Orai3 cell lines is quite typical and reflects a decrease in the rateof the Ca2+ response. Since the endogenous STIM2 protein is presentin Orai3 KO and STIM1Orai3 cells,4-MPTC can reduceits activity and, thereby, change the dynamics of both the release ofCa2+ from the store into the cytoplasm and the entry ofextracellular Ca2+ ions. Knockout of STIM2 using short interferingRNAs results in a similar effect on the Ca2+ response; it decreasesCa2+ release from the store [2+ entry [Fig. 1A,B),which complicates datainterpretation. Therefore, we further used STIM1 (the STIM1 KO cell line) andSTIM2 knockout cells (the STIM2 KO cell line), which are devoid of this drawback.The activity of store-operated channels in a cell is known to be modulated byboth the STIM1 and STIM2 proteins .The prehe store and subs2+ entry .Cell li2+ entry[2+ response by 57 ± 8%compared to that in control cells (incubation with 1% DMSO).It should be noted that 4-MPTC more effectively inhibitsstore-operated Ca2+ entry under these conditions. For example, theTg-induced Ca2+ response was inhibited by 57% in STIM1 KO cells andby only 39% in STIM2Orai3 cells. A significant change in the shape of theTg-induced response is observed after incubation of STIM2-knockout cells inwhich the STIM1 protein is the only activator of store-operated Ca2+entry with 4-MPTC. The calcium concentration increases more slowly in thesecells than in the control cells, with the maximum Ca2+ responseamplitude being 61 Ѓ} 5% higher compared to that in the control.4-MPTC was experimentally demonstrated to actdivergently in STIM1 KO and STIM2 KO cell lines: it inhibits theCa2+ response through the STIM2-dependent pathways and enhances itthrough the STIM1 pathways. Thus, the selected compound, 4-MPTC, enablesdifferentiation between the pathways activating store-operated Ca2+entry through different STIM proteins; however, the mechanism of action of thiscompound requires further clarification.When STIM1 expression is completely suppressed, the STIM2 protein becomes thekey and only activator of store-operated Ca2+ entry. Pre-incFig. 4).We analyzed the effect of 4-MPTC at a concentrationof 0.001, 0.1, 1, 10, and 100 μM on the Tg-induced Ca2+response in STIM2Orai3 cells. The half-maximal inhibitory concentration(IC50), calculated by curve-fitting, equals 1 μM.The 4-MPTC compound has a typical concentration– effect curve, on the contrary, changes the shape of theCa2+ response curve, without decreasing its amplitude. Thus, 4-MPTCselectively inhibits storeoperated Ca2+ entry via the STIM2-mediatedpathway, but not the STIM1-mediated pathway.Thus, given our findings, we may conclude that the use of 4-MPTC in cell linesexpressing predominantly the STIM2 protein (STIM1 KO and STIM2Orai3)significantly inhibits the amplitude of the Tg-induced Ca2+entry inhibitor, it does not appear to selectively inhibit store-operatedCa2+ entry and also has a divergent concentration-dependent effect.2-APB derivatives have been investigated in the search for an inhibitor lackingthese disadvantages [2+ entry at lower concentrations than 2-APB but areat the same time unable to activate Ca2+ entry at certainconcentrations; in other words, they have better inhibitory properties than theparent compound. More attention in the search for new inhibitors ofstore-operated Ca2+ entry has been paid to the STIM1-dependentpathway of activation, while the STIM2-mediated pathway often has remainedunexplored. For example, MDA-MB-231 cells, in which STIM1 and Orai1 proteinsplay a key role in store-operated Ca2+ entry, as well as HEK293cells expressing STIM1 and Orai-family proteins, have been used as model celllines in experiments [2+ entry through both proteins[2+ entry through the STIM1- dependent pathway.Despite the fact that 2-APB is widely used as a store-operated Cavantages -41. Mosteriments , 43. A sproteins. The 4-M2+response through the STIM2-dependent pathway of Ca2+ influx but doesnot inhibit Ca2+ entry through the STIM1-dependent pathway. Themechanism of action of this compound on the STIM2 protein requires furtherinvestigation.Screening of a library of structure 2-APB analogs has yielded an 4-MPTCcompound that has an inhibitory effect on the Tg-induced Ca


Despite significant recent advances in our understanding of the biology and genetics of acute myeloid leukemia (AML), current AML therapies are mostly based on a backbone of standard chemotherapy which has remained mostly unchanged for over 20 years. Several novel therapies, mostly targeting neomorphic/activating recurrent mutations found in AML patients, have only recently been approved following encouraging results, thus providing the first evidence of a more precise and personalized approach to AML therapy. Rewired metabolism has been described as a hallmark of cancer and substantial evidence of its role in AML establishment and maintenance has been recently accrued in preclinical models. Interestingly, unique metabolic changes are generated by specific AML recurrent mutations or in response to diverse AML therapies, thus creating actionable metabolic vulnerabilities in specific patient groups. In this review we will discuss the current evidence supporting a role for rewired metabolism in AML pathogenesis and how these metabolic changes can be leveraged to develop novel personalized therapies. Acute myeloid leukemia (AML), the most prevalent acute leukemia in adults , is a hiAML arises either in a hematopoietic stem cell (HSC) or a more differentiated progenitor which has acquired the ability to self-renew indefinitely as a result of specific mutations . This ceThe association between changes in metabolic status and prognosis or severity of disease in patients with AML has been long known. In hospitalized patients, hyperglycemia is associated with adverse prognosis independently of their cytogenetics profile or advanced disease status, and this effect can be only partially explained by higher risk of sepsis . AdditioIt’s been almost a century since Otto Warburg first described aerobic glycolysis as one of the hallmarks of the metabolism of cancer cells . AML is However, typical descriptions of the Warburg effect, as well as the use of 18-fluorodeoxyglucose as a marker of cancerous cells in positron emission tomography, often lead to a common misconception of cancer cells switching their energy metabolism to glycolysis on the expense of tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OxPhos), while in reality, both pathways are dysregulated and often increased in comparison to healthy cells of origin, which makes them both interesting for potential therapeutic targeting . Infact,de novo nucleotide synthesis. When used in combination with drugs that increase ROS formation, like arsenic trioxide or homoharringtonine, inhibitors of glutaminase (GLS), the first enzyme in glutaminolysis, significantly decreased the viability of AML cells and leukemia burden in treated mice .Glutamine is a highly abundant non-essential amino acid that can serve as a source of precursors for TCA cycle through conversion to glutamate and α-ketoglutarate (αKG). Glutamine metabolism thus carries a great potential for therapeutic targeting in AML. Indeed, inhibition of glutamine utilization, either through uptake inhibition , 50 or ated mice . Glutamiynthesis . Targeti pathway  or pyrim pathway –57, whicThe greatest success in metabolic targeting, and a proof of concept that specific modulation of metabolic pathways could result in therapeutic benefit for specific subgroups of AML patients, is differentiative therapy of patients carrying isocitrate dehydrogenase (IDH) mutations with IDH inhibitors. IDH1 and IDH2 mutations are present in 15–20% of patients with AML . The proBased on this, it is clear that the number of potential metabolic targets in AML is large. The question that naturally arises is how this extensive knowledge of metabolic changes in AML cells can help us identify the subgroups of patients who could benefit the most from modulation of specific metabolic pathways, thus leading to the development of tailored metabolic therapeutic approaches.de novo AML showed that when stratified by morphologic properties, genetic markers, differentiation status and European LeukemiaNet (ELN) 2017 risk groups, AML patients demonstrated a strong diversification of their metabolic properties , the enzyme involved in branched-chain amino acids  catabolism by transferring α-amino groups to αKG, producing glutamate and corresponding α-ketoacids. In IDH1 levels . Interes1 levels , as well1 levels , which fde novo serine synthesis, most likely through decrease in purine synthesis for which serine is a physiological precursor, decreases proliferation of FLT3-ITD mutated AML cells and increases their sensitivity to cytarabine  and increased dependency to extracellular serine  translocation often present with mutations in transcription factor ZBTB7A , which hpathways . Specifirognosis , again poduction , which mr serine , which ir serine . Finally therapy , they st therapy . TP53 haism etc. , but verThe group of patients that could most likely benefit from metabolic modulations are patients resistant to available therapies. As already mentioned, resistance to both cytarabine  and venNo compounds directly targeting an unmutated metabolic pathways has been approved for the treatment of AML, but there are several currently in clinical trials. Phase I clinical trials are underway for the glutaminase inhibitor CB-839 (NCT02071927) and oxidative phosphorylation inhibitor IACS-010759 NCT02882321) in patients with relapsed or refractory AML 321 in pa and a raAnother intriguing therapeutic strategy for targeting metabolic pathways in AML could be through pharmacological modulation of their upstream regulatory signaling pathways, many of which have inhibitors already in clinical use . As one de novo serine synthesis pathway in high fructose conditions, and the inhibition of one of the components of this pathway, phosphoglycerate dehydrogenase, can block the reliance on fructose utilization (Finally, the potential of dietary modifications and nutritional supplementation in unmasking the vulnerabilities of AML cells is recently being increasingly investigated. High fructose diet is known to contribute to cancer development. However it can also unmask novel vulnerabilities as AML cells upregulate lization . It is ylization , or it ilization . Additiolization  or histalization  show benPrecision medicine has become an increasingly desirable goal in oncology. Using available knowledge banks of matched genomic-clinical data has already been shown to facilitate personally tailored therapeutic decisions . MoreoveVD and PG carried out primary literature search and wrote the manuscript. All authors contributed to the article and approved the submitted version.Both authors are currently funded through a CRUK Advanced Clinician Scientist Fellowship reference C57799/A27964.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.


In addition, hyperpolarization via the activation of K+ channels indirectly promotes Ca2+ signaling in osteoblast lineage cells. An improved understanding of the fundamental physiological and pathophysiological processes in bone homeostasis requires detailed investigations of osteoblast lineage cells. This review summarizes the current knowledge on the functional impacts of K+ channels and Ca2+-permeable channels, which critically regulate Ca2+ signaling in osteoblast lineage cells to maintain bone homeostasis.Bone-forming cells or osteoblasts play an important role in bone modeling and remodeling processes. Osteoblast differentiation or osteoblastogenesis is orchestrated by multiple intracellular signaling pathways  and Wnt signaling pathways) and is modulated by the extracellular environment , vitamin D, transforming growth factor β (TGF-β), and integrins). The regulation of bone homeostasis depends on the proper differentiation and function of osteoblast lineage cells from osteogenic precursors to osteocytes. Intracellular Ca Bone modeling initially occurs during skeletal development and comprises two modes: intramembranous ossification and endochondral ossification . During Col1a1, Spp1, Ibsp, Bglap2, and Fn1, and regulates multiple steps during osteoblast differentiation i) signaling has a critical role in osteoblast proliferation and differentiation , a Ca2+/CaM-dependent protein phosphatase, calcineurin (Cn), and a Ca2+-dependent endoplasmic reticulum (ER) chaperone, calreticulin (CRT). Several studies have shown the significance of Ca2+ signaling in osteogenic functions. CaMKII activates cAMP response element-binding protein (CREB)/ activating transcription factor (ATF) and ERK signaling pathways in osteoblasts i mobilization is controlled by intracellular Ca2+ store activation and Ca2+ influx. Membrane hyperpolarization induced by K+ channel activation increases the electromotive driving force of Ca2+ entry through voltage-independent, store-operated, Ca2+-permeable channels in non-excitable cells i is a ubiquitous second messenger regulating various cellular functions, such as proliferation, secretion, differentiation, migration, and apoptosis [2+]i is maintained at very low levels (50–100 nM). The stimulation of cells with agonists, such as cytokines, hormones, or growth factors, results in [Ca2+]i rises via Ca2+ influx and/or Ca2+ release from the ER and mitochondria. In electrically non-excitable cells, including osteoblast lineage cells, membrane hyperpolarization increases the driving force for Ca2+ signaling. A direct link has been demonstrated between membrane hyperpolarization and Ca2+ signaling in epithelial cells i levels by voltage-gated Ca2+ channel blockades, thereby inhibiting neurotransmission and muscle contraction. In contrast, in non-excitable cells, hyperpolarization followed by KV channel activation increases [Ca2+]i levels by facilitating Ca2+ influx through voltage-independent, store-operated Ca2+-permeable channels, thereby promoting cell proliferation, migration, and differentiation i elevation. Due to these properties, the KCa1.1 activation results in repolarization and the closing of voltage-dependent Ca2+ channels in neurons and smooth muscle cells [Ca1.1 serves as a negative feedback regulator of membrane potential and [Ca2+]i in excitable cells. In contrast, KCa2.2 activation hyperpolarizes and thereby promotes Ca2+ influx through TRPC in brain capillary endothelial cells [Ca3.1 activation results in elevated [Ca2+]i in human cancer cells i increase, which is critical in regulating a variety of cellular functions, including proliferation and, more specifically, differentiation of osteoblast lineage cells  channel that mediates Ca2+ influx in most non-excitable cells via a store-operated Ca2+ entry (SOCE) mechanism [2+]i changes are important signaling pathways in the regulation of osteoblast linage function [Orai1 is a pore-forming subunit of the Caechanism . [Ca2+]ifunction , but cauPhex), a mineralizing osteocyte marker gene, dentin matrix protein 1 (DMP1), and a mature osteocyte marker gene, fibroblast growth factor 23 (FGF23) in mouse cortical long bone i in MG-63 cells. The localization of TRPM7 in lipid rafts as Ca2+ microdomains was critical for Ca2+ flicker activity [2+]i, resulting in osteogenic differentiation i rise through TRPM3 and TRPV4 as a mechanosensor are critical to NFATc1 activity and the subsequent physiological activity of osteoblasts.Mechanical stresses regulate bone formation by upregulating RANKL expression in osteoblasts. Acute mechanical stress induced by hypoosmotic shock promoted TRPM3 and TRPV4 activation, resulting in increased Caeoblasts . NFATc1 eoblasts . The [Ca2+]i, levels; thus, calcification was enhanced with upregulated Runx2 and osteocalcin expression. Indeed, the expression and nuclear translocation of NFATc1 were upregulated in osteoblasts expressing TRPV4 L619F. These findings indicate that MD-associated disorganized endochondral ossification is induced by disordered osteogenic differentiation through the TRPV4-Ca2+/NFATc1 signaling axis i and impaired response to flow-induced Ca2+ influx [2+ signaling to shear fluid stress in MG-63 cells [2+-permeable channel TRPP2 is coupled to mechanosensing TRPP1 in response to mechanical stress-loaded osteoblasts during osteoblast differentiation.The osteoblast-specific depletion of TRPP2 resulted in reduced bone volume associated with decreased bone mineral density, mineral apposition rate, trabecular bone volume, and cortical thickness, and impaired biomechanical properties of bone. TRPP2-deficient osteoblasts exhibited lower basal [Ca+ influx . Additio63 cells . These s2+, as well as to monovalent ions [Piezo channels are emerging as important regulators of various aspects of mechanosensing ,127,128.ent ions . Recent ent ions  and reduent ions . Sugimotent ions  showed tent ions , and Pieent ions . Indeed,ent ions   showed severely impaired bone formation and blunt, mechanical unloading-induced bone loss [Prrx1CrePiezo1, exhibited multiple skeletal fractures in long bones, the radius, and ulna; however, Piezo2 conditional-deficient mice (Prrx1CrePiezo2) had normal bone development with no fractures. Piezo1/2 double conditional knockout neonatal mice exhibited more severe skeletal defects in the appendicular skeleton [Dmp1CrePiezo1. The shRNA-mediated blockade of Piezo1 in MLO-Y4 osteocyte-like cells reduced the expression of Ptgs2 and Tnfrsf11b [Osteoblast lineage-specific, Piezo1-deficient mice (one loss . Moreoveskeleton . Piezo1 skeleton . In addiskeleton  reportednfrsf11b , well-knnfrsf11b ,133. Furnfrsf11b  reportedRunx2CrePiezo1, demonstrated a more severe osteoporotic phenotype than Dmp1CrePiezo1 [Runx2CrePiezo1 mice [Piezo1 knockout mice, 1Dmp1Cre . BecauseCre mice . Lee et Cre mice  have desPrx1CrePiezo1 mice. Piezo1 in osteoblasts regulated the YAP-dependent expression of Col types II and IV, which control osteoclast differentiation, suggesting that Piezo1 can coordinate the crosstalk between osteoblasts and osteoclasts by directly sensing mechanical stress in osteoblast lineage cells.Wang et al.  reported2+ signaling, as well as on the external calcium balance that regulates the function and differentiation of chondrocytes, osteoclasts, and osteoblast lineage cells. A number of ion channels critically contribute to the regulation of these processes, including intestinal calcium absorption and renal calcium reabsorption [Osteoporosis is a major skeletal disorder that influences bone structure and composition. Gradual bone loss results in bone fragility and significantly increases the risk of bone fracture. An estimated 1.5 million fractures occur per year in the United States . Bone hosorption .+ channels and Ca2+-permeable channels in osteoblast functions. However, the lack of understanding of their specific functions in bone formation poses a definite limit. Elucidating their involvement in bone signaling pathways could help identify molecules targeting bone biology. Emphasis should be given on data describing the cooperative coupling of K+ and Ca2+ signaling in regulating the physiological and pathophysiological frameworks of bone homeostasis.This review has highlighted the critical role of K


An increase in [Ca2+]i and a decrease in ΔΨm were observed ~10 s after the injury in cells located no further than 150–200 µm from the scratch border. Ca2+ entry into cells during mechanical damage of the primary neuroglial culture occurred predominantly through the NMDA-type glutamate ionotropic channels. MK801, an inhibitor of this type of glutamate receptor, prevented an acute increase in [Ca2+]i in 99% of neurons. Pathological changes in calcium homeostasis persisted in the primary neuroglial culture for one week after injury. Active cell migration in the scratch area occurred on day 11 after neurotrauma and was accompanied by a decrease in the ratio of live to dead cells in the areas adjacent to the injury. Immunohistochemical staining of glial fibrillary acidic protein and β-III tubulin showed that neuronal cells migrated to the injured area earlier than glial cells, but their repair potential was insufficient for survival. Mitochondrial Ca2+ overload and a drop in ΔΨm may cause delayed neuronal death and thus play a key role in the development of the post-traumatic syndrome. Preventing prolonged ΔΨm depolarization may be a promising therapeutic approach to improve neuronal survival after traumatic brain injury.In vitro models of traumatic brain injury (TBI) help to elucidate the pathological mechanisms responsible for cell dysfunction and death. To simulate in vitro the mechanical brain trauma, primary neuroglial cultures were scratched during different periods of network formation. Fluorescence microscopy was used to measure changes in intracellular free Ca Pathological mechanisms underlying the detrimental effects of traumatic brain injury (TBI) are divided into primary and secondary injury mechanisms. Primary damage is due to the mechanical impact itself  and develops in the first few seconds after the trauma. The most important aspect of this stage is necrotic cell death in the area of the injured brain tissue. Secondary damage is longer-term (hours-days) and is mediated by primary damage mechanisms . They inAt the same time, the central mechanism of long-term neuronal death induced by TBI is glutamate excitotoxicity . Briefly2+-dependent neurotoxicity due to prolonged activation of NMDA-type ionotropic glutamate receptors, which have a high permeability to extracellular Ca2+ and Na+. A sharp increase in intracellular free Ca2+ concentration ([Ca2+]i) activates various mechanisms of cell death, leading to additional loss of neurons long after the primary damage. Therefore, a decrease in [Ca2+]i after mechanical damage to neurons in vitro correlates with a decrease in neuronal damage and an increase in their viability. A reproduction of such results in general indicates the neuroprotective activity of the studied preparations  changes, and can be prevented by blocking depolarization-dependent Cl- influx.The increase in intracellular Na concentration near the NMDA channel also plays a substantial role in the pathophysiology of TBI. This leads to a massive membrane depolarization and increase in Hll death . Anotheren shown  to be upmulation . Howevermulation  suggesteThere are various in vitro models of mechanical neurotrauma. Models of acute penetrating impact allow the reproduction of the process of neuronal damage using a flexible plastic pipette tip , a circu2+]i, and mitochondrial potential (ΔΨm) changes in neurons. Studying these parameters, we traced the dynamics of the scratch healing and determined which neuroglial culture cells were the first to migrate to the zone of mechanical damage. Thus, we substantiated a new in vitro model of TBI, which can be further used to study the neuroprotective compounds we previously tested in an experimental model of glutamate excitotoxicity i in response to Glu [2+]i during the period of incubation in Ca2+-free media in experimental stage (e)  C. Cell “tage (e) B, had antage (e) A and alme) , the fraction of cells with a strong increase in [Ca2+]i and a significant drop in ΔΨm decreased 8.5 ± 1.5 times .We checked whether the mechanical damage-dependent rapid increase in i and ΔΨm dynamics in response to the Glu; the lag-period of the development of delayed calcium dysregulation (DCD) in the damaged culture did not differ from the control (In the first series of experiments (7 DIV), no significant differences were found between the groups in the parameters of i to initially lower values than control cells (p < 0.01) . Consequently, higher rates of [Ca2+]i against EGTA in cells from injured cultures may indicate that the activity of transport systems that pump calcium from the neuronal cytoplasm was deteriorated.The integrated fluorescent response reflects the change in fluorescence intensity of Fura-FF in cells during the 15 min action of Glu. It was significantly higher in the neurons from the injured cultures than in the intact ones ( < 0.01) B2. A sim < 0.01) C2. In ad < 0.01) D1,2. Con2+]i, we measured the mitochondrial potential (ΔΨm).In order to assess the contribution of mitochondria to the calcium-dependent processes occurring during the experiment, in parallel with the assessment of the dynamics of [Ca1). The rate of mitochondrial potential recovery in neurons of injured cultures was significantly lower than in neurons of intact cultures (2).The recovery rate of ΔΨm during the “washout” period (administration of EGTA) was estimated by the slope of the graph reflecting the change in Rh123 E1. The rcultures E2.2+]i and ΔΨm changes were synchronous, but also confirms the hypothesis of the participation of mitochondria in glutamate-induced changes in calcium homeostasis i and mitochondrial potential (ΔΨm) of neurons. The closest study is the one in which a stretch injury was applied to neurons and then their excitability was assessed using a patch clamp 30–60 min after the injury [There is a limited number of models that allow the assessment of the effect of mechanical trauma on Ca neurons . Moreovee injury . Anothere injury .2+]i and ΔΨm) of cells (2). In contrast, the recovery of initially low values of [Ca2+]i is reduced (2).In the present work, we studied the healing dynamics of the mixed primary neuroglial culture under the conditions of in vitro experimental mechanical neurotrauma  and how of cells , which mof cells A. The neof cells B. Increaof cells B2. In co reduced D2.These pathological processes may contribute to a decrease in cell survival in the areas located near the scratch. At the same time, it was shown that neurons and astrocytes migrate to the damaged area within two weeks after scratching . Immunoh2+]i, which is mirrored by the rapid increase in the Fura-FF fluorescent signal. The maximum fluorescence level in this phase of the experiment also depends on the efficiency of extra mitochondrial systems responsible for Ca2+ removal from the cytoplasm, such as membrane transporters PMCA, plasma NCX. The magnitude of the integral fluorescent response and the degree of [Ca2+]i recovery during EGTA solution administration are additional parameters to assess the degree of Ca2+-homeostasis stability. Replacing the glutamate solution with EGTA solution not only reduces the deleterious activation of NMDA receptors activation, but also changes the Ca2+ ion concentration gradient between the cytoplasm and the external environment in the opposite direction, which allows cells that have not completely lost their homeostatic potential to remove Ca2+ from the cytoplasm. In Ca2+-free buffer, neurons from mechanically injured cultures poorly recovered the initial [Ca2+]i compared to controls.Several parameters influence the magnitude of the integral fluorescence response during glutamate administration. The most important of them is the rate of the DCD onset, i.e., the multistep increase in [Ca2+ concentration [2+]i recovery can be promoted by the closure of mitochondrial permeability transition pores i spike and synchronous depolarization of mitochondria could be caused by NMDA channel activation, as well as by other, yet unidentified mechanisms  indicate that Ca2+ may enter neurons from the buffer at axon and/or dendrite cutting sites, diffusing through them and causing small changes in [Ca2+]i in the soma. Of all the low-molecular-weight compounds, Glu and ATP are the most abundant in the cytosol (their concentration reaches 3–10 mM) i and ΔΨm in cells close to the damage zone. These changes are comparable with those caused by neurotoxic doses of glutamate. In some neurons, these changes are indeed caused by stimulation of Glu-directed NMDA receptors, whereas in other cells, ATP serves as the signal molecule. On the other hand, the maximal level of [Ca2+]i in the glutamate washout phase depends on the efficiency of extra mitochondrial systems for Ca2+ removal from the cytoplasm, such as PMCA, and plasma NCX. Their efficiency is dependent directly or indirectly on the ATP concentration in the cytosol, which is determined by the intensity of cellular respiration. An increase in the [Ca2+]i during exposure to glutamate in neurons from injured cultures compared to control neurons may indicate impaired ATP synthesis. Thus, mechanical trauma disrupts Ca2+ homeostasis systems in adjacent neurons. Impairment of calcium homeostasis in surviving neurons after TBI is not immediately apparent  and the Declaration on Humane Treatment of Animals and in accordance with the Order of the Ministry of Health and Social Development of Russia no. 708n of 23 August 2010 “On Approval of Laboratory Practices.” Animal care, breeding, and experimental procedures were carried out as required of the Ethical committee of the Institute of General Pathology and Pathophysiology. Protocol no. 05-06/12 of 14.12.2017.6 cells/mL) was obtained by treating brain tissue with papain (10 units/mL), dissociating by pipetting, washed from the cell debris by sedimentation in a centrifuge (200× g) in a Ca2+-free solution, then in Ca2+-containing solution and finally in the neurobasal medium . Cells were seeded in 48-well flat-bottom plates (2.5 × 105 cells/well)  and in Petri dishes (ᴓ35 mm) with a glass insert (ᴓ14 mm) at the bottom . Plates and Petri dishes were pre-coated with polyethyleneimine . After one hour, 1.5 mL of NBM containing 2% Supplement B-27, 0.5 mM L-glutamine, and an antibiotics/antimycotic mixture  was added. The cells were incubated for 18 days in vitro (DIV) at 37 °C in an atmosphere of 5% CO2/95% air at 100% humidity.Wistar rat pups (P1–P2) were used for preparation of a primary mixed neuronal and glial culture of the cerebral cortex  as previously described . Briefly2+]i and mitochondrial potential (ΔΨm) immediately after the trauma (the acute effect), we inflicted a single scratch trauma across the neuroglial culture with an insulin injection needle attached to the tip of a rod-like metal holder connected to a micromanipulator . In other experiments, the neuroglial cultures (4 DIV) were scratched across the diameter of the well bottom by a disposable sterile tip . Then, we evaluated the delayed effect of mechanical damage on the cells viability and examined the long-term changes in [Ca2+]i or ΔΨm on the 3rd (7 DIV) and 7th (11 DIV) days after scratching.To measure changes in i, cells were loaded with acetoxymethyl ester of low-affinity Ca2+ indicator Fura-FF (Fura-FF/AM). Stock solutions of Fura-FF/AM in dimethyl sulfoxide preliminary mixed with nonionic detergent Pluronic F-127 were added to cells in 1 mL of NBM to final concentrations 4 µM and 0.02%, respectively . For simultaneous monitoring of the [Ca2+]i and ΔΨm changes, cells were loaded with a potential-sensitive dye rhodamine 123  sensor. Fura-FF fluorescence was excited at 340 and 380 nm, and that of Rh123 at 485 nm. The accumulation of Rh123 in polarized mitochondria quenches the fluorescent signal. In response to depolarization, the fluorescence is dequenched i and ΔΨm kinetics (described in detail in the figure captions).Statistical analysis was performed using GraphPad Prism 6 . At least three experiments were carried out in each series. The data were checked for normality using three tests: D’Agostino-Pearson omnibus normality test, Shapiro–Wilk normality test, and KS normality test. The data are presented according to statistical rules depending on the type of data distribution. The Kruskal–Wallis test with Dunn’s multiple comparisons correction, the Mann–Whitney test, or the t-test was used to analyze the [Ca2+]i and ΔΨm both in the first seconds, minutes, and several days after injury. NMDA receptor ion channels serve as the main pathway for Ca2+ entry into neurons in the event of mechanical damage to a neuronal culture. Ca2+ overload of mitochondria and a prolonged decrease in ΔΨm can cause delayed neuronal death and thus play a key role in the development of the post-traumatic syndrome. We were also able to visualize the process of the neuroglial culture repair, where neurons were the first in the damaged area and were followed by astrocytes. Nevertheless, the viability of neurons inevitably decreased two weeks after injury. This effect may be attributed to the long-term metabolic dysregulation of neurons caused by trauma. Therefore, the use of neuroprotective compounds would allow us to test this hypothesis.We developed a model of mechanical neurotrauma that allows us to measure the dynamics of [Ca


Then, STIM1 translocates into discrete ER-plasma membrane (PM) junctions where it directly interacts with and activates plasma membrane Orai1 channels to refill ER with Ca2+. Furthermore, Ca2+ entry due to Orai1/STIM1 interaction may induce canonical transient receptor potential channel 1 (TRPC1) translocation to the plasma membrane, where it is activated by STIM1. All these events give rise to store-operated calcium entry (SOCE). Besides the main pathway underlying SOCE, which mainly involves Orai1 and TRPC1 activation, STIM1 modulates many other plasma membrane proteins in order to potentiate the influxof Ca2+. Furthermore, it is now clear that STIM1 may inhibit Ca2+ currents mediated by L-type Ca2+ channels. Interestingly, STIM1 also interacts with some intracellular channels and transporters, including nuclear and lysosomal ionic proteins, thus orchestrating organellar Ca2+ homeostasis. STIM1 and its partners/effectors are significantly modulated in diverse acute and chronic neurodegenerative conditions. This highlights the importance of further disclosing their cellular functions as they might represent promising molecular targets for neuroprotection.Located at the level of the endoplasmic reticulum (ER) membrane, stromal interacting molecule 1 (STIM1) undergoes a complex conformational rearrangement after depletion of ER luminal Ca Desutophagy , and in utophagy ,166,167,utophagy ,169, micutophagy , stronglutophagy .2+ signaling as demonstrated both in experimental models and in patients [2+ current in disease progression [Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin (HTT) protein, thus resulting in striatal degeneration through transcriptional dysregulation of a number of genes, including those regulating Capatients ,172,173.patients ,175. In gression ,176. In gression . By contgression ,178. 2+ release from ER stores, STIM1 aggregation and SOCE stimulation in diverse experimental contexts [2+ signalling dysregulation in HD [Accumulating evidence suggests crosstalk between STIMs and NMDARs that may contribute to dendritic spines pathology in HD ,95. Indecontexts . Althougon in HD .2+ homeostasis is believed to be crucially involved in the selective loss of dopaminergic neurons of the substantia nigra pars compacta [V1.3 channels, and pharmacological inhibition of these currents restores Ca2+-independent ‘juvenile’ pacemaking activity and protects dopaminergic neurons in animal models of the disease [Although the exact mechanisms involved in the pathogenesis of sporadic and familial Parkinson’s disease (PD) are still unclear, the dysregulation of ER Cacompacta ,181. Rhy disease .+) that mimic PD lead to degeneration of dopaminergic neurons by increasing Ca2+ influx through CaV1.3 channel, by reducing TRPC1 expression, while inhibiting stimulation-dependent STIM1-CaV1.3 interaction [V1.3 activity by providing a STIM1-based scaffold, which is crucial for proper firing and survival of dopaminergic neurons [A recent work has demonstrated that neurotoxins  in dopaminergic cells, whereby downregulation of σ1Rs expression or inhibition of σ1R activity substantially prevented MPP+-induced cell death by preserving Ca2+ homeostasis, mitochondrial and ER function, and inhibiting apoptosis [2+, causes the dissociation of σ1R-STIM1 complexes while facilitating STIM1-TRPC1-Orai1 interactions, which could also play a critical role in neuronal demise [+ decreases TRPC1 expression, its interaction with STIM1 and Ca2+ entry in SH-SY5Y cells [In PC12 cells treated with MMPis model . In linepoptosis . In partl demise . Neverth5Y cells , undersc2+ homeostasis [2+ channel TRPML1 has been demonstrated, possibly to regulate lysosomal Ca2+ release, autophagy defects and cell survival in β-methylamino-l-alanine (l-BMAA)-treated motor neurons [G93A astrocytes causing ER Ca2+ overload [G93A transgenic mice [Amyotrophic lateral sclerosis (ALS) is a devastating and fatal neurodegenerative disease characterized by a progressive loss of both upper and lower motor neurons. Molecularly, the disease has a complex pathogenesis including the deregulation of multiple intrinsic pathways and dysfunctional Caeostasis . In moto neurons . Howeveroverload . Furthernic mice . While Snic mice . Interesnic mice .2+ signals that are used by both excitable and non-excitable cells for regulating critical physiological processes. The ER Ca2+ sensor STIM1 is responsible for SOCE machinery orchestration mainly through the interaction with Orai1 channel. However, several other STIM1 targets located in different cell compartments may serve to handle intracellular calcium concentration including plasmalemmal and intracellular channels , and pumps . Of note, the interaction between STIM1 and each of these ionic targets allows the regulation of both cytosolic and organellar Ca2+ homeostasis. Moreover, through the modulation of STIM1 activity and interaction, many adaptors indirectly intervene in intracellular calcium homeostasis regulation.SOCE elicits specific cytosolic Ca2+ homeostasis and, most notably, STIM1 targets may be dysregulated in both acute and chronic neurodegenerative diseases. In fact, dysfunction of SOCE may contribute to the progression of several neurodegenerative diseases. For instance, SOCE activation appears to be neuroprotective in PD and AD, while during HD progression, neuroprotection could be achieved by reducing SOCE. With stroke injury being a multifactorial pathology dependent on the dysfunctional activity of several cell types, the role of SOCE in this neurological disease remains controversial. Therefore, dissection of the molecular components sustaining SOCE should be performed at the level of each specific cell type in order to identify the exact role played by each of them. This is true especially at the level of central nervous system (CNS) cells, where SOCE plays a dualistic role in the different forms of neurodegeneration. On the other hand, STIM1 interacts with other proteins not canonically involved in SOCE and this interaction may be altered during the neurodegenerative process. For instance, dysfunction in STIM1/TRPML1 interaction participates in ALS and stroke pathogenesis through organellar ionic dysfunction. This should be carefully taken into account since each element able to interact with STIM1 should be considered as a putative target for the development of new pharmacological entities.Several lines of experimental evidence clearly demonstrate that Ca2+ dyshomeostasis in neurodegeneration, the validation of new drugs toward STIM1 targets may result in successful treatment strategies for AD, PD, HD, ALS and stroke.Collectively, considering the relevance of Ca


Furthermore, global gene expression profiling analysis was performed on samples from the clinical protocol. Correlation analyses were performed between p53 protein isoform expression and in vitro VPA sensitivity and FAB (French–American–British) class in primary AML cells. The results show downregulation of p53β/γ and upregulation of p53FL in AML cell lines treated with VPA, and in some of the patients treated with differentiation therapy. p53FL positively correlated with in vitro VPA sensitivity and the FAB class of AML, while p53β/γ isoforms negatively correlated with the same. Our results indicate that p53 protein isoforms are modulated by and may predict sensitivity to differentiation therapy in AML. p53 protein isoform expression has been found to correlate with prognosis and chemotherapy response in acute myeloid leukemia (AML). We aimed to investigate how p53 protein isoforms are modulated during epigenetic differentiation therapy in AML, and if p53 isoform expression could be a potential biomarker for predicting a response to this treatment. p53 full-length (FL), p53β and p53γ protein isoforms were analyzed by 1D and 2D gel immunoblots in AML cell lines, primary AML cells from untreated patients and AML cells from patients before and after treatment with valproic acid (VPA), all- Ttrans retinoic acid (ATRA) in the treatment of acute promyelocytic leukemia, as well as the extensive biological effects in acute myeloid leukemia cells in general [Differentiation therapy has been considered a promising approach for the treatment of AML for several decades, inspired by the tremendous effect of all- general . However general ,22,23. IThe main aim of this work was to investigate if p53 protein isoform expression could be a potential independent biomarker for predicting a response to differentiation therapy in AML. We used 2D gel immunoblots of p53FL and p53β/γ protein isoforms and an in-house-developed pixel-by-pixel correlation method, and showed that in vitro VPA sensitivity of primary AML cells positively correlated with the expression of p53FL and negatively correlated with the expression of p53β/γ protein isoforms. The human AML cell lines MOLM-13 and MV4-11 were purchased from ATCC  and cultured according to the manufacturer’s procedure. For patient material, all studies were performed in accordance with the Helsinki declaration and approved by the Regional Ethics Committee . Samples were collected after informed consent, and peripheral blood mononuclear cells (PBMC) were isolated and stored frozen in liquid nitrogen as previously described . Only paValproic acid  (100 mg/mL in solution) was stored at −80 °C for cell culture work.1D and 2D gel electrophoresis and immunoblotting were performed as previously described ,26. The Histone acetylation, proliferation, differentiation and apoptosis markers in primary AML cells were analyzed by flow cytometry as previously described . Samples3H-thymidine  incorporation assay as previously described [5 cells/mL and treated with VPA (0 and 0.5 mM) for 48 h. 3H-thymidine (5 mCi in 100 mL NaCl) was added (20 µL 3H-thymidine to 200 µL cell suspension) 8 h before harvesting and analysis of the cells using a Packard Microplate Scintillation and Luminescence counter . Triplicates were analyzed for each sample.An evaluation of proliferation in primary AML cells after drug treatment was performed using escribed . Cells wGlobal gene expression profiling of primary AML cells before and during in vivo treatment was performed as previously described . All micCorrelation analysis was performed as previously described ,29. 2D gp < 0.05 was considered significant. Graphs and calculations were obtained using GraphPad Prism® version 6.0  and SPSS software.In addition to this main method for correlation analysis, we also performed a more traditional correlation analysis where we quantified regions of interest in 2D gel images using ImageJ software. Separate values for p53FL, p53β/γ and the ratio of p53FL:p53β/γ were obtained , and Spearman correlations or partial correlations were performed using SPSS software . Spearman correlations were performed on original values, while partial correlations were performed on log2-transformed values for p53FL, p53β/γ and the ratio of p53FL:p53β/γ. For all statistical analysis, n = 21) (see 3H-thymidine incorporation), while an analysis of p53 isoforms was performed in untreated samples. The samples were analyzed by p53 2D gel immunoblotting , images were aligned and normalized and correlation analysis was performed as previously described [t-value = 5.96, p-value < 0.005), and a significant inverse correlation between VPA sensitivity and the expression of p53β/γ isoforms   B  and between the VPA response and the ratio of p53FL:p53β/γ . No significant correlation was found between the VPA response and p53β/γ . Pearson correlations using log2-transformed values for p53FL, p53β/γ and the ratio of p53FL:p53β/γ demonstrated significant correlation only between the VPA response and the ratio . Furthermore, partial correlations between the VPA response and the ratio demonstrated significant positive correlations when controlling for p53FL , and when controlling for p53β/γ   See .t-value = 4.51, p-value < 0.005), indicating that AML patient samples of a less differentiated phenotype would express higher levels of p53β/γ isoforms than the more differentiated phenotype (higher FAB classification). Furthermore, significant positive correlations were observed for the more acidic (lower pI) regions of p53 FL , and a significant negative correlation was observed for the less acidic region of p53 FL , indicating that patient samples with higher differentiation stage would express higher levels of more acidified (putatively phosphorylated and activated) forms of p53 FL  classification (morphology and differentiation stage) in 29 AML patient samples previously not analyzed , and no significant correlations between the FAB class and p53FL  or between the FAB class and the ratio of p53FL:p53β/γ .Additional correlation analyses were performed with the quantification of regions of interest in 2D gel images using ImageJ software and Spearman correlation analysis in SPSS. The results demonstrated a significant negative correlation between the FAB class and p53β/γ  and MV4-11 (AML FAB M5) were treated with VPA  for 48 and 72 h, followed by 2D gel immunoblot analysis with an antibody against p53  A, or 1D 2 twice daily for the first 14 days, and VPA together with theophylline from day 3 until disease progression, as previously described [We investigated p53 isoform expression in patient material from a clinical protocol where patients were treated with oral ATRA 22.5 mg/mescribed . SamplesTwo of the patients responded with downregulation of p53β/γ isoforms on day 8, similar to AML cell lines treated with VPA in vitro, one of them also accompanied by a significant increase in p21. As patient material may differ in levels of actin and other commonly used loading controls, the ratio between p53 FL and p53β/γ isoform expression was calculated, demonstrating an increased p53 FL: p53β/γ ratio in some of the samples during treatment. Clinical parameters of the patient material and response to treatment are described below, and were determined previously   of AML, indicating that patient AML blasts with a low level of differentiation would express high levels of p53β/γ isoforms compared to patients with a high level of differentiation. This is consistent with findings of Hofstetter and colleagues, who reported an association between expression of p53β with poorly differentiated ovarian cancers . In thisThe advantage of the self-developed pixel-by-pixel correlation method is that it is sensitive and gives a detailed correlation for specific parts of the p53 protein isoform profile. This method showed a stronger correlation between the VPA response and p53FL compared to the VPA response and p53β/γ (correlation coefficients 0.80 vs. −0.61). The alternative method used for correlation analysis  was not as sensitive. However, we could also calculate correlations between the VPA response and the ratio of p53FL: p53β/γ using this method. The ratio was a stronger predictor of the VPA response compared to p53FL (correlation coefficients 0.610 vs. 0.384), and remained significant also after controlling for p53FL or p53β/γ. However, p53FL did influence the relationship between the VPA response and the ratio of p53FL: p53β/γ to some degree.In AML cell lines expressing wild type p53, there was a marked downregulation of p53β/γ isoforms after VPA treatment, as well as an upregulation of p53FL. Interestingly, a modulation of p53 protein isoforms was also detected in vivo during differentiation therapy. A noticeable downregulation of p53β/γ isoforms was observed in some of the patients, while the increase in p53FL was (as expected) not as evident as for in vivo chemotherapy . p53FL aAltogether, our results demonstrate that p53 isoform expression is modulated by differentiation therapy in AML, both in vitro and in vivo, and that p53 isoform expression could affect sensitivity to differentiation-inducing agents. We anticipate that p53 protein isoform expression analysis may add independent information on predicting therapy response. Future studies should include a correlation analysis of p53 isoform expression with response to in vivo differentiation therapy, as well as explore the potential of p53 isoform-targeted therapy in aggressive blood cancers like AML.


Whether these basic aspects of presynaptic calcium homeostasis show any consistent trend along the axonal path, and how they are controlled by local network activity, remains poorly understood. Here, we take advantage of the recently advanced FLIM‐based method to monitor presynaptic Ca2+ with nanomolar sensitivity. We find that, in cortical pyramidal neurons, action potential‐evoked calcium entry (range 10–300 nM), but not the resting Ca2+ level (range 10–100 nM), tends to increase with higher order of axonal branches. Blocking astroglial glutamate uptake reduces evoked Ca2+ entry but has little effect on resting Ca2+ whereas both appear boosted by the constitutive activation of group 1/2 metabotropic glutamate receptors. We find no consistent effect of transient somatic depolarization or hyperpolarization on presynaptic Ca2+ entry or its basal level. The results unveil some key aspects of presynaptic machinery in cortical circuits, shedding light on basic principles of synaptic connectivity in the brain.Brain function relies on vesicular release of neurotransmitters at chemical synapses. The release probability depends on action potential‐evoked presynaptic Ca 2+‐dependent neurotransmitter release are key to understanding organisational principles of synaptic connectome. Monitoring presynaptic Ca2+ with nanomolar‐sensitive FLIM methods has revealed that, in cortical pyramidal neurons, spike‐evoked Ca2+ entry tends to increase with higher axonal branch orders. Blocking astroglial glutamate uptake reduces evoked Ca2+ entry with little effect on resting Ca2+ whereas both appear boosted by the constitutive activation of group 1/2 metabotropic glutamate receptors. We find no consistent effect of transient somatic depo‐ or hyperpolarisation on presynaptic Ca2+ entry or its basal level. The results unveil some principal aspects of presynaptic machinery in cortical circuits.Mechanisms of Ca Classical studies in preparations of giant synapses that enable direct experimental access, have revealed key mechanistic relationships between neurotransmitter release, evoked Ca2+ entry, and resting presynaptic Ca2+  measurements whereas the accuracy of ratiometric Ca2+ indicators in optically turbid media, such as brain tissue, is compromised by the strong dependence between the wavelength and scattering/absorption of light. Thus, monitoring [Ca2+] inside individual axons, in particular the nanomolar range basal Ca2+ levels, has been a challenge.Information processing and storage in the brain relies on Ca2+ indicators to free Ca2+  has suggested that, in hippocampal granule cells, somatic depolarization reduces spike‐evoked presynaptic Ca2+ entry in proximal axonal segments  of the host neuron affects its axonal Ca22.1ab libitum. This study was not pre‐registered.All experiments involving animals were carried out in accordance with the European Commission Directive (86/609/EEC) and the United Kingdom Home Office (Scientific Procedures) Act (1986) under the Home Office Project Licence PPL P2E0141 E1. C57BL/6 mice  of both sexes  were group housed in a controlled environment as mandated by the locally approved guidelines, on a 12 hr light cycle and with food and water provided 2.22PO4, 1.25 ; HEPES, 20 ; thiourea, 2 ; sodium ascorbate, 5 ; sodium pyruvate, 3 ; MgCl2, 10 ; D‐glucose, 25 ; NaHCO3, 30 ; CaCl2, 0.5 ; and sucrose, 1 . For recovery slices were left for 15–20 min in slicing solution and for 40 min at 34°C ACSF solution, before being transferred to a submersion chamber for storage in an extracellular solution containing (in mM): NaCl, 125 ; KCl, 2.5; NaH2PO4, 1.25; NaHCO3, 26 ; D‐glucose, 18; CaCl2, 2; MgSO4, 1.3 (osmolarity adjusted to 295–310 mOsM with D‐glucose). All solutions were continuously bubbled with 95% O2/5% CO2. Slices were allowed to rest for at least 60 min before recordings started.Acute 300 μm thick coronal brain slices were obtained from 47 3–4 week old C57BL/6 mice (15–20 g), in full compliance with national guidelines on animal experimentation, in accord with Schedule I humane procedures. Animals were anaesthetized by 5% isoflurane inhalation, deep anaesthesia was ensured by a lack of pedal reflexes; after cessation of breathing animals were decapitated for brain isolation and removal. The locally approved isoflurane anaesthesia is sufficiently potent to provide muscle relaxation adequate for ascribed procedure and produces less cerebral vasodilation than analogues ; absorption and elimination of isoflurane inhalation occur through the lungs and allow rapid and reliable aesthetic induction. Slices were prepared in an ice‐cold slicing solution containing (in mM): NMDG, 92 ; KCl, 2.5 ; NaH2.33O3S, 130 ; NaCl, 8; HEPES, 10; phosphocreatine disodium, 10 ; Na2GTP, 0.4 ; MgATP, 4 ; sodium ascorbate, 3 , and supplemented with the morphological tracer dye Alexa 594  with addition of Oregon Green BAPTA‐1  for FLIM recordings. Following whole‐cell break‐in, 40–60 min were allowed for the dyes to equilibrate across the cell, and then the axonal arbour was traced in frame‐scan mode, also using z‐axis browsing, until the first axonal bouton had been identified as described previously  using an adaptation of pre‐synaptic Ca2+ imaging methods previously described  line scans were adjusted to cover the visible bouton profile, and recorded as described below. Depending on the bouton size, one spiral scan typically takes 1–1.5 ms, thus providing readout of axonal fluorescence with high temporal and spatial resolution. Individual action potentials were evoked by a 2 ms pulse of depolarizing current (0.9–1.5 nA), in current clamp mode, as detailed previously  imaging system , integrated with patch‐clamp electrophysiology  and optically linked to two femtosecond pulse lasers MaiTai (SpectraPhysics‐Newport), with independent shutter and intensity control. Patch pipettes were prepared with borosilicate–standard wall filament glass , with 4–5 mOsM resistance. Internal solution contained (in mM): KCH2.4t × x × y × T data cube representing an x‐y image with the distribution of nanosecond decay timestamps (t) of individual photons, pixel‐by‐pixel over the frame duration (T). However, for the purposes of this study, we collapsed all spatial information thus boosting photon counts per scan cycle. The FLIM data represented therefore the average signal over the bouton area (approximately the entire profile) covered by the scan. Post‐hoc FLIM analyses were performed in a custom‐made data analysis toolbox, which is available online . The fluorescence decay curve (lifetime photon counts) was integrated over the 9 ns period post‐pulse, and normalized to the maximum value, as detailed earlier , continuous tornado line scans were collected. The scan data were recorded by the standard analogue integration in Femtonics MES (RRID: SCR_018309), and by TCSPC in Becker and Hickl SPCM (RRID: SCR_018310) using dual HPM‐100 hybrid detectors. Next, we used the fast‐FLIM analysis procedure described previously  was faster than the averaging time of FLIM recording (5–10 ms). Therefore, to improve the signal‐to‐noise ratio in measuring presynaptic Ca2+ entry Δ[Ca2+], the spike‐evoked peak presynaptic [Ca2+]peak was estimated using both FLIM and intensity recordings as follows. First, the saturated OGB‐1 fluorescence value Fmax was estimated asFmax=Frest[Ca2+]0+Kd[Ca2+]0+(Kd/γ) where [Ca2+]0 is measured directly with FLIM, and Kd = 0.24 µM and γ = 6 are Ca2+ affinity and dynamic range of OGB‐1, respectively . Second, [Ca2+]peak (equilibrated over 1–2 ms) was calculated as[Ca2+]peak=KdFpeak-(Fmax/γ)Fmax-Fpeak  basal presynaptic  measurements on a designated two‐photon excitation (2PE) microscopy imaging system, we employed the protocol established for OGB‐1 previously , and the result is related to the peak value  mode Figure . Once foa Figure .3.22+] ([Ca2+]0) and spike‐evoked Ca2+ entry (concentration increment Δ[Ca2+]) from 61 axonal boutons in 25 pyramidal cells. First, the results indicated no overall dependence of either [Ca2+]0 or Δ[Ca2+] on the distance from the soma  and, especially, Δ[Ca2+] (detected range ~10–300 nM) across the axonal population , but had an effect on Δ[Ca2+]   Figure , right. 3.32+ signalling, we documented changes in [Ca2+]0 and Δ[Ca2+] in response to the pharmacological blockade of astroglial glutamate transporters. Application of the transporter inhibitor TBOA  mechanism, such as membrane depolarization, or through the action of presynaptic metabotropic glutamate receptors, or both. To distinguish between these two mechanisms, we recorded [Ca2+]0 and Δ[Ca2+] in individual axonal boutons in baseline conditions, 15 min after washing in the selective group 1/2 mGluR blocker S‐MCPG, and 15 min after the subsequent application of TBOA.To understand whether and how glutamate uptake affects presynaptic Ca% Figure  and b. T2+]0 by ~ 25% . Overall, these findings may reflect a complex nature of presynaptic Ca2+ control by different mGluR subtypes (see Discussion).S‐MCPG application reduced  Figure ; right d3.42+ dynamics, we documented [Ca2+]0 and Δ[Ca2+] in individual axonal boutons when the presynaptic cell was either depolarized, or hyperpolarized, by ~15 mV either way for 500 ms prior to evoking an action potential  local 'hotspot' entry, and is fully in line with previous estimates based on fluorescence‐intensity measures  boutons   somatic depolarization of cortical pyramidal cells, which should mimic subthreshold excitation of the cell, does not consistently affect [Cas Figure . Previous Figure . HoweverThe authors declare no known conflict of interest.