Acute myeloid leukemia (AML) with balanced translocation t  is one of the most frequent chromosomal abnormalities and carries a favorable clinical outcome. However, according to a literature review, additional chromosomal aberrations can affect the overall disease prognosis. Trisomy 4 is a rare numerical abnormality in AML patients with t , which can be associated with c-KIT gene involvement. In adults, c-KIT mutation carries an unfavorable clinical outcome; however, its incidence and clinical importance in the pediatric population are still under scrutiny. Here, we report a case of AML with t and trisomy 4 in an eight-year-old female child and the clinical course of the disease. Acute leukemia is one of the most common childhood cancers, however, acute myeloid leukemia (AML) constitutes only 15% to 20% of such cases [An eight-year-old female presented with a three-day history of epistaxis and fever. On examination, there was no hepatosplenomegaly or lymphadenopathy. She underwent a bone marrow biopsy at an outside institution, where she was diagnosed as acute promyelocytic leukemia (APML) on morphology. Subsequently, she received three doses of all-trans-retinoic acid (ATRA). Her bone marrow aspiration slides and trephine block was referred to our institution for a second opinion. On presentation to our institution, laboratory investigations, including a complete blood count and coagulation studies, were ordered. Her complete blood count showed leukocytosis, with the differential leukocytic count revealing 89% blasts. This was compounded with moderate anemia according to the World Health Organization (WHO) guidelines for her age  and throOn review, her bone marrow aspirate revealed a hypercellular specimen with 76% blasts. These blasts were medium to large in size, with immature chromatin and abundant, hypergranular cytoplasm. They also showed cytoplasmic vacuolations, prominent nucleoli, and Auer rods. However, abnormal promyelocytes and Faggot cells Figure  diagnostA flow cytometric analysis was done on peripheral blood, which showed these blasts to be positive for CD13, CD33, and CD117, strongly positive for myeloperoxidase (MPO), had low expression of CD34, and were negative for HLA-DR. Chromosomal analysis was performed on unstimulated short-term peripheral blood cultures 24 hours), followed by Giemsa trypsin banding. Cytogenetic findings were described according to the International System for Human Cytogenetic Nomenclature (ISCN 2013). An analysis was performed on 16 metaphases, which showed 46,XX,t/47,idem,+4/46,XX. Figure 4 hours, Peripheral blood was used for RNA extraction using reverse transcription polymerase chain reaction (RT-PCR) protocols for PML-RARα and RUNX1/RUNX1T1 amplification. Polymerase chain reaction (PCR) for RUNX1/RUNX1T1 was positive and PML-RARα and core-binding factor subunit beta (CBFB) were negative. Deoxyribonucleic acid (DNA) analysis for c-KIT mutation was not performed due to the non-availability of the test.Consequently, the patient was labeled as WHO category acute myeloid leukemia with t;RUNX1/RUNX1T1 and was admitted to the oncology ward. She received conventional chemotherapy through the standard protocol. A repeat bone marrow biopsy was performed on day 29 post-chemo-induction, which revealed bone marrow in morphological and cytogenetic remission.Following this, the patient was put on maintenance therapy. However, she presented 10 months later to our emergency department with disease relapse. Cytogenetics showed the persistence of the t clone with no trisomy 4 seen. The patient was advised regular follow-up at the hematology clinic where workup for hematopoietic stem cell transplant was started.The balanced translocation t , RUNX1-RUNX1T1 is one of the most frequent chromosomal abnormalities in pediatric AML cases and confers a good prognosis. However, its occurrence with trisomy 4 is rare and studies elucidating the prognostic significance of this aberration are limited. In our study, the patient initially went into remission but the disease relapsed after 10 months. A similar case was reported in Malaysia, where an adult woman was suspected as having AML M3 according to the French-American-British (FAB) classification based on the initial presentation. However, her cytogenetic studies excluded the diagnosis of AML M3 and confirmed AML with trisomy 4 in addition to t. The patient received all-trans-retinoic acid but died soon afterward. This raises concerns over the clinical outcome of patients with the aforementioned chromosomal abnormalities [Pan et al. performed a retrospective analysis of 21 cases of acute leukemia with trisomy 4 published in 2007. It showed that acute leukemia with trisomy 4 has distinctive clinical and laboratory features and a poor prognosis. The most common AML subtype with trisomy 4 was identified as M2 .A study was conducted on 94 adults (age 15 or above) in Japan where patients with t AML were further categorized according to additional chromosomal aberrations. Twenty-seven cases (29.3%) of t AML with loss of the sex chromosome and 10 cases 10.6%) of t AML with chromosome 9 abnormalities were identified. However, the study observed no significant difference in the clinical outcome of the aforementioned patients with those that had t AML with no additional chromosomal aberrations. Trisomy 4 was found in only three cases (3.2%) and all these patients died within 2.4 years. These observations suggest that t with trisomy 4 has a distinctive clinical outcome and may constitute a unique subtype of t AML [% of t AML. The results concluded that trisomy 4 was found in 21 patients and these patients showed an inferior cumulative incidence of relapse and survival . Chilton;21 AML. While the poor prognosis of patients of AML with t and trisomy 4 is still being researched, some studies attribute this to the presence of the c-KIT gene located at chromosome 4. Trisomy 4 may lead to duplication and increased dosage of the mutated c-KIT allele, leading to a distinctive mechanism of leukemogenesis . BeghiniAML in the pediatric population shows a high degree of relapse, therefore, accurate prognostic markers are required to allow treatment modification. AML with different cytogenetic subgroups may respond differently to specific therapies. Therefore, performing cytogenetic studies in all patients with pediatric AML at diagnosis and during the course of the disease may lead to minimizing the risk of disease relapse.Chromosome abnormalities are important parameters for diagnosis, determining prognosis and treatment modalities in pediatric AML. Our findings illustrate the importance of cytogenetic studies in the diagnosis and risk stratification of acute myeloid leukemia in countries where comprehensive molecular testing is not available. In light of the literature review, AML with t and trisomy 4 in pediatric patients should be closely monitored for risk of relapse.


Mouse models exhibit increased Ca2+ buffering and arrhythmias, and we hypothesized that these changes are primary effects of the mutations (independent of compensatory changes) and that increased Ca2+ buffering and altered Ca2+ handling contribute to HCM pathogenesis via activation of Ca2+-dependent signaling. Here, we determined the primary effects of HCM mutations on intracellular Ca2+ handling and Ca2+-dependent signaling in a model system possessing Ca2+-handling mechanisms and contractile protein isoforms closely mirroring the human environment in the absence of potentially confounding remodeling. Using adenovirus, we expressed HCM-causing variants of human troponin-T, troponin-I, and α-tropomyosin  in isolated guinea pig left ventricular cardiomyocytes. After 48 h, each variant had localized to the I-band and comprised ∼50% of the total protein. HCM mutations significantly lowered the dK of Ca2+ binding, resulting in higher Ca2+ buffering of mutant cardiomyocytes. We observed increased diastolic [Ca2+] and slowed Ca2+ reuptake, coupled with a significant decrease in basal sarcomere length and slowed relaxation. HCM mutant cells had higher sodium/calcium exchanger activity, sarcoplasmic reticulum Ca2+ load, and sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) activity driven by Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of phospholamban. The ryanodine receptor (RyR) leak/load relationship was also increased, driven by CaMKII-mediated RyR phosphorylation. Altered Ca2+ homeostasis also increased signaling via both calcineurin/NFAT and extracellular signal–regulated kinase pathways. Altered myofilament Ca2+ buffering is the primary initiator of signaling cascades, indicating that directly targeting myofilament Ca2+ sensitivity provides an attractive therapeutic approach in HCM.Mutations in thin filament regulatory proteins that cause hypertrophic cardiomyopathy (HCM) increase myofilament Ca For example, reduction of SERCA levels e fibers –7. Furth of cTnC , 9. Othe in MYH7  and MYBP (MYBPC3 ) are als systole . We preduffering , 14, wheuffering , 16. In A levels  and myofA levels , both weinea pig . The gen is ∼30% . Cardiac is ∼30% , indicat2+ handling in HCM. Furthermore, we present for the first time a novel analytical paradigm to fully incorporate the consequences of Ca2+ buffering on cardiomyocyte Ca2+ transients and Ca2+ handling protein activities. We consider the dynamics of total intracellular Ca2+  derived from NCX integral measurements rather than free Ca2+ ([Ca2+]i) from fura2 fluorescence when assessing SR load, fractional SR release, and SERCA activity. When applied, our data show that the primary effects of HCM mutant gene expression include altered Ca2+ transients and increased myofilament Ca2+ buffering, increased [Ca2+] in both the SR and cytoplasm, and activation of Ca2+-dependent signaling mediated by Ca2+/calmodulin-dependent protein kinase II (CaMKII), calcineurin/nuclear factor of activated T-cells (NFAT), and mitogen-activated protein kinase/ERK.We have engineered adenoviruses to express WT and R92Q cTnT, WT, and R145G cTnI, and WT and D175N α-TM. By measuring the impact of mutations affecting three different sarcomeric proteins, we aimed to identify hallmark changes in CaA and C). The relative expression of thin filament regulatory protein subunits was determined by Western blotting and subsequently adjusted by the infection levels for each cell preparation tested. Conjugation of an N-terminal (cTnT and cTnI) or C-terminal (α-TM) FLAG tag increased the molecular weight of the recombinant proteins sufficiently to allow differentiation from the endogenous subunit (E). The relative expression level of recombinant protein in purified cardiomyocytes coexpressing human recombinant GFP was found to be 54.3 ± 9.5% (cTnT R92Q), 49.3 ± 8.5% (cTnI R145G), and 57.6 ± 7.5% (TM D175N) (B and D), thus likely to closely reflect the levels of mutant protein found in patients with autosomal dominant cardiomyopathy. The recombinant FLAG-tagged thin filament proteins were localized to the I-band of the myofilaments of infected cardiomyocytes . To assess the impact of culture time on cardiomyocyte function, we compared the contractility and Ca2+ transients of cardiomyocytes cultured for 1, 24, and 48 h along with the extent of t-tubular dedifferentiation . We observed a small but significant reduction in the number of t-tubules and a prolongation in the relaxation and Ca2+ reuptake after 48 h .Isolated guinea pig left ventricular cardiomyocytes were infected with ∼1000 MOI of recombinant adenovirus. The level of infection was between 32.1 ± 7.2 (WT TnI) and 87.6 ± 9.1% (WT TM), calculated from the coexpression of human recombinant GFP , A and C subunit E. The reM D175N) , B and D2+]total and [Ca2+]i were measured upon the application of 10 mm caffeine using simultaneous measurement of fura2 fluorescence ratio and whole-cell voltage-clamp current in a method adapted from Trafford et al. . We postulated that increasing the Ca2+ affinity of myofilaments by introducing a Ca2+ sensitizing HCM mutation will in turn increase total Ca2+ buffering in the intact cardiomyocytes. We fitted the calibrated total versus free [Ca2+]i data to a Michaelis–Menten type equation (A). The dK of Ca2+ buffering was significantly decreased in the presence of each HCM-causing mutation . However, the total Ca2+ occupancy of the myofilaments (Bmax) was not significantly altered between groups (Table S2). The relative buffering at low [Ca2+] is described by Bmax/dK, and this ratio was increased for all three mutants compared with WT . The calculated relationship between [Ca2+]total and [Ca2+]i over the full range of [Ca2+] is shown in Fig. 2D. We have used these fits to convert observed [Ca2+]free to [Ca2+]total for each cell and calculate changes in SR load and SERCA activity. This incorporates the effect of altered Ca2+ buffering when considering these key Ca2+-handling parameters.i (Δ[Ca2+]i = 0.175 ± 0.007 to 0.307 ± 0.011 μm)  and significantly prolonged rates of Ca2+ reuptake (Δτ = 0.027 ± 0.003 to 0.041 ± 0.002 s−1)  compared with cardiomyocytes expressing the corresponding WT protein. The time to 50% Ca2+ release from the SR and the Ca2+ transient amplitude were not significantly different between groups. In measurements of sarcomere shortening at 1-Hz pacing, all three HCM mutations were found to decrease basal sarcomere length compared with WT (ΔSL = −0.030 ± 0.007 to −0.094 ± 0.007 μm) in line with the higher levels of diastolic [Ca2+]i. Cardiomyocytes expressing HCM mutations also had significantly prolonged relaxation (ΔT50 = 0.011 ± 0.002 to 0.037 ± 0.003 s) . Cardiomyocytes expressing the R145G mutation in cTnI displayed reduced fractional shortening compared with WT; these cells also had the shortest diastolic sarcomere length (1.776 ± 0.007 versus 1.865 ± 0.009 μm in WT), and this may impose some limit on the extent of shortening compared with the other two mutations, because of fura2 accumulation .In analyses of cytosolic i transients, SR load, fractional SR Ca2+ release, and SERCA activity were calculated and found to be decreased, increased, and unchanged, respectively . Furthermore, we found that these alterations were independent of pause duration prior to caffeine spritz (Table S6). However, we also calculated caffeine and Ca2+ transients expressed as [Ca2+]total derived from the NCX current integral, thus incorporating any alterations to Ca2+ buffering, and used this approach to recalculate SR load, fractional SR Ca2+ release and SERCA activity . HCM mutant cells consistently displayed a higher SR load  . The preceding [Ca2+]i transient amplitude (ATable S5) was also converted to [Ca2+]total (BTable S5) to calculate the fractional SR Ca2+ release and SERCA2 activity. HCM cells had unchanged SR fractional release compared with WT  and had increased SERCA2 activity (Δrate = 1.53 ± 0.30 to 3.87 ± 0.42 s−1) .Using a standard method  that utiE), whereas the PKA site at serine 16 of PLN and absolute levels of NCX and SERCA2 remained unaltered . This result also confirms the need to consider the [Ca2+]total transient amplitudes when investigating SERCA2 activity and SR load in cardiomyocytes with altered Ca2+ buffering.The increases in SR load and SERCA2 activity appear to be driven by increased CaMKII-dependent phosphorylation of phospholamban (PLN). Relative phosphothreonine 17 PLN levels were increased in HCM cells (between 87 ± 27 and 220 ± 72% more than WT) E, wherea2+]i in HCM cardiomyocytes suggest there may be Ca2+ leak from the RyR. To test this, we used a method adapted from Shannon et al. (A) and found that RyR leak is profoundly increased in cells expressing HCM mutations (B). It was also observed that RyR blockade with tetracaine increased the [Ca2+]i caffeine transient amplitude by 12–25% in HCM cells (C) but had no effect on WT-infected or uninfected control myocytes. Despite the combination of increased leak (B) and [Ca2+]i SR load  (C), the relative leak load relationship was still higher in HCM cardiomyocytes (Δμm/s/μm = 5.24 × 10−5 ± 1.65 × 10−5 to 2.22 × 10−4 ± 3.75 × 10−5) (D), suggesting that other factors may be driving RyR channel open probability. We measured the phosphorylation status of each of the known regulatory sites on the RyR  and found that (consistent with changes in PLN phosphorylation), there was higher phosphorylation at the RyR CaMKII site serine 2814 (20 ± 11 to 87 ± 27% greater than WT) (E); phosphorylation at the PKA/PKG sites (serine 2030 and 2808) did not differ between WT and HCM mutant cardiomyocytes .Increased SR load and diastolic i and [Ca2+]total to be calculated. To exclude the other flux mechanism's contribution to the extrusion of Ca, the measured NCX currents were integrated and corrected as previously described  the change of integral of NCX current. This gave the Δ[Ca2+]total, whereas the change of intracellular Ca2+ transient measured by fura2 gave Δ[Ca2+]i during caffeine application. Thus, the relationship between Δ[Ca2+]i and Δ[Ca2+]total represents the Ca2+ buffering regardless of differential bound myofilament Ca2+ in control and mutant cells at baseline. Plots of [Ca2+]i versus [Ca2+]total were fitted to a Michaelis–Menten equation to give estimates of buffering dK and Bmax. NCX amplitudes were taken from currents obtained during the buffering measurement protocol. Relative changes in SR load were calculated using [Ca2+]total transients upon caffeine spritz to account for the Ca2+ buffering of each cell. SERCA2 activity was determined from the decay constants from the caffeine transient and the preceding Ca2+ transient using standard methods . Sarcomere shortening was captured by Fourier transform of the cardiomyocyte striations under phase contrast microscopy using a switching rate of 100 Hz. Ca2+ transients were captured simultaneously, using the ratio of fura2 fluorescence emission at 365/380 nm at a switching rate of 1000 Hz. All contracting cardiomyocytes were measured for contractility and fura2 Ca2+; any cells displaying asynchronous contractility or excessive blebbing/dysmorphology were ignored for acquisition. fura2 fluorescence ratio was subsequently calibrated to [Ca2+]i by selective membrane permeabilization using ionomycin .fura2-loaded guinea pig left ventricular cardiomyocytes were then allowed to settle to the bottom of a perfusion chamber with a 0 thickness coverslip base, which was mounted on an inverted fluorescence microscope. The cells were perfused with buffer containing 150 mm CaCl2. RyR leak was measured for 50 s in the absence of field stimulation in a Na+- and Ca2+-free solution. Field stimulation and perfusion of 1.8 mm CaCl2 were restarted to allow verification that basal functional conditions were as at the start of the experiment. Perfusion was again switched to Na+- and Ca2+-free solution containing 1 μm of tetracaine, and field stimulation was stopped to make a baseline reading that was subtracted from the preceding RyR leak measurement. Leak/load relationships were measured by 10 mm caffeine spritz following each perfusion switch.RyR leak was calculated using the RyR channel blocker tetracaine as previously reported . Brieflyversus α-actinin. NFAT and ERK nuclear localization was also measured versus nucleoplasmic marker 4′,6′-diamino-2-phenylindole.Western blots were performed to detect cTnT, cTnI α-TM and FLAG tag, PLN, phosphoserine 16 PLN, phosphothreonine 17 PLN, RyR, phosphoserine 2030 RyR, phosphoserine 2808 RyR, phosphoserine 2814 RyR, phosphoserine 22/24 cTnI, NCX, SERCA2A, NFAT, phosphoserine 165 NFAT, ERK, or phosphothreonine 202/tyrosine 204 ERK in WT and mutant-infected cardiomyocytes. In immunolocalization experiments, recombinant FLAG-tagged protein was detected n experiments ± S.E. throughout. Statistically significance was determined using unpaired Student–Newman–Keuls analysis for nonnormally distributed data , with significance values defined as p < 0.05.The data are expressed as the average of P. R., Y.-H. Z., B. C., H. W., and C. R. conceptualization; P. R., X. L., A. S., S. P., and C. R. data curation; P. R., X. L., and S. P. formal analysis; P. R., B. C., H. W., and C. R. supervision; P. R., X. L., A. S., S. P., and Y.-H. Z. investigation; P. R. and X. L. methodology; P. R. and C. R. writing-original draft; P. R. project administration; P. R., X. L., Y.-H. Z., B. C., H. W., and C. R. writing-review and editing.


SET-overexpressing AMLs. Here, we show that upregulation of protein kinase p38β is a common event in AML. We provide evidence that p38β potentiates SET-mediated PP2A inactivation by two mechanisms: facilitating SET cytoplasmic translocation through CK2 phosphorylation, and directly binding to and stabilizing the SET protein. We demonstrate the importance of this new regulatory mechanism in primary AML cells from patients and in zebrafish xenograft models. Accordingly, combination of the CK2 inhibitor CX-4945, which retains SET in the nucleus, and FTY720, which disrupts the SET-PP2A binding in the cytoplasm, significantly reduces the viability and migration of AML cells. In conclusion, we show that the p38β/CK2/SET axis represents a new potential therapeutic pathway in AML patients with SET-dependent PP2A inactivation.Acute myeloid leukemia (AML) is an aggressive hematologic malignancy. Although novel emerging drugs are available, the overall prognosis remains poor and new therapeutic approaches are required. PP2A phosphatase is a key regulator of cell homeostasis and is recurrently inactivated in AML. The anticancer activity of several PP2A-activating drugs  depends on their interaction with the SET oncoprotein, an endogenous PP2A inhibitor that is overexpressed in 30% of AML cases. Elucidation of SET regulatory mechanisms may therefore provide novel targeted therapies for  For decades, chemotherapy consisting of cytarabine and anthracyclines has been the standard in AML care. Emerging drugs show promising results3; however, the outcome for AML remains poor and most patients ultimately relapse and die from disease progression despite initial sensitivity to chemotherapy. Patients older than 60 years old, who represent the main group, are refractory to cytotoxic intensive chemotherapy because of biological disease-related factors, such as increased frequency of adverse-risk cytogenetic and molecular features, and secondary AML3. Moreover, they present comorbidities that reduce their tolerance of intensive therapies, leaving few treatment options in most cases1. Even in younger patients the outcome is dismal. In patients ≤60 years old complete remission is achieved in around 70%, but a subset of patients relapse, depending on the prognostic factors, and only 5–10% survive after relapse4. Current efforts directed towards the genetic characterization of AML have led to the development of new targeted therapies, including FLT3, BCL2 and IDH1/2 inhibitors10. However, monotherapy with these drugs does not result in durable responses. Thus, further research is necessary to develop new personalized therapeutic strategies for the treatment of this aggressive disease.Acute myeloid leukemia (AML) is a highly heterogeneous fatal disease that results from the enhanced proliferation and impaired differentiation of hematopoietic stem and progenitor cells12. Protein phosphatase 2A (PP2A) is a tumor suppressor that regulates several essential cell functions and counteracts most of the kinase-driven intracellular signaling pathways13. Previous results from our group and others showed that PP2A inactivation is a recurrent event in AML, and that its pharmacological activation by PP2A-activating drugs  effectively antagonizes leukemogenesis15. Furthermore, preclinical studies show that these drugs have synergistic effects with conventional chemotherapy and tyrosine kinase inhibitors, opening new possibilities for personalized medicine in AML17. Interestingly, the anticancer activity of several PP2A-activating drugs depends on their ability to interact with the endogenous PP2A inhibitor SET, an oncoprotein overexpressed in ~30% of AML patients and associated with poor outcome19. Therefore, targeting SET allows PP2A to be reactivated indirectly, avoiding toxicity problems related to the direct activation of this complex holoenzyme. SET is a multitask oncogenic protein involved in many cellular processes23. However, despite the prognostic impact of SET overexpression in both hematologic and solid tumors, the mechanisms by which SET is regulated remain poorly understood. We have previously reported a novel multi-protein complex that activates SET transcription in AML24. Here, we explore the post-transcriptional regulation of SET, which may help us to develop novel targeted therapies in AML patients with PP2A inactivation and high expression of SET. Using genetic and pharmacological approaches, we found that p38β, one of the p38 family members whose function is not well known, has a dual role in the regulation of PP2A activity in AML. p38β regulates SET phosphorylation and intracellular localization through the activity of casein kinase 2 (CK2). Furthermore, p38β stabilizes the SET protein, facilitating its PP2A inhibitory role. Importantly, we validated this mechanism in vivo by demonstrating that the combination of the CK2 inhibitor CX-4945, and the PP2A-activating drug FTY720 significantly reduces the viability and migration of AML cells. This novel mechanism may constitute the basis for targeted therapy in AML patients with SET overexpression.Reversible phosphorylation allows the cell to maintain a proper homeostasis regulation; therefore, the balance between kinases and phosphatases is essential to control correct proliferation, apoptosis, and differentiation. Many studies have analyzed the abnormal behavior of protein kinases in AML, but the role of phosphatases remains underexploredComité Ético de Investigación Clínica, Gobierno de Navarra (2018/32). The experiments conformed to the principles set out in the WMA Declaration of Helsinki. AML patient sample cells (CD34+) were cultivated in the semisolid medium MethoCult  supplemented with penicillin G (100 U/ml) and streptomycin (0.1 mg/ml). In the medium, different concentrations of FTY720, CX-4945 and combination were added. After 12–14 days growing at 37 °C in a 5% CO2 atmosphere, the present colonies were counted at an inverted light microscope  using a grid .The study comprised peripheral blood mononuclear cells (PB-MC) samples of 27 patients with AML at diagnosis who stated an informed consent  containing 100 μM cold ATP and 2μCi of [γ-32P]ATP (3 000 Ci/mmol) for 40 min at 30 °C. Reactions were stopped by adding sample loading buffer and boiling 5 min. Proteins were resolved by SDS-PAGE, stained with Coomassie, and analyzed by autoradiography.Bacterially-expressed p38α or p38β (0.2 µg) were pre-incubated with purified MKK6 (40 ng) and then incubated with purified GST, GST-ATF2 or GST-SET (1 µg) in kinase buffer , MAPK11/p38β (#1:s11155 and #2:11156), MAPK14/p38α (#1:s3586 and #2:s3585), and CK2 (s3638). SET siRNAs were siSET#1  and siSET#2 . Due to the high efficiency obtained with siRNAs #1 from p38α and p38β we used them for all experiments. For silencing experiments, cells were transfected using GenePulser XcellTM  with 300 V and 1000 µF. The shRNAp38β cloned in the pINDUCER 11  was shRNA1: CACGTTCAATTCCTGGTTT and shRNA2: GCGCCAGAAGGTGGCGGTGAAG.siRNAs were from Ambion : scramble siRNA 25. Reagents and antibodies used are displayed in Supplementary Tables Details on general methodology as western blot, protein immunoprecipitation, apoptosis, and MTS assay have been previously described2 atmosphere. Prior treatments, cells were plated at 100,000 cells/ml.HL60, MOLM-13, and HEK293T cells were maintained in RPMI-1640  supplemented with 10% FBS, penicillin G (100U/ml) and streptomycin (0.1 mg/ml). Cell lines were grown at 37 °C in a 5% CO26. For colocalization, the red, green and red-green colocalization volumes (umÂ³) were quantified and referred to total cell volume. For nuclear and cytoplasmic quantification, green volume (umÂ³) was measured in the cytoplasm and the nucleus and referred to total cell volume.100,000 cells were seeded on cover slips coated with poly-L-lysine , fixed with 4% paraformaldehyde (Thermo-scientific) and permeabilized with 0.1% Triton-X-100. After blocking with 5% FBS, incubation with primary and secondary antibodies were performed  using the Tank blotting system . As a control, an aliquot of the cell lysate (15 µg) was incubated with 100 units of λ-phosphatase  for 1 h at 30 °C in a shaking thermoblock.10% acrylamide gels were prepared in the presence of 40 µM of Phos-tag  and 20 µM of MnClMigration assay was performed in a 24-transwell permeable plate with 8.0 μM pores . The lower compartment contained RPMI supplemented with 10% FBS. 500,000 treated cells were seeded in the upper insert in medium without serum and allowed to migrate for 3 h. The volume of the bottom well was collected and mixed with perfect-count microspheres . The amount of viable migrated cells was determined by flow cytometry, counting 5000 microsphere-events and expressed as a percentage of the control.Danio rerio, AB strain), from the Zebrafish International Resource Centre, were maintained in re-circulating tanks according to the standard procedures. Adult fishes were maintained at 26 °C, with a light/dark cycle of 14/10 h, and were fed twice daily, once with dry flake food  and another with live Artemia salina . Zebrafish embryos were maintained in egg water at 28.5 °C, fed for 5 days with Novo Tom and with live Artemia salina at 11 days of life. All experiments were performed in compliance with the Guidelines of the European Union Council for animal experimentation (86/609/EU).Wild-type zebrafish (Wild-type zebrafish embryos at 48hpf were anesthetized with 0.04% Tricaine (Sigma–Aldrich). Treated leukemia cells were stained with red fluorescent CM-DiI (Invitrogen) prior the injection. 50–75 labeled cells were injected into the yolk sac of dechorionated zebrafish embryos using a manual injector (Narishige). Fish with fluorescently labeled cells appearing outside the implantation area at 2hpi were excluded from analysis. All other fishes were incubated at 35 °C for 72 h and analyzed with the SteReo Lumar V12 stereomicroscope with an AxioCam MR5 camera . Positive embryo colonization was considered when more than five human leukemia cells were present outside the yolk sac at 72hpx. Zebrafish colonization index was calculated as the proportion of embryos colonized in the treatment condition divided by the proportion of invaded embryos in the control condition. Tumor growth and proliferation were evaluated at 2 (reference) and 72hpx in a M205-FA fluorescence microscopy with a DFC365FX camera (Fujifilm Leica). Proliferation index  and area were measured with a Leica Application Suite-X software.p < 0,05). Two-way ANOVA (Tukey’s multiple comparisons test) when the distribution was normal (zebrafish proliferation experiments). Chi-square statistical analysis was done for the invasive potential calculation in zebrafish experiments. Significance was considered when p < 0.05. For AML patient samples tested, 20% decrease in viability was chosen as the threshold as a response to the treatments.Data represented are the mean of three independent experiments ±S.D. Statistical comparisons were carried out using the nonparametric method Kruskal–Wallis test for more than two independent samples, followed by Mann–Whitney U test to compared two groups when the distribution was not normal . However, no correlation was found between p38α and SET . Quantitative analysis confirmed that p38α was expressed at similar levels in PB and AML cell lines . However, p38β was expressed at lower levels than p38α in HL60 and MOLM-13 cells , but it was undetectable in PBMC , and in 23 out of 27 AML patient samples (85%); whereas the p38α protein was almost equally expressed in PB and AML specimens Fig. . CorrelaWe next focus on dissecting the mechanisms through which p38β regulates the SET protein. Co-immunoprecipitation experiments indicated that SET bound to p38β in both HL60 and MOLM-13 cells, and to a lesser extent, to p38α in HL60 cells Fig. . Immunof25. Here, we show that SETBP1 and p38β colocalized along with SET in the cytoplasm  cascades are important signaling pathways used by eukaryotic cells to transduce extracellular signals. Using chemical inhibitors of several MAPKs, we found that only p38 inhibitors were able to increase PP2A activity by decreasing SET protein, suggesting post-transcriptional regulation of SET. The p38 family is involved in many cellular processes, and plays a key role in the stress response45. However, the nature of their connection varies depending on the context. Upon TNF-induced stress conditions in endothelium-derived cell lines, p38 positively regulates PP2A activity42, whereas under hypoxia and survival conditions in colorectal cancer cell lines, PP2A negatively regulates p38MAPK activity44. Here, we show for the first time that p38β controls PP2A activity through the regulation of its endogenous inhibitor SET.It has been reported that PP2A can regulate p38 signaling pathway, and that PP2A and p38 form complexes in the cytoplasm in basal conditions; in fact, p38 may act as scaffold protein for NMP and PP2A25, and this result has been confirmed in other reports47. Here we further characterize this mechanism by showing that p38β co-localizes with SET, SETBP1, and PP2A, regulating PP2A activity in AML cells. Additional studies will be needed to elucidate how the interplay among these proteins regulates SET stability.Pyridinyl-imidazole inhibitors have allowed the identification of many functions regulated by p38 beyond the stress response. However, these compounds do not permit us to distinguish functions mediated by p38β from those regulated by p38α. Therefore, we used RNA interference to decipher the specific role of p38β in AML cells. Our immunoprecipitation and immunofluorescence analysis support the notion that p38β interacts with SET in AML cells, and knockdown of p38β, but not p38α, decreases SET protein levels and enhances PP2A activity. Moreover, we found that p38β binding stabilizes the SET protein in the cytoplasm, demonstrating a new role for p38β. We had previously demonstrated that SETBP1 binds to and stabilizes SET, facilitating PP2A inhibition20, DNA repair21, migration22, and cell-cycle progression23. Here, we report a robust accumulation of SET into the cytoplasm of primary and patient-derived AML cells. Several studies in Alzheimer’s disease show that CK2 phosphorylates SET on Ser9, in the nuclear localization signal, which is key for SET cytoplasmic localization and inhibition of PP2A, leading to tau hyperphosphorylation48. Here we demonstrate in AML cells that p38β is involved in SET trafficking to the cytosol and PP2A inactivation through the activation of CK2, and that silencing of p38β but not p38α decreases CK2-dependent phosphorylation of SET. Moreover, overexpression of p38β decreased PP2A activity in a CK2-dependent manner. Consistent with these findings, pharmacological inhibition or silencing of CK2 increased the nuclear localization of SET, as well as PP2A activity, without altering total SET protein levels. Interestingly, in the absence of SET, inhibition of CK2 has no effect in PP2A activity, supporting the new mechanism described here. It should be noted that CK2 overexpression has been associated with poor prognosis in AML patients with normal karyotype49. Thus, our results support a model in which p38β overexpression activates CK2, which in turn phosphorylates SET, facilitating its trafficking to the cytoplasm where it inactivates PP2A. Therefore, our study identifies a novel p38β-CK2-SET signaling pathway in leukemogenesis that mediates PP2A inactivation. Interestingly, the same pathway would be probably activated in AML cases with CK2 overexpression, opening new insights into the role of CK2 in AML.SET is mostly located in the nucleus where it regulates DNA replication, chromatin remodeling, gene transcription34 supported the importance of p38β in AML cells, and confirmed that the combination of CX-4945 and FTY720 is more effective than either treatment alone at reducing tumor growth, as well as impairing cell migration and invasion. Consistent with our results, SET phosphorylation would allow its interaction with the GTPase Rac1 and cytoplasm localization, where SET inactivates PP2A. In fact, it has been reported that Rac1 stimulated signaling required for efficient cell migration involves SET-mediated inhibition of PP2A, and that cytoplasmic targeting of SET inhibits Rac1-induced cell spreading and migration22. Taken together, our in vivo results confirm the value of targeting multiple components of the same pathway, and support the use of zebrafish xenografts to predict drug sensitivity for personalized treatments in AML cases.We validated the importance of this new pathway by showing that the combination of CX-4945, which inhibits CK2 allowing nuclear SET retention, and FTY720, which disrupts SET-PP2A binding, is more effective in decreasing viability and inducing apoptosis of AML cells from patients than either single treatment. In vivo studies using zebrafish xenografts as a preclinical modelIn conclusion, we have identified a new role of p38β MAPK and CK2 in AML leukemogenesis. We show that p38β overexpression is a recurrent event in AML cases that contributes to PP2A inactivation by regulating the SET oncoprotein through two mechanisms: (i) p38β controls CK2-mediated phosphorylation of SET facilitating its cytoplasmic localization, and (ii) p38β binds to and stabilizes SET in the cytoplasm. Furthermore, we provide in vivo evidence of this mechanism by targeting the same pathway at different levels. We show that a combination therapy using the CK2 inhibitor CX-4945, which retains SET in the nucleus, and FTY720, which disrupts the SET-PP2A binding in the cytoplasm, re-activates PP2A, reducing the viability of AML cells. Our results therefore provide the rationale for using a combination of PP2A-activating drugs and CK2 inhibitors as a novel therapeutic option for treating a subgroup of 30% AML cases characterized by SET-dependent PP2A inactivation.Supplemental MaterialReporting Checklist


TRPM7 is a Ca2+/Mg2+‐permeable non‐selective cation channel. TRPM7 mRNA is highly expressed in adipose tissue, however, its functional expression in adipocytes remains to be elucidated. In this study, we demonstrated for the first time that TRPM7 was functionally expressed in both freshly isolated white adipocytes and in 3T3‐L1 adipocytes differentiated from a 3T3‐L1 pre‐adipocyte cell line by whole‐cell patch‐clamp recordings. Consistent with known properties of TRPM7 current, the current in adipocytes was activated by the elimination of extracellular divalent cations and the reduction of intracellular free Mg2+ concentrations, and was inhibited by the TRPM7 inhibitors, 2‐aminoethyl diphenylborinate (2‐APB), hydrogen peroxide (H2O2), N‐methyl maleimide (NMM), NS8593, and 2‐amino‐2‐[2‐(4‐octylphenyl)ethyl]‐1,3‐propanediol (FTY720). Treatment with small‐interfering (si) RNA targeting TRPM7 resulted in a reduction in the current to 23 ± 7% of nontargeting siRNA‐treated adipocytes. Moreover a TRPM7 activator, naltriben, increased the TRPM7‐like current and [Ca2+]i in 3T3‐L1 adipocytes but not in TRPM7‐knockdown adipocytes. These findings indicate that TRPM7 is functionally expressed, and plays a role as a Ca2+ influx pathway in adipocytes.In adipocytes, intracellular Ca TRP melastatin (TRPM) 7 channel permeates Ca2+ and Mg2+, and is expressed in most cell types  proteins are a superfamily of cation channels, most of which are Ca2+]i affects insulin signaling .2‐aminoethyl diphenylborinate (2‐APB), N‐methyl maleimide (NMM), NS8593, naltriben, dexamethasone (DEX), and 3‐isobuthyl‐1‐methylxanthine (IBMX) were purchased from Sigma‐Aldrich . 2‐amino‐2‐[2‐(4‐octylphenyl)ethyl]‐1,3‐propanediol (FTY720) was purchased from Cayman Chemicals . Recombinant human insulin was purchased from Wako . Fura‐2 acetoxymethyl ester (AM) was purchased from Thermo Fisher Scientific .2 under humidified conditions. Three days after confluence, cells were differentiated into adipocytes with a differentiation medium, consisting of DMEM supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, penicillin‐streptomycin, and a differentiation cocktail . After 42 h, the medium was changed to a fresh differentiation medium without the differentiation cocktail, and then replaced with fresh differentiation medium every 2 days. Differentiated 3T3‐L1 cells were harvested by trypsin digestion and plated on Matrigel® ‐coated glass coverslips for patch‐clamp experiments and Ca2+ imaging experiments, or for siRNA transfection.Freshly isolated murine white adipocytes were prepared as described previously  and 10 μL of 2 μmol/L siRNA  were mixed and incubated for 5 min at room temperature. TRPM7‐targeted siRNA and nontargeted siRNA were used (Silencer® Select s81666 and negative control 1 from Thermo Fisher Scientific). Subsequently, 18.6 μL Opti‐MEM and 1.4 μL of a lipid‐based transfection reagent, DharmaFECT 4 , were added and incubated for 20 min at room temperature. A suspension of differentiated 3T3‐L1 adipocytes was then added to the plate containing siRNA complex at a density of 1.02–1.16 × 105 cells/cm2 and cultured for 24 h. To visualize cells transfected with siRNA, siGLO Green Transfection Indicator (Thermo Fisher Scientific) was co‐transfected. Twenty‐four hours after transfection, cells were re‐plated onto Matrigel‐coated glass cover slips and then cultured an additional 18 to 24 h before patch‐clamp experiments or Ca2+ imaging experiments.Transfection of TRPM7‐targeted siRNA into 3T3‐L1 adipocytes was performed as described previously  following manufacturer’s instructions. The expression level of TRPM7 was determined by quantitative real‐time polymerase chain reaction (PCR) in each sample using the ABI 7500 Real‐Time PCR system (Thermo Fisher Scientific). The expression level of the house‐keeping gene TATA box binding protein was used as an internal control. Primers and probes used for target genes were TaqMan Gene Expression Assays  purchased from Thermo Fisher Scientific.2, 1 CaCl2, 1.2 NaH2PO4, 10 HEPES, 2 glucose, and 27 mannitol . Divalent‐free bath solution (DVF) was made by omitting MgCl2 and CaCl2, and adding 0.5 mmol/L EDTA and 0.2 mmol/L EGTA. The intracellular (pipette) solutions were as follows (mmol/L): 25 CsCl, 135 CsOH, 135 glutamate, 5 HEPES, 1 MgCl2, 5 Na2ATP, and 0.1 NaGTP . When [Mg2+]i was varied to 0, 7.35, 97, and 217 μmol/L . MgSO4 was added to vary [Mg2+]i. [Mg2+] was calculated by Maxchelator software (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/).All experiments were conducted at room temperature (23–25°C). A salt bridge containing 3 mol/L KCl in 2% agarose was used to connect a reference Ag–AgCl electrode. The patch electrodes, prepared from borosilicate glass capillaries, had a resistance of 1.5–2.5 MΩ when filled with a pipette solution (see below). Series resistance (<5 MΩ) was compensated to 80% to minimize voltage errors. Currents were recorded using an Axopatch 200B amplifier  coupled to a DigiData 1321A A/D and D/A converter (Molecular Devices). Current signals were filtered at 1 kHz using a four‐pole Bessel filter and were digitized at 5 kHz. pClamp 10.4 software was used for the command pulse protocol, data acquisition, and analysis. The time courses of current activation and recovery were monitored by repetitively applying (every 10 sec) ramp pulses from −100 to +100 mV (1 sec duration) from a holding potential of 0 mV. The control bath solution consisted of (mmol/L): 135 NaCl, 5 KCl, 1 MgClμmol/L Fura‐2 AM for 30 min at 37°C in HEPES‐buffered saline (HBS) consisting of (mmol/L): 135 NaCl, 5 KCl, 2 CaCl2, 1.2 NaH2PO4, 10 HEPES, and 2 glucose (pH 7.4 adjusted with NaOH). After loading, cells were superfused with HBS for 15 min at room temperature. Calcium‐free HBS was made by omitting CaCl2 and adding 0.2 mmol/L EGTA. Fluorescent images of Fura‐2 excited at 340 and 380 nm were acquired using a scientific CMOS camera  with 40× objective  of an inverted microscope . Data were collected and analyzed with image analysis software . [Ca2+]i was monitored by fluorescence ratio (F340/F380).Differentiated 3T3‐L1 adipocytes were loaded with 1 t‐test  of observations. Statistical analysis was performed between the two groups using a paired st Figs. E–G or anest Fig. A and B. 2+  relationships  inhibited by intracellular Mgμmol/L [Mg2+] in control bath solution , these currents may contain TRPM7 current, which is activated due to a reduction in intracellular Mg2+ concentration to 29 μmol/L by intracellular perfusion with the pipette solution   of 187 μmol/L , NMM (50 μmol/L), or FTY720 (10 μmol/L) inhibited the DVF‐activated current in white adipocytes  as well as NS8593 (20 μmol/L), which has been reported to inhibit TRPM7 current at low concentrations with an IC50 of 1.6 μmol/L  Fig. A and B. 2O2 (500 μmol/L), 2‐APB (200 μmol/L), NMM (100 μmol/L), and NS8593 (20 μmol/L) in differentiated 3T3‐L1 adipocytes compared to native white adipocytes  and the DVF‐activated currents were dramatically reduced in siTRPM7‐transfected adipocytes compared to siControl‐transfected adipocytes   resulted in the reduction of TRPM7 mRNA expression to 22.6 ± 1.7% of nontargeting siRNA (siControl) at post‐transfection day 2  in differentiated 3T3‐L1 adipocytes , and was reversed to the levels before application  in the presence of 2 mmol/L [Ca2+]o  and −0.005 ± 0.006 in siTRPM7‐adipocyte (n = 50), but the resting levels of [Ca2+]i did not differ between siControl‐adipocytes and siTRPM7‐adipocytes  which is necessary for TRPM7 channel activity (Runnels et al. 2+]i by activation or inactivation of TRPM7. Insulin has been reported to activate PLC in adipocytes; however it remains controversial whether insulin induces Ca2+ flux (Draznin et al. 2+] near TRPM7 channel can be induced by insulin via PLC in adipocytes. Further study is needed to clarify if there are physiological stimuli that cause Ca2+ influx via TRPM7 in adipocytes.It is well established that TRPM7 channel activity is tonically regulated by intracellular free Mg2+/Mg2+‐permeable channel that contains a protein kinase domain, it likely exerts functions via Ca2+/Mg2+ influx and/or its kinase activity. In adipocytes, Ca2+ modulates lipolysis (Tebar et al. 2+ content is related to impaired glucose metabolism and insulin resistance (Kandeel et al. In pre‐adipocytes, it has been reported that TRPM7 is functionally expressed and involved in proliferation and adipogenesis (Chen et al. 2+ imaging experiments in adipocytes. TRPM7 may modulate physiological functions of adipocytes by permeating Ca2+/Mg2+ and/or phosphorylating the substrates.In conclusion, we determined the functional expression of TRPM7 by whole‐cell path‐clamp and CaNo conflicts of interest are declared by the authors.


The mechanisms that permit highly localized Ca2+ changes to evoke cell‐wide activity are incompletely understood. Here, we tested the hypothesis that TRPV4‐mediated Ca2+ influx activates Ca2+ release from internal Ca2+ stores to generate widespread effects.The TRPV4 ion channels are Ca2+ signals in large numbers (~100) of endothelial cells in intact arteries were imaged and analysed separately.Ca2+ responses comprised localized Ca2+ changes leading to slow, persistent, global increases in Ca2+ followed by large propagating Ca2+ waves that moved within and between cells. To examine the mechanisms underlying each component, we developed methods to separate slow persistent Ca2+ rise from the propagating Ca2+ waves in each cell. TRPV4‐mediated Ca2+ entry was required for the slow persistent global rise and propagating Ca2+ signals. The propagating waves were inhibited by depleting internal Ca2+ stores, inhibiting PLC or blocking IP3 receptors. Ca2+ release from stores was tightly controlled by TRPV4‐mediated Ca2+ influx and ceased when influx was terminated. Furthermore, Ca2+ release from internal stores was essential for TRPV4‐mediated control of vascular tone.Responses to the TRPV4 channel agonist GSK1016790A were heterogeneous across the endothelium. In activated cells, Ca2+ influx via TRPV4 channels is amplified by Ca2+‐induced Ca2+ release acting at IP3 receptors to generate propagating Ca2+ waves and provide a large‐scale endothelial communication system. TRPV4‐mediated control of vascular tone requires Ca2+ release from the internal store.Ca Central therefore to an understanding of endothelial function is an appreciation of the control of intracellular Ca2+ concentrations.The endothelium plays a critical role in the regulation of numerous vascular processes such as maintaining vascular tone, regulating the passage of macromolecules and oxygen to tissues, modulating immune responses, initiating angiogenesis, and controlling vascular remodelling. The control by the endothelium of each process is mediated by the generation of various signalling molecules that include NO, prostacyclin, von Willebrand factor, tissue plasminogen activator, and endothelial‐derived hyperpolarizing and contracting factors  or IP3 receptors (IP3Rs). While RyRs may be expressed in endothelial cells  which expand by Ca2+‐induced Ca2+ release opening neighbouring IP3Rs generating a Ca2+ puff (∼4 μm spread). Puffs may further propagate via Ca2+‐induced Ca2+ release to form a transient global Ca2+ elevation throughout the cell or a Ca2+ “wave” , from an in‐house colony, were used for the study. Sprague–Dawley rats are a widely used experimental model with a wealth of background information to aid interpretation of results. The animals were housed three per cage, and the cage type was North Kent Plastic model RC2F with nesting material “Sizzle Nest.” A 12:12 light dark cycle was used with a temperature range of 19–23°C (set point 21°C) and humidity levels between 45% and 65%. Animals had free access to fresh water and SDS diet RM1 (rodent maintenance). The enrichment in the cages was aspen wood chew sticks and hanging huts.All animal care and experimental procedures complied with the relevant UK Home Office Regulations,  and were approved by the University of Strathclyde Animal Welfare and Ethical Review Body. Animal studies are reported in compliance with the ARRIVE guidelines  on a Sylgard block. The endothelium was then preferentially loaded with Cal‐520/AM  in physiological saline solution (PSS) at 37°C for 30 min  or GSK1016790A . GSK1016790A is a selective TRPV4 channel agonist and evokes Ca2+ influx in wild type but not TRPV4 channel knockout mice  were isolated from the mesenteric bed, cleaned, then cut and pinned flat 2+ signalling, drugs were added to the perfusate and remained thereafter. Images were acquired at 10 Hz using an inverted fluorescence microscope  equipped with a 40× objective  and an electron‐multiplying charge‐coupled device camera  or on an upright epi‐fluorescence microscope  equipped with a 60× objective . Cal520/AM was excited at 488 nm using a monochrometer  or an LED illumination system .In experiments examining the effects of various pharmacological agents on stimulated endothelial Ca3 (5 μM), and the Ca2+ response to local photolysis of caged IP3 examined. In these experiments, the endothelium was imaged as above, and a xenon flash lamp  was used to uncage IP3  and a membrane permeant, caged IP2.32+ signals were extracted automatically from fluorescence recordings using custom written Python software  were expressed as fractional changes in fluorescence intensity (F/F0) by dividing each intensity value by the average intensity of a 100‐frame period exhibiting the least activity/noise  SD of the derivative, and the rolling (100‐frame) summation of the rolling SD. The minimum value of the rolling summation indicates the centre of the portion of the signal with the least activity/noise.Cellular Ca2+ response to activation of TRPV4 channels contained two main components: a “slow” persistent Ca2+ elevation rise in the baseline Ca2+ levels and fast intracellular Ca2+ waves. To determine the underlying mechanisms, we isolated the two components from each F/F0 trace. The slow persistent Ca2+ elevation (baseline) component of each signal was extracted by applying an asymmetric least squares (ALS) smoothing function  were then calculated from a fitted sigmoidal curve  component of each signal was extracted by dividing each F/F0 trace by the ALS‐smoothed signal. This procedure effectively flattens the baseline and removes any slow drift from the signal  and Fmin the signal in the absence of light to the camera.In some experiments, Ca2.4en face artery preparations. This technique permits simultaneous visualization of the endothelium and assessment of functional responses (contraction and dilation) and was used so that we could confirm the removal of the endothelium as required.To assess the endothelium dependence of contractile and dilation responses, vascular reactivity to various agonists were assessed in −1) when mounted on an upright microscope . Handling and pinning of the arteries were restricted to the outermost corners, leaving the central portion of the vessels with intact endothelium and able to contract/dilate freely.Arteries of 200–230 μm diameter were loaded with Cal‐520/AM as above, except that they were pinned to the bottom of a custom, Sylgard‐coated flow chamber. This flow chamber was designed to facilitate the perfusion of solutions (1.5 ml·min−1 using a syringe pump). Contraction was stimulated by swapping the PSS for one containing phenylephrine. The concentration of phenylephrine was titrated to contract arteries to ~20% of resting diameter. Relaxation was stimulated by changing the phenylephrine‐containing syringe for one containing phenylephrine (at the same concentration) and either ACh (100 nM), GSK1016790A , or sodium nitroprusside . Contractile and dilator responses were assessed before and after (paired) various treatments in the same artery, as described in the text. After treatment, antagonists were continuously present in the PSS. The removal of the endothelium was achieved by gentle mechanical disruption with a human hair that was superglued onto a pipette tip. Endothelium removal was confirmed by the absence of endothelial cells stained with the Ca2+ indicator Cal‐520/AM. After the removal of the endothelium, arteries were left to equilibrate for 60 min.Following incubation, arteries were gently washed with PSS and left to equilibrate for 30 min in PSS  using an upright fluorescence microscope equipped with a 16× objective lens  and large format  back‐illuminated electron‐multiplying charge‐coupled device camera  and stored for offline analysis. An edge‐detection algorithm , Phenylephrine‐induced contractions were transient, and it was not possible to achieve a stable contraction. Therefore, assessing the effect of store depletion on TRPV4‐induced dilation was not feasible. Instead, vessels were pretreated with GSK and CPA, and the magnitude of the transient contraction induced by phenylephrine was assessed. Furthermore, after depletion of Ca2+ stores using CPA, a significantly higher concentration of phenylephrine was required to generate comparable levels of vascular tone  established by a 10 cmH2O pressure gradient.Diameter measurements of pressurized arteries were performed using the VasoTracker open source pressure myograph system  and recorded by VasoTracker acquisition software. Arteries were contracted using phenylephrine (~500 nM) to ~80% of resting diameter, which was added to the perfusion solution. All other drugs  were applied intraluminally. Summary relaxation data are expressed as the percentage increase in diameter compared to resting diameter.2.73  was obtained from Sichem (Germany). Pluronic F‐127 was obtained from Invitrogen . U73343 was obtained from Tocris . All other drugs and chemicals were obtained from Sigma . GSK, HC067047, CPA, U73122, U73343, 2‐aminoethoxydiphenyl borate (2‐APB), and ionomycin were dissolved in DMSO and diluted to working concentration in PSS such that the total volume of DMSO was less than or equal to 0.1%. All other drug stocks were dissolved in water. The PSS consisted of the following (mM): 145 NaCl, 4.7 KCl, 2.0 MOPS, 1.2 NaH2PO4, 5.0 glucose, 0.02 EDTA, 1.17 MgCl2, and 2.0 CaCl2 (adjusted to pH 7.4 with NaOH). In experiments using a Ca2+‐free PSS, Ca2+ was substituted with Mg2+ on an equimolar basis, and EGTA (1 mM) was included. All solutions were freshly prepared each day.Cal‐520/AM was obtained from Abcam . Caged‐IP2.8n vales shown are the numbers of biological replicates . For each replicate, a single field of endothelial cells (Ca2+ imaging experiments) or a single artery segment (vascular reactivity studies) was studied. Summary data are presented graphically as paired mean responses, or as the grand mean ± SEM of n biological replicates. Two‐tailed, paired t test (for paired observations), independent two‐sample t tests or repeated measures one‐way ANOVA with multiple comparisons were used as indicated in the text. All statistical analyses were performed using GraphPad Prism version 6.0 . A P value of <.05 was considered statistically significant.The data and statistical analysis in this study comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology The 2.9http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY  or the TRPV4 channel agonist GSK (20 nM). Ca2+ activity was then visualized in the fields of ~100 endothelial cells. Cellular responses were analysed individually . Finally, large propagating Ca2+ waves developed .Cat Figure . ACh evow Figure . Activats Figure  that cons Figure  and 2. T Figures . The GSKd Figure  and S2. 1 Figure . GSK‐evo2+ signal arising from activation of TRPV4 channels: (a) initial local signals which lead to a slow global rise in Ca2+ and (b) large fast propagating Ca2+ waves.These results suggest there are at least major two components of the Ca2+ signals. We first confirmed the reproducibility of GSK‐evoked responses (20 nM) in the same preparation so that a paired experimental design could be used to examine the effects of blockers. There was no significant difference in any of the Ca2+ signal parameters measured  on repeated applications of GSK  component from raw Ca2+ signals in each cell . However, removal of external Ca2+ significantly reduced the amplitude  and the frequency of occurrence  of these residual propagating Ca2+ waves.To determine if each component is dependent on CaS Figure a,b. The K Figure c. The ve2+ influx across the plasma membrane is essential for the slow global increase in Ca2+ and for the propagating Ca2+ waves evoked by GSK.These results suggest that Ca2+ rise and propagating Ca2+ waves evoked by GSK arose from the activation of TRPV4 channels, we pre‐incubated endothelial cells with the non‐selective TRPV channel antagonist, ruthenium red  and the frequency of occurrence  of propagating Ca2+ waves but had no effect on the velocity of propagating waves . A similar reduction in GSK‐evoked Ca2+ signalling was observed using the selective TRPV4 channel antagonist, HC067047  and the frequency  of propagating Ca2+ waves. The velocity of residual Ca2+ waves was unaffected by HC067 .To test whether or not the slow global Ca2+ influx via TRPV4 channels is required to induce both the slow global rise component and the propagating Ca2+ waves induced by GSK.Together, these results suggest that Ca3.32+ influx in GSK‐stimulated Ca2+ signals, we next investigated the contribution of the internal Ca2+ store using the sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase (SERCA) inhibitor, CPA , amplitude , and frequency  of Ca2+ waves. The amplitude of the slow global Ca2+ rise initially appeared to be reduced by CPA , showing no significant effect of CPA. CPA itself evoked an increase in Ca2+ concentration from a resting value of 176 ± 52 to 335 ± 58 nM (n = 3). The elevated Ca2+ may account for the reduced number of cells activated by GSK , there was a significant reduction in the propagation velocity  and frequency of those events , amplitude , nor the frequency  of propagating Ca2+ waves. Moreover, although U73122 inhibits the internal store Ca2+ pump in colonic smooth muscle cells  but significantly reduced propagation velocity  and frequency  of fast Ca2+ waves . Relaxations to GSK were reversed by the selective TRPV4 channel antagonist, HC067. However, the dilations to ACh were preserved after blockade of TRPV4 channels with HC067 . These results suggest that these channels do not contribute to ACh‐evoked relaxations in rat mesenteric arteries , ACh and GSK each relaxed phenylephrine‐constricted arteries back to pre‐constricted levels . The residual ACh‐evoked response may have arisen from the incomplete removal of the endothelium. The functional removal of the endothelium also prevented GSK‐evoked relaxations. Indeed, after the removal of the endothelium, GSK caused an increase in tone . Endothelium removal did not prevent relaxation to the endothelium‐independent vasodilator SNP  prevents stable contractions such that it was not possible to reliably assess vasodilator responses in these arteries. Instead, we examined whether pretreatment with GSK was capable of modulating PE‐evoked contraction  was required to achieve a level of contraction  equivalent to control conditions . Rather, GSK tended to enhance the contraction to phenylephrine .In this study, we analysed individual TRPV4‐mediated Ca2+ changes occurring as a result of activation of TRPV4 channels was critically dependent on Ca2+ influx across the plasma membrane. The Ca2+ changes were abolished after the removal of external Ca2+ or by the inhibition of TRPV4 channels, with RuR or HC067047. These results suggest that Ca2+ influx via TRPV4 channels generates the persistent global Ca2+ rise. Inhibiting IP3‐mediated Ca2+ release by depleting the internal Ca2+ store, or inhibiting IP3Rs, had little effect on the slow persistent global elevation in Ca2+ but prevented the large transient increases arising from propagating Ca2+ waves. This result suggests that Ca2+ entry via TRPV4 channels triggers activation of IP3Rs which amplifies the initial Ca2+ rise and initiates regenerative release of Ca2+ in the form of propagating waves. In support of the active nature of the Ca2+ waves, the velocity of wave progression away from the release site was relatively constant within and between cells (~5 to 15 μm·s−1). If diffusion alone was responsible for wave progression, velocity and amplitude would decline with distance from the release site. The mechanisms contributing to TRPV4‐mediated activation of Ca2+ release and propagation may include direct activation of IP3Rs (in a Ca2+‐induced Ca2+ release like process) or indirect Ca2+‐dependent activation of PLC. The partial inhibition of the propagating Ca2+ waves by the PLC‐blocker, U73122, is consistent with being a Ca2+‐induced Ca2+ release‐like process at IP3Rs. Basal PLC activity has been reported for many PLC isoforms in biochemical experiments . Previous work has shown that TRPV4 channels are inhibited by the phospholipid, phosphatidylinositol 4,5‐bisphosphate  because IP3Rs are activated by Ca2+. TRPV4 channels are also activated by Ca2+  TRPV4 channel activities are both required for these intercellular waves to occur.As well as propagating at a constant velocity within cells, TRPV4‐mediated Ca2+ waves . These w2+ waves may be a fundamental mechanism for coordinating multicellular responses  than those in mouse cremaster arterioles, suggesting a different physiological control mechanism.In other tissues, intercellular Ca3R‐mediated signalling was eliminated by depletion of the internal Ca2+ store, activation of TRPV4 channels induced a large but highly localized (few square micrometres) increase in Ca2+ concentrations  Ca2+ image displaying a field of 97 native mesenteric artery endothelial cells. The centre of each cell is marked with a red dot. B) F/F0 Ca2+ traces for each of the cells identified in (A). The baseline region (red) was automatically identified for each cell, a trace from a single cell is highlighted in bold. C‐E) To extract signalling metrics, fast and slow Ca2+ signal components were extracted from F/F0 traces. C) Example of Ca2+ signal de‐multiplexing for the cellular trace (#57) highlighted in B. The time‐dependent baseline  of the F/F0 signal (black) was calculated using an asymmetric least‐squares (ALS) function, and modelled by a sigmoid function (green). The fast component of the signal (red) was isolated by dividing the F/F0 signal by the ALS signal. D) Sigmoid function, used to extract the rise time of the slow signal component, and fast signal component (shown in C) shown on an expanded time scale. Peaks were identified in the fast signal using an automated peak detection algorithm. E) Original F/F0, de‐multiplexed signal components, and sigmoid fits for each of the signals shown in B. Each trace is coloured according to the magnitude of the ALS signal, mean signal is overlaid in black.n = 10). B) Mean PE‐contracted vessel diameter  and corresponding concentration of PE (right), in the absence (‐) and presence (+) of cyclopiazonic acid . C) Summary data showing the effect of ACh (blue) and GSK (red) on the diameter of PE‐constricted arteries. Statistical analyses were performed using paired Student's t‐test (A), unpaired Student's t‐test (B) and repeated measures one‐way ANOVA with multiple comparisons (C). * = p < 0.05.Figure S2: ACh and GSK dilate PE‐constricted mesenteric arteries. A) Mean PE‐contracted vessel diameter  and corresponding concentration of PE (right), before (+ endothelium) and after (‐ endothelium) mechanical removal of the endothelium (p < 0.05 (n=13) using paired Student's t‐test.Figure S3: Repeat application of GSK elicits reproducible Ca2+ signals. A) Schematic of the experimental protocol. Mesenteric artery endothelial cells were subjected to perfusion with GSK1016790A  prior to washing, and a second application of GSK (20 nM). B) Composite images showing basal Cal‐520/AM fluorescence (grey) with Ca2+ activity overlaid (red). Ca2+ activity was derived from the 120 sec periods indicated by the numbered arrows in A. Scale bars = 50 μm. C) GSK‐evoked Ca2+ signals for the 1st (left) and 2nd (right) application of GSK. D) GSK‐evoked Ca2+ signalling parameters. Upper panels show the mean response for individual cells (grey dots) for a single experiment. The experimental mean is overlaid in red or blue. Lower panel show paired summary data (grand mean) for all experimental replicates. Parameters extracted include: the percentage of cells activated, the mean ΔF/F0 of Ca2+ peaks, the number of peaks detected per cell (peaks/cell), the amplitude of the fitted sigmoid, as well as the duration, rise time, fall time, area under the curve (AUC) of all Ca2+ signal peaks. * Figure S4: TRPV4‐mediated local Ca2+ activity. A) Local Ca2+ events elicited by GSK101679A . Panels show a ΔF/F0 time series of a single endothelial cell. Ca2+ events initiate at, but remain localized within, either end of the cell. Scale bars = 20 μm. B) Ca2+ traces from the entire cell (red) or from circular ROIs (~2.5 μm diameter) positioned at each of the Ca2+ event initiation sites. Local Ca2+ events are inconsistently reflected in global (whole‐cell) Ca2+ traces.Figure S5: Acetylcholine and GSK101679A stimulate different patterns of signalling across multiple cells. Left; Image showing the basal Cal‐520/AM fluorescence (grey) in a field of mesenteric artery endothelial cells. Scale bar = 50 μm. A transect (x‐y) was drawn through three adjoining cells (1‐3). Right) Ca2+ signals (F/F0) and kymographs illustrating Cal‐520/AM fluorescence (F) along the transect  for cells 1‐3, when stimulated with acetylcholine  and then, after washout and recovery, GSK101679A . Traces are coloured according to the assigned cell number and kymographs are coloured on a scale where dark blue = min F and dark red = max F.p < 0.05 using paired Student's t‐test (n = 5).Figure S6: U73343, the inactive analogue of U73122, did not alter GSK induced Ca2+ signals. A) Composite images showing GSK1016790A (GSK)‐evoked Ca2+ activity in the absence (left) and presence (right) of U73343 , the inactive analogue of the PLC inhibitor U73122 . Images show basal Cal‐520/AM fluorescence (grey) with Ca2+ activity overlaid (red). Scale bars = 50 μm. B) GSK‐evoked  Ca2+ signals  were decomposed into propagating Ca2+ waves  and slow global Ca2+ rise  components. C) Summary data illustrating the percentage of cells displaying propagating Ca2+ waves and slow global Ca2+ rises. D‐F) Paired summary data showing the effect of U73343 on Ca2+ wave propagation velocity (D), peak amplitude  and the number of peaks per cell (F). The left plots in E‐F are scatter plots showing the mean Ca2+ event amplitude, or oscillation frequency, within each cell visualised in the experiment shown in panels A‐B. Individual data points are coloured  according to the density (i.e. occurrence) of particular values. * p < 0.05; NS, no statistically significant difference detected (i.e. p > 0.05) using paired t test. Scale bars = 10 μm.Figure S7: U73122 does not alter IP3‐mediated Ca2+ signals. A) Composite image (max F/F0 intensity projection) illustrating the Ca2+ response to photolysis of caged‐IP3 in the absence (upper) and presence (lower) of U73122 . B) Whole‐cell Ca2+ traces extracted from the data shown in A. C) Summary data showing the effect of U73122 on the peak amplitude and number of cells that responded to photorelease of caged IP3. Each data point indicates the mean from a single field of endothelial cells . *, p < 0.05 using paired Student's t‐test (n = 5).Figure S8: Caffeine inhibits propagating Ca2+ waves induced by TRPV4 activation. A) Composite images showing GSK1016790A (GSK)‐evoked Ca2+ activity in native mesenteric artery endothelial cells in the absence (left) and presence of caffeine . Images show basal Cal‐520/AM fluorescence (grey) with Ca2+ activity overlaid (red). Scale bars = 50 μm. B) Raw F/F0 (top), propagating Ca2+ waves , and slow Ca2+ rises  GSK‐evoked  Ca2+ signal components in the absence (left) and presence (right) of caffeine. C) Summary data illustrating the percentage of cells exhibiting fast and slow signal components. D‐F) Paired summary data showing the effect of IP3R inhibition on Ca2+ wave propagation velocity (D), peak amplitude  and the number of peaks per cell (F). The left plots in E‐F are scatter plots showing the mean Ca2+ event amplitude, or oscillation frequency, within each cell visualised in the experiment shown in panels A‐B. Individual data points are coloured  according to the density (i.e. occurrence) of particular values. * Figure S9: GSK‐evoked Ca2+ signalling returns towards basal levels during a washout period. Ca2+ signals  extracted from mesenteric artery endothelial cells stimulated with GSK1016790A . When GSK was washed out, signals returned to resting levels. Composite images (bottom) showing basal Cal‐520/AM fluorescence (grey) with Ca2+ activity overlaid (red). Ca2+ activity was derived from the 60 sec periods indicated by the dashed lines. Scale bars = 50 μm.n=5; * p<0.05).Figure S10: Acetylcholine (ACh) evoked relaxation is independent of TRPV4 in rat mesenteric arteries. (A) Representative diameter trace of a second order mesenteric artery  in response to ACh or GSK. Upon exposure to PE , vessels contracted and underwent vasomotion. Upon subsequent exposure to ACh (100 nM), arteries relaxed back towards resting diameter. On wash‐out of ACh, arteries contracted again. GSK (20 nM) also relaxed arteries back towards resting diameter. Dilation to GSK was reversed by the TRPV4 antagonist, HC067047 (10 μM). However, HC067047 did not prevent dilation to ACh. (B) Example images from the artery in A showing the artery prior to PE exposure , after PE, after ACh, after GSK, after GSK and HC067047, and after ACh with HC067047. The red horizontal lines are measurement scan lines created by Vasotracker (see Methods). (C) Summary data showing the percentage relaxation to ACh and GSK1016790A (GSK) in the absence (black bar) and present (open bar) of HC067047  evoked by the TRPV4 agonist, GSK1016790A. Data acquired at 10 Hz. Scale bar = 20 μm.Click here for additional data file.Movie S2: GSK‐evoked multicellular Ca2+ waves. Pseudo‐coloured active Ca2+ wave fronts  showing a GSK‐evoked (20 nM) Ca2+ wave that propagates across four endothelial cells. Cell outlines are overlaid in white. Data acquired at 20 Hz. Scale bar = 20 μm.Click here for additional data file.Movie S3: Ca2+ waves stop upon washout of GSK. The grey channel shows basal Ca2+ levels,while the red channel shows Ca2+ activity . GSK (20 nM) present from start of video and washed out when indicated. Data acquired at 10 Hz. Scale bar = 20 μm.Click here for additional data file.


These non-steady state conditions make the Ca2+-binding kinetics of the Ca2+ sensors for release and their spatial coupling to the Cavs important parameters of synaptic efficacy. In the mammalian central nervous system, the main release sensors linking action potential mediated Ca2+ influx to synchronous release are Synaptotagmin (Syt) 1 and 2. We review here quantitative work focusing on the Ca2+ kinetics of Syt2-mediated release. At present similar quantitative detail is lacking for Syt1-mediated release. In addition to triggering release, Ca2+ remaining bound to Syt after the first of two successive high-frequency activations was found to be capable of facilitating release during the second activation. More recently, the Ca2+ sensor Syt7 was identified as additional facilitation sensor. We further review how several recent functional studies provided quantitative insights into the spatial topographical relationships between Syts and Cavs and identified mechanisms regulating the sensor-to-channel coupling distances at presynaptic active zones. Most synapses analyzed in matured cortical structures were found to operate at tight, nanodomain coupling. For fast signaling synapses a developmental switch from loose, microdomain to tight, nanodomain coupling was found. The protein Septin5 has been known for some time as a developmentally down-regulated “inhibitor” of tight coupling, while Munc13-3 was found only recently to function as a developmentally up-regulated mediator of tight coupling. On the other hand, a highly plastic synapse was found to operate at loose coupling in the matured hippocampus. Together these findings suggest that the coupling topography and its regulation is a specificity of the type of synapse. However, to definitely draw such conclusion our knowledge of functional active zone topographies of different types of synapses in different areas of the mammalian brain is too incomplete.Ca The inflowing Ca2+ diffuses toward the SV, which bears the primary Ca2+ sensor proteins Synaptotagmin (Syt) 1 or 2 on its surface that are required for triggering its fusion with the presynaptic plasma membrane. Ca2+ binding to Syt changes its conformation and the resulting interaction with proteins of the core release machinery, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, and other proteins at the AZ ultimately triggers the fusion of the SV with the presynaptic plasma membrane . An action potential (AP) triggers the release of a neurotransmitter filled synaptic vesicle (SV) by opening voltage-gated Ca Südhof, . Thus, apr) can be quantified by way of analyzing fluctuations in postsynaptic current amplitudes (PSCs) under conditions of different pr, e.g., at different concentrations of extracellular Ca2+  is an important factor not only in determining the release fidelity for a single AP but also in setting the properties of short-term plasticity of a synapse , where pr2 is the release probability of the second release process, which may differ from pr1. In general, it can be noted that if pr1 > 0.5 the synapse will depress, i.e., PPR < 1, and only the magnitude of the depression will depend on pr2. However, if pr1 < 0.5 the synapse will show facilitation or depression depending on pr2.The initial pr depends on the Ca2+-binding kinetics of the release machinery, i.e., the Ca2+-binding kinetics of Syt in the context of the SNARE and other proteins at the AZ, and on the amplitude and time course of the Ca2+ signal “seen” by Syt. The latter depends on different factors, including the number and types of Cavs, their diffusional distance to Syt, and the characteristics of other Ca2+-binding proteins present in the terminal. Ca2+ entering the presynaptic terminal builds a steep, short-lasting concentration gradient around the mouth of the open Cavs that rapidly diminishes with increasing distance from the channel. Due to the steepness and short duration of this Ca2+ gradient a chemical equilibrium is never established in this process. This makes the intracellular Ca2+-binding kinetics of the release sensor, rather than its affinity alone, as well as its diffusional distance to the Cavs crucial to the control of speed and reliability of transmitter release  are the known Ca Südhof, . Syt1 an Südhof, . In addi Südhof, . Asynchr Südhof, . Due to  Südhof, .2+-binding and in addition might mediate protein-protein interactions with SNAREs and other Syt proteins or interactions with the membrane. One of the C2 domains is a C2A domain that binds three Ca2+ ions, while the other one is a C2B domain that binds two Ca2+ ions . Second, upon Ca2+ influx fusion is triggered by relieving SNAREs from the clamp (SNARE unclamping). Among the proposed models it is under debate if SNARE clamping is mediated directly by Syt or if and in as much it involves a second protein called Complexin (Cpx), which is discussed to also have a SNARE clamping function  and accounted for the experimental, cooperative power of 4 dependency of the release rate onto the intracellular Ca2+ concentration ([Ca2+]i) as well as brief synaptic delays  allows for calculating the release rates by deconvolution analysis  8-9 and P12-15 at the calyx of Held , indicating differences in the kinetics of Syt1 vs. Syt2 triggered release. Consequently, it has been suggested that the expression of a particular Syt isoform dictates the properties of release at its synapse  calyx of Held but subsequently also at a small CNS synapse, the immature cerebellar basket cell to Purkinje cell (PC) synapse Sakaba, , at whicd Voets, . In this2+ remaining attached to specific sites on the inner axon membrane” causes facilitation. For this Ca2+ the term “active Ca2+” was coined  from the first AP summates with free Ca2+ ([Ca2+]i) from the second AP, thereby, causing amplified release. However, it has been recognized early that the decay of [Ca2+]res deviates from the time course of facilitation, such that [Ca2+]res cannot fully account for facilitation  and nano- or microdomain [Ca2+]i at the release site during the second AP (~20–100 μM) simple Ca2+ summation is unlikely to be the exclusive source of facilitation  is a form of short-term synaptic plasticity important for synaptic computation  and Calbindin-D28k (CB), respectively, PPF was not affected by loss of either of the buffers  typical for AP-evoked [Ca2+]i elevations in the vicinity of Ca2+ channels, while Syt7 can also operate during longer lasting increases in residual Ca2+ in the low micromolar range  if a cluster of open Cavs with overlapping Ca2+ signaling domains controls release. Finally, we will use “nanodomain” as synonym for tight coupling plus SDT and “microdomain” as synonym for loose coupling plus DOT  but different Ca2+-binding kinetics, i.e., different on-rates -N,N,N',N'-tetraacetic acid; KD = 70 nM, kon = 107 M−1s−1; Nägerl et al., KD = 220 nM, kon = 4∗108 M−1s−1; Naraghi and Neher, KD values but BAPTA is ~40 times faster than EGTA. Ca2+ chelators suppress synaptic transmission by reducing the amount of Ca2+ that binds to Syt  to the extracellular bath solution  of a paired pulse experiment irrespective of the domain topography. However, its impact on the PPR markedly depends on whether the release sites operate with SDT or DOT. At a subsaturating concentration of Cd2+ some but not all Cavs will be blocked during an AP. In a DOT, blocking some of the Cavs controlling a synaptic vesicle will have effects similar to reducing [Ca2+]e, i.e., the initial pr will be reduced while the PPR will increase. On the other hand, if a release site is controlled by a single Cav (SDT), release of synaptic vesicles encountering no Ca2+ would be blocked while release or facilitation of vesicles encountering Ca2+ would be the same as in the absence of Cd2+. In consequence, application of Cd2+ will increase PPR in a DOT but leave it unaltered in a SDT  because of the non-linear dependency of release on Ca2+  and matured (~3 weeks old) calyces indicated a substantial developmental tightening of the coupling distance during postnatal development  and Cav2.2 (N-type) concomitantly controlling a release site, with likely 2 Cav2.1 and 1 Cav2.2 triggering release during an AP. The nanodomain at more matured PF terminals comprised only a Cav2.1  did not affect pr  are metal free  are known as central organizer of the AZ (Südhof, v2.1 and Cav2.2 channels to the AZ (Kaeser et al., 2+ influx to transmitter release, in particular since these channel subtypes are the most important ones for AP-mediated fusion (Table Ca Südhof, . Specifion Table . Proteinon Table  and Muncon Table , as posion Table . However2+]e has been established (Dodge and Rahamimoff, 2+]i dependency of Syt2-triggered transmitter release were developed (Bollmann et al., More than 30 years after the steep non-linear dependency of transmitter release onto [Cavs at mammalian cortical synapses. Initially it was thought that only GABAergic synapses in cortical structures make use of tight coupling, while cortical glutamatergic synapses seemed to operate with loose coupling (Ohana and Sakmann, v. At synapses investigated in a developmental context, it was found that tight coupling at matured synapses develops from an initially loose coupling at younger synapses. This latter result provides an explanation why previous studies predominantly found microdomain coupling at glutamatergic cortical synapses. These earlier studies were performed at synapses of very young animals (Eggermann et al., During the past decade several functional studies focused directly or indirectly on the coupling distance between Syts and CaHowever, tight coupling is not universal for synapses of the mature mammalian brain (Vyleta and Jonas, vs. Recent studies indicated RIM-BPs (Acuna et al., v clusters at the AZ and in narrowing the coupling distance. Munc13-3 was identified as a specific developmental mediator of nanodomain coupling at a glutamatergic synapse in cerebellar cortex (Kusch et al., For understanding the rules regulating coupling, it will be also required to identify the proteins that link Syt bearing SVs to CaHS wrote the first draft of the manuscript. GB and HS prepared figures and tables. GB and HS contributed to manuscript revision, read and approved the submitted version.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.


Intracranial hemorrhage (ICH) is a catastrophic complication in patients with acute myeloid leukemia (AML). AML cells, especially in the acute promyelocytic leukemia subtype, may release microparticles (MPs), tissue factor (TF), and cancer procoagulant (CP) to promote coagulopathy. Hyperfibrinolysis is also triggered via release of annexin II, t-PA, u-PA, and u-PAR. Various inflammatory cytokines from cancer cells, such as IL-1β and TNF-α, activate endothelial cells and promote leukostasis. This condition may increase the ICH risk and lead to poor clinical outcomes. Here, we present a case under a unique situation with acute ICH detected prior to the diagnosis of AML. The patient initially presented with two episodes of syncope. Rapidly progressive ICH was noted in follow-up computed tomography (CT) scans. Therefore, we highlight that AML should be among the differential diagnoses of the etiologies of ICH. Early diagnosis and timely intervention are very important for AML patients. Intracranial hemorrhage (ICH) is an uncommon complication in patients with acute myeloid leukemia (AML), and coincides with poor clinical outcome. The mortality rate of AML patients developing acute ICH is high and death can occur within days if not diagnosed and treated appropriately. In a study by Owattanapanich et al. , 4.29% .X-ray revealed no significant pulmonary nodules or pneumonia patch. An influenza A + B rapid screening test was performed, which showed negative results. A brain CT revealed a 13 mm lesion with hyperdensity in the left temporal region, with suspected intracerebral hemorrhage  score was E4V5M6 and bilateral pupil size was 2 mm with light reflex. There was no horizontal or vertical nystagmus. The neck was supple with no limited range of motion. The bilateral breath sound was clear without wheezing or crackle, and tachycardia was noted. There was no Babinski sign, decreased muscle power, or unsteady gait. The chest morrhage . Laboratmorrhage .Results from the bone marrow biopsy report showed 90% cellularity. Blasts accounted for more than 90% of all nucleated cells. Hypercellular and monotonous bone marrow was noted with undifferentiated myeloblasts with prominent, convoluted nuclei, and agranular cytoplasm. There were significantly decreased erythroid and megakaryocyte lineages. The immunohistochemical profile was as follows: CD34(+), CD117(+), MPO(+), CD33(+), CD68(–), hemoglobin A(–), Factor VIII(–), CD19(–), CD3(–), TdT(–), and PAX5(–). The peroxidase and alpha naphthyl acetate esterase (ANAE) test was positive and chloroacetate esterase (CAE) test was negative. Acute myeloid leukemia was diagnosed. Broad-spectrum antibiotic and adequate hydration were administered. An antineoplastic agent, hydroxyurea (15 mg/kg/day), and emergency leukocytapheresis were used to control disease progression. Unfortunately, the patient became drowsy with asymmetric pupil size and no light reflex. The follow-up brain CT showed multifocal intracranial hemorrhage in the bilateral cerebral hemispheres with midline shift involving the brain stem . Progres9/L) was reported. In those populations, there was a significantly higher risk of death from rapidly developed fatal ICH. In the study by Chen et al. [In the AML population, intracranial-hemorrhage-induced syncope at initial presentation is an uncommon but fatal condition. The study by Balmages et al.  showed an et al. , a totaln et al. ,6. The hn et al. . Hyperlen et al. . The mecn et al. ,11. The In the study by Dixit et al. , DIC wasp = 0.011), lower hemoglobin (p = 0.004), and blast crisis (p = 0.004) presented with highly probable leukostasis with a high score. A subgroup was given early leukapheresis treatment which showed a benefit (based on a grading score) in preventing leukostasis-related early death. In our case, hyperleukocytosis with thrombocytopenia and blast crisis was noted. Leukapheresis was performed but the treatment was not very effective. Persistent leukostasis and DIC promoted the progression of ICH with multiple focal hemorrhages in day 2 CT scans.Leukapheresis in AML patients with hyperleukocytosis rapidly removes excessive leukocytes by mechanical separation to control complications. One round of leukapheresis could reduce the WBC count by about 10–70% . The effIn this article, we present the rare and unusual case of a patient with AML-induced ICH initially presenting with syncope. In the AML population, DIC and leukostasis play a critical role in ICH. This detailed pathophysiology may improve physicians’ development of therapeutic strategies for AML-induced ICH. Here, we highlight the clinical features and etiologies of AML-induced ICH. Early diagnosis and timely leukapheresis may prevent fatal progressive complications.