Acute myeloid leukemia (AML) is a debilitating and life-threatening condition, especially for elderly patients who account for over 50% of diagnoses. For over four decades, standard induction therapy with intensive cytotoxic chemotherapy for AML had remained unchanged. However, for most patients, standard therapy continues to have its shortcomings, especially for elderly patients who may not be able to tolerate the complications from intensive cytotoxic chemotherapy. New research into the development of targeted and alternative therapies has led to a new era in AML therapy. For the nearly 20% of diagnoses harboring a mutation in isocitrate dehydrogenase 1 or 2 (IDH1/2), potential treatment options have undergone a paradigm shift away from intensive cytotoxic chemotherapy and towards targeted therapy alone or in combination with lower intensity chemotherapy. The first FDA approved IDH2 inhibitor was enasidenib in 2017. In addition, IDH1 inhibitors are in ongoing clinical studies, and the oral BCL-2 inhibitor venetoclax shows preliminary efficacy in this subset of patients. These new tools aim to improve outcomes and change the treatment paradigm for elderly patients with IDH mutant AML. However, the challenge of how to best incorporate these agents into standard practice remains. Acute myeloid leukemia (AML) comprises a heterogeneous group of hematologic malignancies characterized by blastic and abnormally differentiated myeloid clonal populations. It is the most common acute leukemia in adults, with 19,520 new cases (approximately 76% of predicted acute leukemias) and 10,670 deaths estimated to occur in the United States in 2018 [Compared to younger age groups, treatment outcomes for older adults with AML are dismal, particularly for patients aged 75 years and older. Complete remission (CR) rates, which may be predictive of survival, are noted to be lower 40–50%) and of shorter duration in elderly patients ,4,5,6,7.0–50% andReduced survival in this population is multifactorial. Older patients with AML are more likely to have comorbidities impacting ability to receive and tolerate intensive treatment ,9. AdditIDH, encoding isozymes of isocitrate dehydrogenase that catalyze conversion of isocitrate to α-ketoglutarate (α-KG). Subsequently, whole genome sequencing of patients with AML identified a number of recurring somatic mutations, including IDH1 [IDH1/2 (mIDH) entered the clinic, which have produced exciting results [This search for targeted therapies has also included investigations stemming from the longstanding notion that aberrations in cellular metabolism contribute to carcinogenesis ,16. Initing IDH1 . These o results ,23,24,25R,3S-isocitrate and NADP+ is key to regulation of these metabolic processes.In addition to their canonical roles in oxidative respiration via the mitochondrial tricarboxylic acid (TCA) cycle, isocitrate, α-KG, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) have myriad roles in other physiologic processes . These i+-dependent IDH1 is localized primarily to the cytosol and peroxisomes, while NADP+-dependent IDH2 is localized to the mitochondria. IDH3 is also localized to the mitochondria, but unlike IDH1/2, is nicotinamide adenine dinucleotide (NAD+)-dependent. When cellular energy conditions necessitate oxidative respiration, IDH2 is active in the traditional TCA cycle, catalyzing dehydrogenation of isocitrate to α-KG and producing NADPH. IDH1 does not participate in the TCA cycle. Importantly, unlike IDH3, IDH1/2 can also participate in reverse reductive carboxylation of isocitrate to α-KG. In energy-rich conditions, IDH3 activity is allosterically suppressed by regulatory co-factors (owing to its extra regulatory subunits), producing accumulation of isocitrate. Some intramitochondrial isocitrate is converted by IDH2 to α-KG, generating NADPH which reduces oxidative damage by reactive oxygen species. Remaining isocitrate is converted by aconitase back to citrate, which is shunted into the cytosol for production of acetyl-CoA and NADPH mediated, in part, by IDH1. [The human genome encodes three IDH isozymes . NADP+-dby IDH1. ,27,28,29IDH1 were identified in 2008 during an integrated genomic analysis of a set of human glioblastoma tumor samples, and subsequently IDH2 was identified in 2009 in a set of glioma tumor samples [IDH1/2 mutations were noted in AML in 2009, along with several other solid tumors and myelodysplastic syndrome (MDS) [IDH1 and 8–19% for IDH2, and mutations of either isozyme have been reported in up to 33% of collective cases [IDH1/2 mutations may occur in up to 19% of mIDH patients [IDH1/2 mutations reported in AML are somatic missense mutations affecting highly conserved arginine residues at codon 132 in exon 4 of IDH1 (IDH1R132) and at codons 140 and 172 in exon 4 of IDH2 (IDH2R140 and IDH2R172) [IDH1 has been reported in AML [IDH3 mutations have been reported in AML or other cancers.Recurrent mutations in  samples ,19. Shorme (MDS) ,31,32. Tve cases ,35,36. Cpatients ,38,39. Hd in AML . No oncoIDH1/2 mutations, particularly IDH1, as producing loss of function [IDH1/2 mutations in AML and other tumors are heterozygous and occur in the active catalytic site, suggesting oncogenic gain of new function rather than loss of tumor suppression [R)-2-hydroxyglutarate (2-HG) is produced at low levels by malate dehydrogenase-mediated reduction of α-KG. Accumulation in normal cells does not occur due to “metabolic proofreading” by endogenous 2-hydroxyglutarate dehydrogenases [IDH1/2, as 2-HG in these cells is derived in large proportion from glutamine [Early reports described function ,42. Howepression ,43,44. Togenases ,46. As dogenases ,48. Thisogenases . In addiogenases ,51,52,53lutamine .IDH cells display dependence on BCL-2 [The metabolic and non-metabolic effects of mIDH-mediated elevations in 2-HG have recently been thoroughly reviewed ,56. Accuon BCL-2 ,71,72.ATM) expression leading to impaired homologous recombination, both of which may sensitize IDH-mutated cells to poly(ADP-ribose) polymerase (PARP) inhibitors [+ T-cells are also noted to be inhibited by high concentrations of 2-HG, and in vitro models have indicated potential for immune evasion by tumors with mutated IDH; this has not yet been demonstrated in AML [IDH cells’ reduced capacity for producing NADPH may lead to depletions in glutathione, increasing reactive oxygen species and oxidative stress [In addition to processes linked to the TCA cycle, mIDH isozymes affect other cellular functions that likely contribute to leukemogenesis. Interestingly, 2-HG accumulation has been linked in vitro to inhibition of the AlkB homolog (ALKBH) DNA repair enzymes, as well as decreased ataxia telangiectasia mutated  to produce epigenetic modifications leading to overt leukemic transformation [ITD;Tet2Flt3-mutant AML monomorphic CD48+CD150− multipotent progenitor cells to 5-Aza and the mIDH2 inhibitor enasidenib led to partial normalization of cell differentiation, as well as reversal in many sites of known DNA hypermethylation induced by Tet2. However, in vivo RNA sequencing indicated that neither agent alone significantly suppressed the malignant progenitor clone. When both agents were used in combination in this model, hematopoietic differentiation was again noted, along with significant reduction in the proportion of progenitor cells. Combination therapy also produced more potent reversal of both site-specific and global hypermethylation [The increasingly prevalent role of hypomethylating agents  and growing understanding of mutant ogenesis . These mormation ,81,82. Ihylation . These r®; Celgene Corporation, Summit, NJ, USA) is a selective, non-competitive inhibitor of IDH2 recently approved by the United States Food and Drug Administration (FDA) for relapsed/refractory mIDH2 AML. Enasidenib binds to the divalent cation binding helices at the interface of IDH2 homodimers, allosterically maintaining the enzyme in an open conformation to reduce binding affinity for NADPH and impair catalytic activity [IDH2 wild-type levels [Enasidenib  and expansion (126 patients) phases reported [IDH2-mutated AML in the relapsed/refractory setting. Clinical trial outcomes for enasidenib-based therapy in reported . Median reported ,91. In tOf note, a later analysis of patients in the above trial who maintained stable disease (SD) through day 90 of treatment  noted that 27% of these patients experienced “late response” at a median of 130 days, while 45% maintained SD to a median of 173 days and 28% progressed . This den = 37) with untreated IDH2-mutated AML who were aged 60 or older (median 77 years) were enrolled within the original AG221-001 phase 1/2 dose escalation study, with the dose expansion phase utilizing the 100 mg daily dose in continuous 28-day cycles. ORR was 37.8%, with 19% achieving CR at a median time of 5.6 months ranging up to 12.9 months. Median duration of response was 12.2 months; median duration of CR was not reached. Median OS for responders and non-responders was 19.8 months and 5.4 months, respectively. The most common adverse events were fatigue (43%), nausea (41%), and decreased appetite (41%). DS was reported in 8%, and TLS was reported in 5% [Enasidenib has also been evaluated in the front-line setting for older patients who were not candidates for standard therapy. Patients (ed in 5% .IDH-mutated AML is utilizing standard 7 + 3 chemotherapy (anthracycline for three days and continuous infusion of cytarabine for seven days) in combination with ivosidenib 500 mg daily or enasidenib 100 mg daily for mutant IDH1 or IDH2, respectively [IDH-mutated AML who are ineligible for intensive induction therapy is utilizing 5-Aza in combination with ivosidenib 500 mg daily or enasidenib 100 mg or 200 mg daily for patients with IDH1 or IDH2 mutations, respectively [Additional ongoing studies are evaluating front-line regimens in combination with enasidenib. A phase 1 trial of patients with newly diagnosed ectively . Prelimiectively . PrelimiThe FDA’s review of safety data for enasidenib utilized a pooled safety population of 214 patients with relapsed/refractory AML, all of whom received at least one 100 mg dose of enasidenib . Among tEnasidenib’s approved labeling contains a statement indicating a possible risk of TLS . This istrans retinoic acid and arsenic trioxide for acute promyelocytic leukemia (APL) [Originally dubbed retinoic acid syndrome, DS was first described with the use of the differentiating agents all-ia (APL) . These aia (APL) . The patia (APL) . Clinicaia (APL) ,110,111.ia (APL) ,112.Enasidenib-induced DS was first noted in the initial phase 1 clinical trial in relapsed/refractory AML, approximately 1 month after study initiation ,91. An iManagement was similar to APL treatment-induced DS . DexametIn addition to the DSRC report, the above-described FDA safety review included an independent review of pooled safety data to identify possible DS events . The FDAIDH1-mutated AML [R132HIDH1 cells, treatment with ivosidenib restored erythropoietin-induced differentiation [IDH1-mutated advanced hematologic malignancies, plasma 2-HG levels were reduced by up to 99.7% after multiple doses of ivosidenib and were similar to those observed in healthy volunteers. Similar reduction was noted in bone marrow (up to 99.9%) [Ivosidenib (AG-120) is a selective, reversible, potent inhibitor of mutant IDH1 currently undergoing phase 1–3 trials for treatment of newly diagnosed ated AML ,101. Expntiation . In a sao 99.9%) .IDH-mutated AML, as well as ongoing clinical trials, are summarized in IDH1-mutated hematologic malignancies, including both treatment-naïve and relapsed/refractory AML [n = 125) noted an ORR of 42%, with CR/CRi in 30% of patients for a median duration of 8.2 months. Among all patients enrolled (n = 258), the most common all-grade adverse events were diarrhea (33%), leukocytosis (30%), nausea (30%), fatigue (29%), febrile neutropenia (25%), dyspnea (24%), anemia (23%), QT prolongation (23%), peripheral edema (22%), pyrexia (21%), and decreased appetite (20%). All-grade and grade ≥ 3 DS was noted in 11% and 5% of patients, respectively [Clinical trial outcomes for ivosidenib-based therapy in tory AML . Prelimiectively .IDH1 variant allele frequency to assess the depth of mIDH1 clearance [IDH1 clearance did not occur in any patients who did not achieve best response of CR or CRi. However, the clinical impact of mIDH1 clearance requires further study. While formal studies of ivosidenib-induced hematopoietic cell differentiation using clinical samples have not yet been reported, ivosidenib-induced DS has been reported in a three-patient case series [Bone marrow mononuclear cells from patients in another phase I study of ivosidenib in advanced hematologic malignancies were assessed for mutant learance . At the e series .Additional clinical trials of ivosidenib as part of combination therapy are ongoing. In the above-described phase 1 front-line trial of enasidenib or ivosidenib in combination with standard 7 + 3 chemotherapy, 27 patients have been treated with ivosidenib (median age 60 years), and 23 were evaluable for response at the time of publication . Collect®; AbbVie Inc., North Chicago, IL, USA) is a highly selective oral agent that binds the BH-3 binding groove of BCL-2, preventing BCL-2-mediated inactivation of pro-apoptotic proteins [As described previously, 2-HG suppresses COX and leads to dependence of AML cells on BCL-2 for continued viability . Venetocproteins ,119. Impproteins . While nproteins .IDH-mutated AML, as well as ongoing clinical trials, are summarized in IDH1/2 mutations were detected in 12 (38%) patients [IDH1/2 mutations [IDH1/2 mutations [Clinical trial outcomes for venetoclax-based therapy in patients . Among tutations . Among tutations . In a reutations . Among tIDH-mutated AML studies described above were frequently higher (ranging from 400 mg to 1200 mg daily) than the FDA-approved CLL dose of 400 mg daily [It is important to note that the venetoclax doses used in the small mg daily ,103,122.IDH-mutated AML. Additionally, TLS risk with venetoclax is likely to be highest in treatment-naïve patients, those with high disease burden, and high-risk disease  [Additionally important to note is the risk of TLS associated with venetoclax. TLS is a well-recognized phenomenon caused by deposition of malignant cellular components into the bloodstream following lysis, manifesting as rapidly evolving electrolyte derangements with possible life-threatening clinical sequelae ,124. In g., CLL) . Thus, aThe phase 1b study described above also included a cohort of patients receiving venetoclax and DAC alongside the triazole antifungal posaconazole—a well-known inhibitor of cytochrome P450 isozyme 3A4 (CYP3A4)—to assess posaconazole’s effects on venetoclax pharmacokinetics . VenetocBased on available evidence, patients with mIDH AML appear to have improved response and survival with the use of single-agent oral mutant IDH targeted agents compared to standard therapies in the relapsed setting. The magnitude of improvement and duration of response, as well as evaluation of long-term toxicities, will be further quantified and better elucidated with time. Furthermore, as enasidenib is the first targeted therapy approved for mIDH AML, it is not yet clear how to best incorporate this and other targeted agents into routine clinical practice. It is likely that combination approaches, i.e., with hypomethylating agent therapy or with other oral small molecule inhibitors, will soon become the new standard. Ongoing studies of targeted agents with hypomethylating agents and other combinations will help to answer these questions and may reshape the treatment landscape of elderly patients with AML. As balancing efficacy and toxicity is of special importance in this patient population, this reshaping may take the form of a treatment approach resembling outpatient chronic disease state management.Therapy for AML saw a leap forward in 2017, with the FDA approval of enasidenib and renewed enthusiasm for targeted agents, including IDH1/2 inhibitors and venetoclax. These agents appear to complement and—particularly in elderly patients—may reduce or eliminate use of intensive cytotoxic chemotherapy. It is important to note that this may necessitate a paradigm shift in the approach to AML induction therapy away from quickly achieving CR in elderly patients. Given the promising results of early trials, such a shift may bear significant long-term benefits for this population.


Trichoderma filamentous fungi are increasingly used as biocontrol agents and plant biostimulants. Growing evidence indicates that part of the beneficial effects is mediated by the activity of fungal metabolites on the plant host. We have investigated the mechanism of plant perception of HYTLO1, a hydrophobin abundantly secreted by Trichoderma longibrachiatum, which may play an important role in the early stages of the plant-fungus interaction. Aequorin-expressing Lotus japonicus suspension cell cultures responded to HYTLO1 with a rapid cytosolic Ca2+ increase that dissipated within 30 min, followed by the activation of the defence-related genes MPK3, WRK33, and CP450. The Ca2+-dependence of these gene expression was demonstrated by using the extracellular Ca2+ chelator EGTA and Ned-19, a potent inhibitor of the nicotinic acid adenine dinucleotide phosphate (NAADP) receptor in animal cells, which effectively blocked the HYTLO1-induced Ca2+ elevation. Immunocytochemical analyses showed the localization of the fungal hydrophobin at the plant cell surface, where it forms a protein film covering the plant cell wall. Our data demonstrate the Ca2+-mediated perception by plant cells of a key metabolite secreted by a biocontrol fungus, and provide the first evidence of the involvement of NAADP-gated Ca2+ release in a signalling pathway triggered by a biotic stimulus. Trichoderma is a widely spread genus of free-living filamentous fungi belonging to Ascomycota, increasingly used in agricultural applications as biocontrol agents and biofertilizers cyt) was very similar in L. japonicus wild-type cells and Ljsym4-2 mutant cells, stably expressing cytosolic aequorin , which is consistent with the sum of the perinuclear Ca2+ oscillations that were visualized in Ca2+ imaging experiments in L. japonicus wild-type, but not CASTOR mutant cyt elevation evoked by HYTLO1 was very efficiently blocked (83.2 ± 3.5%) by pre-treating L. japonicus cells with EGTA (600 μM) in combination with Ned-19 (100 μM) in Ca2+-free medium (3 (3 mM), a widely used inhibitor of Ca2+-permeable channels located at the plasma membrane cyt in response to HYTLO1  by pre-treatment with EGTA + Ned-19  and cyclic ADP-ribose (cADPR) [2+ release also in higher plants [2+ release was shown to occur in response to copper excess in the marine alga Ulva compressa [Ectocarpus siliculosus [2+ signalling and highlight the participation of NAADP in a plant response to a biotic stimulus . Interestingly, whereas members of the two-pore channel (TPC) gene family have been demonstrated to encode NAADP receptors in acidic organelles (endo-lysosomes) of animal cells  channel, a Ca2+-permeable channel located at the vacuolar membrane [2+ -localization for NAADP-gated Ca2+ release [A. thaliana TPC1 highlighted the presence of a Ned-19 binding site [The pharmacological approach used in this work suggests that HYTLO1 triggers both a Ca (cADPR) . Althougr plants , its invr plants , a NAADPompressa , as welliculosus . These dlls , lyophilizing and resuspending them in cell culture medium prior to cell treatment, as previously described [Mesorhizobium loti were kindly provided by Makoto Hayashi . All chemicals used in the pharmacological analysis of Ca2+ fluxes  were obtained from Sigma-Aldrich , except trans-Ned-19, that was purchased from ENZO Life Sciences .The hydrophobin HYTLO1 was isolated and purified to homogeneity from in vitro cultures of the biocontrol strain MK1 of o et al. . The purescribed . Crude NLotus japonicus (ecotype Gifu) wild-type and sym4-2 mutant seeds, defective in CASTOR, were kindly provided by J. Stougaard  and M. Parniske , respectively.SmaI digestion. The 600 bp SmaI fragment was subcloned into the pSE380 plasmid  in order to gain additional restriction sites for the next step of subcloning. A BglII-SalI fragment was then ligated between a CaMV-35S promoter sequence and a tNOS terminator into pCAMBIA1300 [BglII-SalI double digested to obtain the pAB1 T-DNA construct  Callus Induction Medium  sucrose, 0.5 µg/mL 2,4-dichlorophenoxyacetic acid , 0.05 µg/mL kinetin), supplemented with 5 µg/mL hygromycin B. After two subculturing steps, well-developed calli were transferred in Gamborg B5 liquid medium, pH 5.5, containing 2% (w/v) sucrose, 2 μg/mL 2,4-D, 5 μg/mL hygromycin B. Suspension-cultured cells were maintained at 24 °C on a rotary shaker at 80 rpm under 16 h light /8 h dark photoperiod. They were subcultured weekly by inoculating 1 mL packed cell volume into 20 mL fresh cell culture medium with, as described by Moscatiello et al. [Aequorin-expressing cell cultures were set up by in vitro dedifferentiation of hypocotyls from transgenic o et al. .3) for two days prior to the onset of the Ca2+ measurement assays.Exponentially-growing cells (five days) were treated with purified HYTLO1 (0.6 μM). The dose applied to cells was chosen on the basis of previous work concerning in vivo bioassays of physiological effects of HYTLO1 on plants . In someL. japonicus cell cultures with 5 µM coelenterazine  in darkness. Cells were then extensively washed with fresh culture medium and allowed to recover from the touch response for at least 15 min. Aequorin-based Ca2+ measurements were performed as recently described [3) or 30 min . For experiments carried out in the absence of extracellular Ca2+, cells were extensively washed with Ca2+-free culture medium and then resuspended in the same medium containing 600 µM EGTA. In the case of molecules dissolved in ethanol (HYTLO1) or DMSO (Ned-19), control cells were treated with the same percentage  of the organic solvent.Aequorin was reconstituted by overnight incubation of mid-exponential phase transgenic escribed , by usinL. japonicus suspension cultured cells as described by Zonin et al. [L. japonicus cells. Immunofluorescence experiments were performed on n et al. . LabelliTEM analyses were performed as described by Tagu et al. . ImmunogFor negative staining, purified HYTLO1 (0.6 μM) was adsorbed on carbon-coated EM grids, washed and stained on a drop of 1% uranyl acetate solution. L. japonicus plants by the procedure reported by Kistner and Matamoros [UBI, AW719589) was used as an internal standard to normalize expression levels .Semi-quantitative RT-PCR analyses of defence gene expression in L. japonicus cell suspension cultures incubated in control conditions or after treatment with HYTLO1 were performed as recently described [ATP synthase [LjMPK3, LjWRKY33, LjCP450, and LjATPsyn, and by [LjPR1. The number of cycles exploited for a linear range of gene amplification in the RT-PCR reactions was experimentally determined. Densitometric analysis of agarose gels stained with 0.5 μg/mL ethidium bromide was carried out with the Quantity One software . RT-PCR experiments were conducted in triplicate on at least three independent experiments. For the screening of primary T1 transformants, total RNA was prepared from leaves of two week-old atamoros  and reveescribed . The defescribed ; CP450, escribed ; WRKY33,escribed ,80; PR1,escribed ,82. Transynthase . The olisynthase  for LjMP, and by  for LjPRLjATPsyn.Quantitative reverse transcription PCR, used to validate the data, was performed in 10 μL using HOT FIREPol EvaGreen qPcr Mix Plus  and 2.5 ng of the different cDNA template. Three replicates were performed for each reaction. The qPCR reaction was conducted in a 7500 Real-Time PCR System (Thermo Fisher) according to the following cycle: 95 °C for 12 min; 95 °C for 15 s, 59 °C for 20 s, for 40 cycles. Differences in the target genes expression were evaluated by a relative quantification method normalizing the data to the endogenous reference gene 2+ chelating agents/Ca2+ channel inhibitors, was determined by the Evans Blue method [Cell viability, after treatment for HYTLO1 for different time intervals, in the absence or presence of Cae method . P < 0.05) between means was evaluated using the Student’s t test.Data are expressed as mean ± SE. The statistical significance of differences (


Our study was spurred by (1) the curious observation that ER is sparsely distributed in dendritic spines, but over‐represented in larger spines that are likely to have undergone activity‐dependent strengthening, and (2) evidence suggesting that ER motility at synapses can be rapid, and accompany activity‐regulated spine remodelling. We constructed a physiologically realistic computational model of an ER‐bearing CA1 spine, and examined how IP3‐sensitive Ca2+ stores affect spine Ca2+ dynamics during activity patterns mimicking the induction of long‐term potentiation and long‐term depression (LTD). Our results suggest that the presence of ER modulates NMDA receptor‐dependent plasticity in a graded manner that selectively enhances LTD induction. We propose that ER may locally tune Ca2+‐based plasticity, providing a braking mechanism to mitigate runaway strengthening at potentiated synapses. Our study provides a biophysically accurate description of postsynaptic Ca2+ regulation, and suggests that ER in the spine may promote the re‐use of hippocampal synapses with saturated strengths.Long‐term plasticity mediated by NMDA receptors supports input‐specific, Hebbian forms of learning at excitatory CA3–CA1 connections in the hippocampus. There exists an additional layer of stabilizing mechanisms that act globally as well as locally over multiple time scales to ensure that plasticity occurs in a constrained manner. Here, we investigated the role of calcium (Ca 2+) entry mediated by NMDA receptors is considered central to the induction of activity‐dependent synaptic plasticity in hippocampal area CA1; this description does not, however, take into account the potential contribution of endoplasmic reticulum (ER) Ca2+ stores.Calcium (Ca2+ signalling between synapses, as suggested by experiments on metabotropic receptor‐dependent long‐term depression.The ER has a heterogeneous distribution in CA1 dendritic spines, and may introduce localized functional differences in Ca2+ dynamics at a CA3–CA1 excitatory synapse characterizes the contribution of spine ER via metabotropic signalling during plasticity induction protocols.A physiologically detailed computational model of Ca2+ release via IP3 receptors modulates NMDA receptor‐dependent plasticity in a graded manner, to selectively promote synaptic depression with relatively diminished effect on LTP induction; this may temper further strengthening at the stronger synapses which are preferentially associated with ER‐containing spines.ER CaAcquisition of spine ER may thus represent a local, biophysically plausible ‘metaplastic switch’ at potentiated CA1 synapses, contributing to the plasticity–stability balance in neural circuits. Hebbian synaptic plasticity involves activity‐driven changes in synaptic strengths Hebb, . This foet al. et al. et al. et al. N‐methyl‐d‐aspartate receptors (NMDARs) and the ensuing entry of calcium (Ca2+) into the dendritic spine  store, may be available to CA1 spines ‐sensitive intracellular stores  elevation and early long‐term plasticity driven by synaptic activation in the presence of an ER/spine apparatus  found experimentally  is described by the following Hodgkin–Huxley (HH)‐type ordinary differential equation:gL), voltage‐dependent AMPA receptor (AMPAR)/NMDA receptor (NMDAR)‐gated currents (IA/IN) in the postsynaptic density (PSD), a high voltage‐activated L‐type Ca2+ current (IL‐VGCC), and passive electrical coupling to the dendritic shaft (RC) with ud denoting the voltage of the dendritic compartment . We assumed standard membrane parameters (capacitance Cm = 1 μF cm−2 and uniform leak conductance density gL = 0.0002 S cm−2), and the resting membrane potential in both the spine and parent dendrite was set to urest = −70 mV    = 1/(1 + 0.28 exp(−0.062u)) . Trains of synaptic stimulation have been modelled as the sum of the above conductance waveforms  = gL‐VGCCm2u(t)hu(t), where the time dependence of the HH‐type activation and inactivation gating variables, mu(t) and hu(t), respectively, is governed by the following equations  = 1/(1 + exp(−(u – um)/km)) and h∞(u) = 1/(1 + exp(−(u – uh)/kh)), with um,h, km,h and τm,h listed in Table 2+) current through the L‐VGCC was described by a modified Goldman–Hodgkin–Katz (GHK) relation  and the exterior of the cell ([Ca2+]ext = 2 mm) under basal conditions. It is given by:qCa is the electric charge per Ca2+ ion, Na is the Avogadro number, and the conductance parameter gL‐VGCC is set according to the value of gN such that the peak L‐VGCC Ca2+ influx rate during a backpropagating action potential (bAP) is comparable to that mediated by NMDAR in response to a glutamate pulse , consistent with individual spine measurements , and was set to zero in the ER− spine head, which in all other respects was identical to the ER+ spine. The Ca2+ influx through the NMDAR channel cluster, JN, was also described by a GHK‐type current term, and as in previous studies, we have assumed that the Ca2+ current constitutes 10% of the total NMDAR current (RT/4F2), with R denoting the universal gas constant (8.314 J mol−1 K−1), F = 96485 C mol−1 being the Faraday constant, and the temperature T set to 30°C.The terms JB encapsulates the net effect of cytosolic buffers . Each CB molecule carries two high‐affinity and two medium‐affinity binding sites for Ca2+. The total concentration of CB was taken to be 45 μm, as measured experimentally in CA1 pyramidal neurons  with total concentration of 80 μm, as inferred previously from a spatial reaction–diffusion model of spine Ca2+ transients fit to single‐spine experimental data . In addition, we included a slow buffer with a total concentration of 40 μm, the kinetic parameters for which were set 10 times slower than those for the immobile buffer , but is expected to make a significant contribution to regulating free Ca2+ levels on longer time scales during persistent stimulation. We set the total CaM concentration to be 50 μm, which represents an average of several estimates found in the literature .The term rs Table . Calbind2+ currents and buffers, the Ca2+ level in the cytosol is also regulated by efflux mechanisms operating at the plasma membrane (denoted by the Jout term in Eq. 2+ transients in the spine head. We modelled efflux via plasma membrane Ca2+‐ATPase pumps (PMCA) and sodium–calcium exchangers (NCX), both of which were approximated by Michaelis–Menten‐like enzyme kinetics . Surface densities and kinetic parameters for PMCA and NCX were adopted from a previous modelling study of CA1 spines , which were set so as to exactly balance the efflux rate per pump molecule when the intracellular Ca2+ is held at 50 nm  (assumed to be present on the postsynaptic membrane), subsequent production of the second messenger IP3, and the downstream activation of IP3 receptors on the ER membrane . We have adapted a previously published kinetic description of mGluR activation leading to phospholipase Cβ (PLCβ)‐mediated hydrolysis of phosphatidylinositol 4,5‐bisphosphate (PIP2) at cerebellar parallel fibre–Purkinje cell synapses  model for mGluR activation and IP3 turnover is unable to reproduce this experimental delay, prompting us to use the detailed kinetic description, which provides more flexibility in tuning the time course of IP3 production.The intracellular Caglu = 1 ms . We asked which of the parameters in the detailed mGluR–IP3 model most affected the latency in IP3 production. The IP3 time course was found to be robust to variation in all except a combination of three parameters , which were scaled down appropriately (a1b and a2b by a factor of 50 and b11 by a factor of 4) to introduce a few hundred milliseconds delay in the peak of the IP3 response following the initial input. All other parameter values governing Gq‐mediated PLC activation and IP3 turnover were taken to be the same as in the original study  as an α function with a time constant of τ3 receptors was modelled with a reduced kinetic scheme adopted from a previous computational study of ER Ca2+ dynamics in neuroblastoma cells  and m2 = [Ca2+]/(d5 + [Ca2+]), with half‐activation constants d1 = 0.8 μm  transporters present on the ER surface, which maintain the steep concentration gradient (∼103–104) across the ER membrane. The SERCA activity was assumed to have a Hill‐type dependence on the cytosol Ca2+ level , with the half‐activation constant KS set to 0.2 μm , where kS is the SERCA leak rate, which balances the SERCA pump activity to help maintain a cytosolic [Ca2+] = 50 nm under resting conditions. The magnitude of maximum SERCA activity in the spine, VS, depends on the density of SERCA molecules on the ER membrane, ER surface area in the spine head and pump efficiency per molecule. As no estimate appropriate for a spine‐sized region was readily available, we obtained an approximate estimate for VS by referring to previous experimental measurements on ER refilling rates in rat sensory neurons  is considered to be in the range of a few hundred nM. For our control synapse (ER− spine) we set the NMDAR conductance parameter gN to 65 pS, which yields a ΔCaEPSP = 0.2 μm, consistent with previous studies . The delay in Ca2+ release from the ER following the NMDAR‐mediated Ca2+ rise is measured with respect to the time of application of glutamate.Unitary synaptic input at the spine head was modelled as a single pulse of glutamate, which transiently binds to postsynaptic mGluR, and concurrently activates AMPAR/NMDAR currents as described above to produce a small (∼few millivolts) depolarization of the postsynaptic membrane. The amplitude of spine Ca−/ER+ spines was simulated with trains of regularly spaced synaptic inputs delivered over a range of different frequencies (0.1–20 Hz). In order to simulate the activation of Schaffer collateral (SC) fibres during the induction of LTP/LTD, we assumed that the local dendritic shaft (modelled as a passive compartment) receives synchronous input from multiple synapses , leading to stronger activation of the NMDAR current at the spine head. The parameter ρS, which sets the magnitude of the total number of co‐active synaptic inputs onto the dendritic compartment during SC stimulation, has been adjusted to evoke a depolarization of ∼15 mV at the spine head in response to a single input  and a slow (τs = 40 ms) exponentially decaying components  = 1 for t ≥ 0, and 0 otherwise, for a bAP arriving at t = 0. A bAP is simulated by feeding the above voltage profile to the dendritic compartment. During repetitive stimulation, each synaptic input is paired with either one or two bAPs (spike doublets/triplets). The timing difference between the pre‐ and postsynaptic firing, Δt, is measured as the interval between the start of the glutamate pulse and peak of the bAP at the dendritic compartment. When a synaptic input is paired with a postsynaptic burst composed of two bAPs (spike triplet), Δt is defined as the interval between the glutamate pulse and the peak of the second bAP; bAPs in a burst are separated by a fixed interval of 10 ms. By convention, glutamate release preceding the bAP is assigned a positive Δt.We also modelled the pairing of synaptic inputs with the strong depolarization of the postsynaptic membrane induced by backpropagating action potentials (bAPs) in the CA1 neuron, mimicking the conditions for the induction of spike timing‐dependent plasticity (STDP) . The model describes the dynamics of a (dimensionless) weight variable, w (a proxy for the postsynaptic AMPAR conductance), and its functional form approximates biophysically plausible descriptions of the regulation of AMPAR number and/or phosphorylation level by a combination of Ca2+‐activated kinases and phosphatases  instead of the free Ca2+. Ca2+‐bound CaM is known to regulate the activation of several downstream effectors such as Ca2+/CaM‐dependent protein kinase II (CaMKII)  is relevant, recent studies indicate that even the partially bound forms are capable of regulatory activity . The dynamic range of aCaM is restricted by the availability of total CaM in the spine, unlike the Ca2+ response, which is not bounded and in principle can grow very large during persistent high‐frequency stimulation. The dynamical equation governing the Ca2+ dependence of w is given byw is modelled as the difference between two sigmoid functions  control the thresholds for the induction of LTP/LTD during synaptic stimulation: no plasticity is induced when aCaM levels remain below θD, LTD is induced when aCaM is restricted by and large to the interval , and LTP induction requires higher levels of aCaM, exceeding the threshold θP. The temporal factor τw is given by:et al. Induction of long‐term modification at hippocampal/cortical synapses is governed by the magnitude and duration of local Caet al. t varied from −100 ms and 100 ms in steps of 1 ms. The weight variable w  integrates over the temporal spine Ca2+ signal evoked by these synaptic activation patterns, resulting in a net (cumulative) change Δw at the end of the stimulation period. Spine ER was assumed to contribute to the Ca2+ pool in the spine driving changes in w, thereby modulating the induction of NMDAR‐dependent plasticity and giving rise to possible differences in Δw relative to the reference ER− spine. We characterized the nature of this differential effect of spine ER, ΔΔw, as a function of various biophysical model parameters. The plasticity profile obtained in our model synapse is shaped by the choice of θP/θD (assumed to be same for the ER+ and ER− spines), which were appropriately set to have agreement with experimentally obtained plasticity curves. For the rate‐dependent plasticity, θP was adjusted such that LTP induction occurs for frequencies ≳15 Hz in the ER− spine head . Different choices of the LTD threshold (θD) for the ER− spine have been similarly explored (fD = 1–6 Hz). In the case of STDP, we referred to a previous experimental study . We also followed this up by examining the sensitivity of the results to variation in ΔtD and ΔtP over ±10 ms windows.Rate‐dependent plasticity was simulated over the 0.1–20 Hz frequency range (in steps of 0.1 Hz) with 900 presynaptic SC inputs applied at each frequency, following earlier studies .All numerical simulations and analysis comprising this study were carried out in Python 2.7 using NumPy, SciPy and Matplotlib modules. Python code for running the model simulations is available at GitHub (+ spine), described by a single‐compartment point model (Methods). The time course of spine Ca2+ evoked by synaptic activation is shaped by the coupled electrical and Ca2+ dynamics at the spine head, which involves contributions from various biophysical components, and passive electrical coupling with a dendritic compartment . The spine ER store, modelled as an intracellular Ca2+ pool , contributes through IP3 receptor‐gated Ca2+ release  and the uptake of cytosolic Ca2+ by SERCA pumps present on the ER membrane.We considered an average CA1 pyramidal neuron dendritic spine head containing ER . We adopted a detailed kinetic scheme . We tuned the parameters regulating IP3 production rate in this model . Figure A and B shows the simulated Ca2+ response to application of a single pulse of glutamate at t = 0 in our model spine head with ER (coloured curves), which is compared with the reference ER− spine (black curve). The latency as well as magnitude of Ca2+ release from ER is dependent on the number of IP3 receptors present. Although Ca2+ at low levels (≲0.3 μm) is a coagonist for IP3R activation , which, again, is consistent with experimental findings  and the IP3 receptor cluster size directly determine the Ca2+ signal in the spine. Changes in the ICCR profile in our calibrated model corresponding to changes in these crucial components are summarized as heat maps in Fig. D and E. These represent the dependence of the delay of ICCR (in ms) and the amplitude of the second Ca2+ peak relative to the first, respectively, over a biologically realistic range of parameter values. These parameter ranges yield a fairly broad distribution of possible outcomes, and for a small subset of parameter choices, the outcomes are consistent with the averaged experimental estimates . Notably, the delay in synaptically evoked release of store Ca2+ has a non‐monotonic dependence on the IP3R cluster size: for a fixed ΔCaEPSP, the delay first increases, and then decreases with increasing number of IP3Rs. This dependence may be understood by noting that the IP3R cluster size sets the strength of the Ca2+‐dependent positive feedback loop driving IP3R activation in the presence of sufficient IP3. For higher IP3R numbers, this feedback can drive a self‐sustained burst of Ca2+ release from ER. The larger the cluster size, the more swiftly ICCR increases, while also speeding up the suppression of the slower Ca2+‐dependent inactivation variable h, which eventually shuts off the IP3R channel flux. Thus, the temporal profile of the ICCR transient is expected to advance (i.e. follow with a shorter lag) with increasing IP3R number. At small cluster sizes, on the other hand, the flux through IP3Rs is inadequate to drive a burst of ICCR even while IP3 is present. In this regime, there is no discernible second Ca2+ peak , and the kinetics of the IP3R open fraction (i.e. the ICCR peak location) in this case is primarily shaped by the decaying NMDAR Ca2+ transient. The relatively minor contribution of ICCR only adds a small delay to this decay of the total spine Ca2+. This additional delay is proportional to the IP3R number, NR, thus accounting for the shift of the peak location of IP3R influx to longer latencies with increasing NR in the regime of small IP3R numbers.The amplitude of the synaptically evoked Ca+ spine head during the induction of synaptic weakening by low frequency afferent stimulation of the CA3–CA1 pathway. Figure A shows an example of the Ca2+ time course evoked by a 1 Hz train of regularly spaced glutamate pulses. Binding of glutamate to postsynaptic AMPARs produces a small depolarization at the spine head (∼few millivolts), resulting in weak NMDAR activation and modest Ca2+ entry. Due to little overlap of successive Ca2+ events at low input rates, there is no build‐up of Ca2+ concentration in the spine over time. Figure A compares the Ca2+ signal in the ER− control spine (black curve) with the responses in the ER+ spine (coloured curves correspond to different numbers of IP3R). mGluR‐mediated Ca2+ release from spine ER contributes to the common pool of Ca2+ in the spine head and augments the NMDAR‐mediated Ca2+ signal.We next simulated the calcium response in an ER2+ time course to plasticity, we followed the activation of calmodulin (CaM), which is included in our model and makes a significant contribution to the Ca2+ buffering capacity in the spine. Ca2+‐bound CaM is known to regulate a number of downstream signalling molecules that collectively determine changes in synaptic strength associated with early LTP/LTD; thus, it provides a suitable choice of input to the dynamical model governing the synaptic weight w . The parameter θD in Eq. 2+–CaM threshold for LTD induction. For the example in Fig. B, a range of θD choices is possible  such that LTD can be induced at the ER+ spine, whereas the synaptic strength associated with the ER− spine remains unaffected. This is illustrated in Fig. C by a comparison between the time courses of w(t) in the ER− and ER+ spines over 900 SC inputs for the specific choice of θD = 2 μm. The elevated Ca2+–CaM response in the ER+ spines (coloured curves) drives a slow induction of synaptic depression (Δw<0) with repeated stimulation, which is absent in the ER− spine (black curve). This particular instantiation of our model thus recapitulates experimental observations regarding the association of mGluR‐mediated store Ca2+ release with LTD induction at low stimulation rates . Figure D shows the dependence of the maximum steady‐state amplitude of active CaM during persistent stimulation on the input frequency. The corresponding total change in the weight variable (Δw) at the end of the stimulus train is shown in Fig. E (fD is fixed at 1 Hz). ICCR robustly enhances CaM activation to facilitate LTD induction over a range of low frequencies, although the contribution of ICCR is non‐monotonic in the input rate. This dependence may be accounted for by noting that the opening of IP3R is regulated by a combination of two factors: the Ca2+‐ and IP3‐dependent activation, and the level of inhibition (h). At low input rates (≲2 Hz), the IP3 level increases with the frequency of glutamate application. At the same time, the slowly changing h variable has less time to recover between successive inputs, the more frequently the inputs arrive; therefore, h decreases with increasing input frequency. The balance between these two competing factors (IP3‐mediated activation and inactivation mediated by h) shapes the overall profile of the IP3R open probability, and hence the IP3R Ca2+ flux, as a function of the frequency of glutamate input.In order to address the dependence of the results on the model parameters, we repeated our simulations across a range of synaptic input frequencies (0.1–5 Hz) and synaptically evoked spine CaF and G compare the responses before and following spine ER acquisition over a range of low frequencies for different choices of the NMDAR conductance parameter (ΔCaEPSP), with a fixed number of IP3Rs (NR = 30). The profiles of the excess CaM activation  and differential plasticity outcome  in the presence of spine ER depend on the amplitude of synaptically evoked Ca2+. However, in general, ICCR contribution enhances LTD induction over a range of low‐frequency inputs.Figure ion Fig. F and diome Fig. G in theH and I summarize the dependence of the maximal Ca2+–CaM response and plasticity output in the ER+ spine, relative to the ER− reference spine, on the input frequency and NMDAR Ca2+ conductance. The vertical axes in both figures are linear in the NMDAR conductance parameter gN; however, to aid interpretation, they have been parametrized in terms of the NMDAR Ca2+ amplitude instead. As before, the frequency threshold for LTD induction is set to fD = 1 Hz, and all results are for an IP3R cluster size of NR = 30. ICCR is found to robustly enhance NMDAR‐driven LTD induction at lower frequencies (f ≲ 1 Hz). The underlying CaM activation in our model exhibits complex dependence on the input rate and ΔCaEPSP : the excess CaM response in the ER+ spine decreases with increasing ΔCaEPSP at very low input frequencies (f ≲ 0.5 Hz), but this trend reverses at higher frequencies (f ∼ 1–2 Hz).Elaborating on the above results, the heat maps in Fig. PSP Fig. H: the ef ≲ 0.5 Hz). Due to the delayed synthesis of IP3, the initial NMDAR Ca2+ elevation only contributes to ICCR by changing the slower inactivation variable h (and not through the activation variable m1), which decreases with increasing ΔCaEPSP. The level of h determines the magnitude of the subsequent ICCR (i.e. the amplitude of the second Ca2+ peak), and as h gets smaller with increasing ΔCaEPSP, so does the maximal IP3R Ca2+ flux . At higher frequencies (∼1–2 Hz), on the other hand, there is insufficient time between one input and the next for IP3 to decay back to resting levels. As IP3 is now present at moderate levels when a glutamate input arrives, the NMDAR‐mediated ΔCaEPSP switches its role: now it directly controls the activation of IP3Rs (through the instantaneous activation variable m1), while the slowly evolving h(t) only has a delayed effect on the IP3R open probability. Due to this switch in its role from inactivation to activation at higher input rates, an increase in gN  is now associated with increased IP3R flux . This explains the increase of ICCR with the NMDAR conductance. In sum, our results demonstrate that store Ca2+ contribution regulated by mGluR–IP3 signalling at a realistic synapse can robustly augment the NMDAR‐mediated Ca2+ response to weak synaptic stimulation, providing a basis to understand compromised hippocampal LTD associated with blocking of ER Ca2+ stores.To see why this difference arises, we first considered the case of very low frequencies  of the IP3R as a joint function of glutamate and spine Ca2+ concentration. Increasing the level of glutamate stimulation (e.g. in the context of rate‐based plasticity) has a direct effect on IP3 synthesis via mGluR–PLCβ activation. At the same time, it also drives increased postsynaptic NMDAR activation. The resulting elevation of spine Ca2+ level can affect both the production (via the mGluR pathway) and degradation (via the Ca2+ dependence of IP3K activity) of IP3, besides directly controlling Popen through the m1 and h variables (Methods). The broad strokes of this non‐linear dependence on glutamate and Ca2+ can be captured by the asymptotic steady‐state response of the IP3R over a range of (constant) glutamate and Ca2+ levels . IP3R shows maximal activation for a Ca2+ concentration of ∼0.3 μm over a range of realistic glutamate levels, and is progressively inhibited with increasing Ca2+. Thus, in an ER+ spine, the contribution of ICCR is anticipated to diminish progressively with increasing NMDAR activation for a fixed glutamate signal (as in the case of STDP induction protocol). Given the weak dependence of Popen on the glutamate level as suggested by Fig. A, we can also anticipate a similar graded contribution of ICCR with increasing NMDAR‐mediated Ca2+ elevation in the context of rate‐dependent plasticity. The interplay between NMDAR‐mediated Ca2+ entry, mGluR signalling and IP3R gating suggested by the above picture is characterized in detail in our synaptic model with realistic parameter settings.Gating of IPels Fig. A. IP3R B compares the frequency–response profile of spine Ca2+ elevation (the maximum amplitude attained during the steady state) in the reference ER− spine (black) with the ER+ spine for different numbers of IP3Rs (coloured curves). ICCR enhances the Ca2+ responses at lower frequencies, and its contribution steadily diminishes with increasing frequency above f ≈ 5 Hz; this is also highlighted by the profile of the excess Ca2+ amplitude in the ER+ spine relative to the ER− spine, shown in Fig. C. Similar dependence on the input rate is also observed for the activation of CaM in the ER− and ER+ spines . The dip in the differential Ca2+ signal below zero at higher frequencies  is due to SERCA pump activity in the ER+ (but not the ER−) spine, which contributes to the extrusion of cytosolic Ca2+ and leads to a net lowering of the Ca2+ levels in the presence of ER. The action of SERCA Ca2+ efflux is revealed only at higher frequencies when ICCR in the spine is suppressed by the Ca2+‐dependent inactivation of IP3R. The inverse dependence of the ER Ca2+ contribution on the level of NMDAR activation is also highlighted by a direct comparison between the NMDAR and IP3R Ca2+ current profiles in the ER+ spine, which are shown in Fig. F.We simulated a standard protocol consisting of repeated presynaptic stimulation (900 spikes) of the SC–CA1 pathway at different rates (0.1–20 Hz in steps of 0.1 Hz) (Methods). Figure 2+ elevation drives a change in the synaptic weight variable w (Eq. − (control) spine in Fig. A, which shows the temporal profile of Ca2+–CaM  and the corresponding evolution of the w variable  over 900 spikes at two different input rates, 5 and 17 Hz, associated with LTD and LTP, respectively. (The plasticity thresholds have been adjusted to have fD = 1 Hz and fP = 15 Hz at the ER− spine.) The differential effect of ER is displayed separately for the 5 Hz  and 17 Hz  examples. At 5 Hz, ICCR makes an appreciable contribution to the spine Ca2+ elevation, and thus to the rate and amplitude of the resulting synaptic changes, with a larger number of IP3Rs associated with stronger synaptic depression . In contrast, at 17 Hz, due to the strong suppression of ICCR by the NMDAR‐driven persistent Ca2+ elevation, there is little difference in the response in the ER− and ER+ spines , resulting in nearly the same plasticity outcome (strong potentiation) at the end of the stimulation .Spine Cale w Eq. . This isCaM Fig. A, top a Hz Fig. B and 17 Hz Fig. C examplnes Fig. C, resulD. The total synaptic weight change (Δw) at the end of the stimulus train is plotted as a function of the input rate f for the reference ER− spine (black) and following ER acquisition for different numbers of IP3Rs (coloured curves). Consistent with our expectation from Fig. A, we found that ICCR enhances plasticity at lower input frequencies, leading to a broadening of the effective LTD window. Due to the Ca2+‐dependent suppression of ICCR at higher frequencies, the profiles of Δw for synapses associated with the ER− and ER+ spines are near‐identical above f ≈ 10 Hz.We quantified the induced plasticity profile across the full range of synaptic input rates (0.1–20 Hz), which is shown in Fig. EPSP = 0.2 μm) is characterized in terms of the relative broadening of the depression and potentiation windows. A simple way to capture the overall modulation of the plasticity curve is by estimating changes in the threshold frequency for LTD induction (ΔfD) and LTP induction (ΔfP) with the inclusion of ER, relative to the ER− spine. The plasticity thresholds for the ER− spine (fD and fP) were repeatedly sampled at random from 1–6 Hz for fD and 10–20 Hz for fP to account for experimental uncertainties in these estimates, as well as to as assess the variability in the model output. The resulting distribution of  values (aggregate of 5000 runs over 10–50 IP3Rs) is visualized as a heat map in Fig. E, and on the whole, it suggests selective enhancement of LTD induction in the presence of ER. In summary, our analysis of the model spine head suggests a graded, frequency‐dependent contribution of ER to spine Ca2+ signalling and plasticity, with steadily diminishing contribution of ICCR at higher input frequencies.The differential modulation of LTD and LTP suggested by the above results for our model synapse , which simulate the conditions for induction of timing‐ dependent synaptic plasticity  or two bAPs (spike triplets), and these paired stimuli are presented 100 times at a fixed rate of 5 Hz (Methods). Figure A illustrates the time course of Ca2+–CaM (top) and the corresponding change in the synaptic weight (bottom) in the ER− (control) spine head during the triplet stimulation, for three different choices of the pre‐post spike timing difference, Δt. By appropriately adjusting the plasticity thresholds ΔD and ΔP, our ER− spine model can show broad agreement with the experimentally reported plasticity profiles. Figure B (top) shows the dependence of the Ca2+–CaM amplitude on the spike timing difference (Δt) in the doublet (blue) and triplet (red) cases. The temporal proximity and ordering of pre‐ and postsynaptic inputs, together, control the NMDAR activation level, which in turn decides the maximum Ca2+ elevation in the spine during persistent stimulation. The corresponding synaptic weight changes (Δw) induced by the STDP inputs are displayed in Fig. B (bottom). Consistent with experimental data, the doublet protocol only induces synaptic weakening over a restricted range of Δt values . On the other hand, the triplet protocol induces potentiation over an ∼35 ms window of positive Δt values, flanked by two ∼40 ms windows of depression, one for Δt < 0 and a second for causal pre/post pairings with longer time differences (Δt ≥ +35 ms) .We simulated the Ca2+ signalling during STDP induction? Figure C–E compares the activity‐driven responses of an ER+ spine (with different IP3R cluster sizes) with the ER− reference spine, as a function of the spike timing difference. Release of ER store Ca2+ augments both the Ca2+ and Ca2+–CaM in the spine head , and this excess response in the ER+ spine relative to the ER− spine indirectly depends on the spike timing difference, which regulates the NMDAR‐mediated Ca2+ entry into the spine . This inverse dependence of the ICCR contribution on NMDAR activation level is reflected in the NMDAR and IP3R Ca2+ current profiles, shown in Fig. E. Stronger NMDAR activation, particularly at small positive spike timing differences (0 < Δt ≤ +40 ms), is associated with reduced ICCR, which follows from the Ca2+‐dependent inhibition of IP3 receptors at sustained Ca2+ levels above ∼0.3 μm. These trends broadly agree with the results from our previous simulations of rate‐based plasticity . The additional release of ER store Ca2+ increases the total CaM activation above the LTD threshold, inducing strong synaptic weakening (Δw < 0) in the presence of ER . When Δt = +10 ms, ICCR makes a relatively modest contribution to the total Ca2+ response in the spine head , and this yields a small net enhancement of synaptic strengthening compared to the ER− spine . We simulated STDP inputs to our synaptic model over the full range of allowed spike timing differences (−100 ms ≤ Δt ≤ +100 ms), and the plasticity profiles obtained for the triplet and doublet protocols are shown in Fig. C. Ca2+ release from spine ER modulates the overall STDP curve for triplet inputs , and synaptic weakening is elicited over a broader range of spike timing differences compared to the ER− spine. Consistent with the reduced contribution of ICCR at small positive Δt , presence of ER introduces relatively less broadening of the LTP induction window. In the case of doublet inputs , ICCR extends the window for induction of synaptic depression over a broader range of spike timing differences relative to the ER− spine, the extent of which scales with the number of IP3R present.Transient elevation of Caead Fig. B, top, uts Fig. C, top,  Δt Fig. D and E,D and ΔP) in the presence of ER. Repeated random sampling of the STDP thresholds ΔtD/ΔtP for the ER− spine (from ±10 ms windows centred on ΔtD = −35 ms and ΔtP = +35 ms) yields a distribution of possible  pairs. The results  are represented by separate heat maps for the triplet and doublet input patterns . The overall distribution, which, by and large, is confined to the lower triangle (ΔD > ΔP), indicates that the contribution of ICCR can potentially extend the LTD window by several tens of milliseconds, whereas its effect on the LTP window is comparatively less. Taken together, Fig. C and D demonstrates the relative broadening of the LTD window in the presence of ICCR, which is fairly robust to variation of the plasticity thresholds and the IP3R cluster size. In summary, we found that mGluR‐mediated Ca2+ release from spine ER during correlated activation of pre‐ and postsynaptic neurons promotes the induction of synaptic depression over a broader range of temporal activation patterns (Δt), with relatively less influence on LTP induction.The differential modulation of the depression and potentiation windows seen in the above data was quantified in terms of the relative change in widths of the LTD and LTP windows .The foregoing analysis of our detailed synaptic model highlights the potential contribution of ICCR to activity‐driven CaA summarizes the differential responses in the ER+ spine (for NR = 30 IP3Rs) relative to the ER− (control) spine during trains of synaptic (SC) input at different rates. The heat map in Fig. A (left) represents the excess Ca2+–CaM amplitude as a function of the input frequency  and gN . ER contributes robustly to spine Ca2+ elevation at low input frequencies (f ≲ 5 Hz). The contribution of ICCR steadily declines at higher input rates due to Ca2+‐dependent inhibition of IP3 receptors, and this frequency‐dependent suppression of the difference between the ER− and ER+ spines is more pronounced at higher ΔCaEPSP. The corresponding results for the differential induction of plasticity in the ER+ spine are displayed as a heat map in Fig. A (right). For each gN, the plasticity thresholds θD and θP have been adjusted to have fD = 1 Hz and fP = 15 Hz in the ER− spine, and the excess plasticity (ΔΔw) in the presence of ER has been estimated for every input frequency. Figure A (right) indicates that ICCR robustly augments NMDAR‐mediated Ca2+ responses to facilitate the induction of synaptic depression at low input frequencies (f ≲ 1 Hz). Due to the inhibition of ICCR with increasing NMDAR activation , there is little difference between the plasticity curves for the ER− and ER+ spines at higher input frequencies. Except in a limited range of small gN values (ΔCaEPSP ≲ 0.15 μm), ICCR is strongly suppressed at stimulation rates above ∼10 Hz, and the LTP threshold (fP) in the ER+ spine remains nearly unchanged relative to the ER− spine.Figure ion Fig. A, left,+ spine during STDP input patterns . For each gN, the plasticity thresholds θD and θP in our model were adjusted so as to yield (for spike triplets) an LTD window for −35 ms < Δt < 0 ms and a potentiation window for 0 ms < Δt < 35 ms in the ER− spine. Figure B (left) summarizes the dependence of the excess Ca2+–CaM response in the ER+ spine (with NR = 30 IP3Rs) on gN and the spike timing difference (Δt) in the triplet case, showing a general reduction in the ICCR‐mediated enhancement of spine Ca2+ signals with increasing ΔCaEPSP. The corresponding differences in the plasticity outcome  show significant broadening of the window of LTD induction over a fairly wide range of gN values. The ER‐associated broadening of the LTP window is restricted to a comparatively narrow range of gN values and spike timing intervals. The differential Ca2+–CaM response and plasticity outcome in the doublet case  similarly highlight a general broadening of the window of spike timing differences eliciting synaptic depression, the extent of which scales inversely with ΔCaEPSP.We similarly characterized the differential responses and plasticity outcomes in the ERrns Fig. B and C.ase Fig. C simila3R cluster size, we repeated these comparisons with variable number of IP3 receptors (NR = 10–50) and plasticity thresholds, for both frequency‐dependent plasticity and STDP (Methods). For each combination of parameter values , the overall modulation of the NMDAR‐only plasticity curve by ICCR was quantified in terms of the shifts in the LTD and LTP thresholds for rate‐dependent plasticity, and changes in the LTD/LTP window widths in the case of STDP. The summary statistics  provide a sense of the variability in model outcomes, and taken together, suggest graded modulation of NMDAR‐based bidirectional plasticity by ICCR, with heightened LTD induction. In sum, our model simulations over realistic parameter ranges provide support for an ER‐linked form of synaptic metaplasticity on the level of individual spines, the nature of which is regulated by mGluR–IP3 signalling in concert with the temporal profile of NMDAR activation.As the above results were obtained for a specific choice of the plasticity thresholds and IPrs NR = –50 and p2+‐driven plasticity on the scale of individual excitatory CA1 synapses. Results from our model simulations of rate‐based plasticity and spike timing‐dependent plasticity, taken together, suggest that the presence of ER selectively enhances the propensity for LTD induction with a relatively diminished effect on LTP induction. Targeting of ER to larger spine heads can thus mediate a ‘metaplastic switch’ at stronger synapses that specifically relies on ER Ca2+ handling and metabotropic glutamatergic signalling. The graded contribution of the IP3‐sensitive ER store to spine Ca2+ elevation as a function of NMDAR activation that we have characterized here offers a synapse‐specific mechanism to differentially modulate the windows for depression/depotentiation and potentiation, yielding a net enhancement of activity‐driven synaptic depression . We calibrated the parameters in our model of ICCR to be consistent with salient features of experimentally reported Ca2+ responses in ER‐containing CA1 spines to unitary synaptic events  . As noted before, this arises from a balance between the contrasting effects of the input rate on (1) recovery of IP3Rs from inactivation between inputs , and (2) the availability of IP3, which increases with input rate and promotes the opening of IP3R channels. Analogous to the profile of ER Ca2+ contribution, our model also predicts a non‐monotonic rate dependence for the time delay in ICCR following glutamate release, which initially increases before decreasing with the input frequency as ICCR gradually synchronizes with the NMDAR‐mediated Ca2+ transient. The foregoing properties of spine Ca2+ response as functions of the synaptic input frequency, inferred from our numerical simulations, are potentially informative readouts of the model which could be compared with similar readouts from Ca2+ imaging experiments at individual CA1 spines. Such a comparison would help validate the interplay of different kinetic time scales in the ER+ spine model used in this study and constrain key model parameters.The response of an ERHz) Fig. D. As no2+ signalling can be broadly understood in terms of NMDAR‐mediated spine Ca2+ regulation and the kinetics of IP3R with its characteristic bell‐shaped dependence on the cytosolic Ca2+ level. With repeated low‐frequency stimulation, there is no sustained build‐up of Ca2+ evoked by the sequence of glutamate pulses. Every synaptic input triggers transient mGluR activation, evoking a short burst of Ca2+ release from ER which is initiated by fast activation (m1 and m2) of IP3R influx, followed by the slower Ca2+‐dependent inactivation (h) of the IP3R which brings the Ca2+ level down. This allows the IP3R to recover from inactivation between successive inputs when Ca2+ has decayed back to near‐resting levels, and explains the robust augmentation of the NMDAR‐mediated Ca2+ signal by ICCR at low stimulation frequencies .The contribution of ICCR to spine Caies Fig. . At highies Fig. , and impges Fig. D and E.t) on the millisecond scale. The amplitude of the sustained Ca2+ elevation driven by NMDA receptor activity is thus a function of the spike timing difference, and it directly controls the extent of inhibition (h) of the IP3R .An analogous situation arises during trains of pre‐ and postsynaptic spikes at a constant rate (in the theta band), mimicking the experimental induction of spike timing‐dependent plasticity. The activation of NMDARs in this case is controlled by the relative timing of pre‐ and postsynaptic firing , were kept fixed based on literature estimates; however, our results are not sensitive to these specific values and higher values yield similar plasticity profiles. Further, our main conclusions regarding the contribution of ER to spine Ca2+ dynamics are also valid when IP3Rs are modelled stochastically to account for fluctuations arising from small numbers of receptors.The key findings following from our simulations are fairly general, and hold for variation in important model parameters such as NMDAR conductance and number of IPet al. et al. et al. 2+‐based plasticity adds to the repertoire of dynamically regulated biophysical mechanisms that may be in place to control plasticity and function at excitatory hippocampal synapses  may evoke Ca2+ release from ER in the dendritic body as well  to spatially confined Ca2+ dynamics in spines, and it is in this specific context that our analysis predicts decreasing ER contribution with stronger synaptic (NMDAR) activation due to the Ca2+‐dependent suppression of IP3R activity. We note that an early experimental study of STDP at CA3–CA1 synapses  induction, as a possible explanation for the absence of associated heterosynaptic plasticity mediated by regenerative Ca2+ release (Ca2+ waves) from IP3‐sensitive stores. The predicted graded effect of spine ER on plasticity curves could be potentially tested by imaging spine Ca2+–CaM activity during different forms of persistent stimulation at individual CA3–CA1 synapses contacting ER+ spines, and assessing the effect of pharmacological blockade of either stores (SERCA inhibitors such as thapsigargin) or specific inhibition of the IP3 pathway (e.g. with heparin) on their response profiles. Such a comparison should ideally control for the possible confounding effects of changes in spine volume/synapse strength that may concurrently occur due to activity‐induced plasticity, through targeted inhibition of key effectors downstream of CaM, e.g., CaMKII and calcineurin.Although several lines of evidence link group I mGluR activation and ER Ca2+ signals and plasticity at hippocampal dendritic spines by ER. Our analysis quantifies a unique role for ER in dynamically altering the long‐term plasticity profile at individual CA1 spines and is supported by experimental evidence  in biochemical signalling likely extends to other aspects of dendrite function, such as in mediating some forms of heterosynaptic plasticity . G.M.: Wellcome Trust/DBT India Alliance (grant number IA/I/12/1/500529) and Science & Engineering Research Board, India (grant number PDF/2017/001803)


Current strategies are not especially successful in the treatment of acute myeloid leukemia (AML). The identification and characterization of oncogenes crucial to the survival and growth of leukemia cells will provide potential targets for the exploitation of novel therapies. Herein, we report that the elevated expression of SH3 domain-binding protein 5 (SH3BP5) significantly correlates with poor outcomes of AML patients. To test whether SH3BP5 contributes to the growth and survival of AML cells, we use the shRNA-encoding lentivirus system to achieve the knockdown of SH3BP5 expression in human AML cell lines U937, THP-1, Kasumi-1, and MV4-11. Functionally, the knockdown of SH3BP5 expression markedly inhibits the cell viability and induced apoptosis of these leukemia cells. Mechanistically, western blot analysis indicates that the knockdown of SH3BP5 expression decreases the phosphorylation of JNK and BAD. Moreover, the JNK agonist anisomycin rescues the growth inhibition phenotype of SH3BP5 deficiency in THP-1 cells. Moreover, the expression of SH3BP5 positively correlates with CD25 and CD123 levels. Finally, our study highlights the crucial role of SH3BP5 in promoting the survival of AML cells, and its suppression may be a potential therapeutic strategy for treating human AML. Despite the rapidly growing armamentarium of effective biologic agents and targeted therapies, AML remains a serious challenge for patients and hematologists. There were about 21,380 new diagnoses of AML in 2017 in USA, but conjected death cases were over 10,590, making AML the 6th highest cancer-related death in the male population [To identify novel therapeutic targets in human AML, we have established a systematic approach to explore potential genes important for leukemia propagation. Firstly, we determine new genes that correlate with the outcomes of AML patients using clinical databases. Then, we determine the biological function of the candidate genes using a loss-of-function approach in vitro. Furthermore, we validate the function of target candidate genes in leukemia mouse models in vivo. The candidate genes and their downstream signaling pathways will provide potential targets for the development of therapeutic approaches. We have identified several specific receptors, metabolizing enzymes, and some membrane proteins that support AML propagation using the above approach ,3,4,5.SH3BP5, a mitochondrial outer membrane scaffold protein, has a N-terminal SH3 domain binding site, a membrane spanning domain, and a two D-motif (KIM) on C-terminus ,7. The tRoswell Park Memorial Institute 1640 (RPMI-1640) and high glucose Dulbecco’s modified Eagle’s (DMEM) medium were purchased from Hyclone . Fetal bovine serum (FBS) was purchased from Gibco . JNK agonist anisomycin was purchased from Tocris biosciences . Anti- SH3BP5 (#11127-2-AP), Caspase 3 (#19677-1-AP), JNK (#24164-1-AP), BAD (#10435-1-AP), and Actin (#60008-1-Ig) antibodies were purchased from Proteintech Group . Anti- p-JNK Thr183/Tyr185 (#9255) and p-BAD Ser112 (#5284) antibodies were purchased from Cell Signaling Technology .All the leukemia cell lines—MV4-11, THP-1, U937, Kasumi-1, Rch-Acv, Kasumi-2, and NALM-6—were cultured in PMI-1640 supplemented with 10% FBS and 1% pen/strep. HEK-293 T cells were maintained in high glucose DMEM supplemented with 10% FBS and 1% pen-strep. All cell lines were maintained in the humidified incubator containing 5% CO2 at 37 ℃. All the cell lines were kindly provided by Prof. Cheng Cheng Zhang .https://tcga-data.nci.nih.gov/tcga/). Transcriptomic profiles of SH3BP5 gene were measured in 200 primary AML patients. However, only 151 AML patients with intact clinical information were analyzed in this study.We analyzed gene expression datasets from human AML studies as previously described ,12,13. SThe targeting SH3BP5 shRNAs were designed online as we previously described . The tarReal-time (RT) PCR primers were designed online as previously described . BrieflyFor cell growth assay, lentivirus-infected cells (GFP positive) were sorted by flow cytometry at 48 h post-infection. Then, cells were seeded in 24-well plates at 10,000 cells per well in triplicate wells. Then, cell numbers were counted at 2, 4, and 6 days. Cell apoptosis was evaluated using the Annexin V-PE/7-AAD apoptosis detection kit , as previously described [Cells were harvested and lysed in cold RIPA lysis buffer containing protease and phosphatase inhibitor cocktails. The proteins were resolved by SDS-PAGE and then transferred to a PVDF membrane via the permeant blotting method. Then, the membrane was blocked with 5% BAS and incubated with appropriate primary antibodies at 4 °C overnight. Thereafter, the membranes were incubated with the appropriate HRP-conjugated 2ed antibodies after being washed three times. Detection was performed with ECL chemiluminescent agent. Densitometric analysis of blots were performed using Image J. The levels of protein expression were normalized to Actin.All statistical analyses were performed with the SPSS 24.0 software package. Difference between SH3BP5 expression and cell growth/apoptosis was compared using two-tailed student’s t-test or two-way Anova. Associations between SH3BP5 expression and clinicopathologic parameters were analyzed with a chi-square test. Overall, survival curves were determined by the Kaplan–Meier technique and contrasted via log-rank test. Univariate and multivariate Cox analyses were used to determine risk factors for overall survival. For all experiments, differences were considered statistically significant if p < 0.05.To investigate the expression profile of SH3BP5 in human AML cells, microarray data derived from the GEO database  were analyzed. The results suggested that expression of SH3BP5 in AML patients was significantly higher than that in healthy samples A. FurtheTo further investigate the potential function of SH3BP5 on AML propagation, Kaplan–Meier analysis of the relationship between SH3BP5 expression level and clinical endpoints of AML patients was performed. Analysis demonstrated that the higher expression of SH3BP5 was significantly associated with shorter overall/disease free survival in AML patients A,B. To eTo explore the potential role of SH3BP5 in AML, expression of SH3BP5 was knocked down in AML cells using lentivirus-encoded shRNAs. Firstly, the efficiency of the designed shRNAs targeted SH3BP5 was examined. RT-PCR results suggested that shRNA 3 and 4 efficiently inhibit SH3BP5 expression at mRNA level compared to scramble shRNA A. We furPrevious studies have suggested that SH3BP5 physically interacts with JNK by its N-terminal SH3 domain binding site, and plays unique roles in JNK-mediated signal transduction ,7,8,9. WHerein, our data revealed that elevated expression of SH3BP5 significantly correlated with poor survival of AML patients. Moreover, SH3BP5 was highly expressed in many AML cell lines both at mRNA and protein levels. Using a loss-of-function approach, we further reported that SH3BP5 promoted AML propagation through suppressing cells apoptosis.SH3BP5, as a novel JNK-interacting protein, was associated with disturbed mitochondrial function and elevated mitochondrial ROS production . Recent JNK-mediated pathways regulate a wide range of developmental function depending on the cell type and lineage, although they are somewhat contradictory ,19. On tThe opposing functions of JNKs attributed to the findings that JNKs activate different substrates, based on cell type temporal aspects, or specific stimulus . EvidencIn conclusion, we found that expression of SH3BP5 was enhanced in AML cells and was negatively correlated with survival in AML patients. Elevated expression of SH3BP5 was an independent prognostic factor for AML patients. Furthermore, SH3BP5-mediated activation of JNK-BAD signaling contributes to the survival of AML cells. Taken together, our data suggested that SH3BP5 played vital roles in progression of AML and demonstrated SH3BP5 might be a potential prognostic factor and treatment target for human AML.