When cells are irradiated, their DNA can become damaged, this causes different types of repair processes to try and fix the DNA breaks. One of the most lethal types of DNA damage is double-strand breaks (DSBs). This work models the most used DSB repair process called Non-Homologous End Joining (NHEJ) and includes both its resection-independent and resection-dependent pathways. The models produced are benchmarked against experimental normal and deficient cell-types across a wide range of radiation qualities. We compare two approaches of modelling, the first is where the DSBs can repair in parallel and the second is where the DSBs repair is entwined. We find that it is necessary to consider both the resection-independent and resection-dependent pathways as entwined to produce a model which robustly matches experimental work. Through better modelling of NHEJ repair, it can improve our understanding of radiation response which has potential in biological optimisation for radiotherapy.Mechanistic in silico models can provide insight into biological mechanisms and highlight uncertainties for experimental investigation. Radiation-induced double-strand breaks (DSBs) are known to be toxic lesions if not repaired correctly. Non-homologous end joining (NHEJ) is the major DSB-repair pathway available throughout the cell cycle and, recently, has been hypothesised to consist of a fast and slow component in G0/G1. The slow component has been shown to be resection-dependent, requiring the nuclease Artemis to function. However, the pathway is not yet fully understood. This study compares two hypothesised models, simulating the action of individual repair proteins on DSB ends in a step-by-step manner, enabling the modelling of both wild-type and protein-deficient cell systems. Performance is benchmarked against experimental data from 21 cell lines and 18 radiation qualities. A model where resection-dependent and independent pathways are entirely separated can only reproduce experimental repair kinetics with additional restraints on end motion and protein recruitment. However, a model where the pathways are entwined was found to effectively fit without needing additional mechanisms. It has been shown that DaMaRiS is a useful tool when analysing the connections between resection-dependent and independent NHEJ repair pathways and robustly matches with experimental results from several sources. Radiotherapy is the standard of care for many cancer treatment sites. The goal of radiotherapy treatment planning is to maximise the absorbed dose—a surrogate for biological effects—in the tumour volume, whilst sparing nearby healthy tissues. The fate of irradiated cells mainly depends on the generation of double-strand breaks (DSBs) and the accompanying DNA repair activation . There aFor over a century, mathematical models of experimental or clinical observations have been used to investigate potential mechanisms of biological responses following irradiation . The linDSBs induced by irradiation have been observed to repair with biphasic kinetics in all phases of the cell cycle . In lateDespite the current understanding of DNA DSB repair proteins involved in different repair pathways, the underlying temporal processing and combined action at DSB sites requires further probing. Mechanistic models for DNA repair have potential to facilitate the evaluation of biological uncertainty in radiotherapy treatment planning ,28,29. RThis work aims to address the current gap in modelling the slow component of NHEJ DSB repair in the G0/G1 cell cycle phase using a Monte Carlo based method. Utilising the DNA Mechanistic Repair Simulator (DaMaRiS), within which the mechanisms arrived at by analysing in vivo/vitro experiments can be arranged and tested against known behaviours . We inveThe mechanistic modelling in this study uses DaMaRiS , a framework that has been developed at the University of Manchester ,18,19. TThe DNA damage model used in this work is a well-established model developed by our group at the University of Manchester ,18,19,32The DaMaRiS framework is a user-defined DNA repair model that can simulate the motion and reaction of DSB objects and repair proteins ,19. DaMaWhilst initially co-located in space, the two ends from a DSB can move independently from each other through a Continuous Time Random Walk (CTRW) model of sub-diffusive motion which is implemented by having a trapped state where diffusion is disabled, as there are not yet any bridging proteins present to physically link them. Two DSB ends in proximity and with the appropriate proteins attached can form a synaptic complex. Two DSB ends that have formed a synaptic complex are treated as the same object and will move together. Two DSB ends in a synaptic complex may dissociate, breaking the protein–protein interaction bridging the two strands of DNA, at which point they are again treated independently. In this work we have not yet implemented any large-scale chromatin rearrangements which could move both ends of a DSB together, regardless of their physical attachment to each other. This type of motion, however, has less influence on the probability of two DSB ends meeting than their local motion, and as such has less impact on repair kinetics.In DaMaRiS, DSB objects progresses through the user defined pathways by changing state in a step-by-step manner. For example, a DSB end may transition into a DSB end with Ku70/80 attached, representing the loading of that specific protein. These state transitions are governed by user defined time constants that represent the average time for that transition to occur, with actual transition times being drawn randomly for each individual DSB object. When a DSB object has multiple possible paths for progression, a random transition time is drawn for each possible state change and the shortest time is selected. In this manner, the repair pathway used is dictated by which proteins are recruited to the DSB end. Influence on pathway choice can then be modelled by linking specific factors to critical time constants; shortening them to increase likelihood or lengthening them to reduce the frequency of that pathway.The reaction of a repair protein with a DSB end is a chemical process and as such the state progression of one DSB end has no influence on another, physically separate, DSB end. This is incorporated into the model by treating DSB end progression through a defined pathway independently to its partner end. To influence pathway choice for both ends of a DSB other factors must be considered. Both the chemical state of the DSB ends and local protein concentrations could be factors that influence pathway choice; however, these have not yet been implemented in DaMaRiS, but are important avenues for future investigations. In this work we have considered the possibility for local chromatin environment to dictate or influence pathway choice ,36,37,38No experimental data has been found to fit to on differing recruitment kinetics dependent on chromatin status. As such, these are treated as free parameters in order to investigate possible influence. It is important to note that due to the lack of data, although we have modelled this as the influence of chromatin compaction, the true mechanism could be one of those discussed previously. As an extreme example, it could be that chromatin compaction has no effect on pathway choice and instead there is a proportion of complex breaks which dictate pathway choice that happens to be the same as the proportion of hetero-/euchromatin we have modelled. Models where chromatin compaction have not been included can be thought of as reporting on the average behaviour of protein recruitment in both heterochromatin and euchromatin.The NHEJ repair model in this work consists of both the fast, resection-independent, and slow, resection-dependent, processes in G0/G1 normal tissue cells. We investigate the relative feasibility of two potential models for this pathway, a “Parallel” model A where tParallel” repair pathway (Model A) is that DSBs ends processing through the slow and fast repair process are treated separately in the whole process. That is, a DSB end handled by resection-dependent process cannot enter synapsis with another end handled by the fast process. However, in “Entwined” repair pathway (Model B), DSB ends resected by CtIP and EXO1 are capable of forming synaptic complexes with DSB ends handled by the fast process. One of the distinct features of the “The experimental data selected to fit model parameters, or to assess model performance, has been limited to G0/G1 cell cycle phase. In this way we avoid confounding factors due to other cell cycle effects (such as Homologous Recombination). Time constants in the models, which govern the recruitment of repair proteins to DSB ends, are fitted to reproduce in vitro experiments from literature with cells irradiated by lasers ,62,63,64The repair kinetics of DSBs for all proposed models are compared to γ-H2AX foci or 53BP1 foci experiments in literature with seven different G0/G1 phase fibroblast wild-type cells  and lymphocytes derived from healthy human volunteers exposed to doses of photons and protons between 0.5 and 4 Gy ,66,67,68Given the dependence on Artemis, the proposed models were evaluated against Artemis inhibited repair data. In DaMaRiS this can be simply achieved by removing time constants governing the loading of Artemis and leaving all other parameters the same. The Riballo data set is used to evaluate the agreement between DaMaRiS and experiment where two Artemis-deficient fibroblast cell lines (CJ179 and MEF) were exposed to 1.3 and 2 Gy photons . The robIn these models, the quantity of unrepaired DSBs simulated at 24 h has a strong dependence on end mobility . TherefoIt should be noted that all experimental data used in this work are reproduced from published literature based on various cell lines. As such the parameters of the NHEJ pathway in this work is a general mechanistic description, and not an attempt to recreate the specific response of any one cell line exactly. If the hypothesised models can fit all data adequately, then this gives confidence in the general arrangement of mechanisms, which in reality will be perturbed by cell line specific factors.2/DF). Model results are compared to each experimental data set separately, giving a number of Chi-Square statistics per protein. The final statistic is then quoted as the unweighted average of each Chi-Square, giving an overall impression of goodness-of-fit to the entire experimental data. Alongside this average, we quote the “best” and “worse” reduced Chi-Square, giving an impression of the model’s ability to fit an individual dataset. Given the previously mentioned limitations, the fit to literature data cannot conclusively determine which of the models tested represents reality, but an indication of likelihood can be given. Additionally, the models can be used to determine acceptable ranges for additional parameters that would increase or decrease confidence in a particular model, thus highlighting valuable future experimental tests.The model’s ability to reproduce experimental results was assessed through reduced Chi-Square (χ2/DF) is summarised in To evaluate the potential repair pathways in G0/G1 phase cells, the protein recruitment kinetics of the models and experiments are compared and shown in There is a slight model difference between the recruitment kinetics for the responses of relatively high LET ionizing radiation and laser induced damage as shown in Expanding on our previous work modelling the fast NHEJ repair pathway, we assume that CtIP is recruited after loading Ku  has a fast repair rate in the first 1 to 2 h which fits well with most literature data, and then a plateau beyond 2 h where relatively more DSBs exist. The “Entwined” process (Model B) always produces less unrepaired DSBs than the “Parallel” process at longer repair time, which fits better with experimental data. However, with low-dose cases c,d, it pWhilst Model B tends to overestimate the repair rate for foci based assays, when considering the more direct measurements of damage  the model tends to predict a slower repair rate compared to the experimental data e–f.10 nm2·s−1. Detailed linear fit parameters are given in Furthermore, the relationship between the yield of unrepaired DSBs at 24 h following 1 Gy proton exposure across a range of LET for the models was investigated. As in Warmenhoven et al.  the mainTo investigate this further we considered if the chromatin compaction in the local area of the break could influence both ends of the DSB to take the same path in the “Parallel” process (Model A). At the extreme, forcing DSB ends located in heterochromatin to recruit CtIP and abolishing its recruitment in euchromatin, causes the model to fit repair kinetics for both 25% heterochromatin  and 48% The “Entwined” model was further tested for rationality and robustness by comparing the kinetics of DSB repair of Artemis-deficient and XLF-deficient cells to literature reported experimental data.The Artemis-deficient cell system and wild-type cell system have a similar repair rate within around 1–2 h both for experimental and simulated results of 2 Gy Photon irradiations a. At lowIn this work, two hypothesised NHEJ repair pathways, consisting of both resection-dependent and -independent processes, have been modelled. Model parameters were fitted to literature reported experimental data of protein recruitment, wild-type repair, and protein-deficient repair kinetics, as listed in The initial investigation of repair protein recruitment kinetics , shows t2 produced different results of 7.5 MeV proton irradiation and found no difference . Additionally, ATM is suggested to have a role in loosening highly-compacted chromatin during repair , which mDNA repair has a close relationship with cell survival and NHEJ is the main repair pathway, so understanding the temporal functionality of key NHEJ proteins is crucial. Why certain breaks undergo the resection route, when no HR is available, is still an open question worthy of further investigation.This study has investigated the NHEJ DSB repair pathway within a mechanistic model for the G0/G1 phase of the cell cycle following irradiation. The parameters used in the models are verified by experimental observations taken from the literature. The “Entwined” model B could match the experimental results from several sources for protein recruitment kinetics and overall repair kinetics, in both wild type and specific protein deficient cell lines. For the “Parallel” model A to fit experimental results additional mechanisms had to be considered. This gives less confidence in model A being representative of the true biological mechanisms but does highlight specific areas of investigation that could support it.For this reason we favour the “Entwined” description of the pathway and propose that it is more likely that the resection-dependent NHEJ process could be considered a corrective mechanism for getting double strand breaks back into the NHEJ pathway, rather than as a separate pathway altogether.


We showed that the amino acid-mediated increase of intracellular Ca2+ is important for mTORC1 activation and thereby contributes to the promotion of nascent protein synthesis. We found that Ca2+/CaM interacted with TSC2 at its GTPase activating protein (GAP) domain and that a CaM inhibitor reduced binding of CaM with TSC2. The inhibitory effect of a CaM inhibitor on mTORC1 activity was prevented by loss of TSC2 or by an active mutant of Rheb GTPase, suggesting that a CaM inhibitor acts through the TSC2-Rheb axis to inhibit mTORC1 activity. Taken together, in response to amino acids, Ca2+/CaM-mediated regulation of the TSC2-Rheb axis contributes to proper mTORC1 activation, in addition to the well-known lysosomal translocation of mTORC1 by Rag GTPases.Mechanistic target of rapamycin complex 1 (mTORC1) is a master growth regulator by controlling protein synthesis and autophagy in response to environmental cues. Amino acids, especially leucine and arginine, are known to be important activators of mTORC1 and to promote lysosomal translocation of mTORC1, where mTORC1 is thought to make contact with its activator Rheb GTPase. Although amino acids are believed to exclusively regulate lysosomal translocation of mTORC1 by Rag GTPases, how amino acids increase mTORC1 activity besides regulation of mTORC1 subcellular localization remains largely unclear. Here we report that amino acids also converge on regulation of the TSC2-Rheb GTPase axis via Ca Cells appropriately regulate cell growth, metabolism, and autophagy in response to environmental cues such as amino acids, glucose, growth factors, and stresses. A key mediator of these responses is a multiprotein kinase called mechanistic target of rapamycin complex 1 (mTORC1), which includes the protein kinase mTOR and two core subunits, Raptor and mLST8. In addition to mTORC1, mTOR can form another complex called mTORC2, which contains Rictor as a defining component, instead of Raptor, and has distinct functions such as regulation of actin organization . It has 2+ concentration [2+ chelator, resulted in reduced mTORC1 activity [2+ concentration through an unidentified mechanism and modulates mTORC1 activity [2+ concentration and in turn binds to hVps34, a class III phosphatidylinositol-3-kinase, to activate mTORC1. Consistently, hVps34 has been reported to be activated by amino acids [2+/CaM in the activation process of hVps34 remains controversial [It was reported that mTORC1 activity can be regulated by the intracellular Cantration ,14. Indeactivity . Furtheractivity ,15. Moreno acids . Howeveroversial , and detFrom the current view of the proposed mTORC1 activation mechanism, which involves Rags and Rheb, it is reasonable that amino acids are primarily required for mTORC1 activity at lysosomes due to the rate-limiting step for mTORC1 activation ,3,4. In 2+ entry that leads to a rise in intracellular Ca2+ concentration. The elevation of Ca2+ contributes to mTORC1 activation and to cellular functions such as promotion of protein synthesis. We revealed that CaM plays a key role linking Ca2+ signaling to TSC2 to promote mTORC1 activation. Ca2+/CaM interacts with the GAP domain of TSC2 and might thereby regulate its GAP activity toward Rheb. Thus, our findings revealed a new regulatory pathway of amino acid-induced activation of the mTORC1 pathway through the TSC2-Rheb GTPase axis by controlling intracellular Ca2+ levels.Here we show that amino acids activate mTORC1 by modulating the TSC2-Rheb pathway in addition to Rag GTPases. We found that amino acids stimulate extracellular Ca2+ level [2+ rise in mTORC1 signaling, we established HeLa cells stably expressing R-GECO1 [2+ indicator. As previously reported [2+ rise . These results suggest that the addition of amino acids causes intracellular Ca2+ rise by promoting Ca2+ entry from the extracellular medium.Previous studies including our studies demonstrated that addition of amino acids induces a rise in intracellular Ca2+ level ,15,20,21 R-GECO1 , a red f[2+ rise A. To exa2+ rise affects amino acid-mediated mTORC1 signaling. As shown in 2+ rise, although the effects of EGTA were slightly different among cell lines. We also tested mouse embryonic fibroblast (MEF) cells and found that Ca2+ chelation by EGTA dramatically prevented mTORC1 activation by amino acids as shown by decreased phosphorylation of both S6K1 and 4E-BP1 , Arg and Lys potently induced intracellular Ca2+ rise  is responsible for induction of intracellular Ca, we found that TSC2 was bound to Strep-CaM in a Ca2+-dependent manner  and found that Strep-CaMDA reduced binding to TSC2  and screened proteins in the presence or absence of Cat manner A, consist of CaM   showed much greater resistance to the CaM inhibitor-mediated decrease in phosphorylated S6K1. These results indicate that CaM inhibitors likely decreased GTP-loading of Rheb, leading to reduction of S6K1 phosphorylation. Taken together, our results suggest that Ca2+/CaM binding to TSC2 likely results in the inhibition of TSC2 action toward Rheb GTPase, which ultimately leads to an increase of mTORC1 activity. To confirm that TSC2 is indeed involved in mTORC1 regulation in response to amino acid availability, TSC2 KO cells were tested for amino acid starvation as well. As shown in Q64L) were resistant to amino acid deprivation-induced mTORC1 inactivation , resulted in reduced muscle growth concomitant with reduced activation of Akt or Erk1/2, indicating that STIM1-mediated regulation of intracellular Ca2+ concentration is required for proper muscle hypertrophy [2+ elevation during amino acid readdition is required for promotion of protein synthesis. This finding might explain muscle hypertrophy in response to exercise and protein supplementation, because it was reported that increase in Ca2+ contributes to muscle hypertrophy [2+ levels might be one of the key mechanistic bases for onset of aberrant hypertrophy. Indeed, consistent with the results of our study, a very recent study on mice with cardiac hypertrophy showed that higher Ca2+ levels induced by aberrant Ca2+ entry by overexpression of SARAF, a negative regulator of SOCE, promotes hypertrophy at least in part through mTORC1 hyperactivation [+ T cells, because it was reported that activation of mTORC1 via Rag GTPase by amino acids [2+ entry via modulation of SOCE [As Ca atrophy . For exaertrophy . The resertrophy . In additivation . Thus, ono acids  and Ca2+ of SOCE  regulate2+ entry from extracellular medium. However, it remains unknown what kinds of Ca2+ channels are involved in these amino acid-induced processes. Multiple different Ca2+ channels might be involved in amino acid-dependent Ca2+ entry. It is also possible that depolarization might be associated with Ca2+ entry because Arg is reported to stimulate Ca2+ entry via depolarization in pancreatic β cells [2+ channels.We find that the addition of amino acids, especially Arg and Lys, promotes Ca β cells . Further2+/CaM was reported to interact with hVps34, a PI3 kinase that produces phosphatidylinositol 3-phosphate (PI3P) [2+/CaM was reported to bind directly to mTORC1, leading to promotion of mTORC1 kinase activity by an unknown mechanism [2+/CaM binding to TSC2 to modulate mTORC1. However, we propose that TSC2 is a major contributor of the Ca2+/CaM action toward mTORC1 signaling, since the inhibitory effect of CaM inhibitors was rescued by TSC2 KO or by the GTP-bound form of Rheb (RhebQ64L)  . The proe (PI3P) . Secondlechanism . We cannhebQ64L) . One of 2+/CaM-mediated repression of GAP activity of TSC2. Very recently, the cryo-EM structure of the human TSC complex, which is a pentameric protein complex consisting of two TSC2 molecules, two TSC1 molecules and one TBC1D7 molecule, was reported [2+/CaM might affect the structure of the helix pair and thus the overall structure of the catalytic domain of TSC2, leading to inactivation of TSC2. In fact, multiple phosphorylation sites that are known to affect TSC2 GAP catalytic activity are located far from the catalytic domain of TSC2 [We demonstrated that deletion of 1717–1732 a.a. in TSC2 resulted in loss of binding with CaM B. Indeedreported . The CaMreported . Althoug of TSC2 ,41,42,432+/CaM. Consistently, several studies suggested that TSC2 activity and thereby GTP-loaded Rheb are regulated by amino acid availability. GTP/GDP-bound forms of Rheb were reported to be changed in response to amino acid availability [2+/CaM appears not to regulate TSC2 subcellular localization during the change of amino acid availability. Therefore, interaction of Ca2+/CaM with TSC2 might primarily affect catalytic activity of TSC2.Our findings suggest that TSC2 activity can be regulated by amino acid availability via Calability ,44,45. Mlability ,19. Howe2+/CaM binding to TSC2 are fascinating for the development of a new compound activating TSC2. As shown in Since TSC2 is one of the most important negative regulators of mTORC1 signaling, our results showing CaAnti-pThr389-S6K1 (#9205 and #9234), anti-S6K1 (#9202), anti-pThr37/46-4E-BP1 , anti-4E-BP1 , anti-mTOR , and anti-TSC2  were purchased from Cell Signaling Technology . Anti-S6K1 , anti-Raptor , anti-Rictor , anti-TSC2 , anti-Myc , and anti-GST  were from Santa Cruz Biotechnology . Anti-HA , and anti-FLAG  were from Sigma-Aldrich . Anti-Strep tagII (PAB16603) was from Abnova . Anti-puromycin  was from Millipore . The anti-LAMP1 (H4A3) antibody was from BD Transduction Laboratories . Horseradish peroxidase (HRP)-conjugated goat antibodies against mouse IgG and rabbit IgG and an HRP-conjugated rabbit antibody against goat IgG were from Jackson ImmunoResearch . For immunofluorescence, AlexaFluor488, AlexaFluor555, or AlexaFluor647-conjugated donkey anti-mouse IgG, AlexaFluor488, AlexaFluor555, or AlexaFluor647-conjugated donkey anti-rabbit IgG, and AlexaFluor488, AlexaFluor555, or AlexaFluor647-conjugated donkey anti-goat IgG antibodies were purchased from Invitrogen  and used as secondary antibodies. BAPTA-AM solution (348-05451) was purchased from Wako . W-7 (14826), W-13 (14277) and Calmidazolium (14442) were from Cayman Chemical . Torin1 (T5870) was from LKT laboratories .The expression plasmid for HA-S6K1 (pRK5-HA-S6K1) was described previously . The expQ64L (pcDNA3.1-Myc-RhebQ64L) was generated by site-directed mutagenesis using primers (agccggtctagatgaatattctatctt and tcatctagaccggctgtgtctacaagt). The expression plasmid for Strep-CaM (pEXPR-IBA105-B-CALM1) was constructed by inserting a PCR fragment of human CALM1 amplified using primers (ccgcgggaattcggggatccatggctgatcagctgaccgaag and cgcaagcttggtacctcgagtcattttgcagtcatcatctgtacg) and cDNA from HeLa cells into BamHI and XhoI sites of pEXPR-IBA105-B (laboratory stock). Note that the amplified CALM1 sequence had many nucleotide differences from the cDNA database but no amino acid substitution. Therefore, we used the amplified CALM1 cDNA for expression of CaM. The expression plasmid for Strep-CaMD21A, D57A, D94A, D130A  was created by site-directed mutagenesis using primers . pcDNA3.1-FLAG-A-CALM1 was generated by inserting a PCR fragment of CALM1 using primers (cgataaggtacctaggatcctcgctgatcagctgaccgaaga and cctctagactcgagcggccgctcattttgcagtcatcatctg) into pcDNA3.1-FLAG-A (a gift from Dr. T. Maeda). Then the expression plasmid for FLAG-CaM (pHUEV-FLAG-CALM1) was constructed by inserting a PmeI-XbaI fragment of pcDNA3.1-FLAG-A-CALM1 into SmaI and XbaI sites of pHEK293 Ultra Expression Vector I . The expression plasmid for FLAG-CaMD21A, D57A, D94A, D130A  was constructed similarly.The expression plasmid for Myc-Rheb (pcDNA3.1-Myc-Rheb) was constructed by insertion of a PCR fragment of human Rheb1 amplified from human cDNA using primers (aatggatccatgccgcagtccaagtcccggaag and cccaaatgatatctttcaggttaacagaag) into BamHI and EcoRV sites of pcDNA3.1-Myc-C (a gift from Dr. T. Maeda). The expression plasmid for Myc-RhebΔ1717–1732 (pcDNA3-FLAG-TSC2Δ1717–1732), a PCR fragment having the corresponding deletion was amplified using primers (gctccaaccccaccgatatctacccttcgaaggaggaagccgcctactcc and atagaatagggccctctagaactagtggatc) and inserted into EcoRV and XbaI sites. The expression plasmid for a patient-derived mutation of TSC2Δ1723–1728 (pcDNA3-FLAG-TSC2Δ1723–1728) was constructed similarly using a PCR fragment with primers (ctccaaccccaccgatatctacccctccaagtggattgcccggctccgtcaacggatctgcgaggaagcc and atagaatagggccctctagaactagtggatc).To generate an expression plasmid for FLAG-TSC2To generate the expression plasmid for GFP-TSC2 (1717–1732 a.a.), oligonucleotides (tcgaggatggattgcccggctccgccacatcaagcggctccgccagcggatctgctag and gatcctagcagatccgctggcggagccgcttgatgtggcggagccgggcaatccatcc) were annealed and ligated into XhoI and BamHI sites in pSGFP2-C1 .For PCR cloning, PrimeSTAR Max DNA polymerase (Takara) was used for amplification, except for cloning human Rheb1 cDNA, which was amplified with Pyrobest DNA polymerase (Takara). pCX4pur-R-GECO1 was a laboratory stock.Q64L was constructed similarly.pEXPR-IBA105-B-Rheb was generated by inserting a BamHI-XhoI fragment of pcDNA3.1-Myc-Rheb into BamHI and XhoI sites of pEXPR-IBA105-B. pCX4pur-Strep-tagII-Rheb was constructed by using primers (tcctctagactgccggatccgccaccatggctagctggagcc and cacgcgtcggtccggaattctcacatcaccgagcatgaag) and pEXPR-IBA105-B-Rheb as a template. pCX4pur-Strep-tagII-RhebpRK5-FLAG-RagB was constructed by inserting annealed primers (cgatgccaccatggactacaaggatgacgatgacaagggtggtggtggtggtgcg and tcgacgcaccaccaccaccacccttgtcatcgtcatccttgtagtccatggtggcat) into SalI and ClaI sites of pRK5-HA-GST-RagB. pCX4pur-FLAG-RagB was generated by inserting a ClaI-NotI fragment of pRK5-FLAG-RagB into BamHI and NotI sites of pCX4pur.eSpCas9(1.1)-2A-Puro-TSC2-sg2 was constructed by inserting annealed oligos (caccggacggagtttatcatcaccg and aaaccggtgatgataaactccgtcc) into the BbsI site of eSpCas9(1.1)-2A-Puro . All seq2 atmosphere. Transient transfection of the indicated plasmids was performed using Polyethylenimine “Max” (PEImax)  for HEK293T cells and FuGENE6  for HeLa cells according to the manufacturer’s instructions. HeLa cells stably expressing R-GECO1 and HEK293T cells stably expressing FLAG-RagB, Strep-Rheb, or Strep-RhebQ64L were generated by retroviral infection and selected with 1 μg/mL puromycin as essentially described previously [Q64L, pCX4pur-SGFP2-Rheb, or pCX4pur-SGFP2-RhebQ64L using FuGENE6.HeLa, MEF, and HEK293T cells were grown at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) with 1000 mg/L glucose  or with 4500 mg/L glucose (for HEK293T cells) supplemented with 5% fetal bovine serum under a 5% COeviously . RetroviAmino acid starvation and readdition were done as described previously  with modGeneration of TSC2 knockout HEK293T cells by the CRISPR/Cas9 system was done as described previously . BrieflyThe Surface Sensing of Translation (SUnSET) method was used for monitoring protein synthesis rate. After treatments with various conditions shown in Live-cell imaging analyses were performed essentially as described previously . BrieflyHeLa and HEK293T cells were seeded onto cover glasses coated with poly L-Lysine. After 24 h, the cells were washed once with PBS(−) and fixed with 4% PFA/PBS(−) for 15 min, followed by washing three times with PBS(−). Then the cells were permeabilized with 0.05% Triton X-100/PBS(−) for 5 min, followed by washing three times with PBS(−). After blocking with 0.25% BSA/PBS(−) for 60 min, the cells were incubated with primary antibodies at 4 °C overnight and washed five times with 0.25% BSA/PBS(−). The cells were then incubated with secondary antibodies at room temperature for 60 min in the dark and then washed five times with 0.25% BSA/PBS(−) and mounted. Images were acquired under an FV1000-D confocal laser-scanning microscope.Colocalization (Pearson’s correlation coefficient) of mTOR with LAMP1 was calculated using ImageJ Coloc 2 Plugin. After background subtraction using Sliding paraboloid and Disable smoothing settings, individual cells were manually selected and calculated.g for 10 min to obtain cell lysates.Cells were washed once with ice-cold PBS(−) and lysed with lysis buffer TX (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 50 mM NaF, 10 mM β-glycerophosphate) containing 3 μg/mL leupeptin, 1 μM pepstatin A, and 0.1 mM pefabloc and centrifuged at 9000× g for 10 min, the supernatant was incubated overnight with anti-mTOR (N-19). The immunocomplexes were collected by the addition of Dynabeads protein G (Invitrogen) and washed three times with lysis buffer A.In g for 10 min. The cell lysates were incubated overnight with MagStrep “type 3”  in the presence of 100 μM CaCl2 or 5 mM EGTA and washed three times as above. In In In g for 10 min. The cells lysates were incubated with FLAG M2 agarose beads for 3 h with rotation at 4 °C and washed three times with RIPA buffer.Crosslinking was done essentially as described previously . DSP wasp-values less than 0.05 are considered statistically significant.Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey’s test using Origin 9.1 . We followed biosafety guidelines for recombinant DNA research at Nagoya University. Experimental proposals were approved by the Recombinant DNA Biosafety Committee of the Graduate School of Bioagricultural Sciences, Nagoya University: Nou15-067 (approved on 24 March 2006) and Nou19-002 (approved on 12 April 2019).


Here, we explored the spatiotemporal changes in Ca2+ signaling in lens epithelial cells (LECs) upon local mechanical stimulation, to better understand the LECs’ intercellular communication and its association with cataractogenesis. We were interested in if the progression of the cataract affects the Ca2+ signaling and if modifications of the Ca2+ homeostasis in LECs are associated with different cataract types. Experiments were done on the human postoperative anterior lens capsule (LC) preparations consisting of the monolayer of LECs on the basement membrane. Our findings revealed that the Ca2+ signal spreads radially from the stimulation point and that the amplitude of Ca2+ transients decreases with increasing distance. It is noteworthy that a comparison of signaling characteristics with respect to the degree of cataract progression revealed that, in LCs from more developed cataracts, the Ca2+ wave propagates faster and the amplitudes of Ca2+ signals are lower, while their durations are longer. No differences were identified when comparing LCs with regard to the cataract type. Moreover, experiments with Apyrase have revealed that the Ca2+ signals are not affected by ATP-dependent paracrine communication. Our results indicated that cataract progression is associated with modifications in Ca2+ signaling in LECs, suggesting the functional importance of altered Ca2+ signaling of LECs in cataractogenesis.Ca The ocular lens needs to be transparent to transmit and focus light to the retina. Cataracts are opacities of the lens and are the most common cause of vision impairments. Cataracts caused 33.4% of all blindness worldwide in 2010 (10.8 million people) and 18.4% of all moderate to severe vision impairments (35.1 million people) i, while the 360/380 nm ratio allowed for the visualization of the Ca2+ concentration changes in the cytoplasm. Image acquisition, timing, and filter wheel operation were all controlled by WinFluor software via a PCI6229 interface card . The criteria for selecting the regions for imaging were the presence of adherent cells and good cell morphology, both assessed by observation of transilluminated and 360-nm fluorescent images. Several repetitions of the mechanical stimulation on the different regions of on the same LC were done. Image frames were acquired every 500 ms, resulting in frame cycles being 1 s long . The manual selection of individual cells  and the subsequent exportation of Ca2+ traces were performed with ImageJ software .Image acquisition was done with the 12-bit cooled CCD camera SensiCam . The software used for the acquisition was WinFluor . Objectives used were: 40X/0.75 Plan-NeoFluar and 63X/1.25 oil Plan-NeoFluar . The light source used was XBO 75 W  Xe arc lamp. The light intensity was attenuated when necessary with grey filters with optical densities of 0.5, 1, and 2 . The excitation filters used, mounted on a Lambda LS-10 filterwheel , were 360 and 380 nm . Excitation with the 360-nm filter (close to the Fura-2 isosbestic point) allowed for the observation of the cells’ morphology and of the changes in the concentration of the dye, irrespective of changes in i signaling in individual LECs, as indicated in i-th cell, tires,, symbolizes the time at which the cells started to respond to the mechanical stimulation. The time at which the maximal amplitude of [Ca2+]i was reached is labeled with timax,. Lastly, the time tihalf, symbolizes the half decay time, i.e., the time necessary for the 360/380 ratio to decay by 50% from the maximum towards its pre-stimulation value. These three times were then used to define the [Ca2+]i signaling characteristics by means of the activation time, 2+ signal. In our analyses, we predominantly investigate the characteristics of Ca2+ signaling with regard to the distance from the point of mechanical stimulation. More specifically, we calculated the average response times, average activation times, the average signal durations, and the average relative amplitude for all cells located in ring (annulus) with a given radius. The difference between the larger and the smaller circle defining the annulus was always 25 µm. In this manner, we calculated the signal characteristics for all cells that were present with respect to stimulation point: (1) closer than 25 µm, (2) between 25 µm and 50 µm away, (3) between 50 µm and 75 µm away, etc., as illustrated in Time traces from individual ROIs were analyzed offline with custom-made scripts in C/C++. The 360/380 nm fluorescence ratio was used to assess the Caeviously . Moreovet-test was applied to test the statistical differences between two subgroups referring to different stages of the cataract if data was normally distributed, whilst a Mann–Whitney rank-sum test was used if data were not normally distributed. The statistical significance between three subgroups referring to different types of cataract was evaluated with a one-way ANOVA test. If data failed the normality test, the Kruskal–Wallis ANOVA test on ranks was used instead. For all analyses, p < 0.05 was considered as statistically significant.A standard 2+ signaling in the human anterior lens epithelium, built from LECs, whereby we compared different signaling characteristics with regard to the degree of cataract progression, where 1 was the lowest and 5 was the highest level of cataract development, and we considered the type of the cataracts as C, N, and C&N. The former encompasses the comparison between mild (CN1–2) and severe (CN3–5) cataracts, irrespective of the type of cataract. The results were based on 17 LC from mild cataracts  and 13 LC from severe cataracts . The experimental protocol consisted of the time before stimulation, the time of mechanical stimulation, and the subsequent recovery time. We systematically analyzed different aspects of intra- and intercellular Ca2+ , very similar results were obtained conceptually and are presented in 2+ responses more precisely, we present the color-coded values of times of interest for the particular LC from a mild  and severe cataracts  in 2+ transients followed a very similar trend in both groups. However, the results in tres, were more pronounced in the LC from mild cataracts, thereby indicating that the signal propagates more rapidly in LCs from more developed cataracts. To be more precise, the Ca2+ waves were on average roughly 30% faster in LCs from severe cataracts. On the other hand, no significant differences were inferred by the activation times, Δtact, of LECs at any distance  are shown for five different LCs with mild cataracts. For each LC, two experiments were recorded: first, the control mechanical stimulation response, and then, after the incubation with Apyrase, the second mechanical stimulation response. There was no significant difference in any of the parameters between Apyrase and the control group, indicating that paracrine ATP does not considerably affect the characteristics of Ca2+ signals in LCs.Finally, we examined if the spreading phenomenon depends on paracrine ATP. In 2+ signaling in human postoperative anterior LC’s LECs with the aim to gain further insight into its relation to cataract formation. More specifically, we studied the Ca2+ signaling characteristics after local mechanical stimulation with special focus on the role of the degree of cataract progression. Our results revealed that a point of mechanical stimulation induced a Ca2+ wave that propagated radially outwards from the stimulation site. By tracking the dynamic changes of the intracellular Ca2+ concentration in all cells in the lens epithelial monolayer, we characterized different Ca2+ signal parameters as a function of distance from stimulation site. It turned out that Ca2+ waves propagate faster in LCs from more developed cataracts than in LCs from less severe ones. Moreover, the relative amplitudes of Ca2+ transients were found to be always decreasing with increasing distance from stimulation point, but the amplitudes were found to be significantly lower in LCs associated with higher degrees of cataract pathology. The durations of Ca2+ transients were found to be longer in LCs from more developed cataracts, which essentially goes on account of a slower Ca2+ decay rate, since a significant difference in the time of Ca2+ increase (tact) between both groups was not detected. However, it should be noted that the relative amplitudes of Ca2+ transients in LCs from severe cataracts were considerably lower, and hence, the absolute slope of decay in Ca2+ was therefore still steeper in LECs associated with less developed cataracts. With regard to the cataract type, we found no significant differences between the two most frequently present types of cataract, i.e., the cortical, C, and nuclear, N, cataracts, in any examined aspect of Ca2+ signaling after mechanical stimulation. Notably, these findings partially parallel our previous investigation about the general Ca2+ signaling characteristics after stimulation with ACh, where no differences between different types of cataracts were detected as well i increase in the cell, be it via Ca2+ influx channels or release from intracellular stores, which are very tightly regulated and are an integral part of Ca2+ signaling under normal circumstances i decrease in the LECs, as there was no significant difference in the activation time, but there was in the duration time. In more developed cataracts there is most probably more intracellular Ca2+ due to its lower extrusion from the cytoplasm, which was also reflected and visible after mechanical stimulation in our study.At a first glance it seems a bit counterintuitive that the mechanically induced intercellular Caormation ,9, and mmstances . With anOur understanding of the dynamics of spatiotemporal cell signaling were enhanced by analysis in experimental systems that recreated in vivo environments, where cells respond to an extracellular environmental cue as a local mechanical stress. Extracellular mechanical events are translated and processed into intracellular biochemical events. If this mechanochemical translation is impaired, resulting deficiencies in cellular mechanosensation can have an effect on pathologies, as the cataract is.2+ wave propagation triggered by local mechanical stimulation can be used for studying gap junctions and hemichannels [2+]i, which, in the form of a wave, extends from the mechanically stimulated cell to adjacent cells. Ca2+ signal propagation is studied in many different tissues, reflecting its importance in their functionality. The first major reports of intercellular Ca2+ waves following mechanical stimulation of a single cell appeared in 1990, and described Ca2+ waves propagating through cultured airway epithelial cells i changes, including Ca2+ oscillations [3-mediated Ca2+ release has also been demonstrated in LECs [2+ entry into the cell and the liberation of cell constituents, such as ATP or other messengers, from the cell [Mechanical stimulation is proposed to result in membrane stress that triggers the production of inositol 1,4,5-trisphosphate , thereby substantiating an important role of intercellular signaling mechanisms between LECs for the normal functioning of the lens. Importantly, significant changes in Ca2+ signalization were obtained when comparing LC from different stages of the cataract. Our findings thus indicate that cataract progression entails the impairment of specific Ca2+ signaling pathways, which opens some new issues that will need to be addressed during future efforts within lens pathophysiology research. Moreover, we also showed that the paracrine ATP-mediated pathway does not affect the spatiotemporal Ca2+ signaling characteristics, indicating the predominant importance of gap junctions in intercellular signaling and communication in human anterior lens epithelium.To conclude, we demonstrated that the localized mechanical stimulation of human postoperative anterior LC epithelial cells induced a wave of increased Ca


Overall, this study identified that, by high-throughput screening, the most KMT2A-selective drugs converged in different but all mitochondrial apoptotic network activation, supporting the use of venetoclax in this AML setting. The novel drug combinations here unveiled provide a rationale for evaluating these combinations in preclinical studies to accelerate the introduction of targeted therapies for the life-threatening KMT2A-AML subgroup of pediatric AML.In pediatric acute myeloid leukemia (AML), fusions involving lysine methyltransferase 2A (KMT2A) are considered hallmarks of aggressive AML, for whom the development of targeted specific therapeutic agents to ameliorate classic chemotherapy and obtain a complete eradication of disease is urgent. In this study, we investigated the antiapoptotic proteins in a cohort of 66 pediatric AML patients, finding that 75% of the KMT2A-r are distributed in Q3 + Q4 quartiles of BCL-2 expression, and KMT2A-r have statistically significant high levels of BCL-2, phospho-BCL-2 S70, and MCL-1, indicating a high anti-apoptotic pathway activation. In an attempt to target it, we tested novel drug combinations of venetoclax, a B-cell lymphoma-2 (BCL-2) inhibitor, in KMT2A-MLLT3, for being the most recurrent, and KMT2A-AFDN, for mediating the worst prognosis, rearranged AML cell lines. Our screening revealed that both the bromodomain and extra-terminal domain (BET) inhibitor, I-BET151, and kinase inhibitor, sunitinib, decreased the BCL-2 family protein expression and significantly synergized with venetoclax, enhancing KMT2A-r AML cell line death. Blasts t  KMT2A-AFDN rearranged, both from cell lines and primary samples, were shown to be significantly highly responsive to the combination of venetoclax and thioridazine, with the synergy being induced by a dramatic increase of mitochondrial depolarization that triggered blast apoptosis. Finally, the efficacy of novel combined drug treatments was confirmed in KMT2A-r AML cell lines or  Leukemia is the most common type of cancer in childhood, accounting for 25–30% of cancers in children and adolescents aged 0–18 years .Despite the refinement of risk stratification on the basis of clinical characteristics, molecular profiling, and detection of minimal residual disease after induction therapy, to date the overall survival for children with acute myeloid leukemia (AML) has not exceeded 70% . CollaboIn the last decades, data that emerged from sequencing and molecular profiling have identified molecular subsets with prognostic significance, with a pediatric AML molecular landscape that remains quite distinct from adults . Among tNevertheless, venetoclax showed limited activity as a single agent in high-risk AML , encouraex vivo primary KMT2A-r AML samples in a three-dimensional (3D) scaffold mimicking the bone marrow niche, which strengthen the drug efficacy prediction. Our results documented that I-BET151, sunitinib, and thioridazine act synergistically with venetoclax, all converging in different but mitochondrial apoptotic network activation, enhancing leukemia cell death.In this study, we pursued the identification of newly targeted opportunities for KMT2A-rearranged AML that still represent a challenge in the oncohematology field by selecting novel drug combinations. To accomplish this goal, we evaluated the proteomic profile of KMT2A-r AML pediatric patients, unveiling the hyper-activation of BCL-2 antiapoptotic pathway. We re-analyzed the data of high-throughput drug screening previously performed on AML cell lines, identifying AML-KMT2A-r selective drugs. Thus, to tackle KMT2A-r AML, we tested combinations of the novel KMT2A-r identified drugs with the BCL-2 inhibitor venetoclax. Synergistic combinations have been validated in KMT2A-r AML cell lines and Reverse-phase protein array (RPPA) analysis was performed as previously described .The cell lines NOMO-1, THP-1, and HL-60  were maintained in RPMI 1640  and SHI-1 (DMSZ) in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific). All the media were supplemented with 10% fetal bovine serum , 2 mM glutamine , and 100 U/ml streptomycin/penicillin .Ex vivo cells were obtained from the bone marrow of pediatric patients affected by de novo AML with KMT2A rearrangements, provided by the pediatric OncoHematology Lab of Padova Hospital. Primary cells were cultured in RPMI Medium 1640 with 10% FBS, 2 mM glutamine, and 100 U/ml streptomycin/penicillin, supplemented with cytokines .6/ml at concentrations selected based on the dose–response curve of each drug. In particular, in synergy evaluation, we tested multiple doses as follows: for venetoclax, we tested three doses, including concentrations between IC40 and IC60; for the KMT2A-r AML cell lines, we used 0.2, 1, and 5 μM; and for HL-60, we used 1, 10, and 100 nM. For KMT2A-specific drugs , we included concentrations ranging from IC25 to IC50 since these drugs would be used in combination for the evaluation of their synergistic abilities. For KMT2A-AFDN-specific drugs, thioridazine and PD98059, we selected the concentration based on our previously published data   .® assay  following the guidelines of the manufacturer. Briefly, at the experimental endpoint, 100 μl of treated cells was transferred into the wells of white, flat-bottomed, opaque 96-well plates , then 100 μl of CellTiter-Glo® reagent was added, and the plates were shaken for 2 min and incubated for 20 min at room temperature. Luminescence was recorded using the Spark® multimode microplate reader , with an integration time of 0.1 s per well. Cell viability was evaluated up to 72 h after treatment in dose–response curves, 48 h after treatment in drug combination experiments on AML cell lines, and 24 h after treatment in drug combination experiments on ex vivo primary AML cells.Cell viability was evaluated using CellTiter-GloHigh-throughput screening (HTS) was performed as previously described . Drugs sTotal RNA was isolated using Trizol (Invitrogen—Thermo Fisher Scientific). Then, 1 μg of RNA was reverse-transcribed into cDNA using the SuperScript II system (Invitrogen—Thermo Fisher Scientific) according to the instructions of the manufacturer. The expression of BCL2 mRNA was measured by real-time PCR (RQ-PCR) on an ABI 7900HD platform  using the Platinum™ SYBR™ Green qPCR SuperMix (Invitrogen—Thermo Fisher Scientific) and normalized on GUS housekeeping gene using the 2^-ΔΔCt method. The primers are as follows: BCL2 F: GGCCGTACAGTTCCACAAA; BCL2 R: AGTACCTGAACCGGCACCT; GUS F: GAAAATATGTGGTTGGAGAGCTCATT; GUS R: CCGAGTGAAGATCCCCTTTTTA.For Western blot analysis, whole cells were lysed in RIPA buffer  and processed for protein expression by Western blot. The following primary antibodies were used: BCL-2 , MCL-1 , and GAPDH . The horseradish peroxidase–conjugated secondary antibody was either anti-rabbit or mouse , and signal was quantified using ImageJ software.Mitochondrial membrane potential was measured by using TMRE Assay Kit , following the instructions of the manufacturer. Briefly, TMRE (200 nM) diluted in BSA (0.2%)-PBS 1X was added for 20 min at 37°C. The cells were analyzed by flow cytometry using FC500 .3ex vivo AML cells were seeded into 500 μl of MethoCult™  in 24-well plates and incubated at 37°C. After 2–4 weeks of culture, for colony counting, an adequate volume of a 1:6 solution of 3--2,5-diphenyltetrazolium bromide (Sigma-Merck Millipore) in Hanks’ balanced salt solution was added to the semisolid medium, and images were acquired by an optical microscope with camera.At 24 h after treatment, 2 × 10The AML-MSC isolation and culture, 3D scaffold specifications, and 3D culturing setup were previously described . Briefly® 3D reagent (Promega) according to the instructions of the manufacturer at 48 h after treatment. Briefly, the scaffolds were individually transferred into wells of white, flat-bottomed, opaque 96-well plates (Corning Life Sciences) with 100 μl of RPMI; then, 100 μl of CellTiter-Glo® 3D reagent was added into each well. The plates were shaken for 5 min to induce scaffold and cell lysis. The samples were then incubated for an additional 20 min in the dark, at room temperature, to stabilize the bioluminescent signal, which was then recorded using Spark® multimode microplate reader (TECAN), with an integration time of 0.1 s per well. Cell viability was compared to the control sample [scaffold with the same cells but treated with dimethyl sulfoxide (DMSO)].Cell viability was measured using CellTiter-Gloet al.   .t-test was adopted for significance between differences in means when two groups were evaluated, after a preliminary testing of normal distribution of data. ANOVA test was performed when comparing more than two groups, applying Bonferroni correction for multiple statistical hypotheses testing. Graphs and associated statistical analyses were generated using GraphPad Prism 8 . All data are presented as mean ± standard error of the mean (SEM), with *p-value <0.05, **p-value <0.01, ***p-value <0.001, and ****p-value <0.0001 considered statistically significant.The n = 12, 75% vs. Q1 + Q2, n = 4, 25%; p < 0.01, ***p < 0.001). Of note is the fact that we found a significant correlation between BCL-2 and BCL-2S70  and between BCL-2 and MCL-1 , describing that there was an active anti-apoptotic pathway in KMT2A-AML .We analyzed the BCL-2, BCL-2 S70, and MCL-1 protein levels in a cohort of 66 pediatric AML by RPPA analysis , and we MT2A-AML . ConversWe tested the BCL-2 inhibitor venetoclax in a panel of three KMT2A-r AML cell lines, namely, t  SHI-1 and t  NOMO-1 and THP1 and in a non-KMT2A cell line, HL60. We found that the SHI-1 and THP1 cell lines were resistant (IC50 >5 µM) to the treatment compared to the HL60 that was sensitive (IC50 <0.0016 µM), with NOMO1 cells responding at intermediate levels . Thus, we explored BCL-2 RNA and protein expression after I-BET 151 treatment, as BCL-2 is a direct KMT2A target whose transcription is dependent on BET family protein placement on chromatin  in both RNA and protein expression, sensitizing KMT2A-AML to venetoclax treatment.To assess the pharmacological interactions between venetoclax and KMT2A-specific drugs, incremental doses were applied based on the IC50 value of each drug  on SHI-1hromatin , and we p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). In this case, a more in-depth molecular analysis of the mechanisms underlying the cellular response showed that sunitinib decreased the MCL-1 expression more than the BCL-2 , supporting that KMT2A-AML cells were susceptible to venetoclax treatment due to the effects induced in MCL-1.We tested the venetoclax and sunitinib combination with the same approach, finding a strong synergism in KMT2A-AML , excluding this combination of drugs from further analysis.Consistently, the combination treatment resulted to be synergistic only in NOMO-1 . We previously demonstrated that apoptosis was driven by Ca2+ influx-induced mitochondrial depolarization that occurred after thioridazine treatment. Therefore, we looked at the mitochondrial potential status, finding that the combined treatment led to a more rapid mitochondrial depolarization, triggering greater cytotoxicity . A dose–combination curve confirm that the t -AML samples were more sensitive to thioridazine treatment compared with the non-t  ones . Notably, we revealed that the combination treatment of venetoclax + thioridazine completely abrogated the cell clonogenic capacity of t -rearranged AML cells, supporting this strategy to be useful for a complete blast clearance -r AML is known to be a peculiar KMT2A-r subgroup , we prev11) ones , and thelearance .On the contrary, although the combination of PD98059 + venetoclax was more effective in the t  cell line, it did not result to be synergistic , recapitulating the crucial physiological aspects of leukemia niche , confirming the strong synergy of these drug combinations. Finally, we performed treatments in the 3D system seeded with ex vivo primary AML samples and demonstrated a greater effect in reducing leukemia cell proliferation when drugs are used in combination on t  and t  KMT2A-r AML  culture model that we recently developed; it consists of a 3D biomimetic scaffold made of hydroxyapatite/collagen (70/30%)  that allia niche . This 3Des in 3D . Of notees in 3D . Blasts In the field of pediatric AML, the need to improve survival while decreasing relapse occurrence and treatment-related toxicity dictates the great effort that is continually being spent to find novel therapeutic approaches. To reach this goal, it is necessary to consider the high intra- and inter-AML complexity both at the genetic and clinical points of view.BCL2 gene, lowering the BCL-2 protein and significantly sensitizing the cells to venetoclax treatment. Differently, sunitinib is a small molecule with selectivity for PDGFR, VEGFR1, VEGFR2, KIT, and FLT3  KMT2A-AFDN-rearranged cases that allowed the identification of thioridazine as the most selective compound inducing the cytoskeletal remodeling of blast cells that led to Ca nucleus . Given t nucleus . For thi nucleus . TherefoIn summary, the combination of venetoclax with I-BET151, sunitinib, or thioridazine dramatically decreases cell viability in KMT2A-r AML. This anti-leukemia efficacy is associated with the simultaneous inhibition of BCL-2 by venetoclax and the downregulation of anti-apoptotic proteins or disrupting mitochondrial homeostasis, supporting that enhancing the mitochondrial pathway targeting could be a good strategy to sensitize resistant AML to venetoclax. Our 3D system co-cultured with primary MSCs and KMT2A-r blasts confirmed that our combinations are effective and predictable, allowing further rational prioritization of these compounds to be included in preclinical trials.Overall, this study provides a rationale for evaluating therapy cocktails in 3D models and preclinical studies to accelerate the introduction of new compounds to treat the life-threatening KMT2A-AML subgroup of pediatric AML.


The cER is dynamically regulated by STIM and tethering proteins during SOCE, but the ultrastructural rearrangement and functional consequences of cER remodeling are unknown. Here, we express natural (E-Syt1 and E-Syt2) and artificial (MAPPER-S and MAPPER-L) protein tethers in HEK-293T cells and correlate the changes in cER length and gap distance, as measured by electron microscopy, with ionic fluxes. We found that native cER cisternae extended during store depletion and remained elongated at a constant ER-plasma membrane (PM) gap distance during subsequent Ca2+ elevations. Tethering proteins enhanced store-dependent cER expansion, anchoring the enlarged cER at tether-specific gap distances of 12-15 nm (E-Syts) and 5-9 nm (MAPPERs). Cells with artificially extended cER had reduced SOCE and reduced agonist-induced Ca2+ release. SOCE remained modulated by calmodulin and exhibited enhanced Ca2+-dependent inhibition. We propose that cER expansion mediated by ER-PM tethering at a close distance negatively regulates SOCE by confining STIM-ORAI complexes to the periphery of enlarged cER sheets, a process that might participate in the termination of store-operated Ca2+ entry.Recruitment of STIM proteins to cortical endoplasmic reticulum (cER) domains forming membrane contact sites (MCSs) mediate the store-operated Ca Summary: ER-PM tethering at close distance limits Ca2+ entry by confining STIM-ORAI complexes to the periphery of contact sites. The ensuing Ca2+ depletion of the ER is sensed by the ER membrane-resident protein stromal interaction molecule 1 (STIM1)  pumps (2+ entry is prevented by fast (in the order of milliseconds) and slow (in the order of seconds) Ca2+-dependent inactivation (CDI) mechanisms mediated in part by calmodulin and SARAF, which interact with STIM-ORAI complexes to regulate channel activity (Store-operated Caferation . Upon ag (STIM1)  via its  (STIM1) . This in (STIM1) . The swi (STIM1) . Exposurchannels . The floA) pumps , therebyactivity .2+ depletion, a remodeling that is recapitulated by STIM1 expression in cells with replete stores (ASPH) stabilizes the intracellular MCSs generated by STIM1, thereby contributing to prophagocytic Ca2+ signals by recruiting inositol-trisphosphate receptors (InsP3R) to phagosomes  to replenish PM phosphatidylinositol 4,5-bisphosphate (PIP2) following receptor-induced hydrolysis  between the ER and plasma membrane (PM)  that enae stores . Expresse stores . Other tagosomes . The extevations , and itsevations . The two2+ entry . The mai2+ entry , whereasdrolysis . A subsenishment .2+ signaling and lipid trafficking motivated several groups to develop artificial ER-PM tether proteins to label and manipulate MCSs. Using the transmembrane domain of STIM for ER retention and the polybasic motif of the small G protein Rit for constitutive binding to PM phosphoinositides, the group of Jen Liou developed a genetically encoded marker for ER-PM junctions termed ‘MAPPER’ for ‘membrane-attached peripheral ER’ . Quantification of the cER morphometric parameters revealed that very large cER structures were generated by the expression of E-Syt2 or MAPPER-L during ER Ca2+ store depletion, we analyzed by EM the morphology of individual cER structures in cells fixed and embedded in Epon resin in situ. This procedure optimally preserves cellular architecture and maintains the orientation of cells relative to the substrate. Cells were divided into three groups: (1) non-treated cells (NT), (2) cells exposed to 1 µM thapsigargin (Tg) for 10 min in the absence of Ca2+ to deplete ER Ca2+ stores  and favor the formation of STIM1-mediated ER-PM MCSs, and (3) cells treated with Tg as in (2) and re-exposed to 1 mM Ca2+ for 2 min to enable Ca2+ influx  and evaluate the impact of Ca2+ elevations on MCS morphology (Table S1). In cells treated with Tg, a massive elongation of the cER was observed, the average length increasing by 2.3-fold, from 66 to 156 nm, while the ER-PM gap distance remained unchanged (Tables S1 and S2). We then calculated the volume of the cytosolic cleft contained between the cER and PM membranes, assuming that cER sheets form cylindric structures close to the PM . The cleft volume increased by 4.8-fold upon Tg treatment (Tables S1 and S2). Interestingly, the gap distance, cER length, and cleft volume were not impacted by the readmission of Ca2+ to cells treated with Tg . These data indicate that all four tether proteins accumulate in the TIRF plane into structures of similar apparent size when observed by optical microscopy.To establish how ER-PM tethering impacts the cER structure and dynamics we transiently transfected HEK-293 T cells with a plasmid expressing GFP-tagged versions of natural (E-Syt1 or E-Syt2) or artificial (MAPPER-S or MAPPER-L) tether proteins. We first verified by total internal reflection fluorescence (TIRF) microscopy that these tether proteins are recruited to regions of the ER close to the PM in cells exposed to Tg. GFP–E-Syts and GFP–MAPPERs were detected in the TIRF plane as punctate fluorescent structures of 0.3 µmFig. S3). Cells with low to moderate overexpression were selected to avoid the formation of artificial cortical ER structures in the absence of store depletion. In resting cells, the average gap distance and cER length were not impacted by the expression of any of the tether proteins (Tables S1 and S2), but MAPPER-L and E-Syt2 induced the formation of cER cisternae longer than 300 nm . Interestingly, as the length of individual cER structures increased, the gap distance decreased to values specific for each family of tether protein, of 12-15 nm for the E-Syts and 5-9 nm for the MAPPERs . After readmission of Ca2+ to enable Ca2+ influx, the cER length decreased significantly in cells expressing all tethers except MAPPER-S, while the gap distance returned to pre-stimulatory values in cells expressing E-Syt1 . In cells expressing the other tethers, the gap distance remained unchanged and cER cisternae longer than 300 nm were still clustered at gap distances specific for each tether . These observations indicate that ER-PM tethering enhances cER expansion during store depletion by anchoring elongated cER cisternae at fixed gap distances, and that this elongation process is partly reversed during the Ca2+ entry phase. The expanded cER sheets are anchored at 12-15 nm from the PM by E-Syts and at 5-9 nm by MAPPERs and these gap distances are minimally affected by changes in cytosolic Ca2+ concentration.We then assessed the impact of the expression of E-Syts or MAPPERs on MCS ultrastructure. Transfected cells were sorted by flow cytometry to ensure that equal amounts of tether proteins were expressed . The SOCE responses, however, were significantly reduced by ER tethering and the Ca2+ entry rates measured following readmission of 1 mM Ca2+ were decreased by 60% and 40% in cells expressing E-Syt2 and MAPPER-L, respectively . Since these two protein tethers induce a maximal cER expansion that is Ca2+ dependent, we tested whether the SOCE defect was also Ca2+ dependent by varying the concentration of Ca2+ re-added following Tg exposure. The SOCE defect associated with MAPPER-L expression persisted when the Ca2+ concentration was decreased to 0.5 mM or increased to 2 mM Ca2+, and lost when Ca2+ concentration was increased to 10 mM . This indicates that the SOCE defect associated with ER tethering is not restricted to a specific cell type or to proteins tethering the ER at a specific gap distance, but is likely related to the cER expansion. To better assess the contribution of local Ca2+ fluxes in the functional effects of the MAPPERs, we then used the reversible SERCA inhibitor cyclopiazonic acid (CPA) to allow the refilling of depleted ER Ca2+ stores during Ca2+ readmission. SOCE was equally reduced by the two MAPPERs when CPA was used to transiently deplete ER Ca2+ stores in HEK-293 T cells   calmodulin (CaM) constructs together with the MAPPER tethers. Expression of DN-CaM increased SOCE  . The incsed SOCE A,B, confsed SOCE . Regardlsed SOCE B, indica2+, which permeates SOCE channels and quenches the fluorescence of Fura-2, measured at the isosbestic wavelength of 360 nm. The basal rates of Mn2+ quenching were slightly increased by MAPPER-L expression when Mn2+ was added in Ca2+-containing medium . In non-transfected cells, Tg increased Mn2+ quench rates by 2.4-fold in nominal Ca2+-free medium, consistent with SOCE activation . Following readmission of 1 mM Ca2+ the quench rates remained elevated in control cells but were close to basal levels in cells expressing the MAPPERs  cells]. Fura-2 quenching by Mn2+ was abrogated in TKO cells and restored by ORAI1 re-expression , indicating that Mn2+ fluxes are mediated by the ORAI1 channel. Importantly, MAPPER-L expression reduced Mn2+ influx to the same extent in wild-type cells and in TKO cells expressing mCherry (mCh)-ORAI1 . These data indicate that Mn2+ enters HEK-293 T cells predominantly via ORAI1 channels, whose activity is decreased by enforced ER tethering.To verify that cER tethering altered the permeability of SOCE channels, we added the divalent cation Mng medium C,D, but  MAPPERs C,D. Corrxpansion D. These Fig. S8A) without altering the localization and morphometric features of mCh-ORAI1 clusters . MAPPER-L thus accumulates in cER domains adjacent to STIM-ORAI interaction sites. To test whether this localization depends on the length of the tether, we expressed the SNARE protein Sec22b and its extended version Sec22b-P33 to anchor the ER at 20 and 30 nm from the PM, respectively  and their overexpression did not impact SOCE . Expression of a long linker that colocalizes with STIM1 thus fails to inhibit SOCE, while the shorter MAPPERs that inhibit SOCE form clusters juxtaposed to STIM-ORAI complexes.We next determined the localization of the ER tethers relative to STIM-ORAI complexes by TIRF imaging. CFP-STIM1 and mCh-ORAI1 colocalized extensively while GFP–MAPPER-L formed adjacent clusters  cER cisternae, which ranged from 5 to 30 nm regardless of tether expression. Another unexpected finding was that the cER elongation associated with tether expression decreased during the Ca2+ influx phase whereas the ER-PM gap distances remained constant. E-Syt1 expression in HEK-293 T cells was previously reported to reduce the ER-PM gap distance by 12 nm upon Ca2+ entry, as estimated from changes in fluorescence intensity in variable-angle TIRF imaging , Mg-ATP (Sigma), MnCl2 , Fura-2-AM , Deep Red CellMask  and Lipofectamine 2000 . Ca2+ recording buffers contained: 140 mM NaCl, 5 mM KCl, 1 nM MgCl2, 20 mM HEPES, 10 mM glucose, supplemented with 1 mM EGTA or 0.5-10 mM CaCl2 as indicated.The following reagents were used in this article; thapsigargin , cyclopiazonic acid from ER (#36325), EGFP-E-Syt1 (#66830), EGFP-E-Syt2 (#66831) and pCMV-R-CEPIA1er (#58216) were from Addgene. MAPPER constructs were a kind gift from Jen Liou, UT Southwestern, Texas; CaM constructs ; pcDNA-D12 and regularly tested for mycoplasma. MEF cells  were grown in identical conditions. HeLa cells purchased from the European Collection of Authenticated Cell Cultures (ECACC) were grown in MEM Gibco (41090). Cell lines used in this study tested negative for mycoplasma contamination, which was performed on a monthly basis. HEK 293 T and HeLa cells were genetically confirmed (by genomic profiling [STRs]) prior to expansion and stockage. Prior to experiments, cells were seeded on coverslips of 25 mm coated with poly-L-lysine  and transfected 1-day post-seeding using 200 ng of plasmids for HEK-293 T, 0.5 µg for MEF and 1 µg for HeLa cells mixed with 3 µl Lipofectamine 2000. The DNA-Lipofectamine mix was added to cells for 4 h and the culture medium was replaced.Human embryonic kidney (HEK-293 T) cells were obtained from ATCC  maintained in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, grown at 37°C and 5% CO2+ (CF) or treated with Tg as above and re-exposed to 1 mM Ca2+ for 2 min (CA), fixed with 2.5% glutaraldehyde, stained with uranyl acetate, postfixed with osmium tetroxide, and embedded in Epon resin. After sectioning at 30 nm, samples were imaged on a Technai 20 transmission electron microscope  at 92,000 times magnification. For each cell identified on the grid every ER structure located within 30 nm of the PM was imaged. The cER length and the ER-PM gap distance were determined on EM images using ImageJ/Fiji and MATLAB, respectively. A segmented line was drawn in ImageJ/Fiji across the cER long axis to extract the cER length. The ER-PM gap distances was obtained from two lines drawn on the PM and proximal cER membranes whose pixel-to-pixel geodesic distances were extracted and averaged for each cER. Only structures located at less than 30 nm from the PM were analyzed.Cells were sorted by flow cytometry  for similar GFP fluorescence levels 24 h post transfection and re-seeded for 24 h on plastic dishes coated with poly-L-lysine. Cells were left untreated (NT), exposed to 1 µM (Tg) for 10 min in the absence of Ca2+ imaging was performed as described previously  using a Python script to better control the time resolution during the protocol (https://github.com/Carandoom/VisiviewFura2). Ca2+ images were analyzed using an ImageJ script . Using regions drawn on the background and on each cell of interest, the script removes the mean background fluorescence and extracts the mean fluorescence of each cell region for each frame to calculate the fluorescence ratio and graph it as R/R0 in Excel. The rates of Ca2+ entry were calculated with a MATLAB script . Briefly, the script plots the recording, allowing the user to analyze a positive or negative slope within a given range of timepoints. The script calculates the first derivative to define a region to fit a linear regression and extracts a slope parameter.Caeviously . Brieflyhttps://github.com/Carandoom/STIM-ORAI-Segmentation; https://zenodo.org/badge/latestdoi/373828941).TIRF imaging was performed on a Nikon Eclipse Ti microscope equipped with a Perfect Focus System (PFS III) and a 100× oil CFI Apochromat TIRF Objective . For 488 nm excitation, the filter cube contained a ZET488/10 excitation filter (Chroma), a 502 nm dichroic mirror (H 488 LPXR superflat) and a 530/43 Bright Line HC emission filter . For CellMask imaging, we used a 640 nm laser and Cy5 700/75 emission filter. The fluorescence was collected by an EMCCD camera cooled at −80°C  piloted with NIS-Elements Ar software V4.13 (Nikon). The PM was labeled with deep red CellMask  and used to adjust the TIRF angle. Fluorescence puncta on TIRF images were segmented using a custom ImageJ/Fiji script available on GitHub  within a minimum of three biologically independent experiments, considered as an independent transfection recorded on a different day. Samples allocation/randomisation were not pre-defined for this article.Statistical analyses were performed with GraphPad Prism 9,


In this study, recognizing the importance of 1,25VD3 as one of only 2 molecules  photosynthesized for energy during the beginning stage of life on this planet, we explored the functional role of 1,25VD3 in AML metabolism.Acute myeloid leukemia (AML) has the lowest survival rate among the leukemias. Targeting intracellular metabolism and energy production in leukemic cells can be a promising therapeutic strategy for AML. Recently, we presented the successful use of vitamin D  gene therapy to treat AML mouse models in vivoTranscriptome database (RNA-seq) of four different AML cell lines revealed 17,757 genes responding to 1,25VD3-treatment. Moreover, we discovered that fructose-bisphosphatase 1 (FBP1) noticeably stands out as the only gene  with a 250-fold increase in gene expression, which is known to encode the key rate-limiting gluconeogenic enzyme fructose-1,6-bisphosphatase. The significant increased expression of FBP1 gene and proteins induced by 1,25VD3 was confirmed by qPCR, western blot, flow cytometry, immunocytochemistry and functional lactate assay. Additionally, 1,25VD3 was found to regulate different AML metabolic processes including gluconeogenesis, glycolysis, TCA, de novo nucleotide synthesis, etc. In summary, we provided the first evidence that 1,25 VD3-induced FBP1 overexpression might be a novel therapeutic target to block the “Warburg Effect” to reduce energy production in AML blasts.The online version contains supplementary material available at 10.1186/s40364-022-00367-3. To the Editor,Acute myeloid leukemia (AML) is the most common type of leukemia in adults . DespiteVitamin D is known to be the oldest hormone on earth . Some ofFirst, we performed transcriptome analyses (RNA-seq) of 4 different AML-FLT3 cell lines including MV4–11, MOLM-14, MV4–11-midostaurin-resistant cells and MOLM-14-midostaurin-resistant cells which were previously reported . Among mIn addition to the central pathway of metabolizing glucose to pyruvate via glycolysis, AML metabolism involves diverse processes of nucleotides, amino acid, lipids and their end metabolites to perform signaling functions and produce energy to support tumorigenesis . Here, wAdditional file 1.


In particular, toxic β-amyloids (Aβ) perturb the activities of numerous Ca2+ transporters or channels. Because of the tight coupling between Ca2+ dynamics and the membrane electrical activity, such perturbations are also expected to affect neuronal excitability. We used mathematical modeling to systematically investigate the effects of changing the activities of the various targets of Aβ peptides reported in the literature on calcium dynamics and neuronal excitability. We found that the evolution of Ca2+ concentration just below the plasma membrane is regulated by the exchanges with the extracellular medium, and is practically independent from the Ca2+ exchanges with the endoplasmic reticulum. Thus, disruptions of Ca2+ homeostasis interfering with signaling do not affect the electrical properties of the neurons at the single cell level. In contrast, the model predicts that by affecting the activities of L-type Ca2+ channels or Ca2+-activated K+ channels, Aβ peptides promote neuronal hyperexcitability. On the contrary, they induce hypo-excitability when acting on the plasma membrane Ca2+ ATPases. Finally, the presence of pores of amyloids in the plasma membrane can induce hypo- or hyperexcitability, depending on the conditions. These modeling conclusions should help with analyzing experimental observations in which Aβ peptides interfere at several levels with Ca2+ signaling and neuronal activity.Alzheimer’s disease is characterized by a marked dysregulation of intracellular Ca In neur2+ as an ion involved in signaling and in neuronal activity is highly relevant for the understanding of the development of neurodegenerative diseases and, in particular, Alzheimer’s disease (AD). Together with the hyperphosphorylation of τ proteins and the accumulation of amyloid-β peptides (Aβ) inside neurons, the progression of AD is indeed characterized by a perturbation of intracellular Ca2+ homeostasis [2+ dysregulation associated with neurodegenerative diseases is multifactorial. Aβ can indeed affect Ca2+ exchanges with the extracellular medium and with internal Ca2+ stores [2+. It is now widely accepted that disruption of ER calcium mediates the most significant signaling pathways that are associated with Alzheimer’s disease, and is a main driving force in the development of the disease [2+ increase can lead to apoptotic neuronal death. Ca2+ also stimulates the activity of γ-secretase, a key enzyme involved in the production of toxic Aβ peptides. Such a vicious circle between amyloids and Ca2+ has been proposed to play a key role in the progressive and irreversible character of the disease [This dual role of Caeostasis . Ca2+ dy+ stores . These m disease ,6,7. Amo disease ,8.2+, and neuronal excitability are related to each other at the single cell level is not well understood and is difficult to assess experimentally. Intuitively, increases in Ca2+ are expected to be associated with a decrease in neuronal activity via the activation by Ca2+ of a hyperpolarizing K+ current mediated by SK or BK channels, which represent the major link between Ca2+ concentration and the electrical activity of the plasma membrane. The question thus arises as to how AD’s progression can be associated at the same time with the increase in intracellular Ca2+ that follows the calcium hypothesis of AD, and with an increase in neuronal electrical activity. Computational modeling is well suited to gain insight into this paradox, because it allows for individually assessing the consequence of Aβ peptides on their various targets. It can also predict their relative importance.At the level of neuronal activity, extracellular plaques of Aβ peptides are reported to induce electrical hyperexcitability . Such an2+ signaling have been described in specific cell types, such as astrocytes [2+ dynamics in a prototypical neuronal cell. An early generic model of a neuron shows that less current is necessary to generate an AP when Aβ peptides are considered to block the fast-activating potassium current [2+ dynamics, to study the impact of tonic NMDA receptor activity on neuronal excitability [2+ influx induced by this tonic activity can lock previously active neurons in a hyperactive state. This result agrees with the observed hyperactivation of neurons in presence of Aβ-induced glutamate spill-over [From a mathematical point of view, interactions between membrane electrical activity and Catrocytes ,14, panctrocytes ,16, GnRHtrocytes  motoneurtrocytes , and cartrocytes ,20. To t current . In a prtability . Modelinill-over ,24.2+ dynamics to consider a more accurate description of the evolution of intracellular Ca2+, including exchanges with the ER. The model involves three compartments: a fictious, sub-plasmalemmal shell that controls PM electrical activity, the cytosol, and the ER. It considers Ca2+ fluxes through the plasma membrane Ca2+ ATPase (PMCA), the sodium calcium exchanger (NCX), the sarco- or endoplasmic reticulum Ca2+ ATPase (SERCA) pumps, and the inositol 1,4,5-trisphosphate receptor (IP3) and ryanodine receptors (IP3R and RYR), grouped together in a single phenomenological flux. In this first approach, we indeed do not incorporate Ca2+ fluxes resulting from stimulation by IP3-inducing agonists. Our aim is to investigate how long-term alterations in Ca2+ fluxes modify the resting concentrations of Ca2+ in the different compartments and neuronal excitability. The model is then used to systematically investigate the various effects of Aβ peptides that have been reported in the literature. We found a strong independence between changes in cytosolic and subplasmalemmal Ca2+ concentrations, allowing cytosolic Ca2+ to vary without affecting neuronal excitability. In contrast, the latter is much affected by changes in subplasmalemmal Ca2+, with decreases in PMCA activity provoking neuronal hypo-excitability. Hyperexcitability is however predicted by the model when simulating the Aβ peptides reported changes in the L-type Ca2+ current [2+-sensitive K+ channels [2+ flux through the pores of Aβ oligomers [2+ and provoking neuronal hyperexcitability. It also provides a simple description of intracellular Ca2+ dynamics and membrane excitability that can be further adapted to describe Ca2+ dysregulation in neurodegenerative diseases in a finely tuned manner.In the present study, we extend the same minimal model of granule cell electrical activity and membrane Ca current  and the channels . Lastly,ligomers ,28 can aThe core model used in this study is a previously proposed simple mathematical description of the electrical activity of cerebellar granule cells ,29. This2+ concentration in a sub-plasmalemmal compartment that can be seen as a thin shell just below the plasma membrane (Ca). The currents included in the model, schematized in black in + current (NaIV), a delayed rectifier K+ current (KIV), a high-threshold voltage dependent Ca2+ current (CaIV), a Ca2+-activated K+ current (KICa), and injI is the current injected into the cell that allows for studying its excitability properties. The mathematical expressions used for the currents, together with the evolution equations for the gating variables, are given in the K(Ca)I current is considered explicitly. In reality, most excitable cells express both large-conductance (BK) and small-conductance (SK) calcium-activated potassium channels. The former is sensitive to Ca2+ only, while the latter is sensitive to both voltage and calcium. For simplicity, Equations (S7), (S18) and (S19) empirically describe the two types of channels. The equations and parameter values that govern the dynamics of opening/closing of the ion channels are the same as in the previous studies [mC is the cell capacitance. When active, the CaIV). The rate of Ca2+ transport through the ATP-consuming plasma membrane Ca2+ ATPase (PMCA) is given by:PMCAV and PMCAK stand for the maximal velocity and the half saturation constant of the PMCA, respectively. Note that export of Ca2+ by the PMCA is accompanied by a concomitant influx of H+ [+/Ca2+ exchanger removes Ca2+ from the cytoplasm at the expense of Na+ entry, with a 1:3 stoichiometry. It is thus electrogenic. Following Gall et al. (1999) [NCXg is the NCX conductance, NCXK the half saturation constant for NCX activation by Ca2+, and NCXV the resting potential for this channel calculated using resting Na+ and Ca2+ concentrations. This current is added (with a negative sign) to the right-hand side of Equation (1) and with the appropriate factors to the evolution equation for Ca (see below).Here, we extend the model to consider a cytosolic and an endoplasmic reticulum (ER) Caux of H+ , in such. (1999) , we desc2+ diffusion from the sub-membrane region to the cytosol, we constructed a compartmental model wherein the cytoplasm and the shell are assumed to be homogeneous compartments. In such an approach, the corresponding efflux of Ca2+ from the cytosol considers the Ca2+ diffusion coefficient and the respective volumes of the compartments. It can be written as:D stands for the effective Ca2+ diffusion in the cytosol and h for the distance between the shell and the cytoplasmic compartment. Thus, for a typical granule cell with a radius of ~4 μm, γ is around 0.001 ms−1 considering that D = 15 μm2s−1 [D decreases together with f.To introduce Ca5 μm2s−1 . It shou2+ concentration in the cytosol (cytCa) is given by Assuming that the volume of the shell is 1/10 of the volume of the cytosol, the corresponding change of Ca2+ with the ER, in which the Ca2+ concentration is much larger, around 500 μM. Ca2+ exchanges between these two compartments have been much studied, both experimentally and by modelling, because they can produce cytosolic Ca2+ oscillations [2+ concentration are much smaller in the ER than in the cytoplasm. In the present study where only basal, non-stimulated Ca2+ release from the ER is simulated, we consider that the ER Ca2+ concentration remains constant [ER at 500 μM [2+ efflux from the ER is given byink gathers basal ER Ca2+ release through the IP3R and the RyR, as well as unspecific leaks. The opposite flux, i.e., Ca2+ pumping from the cytosol to the ER, is mediated by the sarco- or endoplasmic reticulum Ca2+ ATPase (SERCA) and is modelled asSERCAV and SERCAK stand for the maximal velocity and the half saturation constant of SERCA, respectively.The cytosolic compartment also exchanges Callations ,33. It iconstant ,35 and ft 500 μM . Basal CWhen considering these additional processes, the core model originally described by Equations (1) and (2) takes the form:https://github.com/genedupont/amyloids, accessed on 26 November 2021).Together with the equations for the currents (Equations (S1)–(S4)) and for the fluxes (Equations (3)–(7)), the evolution Equations (8)–(10) define the model used throughout the whole study. The values of parameters are given in injI, the model displays repetitive action potentials ), which reflect the slow diffusion of Ca2+ in the cytoplasm because of buffering. Because sub-plasmalemmal Ca2+ is mainly removed by the PMCA, when the cell is electrically active the Ca2+ concentration in the cytoplasm remains practically constant at 40 nM. This value of cytCa is determined by the exchanges with the ER.We first checked if the extended model described in tentials A, which ment Ca, B. These the PMCA C, with at, −5 pA D, which rvations . The thi2+ diffusion potentially affects the firing threshold of the injected current  current. Membrane polarization is therefore attenuated, which decreases the value of the depolarizing current that must be injected to initiate an action potential.The bifurcation diagram E of the  current F. When d2+ dynamics. In the following, we use this model to investigate how changes in Ca2+ dynamics influence neuronal excitability. In particular, we focus on how known targets of Aβ peptides modify (1) the threshold value of injected current triggering repetitive action potentials, i.e., the value of injI at the bifurcation point, and (2) the frequency of repetitive APs. A decrease of the threshold value of injI or an increase in frequency from the control situation  indeed provokes an increase in the amplitude of the current that must be injected to initiate neuronal firing (KICa)( with decreasing PMCA activity is responsible for the observed change in injI at the bifurcation point. KICa) also affects the domain of repetitive firing /10, is influenced in both cases. As far as neuronal activity is concerned, excitability decreases together with the activity of the PMCA’s, while it is not influenced by the SERCAs.The strong influence of PMCA on electrical activity contrasts with the lack of effect of changing the kinetic parameters of the SERCA visible in 2+ is released from the ER via ryanodine and IP3 receptors and there is compelling evidence that Aβ peptides mobilize internal Ca2+ via these two receptors [3 [2+ responses, but also provoke a sustained increase in the basal rate of ER Ca2+ release that can be modeled by an increase in the value of parameter ink that appears in Equation (6). Bifurcation analysis of the influence of this parameter ), while PMCAs are electroneutral [AbetaV  channels. However, the threshold value of injected current is not affected by the pores.Thus, ions permeable pore of Aβ peptides provoke an increase in sub-plasmalemmal Ca2+ channel has attracted much attention in the field of Alzheimer’s disease. Over-activation of these channels by Aβ peptides has been observed in rat cultured cortical and hippocampal neurons [2+ channels in rat hippocampal neurons [2+ current, noted αs and βs in Equations (S18) and (S19). When consideringThe L-type Ca neurons . Injecti neurons , a pheno neurons . In part neurons . Such a injI, the frequency of action potentials is larger than in the control situation. As can be seen in 2+ in the sub-plasmalemmal compartment .Simulations indicate that repetitive APs occur in the absence of injected current with these modified values of parameters for the L-type channel B. Moreovpartment D. As for2+ channel results into marked neuronal hyperexcitability. This is reflected by an increase in both the firing frequency and the propensity of the cell to become electrically active. It also significantly raises the concentration of the sub-plasmalemmal Ca2+ during activity.Thus, Aβ peptides induced up-regulation of the L-type Ca2+-sensitive potassium channels has also been shown to be modified by the presence of Aβ peptides [The activity of the Capeptides . In mouspeptides . The sampeptides . More inpeptides  observedK(Ca)g (K(Ca)g  channels provokes a marked decrease in the threshold value of current that must be injected to induce repetitive firing A. As visgK(Ca) B. Simula (gK(Ca) B, as obsThus, a reduction in the conductance of K(Ca) points to electrical hyperexcitability at all levels: reduction of the threshold current intensity required to induce repetitive AP’s, and an increase in the frequency and broadening of the AP’s.2+ microdomains, for example [Mathematical modeling provides a unique tool to derive insight into the possible molecular mechanisms that may be relevant in AD. We therefore used a modeling approach to systematically investigate the various effects of Aβ peptides on calcium dynamics and neuronal excitability. We extended a previously proposed mathematical description of the electrical activity of cerebellar granule cells  to propo example .2+ concentration just below the PM and in the bulk of the cytosol. Indeed, Ca2+ changes in the sub-plasmalemmal compartment are governed by Ca2+ exchanges with the extracellular medium that occur with a much larger rate than passive diffusion from and to the bulk of the cytosol. On the other hand, these changes have a moderate influence on the Ca2+ concentration in the much larger cytosolic space. In indirect agreement with this independence between sub-plasmalemmal and bulk cytosolic Ca2+, Yao et al. (2020) [2+ on the activity of a Ca2+-dependent voltage gated channel located in the PM. In the context of AD, the decoupling between membrane and bulk cytosolic Ca2+ observed in the simulations suggests that disruptions of Ca2+ homeostasis that interfere with signaling and promote the progression of the disease are unrelated from changes in the electrical properties of the neurons. It should be kept in mind, however, that this conclusion only holds at the single cell level. At the network level, Ca2+ could affect electrical activity because AD-related synaptic loss may be partly due to Ca2+ dysregulations [Our simulations point to a clear decoupling between the evolution of Ca. (2020)  reportedulations .2+, our results predict that the reported modifications induced by Aβ peptides on the activities of transporters affecting deep cytosolic Ca2+ have no impact on neuronal excitability in the absence of the ER-Ca2+ mobilizing agonist. The fact that we considered conditions corresponding to the absence of stimulation of IP3 or RyR receptors is in contrast with the approaches followed by Latulippe et al. [2+ dynamics. They concluded that amyloids affect the existence, frequency, and shape of agonist induced Ca2+ oscillations. The latter conclusion is significant in view of the observation that Aβ oligomers can stimulate IP3 production [2+ and consequently on mitochondrial metabolism has also been investigated by modelling [As a direct consequence of the decoupling between sub-PM and bulk cytosolic Cae et al.  and Liu e et al. . In the oduction , a pointodelling .2+ fluxes across the PM, they have a significant influence on the membrane neuronal activity in the model. It was shown that Aβ peptides bind to the catalytic site of the PMCA, which corresponds to an increase in the value of PMCAK appearing in Equation (3). As shown in 2+ by forming pores in the PM. In the model, they have rather mixed consequences on neuronal activity (2+ through the pores stimulates the hyperpolarizing K(Ca) current, but also depolarizes the cell membrane. Indeed, hyperexcitability is more pronounced when decreasing the conductance of the K(Ca) channel. In addition, if Ca2+ influx through the pores is very large, aberrant spiking in the form of complex neuronal activity can also be observed in the model. As a perspective, it would be interesting to test if the same simulated behaviors can be recovered when considering a stochastic Markov model validated against experimental data [2+ channels and Ca2+-sensitive K+ channels in the presence of Aβ peptides clearly point to hyperexcitability when considered in the model. These results are in agreement with the numerous studies reporting neuronal hyperexcitability in the presence of Aβ peptides. In particular, limiting the open time duration of RYR2 in the hippocampal CA1 neurons of an AD mouse model upregulates the surface expression of the K(Ca) channel and prevents electrical hyperactivity, but does not directly modulate the activity of the K(Ca) channels [2+ fluxes or Ca2+-regulated currents have qualitatively different outcomes. However, it should be noted that although Aβ peptides are most of the time associated with neuronal hyperexcitability, some studies however report opposite consequences [In contrast, when Aβ peptides affect Caactivity . On the tal data  to descrchannels . Altogetequences ,51.The seemingly contradictory effects of Aβ peptides reported in this study may also be due to the limitations of the proposed model. For example, it does not consider the specificities of the various neuronal cell types. Additionally, we considered one target of Aβ peptides at a time, which is important to dissect the pathological consequences of the oligomers of amyloids, but does not correspond to a realistic situation. Because they are limited to a single neuron, simulations do not include network effects, which play a crucial role in the cognitive defects related to the disease . It is c