PARK2, coding for parkin, which plays an important role in mitochondria function and calcium homeostasis. Cyclic adenosine monophosphate (cAMP) is a major second messenger regulating mitochondrial metabolism, and it is strictly interlocked with calcium homeostasis. Parkin-mutant (Pt) fibroblasts, exhibiting defective mitochondrial respiratory/OxPhos activity, showed a significant higher value of basal intracellular level of cAMP, as compared with normal fibroblasts (CTRL). Specific pharmacological inhibition/activation of members of the adenylyl cyclase- and of the phosphodiesterase-families, respectively, as well as quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis, indicate that the higher level of cAMP observed in Pt fibroblasts can contribute to a higher level of activity/expression by soluble adenylyl cyclase (sAC) and to low activity/expression of the phosphodiesterase isoform 4 (PDE4). As Ca2+ regulates sAC, we performed quantitative calcium-fluorimetric analysis, showing a higher level of Ca2+ in the both cytosol and mitochondria of Pt fibroblasts as compared with CTRL. Most notably, inhibition of the mitochondrial Ca2+ uniporter decreased, specifically the cAMP level in PD fibroblasts. All together, these findings support the occurrence of an altered mitochondrial Ca2+-mediated cAMP homeostasis in fibroblasts with the parkin mutation.Almost half of autosomal recessive early-onset parkinsonism has been associated with mutations in  Although most cases of PD are sporadic, several genes involved in Mendelian forms of PD have been identified. Specifically, almost half of all the early-onset familial PD cases have been associated with mutations in PARK2, which codes for parkin, a multifunctional E3 ubiquitin ligase [2+) homeostasis [parkin-mutant patients, affected by early-onset autosomal recessive PD, displayed severe ultrastructural abnormalities, (mainly in the mitochondria) [2+ homeostasis, oxidative stress response protein, and RNA processing [parkin-mutant fibroblasts [Parkinson’s disease (PD) is one of the most common neurodegenerative diseases, characterized by the selective death of dopaminergic neurons in the n ligase ,2, whichn ligase ,5. Furthn ligase ,7. Functn ligase , as welln ligase ,10,11. Pn ligase ,13 and ieostasis ,15,16,17ocessing ,19 as weocessing . In addi2+ homeostasis [2+ is known to regulate sAC activity and that of specific tmACs [cAMP is one of the major second messengers that regulates mitochondrial metabolism ,25,26,27eostasis . cAMP iseostasis  and by seostasis ,30,31; teostasis ,33,34. Deostasis ,36,37. Cic tmACs ,39,40 anic tmACs .parkin-mutant (Pt) fibroblasts, a higher basal level of cAMP resulting from a greater contribution by sAC and a low expression of the isoform PDE4. Moreover, cytosolic and mitochondrial Ca2+ measurements in Pt fibroblasts show a high basal Ca2+ level in the cytosolic and, in particular, in the mitochondrial compartments resulting in increased mitochondrial Ca2+-mediated cAMP level.In this study we show, in Primary skin fibroblasts from three patients P1, P2, and P3 (or Pt), affected by early-onset PD, with different parkin compound heterozygous mutations  fetal bovine serum (FBS), 1% (v/v) ʟ-glutamine, 1% (v/v) penicillin/streptomycin, at 37 °C in a humidified atmosphere of 5% CO2. All experiments were performed on cells with similar passage numbers, ranging from 5 to 14, to avoid an artefact, due to senescence, known to occur at passage numbers greater than 30. In the passage range used, the fibroblasts were β-Gal-negative.Cells were grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% ). The cAMP values were normalized to the protein concentration, and expressed as pmol/mg protein.The cAMP levels were measured using the direct cAMP ELISA Kit . The cells were treated in the presence of dimethyl sulfoxide (DMSO) (0.02%), used as control vehicle, or 10 µM Rolipram, 100 µM 3-Isobutyl-1-methylxanthine (IBMX), 10 µM forskolin, 1 µM isoproterenol, 100 µM SQ22536, 25 µM of KH7 or 1 µM carbonic anhydrase inhibitor (CAI) for 30 min at 37 °C where indicated in the figures. For cAMP assays, the culture medium was removed and 1 mL of 0.1 M HCl was added to the cell layer, followed by 10 min incubation at 37 °C. The lysed cells were scraped and transferred into Eppendorf tubes. The samples were centrifuged at 1300× 2+ and 4 µM X-Rhod-1 AM  for mitochondrial Ca2+ level measurements. Stained cells were washed with Phosphate Buffered Saline (PBS), fixed in PBS containing 4% paraformaldehyde for 10 min at room temperature, and cell nuclei were stained with 1 μg/mL 4′,6-Diamidino-2-phenyindole, dilactate (DAPI) Sigma-Aldrich, St. Louis, MO, USA) for 10 min at room temperature. Stained cells were examined with a Leica TCS SP8 microscope (images collected using a 40× and 60× in oil immersion objective) coupled to the Laser Scanning Confocal Microscopy (LSCM) system. Acquisition, storage, and data analysis were performed using Leica software LAS AF 3 (https://www.leica-microsystems.com/products/microscope-software/).CTRL and Pt primary fibroblasts were cultured onto 24 × 24 mm glass slides in six-well plates and incubated for 30 min at 37 °C, with the following probes: 2 µM Fluo-4 AM  for cytosolic Ca2+ concentration was measured by using, respectively, the fluorescent indicator Fluo-4 AM and X-Rhod-1AM . CTRL and Pt primary fibroblasts were grown in a T25 Flask. Cells at 80% confluence were incubated with a fluorescent probe for 30 min at 37 °C. Cell monolayers collected by trypsinization and centrifugation were resuspended in a buffer containing 10 mM HEPES and 6 mM d-Glucose (pH 7.4) at an approximate concentration of 1 × 105 cells in 1 mL. Fluorescence intensity was measured at 25 °C in a spectrofluorometer , equipped with a stirrer and temperature control, by the subsequent addition of 5 mM CaCl2, 0,1% Triton X-100 (for cytosolic Ca2+ levels), 0.1% Na-Cholate  and 40 mM EGTA. The excitation/emission wavelengths were 495 nm/506 nm for Fluo-4 AM and 580 nm/602 nm for X-Rhod-1 AM. The cytosolic and mitochondrial Ca2+ levels were evaluated by using an apparent Kd (443 nM for Fluo-4AM and 700 nM for X-Rhod-1AM) according to the equation described by Grynkiewicz et al. [Cytosolic and mitochondrial Caz et al. . Where iADCY and PDE messenger RNA (mRNA) levels were performed by real-time qRT-PCR, using SYBR Green (Bio-Rad). Reactions were performed in duplicate for each sample in three independent experiments. Multiple reactions were performed in a volume of 25 μL containing 12.5 μL of 2 × PCR master mix, 0.2 μM of specific primers, and 200 ng of cDNA template. Amplifications were performed in the BioRad iCycler iQ Real-Time PCR Detection System , using the following cycle program: denaturation step at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 1 min, and extension at 72 °C for 30 s. The relative mRNA expression levels were calculated by using the comparative CT method (ΔΔCT) [The purification of total RNA from fibroblasts was carried out by using RNeasy Mini Kit , according to the manufacturer’s protocol. One microgram of total RNA was then reverse-transcribed to generate cDNA for PCR by using the iScript cDNA Synthesis kit . Semi-quantitative determination of d (ΔΔCT) . Quantithttps://imagej.nih.gov/ij/index.html).Whole cell extracts (30 μg) were separated on a 13% Sodyum-Dodecyl-Sulphate Polyacrilamide Gel Electrophoresis (SDS-PAGE) according to , and traTotal protein concentration was determined by the Bio Rad Bradford protein assay, using bovine serum albumin as the standard.p-value) was set to 0.05.Data are shown as mean ± SEM. The significance of any differences throughout the data sets presented  was determined by one-way Analysis of Variance (ANOVA) with the Bonferroni post-hoc test and with Student’s t-test. The threshold for statistical significance (parkin-mutant fibroblasts [parkin gene (del exon7-9/Glu409X). parkin affect the cAMP metabolism, irrespective of the genetic background. To gain deeper insights into cAMP metabolism, we performed further experiments only on P3 fibroblasts, (later referred as Pt fibroblasts) as cellular model of parkin null-cells.The second messenger cAMP is involved in many important cellular signaling pathways, and several of these have been demonstrated to be defective in roblasts . We prevroblasts . Here, wIn mammals, cAMP is generated from ATP by adenylyl cyclases (ACs), and degraded by specific phosphodiesterases (PDEs). The treatment of fibroblasts with 10 µM Rolipram, a selective inhibitor of PDE4, the mostly expressed isoform in fibroblasts ,46,47,482+ and bicarbonate cellular levels [Intracellular cAMP is synthesized either by the hormonally responsive, G protein-regulated transmembrane adenylyl cyclases (tmACs) , or by sr levels ,32,49. Or levels  and KH7 r levels ,52, inhiSince sAC is stimulated by bicarbonate  we also In order to assess whether the differences described in the cAMP basal level between CTRL and Pt fibroblasts could be due to differential gene expression of the main players in cAMP metabolism, i.e., adenylyl cyclase and phosphodiesterases, we analyzed their transcription levels. RT-qPCR analysis of mRNA transcript levels revealed that the PDE4A mRNA level was significantly lower in Pt fibroblasts, as compared with CTRL fibroblasts , whilst 2+ in a calmodulin-independent way, by lowering the Km for Mg2+ ATP [2+-dependent activation of sAC, we measured the cytosolic and mitochondrial Ca2+ steady-state levels. The fluorescence intensity analysis of the confocal microscopy images in the presence of specific cytosolic (Fluo-4 AM)  A and mituo-4 AM) B Ca2+ prroblasts A,B. Quanroblasts C and mitroblasts D steady-2+ level measured in the Pt cells could result from altered Ca2+ fluxes from intracellular sources. Both the mitochondria and the endoplasmic reticulum (ER) are the major regulators of Ca2+ homeostasis, therefore controlling Ca2+-dependent cellular signaling. Treatment with RR, inhibitor of the mitochondrial Ca2+ uniporter (MCU), resulted, as expected, in an enhanced cytosolic  in Ca2+ homeostasis, and the mitochondrial and cytosolic Ca2+ levels were measured in the presence of thapsigargin (TG), a specific irreversible inhibitor of ER Ca2+-ATPase (SERCA) [2+ uptake by the ER, and dantrolene, a specific inhibitor of ryanodine receptor (RyR), the major ER Ca2+ release channel [2+ levels, both in CTRL (184 ± 3%) and Pt (158 ± 9%) fibroblasts, and a slight but significant increase of the mitochondrial Ca2+ levels in the CTRL cells  and Pt (17 ± 3%) fibroblasts  , which m channel . As showRL cells D, in conn points . Unexpecroblasts E, and a RL cells F.2+ entry is largely contributed to by the ER at the ER–mitochondria contact sites, where the local concentration of Ca2+ is exceptionally higher than that which is reachable in the cytoplasm, and (ii) the major ER Ca2+ channel at the inter-organelle contact sites is contributed to by systems that are different from the RyR  [These apparently contradictory results might be reconciled with the notion that: (i) the mitochondrial Caeceptor) .2+ and the high level of cAMP, CTRL and Pt fibroblasts were treated with RR or Ru360, a more selective inhibitor of MCU, and the cellular cAMP content was assayed. The results show, for both the MCU inhibitor treatments, that while the CTRL cells showed a significant increase in cAMP content, in Pt fibroblasts, this was significantly reduced  in parkin-mutant fibroblasts, the mitochondrial cAMP level was higher, and the overall cellular cAMP level was is affected more significantly by the partial inactivation of sAC, following the inhibition of mitochondrial Ca2+ uptake.To test the possible link between mitochondrial Ca reduced . These o2+ induced by dantrolene in CTRL fibroblasts to verify, if independently from the parkin-null context, this was sufficient to cause an increased level of cAMP production. As shown in 2+-mediated activation of the mitochondrial sAC. This condition would mimic what constitutively observed in Pt fibroblasts, supporting the hypothesis that the observed higher cAMP level in the Pt fibroblasts is indeed the result of Ca2+-mediated activation of sAC.As a proof of principle, we took advantage of the observed increase of mitochondrial CaIn order to assess whether the high calcium level measured in dantrolene-treated CTRL might led to differential ADCY10 and PDE4A gene expression, as observed in Pt cells, we measured the mRNA levels. In this setting, the ADCY10 mRNA levels were significantly higher B, as wel2+ [In mammalian cells, the cAMP produced in the cytosol by tmAC, acts as a second messenger (cAMP cascade), following binding to specific receptors of the extracellular signal molecules, like hormones and neurotransmitters . On the 2+ .parkin gene (del exon7-9/Glu409X) [2+, which are causally involved in the observed high level of the basal cAMP in parkin-mutant fibroblasts (Pt cells), taken as a cellular model of parkin null-cells.In the present work, we confirmed the presence of high cAMP level in fibroblasts harboring a heterozygous PARK2 mutation (del exon2-3/del exon3)  and exteGlu409X) . The stuThe direct activation of tmAC by FSK raised cyclic AMP levels, as expected, both in CTRL, and even if to a lesser extent, in Pt cells, whereas isoproterenol, a β-adrenergic receptor agonist coupled to stimulatory Gs proteins, which determine the activation of the transmembrane adenylate cyclase, resulted in a significant increase of cAMP levels, only in CTRL cells. The lack of effect by isoprotenerol in Pt cells, not due to an alteration of tmAC intrinsic activity, as shown by the responsiveness to FSK stimulation, deserves further investigation.Alongside with the higher cAMP content in Pt cells, we showed that a specific lack of response occurred toward Rolipram, a specific PDE4 inhibitor, but not to IBMX, a PDEs pan-inhibitor. On the contrary, the treatment of CTRL fibroblasts showed a significant increase in the cAMP level, since the degradation path was inhibited not only by the PDE4 inhibitor, but also by IBMX treatment. As IBMX treatment resulted in similar level of cAMP increase in both the CTRL and Pt cells, we could deduce that similar PDE activity is present in both cell types, except for the PDE4A isoform, whose expression is reduced in Pt cells. This is in line with recent work showing that the loss of PDE4 expression in the striato-thalamo-cortical circuit, which is associated with deficits in spatial working memory, in PD patients .2 [2+ entry [In the present work, the results of treatment with KH7, a specific inhibitor of sAC, which has been shown to be able to suppress the mitochondrial cAMP content almost completely in isolated mitochondria , reveale2 , as well2+ entry , highlig2+ entry ,64. Notaparkin-mutant fibroblasts were likely be a result of high levels of Ca2+ in the cytosol, which, buffered by the mitochondria, would in turn lead to the activation of mitochondrial sAC. Although the exact mechanism of action is yet to be established, studies on human SH-SY5Y neuroblastoma cells expressing mutant parkin showed a significant increase in the Ca2+ level, caused in particular, by Ca2+ release from ER [2+ cytotoxicity in Drosophila [2+ in the Pt fibroblasts, as compared to the CTRL cells, might be a consequence of the increased activation of the RyR and IP3 receptors by PLCγ1, which are more active in the absence of parkin [2+, modulating the release of Ca2+ from the ER by directly regulating the ryanodine receptors. Proteomics studies conducted on CRTL and parkin-mutant fibroblasts have shown the involvement of parkin in the regulation of Ca2+-binding proteins, which were downregulated in Pt fibroblasts [The high basal levels of cAMP in the  from ER , and a posophila  has beenosophila , which posophila ,68, causf parkin , or it mroblasts .2+ homeostasis, in the Pt fibroblast context, we tested the effects of thapsigargin, a specific irreversible inhibitor of all SERCA isoforms and dantrolene, which inhibits the RyR receptor. In both cases, following the treatment, major differences between CTRL and Pt cell Ca2+ levels were found in the mitochondria. Notably the changes observed in the cytosolic Ca2+ of treated Pt fibroblasts were not reflected in the mitochondrial Ca2+, despite them sharing the same responses to the MCU inhibitor observed in the CTRL fibroblasts. Physical contacts between the ER and mitochondria, called mitochondria-associated membranes (MAMs), provide a controlled Ca2+ flux from the ER store into the mitochondria without raising their concentration in the cytosol [2+ homeostasis negatively affecting mitochondrial functions and biogenesis.To assess the contribution of ER to the cellular Ca cytosol ,70,71. T cytosol ,74,75,76 cytosol , and end cytosol . The alt2+ and cAMP levels in Pt fibroblasts, we investigated whether the two events were causally linked. To this aim, we reduced the mitochondrial Ca2+ level by treating both the Pt and CTRL cells with RR, and we found a specific reduction of cAMP only in the Pt fibroblasts. Further we mimicked an increased mitochondrial Ca2+ content, exploiting the effect of dantrolene treatment in the CTRL fibroblasts. Interestingly, in these conditions, we detected an increase of cAMP level, with a major contribution by sAC, and furthermore, a higher ADCY10, as well as PDE4A mRNA expression level, to balance the cAMP increase. This feedback regulation of cAMP levels, expected in CTRL cells, might be absent or dysregulated in Pt fibroblasts.Taking into account the higher steady-state Caparkin-mutant fibroblasts, resulting from a greater contribution by sAC, and/or a low expression of PDE4, whose loss of function could be, however, compensated by other members of the PDE family, as shown by the results of IBMX treatment. Furthermore, our study confirms, as previously described, the involvement of parkin in the control of cellular Ca2+ levels, suggesting altered interactions between the endoplasmic reticulum and the mitochondria, and a potential different efficiency for the main Ca2+ transport mechanisms. An additional element to the link between parkin and mitochondria-related calcium homeostasis is provided by a recent study, showing that parkin selectively regulates the turnover of the MCU accessory protein MICU1 [2+ uptake [2+ into the mitochondria. The mitochondrial calcium overload could lead to the activation of sAC and PKA, via ensuing the elevation of the steady-state cAMP content as a possible compensatory mechanism to counteract Ca2+-induced ROS production, and the accumulation of damaged mitochondria (In conclusion, we showed for the first time to our knowledge, a significantly high basal level of cAMP in in MICU1 . MICU1 i+ uptake . Parkin + uptake . On thischondria .PKA was found to induce phosphorylation-mediated activation of the respiratory chain complexes I and IV, thus enhancing mitochondrial respiration ,80. Thou


These disruptions can cause changes in homeostasis levels that can have detrimental effects on cell function and survival. Although studies have shown that Aβ can interfere with various Ca2+ fluxes, the complexity of these interactions remains elusive. We have constructed a mathematical model that simulates Ca2+ patterns under the influence of Aβ. Our simulations shows that Aβ can increase regions of mixed-mode oscillations leading to aberrant signals under various conditions. We investigate how Aβ affects individual flux contributions through inositol triphosphate (IP3) receptors, ryanodine receptors, and membrane pores. We demonstrate that controlling for the ryanodine receptor’s maximal kinetic reaction rate may provide a biophysical way of managing aberrant Ca2+ signals. The influence of a dynamic model for IP3 production is also investigated under various conditions as well as the impact of changes in membrane potential. Our model is one of the first to investigate the effects of Aβ on a variety of cellular mechanisms providing a base modeling scheme from which further studies can draw on to better understand Ca2+ regulation in an AD environment.The accumulation of Alzheimer’s disease (AD) associated Amyloid beta (A Sustained Ca2+ disregulation can trigger apoptosis leading to premature neuronal death, a characteristic feature seen in AD. Although the accumulation of Aβ has been linked to the progression of AD by altering Ca2+ signaling processes within neurons and neuroglia, the mechanisms for how and why this occurs are not fully understood.Alzheimer’s Disease (AD) is a devastating neurodegenerative illness affecting over 40 million people worldwide. AD is the leading cause of dementia and is characterized by a progressive and irreversible decline in memory and cognitive skills . The pre plaques . Because+ levels , studyinlum (ER) –8. As suβ can lead to aberrant Ca2+ signaling. The amyloid hypothesis suggests that the accumulation of Aβ in the brain is the primary driving force of AD pathogenesis [β plaques and fibrils are a consequence of the imbalance between the formation and sequestration of Aβ. The slow accumulation of Aβ peptides can alter Ca2+ signaling processes leading to synaptic failure and neuronal death. Although it is unclear how Aβ disrupts intracellular Ca2+ homeostasis, there is growing evidence that Aβ directly affects the production of inositol triphosphate (IP3) [β affects various Ca2+ signaling mechanisms in a simplified whole-cell model.Our goal is to use mathematical modeling to describe various conditions under which Aogenesis –12. In tte (IP3) , calciumte (IP3) , 14, andte (IP3) , 16. We 2+ oscillatory patterns within an AD environment. By developing a mathematical model for intracellular Ca2+ regulation, we can begin to study how Aβ affects Ca2+ flux through various individual channels and pumps. Investigating model solutions can also provide important information on the impact of Aβ on Ca2+ basal levels over various timescales. Due to current experimental limitations, mathematical and computational models can provide insights for targeting specific mechanisms in order to restore neuronal function, and to suggest symptomatic improvement strategies. In fact, according to Liang et al. (2015) there is growing momentum to study Ca2+ dynamics in AD, specifically through computational and mathematical modeling, and this work outlines such an approach.In this study we present a theoretical approach to better understand the driving mechanisms for various Caβ on intracellular Ca2+, we first build a simplified whole-cell Ca2+ model by making use of the vast array of work on modeling Ca2+ dynamics and the calcium signaling “toolkit”  are those corresponding to IP3 receptors (IPR), RyRs, a general membrane leak Jin, and we include a fast Voltage Gated Ca2+ Channel (VGCC) in Jvca to account for membrane permeability. The out-fluxes (out of the cell or sequestered into Ca2+ pools such as the ER) are modeled using a plasma membrane pump Jpm, and a sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) pump JSERCA. A diagram of the major fluxes outlined in the model is given in β with respect to individual flux term.Our model structure was selected to account for the major components that have been shown to be influenced by A2+ through the IPR and RyR are proportional to the difference in the concentration of Ca2+ between the ER and the cytoplasm. Under these standard assumptions, using conservation of fluxes, our general Ca2+ model takes the formce denote the concentration of Ca2+ in the ER, and γ is the ratio of cytoplasmic volume to the ER volume. The individual contributions of each flux can vary from a simple Hill function to more complicated forms involving numerous parameters and additional terms. We provide a description of each of the flux terms we use in our model below. We also provide background information on their development and why it may be well suited for our purposes. Each term was chosen in order to balance meaningful biophysical quantities while maintaining a tractable mathematical structure.We assume that the ER is homogeneously distributed throughout the interior of the cell and that the fluxes of CaJvca in . Becausfound in ).3 is a ligand produced by phospholipase C (PLC) when activated by a G-protein coupled receptor reacting to an external stimulus such as neurotransmitters, hormones, and growth factors [3 regulates many important cellular functions, including the release of Ca2+ from the ER through the IP3 receptor [3 receptor follows a saturating binding rate model of the form found in [kf controls the density of the IP3 receptor, Jer is a leak from the ER to the cytoplasm, and P0 is the open probability of the IPR. The leak term is necessary to balance the ATPase flux at steady state. To model P0, we use a version of the Sneyd and Dufour (2002) model that is based on previous models found in [R the resting state, O the open state, A the active state, S the shut state, and I1 and I2 both represent inactive states. The transitions between states can depend on Ca2+ and IP3 concentration p. As such, the IP3 receptor will be in various states depending on the value of p.IP factors . As a kereceptor . We assufound in , 25. Thufound in –28. The α1 and α2 are parameters that control the individual contributions of the open and active states of the receptor. As in [α1 = 0.1 and α2 = 0.9. In the model, it is assumed that the receptor has four identical and independent subunits and that Ca2+ flows when all four subunits are in either the O or A state. The equations for the transitions between the states are given in [2+ concentration and IP3 concentrations [In this model, the receptor open probability is given byr. As in , we assugiven in , but havtrations .2+ oscillations in a sympathetic neuron. Thus, the flux through the RyR is given byk3 is defined as an increasing sigmoidal function of intracellular Ca2+ concentration and takes the formk1, k2, and kd are parameters. We use n = 3 to match the experimental results obtained in [k1 in . The receptor is modeled as a simple leak channel, with a flux through the channel proportional to the concentration difference between the ER and cytosol. This model was used to investigate Caained in . The parcnkdn+cn,where k12+ level . The terJin is modeled using a linearly increasing function of IP3 concentration [2+ concentration depend on p. Thus,a1 and a2 are parameters.The membrane leak ntration . Althoug2+ dynamics both the SERCA pump and the plasma membrane pump play an important role in maintaining concentration gradients. Different models of these pumps exist and vary in complexity. Here we model the plasma membrane pump using a simple Hill equation of the formVpm is the maximal velocity and Kpm is the channel sensitivity. We have chosen a standard Hill coefficient of 2 as is found in [When modeling Cafound in .Ki for i = 1, …, 5 are constants. Notice that this choice of model is more complicated than a simple Hill equation. Experimental evidence show that the rate of the pump may be modulated by the level of Ca2+ in the ER [c and ce.To describe the behavior of the SERCA pump we utilize a simplified four state bidirectional Markov model (as found in ) of the 4ce+K5cceto modeln the ER , 33, andJvca. When the volume of the cell is constant, Ca2+ flux and current are related by the equationIca is the current through the VGCC, F is Faraday’s constant, and w is the cell volume. Since Ica depends on the membrane potential V and Ca2+, we need a way to track membrane potential. Here, we assume that the membrane potential follows a Hodgkin and Huxley formulation [Cm is the cell capacitance, V is the membrane potential, and the sum is over all ionic currents across the cell membrane. In the case of non-excitable astrocytes, current flow across its membrane is characterized by potassium (K+)-selective membrane conductance [Ikir, Ina, Il, and Ica correspond to an inward rectifying potassium, sodium, leak, and Ca2+ current, respectively, and Iapp is an applied current. In (Ikir(V) similar to [gkir is the maximal channel conductivity, Vka is the Nernst K+ potential, K0 is the extracellular K+ concentration, and Va1, Va2 and Va3 are constants. In the model of [K0 is time dependent and depends on proximal neuronal activity (further details of this formulation can be found in [+ and as such consider K0 to be constant. Each of the remaining currents have the form Ix = gx(V − Vx), where gx is the conductance and Vx is the Nernst potential of each channel.The flux due to changes in membrane potential is given in our model by mulation  that satductance  and voltductance , 37. As (V)+Iapp,where Ikmilar to  and writmodel of , K0 is tfound in ). Here, Ica exist (such as the Goldman-Hodgkin-Katz current approximation), we use a T-type like channel  can also be included in the model, here we simply illustrate how one can link membrane potential with other Ca2+ regulatory mechanisms together into a single model and use that model to investigate the role of Aβ on Ca2+ dynamics. As such, the T-type Ca2+ channel described in  of the T(V-Vca),where g¯β on individual pumps, channels, and exchangers remains largely unknown. However, some studies provide insight on the effects of Aβ on Ca2+ regulation and we utilize these findings in making our assumptions about the effects of Aβ. For example, De Caluwé and Dupont (2013) formalized a theoretical model to describe a possible feedback loop between Ca2+ and Aβ. Their model showed that the existence of a bistable steady-state region can exists in the presence Aβ. The model suggests that over time, cytosolic Ca2+ proliferation as a result of Aβ could trigger the onset of AD. In this study, we are interested in the generation of aberrant Ca2+ signals on a relatively short timescale. Since the accumulation of Aβ can occur over months, years, and even decades, Aβ concentrations changes occur on a very long timescale compared to changes in Ca2+. As such, we assume that Aβ may be present in the environment, but make no attempt to track changes in Aβ concentration over time.The influence of Aβ plasma membrane pores can alter Ca2+ signaling by creating additional influx into the cytoplasm [β generated plasma membrane pores, we use a similar mechanism as found in [a represent a fixed level of Aβ concentration present in the environment. Then, we include the term kβam in Jin and write an altered membrane leak flux asm represents a cooperativity coefficient, and kβ is a rate constant , three controlling the membrane potential , and an additional two variables controlling the gating variables mcaT and hcaT . We have also added a scaling and control parameter ps to simplify the contribution from the VGCC flux. When ps = 0, the impact of membrane potential on Ca2+ signaling are ignored. When ps > 0, changes in membrane potential do influence the dynamics of intracellular Ca2+. The large set of parameter values was selected to closely match those of previous studies whenever possible and can be found in The simplified whole-cell Caβ can increase membrane permeability by creating plasma membrane Aβ pores, and have used the work of [β increases RyR sensitivity and have incorporated additional terms in the RyR component of the model. Currently in our model, IP3 acts as the primary agonist that can then trigger Ca2+ release from IP3 receptors. In addition to affecting the IP3 receptor open probability, Ca2+ release activates RyR and subsequent CICR. In the next chapter we separate our analysis for the model under three assumptions. We first characterize the solutions of the model for constant levels of IP3 and when the effects of membrane potential are ignored. Second, also in the absence of membrane potentials, we expand our model to include a dynamic variable for IP3 production. Lastly, we include the effects of membrane potential on Ca2+ dynamics by incorporating flux through VGCC under various levels of fixed IP3. We further look to characterize the impact of VGCC on model solutions in the presence of Aβ.In the formulation of our model we have assumed that the presence of A work of  to alterx-axis and the projection of stable and unstable steady-state solutions, upper and lower branches of periodic orbits, and critical transitions points (such as Hopf and period doubling) are labeled accordingly. Hopf bifurcations occur when a steady-state solution loses it’s stability as a result of small changes in a parameter. Mathematically, this occurs when complex eigenvalues (of the linearization) cross the imaginary axis away from the origin. Period doubling bifurcations occur when a small change in a bifurcation parameter causes the period of an oscillatory system to double. These and other types of bifurcations can be used to describe systemic changes in dynamics and in our case, transitions from single-mode oscillations to mixed-mode oscillations (MMOs). The program AUTO allows us to readily compute the location of these bifurcations as model parameters are varied. Initial conditions for all simulations were set at c0 = 0.05 and ce = 10 with R0 = 1 and all other IP3 receptor initial conditions are set to zero. Initial conditions for the membrane potential are V0 = −65, m0 = 0.05, n0 = 0.32, h0 = 0.6, mcaT0 = 0.29, and hcaT0 = 0.01.Numerical solutions of the differential equations systems were obtained using explicit Runge-Kutta  algorithms implemented in Matlab . Bifurca2+ can emerge in the presence of Aβ. These aberrant signals can occur under various conditions suggesting a complex link between the influence of Aβ and the model components. As such, we break down the dynamics of the model by tracking solutions as a result of altering one or two parameters within a specific signaling component. We first look at model solutions when IP3 concentration is fixed and persists and is not influenced by membrane potential. We then allow for IP3 to change dynamically and simulate the response in Ca2+ with and without the influence of Aβ. Lastly, we incorporate the influence of membrane potentials and investigate model solutions for various levels of Aβ when IP3 concentration is fixed.In all three sections below, the model dynamics show that aberrant Caps = 0 show that incorporating Aβ in a simplified Ca2+ model can lead to aberrant Ca2+ signaling through dynamical transitions into MMOs. Particularly, we look to track the influence of Aβ on the location of Hopf points (labeled HB), period doubling points (labeled PD), and regions of MMOs (shaded) in order to better understand Ca2+ steady-states and oscillatory patterns. From a phenomenological perspective, a transition through MMO may explain why Aβ can trigger persistent aberrant Ca2+ signals. Although the long-term accumulation and influence of Aβ remains difficult to model, our approach illustrates that aberrant Ca2+ signals can occur under the influence of Aβ for a variety of parameter regimes.Simulations of  and 19)19) with 3 can be photoreleased simultaneously through out a cell. In such conditions, IP3 diffusion is minimized and can be treated as constant. By varying the amount of IP3 available in the cytosol, the model can exhibit Ca2+ oscillations as typically found in various cell types. These oscillatory patterns are critical in order for cells to maintain appropriate concentration gradients and to re-establish homeostasis levels after a triggering event. In the absence of Aβ, model Ca2+ oscillations appear and disappear as a result of transitions through Hopf bifurcations as the parameter p increases. Although these stable oscillatory patterns are predictable for a large domain of the parameter p, the model does exhibit MMOs for small parameter regions near the Hopf points. It is these MMO patterns that we are most interested in. Our results show that in the presence of Aβ, the regions of MMOs can grow and become larger. This leads to an increased possibility for aberrant Ca2+ signals. We hypothesize that aberrant Ca2+ signals observed experimentally can be described as dynamic transitions through MMOs. The complicated patterns in MMOs are often characterized by sub-threshold oscillations interspersed within large relaxation types of oscillations [Experimentally, IPllations –57.a = 0 that exhibit sustained Ca2+ oscillations as the amount of p is increased from p = 5 to p = 10. An increase in frequency occurs as p increases within the predictable region and can be seen by comparing p = 18.5. For this p-value, the model undergoes MMOs with varying oscillatory patterns of mixed amplitude, but we do not characterize it as aberrant Ca2+ signaling since it is periodic. Notice that no Ca2+ oscillations occur for small and large values of p. This is due to the assumption that the IP3 receptor is unlikely to open at high and low concentrations of IP3. Although our model does not exhibit a typical Hopf bubble, it has the overall qualitative patterns of a robust Ca2+ model as described in [Illustrated in β has occurred but assume that this amount is fixed during the timescale of our simulations. As such, we assume that a is constant and that the value correlates to the concentration of Aβ in μM. That is, the value of a in the model may be thought of as reflecting the stage of the progression of the disease. For example, a small value of a corresponds to a small amount of Aβ that may have accumulated in the early stages of AD, while a larger value of a may correspond to a late stage AD. As the parameter a is varied, we can then study how the dynamics of the model change as the level of Aβ changes. Our ultimate goal is to better understand the implications of various levels of Aβ on Ca2+ so that we may gain knowledge about the possible progression of AD.We consider an AD environment where some accumulation of Aβ can lead to increased steady-state levels as well as large amplitude oscillations even in the absence of IP3 signaling. To better understand the effects of Aβ on model solutions, we simulate the model and look at the effects of changing the parameter a on steady-state levels. Various experimental studies have used Aβ levels of 0.5 and 1.0 [a that matches these types of levels.The accumulation of A and 1.0 , 50, 51.3 is present, the accumulation of Aβ increases the steady-state of Ca2+ and can even lead to large-amplitude Ca2+ oscillations. Illustrated in a when p = 0. Notice that the steady-state value slowly increases as a increases until solutions transition into stable periodic oscillations between the two Hopf points HBa1 and HBa2. As a increases towards the value of 1.293, the steady-state asymptotes and solutions quickly become non-physical. a = 1.15. a = 1.276. Notice that in this case, the solution first undergoes a large Ca2+ increase before settling in on a steady-state value. When the model is presented with levels of Aβ greater than a = 1.29, solutions become intractable and are not analyzed.In the case when no IPa and p can affect the dynamics of the model, we look at their effects together. Given in ps = 0 using p and a as the bifurcation parameters. The solid lines correspond to the Hopf bifurcation manifolds, while the dashed lines correspond to the period-doubling manifolds. The shaded area in 2m. These MMOs vanish for values of a > 0.275. This bifurcation diagram has a number of interesting features that can bring to light important behavior in Ca2+ regulation. For example, when 0.415 < a < 0.472 the bifurcation diagram has four Hopf points. This leads to small amplitude oscillations for certain ranges in the parameter p. a = 0.45. In this diagram, three of the Hopf bifurcations have been labeled as HB1 since all three points lie on the Hopf manifold labeled HB1m in Since both a = 0.45 is kept fixed while various values of p are used. The corresponding model solutions show different types of behaviors ranging from steady-state Ca2+ levels, stable periodic solutions, aberrant Ca2+ signals, and MMOs. Illustrated in p = 5. This solution corresponds to the region where the small Hopf bubble exists as shown in p = 20. p = 26 and p = 45.5, respectively. 2+ signals when p = 45.8. Notice that this solution enters aberrant oscillations as the bifurcation structure transitions through period doubling points within the MMO region. 2+ oscillations when p = 50.To help us understand this bifurcation structure, various solutions are plotted in β not only increases the steady-state level, but also influences the regions of MMOs. Particularly, the internal range where MMOs emerge between the Hopf manifolds increases for a large parameter range of a. As such, the model suggests that Aβ may drive aberrant Ca2+ signals as transitions through MMOs. However, as the simulated amount of Aβ increases towards a = 1, the region of MMOs decreases and stable periodic orbits exist for most of the p parameter range. This condition is illustrated in a. a = 1 with p = 30, while B shows the solution for a = 1.2 and p = 20. In these cases, Ca2+ oscillations have much larger amplitudes than in the absence of Aβ.The results of the two parameter bifurcation analysis seem to suggest that Aβ on Ca2+ signaling, we now turn our attention to the contributions of the RyR. Recall that in our model we assume that Aβ affects the RyR by altering the receptor’s sensitivity for CICR. To determine the specific contribution of this altered sensitivity, we first simulate the model for fixed a and p and describe the dynamics of varying the parameter ka. Our simulations show that in the presence of Aβ, changing the parameter kα can produce aberrant Ca2+ signals. These aberrant signals result as transitions through MMOs for fixed Aβ levels. However, we also show that we can control these signals by increasing k2, the maximal kinetic rate of the RyR.To better understand the influence of A2+ traces with kα = 0.5, 0.9, 1, and 1.25 when a = 0.25 and p = 10 are fixed. Notice that for kα = 1 aberrant Ca2+ signals emerge. Also included in a = 0.25 using kα as the bifurcation parameter. Notice that MMOs exist for a large parameter set of kα. In kα = {0.5893, 0.7599, 1.01, 1.026}, respectively. The single Hopf bifurcation occurs around the parameter value kα = 1.313. The bifurcation diagram in A provides us with a way to predict the Ca2+ signals for a large set of the parameter kα. Notice that stable periodic solutions occur for the parameter intervals kαs =  ∪ . We have MMOs for the parameter interval kαmmo =  with various sub-threshold oscillations patterns. 2+ signals as the parameter kα remains close to the period doubling value 1.01. kα = 1.25 near the Hopf point.2+ response based on the value of kα. One possible way of dealing with the aberrant signals and MMOs is to drive the maximal reaction rate of the RyR up. This corresponds to increasing the value of k2 in the model. kα on the x-axis and k2 on the y-axis. What this diagram illustrates is the region of MMOs produced by the model for the parameter ranges of kα and k2 given in the figure. Suppose that for a fixed kα value, Ca2+ signals undergo aberrant signals or MMOs. By increasing the parameter k2 sufficiently, passage into stable periodic solutions will occur. This suggests that the maximal rate of the RyR kinetics may help to control both aberrant Ca2+ signals and MMOs. To see this, kα = 0.9 when k2 = 0.18 is increased to k2 = 0.5. Similarly, kα = 1 when k2 = 0.18 is increased to k2 = 0.65. Notice that although solutions do settle into stable periodic orbits that the amplitude of the signals increase. Thus, increasing the parameter k2 may help to stabilizes aberrant oscillations at the cost of increasing Ca2+ oscillation amplitude. A similar stable region also exists below the region of MMOs. This region would also help control signals but may be more difficult to precisely isolate the appropriate range of k2.Each trace illustrated in 3 in the cytosol. Recall that in vivo, IP3 production is typically a result of an agonist activated G-couple protein. The rates of IP3 production and degradation are both modulated by intracellular Ca2+, and as such, the release of Ca2+ through the IP3 receptor can directly alter the IP3 signaling pathway. Furthermore, there is growing evidence that Aβ also affects the IP3 signaling pathway [3 production and degradation, and look to include the influence of Aβ on this signaling mechanism when ps = 0. More specifically, we include an additional equation for IP3 and model the influence of Aβ on the production of IP3.The model dynamics exhibited in the figures above are triggered by a constant value of IP pathway . Thus, w3 production and degradation. Their model takes the form3 and the second term is the degradation. In  to track IP06 to trasms (see  for detaβ directly into Xenopus oocytes causes an increase in Ca2+ dependent fluorescence (a measure for the amount of intracellular Ca2+). Even though their experiments are in oocytes, the ubiquitous properties of IP3 signaling may make their results relevant to other cells including neurons. Their findings suggest that Aβ does not interact directly with the IP3 receptor, but instead they propose that intracellular Ca2+ liberation evoked by Aβ involves opening of IP3 receptors as a result of stimulated production of IP3 via G-protein-mediated activation of PLC. As such, in the presence of Aβ, IP3 are actively stimulated and persist for many minutes or hours even though IP3 is metabolized within tens of seconds [VPLC takes the formKPLC can be written similarly asμPLC and κPLC control the strength of the linear influence of Aβ on each term, respectively. For our purposes, we set both of these values to be μPLC = κPLC = 1. Thus, we add the following equation to  showed that introducing A seconds . Based oation to  and 19)β directlβ on multiple Ca2+ signaling mechanisms. Although the model has a large number of variables and parameters, we seek to characterize model solutions by investigating the dynamical properties of the model in the presence of Aβ.In our simulation we use the work of  to set aβ, model solution with initial condition for IP3 = 0.01 shows a small Ca2+ influx followed by a transition to it’s steady-state level close to 0.5 μM. This is consistent with what we expect as the amount of IP3 is dynamic and depends on Ca2+. It will take sometime for enough IP3 to be present in order to trigger a signaling event throught the receptor. 2+ and IP3, respectively in the absence of Aβ. Notice that the amount of IP3 also reaches a steady-state level.In the absence of Aβ on Ca2+ signals, we use a as a bifurcation parameter and investigate model solutions. a as the bifurcation parameter. This diagram has two oscillatory regions separated by a region of a single stable-steady state. The four Hopf bifurcations are labeled along with a number of period doubling points. Notice that there are four regions of MMOs (shaded regions) that appear close to each Hopf point. In addition to the Hopf bifurcation points, four period doubling points have also been labeled with two limit points (LP). We provide the value of each of these points in a increases to LP3 = 1.274, solutions become unphysical. Thus, we limit our investigation for values of a between 0 and 1.274. 2+ traces predicted by a. Notice that aberrant signals are also present for the model with a dynamic equation for IP3.Since we are interested in understanding the potential effect of AV and the resulting Ca2+ current are shown in t = 0.1 seconds initiated at t = 2 generates the potential response shown in t = 0.1, t = 1, and t = 10 seconds, respectively. Notice that in 2+ through the VGCC are included in the flux term Jvca and as such, allows us to study the impact of membrane potentials on Ca2+ signals. In all subsequent figures, we have applied a constant current pulse at t = 100 for a duration of 50 seconds. Such an applied current does not capture an in vivo-like representation of membrane potentials, but it does offer a way to link the model with typical voltage clamp experiments where the amplitude and duration of the applied current can be controlled.In our simulations below, we examine the effects of changes to membrane potentials by first incorporating the membrane into the model and by stimulating the membrane with a constant applied current pulse. The impact of such an applied current on the voltage 2+ signaling, we first simulate 2+ signal2+ signals seem to stabilize in single-mode oscillations upon the release of the stimulus before transitioning back into MMOs. This may provide one possible path for stabilizing Ca2+ signaling. In an in-vivo like environment, membrane potentials vary based on intrinsic response mechanisms to a stimulus. Our results show that artificially triggering membrane stimulation could potentially help to stabilize Ca2+ signals. Furthermore, 2+ signals and play an important role in Ca2+ regulation. A full analysis for understanding these transitions is beyond the scope of this work but could prove useful for predicting how membrane stimulation may be used to control and/or stabilize aberrant Ca2+ signals.Notice that in 3 and the inclusion of membrane potential, but also by the amount of Aβ present in the model. To better understand the roles of IP3, membrane potentials, and Aβ on Ca2+ signaling, we simulate the model and provide the solutions when p = 10 and p = 15 with various levels of Aβ for ps = 1. Illustrated in β and membrane potentials on Ca2+ signaling. When p = 10 is fixed and a is altered, model behavior is directly impacted by the application and removal of a constant current pulse. This is illustrated in a = 0.2, a = 0.25, and a = 0.28. Specifically, 2+ signals do not go back to MMO patterns but instead transition to stable single-mode oscillations. This shows that stimulation of the membrane can alter intrinsic dynamical patterns and be used to stabilize various types of Ca2+ signals. p = 15 with a = 0.2, a = 0.25, and a = 0.28, respectively. It is interesting to note that when we apply a constant current pulse, Ca2+ solutions can transition into steady MMOs as the level of Aβ increased towards a = 0.28. Although we do not analyze the bifurcation structure, the inclusion of membrane potentials in the model appears to have altered the parameter dependance where regions of MMOs can occur as well as transitions from single- to mixed-mode oscillations. Further analysis may be beneficial for drawing out the underlying mechanisms in the stable oscillatory patterns when a constant current is applied in the model.Model solution patterns are not only linked to the amount of IPps, we have also provided model simulations when this parameter is altered. Small values of ps correspond to small influence of membrane potential while larger values can be used drive the amplitude of Ca2+ signals. ps. When ps = 0.5, 2+ are observed other than a slight increase in the subtreshold oscillations. When ps = 0.75, 2+ oscillations during the applied current. p = 1, contributions from membrane potentials increase overall Ca2+ signaling amplitude. With these values of p and a, the solution transitions from single mode oscillations with amplitude around 0.45 to MMO with an increased amplitude to around 0.7. p = 1.5) does not alter the signal amplitude or oscillatory mode significantly.Because the impact of membrane potentials are controlled by β can alter Ca2+ regulatory mechanisms in a way that leads to both MMOs and aberrant signaling. We have shown that bifurcation regions for dynamical transitions from single mode to MMOs can increase in the presence of Aβ. By altering RyR receptor dynamics, we show that transitions from MMO back to single-mode oscillations can occur. Furthermore, we show that stimulation of the membrane can also be used to control various types of Ca2+ signals. Although we have made a number of simplifying assumptions in the model development, our approach can be easily altered to include other more complex interactions and mechanisms that influence Ca2+ regulation.Although much analysis remains to fully understand the dynamics of the model, the results of our simulations suggest that A2+ is a critically important second messenger within the nervous system. In neurons, Ca2+ is known to mediate the signaling pathways that control neurotransmitter release, gene expression, metabolism, plasticity, development, proliferation, and cell death [2+ may play a major role in the pathogenesis of AD. Unfortunately, the complexity of Ca2+ signaling makes it difficult to precisely understand how Aβ impacts different intracellular regulation mechanisms and components. Various studies have decoupled particular components and merging these theories together to form a whole-cell computational model can help us better understand intracellular Ca2+ regulation and what leads to aberrant signaling. One of our goals is to model Ca2+ in a simplified whole-cell environment that has predictable qualitative structure so that we can study the effects of Aβ on the dynamics. As such, the qualitative features described in this study give us a way to track Ca2+ patterns using dynamical systems theory.Intracellular Call death . As suchβ to describe the potential stage in the evolution of AD. Even before toxic Aβ plaques can aggregate, the slow accumulation of Aβ peptides can trigger alterations in Ca2+ signaling patterns. Our model shows that aberrant signals and changes in homeostasis levels can emerge as the amount of Aβ is increased. In an in vivo environment these changes may be subtle and actually evolve over days or months. Any alterations in intracellular Ca2+ homeostasis can affect the apoptotic signaling cascade. Both the mitochondria and the ER play a significant role in apoptosis and are sensitive to changes in Ca2+ levels. Although we have not considered mitochondrial effects, we do track ER Ca2+. Further analysis that looks at the time evolution of ce could be useful in predicting chronic changes of Ca2+ homeostasis in AD.The simulations presented in this study occur on the order of seconds to minutes while the progression of AD occurs on the timescale of months to years. However, in our model we are using the accumulation of A2+ model that has predictable qualitative structure in the presence of Aβ is challenging. Although Aβ influences many Ca2+ regulatory mechanisms, the particular way which Aβ affects these mechanisms is generally not known. Additionally, the temporal influence of Aβ on certain mechanisms could occur on the order of milliseconds, seconds, days, months, or years. As such, any computational model will necessarily make a number of simplifying assumptions. Even by exploiting these simplifications, our model includes a large number of parameters that make mathematical analysis limited. Unfortunately, we do not have robust estimates for many of the parameters involved in the model. However, we have attempted to provide justification for many assumptions and parameter choices based on the literature and the experimental data currently available. We do recognize that many of these assumptions may need to be altered as we continue to improve our understanding of the effects of Aβ in an AD environment.Developing a whole-cell Ca2+ regulatory mechanisms used in our model makes it easily adaptable for studying various cell types with spatial components. Specifically, Aβ has been shown to cause complex Ca2+ signals in astrocytes [2+ waves and oscillations signals can occur on timescales even slower than those typical of other non-excitable neuroglia. Although our modeling approach does not include spatial components, additional mechanisms can be constructed to account for wave generating behaviors. Furthermore, astrocytes can facilitate synaptic transmission and plasticity through the uptake of neurotransmitter [β clearance and degradation [The ubiquitous nature of the Catrocytes , 62. In nsmitter  and compnsmitter , 64, 65.radation  and our β plays an essential role in the cognitive decline in AD by directly affecting synaptic transmission [2+ ions to flow into the cell through VGCC. The contributions of fast local Ca2+ signals with slow global Ca2+ patterns, especially under the influence of Aβ, may help explain why breakdowns in synaptic efficacy can occur in an AD environment [β may directly, or indirectly, impact various Ca2+ driven mechanisms during synaptic transmission. The simplified whole-cell Ca2+ model presented here could be linked with a synaptic Ca2+ model to investigate how global aberrant Ca2+ signals may impact synaptic transmission on multiple timescales. Simulations over long timescales may help explain how slow global whole-cell Ca2+ signals interfere with fast local Ca2+ signals at the synapse.It is clear that Asmission , 67–70. ironment , 71, 72.3 receptor model. Although this model is sound and well-suited for our purposes, it does increase the number of necessary variables considerably. One could use a two equation model for IP3  formulation for the IPribed in , 73), buel as in ). As suc2+ regulation along with understanding the impact of Aβ, any computational model would benefit from both local and global sensitivity analysis. Although we have not performed any sensitivity analysis, we do understand that much of the analysis and many of our conclusions may be valid for a small set of parameters. Further, the sensitivity of a particular parameter may influence how the model transitions into aberrant Ca2+ signals. As such, we recommend that sensitivity analysis be performed as a next step in order to better understand the role of parameters on model dynamics.Our model solutions are highly sensitive to certain parameters and the oscillatory responses presented here only occur under certain scenarios. Because of the complexity of Ca2+ signals can occur in a simplified whole-cell model under the influence of Aβ. Furthermore, we showed that regions of MMOs can expand as a consequence of increasing the amount of Aβ in the model. This may partially explain how Ca2+ signals are impacted by Aβ from a dynamics perspective within an in vivo like environment. Continued refinement of the model in conjunction with experimental data matching will help make the model more useful. In turn, this can help us determine how to control for both aberrant signals and increased homeostasis Ca2+ levels. The model can then be used to better understand the impact of Aβ on Ca2+ fluxes through individual regulatory components . This computational model can help us study complex cellular behavior in an AD environment by tracking the influence of many interconnected biological mechanisms.In conclusion, we have shown that aberrant CaS1 AppendixAlso provided in the appendix are the model parameters used in the Hodgkin and Huxley formulation of the membrane potential.(PDF)Click here for additional data file.


Fertilization-induced Ca2+ oscillations are essential for activation of the embryonic development, therefore maintaining proper Ca2+ homeostasis is crucial for the oocyte quality. In the present paper, we show that the mechanism underlying age-dependent alterations in the pattern of sperm-triggered Ca2+ oscillations is more complex and multifaceted than previously believed. Using time-lapse imaging accompanied by immunostaining and molecular analyses, we found that postovulatory ageing affects the amount of Ca2+ stored in the cell, expression of Ca2+ pump SERCA2, amount of available ATP and distribution of endoplasmic reticulum and mitochondria in a manner often strongly depending on ageing conditions (in vitro vs. in vivo). Importantly, those changes do not have to be caused by oxidative stress, usually linked with the ageing process, as they occur even if the amount of reactive oxygen species remains low. Instead, our results suggest that aberrations in Ca2+ signalling may be a synergistic result of ageing-related alterations of the cell cycle, cytoskeleton, and mitochondrial functionality.Postovulatory ageing of mammalian oocytes occurs between their ovulation and fertilization and has been shown to decrease their developmental capabilities. Aged oocytes display numerous abnormalities, including altered Ca Aged oocytes display a wide range of various abnormalities, such as dysfunction of actomyosin and microtubular cytoskeleton13, decreased activity of M-phase promoting kinases15, deregulation of energy metabolism18 and epigenetic alterations20. The extent of these abnormalities depends on the length of ageing period and, most likely, the genetic background of the cell. It has been also reported that postovulatory ageing disturbs Ca2+ signalling in oocytes and alters the pattern of sperm-induced Ca2+ oscillations27. The exact mechanism of this particular disturbance is not known, although it has been suggested that it may be caused by ageing-dependent oxidative stress25.Postovulatory ageing in mammals occurs between ovulation of mature oocytes and their fertilization and has been shown to decrease oocytes’ ability to react properly to sperm penetration and to develop further into the embryo2+ oscillations are essential for activation of the embryo development. They trigger processes such as completion of meiosis, establishment of the block to polyspermy and recruitment of maternal mRNAs required for activation of the embryonic genome29. In addition, Ca2+ oscillations regulate functioning of mitochondria32 and influence gene expression during further development of the embryo34. It has been shown that Ca2+ must be elevated for different time to induce properly each of these processes. If the number of Ca2+ transients is too low , the embryo implantation is impaired; on the other hand, an excessive number of Ca2+ transients (i.e. Ca2+ is elevated for too long) affects postimplantation development of the embryo35.Fertilization-induced Ca2+ oscillations are triggered in oocytes by phospholipase C zeta (PLC zeta), introduced by a fertilizing sperm36. PLC zeta cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to 1,4,5-inositol triphosphate (IP3) and diacylglycerol. IP3 binds to its receptor  located in the endoplasmic reticulum (ER) membrane. IP3R1 acts as a Ca2+ channel and opens upon IP3 binding, enabling the release of Ca2+ ions from the ER lumen into the cytoplasm. When the cytoplasmic Ca2+ concentration increases above a threshold level (low micromolar concentrations), the Ca2+ channel closes; the release of Ca2+ stops and the uptake of Ca2+ ions to the ER lumen is facilitated. The uptake of Ca2+ ions is mediated by ATP-dependent Ca2+ pumps . Restoration of the Ca2+ level in the ER is required for the next Ca2+ release39. This interplay between IP3R1-mediated Ca2+ release and SERCA-mediated Ca2+ uptake constitutes the main mechanism of cytoplasmic Ca2+ oscillations observed in fertilized oocytes. Ca2+ oscillations cease after few hours when oocytes enter interphase42.Cain vivo conditions has significantly more detrimental effect on Ca2+ homeostasis in mouse oocytes than in vitro ageing. In both conditions, postovulatory ageing affects several elements of the mechanism generating fertilization-induced Ca2+ oscillations, such as the amount of Ca2+ stored in the cell, expression of IP3R1 and SERCA2, the amount of available ATP and distribution of ER and mitochondria, but in a different way or to a different extent. We also argue that those changes are not necessarily caused by oxidative stress, as they occur even if the reactive oxygen species (ROS) level is not increased. Instead, our data suggest that aberrations in Ca2+ oscillatory pattern are a synergistic result of many ageing-related modifications, including alterations of the cell cycle, cytoskeleton, and mitochondrial functionality.In the present paper, we show that postovulatory ageing in in vitro and in vivo) on the pattern of Ca2+ oscillations generated in fertilized mouse oocytes, we subjected mature, metaphase II oocytes to 9- or 25-hour-long aging, either in in vitro culture or in female oviducts. Then, we labelled oocytes for Ca2+ ions using a fluorescent dye Oregon Green 488 BAPTA-1AM (Oregon Green BAPTA), fertilized them in vitro and subjected to time-lapse imaging. Representative Ca2+ traces are presented in Supplementary Fig. in vitro and in vivo conditions, increased the frequency of Ca2+ oscillations, as compared to freshly ovulated counterparts. However, only in vivo ageing shortened their total duration as well . The same rule applied to the total length of Ca2+ oscillations: in some aged oocytes they were shorter and in some significantly longer than in freshly ovulated ones , suggesting that the 25-hour-long ageing may prolong Ca2+ oscillations. It has been shown that in mouse Ca2+ oscillations cease when fertilized oocytes enter interphase of the 1st embryonic division. This decline may be caused by PLC zeta sequestration into the newly formed pronuclei42  or/and decrease in IP3R1 phosphorylation by M-phase kinases, IP3R1 degradation, and change in ER distribution47. Oocytes aged for 25 hrs often did not form pronuclei during 7 hrs of the recording (data not shown), and instead they remained arrested in M-phase with disarrayed spindles, and, in consequence, displayed most likely high activity of M-phase kinases. It has been reported before that oocytes arrested in metaphase II by microtubule depolymerizing drugs, such as nocodazole or colcemid, generated significantly prolonged Ca2+ oscillations  of low frequency and constant amplitude49. It seems that the inability of oocytes aged for 25 hrs to enter interphase is related to the spindle abnormality present already at the time of fertilization that inhibits M-phase/interphase transition through the spindle assembly checkpoint (SAC)50 50 Fig. . It has in vivo decreased the amplitude of Ca2+ oscillations , both in the 9- and the 25-hour-long variants. In case of in vitro ageing, Ca2+ transients had lower amplitudes only after 25 hrs. We also noticed that the duration of the 1st Ca2+ peak in oocytes aged for 9 hrs in vitro, but not in vivo, was shorter than in freshly ovulated ones, whereas in oocytes aged for 25 hrs the shortening occurred in both conditions , the concentration of cytoplasmic Ca2+ increased at a slower rate during the 1st Ca2+ transient and decreased at a slower rate during the 1st and the 3rd Ca2+ transients while compared to fresh oocytes. In in vivo conditions, the increase and decrease of Ca2+ cytoplasmic levels during the 1st and the 3rd Ca2+ spikes slowed down already after 9 hrs and remained like this after 25 hrs  do not seem to be related to their M-phase kinase activity: they are comparable in oocytes that progressed to interphase after fertilization and in those that remained in M-phase 51. Nevertheless, the tendencies we observed in the analysed groups seem to be real, as they accord with the previously reported data25. Due to the characteristics of Oregon Green BAPTA susceptibility to Ca2+, the actual differences in the Ca2+ concentrations between the groups may be just more pronounced than what we have shown.Postovulatory ageing ons Fig. . Ageing hrs Fig. , S2C,D. in vivo postovulatory ageing alters the Ca2+ oscillation pattern faster than in vitro ageing: it changed 8 out of 11 analysed parameters after 9 hrs, comparing to 2/11 in the in vitro variant. After 25 hrs in both variants more than half of the parameters were changed: 9/11 in in vivo and 6/11 in in vitro ageing. Ageing affected the frequency of Ca2+ transients, their amplitude, duration and dynamics of Ca2+ increase/decrease, and most likely, the total duration of Ca2+ oscillations. Interestingly, in vivo and in vitro aging affects differently not only Ca2+ oscillations, but also activities of M-phase kinases (MPF and MAPK) and profile of protein synthesis; in this case, the same as in our experiments, the effect of in vivo ageing is much more severe than that of in vitro aging52.In summary, 2+ oscillation pattern may be caused by the modified amount of Ca2+ stored in oocytes or/and altered expression of proteins involved in generation of the oscillations. To assess the amount of Ca2+ stored in the ER, we labelled oocytes with Oregon Green BAPTA and treated with thapsigargin (TG), a SERCA inhibitor, which induces a release of Ca2+ from the ER cisterns to the cytoplasm. Increase in the Oregon Green BAPTA fluorescence observed afterwards reflects the size of the ER Ca2+ store. Both in vitro and in vivo ageing, for 9 and 25 hrs, led to a decrease in the amount of Ca2+ available from the ER  pathway, and in porcine oocytes overexpressing ORAI1 (but not STIM1)63.Importantly, the decreased expression of SERCA2 protein, especially in 66. We noticed that indeed, Ca2+ oscillations in oocytes aged for 9 hrs in vivo, i.e. in the company of cumulus cells, were shorter, displayed less Ca2+ spikes, and the 1st Ca2+ transient had on average lower amplitude and slower rate of Ca2+ release than oocytes aged for the same time in vitro. Those differences between the ageing conditions diminished or disappeared after 25 hrs. We, therefore, examined whether the presence of cumulus cells during the 9-hour-long in vitro culture enhanced the effect of ageing on Ca2+ oscillations. Interestingly, we did not notice such a tendency. Although the amount of Ca2+ stored in the ER was depleted in oocytes cultured with cumulus cells to the level typical for oocytes aged in vivo  in oocytes aged in vitro and in vivo for 9 or 25 hrs. Measurements of fluorescence intensity of CellROX Orange, a ROS-sensitive dye, indicated that 9 hrs of in vitro ageing did not affect the abundance of ROS in oocytes, and a significant increase in ROS amount was observed only after 25 hrs. In contrast, both 9 and 25 hrs of in vivo ageing led to a decrease in ROS level, as compared to freshly ovulated oocytes 70. The amount of ROS in such oocytes was significantly lower than in oocytes aged without NAC and similar to the ROS level in fresh oocytes  for Mg2+ and Rhod-2 for Ca2+ ions , fertilized them and subjected to time-lapse imaging. The specificity of MgGreen dye towards Mg2+vs. Ca2+ ions was tested by challenging the labelled oocytes with FCCP or TG. As expected, FCCP led to an abrupt increase in the MgGreen fluorescence reflecting a decrease in the ATP amount caused by uncoupling oxidation from phosphorylation in mitochondria75, whereas TG did not alter significantly the MgGreen fluorescence  oocytes, the general decrease in the MgGreen signal was lower than in freshly ovulated oocytes. A similar decrease in the ATP production was also observed in oocytes aged in vitro for 25 hrs  displayed difficulties in readjusting ATP levels after fertilization-induced Ca2+ transients , we observed it also in oocytes aged in vivo, in which ROS levels were actually decreased in comparison to the fresh oocytes.As shown beforeion Fig. . In in vhrs Fig. . Additio78. Indeed, we showed that ageing alters the structure of ER and mitochondria network, facilitating formation of the cistern aggregates , an actin-depolymerizing agent. As expected, we observed aggregates of ER cisterns and mitochondria similar to those in postovulatory aged oocytes, although never as pronounced as in oocytes aged for 25 hrs in vivo  metabolism83. Moreover, it seems that actin cytoskeleton regulates also ER-mitochondria contact sites that are crucial for Ca2+ signalling in the cell86.Postovulatory ageing often leads to aggregation of organelles, including mitochondria and ER cisternstes Fig. . As ER aivo Fig. . In fertnts Fig. . Indeed,2+ homeostasis in postovulatory aged oocytes may be caused by deregulation of the cell cycle. As we described before, prolonged Ca2+ oscillations in oocytes aged for 25 hrs are likely related to the inability of those oocytes to enter the 1st embryonic interphase. In oocytes aged for 9 hrs in vivo, we observed another cell cycle abnormality: they tend to undergo spontaneous activation before fertilization, which was manifested as formation of the 2nd polar bodies , a parthenogenetic agent. EtOH induces in oocytes a single large Ca2+ transient87. It activated fresh oocytes very effectively, so within approx. 1.5 hrs they reached the same stage of activation as seen in oocytes aged for 9 hrs in vivo  decreased in the EtOH-treated oocytes to the same level as in the aged oocytes. We showed that in the pre-activated oocytes the duration of Ca2+ oscillations, the frequency and the amplitude of Ca2+ transients, as well as the rate of Ca2+ increase/decrease during the transients were similar to those in oocytes aged for 9 hrs in vivo and significantly decreased as compared to the fresh oocytes. Moreover, parameters such as the duration of the 1st and the 3rd Ca2+ transients or the number of Ca2+ transients remained unchanged compared to fresh oocytes, the same as in oocytes aged for 9 hrs in vivo , an inhibitor of protein synthesis. This procedure activated oocytes less uniformly and efficiently than EtOH and at the time of fertilization, usually only a subset of cells formed anaphase bulges (as opposed to the 2nd polar bodies visible in the majority of EtOH-treated oocytes and the oocytes aged for 9 hrs in vivo)  or to the different mechanism of activation. Ca2+-induced activation leads for example to degradation of IP3R1 receptor88 that most likely is not observed in Ca2+-independent mode of oocyte stimulation. Taken together, these results suggest, however, that spontaneous activation may be at least partially responsible for the modification of the Ca2+ oscillation pattern in in vivo aged oocytes.Alterations in the pattern of Ca2+ oscillations caused by postovulatory ageing may lead to oocyte apoptosis and fragmentation89. Pro-apoptotic stimuli provided by postovulatory ageing, such as the decreased amount of BCL-2, ATP or increased oxidative stress91, may push aged oocytes upon fertilization to cell death. Indeed, majority of oocytes aged for 25 hrs (63.3% (19/30) in in vitro and 53.5% (23/43) in in vivo conditions) underwent fragmentation  within few hours after fertilization, while fragmentation occurred neither in freshly ovulated oocytes, nor oocytes aged for 9 hrs ; fertilized oocytes that had remained arrested in metaphase II did not undergo this process  and were performed in compliance with the national regulations. All reagents were purchased from Sigma-Aldrich unless stated otherwise.F1 (C57Bl6/Tar × CBA/Tar) mice were maintained in the Animal Facility of the Faculty of Biology, University of Warsaw at 14:10 light/dark cycle and provided with food and water in vitro aged oocytes); (ii) 24 hrs after hCG (oocytes aged in vivo for 9 hrs); (iii) 40 hrs after hCG (oocytes aged in vivo for 25 hrs). Except for some experiments (ageing with cumulus cells), recovered oocytes were placed in hyaluronidase solution (150 IU/ml in PBS) to remove the cumulus cells. Denuded oocytes were washed in M2 medium (M16 buffered with Hepes97) and either subjected to further experimental procedures or cultured in vitro for 9 or 25 hrs (in vitro ageing for 9 and 25 hrs) in M2 medium and 37.5 °C and 5% CO2 in the air. In some experiments, freshly ovulated oocytes were incubated for 4 hrs with or without cytochalasin D . Some fresh oocytes were also parthenogenetically activated with 8% ethanol solution  or with 2.5 mM 6-DMAP and 10 μg/ml cycloheximide (CHX)  i.e. approx. 1.5 hrs), and others – treated for 15 min with 100 µM H2O2 (in M2 medium). In some experiments, oocytes were aged in vitro for 25 hrs in M2 medium supplemented with 5 mM N-acetylcysteine70.Female mice were superovulated with an intraperitoneal injection of 10 IU of pregnant mare serum gonadotrophin  followed 48 hrs later by 10 IU of human chorionic gonadotrophin . Ovulated oocytes were recovered from oviducts: (i) 15 hrs after hCG  were subjected to acidic Tyrode’s solution (pH 2.5)99 in order to remove zonae pellucidae. Oocytes without zonae were transferred to a glass-bottom dish (MatTek Corporations) with M2 medium without BSA and allowed to stick to the glass bottom (in some experiments M2 medium without BSA but with 2 µg/ml CCD was used). Next, the dish was placed on the time-lapse imaging system (Zeiss Axiovert microscope with AxioCam HRm camera) equipped with an environmental chamber sustaining a temperature of 37.5 °C. 1-2 μl of capacitated sperm suspension was added to the oocytes. The experiment was repeated 3-5 times for each variant with consistent results  mean intensity of fluorescence was calculated for each oocyte and then used to normalize the measurements in this oocyte. The resulting values are ratios: measured fluorescence intensity (F) /initial fluorescence intensity (F0). The rates of increase and decrease of Ca2+ concentration during the Ca2+ transients were calculated as tangents of the rising/decreasing slopes of the Ca2+ transients . Changes in cytoplasmic Ca2+ and Mg2+ ions. After 2 minutes of time-lapse imaging (conditions the same as described in ‘Imaging of Ca2+ oscillations’ section) thapsigargin (TG) or A23187 ionophore were added to the medium . Single-plane images were taken every 10 s for approximately 30 min (the settings were the same as described for imaging of Ca2+ oscillations). Changes in the Oregon Green BAPTA fluorescence were analysed as described for the imaging of Ca2+ oscillations.Oocytes were loaded with 5 μM Oregon Green BAPTA  and transferred to a glass-bottom dish with M2 medium devoid of Ca2+ ions, as described by Igarashi et al.2. The intracellular Mg2+ pool is predominantly present as MgATP2− and the affinity of ATP4− for Mg2+ is about 10-fold greater than that of ADP at physiological pH. Therefore, Mg2+ increases when ATP hydrolysis exceeds the rephosphorylation of ADP to ATP and vice versa74. To simultaneously image changes in the cytoplasmic Ca2+ and Mg2+ concentrations, oocytes were incubated for 30 min with 5 μM Rhod-2 AM  and 5 μM Magnesium Green AM , respectively. Then, their zonae pellucidae were removed with acidic Tyrode’s solution and oocytes were transferred to a glass-bottom dish with M2 medium without BSA and allowed to stick to the glass bottom. Next, they were placed on the time-lapse imaging system (Zeiss Axiovert microscope with AxioCam HRm camera) equipped with an environmental chamber sustaining a temperature of 37.5 °C, and fertilized as described in ‘Imaging of Ca2+ oscillations’ section. To test the specificity of MgGreen towards Mg2+ ions vs. Ca2+ ions, oocytes, labelled as described above, were subjected in separate experiments to 1 μM FCCP or 10 μM TG while imaged. Imaging was performed in normal M2 medium or M2 medium devoid of Ca2+ and Mg2+ ions, respectively.To assess changes in the ATP concentration during fertilization, we visualised Mg2+ concentration were assessed by measuring the mean intensity of Rhod-2 fluorescence in time, whereas changes in the Mg2+ concentration – the mean intensity of MgGreen fluorescence. To avoid any additional variability between the experiments caused by the different extent of dye loading, the initial (pre-fertilization) mean intensity of fluorescence was calculated for each oocyte and then used to normalize the measurements in this oocyte. The resulting values were ratios: measured fluorescence intensity (F) /initial fluorescence intensity (F0). Additionally, a drift caused by the photobleaching of MgGreen was calculated for each experiment (we used traces obtained for unfertilized oocytes) and then subtracted from the experimental measurements for fertilized oocytes.Time-lapse imaging with single-plane images taken every 10 s was performed for approx. 7 hrs for the fertilization experiments and 30 min for the experiments testing MgGreen specificity. Oocytes were illuminated with light passing through 450–490 nm and 538–562 nm excitation filters, and the emitted light was collected with 500–550 nm and 570–640 nm emission filters, for MgGreen and Rhod-2, respectively . Changes in the cytoplasmic Caexp) was normalized with the mean fluorescence intensity in control, freshly ovulated oocytes dyed and imaged simultaneously (Fcontr).Oocytes were loaded with 5 µM CellROX Orange  and transferred to a glass-bottom dish with M2 medium. Single equatorial plane images of the oocytes were taken on a fluorescence microscope (Zeiss Axiovert microscope with AxioCam HRm camera) equipped with an environmental chamber sustaining a temperature of 37.5 °C. Oocytes were illuminated with light passing through a 538–562 nm excitation filter, and the emitted light was collected with a 570–640 nm emission filter . ROS concentration was assessed by measuring the mean intensity of CellROX Orange fluorescence. In each experiment the mean fluorescence intensity in aged/treated oocytes  was normalized with the mean fluorescence intensity in control, freshly ovulated oocytes dyed and imaged simultaneously (Fcontr).Oocytes were loaded with 100 nM TMRE (in M2 medium) for 30 min and transferred to a glass-bottom dish with M2 medium. Single equatorial plane images of the oocytes were taken on a fluorescence microscope (Zeiss Axiovert microscope with AxioCam HRm camera) equipped with an environmental chamber sustaining a temperature of 37.5 °C. Oocytes were illuminated with light passing through a 538–562 nm excitation filter, and the emitted light was collected with 570–640 nm emission filter . As TMRE is a potential-sensitive mitochondrial dyeOocytes were fixed in 4% PFA , permeabilized with 0.5% Triton-X100  and blocked with 3% BSA. Calnexin, an ER marker, was labelled with a rabbit polyclonal antibody  followed by an Alexa Fluor 633-conjugated goat anti-rabbit IgG  and β-tubulin – with a mouse monoclonal antibody conjugated with FITC (1:50). Embryos were incubated in the primary antibodies overnight at 4 °C, washed in PBS and 3% BSA and then, if required, incubated with the secondary antibody for 2 hrs in RT. DNA was stained with propidium iodide . Oocytes were analysed on an inverted confocal microscope (Zeiss and Olympus).100 and with a goat anti-SERCA2 antibody, diluted 1:350 and 1:200, respectively, in 5% non-fat milk in TTBS. A donkey anti-goat antibody and a goat anti-rabbit antibody (Bio-Rad), respectively, conjugated with horseradish peroxidase diluted 1:5000 were used as secondary antibodies in 1-hour-long incubation. Detection was performed by the enhanced chemiluminescence technique using SuperSignal West Dura Extended Duration Substrate reagents  according to the manufacturer’s instruction. Western blot analysis was repeated 3 times for IP3R1 and 2 times for SERCA2.Expression of IP3R1 and SERCA proteins was examined in samples of 50–80 oocytes, depending on the experiment. Cell lysates were mixed with 4× NuPage LDS sample Buffer and 10× NuPage Sample Reducing Agent  and heated for 10 min in 70 °C. The samples were subjected to NuPage Novex 3–8% Tris-Acetate gels  and separated proteins were transferred onto PVDF membranes . The blots were then stained with Ponceau S to confirm the equal sample load, blocked in 5% non-fat milk in TTBS and probed for 1 h with a rabbit polyclonal antibody (Rbt03) raised against a 15 amino acid peptide sequence of the C-terminal end of the IP3R125 bead suspension for 30 min at RT. mRNA was eluted from the beads by adding 10 μl of DEPC-treated water and heated for 10 min at 70 °C with 0.5 μg oligo-(dT)25. The reverse transcription was performed in a total volume of 20 μl using 200 U of Superscript II Reverse Transcriptase, 0.5 mM dNTPs and 40 IU RNase inhibitor  at 42 °C for 50 min. Synthesized cDNA was diluted twice with nuclease-free water (Thermo Fisher Scientific) and subjected to preamplification procedure  with TaqMan PreAmp Mix (Thermo Fisher) and pooled TaqMan Gene Expression Assay probes . The preamplification product was diluted twice and subjected to a real-time PCR using TaqMan Gene Expression MasterMix in StepOne Real-Time PCR System thermocycler . The relative level of expression was calculated using 2−ΔCt method101 with actin B expression used for normalization. PCR analysis was repeated 3–9 times depending on the experimental variant.Oocytes were transferred in groups of 30 into 20 μl of Lysis/Binding Buffer  and stored in −80 °C until further analysis. mRNA was isolated from the samples using the Dynabeads mRNA DIRECT Micro Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. In short, thawed samples were rotated with 10 μl of paramagnetic oligo-(dT)Kruskal-Wallis test with the post-hoc analysis and Mann–Whitney U test were applied to analyse the data. The differences between groups were considered statistically significant for p < 0.05.Supplementary InformationSupplementary Dataset 1


We analyzed antigen expression levels of CD33, CD123, CLL1, TIM3, CD244 and CD7 on AML bulk and leukemic stem cells at initial diagnosis (n = 302) and relapse (n = 54). CD33, CD123, CLL1, TIM3 and CD244 were ubiquitously expressed on AML bulk cells at initial diagnosis and relapse, irrespective of genetic characteristics. For each analyzed target, we found additional expression in different populations of normal hematopoiesis. Analyzing the coexpression of our six targets in all dual combinations (n = 15), we found CD33/TIM3 and CLL1/TIM3 to be highly positive in AML compared with normal hematopoiesis and non-hematopoietic tissues. Our findings indicate that combinatorial targeting of CD33/TIM3 or CLL1/TIM3 may enhance therapeutic efficacy without aggravating toxicity in immunotherapy of AML.Targeted immunotherapy in acute myeloid leukemia (AML) is challenged by the lack of AML-specific target antigens and clonal heterogeneity, leading to unwanted on-target off-leukemia toxicity and risk of relapse from minor clones. We hypothesize that combinatorial targeting of AML cells can enhance therapeutic efficacy without increasing toxicity. To identify target antigen combinations specific for AML and leukemic stem cells, we generated a detailed protein expression profile based on flow cytometry of primary AML ( Despite some advances in the treatment of acute myeloid leukemia (AML) in recent years, overall prognosis remains poor . AllogenIn B-ALL, CD19 can be considered an ideal target antigen owing to its high expression on leukemic cells and its restricted expression profile on normal cells: it is expressed on essentially all B-lineage cells, whereas negative on all other hematopoietic lineages or other normal tissues , 12. In Perna et al.  recentlyn = 302) and relapse (n = 54) as well as bone marrow aspirates from healthy donors (n = 34) were used. Sample size was chosen based on feasibility and experience with previous analysis [For flow cytometric analysis, peripheral blood or bone marrow aspirate samples of AML patients at initial diagnosis . In contrast, we observed no or lower correlation of the MFI ratio with the MFI index for the lymphoid-associated antigens TIM3, CD244 and CD7 (Spearman r < 0.79). The latter was owing to expression of these antigens on lymphocytes, which therefore did not serve as appropriate negative control. For this reason, we chose the isotype-based MFI ratio for our analysis. Positive expression in the majority of cells was defined as MFI ratio ≥ 1.5. MFI values were determined using FlowJo software (Version 9.8.5) .After collection, all samples were analyzed immediately, without prior cryoconservation. Samples were stained with the following fluorochrome-conjugated anti-human monoclonal antibodies: CD7 , CD33 , CD34 , CD38 , CD45 , CD123 , CD244 , TIM3 . Corresponding isotype controls were used for each sample. Surface antigen expression was assessed using a 10-color Navios flow cytometer . Gating was performed as described in Supplemental Fig. Protein expression data for normal tissues were retrieved from the integrated dataset generated by Perna et al. , includiU test for unpaired samples and the Wilcoxon matched-pairs signed rank test for paired samples. Statistical significance was considered for p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****). Results are shown as medians ± 95% confidence interval or as indicated. Graphs were generated using GraphPad Prism 7, R Studio  and Adobe Illustrator CS6 .Statistical analysis was performed using GraphPad Prism 7 . The significance of differences was determined using the Mann–Whitney n = 302) and at relapse (n = 54). We quantified the antigen expression levels of CD33, CD123, CLL1, TIM3, CD244 and CD7 on AML bulk cells   and after allogeneic stem cell transplantation (n = 15)   Fig. .n = 37), NPM1 mut/FLT3-ITD (n = 30) and NPM1 wt/FLT3-ITD (n = 14) AML samples each had significantly higher expression of CD33 and CD123 compared with NPM1 wt/FLT3 wt (n = 40) AML samples, indicating that mutations of NPM1 and FLT3 are independently associated with higher expression of CD33 and CD123 compared with AML with non-mutated NPM1 or FLT3, respectively. Of note, in all evaluated molecularly defined sub-cohorts CD33, CD123, CLL1, TIM3 and CD244 were positive in most samples.To verify potential correlations of AML-associated antigens with genetic characteristics, we analyzed the level of antigen expression in different cytogenetically and molecularly defined AML risk groups according to the MRC and ELN2010 criteria , our analysis revealed that CD33/TIM3 and CLL1/TIM3 were coexpressed in most AML samples, but largely spared in normal hematopoiesis and non-hematopoietic tissues, thus meeting our criteria of suitable target antigen combinations.Starting from our panel of six AML-associated antigens and all possible dual antigen combinations thereof (In this study, we provide a detailed cell surface protein expression analysis of six commonly targeted and leukemic stem cell-associated antigens in AML. For the first time, we directly compare the antigen expression levels and coexpression of CD33, CD123, CLL1, TIM3, CD244 and CD7 on AML bulk cells, LSC and normal bone marrow cells, based on a cohort of > 300 AML patients and 34 healthy donors.We show that CD33, CD123 and CLL1 are highly expressed on AML cells of most patients, which is consistent with previous reports , 34, 43.Beside the commonly targeted AML-associated antigens, CD244 and TIM3 have been reported to be overexpressed on LSC and to have a direct leukemia-promoting effect by maintaining the proliferative capability of LSC , 46–50. Overall, our protein expression data indicate that single-targeting of CD33, CD123, CLL1, TIM3 or CD244 may have antileukemic efficacy in most AML patients. However, on-target off-leukemia toxicity both in hematopoietic and non-hematopoietic tissues as well as high risk of relapse owing to antigen escape and clonal heterogeneity may limit therapeutic success. Among all tested antigens, we report a suitable expression pattern for TIM3, which may qualify for single-targeting or combinatorial targeting approaches in AML.n = 15 possible target antigen pairs, we identify CD33/TIM3 and CLL1/TIM3 as the most suitable antigen combinations, with high coexpression in most AML samples and largely absent coexpression in normal hematopoiesis and non-hematopoietic tissues, excluding tissues with known immune infiltration. Notably, targeting of CD33/TIM3 and CLL1/TIM3 may lead to monocyte depletion. Although the tolerability of monocyte depletion needs to be further evaluated, there may be a rationale for targeting not only AML cells but also monocytes that are involved in disease pathogenesis and progression [We hypothesize that combinatorial targeting strategies with well-matched target antigens enhance therapeutic efficacy without increasing on-target off-leukemia toxicity. Several preclinical reports of combinatorial targeting have been published so far and are either based on triplebodies or combinatorial CAR approaches –57. In agression . The tarIn summary, our comprehensive analysis of > 300 primary AML samples demonstrates antigen positivity of CD33, CD123, CLL1, TIM3 and CD244 in most cases at initial diagnosis and relapse, irrespective of the genetic background. While none of these antigens are truly AML-specific, we describe a suitable expression profile of TIM3 with limited expression on normal cells. Our coexpression analysis of hematopoietic cells and non-hematopoietic tissues identifies CD33/TIM3 and CLL1/TIM3 as promising antigen combinations that should be validated in dual-targeting immunotherapeutic strategies.Supplemental Table 1Supplemental Table 2Supplemental Table 3Supplemental Table 4Supplemental Figure 1Supplemental Figure 2Supplemental Figure 3Supplemental Figure 4Supplemental Figure 5Supplemental Figure 6Supplemental Figure 7Supplemental Figure 8Supplemental Figure 9