In experiments employing myocytes and homogenates from the cardiac left ventricle or soleus muscle, we observed marked inhibition of Ca2+ recycling into the sarcoplasmic reticulum (SR) following incubation with polyarginine CPPs. In both tissues, the rate of SR Ca2+ leak remained unchanged, indicating that protracted Ca2+ removal from the cytosol stemmed from inhibition of the SR Ca2+ ATPase 2 (SERCA2). No such inhibition occurred following treatment with TAT, or in preparations from the SERCA1-expressing extensor digitorum longus muscle. Experiments in HEK cells overexpressing individual SERCA isoforms confirmed that polyarginine incubation specifically inhibited the activity of SERCA2a and 2b, but not SERCA1 or 3. The attenuation of SERCA2 activity was not dependent on the presence of phospholamban, and ELISA-based analyses rather revealed direct interaction between the polyarginines and the actuator domain of the protein. Surface plasmon resonance experiments confirmed strong binding within this region of SERCA2, and slow dissociation between the two species. Based on these observations, we urge caution when employing polyarginine CPPs. Indeed, as SERCA2 is expressed in diverse cell types, the wide-ranging consequences of SERCA2 binding and inhibition should be anticipated in both experimental and therapeutic settings.Cell-penetrating peptides (CPPs) are short peptide sequences that have the ability to cross the cell membrane and deliver cargo. Although it is critical that CPPs accomplish this task with minimal off-target effects, such actions have in many cases not been robustly screened. We presently investigated whether the commonly used CPPs TAT and the polyarginines Arg Cell-penetrating peptides (CPPs) are short peptide sequences of 5 to 30 amino acids, derived from natural or synthetic sources, that have the ability to cross the cell membrane and deliver cargo . The delThe cell membrane is a selective barrier that prevents the entry of large and charged molecules. However, CPPs possess unique properties that allow them to overcome this barrier. Many CPPs exhibit highly hydrophilic and cationic properties that allow them to interact with anionic components of the phospholipid bilayer . Ensuing2+ homeostasis, we presently examined whether the commonly used CPPs Arg9, Arg11, and TAT have off-target effects on intracellular Ca2+ uptake and release. Our results demonstrate that polyarginine CPPs bind to and inhibit the (sarco)endoplasmic reticulum (SR) Ca2+ ATPase type-2 (SERCA2), which complicates their pertinence for both experimental and therapeutic applications.While CPPs have generally been shown to exhibit low cytotoxicity and high cell penetration efficiency, the design and optimization of CPPs for specific applications nevertheless requires the careful consideration of factors such as peptide sequence, cargo type, and cellular targeting to ensure the desired outcomes and minimize potential off-target effects. Given our own interest in intracellular CaAll protocols were approved by the Norwegian Animal Research Authority and performed in accordance with the Norwegian Animal Welfare Act and NIH Guidelines. Experiments employed tissue and cells isolated from adult male Wistar rats (250–350 g), wild-type C57BL/6NT mice (20–30 g), and phospholamban knockout mice (20–30 g) . AnimalsN-terminal biotin tag at >80% purity by GenScript Biotech Corp  for experimental testing:TAT: RKKRRQRRR9: RRRRRRRRRArg11: RRRRRRRRRRRArgCargo: IEKELAQQYQNADAITLE, a scrambled control sequence of RIAD (LEQYNQLADQIIKEATEK) which is a high-affinity disruptor peptide for protein kinase A (PKA) type I .1–9: IEKELAQQYCargo8–18: QYQNADAITLECargo1–11: IEKELAQQYQNCargo111–253, containing the actuator domain, also known as the A-domain, of SERCA2  using the following equation: [Ca2+] = Kd × ((R − Rmin)/(Rmax − R)) × (Sf2/Sb2), where R is the 340 nm/390 nm fluorescence ratio, and Kd is the dissociation constant of Fura-2 and Ca2+ (224 nmol/L) [min is the fluorescence ratio at very low [Ca2+] and Rmax is the ratio at saturating [Ca2+], obtained by sequentially adding 3.3 mmol/L EGTA and 4.8 mmol/L CaCl2 to the well at the end of each recording. Sf2/Sb2 is the ratio of measured fluorescence intensity at 390 nm when Fura-2 is Ca2+-free (3.3 mmol/L EGTA) or Ca2+-bound (4.8 mmol/L CaCl2).Car 12 min . SR Ca2+ mmol/L) . The flu nmol/L) ,19. Rmin2+ recordings were smoothed using TableCurve 2D  prior to analysis. After the addition of Na4ATP, the rate constant for Ca2+ uptake (k) was calculated by fitting the uptake curve to Ca2+ = Ca2+0 + ae−t×k . Ca2+ leak from the vesicles was calculated as the linear rise (slope) of the Ca2+ signal after blockade of SERCA by thapsigargin. Leak measurements were normalized to SR content, determined as the peak [Ca2+] after addition of CmC.Ca2 in a humidified incubator, and transfected using Lipofectamine 2000, as described by the manufacturer . The employed gene constructs included rat SERCA1 (NM_058213.1), rat SERCA2a (NP_001103609.1), rat SERCA2b (NP_001104293.1), and rat SERCA3 (NM_012914.1) with GFP present at the N-terminus (pEGFP-C2 vector) (custom-made by GenScript). Following 24 h transfection, cells were washed with 5 mL 0.9% NaCl before harvesting in 1 mL homogenization buffer  293 cells were cultured to confluence in 10 cm Petri dishes in DMEM supplemented with 10% foetal calf serum, 100 U/mL penicillin, and 1% nonessential amino acids . Cells w111–253  or His-CaMKIIδ1–165  (control) per well and incubated overnight at 4 °C with gentle agitation. Thereafter, wells were washed once in PBS-T  before blocking with 0.5% gelatine  for one hour at room temperature. The coated wells were then incubated for 2 h at 37 °C with 50 µmol/L of biotin-labelled control peptides or CPPs  with gentle agitation. The control peptides consisted of 9–11 amino acids , derived from a scrambled control peptide sequence of 18 amino acids [A 96-well ELISA plate was coated with 1 × PBS (control), recombinant 12xHis-SERCA211 was immobilized at 744.3 resonance units (RUs). Recombinant 12xHis-SERCA2111–253 [111–253 were injected over the sensor surface at a flow rate of 30 µL/minute for 180 s. For the 3 experimental runs performed, the employed concentration range of 12xHis-SERCA2111–253 was 0.7–200 nM, 0.19–50 nM, or 12.5–200 nM. To be able to measure the slow dissociation rate of the interaction, the dissociation time of the instrument was set to 600 or 1000 s. Sensorgrams were analysed using Biacore X100 evaluation software with the assumption of one-to-one binding (Langmuir binding model).A Biacore X100  was employed for surface plasmon resonance analyses. Streptavidin (SA) chips  were conditioned with three consecutive 1 min injections of 1 × Biacore running buffer (BR100826) before biotinylated Argp-value < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism 9.4.1  or Sigmaplot v14.0, Systat Software Inc. .Data are presented as mean ± standard error of the mean. All data were tested for normality of distribution using the Shapiro–Wilk test. Normally distributed data were statistically tested with a one-way ANOVA with Tukey’s post hoc correction for multiple comparisons. Non-normal distributions were examined by Kruskal–Wallis analysis on ranks with Dunn’s post hoc. A 9, Arg11, and TAT . No such effects were noted in cells incubated with TAT or Arg9 , and these values were used to normalize Ca2+ leak measurements. No differences in Ca2+ leak were noted between the treatment groups (2+ uptake in the presence of the polyarginines results from the inhibition of SERCA activity.Following Ca11-cargo continued to significantly slow the Ca2+ reuptake rate in comparison with the untreated control experiments (9-cargo (p = 0.129). Again, we did not observe effects of the peptides on Ca2+ leak rate . However, incubation with the three peptides had similar effects as in wild-type experiments, with Arg11 tending to cause the most marked inhibition of Ca2+ uptake, both in the absence  . We testout mice . As expe absence A and pre absence B of atta2+ uptake in soleus homogenates  fibres  and, likogenates A show thd effect A. Similand Arg11 C. We didble Ca2+ .2+ uptake was observed to be markedly faster in the EDL than the soleus , none of the tested peptides altered the rate of Ca2+ uptake in the EDL  EDL muscle, which expresses SERCA1  Figure B. While  the EDL B,D, Ca2+ble Ca2+ . These fSERCA1, SERCA2a, SERCA2b, or SERCA3 constructs. After growing to confluence, the cells were harvested and vesicles were isolated as for the heart and skeletal muscle tissue. Ca2+ uptake measurements were then performed with Arg9 or Arg11 present in the incubation buffer. Neither Arg9 nor Arg11 altered the Ca2+ uptake rate in cells transfected with SERCA1 or SERCA3  or CPPs . The interactions were measured with PBS   C. We obs or Arg9 C.11 and SERCA2 using surface plasmon resonance to assess the affinity and kinetics of Arg11 binding  of 0.4 ± 0.1 nM, an association rate constant (ka) of 2.1 ± 0.7 × 105 M−1 s−1, and a dissociation rate constant (kd) of 6.6 ± 0.9 × 10−5 s−1  with negatively charged amino acids, such as aspartate and glutamate. While these residues are distributed throughout the A-domain of SERCA2, we note that E238 is present in SERCA2 but not SERCA1 or 3, and that this site is conserved across the three species we have currently examined . Arginine can also participate in hydrogen bonding with amino acids that contain hydrogen bond donor and acceptor groups, such as serine, threonine, asparagine, and glutamine. The threonine residue T166 is thus of particular interest to our current findings, as it is exclusively expressed within the A-domain of the SERCA2 isoforms, and also conserved across humans, rats, and mice. Finally, the guanidinium group of arginine can form pi-cation interactions with aromatic amino acids like phenylalanine, tyrosine, and tryptophan [How do the polyarginine CPPs interact with SERCA2? Our experiments in myocardial preparations from PLB-knockout mice indicate that this off-target action does not arise via effects on PLB, SERCA’s endogenous inhibitor A. Furthepeptides B. Surfacyptophan , althougyptophan ,6, these2+ transport. The A-domain is physically separated from the nucleotide-binding (N) and phosphorylation (P) domains while in the E1-2Ca state, which may allow polyarginine binding. However, ATPase activity requires that the three domains come together [How does polyarginine binding to SERCA2′s A-domain inhibit the pump’s activity? Importantly, this domain serves as SERCA2′s transduction element, critically coupling ATP hydrolysis with Catogether . We postWhile it is perhaps somewhat surprising that the off-target inhibition of SERCA2 has been overlooked in previous work employing polyarginine CPPs, we note that these studies have often only compared the CPP-mediated delivery of cargo and scrambled cargo. Our results stress the need to conduct additional control experiments that include an untreated group. An experimental group treated with the CPP of interest alone (without cargo) should also be considered, since scrambled cargo sequences can have unexpected physiological effects. In our experiments, we observed that Arg-homopolymers inhibited SERCA2 activity both in the absence and presence of scrambled cargo, in comparison with untreated controls . Based o2+ content and release, i.e., systolic dysfunction in this disease [2+ resequestration into the SR. Such effects may not occur at slower pacing rates, as we have employed in our cardiomyocyte experiments, where resting the Ca2+ levels are returned to steady state values between each stimulation  and PTDpeptides ); or thepeptides . In somepeptides . Excitinpeptides .2+ homeostasis can have profound effects on cellular physiology, we urge caution when employing polyarginine CPPs in both experimental and therapeutic settings.In conclusion, our present data show that polyarginine CPPs have previously unrealized off-target effects, as these peptides robustly bind and inhibit SERCA2. Given that SERCA2 is expressed in a broad range of cell-types, and that the manipulation of Ca


Here, using quantitative analysis of both single bouton Ca2+ influx and exocytosis, we uncovered an unexpected non-linear relationship between the magnitude of action potential driven Ca2+ influx and the concentration of external Ca2+ ([Ca2+]e). We find that this unexpected relationship is leveraged by GPCR signaling when operating at the nominal physiological set point for [Ca2+]e, 1.2 mM, to achieve complete silencing of nerve terminals. These data imply that the information throughput in neural circuits can be readily modulated in an all-or-none fashion at the single synapse level when operating at the physiological set point.Control of neurotransmission efficacy is central to theories of how the brain computes and stores information. Presynaptic G-protein coupled receptors (GPCRs) are critical in this problem as they locally influence synaptic strength and can operate on a wide range of time scales. Among the mechanisms by which GPCRs impact neurotransmission is by inhibiting voltage-gated calcium (Ca This sensitivity is driven by the cooperative binding of multiple Ca2+ ions to one or more Ca2+ sensors that form core elements of the exocytic machinery  blocking a fraction of Ca2+ channels, (2) lowering [Ca2+]e, or (3) application of a GPCR agonist, baclofen, known to reduce Ca2+ influx lead to complete silencing of a subset of nerve terminals. Thus, in this operating regime, decreasing presynaptic Ca2+ current may act as a digital switch to eliminate single bouton synaptic throughput.The impact of Caterminal . Thus, tcalcemia . Feedbac mammals . Here, w2+ influx driven by action potentials (APs) at nerve terminals is impacted by changes in [Ca2+]e, we used the genetically-encoded Ca2+ indicator physin-GCaMP6f transfected sparsely into primary cultures of dissociated rat hippocampal neurons (2+]e (2+ binding (determined following ionomycin application) and converted to absolute intracellular Ca2+ concentrations . Previous studies investigating the dependency of Δ[Ca2+]i to [Ca2+]e in nerve terminals of the CNS found the relationship obeyed a simple saturable pore model with a Kd of [Ca2+]e ~2.5 mM . One important confound, however, is that previous measurements were not carried out at physiological temperatures. Here, where the temperature was set to 37 °C, we observed an unexpected sub-proportional relationship of Δ[Ca2+]i to [Ca2+]e as the concentration was lowered below 2.0 mM  but a strikingly disproportionate increase in the mean proportion of silencing below 1.2 mM (2+]e was 48.9% mM–1 transitioning from 1.2 to 0.8 mM compared to 19.0% mM–1 from 2.0 to 1.2 mM (2+]e was 48.9% mM–1 transitioning from 1.2 to 0.8 mM compared to 19.0% mM–1 from 2.0 to 1.2 mM (2+]e 0.4 mM (2+]e 1.2 mM), demonstrating this level of [Ca2+]e potently and consistently shuts down presynaptic function. The change in percentage of silencing versus change in [Ca2+]e transition between 0.8 mM to 0.4 mM is 73.3% mM–1, again highlighting the steep dependence of silencing on [Ca2+]e below the physiologic [Ca2+]e. As expected, responding terminals demonstrated Δ[Ca2+]i that scaled with [Ca2+]e  and closely matched the relationship observed without toxin application .Our observation of steep changes in the proportion of nerve terminals in which Ca protein , tagged  protein . We empl protein . SimilarΔ[Ca2+]i . As expe]i , a genetically-encoded, fluorescent biosensor of extracellular glutamate concentrations with an excellent signal-to-noise ratio and temporal resolution enabling quantification of glutamate release from one AP at individual nerve terminals . For instance, a subset of important presynaptic GPCRs affects neurotransmission by lowering synaptic Ca2+ influx (B receptor (GABABR), which lowers ICa by approximately 50% via interactions of the Gβγ subunit with presynaptic VGCCs  modeled this relationship with Michaelis-Menten kinetics, where Δ[Ca2+]i obeyed a simple saturation of VGCCs, but predicted a simple 1:1 relationship extrapolating to lower [Ca2+]e  exhibited silencing of Ca2+ influx and SV exocytosis at physiologic [Ca2+]e, and this proportion increased steeply as [Ca2+]e was lowered to 0.8 mM and 0.4 mM . In comparison, the change in the proportion of silent terminals in the transition from [Ca2+]e 2.0 mM to 1.2 mM, a difference of ~15%, was smaller despite a larger step in [Ca2+]e. Previous estimates of synaptic silencing were drawn from experiments conducted at [Ca2+]e 2.0 mM and agree with our findings at this concentration  decreases the proportion of silent terminals  or acute (<1 hr) time scales, respectively  of either sex on postnatal days 0–2.All experiments involving animals were performed in accordance with protocols approved by the Weill Cornell Medicine Institutional Animal Care and Use Committee. Neurons were derived from Sprague-Dawley rats  , synapto–1 via a syringe pump  with Tyrode’s solution containing : NaCl 119, KCl 2.5, glucose 30, HEPES 25, CaCl2 0.8–2.0 with MgCl2 adjusted to maintain divalence of 4, D, L-2-amino-5-phosphonovaleric acid (APV) 50 µM, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) 10 µM, adjusted to pH 7.4. Temperature was maintained at 37 °C with a custom-built objective heater under feedback control (Minco). Fluorescence was stimulated with OBIS 488 nm LX or OBIS 561 nm LS lasers (Coherent) passing through a laser speckle reducer . Live-cell imaging was performed with a custom-built, epifluorescence microscope. Emission was acquired with a 40 x, 1.3 numerical aperture objective  and Andor iXon + Ultra 897 electron-multiplying charge-coupled device camera. APs were evoked with platinum-iridium electrodes generating 1 ms pulses of 10 V cm–1 field potentials via a current stimulus isolator . For physin-GCaMP and vGpH, neurons were stimulated with 20 and 200 AP, respectively, delivered at 20 Hz. For iGluSnFR, neurons were stimulated with a single AP, with averaging performed over 15 trials delivered every 20 s. A custom-designed Arduino board coordinated AP and laser stimulation with frame acquisition. Frame rates for AP recordings with vGpH, GCaMP6f, and iGluSnFR were 5, 50, and 100 Hz, respectively. To achieve frame rates of 50 and 100 Hz, a subregion of the EMCCD chip was used . In experiments with neurons expressing vGpH, Tyrode’s solution with NH4Cl 50 mM replacing an equimolar concentration of NaCl was perfused at the end of the experiment to alkalinize intra-vesicular pHluorin molecules . The toxins were applied for 3 min in Tyrode’s solution at concentrations of 1 µM, 400 nM, and 500 nM for ω-conotoxin GVIA, ω-agatoxin IVA, and SNX-482, respectively . BaclofeImages were analyzed with ImageJ . Circulabaseline, 49 frames)  above the baseline (mean of 49 frames) was less than the standard deviation of the baseline (σ frames) . For phyes) (4Cl . For iGles) (4Cl . For phy]i using :[Ca2+]e=max is the peak following saturation of GCaMP6f with ionomycin and Kd (0.38 µM), Rf (51.8), and n (2.3) are the in vitro dissociation constant, dynamic range, and Hill coefficient, respectively .Fluorescent biosensors are available through Addgene  or from the corresponding author by request . Custom-written macros for ImageJ analysis are available through GitHub . Support In this important study Cook and Ryan find that at physiological temperatures, small changes in extracellular calcium concentrations shift individual presynaptic terminals between active and inactive states. This discovery is extended to the action of inhibitory synaptic modulation via GABA, emphasizing potentially broad relevance for our understanding of how synaptic transmission is stabilized and modulated throughout the brain. The evidence for the observed phenomenon is compelling, given the use of multiple functional reporters and rigorous analyses. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Graeme W Davis as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by John Huguenard as the Senior Editor.Thank you for submitting your article "Neuromodulatory silencing of nerve terminals" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.The essential revisions are representative of conversation among reviewers, attempting to synthesize the individual reviews.1. Two reviewers focused on a specific issue: The obvious challenge is distinguishing between a very small response and a non-responder. The data, throughout the paper, are categorized (yes/no) and presented as a percentage. Thus, the crux of the manuscript relies upon thoroughly demonstrating that the discrimination between a responder and non-responder is not confounded. Suggestions for how this might be achieved are presented, but these are not intended to be the only solution to addressing this issue – it is entirely up to the authors.2. The data set could be broadly strengthened by including data from individual release sites, rather than simply presenting averages. This is particularly the case with the GluSnFr data where the probabilistic release of single vesicles could be resolved, inclusive of amplitudes associated with spontaneous release events (when possible).3. Two reviewers highlight a circular argument regarding the classification of silent sites, that are then shown to be silent. This should be addressed either through changes to the figures or the text. Reviewer 3 (comment 3). Reviewer 2 .4. It would be important, in at least one experimental paradigm, to see that release returns to baseline following restoration of external calcium.5. The issue of neuronal excitation can and should be evaluated. It is understood that these are sparse cultures. None-the-less, it remains possible that fields of view contain axons originating in more than one neuron, or in the case that they reside on different branches of the same axon, that branch point failures could 'cause' boutons to appear to function differently. A solution would be to systematically track whether sets of ROI unambiguously residing on single branches still function differently with respect to release .'6. A core feature of the manuscript is the quantification of calcium entry via the genetically encoded calcium sensor. There was a healthy debate among reviewers regarding the necessity of utilizing synthetic dyes. On the one hand, the superior properties of the dyes allow for highly quantitative validation of existing transgenic reporters and central conclusions. On the other hand, this requires loading (likely through patch pipettes). Addressing the robustness of the existing measures, either through dyes or validation with additional variants of transgenic reporters would strengthen the work. Again, however, the precise nature of the experiment is entirely up to the authors.2+]e. This information is essential to see whether there is indeed a shoulder in its effectiveness curve." The title likely should be changed, at a minimum.7. Two reviewers, while agreeing that the GABA data are interesting, commented that the data are somewhat under-developed. On the one hand, the title emphasizes "neuromodulation" and this could be modified to better reflect the single experiment shown regarding GABA. At another level, reviewer #3 argues that "The interesting issue would be to reveal how the effect of GABAB activation depends on the e around the physiological set point.' It seems equally likely that authors have identified a steeper continuum rather than 'all or none' control. As this is a central tenant of the manuscript, it could perhaps be addressed through changes in the text to shore up the argument, differently emphasized through data presentation or through the addition of new data.8. There was discussion about the main conclusion of the manuscript. 'Thus, as with CaReviewer #1 (Recommendations for the authors):While fascinating, the conclusions are most powerful when the data can be distilled to direct observation of single release sites. Yet throughout, the data are presented as all-or-none discriminations at regions of interest (ROI) that may or may not represent single release sites. Specific criticisms are listed below.1. One obvious challenge is distinguishing between a very small response and a non-responder. The data, throughout the paper, are categorized (yes/no) and presented as a percentage. Thus, the crux of the manuscript relies upon thoroughly demonstrating that the discrimination between a responder and non-responder is not confounded. For example, in several figures, nice example traces are provided showing a large initial response that results in a failure upon lowering extracellular calcium. But, beyond these examples, there are no quantitative data demonstrating that this is, indeed, routinely the case. Could the authors bin their data into quartiles based on the amplitude of the initial GCamp signal at 2.0 external calcium. Then, for each quartile, calculate the fractional silencing that occurs. If the fraction of silencing is constant across quartiles, then the authors have a good argument that the method of detection (GCamp) is not contributing to the observed effects.2. Figure 6 uses GluSnFr to directly measure vesicle release, testing synapse silencing. Data are presented as the average of 15 single action potentials. Given that release is probabilistic, a silent synapse is a zero responder over some number of these trials, but we do not know how many. Two issues arise. First, individual successes could be averaged out to appear as a failure in the averaged trace. Second, a true release probability depends on the number of active zones that are present at a single R01. It seems that the true power of GluSnFr is not being deployed. Can the authors utilize spontaneous miniature amplitudes (or sucrose evoked miniatures) to achieve an estimate of single vesicle release event amplitude. Then, if sufficiently above the noise, define the release probability at high and low calcium.3. In general, data from single RO1 would be nice to see, as opposed to averages and percentages per culture dish.4. It would be important, in at least one experimental paradigm, to see that release returns to baseline following restoration of external calcium.Reviewer #2 (Recommendations for the authors):2+]e around 0.8 mM results in zero Ca2+ entry. They also show that at higher [Ca2+]e the terminal has detectable Ca2+ entry. In the following figure, they demonstrate that approximately 25% of the boutons are 'silent' even in 2 mM [Ca2+]e. What does this mean? Is there any evidence that they are functional boutons? This is inconsistent with the literature. In cortical pyramidal neuron axons, each AP results in detectable Ca2+ entry and the Ca2+ entry is 100% reliable from AP to AP. If there is no Ca2+ entry, it is always the consequence of the lack of an active zone .The authors illustrate two terminals in which the [Ca2+] measurements should be repeated with high affinity synthetic dyes.Keeping the superior properties of the synthetic dyes in mind, all e varies from 0.8 to 2.0 mM, while the distribution of responders shifts, as expected, to higher ΔF with increasing [Ca2+]e. This indicates that our cutoff criterion for defining silencing was not artificially being biased by the absolute size of the signal at these different concentrations. (2) We observe a marked, clear separation in these frequency distribution of the silent versus responding populations reinforcing that our cutoff criterium yields distinct populations. Last, our results for silencing at [Ca2+]e 2 mM are consistent with previous publications using the same cutoff criterium or other definitions of presynaptic silencing, bolstering our observations and quantification in this manuscript. Conversely, for the first time to our knowledge, we have extended this methodology to investigate silencing at near-physiologic [Ca2+]e, discovering substantially different proportions of silent terminals within a physiologic range.We thank the reviewers for raising this excellent point regarding the challenge of identifying nonresponding nerve terminals. To address this issue, we compared the frequency distributions of fluorescence responses (ΔF) across all nerve terminals separated into silent or responding populations . This analysis reveals two key aspects: (1) the distribution shape of ΔF signals in the silent population is unchanged as e concentrations. We have added this consideration as a limitation in the discussion of the manuscript . However, the abolishment of Ca2+ influx at terminals despite a robust train of action potentials suggests that all release sites within a terminal are simultaneously silenced by this mechanism. Furthermore, the central importance of nerve terminals to neurotransmission, the ability to readily identify them through various targeted fluorescent proteins, and the widespread use of this marker in other studies of silencing underscore its biologic and experimental relevance.We agree that extending our analysis to the level of single release sites would be interesting to establish whether they act independently within nerve terminals. With our approach, analysis of individual release sites is complicated by the possible activation of iGluSnFr by glutamate exocytosed from neighboring, non-transfected terminals and the variable number of release sites per terminal in CA1-CA3 hippocampal neurons. Thus, we would be limited in our ability to discriminate between neighboring release sites and characterize the proportion of silent release sites between different e 2.0 mM, then 0.8 mM, and finally returned to 2.0 mM. We observed that both the proportion of silent terminals and the net influx of Ca2+ recovered to their initial values as [Ca2+]e was reverted to its starting level . Importantly, these results demonstrate that presynaptic silencing is a reversible process that is under the control of [a2+]e.We thank the reviewers for recommending inclusion of this important control experiment. We quantified the percentage of silent terminals and Ca5. The issue of neuronal excitation can and should be evaluated. It is understood that these are sparse cultures. None-the-less, it remains possible that fields of view contain axons originating in more than one neuron, or in the case that they reside on different branches of the same axon, that branch point failures could 'cause' boutons to appear to function differently. A solution would be to systematically track whether sets of ROI unambiguously residing on single branches still function differently with respect to release .'2+]e 0.8 mM, the concentration we hypothesized would most likely cause branch point failure. To provide robust quantification, we limited our analysis to branches with at least 10 nerve terminals. This analysis demonstrates silent and responding terminals on the same branches of an axon, thereby excluding the possibility that these effects are driven by selective changes in excitability . The overall percentage of silent terminals, though, is similar in this population of branches compared to the results of cell-wide percentages.We thank the reviewers for this suggestion to exclude the possibility that branch point failures are responsible for nerve terminal silencing. We examined this possibility with neurons expressing GCaMP6f in the cytosol to provide a good morphological fill of the axonal volume and co-expressing mRuby-synapsin to mark terminals. We performed the analysis for e 2.0 mM in the current studies to performing experiments at physiologic temperature as opposed to 30°C, which narrows the action potential waveform and thereby reduces Ca2+ influx. However, the overall similarity in our current results with this publication argues against a limitation of GCaMP as responsible for the silencing ofa2+ entry we observed.We are grateful to the reviewers for highlighting this concern. Previous studies investigating presynaptic silencing  utilized a less sensitive genetically-encoded calcium indicator, GCaMP3, and compared these measurements to the low-affinity, synthetic calcium dye, Magnesium Green. There was excellent agreement between the dye and GCaMP3, arguing against a limitation of GCaMP in detecting responses. Given the superiority of GCaMP6f to GCaMP3, we would expect a lower proportion of terminals classified as silent in our current experiments compared to the prior publication. However, we found a modest increase in the percentage of silent terminals (~15-20% compared to ~20-30%). We attribute the higher proportion of silent terminals in [Ca2+]e. This information is essential to see whether there is indeed a shoulder in its effectiveness curve." The title likely should be changed, at a minimum.7. Two reviewers, while agreeing that the GABA data are interesting, commented that the data are somewhat under-developed. On the one hand, the title emphasizes "neuromodulation" and this could be modified to better reflect the single experiment shown regarding GABA. At another level, reviewer #3 argues that "The interesting issue would be to reveal how the effect of GABAB activation depends on the [Ca2+]e on silencing mediated by the GABAB receptor, a GPCR that causes reduced Ca2+ entry via interaction of the Gβγ subunit with voltage-gated Ca2+ channels. As suggested, we investigated the effect of GABABR agonism with baclofen on silencing at [Ca2+]e 2.0 mM to compare to our previous results at 1.2 mM. Interestingly, the net change in percentage of silent terminals is substantially lower at 2.0 mM compared to 1.2 mM . These results demonstrate that [Ca2+]e substantially influences the synaptic response to GABABR agonism, a GPCR that reduces Δ[Ca2+]i through a canonical G-protein subunit pathway.We thank the reviewers for suggesting how to strengthen our conclusions of the impact of e around the physiological set point.' It seems equally likely that authors have identified a steeper continuum rather than 'all or none' control. As this is a central tenant of the manuscript, it could perhaps be addressed through changes in the text to shore up the argument, differently emphasized through data presentation or through the addition of new data.8. There was discussion about the main conclusion of the manuscript. 'Thus, as with Ca2+ influx is not completely silenced, the non-linear nature of the release process would effectively amplify the functional silencing effect .Thank you for this insight. We have modified the text to point out that even if the CaReviewer #1 (Recommendations for the authors):While fascinating, the conclusions are most powerful when the data can be distilled to direct observation of single release sites. Yet throughout, the data are presented as all-or-none discriminations at regions of interest (ROI) that may or may not represent single release sites. Specific criticisms are listed below.1. One obvious challenge is distinguishing between a very small response and a non-responder. The data, throughout the paper, are categorized (yes/no) and presented as a percentage. Thus, the crux of the manuscript relies upon thoroughly demonstrating that the discrimination between a responder and non-responder is not confounded. For example, in several figures, nice example traces are provided showing a large initial response that results in a failure upon lowering extracellular calcium. But, beyond these examples, there are no quantitative data demonstrating that this is, indeed, routinely the case. Could the authors bin their data into quartiles based on the amplitude of the initial GCamp signal at 2.0 external calcium. Then, for each quartile, calculate the fractional silencing that occurs. If the fraction of silencing is constant across quartiles, then the authors have a good argument that the method of detection (GCamp) is not contributing to the observed effects.Please see the response to #1 from the Essential revisions.2. Figure 6 uses GluSnFr to directly measure vesicle release, testing synapse silencing. Data are presented as the average of 15 single action potentials. Given that release is probabilistic, a silent synapse is a zero responder over some number of these trials, but we do not know how many. Two issues arise. First, individual successes could be averaged out to appear as a failure in the averaged trace. Second, a true release probability depends on the number of active zones that are present at a single R01. It seems that the true power of GluSnFr is not being deployed. Can the authors utilize spontaneous miniature amplitudes (or sucrose evoked miniatures) to achieve an estimate of single vesicle release event amplitude. Then, if sufficiently above the noise, define the release probability at high and low calcium.Please see the response to #2 from the Essential revisions.3. In general, data from single RO1 would be nice to see, as opposed to averages and percentages per culture dish.In addition to the inclusion of representative traces for the different biosensors used, we believe that the cumulative frequency distributions  provide the best overall representation of the distribution of ΔF values from individual ROIs.4. It would be important, in at least one experimental paradigm, to see that release returns to baseline following restoration of external calcium.Please see the response to #4 from the Essential revisions.Reviewer #2 (Recommendations for the authors):2+]e around 0.8 mM results in zero Ca2+ entry. They also show that at higher [Ca2+]e the terminal has detectable Ca2+ entry. In the following figure, they demonstrate that approximately 25% of the boutons are 'silent' even in 2 mM [Ca2+]e. What does this mean? Is there any evidence that they are functional boutons? This is inconsistent with the literature. In cortical pyramidal neuron axons, each AP results in detectable Ca2+ entry and the Ca2+ entry is 100% reliable from AP to AP. If there is no Ca2+ entry, it is always the consequence of the lack of an active zone .The authors illustrate two terminals in which the  measurements should be repeated with high affinity synthetic dyes.Keeping the superior properties of the synthetic dyes in mind, all i and the proportion of silencing in [Ca2+]e 0.4 mM and 1.2 mM . These experiments demonstrate that the overwhelming majority of terminals (93 ± 2%) are silent and the mean Δ[Ca2+]i is nearly 0. Thus, these experiments support that the overwhelming majority of terminals have a non-zero threshold of [Ca2+]e for [Δa2+]i.We thank the reviewer for this suggestion and we have conducted additional experiments comparing Δ[Ca3) Page 4, line 23. For the silenced population it is indeed expected, because, by definition, they did not respond and were consequently classified as silenced. As this is circular, the inclusion of these data in Figure 3D (and 6E) is a bit confusing and should probably be removed.As suggested, we have removed the silent bars from these graphs.4) Figure 4 – end of legend – Data are fit as in D (should probably be E)Thank you for identifying this typographical error. It has been corrected.


We found that the virgin tissue contracts more robustly than proven breeder tissue in the absence of oxytocin, yet with oxytocin, proven breeder samples displayed a significantly higher increase in activity. These results could depend on a more elevated expression of oxytocin receptor and/or on an alteration in the intracellular pathways affected by the activation of the oxytocin receptors. Here, we explored the impact of some structures involved in the management of intracellular calcium on the dose response to oxytocin recorded from virgin and proven breeder uterine strips. Specifically, we replicated the dose response experiments in low extracellular calcium (10 μM), in the presence of the intracellular calcium channel blocker ruthenium red (10 μM), and in the presence of the sarcoplasmic-endoplasmic reticulum calcium ATP-ase pump inhibitor, cyclopiazonic acid (10 μM). The results of these experiments suggest that also the expression of proteins that control intracellular calcium availability is affected by the experience of pregnancy. Molecular biology experiments will give us more detail on the magnitude of these expression changes.During pregnancy, the uterus undergoes several modifications under the influence of hormonal and mechanical stimuli. We hypothesize that while most of these modifications are reverted during involution, some of the physiological properties of the uterus are permanently altered. To investigate this hypothesis, we conducted motility experiments to evaluate the contractility response of uterine tissue samples from non-pregnant virgin and proven breeder female rats to oxytocin (10 Although it is well known that the complex array of hormonal and mechanical stimuli of pregnancy exerts profound impacts on the female physiology, our comprehension of the extent and permanency of these impacts is still incomplete. Epidemiologic studies have revealed a correlation between parity and an increase in the incidence of Alzheimer disease . GestatiIn the uterus, pregnancy constitutes a protective factor against fibroids (leiomyomas), the most prevalent tumors in younger women . In termOn the other hand, parity is considered a risk factor for uterine atony, one of the most common causes of potentially fatal hemorrhages in parturition . The exaDysmenorrhea, painful uterine cramping during the menstrual period, is estimated to affect between 15 and 75% of women (with peaks of 90% in women ≤ 25 years old), is inversely associated with age, use of oral contraceptives, and parity . In partA first step in gaining a clearer understanding of these phenomena is to investigate the effects of parity of uterine contractility in a carefully controlled system. In this study, we focused on the response of uteri from non-pregnant virgin (V) and proven breeder rats (PB) to oxytocin, one of the most used pharmacological tools to stimulate and evaluate uterine performance in vivo and in vitro.Oxytocin is a nonapeptide synthesized by the magnocellular neurons of the supraoptic and paraventricular nuclei in the hypothalamus and secreted by the posterior pituitary. More recently, it has been found to be released by other tissues, such as the placenta, the corpus luteum, and the uterus itself . Oxytociαq/11 protein to activate phospholipase C. Phospholipase C hydrolyzes the phospholipid phosphatidylinositol-4,5-bisphosphate into inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG)  curve has a hormetic trend not observed in the AUC/AUC in 40 mM KCl curve per se. After the 50 nM oxytocin dose, successive doses of the drug caused a reduction in both V and PB amplitudes.Throughout the dose response curve, V strips had a significantly more pronounced amplitude than PB strips (P < 0.001). Durations measured for 100 nM oxytocin (see Table P = 0.002).Instead, the frequency vs. [Oxytocin] curve followed a trend very similar to that of AUC. In PB strips, maximum and range were larger than in V strip  and other elements regulating intracellular Ca2+ levels in the uterus could be different after the first pregnancy. Thus, we studied how reducing the availability of extracellular Ca2+, blocking intracellular Ca2+ channels (inositol triphosphate receptors and ryanodine receptors), and blocking the sarcoplasmic-endoplasmic reticulum Ca2+ ATP-ase pump (SERCA) affect the response to oxytocin in V and PB samples.Given the higher responsiveness observed in PB, we hypothesized that expression of OXTR might be more elevated after pregnancy. However, preliminary results from protein expression evaluation with ELISA did not show a significant difference. Preliminary RT-PCR measurements gave contradictory results and are currently being reevaluated. Instead, we explored the hypothesis that pregnancy induces changes in the intracellular targets of the oxytocin-activated pathway, thus causing the observed differences in the motility experiments. We speculated that variations in the availability and activation of calcium channels , a reversal of trend compared to 2.5 mM Ca2+ experiments . Ruthenium red is an unspecific blocker of both inositol triphosphate receptors and ryanodine receptors calcium release channels (IP3R and RyR). RR was added to the regular Krebs solution after strips recovered from the KCl challenge and oxytocin application were started after no less than 30 min incubation. The results of these experiments are presented in Fig. 2+ experiments.To test the hypothesis that Caannels IPR and RyRP < 0.001). The spontaneous AUC/AUC in 40 mM KCl was reduced by 92% in V and by 62% in PB strips after addition of RR. While in 2.5 mM Ca2+ V had significantly higher amplitude than PB, after treatment with RR, PB displayed higher amplitude . RR produced an 84% spontaneous amplitude reduction in V strips and only a 25% reduction in PB. The RR induced inhibition was less dramatic on spontaneous frequency, but V samples were more affected (−62%) than PB samples (−18%). Hence, PB strips displayed higher spontaneous frequency than V strips . Similarly, spontaneous duration was reduced more strongly in V (−68%) than in PB (−54%), which resulted in PB having longer durations than V strip in RR .In addition, in RR, the PB strips displayed a significantly higher degree of spontaneous activity than V strips (N = 5 rat = 30 strips).The maximal AUC/AUC in 40 mM KCl elicited by oxytocin were slightly yet significantly higher in PB, while the range (maximum–minimum) was similar in the two groups  and in both cases, the increase was not as extensive as the one observed in 2.5 mM and 10 μM Ca2+.This trend in the AUC/AUC in 40 mM curves was matched by the correspondent amplitude and frequency curves. Furthermore, in the amplitude curves, values obtained from PB strips were consistently higher than values from V strips. In the frequency curves, instead, low oxytocin doses produced a moderate increase, while the highest doses caused a definitive decline in frequency. Indeed in 10 μM oxytocin, frequencies were lower than in the absence of the drug. On the other hand, 100 nM oxytocin prolonged event durations, as expected. Yet, V strips showed longer duration than PB strips . Compared to the 2.5 mM Ca2+ experiments, amplitude of spontaneous contractions in the presence of CPA did not change significantly in V strips. In PB strips, instead, it increased by 52%. In fact, spontaneous amplitude of V and PB was essentially identical. Frequency and event durations were similar to those registered in 2.5 mM Ca2+ only experiments.In addition to stimulating the activity of voltage gated calcium channels and the release of calcium from the SR, oxytocin reduces the ability of the sarcoplasmic-endoplasmic reticulum calcium ATP-ase (SERCA) to pump cytosolic calcium back into the SR. Thus, the next step was to conduct the dose response to oxytocin in the presence of a blocker of the SERCA pump, cyclopiazonic acid (CPA). Like in the case of the RR experiment, after the KCl challenge, strips were incubated for a minimum of 30 min with 10 μM CPA before treatment with cumulative doses of oxytocin. Figure 2+ removal. Maximal amplitude obtained with oxytocin and spontaneous amplitude were similar in both groups. Maximal frequency was significantly more elevated in V than in PB strips (P < 0.001), and so was the range of activation (max–min). In 2.5 mM Ca2+ only experiments, the frequency trend was completely reverse. In the absence of CPA, oxytocin elicited a maximum frequency of 0.039 ± 0.001 Hz. In the presence of CPA, maximal frequency was significantly (P < 0.001) lower: 0.029 ± 0.001 Hz. The event durations measured at 100 nM oxytocin were longer in PB strips than in V strips (P < 0.001), which is well aligned with the findings in 2.5 mM Ca2+ only experiments , and poorer in SR release channels than V uteri.This work tested the hypothesis that pregnancy induces epigenetic changes in the uterus that permanently modify its contractility properties. A variety of experimental approaches can be adopted to test this idea. We chose to study the motility response of uterine samples from non-pregnant virgin (V) and proven breeder (PB) female rats to oxytocin, since this hormone is both a major regulator of uterine contractility and a uterotonic agent commonly utilized in clinical practice. We found that, although the uterine affinity for oxytocin was not modified by pregnancy, the responsiveness to oxytocin was stronger in PB tissues. This suggested that the experience of pregnancy produces a permanent elevation in the number of available oxytocin receptors and/or elements of the intracellular second messenger-activated pathways triggered by oxytocin. The present paper focused on this latter aspect. In summary, we found evidence that PB uteri might be richer in voltage gated Ca2+ entry as an essential drive of myometrial contractility is well established [2+ entry [2+ availability on myometrial contractility of V and PB tissues. Complete elimination of extracellular Ca2+ abolished any spontaneous or oxytocin-induced contraction. This was an expected result, as the pivotal role of extracellular Ca2+ in myometrial contractility is well established. In as little as 10 μM extracellular Ca2+, uterine samples displayed the ability to respond to oxytocin, but they did not spontaneously contract. The lack of spontaneous contractions suggests that 10 μM extracellular Ca2+ do not sustain an adequate Ca2+ influx. Hence, the main mechanism driving contraction in the presence of oxytocin seems to be Ca2+ release from the SR. The dose response to oxytocin showed a reduction in the amplitude and frequency in both sample types. However, this reduction was more severe in PB strips. Based on this, we hypothesize that PB strips release SR Ca2+ less efficiently because of lower expression/activation of inositol-3-phospate receptor channels (IP3R) or lower expression/activation of ryanodine receptor channels (RyR). In 2.5 mM extracellular Ca2+, this difference is obscured by the strong inward Ca2+ currents through VGCC.The role of extracellular Caablished . Calciumablished . Among t2+ entry . We expl3R channels [2+ pump [2+ uptake in the mitochondria [2+-calmodulin complex [Treatment of the myometrial preparations with 10 μM ruthenium red (RR) induced a robust decrease in AUC, with a stronger depression in amplitude than in frequency and duration. RR is a classical inhibitor of RyRs; however, it has been shown to affect at micromolar concentrations other cellular structures, notably IPchannels , 37 and [2+ pump , Ca2+ upchondria , the for complex , and myo complex . In the 2+, albeit much reduced compared to the experiments conducted in 2.5 mM Ca2+ only. This indicates a substantial reduction of the available intracellular Ca2+. Yet, there was enough Ca2+ influx to support contraction. We cannot completely exclude the possibility that some inhibition of VGCCs occurred; however, the most prevalently affected mechanisms here were Ca2+ release via RyRs and IP3R activation. If RR had blocked VGCCs, no spontaneous activity would have been recorded, as seen in the low Ca2+ experiments. The fact that V samples displayed lower amplitude and frequency than PB supports the idea that V uteri might have a higher expression of SR channels than PB uteri. Thus, RR affects more V than PB samples.Spontaneous activity was stronger than in 10 μM Ca2+ availability. This suggests that the mechanisms inhibited by RR play a major role in the action of oxytocin on the rat myometrium, in accordance with published literature [Exposure of RR-treated strips to oxytocin elicited a weaker response than non-pretreated samples, regardless of extracellular Caterature .A peculiarity of these curves is that they are not sigmoidal. An initial increase in activity is followed by a deflection at higher oxytocin concentrations. In control experiments, the amplitude curve showed this hormetic trend, which we attributed to a partial progressive depletion of the SR under the persistent oxytocin stimulation. In those experiments, though, sustained frequencies compensated for the hermetic trend of the amplitude and the overall AUC/AUC in 40 mM KCl curves were sigmoidal. Instead, in the presence of RR, also frequencies abate significantly at higher oxytocin doses. Furthermore, this decline is identical in V and PB.2+ release could trigger a downregulation of oxytocin receptors. Further investigation with more specific inhibitors of RyRs  and IP3R  might help explain this finding.We first hypothesized that this phenomenon could be time dependent. We speculated that the polycationic nature of RR  would ca2+ ATP-ase (SERCA) contributes to the different responses to oxytocin observed in V and PB samples. Together with the plasma membrane Ca2+-ATPase (PMCA) and the sodium calcium exchanger (NCX), SERCA contributes to intracellular Ca2+ decay and relaxation in the myometrium [3R and RyR channels, Ca2+ leak, and SERCA inhibition [3R activation [2+ homeostasis occurring in the myometrium in the presence of oxytocin.In the last set of experiments, we investigated the hypothesis that the sarcoplasmic-endoplasmic reticulum Caometrium , 45. Furhibition . Oxytocihibition , it can tivation . No diretivation  argued t2+). Amplitude (which reflects cytosolic Ca2+ levels) was the parameter most affected. This is consistent with the decrease efficiency of Ca2+ removal caused by blocking SERCA. This change was significant in PB, not in V strips. In control experiments, the spontaneous amplitude recorded in PB strips was significantly lower than in V strips. The strong increase in PB amplitude observed with CPA could signify that SERCA is more expressed and/or more active in these samples. Without CPA, removal of cytosolic Ca2+ and replenishment of the SR might be quicker, which could also lead to a reduction in the activation of SOCE and an overall reduced availability of cytosolic Ca2+.Application of 10 μM CPA, a reversible SERCA blocker, increased the spontaneous activity of the myometrial samples compared to the control experiments  could explain the stronger response to oxytocin observed in PB strips.2+ uptake in SR vesicles pretreated with oxytocin. It has been shown that 17beta-estradiol upregulates SERCA2a in cultured cells and that in pregnancy and labor, the expression of SERCA in the rat myometrium progressively increases [The oxytocin effects on SERCA and its physiological modulator phospholamban in general are not considered significant in the myometrium . Yet, oxncreases . It is p+/Ca2+-ATPase, the H+/K+-ATPase, or the mitochondrial F1-ATPase [2+ management and specifically elements of the SR could be the main determinants of the different response to oxytocin in V and PB strips.Another consideration is that CPA induces depletion of the SR without affecting the PMCA, the Na1-ATPase . Therefo2+ handling, especially at the SR level. These alterations are likely at the base of clinical observations. For example, the more powerful response to oxytocin observed in PB tissue at least in part explains the more intense postpartum lower abdominal pains experienced by parous mothers. These pains are important for the involution process and are caused by powerful uterine contractions associated with oxytocin release, especially during lactation [In conclusion, this paper presents evidence that pregnancy induces permanent alterations to the uterine tissue. Our focus was on the contractility properties of the myometrium and its response to oxytocin. We presented motility data indicating that the most significant changes in this area concern mechanisms of Caactation , 9. The actation , 52. ThiUndoubtably, more work, especially in the molecular biology area, is required to tease out the specific structures and processes affected, based on the hypotheses we formulated in this discussion. Continued efforts in this line of research promise an advancement of our understanding of both the impacts of pregnancy on the function and health of the uterus and, more generally, of the regulation of myometrial contractility.


Parkinson’s disease (PD) is a degenerative neurological disorder that usually affects people over the age of 60. However, 10%–20% of patients have an early onset of PD (EOPD).To compare disability levels according to the World Health Organization Disability Assessment Schedule 2.0 (WHODAS-2) between people with EOPD and those with late-onset PD (LOPD).We conducted a cross-sectional study with 95 EOPD patients  and 255 LOPD patients . Demographic information, clinical characteristics, cognitive evaluation by Telephone-Montreal-Cognitive-Assessment (T-MoCA), functionality self-evaluation by WHODAS-2 and the Unified-Parkinson’s-Disease-Rating-Scale (MDS-UPDRS), parts I and II, were documented for each patient by an individual remote interview.p-value adjusted <0.009) and activities of daily living related to work/school . T-MoCA scores confirmed more impaired cognition capacity in LOPD . The two groups had no significant differences in levodopa daily dosage, Hoehn and Yahr (H&Y) stages, disease time duration, and MDS-UPDRS I and II scores.Analysis showed a statistically significant difference between EOPD and LOPD in two domains of WHODAS-2 only: cognition (Z-adjusted = 2.60; People living with EOPD face similar disability levels as those with LOPD, except for cognition, where LOPD patients exhibited higher levels of disability than EOPD and for work activities where the EOPD exhibited higher levels of disability than LODP. These results highlight the challenges faced by people with EOPD in interacting with society and living with the disease for a longer time. The WHODAS-2 can be a useful tool to assess disability and tailor interventions for people with PD of different age groups. The disGiven the effect of PD symptoms earlier in life, the interaction of symptoms with potentially more active roles in society, and the longer time-life living with the disease, people with EOPD face more challenges than those with LOPD. Rigidity, and dystonia have been found as more frequently presenting symptoms in EOPD , 8. In aBesides the motor and non-motor aspects, people with EOPD face different social challenges than LOPD. Unemployed men with EOPD are twice more than what observed in the general population , and earThe interaction among the physical, mental, and social aspects may be behind the higher prevalence of suicidal ideas in people with EOPD than in LOPD . DespiteWhen it comes to controlling the severity of PD, objective measures based on motor tests have proven to be quite effective. However, it is crucial to understand that the impact of the disease cannot always be directly linked to motor impairments alone, even though there is a clear relationship . There sThe World Health Organization Disability Assessment Schedule 2.0 (WHODAS-2) was developed to assess disability based on the International Classification of Functioning, Disability, and Health (ICF) . AccordiA seminal study to assess the WHODAS-2 conceptual model and metric properties in a set of chronic and prevalent clinical conditions accounting for a broad scope of disability included people living with early, intermediate, and advanced PD stages among the 1,119 participants recruited in 7 European centers. The results showed very high reliability, good ability to discriminate among known groups, and adequate capacity to detect change over time, confirming that WHODAS-2 is adequate to evaluate disability in patients with chronic conditions, which may help to eliminate barriers to developing policies, giving excellent evidence of these populations’ need .The results from a cross-sectional analytical study examining the metric properties WHODAS-2 short version, which included 168 PD patients with 69.6 mean age, demonstrated their suitable metric properties .Regarding the complex interaction among the motor, mental and social aspects associated with disability level in people living with EOPD and being WHODAS-2, a common, international, and interdisciplinary instrument based on the biopsychosocial model, the present study aimed to investigate the disability level in people living with EOPD in comparison with people living with LOPD.2.2.1.A cross-sectional study of 350 people with idiopathic PD was recruited for this study. The people were divided into the EOPD group  and LOPD group . Additionally, the participants could decide whether to have family assistance during the interview. Then, the researchers applied the questionnaire in a single day, which included: 1—general information , 2—socioeconomic status, 3—information associated with PD, 4—information dosage of medication , 5—self-2.4.2.4.1.It is a practical, generic assessment instrument that can measure health and disability at the population level or clinical practice based on the difficulties presented in the last 30 days. There are several different versions of World Health Organization Disability Assessment Schedule 2.0 (WHODAS-2), which differ in length and intended mode of administration. This study used the full version of 36 items through an interview.WHODAS-2 captures the level of functioning in six domains of life : Domain For all six domains, WHODAS-2 provides a profile and a summary measure of functioning and disability that is reliable and applicable across cultures in all adult populations. Each question is scored between 1 and 5. The higher the score, the worse the functionality of people.2.4.2.The Telephone - Montreal Cognitive Assessment (T-MoCA) is an adapted version of the MoCA 30 administered over the telephone with changes in the score. We used the MoCA items that did not require the use of a pencil and paper, or a visual stimulus were used for the T-MoCA, which has a maximum score of 22.2.4.3.It is a tool to measure the severity and progression of Parkinson’s disease based on the difficulties presented in the last 7 days. It consists of four parts: Part I (non-motor experiences of daily living), Part II (motor experiences of daily living), Part III (motor examination), and Part IV (motor complications). In this study, only parts I and II were used. Part I consists of 13 questions that assess the non-motor impact of PD on patients’ daily living experiences. Part II contains 20 questions that assess motor impact. Each question is scored between 0 and 4. The higher the score, the worse the severity and progression of PD.2.5.Descriptive statistical analysis was used for the demographic and clinical data of the participants. Considering the normal age distribution, test T was used to compare the two groups (EOPD and LOPD).Variables that did not exhibit a normal distribution, including WHODAS, MDS-UPDRS I and II, and T-MoCA, were analyzed using appropriate non-parametric tests.post hoc test. To correct the multiple comparisons, the Bonferroni test was applied.To compare the stage, Kruskal-Wallis ANOVA (KW-ANOVA) was utilized with the H&Y stage  considered as a factor. When statistically significant differences were observed, multiple comparisons of the average ranks for each pair of groups were applied by the Nemenyi U-Test with continuing correction was utilized to compare the two groups (EOPD and LOPD). Normal z-values and value of p were adjusted for each comparison as a correction for the number of comparisons.For onset comparison, the Mann–Whitney Finally, the Spearman Rank Order Correlation was used to test the correlation between MDS-UPDRS I-II and WHODAS-2 scores and between T-MoCA and WHODAS-2 domain cognitive scores.p was below 0.05. The statistical analyses were performed using Statistica Version 13 (TIBCO Software Inc. United States).Differences were considered statistically significant when the value of 3.The demographic and clinical participants’ features are demonstrated in p < 0.00001 and R = 0.65, value of p ≤ 0.00001, respectively).Regarding the disease evolution, there was a significant H&Y stage effect for WHODAS-2, MDS-UPDRS I, and II total scores, as indicated in p < 0.0000001).Regarding the disease onset, there was a statistically significant difference between EOPD and LOPD groups in two domains of WHODAS-2 only: cognition  and ADL In contrast, for ADL associated with work, the participants from EOPD group reported higher disability levels than LOPD. Additional analysis showed that 5% of participants from the LOPD group reported being “unemployed” against 11% from the EOPD group, 36% to be “employed” against 41% from the EOPD group, and 50% were “retired” against 28% from the EOPD group.4.Overcoming a neurodegenerative, progressive, and incurable disease such as PD is a complex challenge in any life cycle. Supposedly, managing the motor and non-motor symptoms and their social consequences is harder for younger people in the more active phase of life. In fact, the results from the current study showed a worse disability level and higher unemployment rate in people living with EOPD. However, the self-perception of disability levels related to mobility, self-care, getting along, life activities, and participation were similar for people living with EOPD and LOPD. In contrast, self-perception cognitive disability level was higher for people with LOPD than EOPD.Several considerations can be made based on our results.First, the WHODAS-2 was able to detect differences between clinical-severity groups: participants classified as intermediate disease stage (H&Y 3) reported worse disability scores than those with early disease stages (H&Y 1 and 2). Additionally, the disability levels according to WHODAS-2 were correlated with motor and non-motor aspects of daily living experiences according to MDS-UPDRS, considered golden standing for PD evaluation . The WHOSecond, despite some previous studies showing the slower disease evolution for early onset , our resThird, differences were found between the two groups for two WHODAS-2 domains: cognition and work/school activity. The disability level related to cognition was higher for LOPD participants. The decline in global cognitive capacity due to the aging process is well-known . The intFinally, the higher disability levels in work activities for EOPD than LOPD was unsurprising. A previous study comparing people with EOPD with LOPD showed that, among those who retired, 97% of the patients with EOPD retired early vs. 73% of those with LOPD . Other sOur study has some limitations. The absence of a motor evaluation of the motor symptoms severely may be considered a limitation of the present study; therefore, we cannot necessarily explore and correlate motor function and self-perception of disability level in the two groups. However, the importance of our findings is still maintained since the self-perception of disability level is a relevant measure that can provide valuable insight into the personal experiences of those living with PD , 31. In Finally, the participants in the study were selected from diverse geographical areas, varying educational levels, socio-economic backgrounds, and different ages, genders, and disability levels. This confirms that our convenient non-probability sampling method was unbiased and representative. However, exclusion criteria based on the presence of dementia and/or speech problems prevented patients in advanced disease stages from being included in the study. As a result, the sample mostly comprised patients in mild to moderate stages (mostly H&Y 2), which may limit the generalizability of the findings to patients in more advanced stages of the disease. The study’s primary inclusion criteria were based on a diagnosis of idiopathic PD by a neurologist who may not be specialized in movement disorders. This may also be considered as a study limitation.Further studies are needed to investigate the association between disability level according to WHODAS-2 and PD motor symptoms severity.5.In our study, we observed similar global disability levels between people living with EOPD and LOPD, according to WHODAS-2, except in the cognition domain that was more impaired in people living with LOPD than EOPD, and in activities related to work that was more impaired in people living EOPD than LOPD, findings that can be partially explained by the productive cycle of life.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving humans were approved by Ethics Committee of the General Hospital of the Faculty of Medicine of the University of São Paulo (#CAAE 19504619.5.0000.0065). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.IN: Conceptualization, Formal analysis, Investigation, Project administration, Supervision, Visualization, Writing – original draft, Writing – review & editing. KN: Investigation, Project administration, Supervision, Writing – original draft. BS: Investigation, Project administration, Writing – original draft. IB: Investigation, Writing – original draft. GC: Investigation, Writing – original draft. VP: Investigation, Writing – original draft. CP: Investigation, Writing – original draft. AP: Investigation, Writing – original draft. NP: Investigation, Writing – original draft. AH: Writing – original draft, Writing – review & editing. AR: Writing – original draft, Writing – review & editing. RS: Writing – original draft, Writing – review & editing. MP: Conceptualization, Formal analysis, Investigation, Project administration, Supervision, Visualization, Writing – original draft, Writing – review & editing.


Refractory or relapsed (R/R) AML is the most challenging form of AML to treat. Due to frequent genetic mutations, therapy alternatives are limited. Here, we identified the role of ritanserin and its target DGKα in AML. Several AML cell lines and primary patient cells were treated with ritanserin and subjected to cell proliferation, apoptosis and gene analyses with CCK-8 assay, Annexin V/PI assay and Western blotting, respectively. We also evaluated the function of the ritanserin target diacylglycerol kinase alpha (DGKα) in AML by bioinformatics. In vitro experiments have revealed that ritanserin inhibits AML progression in a dose- and time-dependent manner, and it shows an anti-AML effect in xenograft mouse models. We further demonstrated that the expression of DGKα was elevated in AML and correlated with poor survival. Mechanistically, ritanserin negatively regulates SphK1 expression through PLD signaling, also inhibiting the Jak-Stat and MAPK signaling pathways via DGKα. These findings suggest that DGKα may be an available therapeutic target and provide effective preclinical evidence of ritanserin as a promising treatment for AML.The online version contains supplementary material available at 10.1007/s12672-023-00737-9. The frequency of acute myeloid leukemia (AML) has been increasing as the population ages, and it is a biologically complex and molecularly heterogeneous disease. Clinically, refractory or relapsed (R/R) AML is the most challenging form of AML to treat, with a very dismal prognosis . It is rInitially developed as a serotonin receptor antagonist, ritanserin has undergone clinical trials for applications including schizophrenia, alcoholism, and insomnia –7. RecenHere, we identified the role of ritanserin and its target DGKα in AML. In vitro experiments have revealed that ritanserin inhibits AML progression by inhibiting cell proliferation and inducing apoptosis, and an anti-AML effect has been observed in xenograft mouse models. To pinpoint the specific function and molecular mechanisms of DGKα in AML, we analyzed the underlying biological mechanisms and their potential as prognostic factors by bioinformatics. Interestingly, this study showed that ritanserin, a DGKα-targeted inhibitor, may participate in the phospholipase D (PLD) signaling pathway, also negatively regulating the Jak-Stat and MAPK signaling pathways and exerting anti-AML tumor activity.2 incubator. Ritanserin (T16759) was provided by TargetMol  and dissolved in dimethyl sulfoxide . When administered in mice, the compounds were diluted with 0.3%(w/v) CMC-Na suspension and injected intraperitoneally.The human AML cell lines Kasumi-1 and KG-1α were provided by the Department of Hematology, The First Affiliated Hospital of Xiamen University  and both cell lines were tested and identified. 10% fetal bovine serum , 100 units/ml penicillin and 100 μg/ml streptomycin  were added to RPMI-1640 medium  for cell culture. Both cell lines were maintained at 37° C in a 5% CO4 per well) were seeded in 100 μl medium in 96-well plates. The cell viability was determined by cell counting kit -8 after 24, 48 and 72 h of treatment with DMSO or ritanserin with specific concentration. Three replicates were presented and the results were expressed as the percentage of living cells compared with the control group. GraphPad Prism 8 was used for statistical analysis and image rendering.AML cells  and NovoCyte Quanteon flow cytometry  were used to analyze cells. Annexin V positive cells were defined as apoptotic cells, and the results of three repetitions were presented as the mean ± SD.According to the manufacturer's instructions, the total RNA was extracted by SteadyPure universal RNA extraction kit , and then reverse transcribed into cDNA using Evo M-MLV RT Master Mix . qRT‒PCR was performed with a SYBR Green Premix Pro Taq HS qPCR Kit  and then amplified and detected by a Light Cycler 480 System . The primer sequences used were as follows: DGKA forward 5’-CACCCACCCACTTCTCGTCTTTG-3’, reverse 5’- CGGAGCCCTATCTCAGGACCATC-3’; β-actin forward 5’-TGTGGCATCCACGAAACTAC-3’, reverse 5’-GGAGCAATGATCTTGATCTTCA-3’. Three replicate experiments were conducted, and the results showed the relative gene expression of β -actin.The western blot experiment was carried out according to the description . After s+ primary AML cells  were sorted by a NovoCyte Quanteon Flow Cytometer (ACEA Biosciences).Twenty human primary AML and five normal hematopoietic stem cell specimens were collected from the First Affiliated Hospital of Xiamen University, Department of Hematology . According to the Helsinki Declaration, this study was approved by the Ethics Review Committee of the First Affiliated Hospital of Xiamen University. Informed consent was obtained from all individual participants in this research. Ficoll-Hypaque density gradient column  was used to isolate monocytes. CD34https://www.cancer.gov/), GTEx  and GSE12417 data [TCGA . A week later, the mice were randomized divided into two groups  and received vehicle (0.3% sodium carboxymethyl cellulose) or ritanserin (5 mg/kg/day) administered by intraperitoneal injection for two successive weeks. The leukemia burden was determined by intravital imaging every week. After 14 days of treatment, 3 mice were sacrificed from each group. The spleen (SP) and bone marrow (BM) were extracted for flow cytometry, HE staining, and immunohistochemistry analysis. The leukemia infiltration level was detected according to the surface markers CD45 , CD34 , and mCD45  by a NovoCyte Quanteon Flow Cytometer (ACEA Biosciences). For immunohistochemistry analysis, the tissue slices were incubated overnight with primary antibodies targeting CD34 , CD45  and DGKA  at 4 °C. Then, DAB  was applied for 30 s for the chromogenic reaction. Analysis was performed under an automatic digital slide scanner, Zeiss AxioScan7 .All animal experiments were approved by the Ethics Committee of Xiamen University. After 1 Gy irradiation, 2 × 10GraphPad Prism 8.0 was applied for statistical analysis. The mean ± SD was used to describe continuous variables. Two independent-sample t-test was used to analyze the differences between groups. Multiple group comparisons were made using one-way ANOVA. Results with the p-value less than 0.05 had statistical significance.50 values for each cell line at 24, 48, and 72 h were also computed and displayed concurrently  being significantly less than those at 24 h .First, we assessed the cytotoxicity of ritanserin against two human AML cell lines, Kasumi-1 and KG-1α. Ritanserin was applied to cells at a variety of concentrations for the specified periods, and CCK-8 assay results were obtained. As shown in Fig. 50 values of the cell proliferation assays, KG-1α cells were found to be more susceptible to ritanserin than Kasumi-1 cells. Given the crucial role that caspases play in the apoptosis execution mechanism, we wondered whether the activation of caspases was necessary for ritanserin-induced cell death. We examined the protein expression of caspase-3, PARP, and anti-apoptosis markers (Mcl-1 and Bcl-xL) through western blotting. Cleaved PARP was substantially increased after receiving ritanserin therapy after 48 h, indicating an apoptotic execution state. Moreover, there was a noticeable downregulation in the expression of total caspase-3, MCL1, and Bcl-xL  and AML samples (n = 173). DGKα expression was markedly elevated in AML samples compared to normal samples Fig. A. We obtWe further divided 173 AML samples into high-risk and low-risk groups according to the expression level of DGKα and discovered that the high-risk group had worse outcomes Fig. C, D. Add+ kinase activity and to promote phospholipid, DAG, and glycerophospholipid metabolic processes . According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, AML, phospholipase D (PLD), MAPK and Jak-Stat signaling pathways were the major biological processes affected by DEGs in the two groups . Additionally, Gene Set Enrichment Analysis (GSEA) enrichment demonstrated that the high-risk group was implicated in the development of leukemia and the metabolic pathways connected to PLD signaling . Consistent with the KEGG results, two crucial pathways, the MAPK and Jak-Stat signaling pathways, were enriched in the high-risk group . Altogether, our findings showed that in the high-risk group (high DGKα expression), DGKα regulated the PLD and Jak-Stat/MAPK signaling pathways, which are highly linked with a worse prognosis, to impact the carcinogenesis of AML.As a pro-oncogene, DGKα promotes proliferation and anti-apoptosis in leukemia , 19. To The main form of DAG metabolism is to convert DAG into phosphatidic acid (PA) through DGKα, and both DAG and PA are critical lipid second messengers in the plasma membrane . SeveralThe sphingosine kinase SphK1 has been identified as a crucial signaling molecule in various growth-related cellular events, such as cell migration, proliferation, and transformation . AlthougThus far, the results have demonstrated that DGKα is essential for negative regulation of the Jak-Stat and MAPK signaling pathways, which leads to the carcinogenesis of AML . We further performed western blotting to discover the critical proteins for two pathways to assess the results of the transcriptome profile in TCGA. As shown in Fig. To better confirm the potential signaling pathway after ritanserin treatment, we performed rescue experiments through the potent MAPK, MEK/ERK agonists C16-PAF , and theTo evaluate whether ritanserin functioned in carcinogenesis in vivo, Kasumi-1 cells were injected intravenously into NSG mice. We randomly assigned these mice to the vehicle and ritanserin groups (5 mg/kg/day) and subjected them to 14 consecutive days of intraperitoneal treatment Fig. A. On theFinally, we collected bone marrow mononuclear cells (BMMCs) from 12 primary AML samples to investigate the clinical application of ritanserin. The clinical characteristics of all samples are summarized in Table The refractory and relapsed forms of AML usually result in death; nevertheless, there are few effective treatment options for R/R AML . AllogenThe discovery of ritanserin as a DGKα inhibitor highlights the value of repurposing medications, as previous clinical trial data established its safety and tolerability in human subjects . Studies+CD38−cells [The expression of DGKα is increased in several cancer cells with poor prognosis, such as hepatocellular carcinoma, melanoma and glioblastoma , 31, 32.38−cells , 36. ConDespite the progress in linking the function of DGKα with the development of cancer and other diseases, it is still challenging to study their biology. After determining the crucial function of DGKα in AML, we carried out a pathway enrichment study. Notably, DGKα mostly activates the PLD pathway and associated pathways in AML samples. PA functions as a second signal to control SphK1 expression , and DGKIn conclusion, we found that selective inhibition of DGKα by ritanserin inhibits AML cell lines and primary patient cells both ex vivo and in in vivo mouse xenografts as a single agent in the clinically achievable range. Moreover, we determined that DGKα may be a promising therapeutic target in AML, and ritanserin not only negatively regulates SphK1 expression through PLD signaling but also inhibits the Jak-Stat and MAPK signaling pathways via DGKα, providing effective preclinical evidence for ritanserin in the treatment of AML.Supplementary file1Supplementary file2Supplementary file3


These localized Ca2+ signals spatially restricted to microdomains are crucial for information processing and synaptic transmission. However, the mechanistic link between astrocytic nanoscale processes and microdomain Ca2+ activity remains hazily understood because of the technical difficulties in accessing this structurally unresolved region. In this study, we used computational models to disentangle the intricate relations of morphology and local Ca2+ dynamics involved in astrocytic fine processes. We aimed to answer: 1) how nano-morphology affects local Ca2+ activity and synaptic transmission, 2) and how fine processes affect Ca2+ activity of large process they connect. To address these issues, we undertook the following two computational modeling: 1) we integrated the in vivo astrocyte morphological data from a recent study performed with super-resolution microscopy that discriminates sub-compartments of various shapes, referred to as nodes and shafts to a classic IP3R-mediated Ca2+ signaling framework describing the intracellular Ca2+ dynamics, 2) we proposed a node-based tripartite synapse model linking with astrocytic morphology to predict the effect of structural deficits of astrocytes on synaptic transmission. Extensive simulations provided us with several biological insights: 1) the width of nodes and shafts could strongly influence the spatiotemporal variability of Ca2+ signals properties but what indeed determined the Ca2+ activity was the width ratio between nodes and shafts, 2) the connectivity of nodes to larger processes markedly shaped the Ca2+ signal of the parent process rather than nodes morphology itself, 3) the morphological changes of astrocytic part might potentially induce the abnormality of synaptic transmission by affecting the level of glutamate at tripartite synapses. Taken together, this comprehensive model which integrated theoretical computation and in vivo morphological data highlights the role of the nanomorphology of astrocytes in signal transmission and its possible mechanisms related to pathological conditions.Astrocytic fine processes are the most minor structures of astrocytes but host much of the Ca These Ca2+ signals appear as “global” and/or “focal” responses within the astrocyte complex  and inspire new insights into therapies for unsolved neurological diseases  so that Ca2+ signals can occur when IP3Rs on the ER membrane are at open state. Shafts contain no organelles but IP3 and Ca2+ can diffuse through them. In the model, the width of nodes was set to greater than or equal to the width of shafts. In summary, these two parts together form the node-based tripartite synapse model.Considering these, we constructed two submodels, a node-shaft model and a tripartite synapse model. The former investigated how the morphology of fine processes affects Ca2+ microdomains can have a pure intracellular or extracellular Ca2+ source  derived from the Li-Rinzel model  to the postsynaptic neuron membrane. The brief descriptions of major neurophysiological steps are presented below, more details can be found in the  are co-localized . Both ofgiven by :IAMPA=gmodel as :MgV=11+2+ current carried by AMPARs, NMDARs, Ca2+ elevations happen in the postsynaptic neuron. We simply modeled this process:Due to fractional Cafound in  and Tabl2+ peaks were detected when 2+ concentration, 2+ fluctuations. The two indicators we used to characterize Ca2+ signals were the total number of peaks and mean amplitude of these peaks, respectively.The CaIn this paper, all simulations were performed with MATLAB  and the code is available in supplementary materials. For each simulation experiment, we executed 20 trials with different random seeds depending on the system clock. So, the results presented in the figure were expressed as the mean ± STD of 20 simulations.2+ signals essentially in agreement with results of experiments in live tissue  . The siz2+ activity. Besides, all changes in morphology of fine processes did not affect Ca2+ signals of the parent process they connected.Shaft length is the distance between two neighbor nodes and ranges from 0.4 μm to 2.4 μm (median length: 1.1 μm) . In our 2+ traces for four example parameter settings. The median values of width of nodes (330 nm), shafts (202 nm) and length of shafts (1.1 μm) measured in the experiments were reset as the control group . This might be due to the increased diffusion fluxed from additional compartments. Although the mean amplitude of these peaks did not rise, the Ca2+ activity seemed to be more active. While the distal node (Node 1) which was away from the changed branched nodes barely changed. 2+ activity of large process was either not affected by the structure of the microdomain.The crosstalk of Carphology . We specrphology . We foun2+ exchange between compartments altering the mutual Ca2+ activity but seemed to need cumulative effects, i.e., enough compartments and fluxes. Hence, we then proposed another model consisting of only those nodes connected to the parent process  and postsynaptic Ca2+ dynamics. Glutamate is the predominant excitatory neurotransmitter, hence the abnormally low level of glutamate probably leads to a decrease in brain excitability.As shown, both the number of Cat manner , therefo2+ and EPSP, eventually leading to NMDAR-mediated excitotoxicity.Also, glutamate can be a potential excitotoxin. We considered another possible situation which is shown in In general, our simulations indicated that astrocyte structural changes could impair the glutamate-dependent synaptic transmission and affect the downstream processes closely connected to memory, cognition, and other advanced brain functions .2+ language” to process information and most of the basal Ca2+ activity occurs in their fine structures where they compose the spongiform domain  than in thick processes or Soma  stop there just like some forms of physical and/or biological barrier are presented at the interfaces (2+ signal of the large structures. This finding may make sense because in many neurodegenerative instances, the change in number of astrocytic processes is a frequent occurrence, for example, reactive astrocytes always have more complex branchy degree while atrophic astrocytes have less processes because of morphological deficits (Astrocytic Caterfaces . How thedeficits .2+ activity thereby affecting the release of glutamate. The degradation of the excitability was hence the possible reason for early cognitive disorder and memory loss in the early stage of AD. This conclusion is an interesting idea but we realize and believe that more wet-lab design and data are required to address this specific clinical question. On the other hand, our model explained that the increase of the astrocytic coverage could enhance glutamate uptake in the synaptic cleft and decrease the high extracellular glutamate levels and overall neuronal hyperactivity which was consistent with the experimental results in the parkinsonian state (Astrocytes are important shapeshifters which allow them to respond to the changing environment during development, injury and disease. Their morphological changes have been observed in various experiments especially in pathological conditions, but most mechanisms remain unknown. For example, hypertrophic reactive astrocytes are always thought to be a hallmark in many neurodegenerative diseases, while the atrophic astrocytes are presented earlier in AD . So far,an state . Overall2+ microdomain is rapidly increasing. A recent study indicates that ER is observed in only ∼45% of fine processes (2+ influx through cell membrane ion channels can contribute to astrocytic microdomain events with fast dynamics, such as TRPA1 and Na+/Ca2+ exchanger (3-dependent release, microdomain Ca2+ transients still occur due to the opening of the mitochondrial permeability transition pore. These various fluxes can also induce cascade reaction. For instance, once store-operated Ca2+ entry is activated, the depletion of ER activates the ER Ca2+ sensors stromal interaction molecules 1 and 2, then the molecules translocate to the junctional ER to interact with and activate store-operated Ca2+ release-activated Ca2+ channels (2+ microdomains. Considering a complete picture of the Ca2+ microdomain would definitely help better understand the theme but also is challenging due to the missing explanation of some mechanisms. In this study, we aimed to provide a biologically plausible theoretical framework that could provide reasonable predictions on the morphology-defined Ca2+ microdomains that are difficultly enabled in experiments.Knowledge about Carocesses  and othexchanger . Besidesxchanger  found thchannels , suggest2+ dynamics. In astrocytes, in addition to glutamate, ATP, GABA and other signaling pathways also affect Ca2+ signalization and contribute to the regulation of a number of both physiological and pathophysiological processes in the nervous system as well (2+ diffusion. Thirdly, Some details of the model are not rigorous enough. The presence of ER in the fine processes is controversial, but there is evidence that Ca2+ microdomains can have a pure intracellular or extracellular Ca2+ source (In fact, our model leaves much to be desired and needs further improvement. Firstly, our node-shaft model is only an approximation of the astrocyte microdomains and cannot fully reflect the morphology of fine processes. Secondly, our model does not fully consider the factors affecting astrocyte Ca as well . The mod+ source . And we 2+ signals in astrocyte microdomains have gained a decade of significant advances, as said, a new decade of great discoveries has just begun (Although many questions and challenges exist, Cast begun . We need


It controls several biological processes: it triggers life at fertilization, and controls the proliferation, development and differentiation of cells, as well as regulating diverse processes such as secretion, metabolism, muscle contraction, neuronal excitability, learning and memory, and cell death , together with green-fluorescent protein-based fluorophores and can be targeted to specific organelles or cellular compartments to measure localised Ca2+ changes , where depletion of intracellular Ca2+ stores—sensed and communicated by stromal interaction molecules (STIMs)—triggers Ca2+ influx via Orai channels, is a fundamental mechanism in maintaining cellular Ca2+ homeostasis. In this Research Topic, Manning et al. review the role of SOCE in skin physiology and pathophysiology. In the skin, SOCE regulates the processes of proliferation, differentiation, melanogenesis and sweat secretion  channels, which mediate the Ca2+ influx that drives the differentiation of keratinocytes ] and a cardiac arrhythmia syndrome [catecholaminergic polymorphic ventricular tachycardia (CPVT)], result from similar molecular mechanisms. Using chemical cross-linking reactions, Zhang et al. demonstrated intra-subunit interactions within the tetrameric structure of the channel are disrupted as a result of mutation which causes amino acid substitution in the N-terminus of the protein [R163C in the skeletal muscle isoform, RyR1  stress can affect this phenomenon, as well as regulation of sarco (SR)/endoplasmic reticulum Ca2+ store homeostasis in general. It seems logical that, since the SR/ER is the site of protein processing, stress-responses in this organelle can have a profound and ultimately pro-arrhythmic effect on Ca2+ handling proteins and ion channels. The unfolded protein response (UPR) is a signal transduction system that upregulates various stress-response proteins  in response to conditions  that impact the efficiency of protein folding in the rough ER (2+ ATPase pump, thereby altering SR Ca2+ homeostasis (2+ handling and SR/ER stress proteins may be when developing new therapeutic approaches for cardiovascular disease.RyR2 dysfunction is a known arrhythmia trigger and rough ER . While ieostasis . This caeostasis —which ineostasis , or by aeostasis . Given teostasis , it will2+ signalling remains a growing area of research with still much scope for the development of both diagnostic and therapeutic tools that target its component parts.The articles included in this Research Topic reinforce that Ca