Cell Calcium

Store-operated calcium entry (SOCE) is a ubiquitous calcium (Ca2+) signalling mechanism that replenishes endoplasmic and sarcoplasmic reticulum Ca2+ stores. We previously identified STIM1L, a skeletal muscle-enriched STIM1 splice variant that enables rapid SOCE activation in human myotubes. To determine its physiological role in vivo, we generated a constitutive knockout mouse model that selectively abolishes STIM1L expression while preserving the canonical STIM1 isoform. Stim1l-KO mice developed normally and exhibited no overt abnormalities, but both sexes showed reduced voluntary wheel-running activity, suggesting neuromuscular perturbation. Skeletal muscle deprived of STIM1L did not show any histological alterations or changes in fibre- type and size. Likewise, force-frequency relationships and fatigue resistance measured ex vivo in isolated extensor digitorum longus and soleus muscles were indistinguishable from control muscles. This lack of difference may be explained by the increased expression of STIM1 in several skeletal muscles and suggests that changes in the voluntary running activity of Stim1l-KO mice may arise from STIM1L loss in non-muscular tissues, including the central nervous system. Notwithstanding, primary myotubes derived from Stim1l-KO mice displayed a marked delay in SOCE activation kinetics, whereas SOCE amplitude remained comparable to controls. This was consistent with a compensatory STIM1 upregulation maintaining overall Ca2+ entry capacity. Interestingly, electrically evoked Ca2+ transients in intact Stim1l-KO flexor digitorum brevis fibres exhibited significantly faster decay kinetics than controls. This revealed another role of STIM1L in Ca2+ handling in skeletal muscle that appears independent of its contribution to SOCE activation. Together, these findings establish STIM1L as a regulator of SOCE activation kinetics and indicate an additional role in myofibre Ca2+ clearance, while suggesting that compensatory upregulation of canonical STIM1 may preserve muscle structure and contractile function in its absence in mice.

Aims: Seizure generation has been strongly linked to the accumulation of extracellular potassium. As astrocytes play a key role in potassium homeostasis, astrocyte targeted optogenetic modulation of extracellular potassium has been proposed as a novel strategy for seizure control. The effectiveness of such modulation is expected to depend strongly on the ion selectivity of the employed opsin. This study aims to mechanistically compare potassium-selective (WiChR) and cation-conducting (ChR2-like, ChR2*) opsins for astrocyte targeted modulation of extracellular potassium, and to determine how astrocytic properties and stimulation parameters influence modulation efficacy. Methods: A conductance-based computational framework was used to model astrocyte-extracellular space interactions, incorporating ion-specific photocurrents, Na+/K+-ATPase activity, and gap junction-mediated coupling within the astrocytic syncytium. Systematic parameter sweeps were performed to evaluate the influence of ion selectivity, astrocytic parameters, and stimulation paradigms on potassium dynamics. Results: WiChR-based modulation consistently produced more effective and robust reductions in [K+]e than ChR2*, via direct K+ inward currents and reduced dependence on pump activity. In contrast, ChR2* primarily limited potassium accumulation rather than reversing it and exhibited greater sensitivity to parameter variations, particularly Na+/K+-ATPase activity. The effectiveness of astrocyte-targeted modulation was strongly dependent on syncytial integrity, with reduced gap junction coupling impairing potassium clearance. Optimal stimulation performance for WiChR was observed for a pulse repetition period of 20 ms, with outcomes determined by the interaction between opsin kinetics and stimulation parameters. Conclusions: These results demonstrate that activation of WiChR opsins in astrocytes can reduce extracellular potassium levels and thereby modulate neuronal excitability. Ion selectivity emerges as a key determinant of modulation efficacy at the astrocytic level, with potassium-selective opsins providing a more direct and reliable approach than non-selective cation-conducting alternatives. Together, these findings establish a mechanistic basis for optogenetic activation of astrocytic potassium buffering as a potential strategy for seizure suppression and other neurological disorders associated with potassium dysregulation.

Skeletal muscle is a dynamic tissue able to regenerate in response to injury. This regeneration relies primarily on the activation of precursor cells, known as muscle stem cells (MuSC). Upon activation, skeletal MuSC re-enter the cell cycle and proliferate as myoblasts, which subsequently either differentiate and fuse to form new fibers or return to quiescence to maintain long-term regenerative capacity. From primary human myoblasts, we generated reserve cells (RCs), quiescent cells similar to MuSC, and investigated their activation after stimulation with a growth medium containing serum. Here, we show that Orai3 calcium channel is primarily expressed in RC, where it promotes RC activation in a calcium-independent manner. We demonstrate that Orai3 does not contribute to store-operated calcium entry (SOCE) or calcium response induced by serum stimulation in RC cells, supporting the hypothesis that Orai3 plays a unique role in RC, distinct from its calcium channel activity. Using a protein proximity assay (BioID), we identified the large scaffold protein AHNAK2 as a potential partner of Orai3. AHNAK2 is expressed at high level in myotubes, and has a similar effect on RC activation than Orai3. Both Orai3 and AHNAK2 are necessary for proper myoblast differentiation and RC activation, while acting through different signaling pathways. Specifically, we show that Orai3 directly influences the RC fate, whereas AHNAK2 facilitates RC activation via signals derived from myotubes. These findings provide new insights into the molecular mechanisms underlying the activation and quiescence of human MuSC.

Traumatic brain injury (TBI) is one of the world's leading causes of death and disability and a major risk factor for dementias. The primary dementia associated with TBI is chronic traumatic encephalopathy (CTE), a neurodegenerative disease classified as a tauopathy, in which toxic tau molecules lead to disease pathologies and degeneration. The processes that lead to tauopathy and subsequent dementia after TBI remain unclear. Here, we built upon the finding that seizures after TBI may be a mechanism leading to tauopathy, by dissecting the functions of the metabotropic glutamate receptor 5 - cellular prion protein (mGluR5-PrPC) pathway. We delivered TBI to larval in a blast paradigm, and quantified aggregation of Tau via a genetically-encoded Tau-GFP fusion reporter. Zebrafish larvae lacking prp2 (homolog of mammalian cellular Prion Protein, PrPC) displayed a 168% increase in post-traumatic seizures activity after TBI. An mGluR5 agonist (CHPG) reduced post-traumatic seizures, whereas an mGluR5 antagonist (MPEP) increased post-traumatic seizures. Moreover, agonizing mGluR5 reduced tau aggregation and antagonizing mGluR5 increased tau burden. Larvae seizing from convulsants, rather than TBI, were treated with CHPG/MPEP and provided a similar pattern of outcomes, suggesting seizures may be a factor needed for mGluR5 activity to influence tau aggregation. The PrPC-mGluR5 pathway is proposed as one candidate pathomechanism linking TBI to subsequent seizures and tauopathy, and thus it warrants investigation as a target for prophylactic interventions.

Cell surface receptors are regulated via a variety of mechanisms including proteolytic inactivation by metalloproteases. Cells often co-express both the receptor and its cognate protease simultaneously on the cell surface, often with the proteases in an active state. Despite this, receptor cleavage is generally not constitutive, and rather must be induced. How a receptor and its inactivating protease can both be present in their active form on the cell surface with no cleavage of the target receptor is unclear. Using the efferocytic receptor MERTK and its inactivating protease ADAM17 as a model, along with super-resolution microscopy approaches, we demonstrate that MERTK and ADAM17 are localized to distinct sub-regions of the plasma membrane. These microdomains are distinct subcellular structures, with MERTK contained within actin-based corrals and ADAM17 contained within caveolae. Induction of MERTK cleavage by PMA results in the reorganization of the membrane-proximal actin cytoskeleton that enables MERTK to diffuse into ADAM17-containing microdomains. Only after localizing to ADAM17-containing microdomains is MERTK cleaved. These results demonstrate that the spatial organization of the plasma membrane is dynamically regulated, with reorganization of membrane microdomains playing a role in the regulation of ADAM17 activity.

Prostaglandin E2 (PGE2) is recognized as a major mediator of inflammatory pain. However, the intracellular signaling mechanisms by which PGE2 mediates pain remain unclear. Here, we show that pain-like responses evoked by PGE2 in dorsal root ganglion (DRG) nociceptors are mediated by internalization and compartmentalized signaling of the EP4 receptor. The EP4-selective agonist L-902,688 induced immediate nocifensive behavior and prolonged mechanical allodynia in mice and sensitized isolated DRG nociceptors. Pharmacological inhibition of clathrin- and dynamin-mediated endocytosis (using pitstop2 and dyngo4a) and siRNA knockdown of Dnm1 prevented EP4-induced nociception and sensitization, implicating receptor trafficking in pain signaling. Using genetically-encoded biosensors, we monitored EP4 trafficking and downstream signaling within subcellular compartments of HEK293 cells. PGE2 stimulated dynamin- and {beta}-arrestin-dependent EP4 trafficking from the plasma membrane to early, late and recycling endosomes and Golgi apparatus, and mobilized intracellular EP4 pools from the endoplasmic reticulum. PGE2 induced the assembly of EP4, G proteins and {beta}-arrestin signaling complexes in endosomes and the Golgi apparatus. Inhibition of EP4 endocytosis suppressed intracellular cAMP production and ERK activation. Our findings reveal that EP4-mediated nociceptive signaling originates from intracellular compartments and suggest that modulating EP4 internalization and subcellular signaling dynamics could offer a novel strategy for inflammatory pain management.

Neuronal intracellular chloride concentration shapes the polarity and strength of GABAA receptor-mediated inhibition. ClC-2 is a hyperpolarization-activated chloride channel proposed to support chloride extrusion, but its acute contribution to EGABA and neuronal chloride homeostasis remains incompletely understood. Here, we used the selective ClC-2 inhibitor AK-42 to test how acute ClC-2 inhibition affects GABAergic chloride signaling in organotypic hippocampal slice cultures. We combined targeted single-interneuron optogenetic activation of stCoChR-expressing GABAergic interneurons with gramicidin-perforated patch recordings to measure optogenetically evoked inhibitory postsynaptic currents and calculate EGABA and we used SuperClomeleon imaging to measure neuronal intracellular chloride. AK-42 depolarized EGABA, with a larger effect when stimulation was delivered 2 s rather than 100 ms after voltage-step onset. This delay-dependent effect is consistent with greater engagement of voltage- and time-dependent ClC-2 activity during prolonged voltage steps. AK-42 also abolished the more hyperpolarized EGABA normally measured at the 2-s stimulation delay and shifted baseline holding currents at hyperpolarized command potentials without measurably changing estimated resting membrane potential. SuperClomeleon imaging showed that AK-42 increased neuronal intracellular chloride in the presence of TTX, supporting a role for ClC-2 in basal chloride regulation. Together, these findings show that acute ClC-2 inhibition depolarizes EGABA, increases neuronal intracellular chloride, and alters the temporal dynamics of GABAergic chloride signaling, identifying ClC-2 as an acute regulator of synaptic GABAergic chloride signaling.

Metacaspases are cysteine proteases and are found in every group of organisms except animals. They are structural orthologs of caspases, which orchestrate apoptosis in animals. Caspases, metacaspases, paracaspases and orthocaspases are class C14 proteases with a similar active site (the hemoglobinase fold) and a catalytic cysteine residue. However, metacaspases cleave after arginine and lysine instead of aspartate and some researchers point out that a name ending with caspase is unsuitable. Also, most metacaspases are activated by calcium, they do not target the same proteins as caspases and they cut multiply instead of once. The Candida albicans metacaspase Mca1p mediates cell death in response to various stresses but also has pro-life functions. Many plant and protist metacaspases lack cell death roles and are involved in development, differentiation and immunity. This article highlights an interesting phenomenon, whereby the promotion of stress sensitivity and reduction of virulence, by Mca1p in C. albicans cells from exponential culture are abolished and reversed, respectively in cells from stationary-phase culture. This hints at the proteases's dual nature and at the conditions that might initiate a switch between pro-life and pro-death functions.

Although MAP1LC3B/LC3B (LC3) is best known as a cytoplasmic marker of autophagosome biogenesis, a substantial pool of LC3 resides in the nucleus and shuttles dynamically between nuclear and cytoplasmic compartments, yet the composition and regulation of this nuclear interactome remain poorly defined. Using mass spectrometry-based proteomic profiling of nuclear GFP-LC3 immunoprecipitates from primary human trabecular meshwork (TM) cells, a mechanosensitive ocular cell type, we identified a reproducible nuclear LC3 interactome enriched for proteins containing LC3-interacting region (LIR) and expanded LIR (xLIR) motifs and reported nuclear localization. Among these, clathrin heavy chain (CLTC) emerged as a previously unrecognized nuclear LC3 partner that localizes to the nucleus and colocalizes with nuclear LC3 puncta. CLTC depletion reduced basal LC3-II levels, consistent with a role in autophagosome biogenesis, and markedly impaired nuclear LC3 accumulation induced by both nuclear export blockade with leptomycin B and cyclic mechanical stretch, without altering total CLTC abundance, indicating that CLTC actively promotes LC3 nuclear translocation rather than serving as a passive scaffold. Strikingly, mechanical stress-induced nuclear LC3 trafficking, but not its basal component, was selectively impaired in TM cells derived from glaucoma patients, despite comparable CLTC levels, pointing to a defect in coupling this trafficking pathway to mechanotransduction rather than in the core transport machinery itself. Together, these findings establish nuclear LC3 trafficking as an actively regulated, CLTC-dependent process linked to cytoskeletal and vesicular machinery, and implicate its dysregulation in mechanically stressed glaucomatous cells, providing a framework for understanding how autophagy intersects with nuclear homeostasis, mechanotransduction, and glaucoma pathogenesis.

Low-intensity ultrasound alters neuronal excitability long after exposure ends, but the basis of this offline effect is unclear and most proposed mechanisms require synaptic receptors. We recorded from cultured mouse hippocampal neurons in whole-cell current clamp, before and 20 min after 1 min of 850 kHz continuous-wave ultrasound at 0.5 W/cm2 or sham, with synaptic transmission blocked throughout, each neuron re-patched. Sonicated neurons remained intact and firing, yet depolarized, with smaller action potentials and more noise. A passive-membrane fit showed reduced capacitance and a depolarized leak reversal potential in every sonicated cell, with a shortened time constant and no detectable change in input conductance, while sham cells showed no significant change. Changes in the time constant and reversal potential, both independent of membrane area, indicate a modification of the membrane itself rather than a loss of area. Brief ultrasound therefore leaves a lasting, receptor-independent imprint on passive membrane properties.

Background: Human induced pluripotent stem cell-derived cardiomyocytes are valuable for cardiac disease modelling and drug testing but remain limited by immature structural and electrophysiological properties in conventional two-dimensional cultures. Three-dimensional cardiac models, including cardioids, provide a more physiological environment through multicellular organization and enable tissue-level functional analysis. Their electrophysiological properties and maturation, however, remain incompletely characterized. Methods: We performed an integrated electrophysiological characterization of human cardioids over the first three weeks of maturation using sharp microelectrode recordings, calcium transient measurements, whole-cell patch clamp recordings, immunofluorescence, transcriptomic analysis, and pharmacological interventions. Results: During maturation, cardioids exhibited progressive electrophysiological maturation, characterized by more physiological resting membrane potentials (-54.0{+/-}3.1mV to -68.1{+/-}1.2mV), increased action potential amplitude (73.5{+/-}4.8mV to 101.7{+/-}2.1mV) and upstroke velocity (44.3{+/-}5mV/ms to 86.6{+/-}8.4mV/ms), and shorter action potential duration (178.9{+/-}8.7ms to 144.0{+/-}4.8ms at 1Hz stimulation). These changes were associated with progressively increased INa density and enhanced SCN5A mRNA and Nav1.5 protein expression, consistent with progressive maturation of cardiac excitability. IKr current and KCNH2/hERG expression were already present at early maturation stages and remained stable, as evidenced by preserved sensitivity to dofetilide-blockade. Pharmacological inhibition of barium- and HMR-sensitive currents altered resting membrane and/or action potential characteristics in intact cardioids, whereas in isolated cardiomyocytes classical IK1 remained undetectable and IKs was small, highlighting a certain degree of persisting immaturity of the model. Conclusions: These findings demonstrate that human cardioids undergo progressive electrophysiological maturation and provide complementary tissue- and cellular-level insights into cardiac function, establishing their suitability for investigating INa and IKr-related cardiac pathologies.

A cell's ability to maintain its volume is a prerequisite for its essential physiological functions. Changes in extracellular osmolarity induce the flux of water and solutes across the cell and nuclear membrane and, therefore, alters cell and nuclear volumes. Here, we investigated the equilibrium and dynamic volumetric responses of cells and nuclei in adherent human hepatoma (HepG2) cell monolayers using live three-dimensional imaging. Through image segmentation, 3D reconstruction, and volume rendering, precise cell and nuclear volumes were quantified. We found that during hypoosmotic stress, cells swell and lose osmolytes through active volume regulation, and nuclei undergo greater relative expansion than cells. During hyperosmotic stress, we found that both cells and nuclei shrink rapidly, develop irregular morphologies and undergo passive volume regulation. Furthermore, we explored the osmotic responses in these cells by restoring isosmotic conditions following osmotic stress and found that nuclei returned to volumes greater than their initial isosmotic volume, whereas cytoplasmic volume returned to a value below its initial isosmotic volume following swelling. By fitting coupled transport models water and solute permeability estimates for both cells and nuclei were made from their dynamic responses to different anisosmotic media with and without the presence of permeating solutes dimethyl sulfoxide and ethylene glycol. Together, these quantitative measurements of cell, cytoplasm, and nuclear volumetric responses and associated transport parameters have important implications for understanding cell physiology and the mechanism of osmoregulation.

Retinal neurons rely on continuous oxygen supply to maintain function, yet some species, like goldfish, withstand prolonged hypoxia due to adaptations that remain unclear. Horizontal cells (HCs) are inhibitory interneurons of the retina that express ionotropic glutamate receptors (iGluRs) and secrete gama-aminobutyric acid (GABA), allowing them to shape visual signalling by feeding back to photoreceptors. We investigated whether iGluRs in dissociated goldfish HCs are suppressed during hypoxia, and whether this process is mediated by oxygen-sensitive activation of mitochondrial ATP-sensitive K+ (mKATP) channels. Through perforated patch-clamp electrophysiology, we determined that 20 min of hypoxia reduced peak inward current density associated with glutamate application by ~25% (P = 0.0059). When mKATP channels were selectively blocked with 100 microM glibenclamide, hypoxic suppression of iGluRs was abolished. Inhibiting mitochondrial Ca2+ uptake via the mitochondrial Ca2+ uniporter with ruthenium red, or blocking Ca2+ release through ryanodine receptors in the endoplasmic reticulum, also abolished the hypoxia-induced suppression. Similarly, inhibition of calmodulin with calmidazolium, or of protein phosphatase 1 and 2A (PP1/PP2A) activity with calyculin A, prevented the hypoxia-induced suppression of iGluRs. Taken together, these results indicate that mKATP activation is required to mediate an intracellular Ca2+ signal that downregulates iGluR activity during hypoxia. Through the reduction of iGluR activity, HCs may lower their metabolic demand, allowing them to maintain cell function during hypoxia. Additionally, GABA release from HCs, as measured by mass spectrometry, was not affected by hypoxia, and we propose that downregulation of iGluR may account for hypoxic suppression of the retina in hypoxia-tolerant organisms.

Background: Ion channels regulate mammalian sperm function, yet chloride channels have received less attention than their cation counterparts. Herein, we investigated the presence, localization, and function of voltage-gated chloride channels (ClC-1, ClC-2, and ClC-K) and calcium-activated channels (ANO1 and ANO2) during sperm capacitation and the acrosome reaction. Results: Using the pig as an animal model, we localized these channels in different sperm regions: ClC-1 in the equatorial and post-acrosomal regions; ClC-2 in the connecting and principal pieces; ClC-K in the acrosome, connecting, and principal pieces; ANO1 in the principal piece; and ANO2 in the mid-piece and post-acrosomal sheath region. Following this, we addressed the roles of these channels in sperm capacitation and acrosome reaction by blocking them with different inhibitors: 9AC, specific for ClCs; BBR, specific for CaCCs; and NPPB, a broad-spectrum blocker that inhibits ClCs, CaCCs, and other chloride channels. Blocking CaCCs with BBR led to a significant reduction in sperm motility and mitochondrial membrane potential, with effects similar to those of the non-selective inhibitor NPPB or to a combination of BBR and NPPB. Moreover, although none of the three inhibitors affected intracellular calcium levels or acrosomal integrity, a significant reduction in protein tyrosine phosphorylation was observed in sperm treated with the combination of NPPB and BBR. Finally, membrane lipid disorder was increased when ClCs were blocked with 9AC and when sperm were incubated with a combination of BBR and NPPB. Conclusions: Our results suggest that CaCCs play a major role in sperm capacitation compared to ClCs. Further research to address the regulatory mechanisms involved and the potential relationship with other ion channels is warranted.

{beta}-arrestins 1 and 2 are intracellular adaptor proteins that regulate the subcellular localisation and signalling of GPCRs including Parathyroid Hormone Receptor 1 (PTH1R), which produces {beta}-arrestin-dependent signalling that may selectively stimulate bone anabolism. {beta}-arrestins can themselves be regulated by post-translational modifications including phosphorylation, which dictates the translocation and signalling of the few GPCRs studied in this context to date. We aimed to investigate whether phosphorylation of {beta}-arrestins modulates their subcellular translocation in response to PTH1R activation. Translocation of {beta}-arrestins to the plasma membrane and early endosomes was quantified using BRET biosensors and immunocytochemistry with high content analysis. Phosphoknockout or phosphomimetic mutations were introduced to previously demonstrated phosphorylation sites in {beta}-arrestins 1 and 2, initially in bulk to assess overall effects. Plasma membrane translocation was altered only subtly, whereas early endosome translocation was markedly reduced for both mutations but to a greater degree for the phosphomimetic. Contributions of individual or paired sites were assessed by restoration to wildtype from the bulk phosphomimetic mutants. Dual restoration of S13 and T275 in {beta}-arrestin 1, or the analogous sites in {beta}-arrestin 2, restored the greatest degrees of early endosomal translocation, with complete restoration for {beta}-arrestin 1 and partial for {beta}-arrestin 2. Restoration of other sites had minimal or no effect. Dephosphorylation therefore appears to be required for PTH1R-stimulated {beta}-arrestin early endosomal translocation, with different phosphorylation sites implicated than some other GPCRs. These findings raise the possibility of differential {beta}-arrestin regulation by distinct stimuli which may have implications in therapeutic targeting of {beta}-arrestin-dependent pathways.

The sarcoplasmic reticulum (SR) is an elaborate membrane network and the primary intracellular Ca2+ store for muscle contraction. SR abundance, morphology, and spatial organization must be remodeled during muscle atrophy and hypertrophy, yet the mechanisms underlying this remodeling remain poorly understood. Autophagy has been implicated in SR homeostasis and may contribute to this process. We previously showed that a subset of Drosophila larval muscles remodels into adult dorsal internal oblique muscles through an autophagy-dependent process accompanied by extensive SR reorganization. Here, we show that autophagy is essential for maintaining SR abundance and architecture during muscle remodeling. Blocking autophagosome formation caused the accumulation of large aggregates of SR membranes, whereas blocking autophagosome-lysosome fusion led to the accumulation of autophagosomes with SR transmembrane proteins localized to their membranes. Moreover, disrupting LC3-interacting region (LIR)-dependent cargo recognition did not impair SR remodeling, arguing against a major contribution from selective autophagic degradation of the SR. Instead, our findings suggest that SR membrane components are incorporated into autophagosomal membranes during muscle remodeling. We propose that this membrane flow couples the removal of excess SR membrane to autophagosome biogenesis during atrophy, thereby facilitating large-scale SR reorganization during muscle remodeling.

The glycolytic regulator PFKFB3 supports dysfunctional {beta}-cell states, but its role in pancreatic Ca2+ signaling remains unclear. We examined responses to PFKFB3 inhibitors (AZ67, AZ26) in pancreatic tissue slices from aged 3xTg-AD mice, a model of Alzheimer's disease that develops impaired glucose tolerance and diabetes as comorbidity. Compared to wild-type controls, {beta}-cells showed weaker glucose-stimulated and acetylcholine-potentiated Ca2+ activity, accompanied by increased nuclear PFKFB3 positivity in insulin-defined islet-cell populations, used here as a PFKFB3-associated stress-state marker. During glucose and acetylcholine stimulation, AZ67 increased {beta}-cell Ca2+ event frequency while maintaining short events. In neighboring acinar cells, PFKFB3 inhibitor exposure was followed by renewed oscillations after acetylcholine-evoked activity had waned. {beta}-cell recruitment was preserved during adrenergic inhibition, and enhanced activity persisted after inhibitor withdrawal, although these recordings were exploratory and residual tissue drug exposure could not be excluded. An effect that survives both drug removal and adrenergic opposition identifies a Ca2+ signaling phenotype in 3xTg-AD {beta}-cells that is acutely inducible yet not transient, and establishes PFKFB3 as a regulator of muscarinic responsiveness in the stressed {beta}-cell.

Hippocampal ripples, accompanied by highly synchronous neuronal firing, are critical for immediate offline memory consolidation, learning, and planning. However, the cellular mechanisms that determine which individual neurons are recruited to participate in action potential (AP) firing during ripples remain unclear. We investigated the membrane potential (Vm) dynamics of CA1 pyramidal cells during ripples using whole-cell recordings in awake, head-fixed mice. We found that ripple participation was strongly associated with concurrent membrane-potential depolarization on an event-to-event basis, whereas between-cell differences in overall participation propensity were jointly explained by ripple-associated depolarization and intrinsic excitability. Applying this framework across CA1 cell populations revealed that differences in recruitment between superficial and deep pyramidal cells were primarily driven by ripple-associated depolarization, counteracted by excitability. Differences between place and non-place cells were similarly driven by ripple-associated depolarization, though this effect acted in the same direction as excitability. Finally, we identified the post-ripple LFP rebound as a prominent marker of ripple diversity linked to post-ripple membrane-potential hyperpolarization at the single-neuron level. Together, these findings provide a mechanistic understanding of how CA1 pyramidal cells are selectively recruited during hippocampal ripples.

Anion exchanger 4 (Ae4), encoded by solute carrier family 4 member 9 (Slc4a9), is required for normal salivary secretion, but how it supports fluid secretion remains poorly understood. Nearly a decade ago, we developed a mathematical model that attributed reduced secretion following Ae4 knockout primarily to Ae4 mediated monovalent cation transport. Here, we reassess that interpretation by developing a dynamic model of salivary fluid secretion informed by subsequent experimental findings. The model incorporates {beta}-adrenergic regulation of Ae4 and an additional apical chloride conductance shared by the control and knockout. To isolate the contribution of Ae4, the NKCC1 and Ae2 cycle rates calculated from the evolving control state are also prescribed in the knockout. Under these conditions, the knockout model secretes 20% less fluid over 600 seconds despite identical apical chloride conductances. The control begins with a larger intracellular chloride store and continues to acquire chloride through Ae4 during stimulation. The intracellular chloride difference increases during stimulation, whilst the weaker apical electrochemical driving force in the knockout reduces chloride export and fluid secretion. The analysis also identifies the alkalinity requirement of sustained Ae4 transport and quantifies the additional NKCC1 and Ae2 transport needed to compensate for its loss. These results support intracellular chloride availability as a mechanism linking Ae4 loss to reduced salivary secretion.

The pace of fracture repair declines with age, increasing the risk of delayed union and nonunion. Delayed union and nonunion are associated with significant morbidity and even mortality with few effective non-surgical interventions. Here, we identify cathelicidin antimicrobial peptide (CAMP) as an age-dependent regulator of bone repair. Single-cell RNA sequencing and immunohistochemistry demonstrated higher CAMP expression during fracture healing in young compared with aged mice. Global or myeloid lineage-depletion of Camp delayed fracture repair in young mice, whereas Camp depletion in aged mice increased callus bone formation. Heterochronic bone marrow transplantation using the conditional genetic knockout further implicated hematopoietic-lineage derived cells in these effects and demonstrated that myeloid-specific Camp expression is important in the rejuvenation of the pace of fracture healing. CAMP promotes osteogenic differentiation through P2X7 receptor (P2X7R)-dependent signaling, influencing the downstream regulation of NLRP3 expression. NLRP3 expression is also correlated with osteogenesis following Camp modulation in both young and old mice, supporting CAMP-P2X7R-NLRP3 signaling as a regulator of osteogenesis. Together, these findings identify CAMP as a hematopoietic-derived regulator of bone repair whose function is age-dependent, promoting fracture healing in young animals while constraining it in aged animals.