Cell Ca2 Research Articles

The development of potent, competitive CXCR4 antagonists for the prevention of cancer metastasis

Cancer metastasis is the cause of up to 90 % of cancer related mortality. The CXCR4 receptor and its cognate ligand, CXCL12, have major roles in enabling cancer metastasis and consequently, the CXCR4 receptor has become an attractive therapeutic target for the prevention of metastasis. Despite this, CXCR4 antagonists have had limited success in clinical trials due to cellular toxicity and poor stability and efficacy. In this study, we developed a novel, competitive CXCR4 antagonist (IS4) that through copper-catalysed-azide-alkyne-cycloaddition can be clicked to other chemical moieties such as fluorescent dyes (IS4-FAM) for CXCR4-based imaging. We determined that these CXCR4 antagonists were non-toxic and could be used to specifically label the CXCR4 receptor. Furthermore, IS4 and IS4-FAM inhibited CXCL12-stimulated cancer cell migration and Ca2+ release in both adherent and suspension cell lines with similar or improved potency as compared to two literature CXCR4 antagonists. Our results highlight the potential of IS4 and IS4-FAM as research tools and as potent CXCR4 antagonists for the prevention of metastasis.

Localized APP expression results in progressive network dysfunction by disorganizing spike timing

Progressive cognitive decline in Alzheimer's disease could either be caused by a spreading molecular pathology or by an initially focal pathology that causes aberrant neuronal activity in a larger network. To distinguish between these possibilities, we generated a mouse model with expression of mutant human amyloid precursor protein (APP) in only hippocampal CA3 cells. We found that performance in a hippocampus-dependent memory task was impaired in young adult and aged mutant mice. In both age groups, we then recorded from the CA1 region, which receives inputs from APP-expressing CA3 cells. We observed that theta oscillation frequency in CA1 was reduced along with disrupted relative timing of principal cells. Highly localized pathology limited to the presynaptic CA3 cells is thus sufficient to cause aberrant firing patterns in postsynaptic neuronal networks, which indicates that disease progression is not only from spreading pathology but also mediated by progressively advancing physiological dysfunction.

Fecal microbiota transplantation and short-chain fatty acids improve learning and memory in fluorosis mice by BDNF-PI3K/AKT pathway

Fluoride, an environmental toxicant, not only arouses intestinal microbiota dysbiosis, but also causes neuronal apoptosis and a decline in learning and memory ability. The purpose of this study was to explore whether fecal microbiota transplantation (FMT) from healthy mice and bacteria-derived metabolites short-chain fatty acids (SCFAs) supplement protect against fluoride-induced learning and memory impairment. Results showed that FMT reversed the elevated percentage of working memory errors (WME) and reference memory errors (RME) in fluorosis mice during the eight-arm maze test. Nissl and TUNEL staining presented that fluoride led to a decreased proportion of Nissl bodies area in the hippocampal CA3 region and an increased apoptotic ratio of nerve cells in CA1, CA3 and DG areas, whereas FMT alleviated those pathological damages. Moreover, the expressions of mRNA in hippocampal BDNF, PDK1, AKT, Bcl-2, and Bcl-xL were downregulated in mice exposed to fluoride, but the levels of PI3K, Bax, Bak, and Caspase-7 mRNA were upregulated. NaF treatment had an increase in PI3K and Caspase-3 protein levels and reduced the expressions of these four proteins, including BDNF, p-PI3K, AKT and p-AKT. By contrast, FMT enhanced the expression of BDNF and thus activated the PI3K/AKT pathway. Besides, the 16S rRNA sequencing revealed that fluoride caused a reduction in certain SCFA producers in the colon as evidenced by a decline in Erysipelatoclostridiaceae, and a downward trend in Akkermansia, Blautia and Alistipes. However, the disordered gut microbiome was restored via frequent FMT. Of note, SCFAs administration also increased BDNF levels and regulated its downstream pathways, which contributed to cell survival and learning and memory function recovery. In conclusion, FMT and SCFAs may activate the BDNF-PI3K/AKT pathway to play an anti-apoptotic role and ultimately improve learning and memory deficits in fluorosis mice.

Effects of low level laser on periodontal tissue remodeling in hPDLCs under tensile stress

This study aimed to investigate the effect of Low-Level Laser Therapy (LLLT) on human Periodontal Ligament Cells (hPDLCs) under tension stress. Primary hPDLCs were obtained using the tissue culture method, and P3 cells were utilized for the subsequent experiments. The study comprised four groups: a blank control group (Group B), a laser irradiation group (Group L), a tension stress group (Group T), and a laser + tension stress group (Group LT). Mechanical loading was applied using an in-vitro cell stress loading device at a frequency of 0.5 Hz and deformation of 2% for two hours per day for two days. Laser irradiation at 808 nm GaAlAs laser was administered 1 h after force loading. Cell samples were collected after the experiment. Bone and fiber remodeling factors were analyzed using PCR and Western blot. Flow cytometry was employed to assess the cell cycle, while ROS and Ca2+ levels were measured using a multifunctional enzyme labeling instrument. The results revealed that laser intervention under tension stress inhibited the expression of osteogenic differentiation factors, promoted the expression of osteoclast differentiation factors, and significantly increased the production of collagen factors, MMPs, and TIMPs. The LT group exhibited the most active cell cycle (P < 0.05). LLLT not only enhanced Ca2+ expression in hPDLCs under tension stress, but also stimulated the production of ROS. Overall, our findings demonstrate that LLLT effectively accelerated the proliferation of hPDLCs and the remodeling of periodontal tissue, possibly through the regulation of ROS and Ca2+ levels in hPDLCs.

T cell Ca2+ microdomains through the lens of computational modeling.

Cellular Ca2+ signaling is highly organized in time and space. Locally restricted and short-lived regions of Ca2+ increase, called Ca2+ microdomains, constitute building blocks that are differentially arranged to create cellular Ca2+ signatures controlling physiological responses. Here, we focus on Ca2+ microdomains occurring in restricted cytosolic spaces between the plasma membrane and the endoplasmic reticulum, called endoplasmic reticulum-plasma membrane junctions. In T cells, these microdomains have been finely characterized. Enough quantitative data are thus available to develop detailed computational models of junctional Ca2+ dynamics. Simulations are able to predict the characteristics of Ca2+ increases at the level of single channels and in junctions of different spatial configurations, in response to various signaling molecules. Thanks to the synergy between experimental observations and computational modeling, a unified description of the molecular mechanisms that create Ca2+ microdomains in the first seconds of T cell stimulation is emerging.

Monosynaptic rabies tracing maps inputs to CA1 cells in old AD model mice (APP-KI).

Monosynaptic rabies tracing maps inputs to CA1 cells in old AD model mice (APP-KI).

Theta-band phase locking during encoding leads to coordinated entorhinal-hippocampal replay

Precisely timed interactions between hippocampal and cortical neurons during replay epochs are thought to support learning. Indeed, research has shown that replay is associated with heightened hippocampal-cortical synchrony. Yet many caveats remain in our understanding. Namely, it remains unclear how this offline synchrony comes about, whether it is specific to particular behavioral states, and how-if at all-it relates to learning. In this study, we sought to address these questions by analyzing coordination between CA1 cells and neurons of the deep layers of the medial entorhinal cortex (dMEC) while rats learned a novel spatial task. During movement, we found a subset of dMEC cells that were particularly locked to hippocampal LFP theta-band oscillations and that were preferentially coordinated with hippocampal replay during offline periods. Further, dMEC synchrony with CA1 replay peaked ∼10ms after replay initiation in CA1, suggesting that the distributed replay reflects extra-hippocampal information propagation and is specific to "offline" periods. Finally, theta-modulated dMEC cells showed a striking experience-dependent increase in synchronization with hippocampal replay trajectories, mirroring the animals' acquisition of the novel task and coupling to the hippocampal local field. Together, these findings provide strong support for the hypothesis that synergistic hippocampal-cortical replay supports learning and highlights phase locking to hippocampal theta oscillations as a potential mechanism by which such cross-structural synchrony comes about.

Fine-tuned photochromic sulfonylureas for optical control of beta cell Ca2+ fluxes.

We previously developed, synthesized and tested light-activated sulfonylureas for optical control of KATP channels and pancreatic beta cell activity in vitro and in vivo. Such technology relies on installation of azobenzene photoswitches onto the sulfonylurea backbone, affording light-dependent isomerization, alteration in ligand affinity for SUR1 and hence KATP channel conductance. Inspired by molecular dynamics simulations and to further improve photoswitching characteristics, we set out to develop a novel push-pull closed ring azobenzene unit, before installing this on the sulfonylurea glimepiride as a small molecule recipient. Three fine-tuned, light-activated sulfonylureas were synthesized, encompassing azetidine, pyrrolidine and piperidine closed rings. Azetidine-, pyrrolidine- and piperidine-based sulfonylureas all increased beta cell Ca2+ -spiking activity upon continuous blue light illumination, similarly to first generation JB253. Notably, the pyrrolidine-based sulfonylurea showed superior switch OFF performance to JB253. As such, third generation sulfonylureas afford more precise optical control over primary pancreatic beta cells, and showcase the potential of pyrrolidine-azobenzenes as chemical photoswitches across drug classes.

Enhanced presynaptic mitochondrial energy production is required for memory formation

Some of the prominent features of long-term memory formation include protein synthesis, gene expression, enhanced neurotransmitter release, increased excitability, and formation of new synapses. As these processes are critically dependent on mitochondrial function, we hypothesized that increased mitochondrial respiration and dynamics would play a prominent role in memory formation. To address this possibility, we measured mitochondrial oxygen consumption (OCR) in hippocampal tissue punches from trained and untrained animals. Our results show that context fear training significantly increased basal, ATP synthesis-linked, and maximal OCR in the Shaffer collateral-CA1 synaptic region, but not in the CA1 cell body layer. These changes were recapitulated in synaptosomes isolated from the hippocampi of fear-trained animals. As dynamin-related protein 1 (Drp1) plays an important role in mitochondrial fission, we examined its role in the increased mitochondrial respiration observed after fear training. Drp1 inhibitors decreased the training-associated enhancement of OCR and impaired contextual fear memory, but did not alter the number of synaptosomes containing mitochondria. Taken together, our results show context fear training increases presynaptic mitochondria respiration, and that Drp-1 mediated enhanced energy production in CA1 pre-synaptic terminals is necessary for context fear memory that does not result from an increase in the number of synaptosomes containing mitochondria or an increase in mitochondrial mass within the synaptic layer.

Unique potential of immature adult-born neurons for the remodeling of CA3 spatial maps

Mammalian hippocampal circuits undergo extensive remodeling through adult neurogenesis. While this process has been widely studied, the specific contribution of adult-born granule cells (aGCs) to spatial operations in the hippocampus remains unknown. Here, we show that optogenetic activation of 4-week-old (young) aGCs in free-foraging mice produces a non-reversible reconfiguration of spatial maps in proximal CA3 while rarely evoking neural activity. Stimulation of the same neuronal cohort on subsequent days recruits CA3 neurons with increased efficacy but fails to induce further remapping. In contrast, stimulation of 8-week-old (mature) aGCs can reliably activate CA3 cells but produces no alterations in spatial maps. Our results reveal a unique role of young aGCs in remodeling CA3 representations, a potential that can be depleted and is lost with maturation. This ability could contribute to generate orthogonalized downstream codes supporting pattern separation.

Decreased adhesion to endothelium leads to elevated neutrophil granulocyte count in hereditary angioedema patients

As many aspects of hereditary angioedema (HAE) due to C1-inhibitor (C1-INH) deficiency (C1-INH-HAE) cannot be explained with elevated bradykinin level alone, it has recently become clear that other factors also play an important role in the pathogenesis. One of these factors could be elevated neutrophil granulocyte (NG) counts, which are associated with increased NG activation in C1-INH-HAE patients; however, their origin has not been elucidated so far. Here, we aimed to investigate whether the excess of NGs is due to disturbed maturation, biased circulating/marginated pool equilibrium or decreased elimination. We enrolled 20 attack-free C1-INH-HAE patients together with 21 healthy controls and collected blood samples. We compared cell surface maturation markers, adhesion molecules, cytokine receptors, and Ca2+-mobilization of NG by flow cytometry, activation markers by ELISA, and NG/endothelial cell adhesion by automated pipetting system. Cell-surface markers showed normal maturation of NGs in C1-INH-HAE patients. Adhesion of NGs to endothelial cells pretreated with lipopolysaccharide or phorbol 12-myristate 13-acetate was significantly weaker in samples from C1-INH-HAE patients and bradykinin had no effect on the adhesion. NGs from C1-INH-HAE patients were in an activated state when assessed by soluble activation markers without any stimulation. Our data support that the maturation of NGs in C1-INH-HAE patients is normal, whereas adhesion properties of patient-derived NGs to the endothelium are reduced compared to those from healthy controls, indicating a bias between the circulating and marginated pools of NGs in patients. Bradykinin may not be responsible for reduced adhesion properties of NGs.

Quantitative analysis of the optogenetic excitability of CA1 neurons.

Optogenetics has emerged as a promising technique for modulating neuronal activity and holds potential for the treatment of neurological disorders such as temporal lobe epilepsy (TLE). However, clinical translation still faces many challenges. This in-silico study aims to enhance the understanding of optogenetic excitability in CA1 cells and to identify strategies for improving stimulation protocols. Employing state-of-the-art computational models coupled with Monte Carlo simulated light propagation, the optogenetic excitability of four CA1 cells, two pyramidal and two interneurons, expressing ChR2(H134R) is investigated. The results demonstrate that confining the opsin to specific neuronal membrane compartments significantly improves excitability. An improvement is also achieved by focusing the light beam on the most excitable cell region. Moreover, the perpendicular orientation of the optical fiber relative to the somato-dendritic axis yields superior results. Inter-cell variability is observed, highlighting the importance of considering neuron degeneracy when designing optogenetic tools. Opsin confinement to the basal dendrites of the pyramidal cells renders the neuron the most excitable. A global sensitivity analysis identified opsin location and expression level as having the greatest impact on simulation outcomes. The error reduction of simulation outcome due to coupling of neuron modeling with light propagation is shown. The results promote spatial confinement and increased opsin expression levels as important improvement strategies. On the other hand, uncertainties in these parameters limit precise determination of the irradiance thresholds. This study provides valuable insights on optogenetic excitability of CA1 cells useful for the development of improved optogenetic stimulation protocols for, for instance, TLE treatment.

Hippocampal ensemble dynamics and memory performance are modulated by respiration during encoding

During offline brain states, such as sleep and memory consolidation, respiration coordinates hippocampal activity. However, the role of breathing during online memory traces remains unclear. Here, we show that respiration can be recruited during online memory encoding. Optogenetic manipulation was used to control activation of the primary inspiratory rhythm generator PreBötzinger complex (PreBötC) in transgenic mice. When intermittent PreBötC-induced apnea covered the object exploration time during encoding, novel object detection was impaired. Moreover, the mice did not exhibit freezing behavior during presentation of fear-conditioned stimuli (CS+) when PreBötC-induced apnea occurred at the exact time of encoding. This apnea did not evoke changes in CA3 cell ensembles between presentations of CS+ and conditioned inhibition (CS−), whereas in normal breathing, CS+ presentations produced dynamic changes. Our findings demonstrate that components of central respiratory activity (e.g., frequency) during online encoding strongly contribute to shaping hippocampal ensemble dynamics and memory performance.

Spatio temporal interdependent calcium and buffer dynamics regulating DAG in a hepatocyte cell due to obesity.

Calcium ions (Ca2+) serve as a crucial signaling mechanism in almost all cells. The buffers are proteins that bind free Ca2+ to reduce the cell's Ca2+ concentration. The most studies reported in the past on calcium signaling in various cells have considered the buffer concentration as constant in the cell. However, buffers also diffuse and their concentration varies dynamically in the cells. Almost no work has been reported on interdependent calcium and buffer dynamics in the cells. In the present study, a model is proposed for inter-dependent spatio-temporal dynamics of calcium and buffer by coupling reaction-diffusion equations of Ca2+ and buffer in a hepatocyte cell. Boundary and initial conditions are framed based on the physiological state of the cell. The effect of various parameters viz. inositol 1,4,5-triphosphate receptor (IP3R), diffusion coefficient, SERCA pump and ryanodine receptor (RyR) on spatio-temporal dynamics of calcium and buffer regulating diacylglycerol (DAG) in a normal and obese hepatocyte cell has been studied using finite element simulation. From the results, it is concluded that the dynamics of calcium and buffer impact each other significantly along the spatio-temporal dimensions, thereby affecting the regulation of all the processes including DAG in a hepatocyte cell. The proposed model is more realistic than the existing ones, as the interdependent system dynamics of calcium and buffer have different regulatory impacts as compared to the individual and independent dynamics of these signaling processes in a hepatocyte cell.

Electroacupuncture improves gout arthritis pain via attenuating ROS-mediated NLRP3 inflammasome overactivation

BackgroundGout results from disturbed uric acid metabolism, which causes urate crystal deposition in joints and surrounding tissues. Gout pain management is largely limited to colchicine and nonsteroidal anti-inflammatory drugs. Constant usage of these medications leads to severe side effects. We previously showed electroacupuncture (EA) is effective for relieving pain in animal model of gout arthritis. Here we continued to study the mechanisms underlying how EA alleviates gout pain.MethodsMonosodium urate was injected into ankle joint to establish gout arthritis model in mice. EA or sham EA was applied at ST36 and BL60 acupoints of model animals. Biochemical assays, immunostaining, live cell Ca2+ imaging and behavioral assays were applied.ResultsModel mice displayed obvious mechanical allodynia, accompanied with gait impairments. EA attenuated mechanical hypersensitivities and improved gait impairments. EA reduced the overexpression of NLRP3 inflammasome signaling molecules in ankle joints of model animals. EA-induced anti-allodynia, as well as inhibition on NLRP3 inflammasome, were mimicked by antagonizing but abolished by activating NLRP3 inflammasome via pharmacological methods. EA attenuated oxidative stress, an upstream signaling of NLRP3 inflammasome in ankle joints of model mice. Exogenously increasing oxidative stress abolished EA’s inhibitory effect on NLRP3 inflammasome and further reversed EA’s anti-allodynic effect. EA reduced neutrophil infiltrations in ankle joint synovium, a major mechanism contributing to oxidative stress in gout. Pharmacological blocking NLRP3 inflammasome or EA reduced TRPV1 channel overexpression in dorsal root ganglion (DRG) neurons. Ca2+ imaging confirmed that EA could reduce functional enhancement in TRPV1 channel in DRG neurons during gout.ConclusionsOur results demonstrate that EA reduces gout pain possibly through suppressing ROS-mediated NLRP3 inflammasome activation in inflamed ankle joints and TRPV1 upregulation in sensory neurons, supporting EA as a treatment option for gout pain.

Multiple PDZ domain protein regulates sperm motility through CatSper channel

Multiple PDZ domain protein (MPDZ) is a component of the crumbs cell polarity complex and is ubiquitously expressed in mammalian tissues. In mouse sperm, MPDZ was identified as a regulator of the sperm acrosome reaction in the past two decades. Here, a relationship between MPDZ and sperm motility was discovered. During breeding, we found that MPDZ-null mice had smaller litter size. The Ca2+ signal in mouse sperm decreased. Computer-assisted semen analysis revealed that MPDZ-null males had reduced mouse sperm motility. In humans, MPDZ expression is positively associated with sperm motility. Considering the regulatory role of the CatSper channel in sperm motility and fertility, we identified the expression profile of CatSper subunits (CatSper1–4) and found a reduction at both the transcriptional and translational levels in MPDZ-null mouse spermatozoa. However, in vitro, only CatSper1/2 expression was upregulated in MPDZ-overexpressing GC2 cells. Meanwhile, we found an upregulated cell Ca2+ signal when MPDZ was overexpressed in GC2 cells. Mechanical analysis revealed that MPDZ bound to the signal transducer and activator of transcription 3 (Stat3) and promoted its phosphorylation at tyrosine 705 (Y705) to upregulate CatSper1/2 expression. Inhibition of Stat3 (Y705) phosphorylation or Stat3 expression attenuated the effect of MPDZ on CatSper1/2 expression. These results suggested that MPDZ was responsible for the transcriptional regulation of the CatSper channel, at least in part, to regulate Ca2+ signal and sperm motility. To the best of our knowledge, this is the first study to highlight the importance of MPDZ acting as a regulator of sperm motility.

Agmatine improves liver function, balance performance, and neuronal damage in a hepatic encephalopathy induced by bile duct ligation.

In the current study, we investigate whether oral administration of agmatine (AGM) could effectively reduce motor and cognitive deficits induced by bile duct ligation (BDL) in an animal model of hepatic encephalopathy (HE) through neuroprotective mechanisms. The Wistar rats were divided into four groups: sham, BDL, BDL+ 40mg/kg AGM, and BDL+ 80mg/kg AGM. The BDL rats were treated with AGM from 2 weeks after the surgery for 4 consecutive weeks. The open field, rotarod, and wire grip tests were used to assess motor function and muscle strength. The novel object recognition test (NOR) was performed to evaluate learning and memory. Finally, blood samples were collected for the analysis of the liver markers, the animals were sacrificed, and brain tissues were removed; the CA1 regions of the hippocampus and cerebellum were processed to identify apoptosis and neuronal damage rate using caspase-3 immunocytochemistry and Nissl staining. The serological assay results showed that BDL severely impaired the function of the liver. Based on histochemical findings, BDL increased the neuronal damage in CA1 and Purkinje cells, whereas apoptosis was significantly observed only in the cerebellum. AGM treatment prevented the increase of serum liver enzymes, balance deficits, and neuronal damage in the brain areas. Apoptosis partially decreased by AGM, and there were no differences in the performance of animals in different groups in the NOR. The study suggests AGM as a potential treatment candidate for HE because of its neuroprotective properties and/or its direct effects on liver function.

1749-P: Influence of the Biomechanical Environment on Islet Function and Maturity

It is well-established that cells respond to mechanical cues via cytoskeletal remodeling. However, it is not known whether the mechanical environment influences β-cell maturity and function. To examine how mechanical cues influence the β-cell, we used biocompatible substrates with tunable mechanical properties synthesized with elastic moduli ranging from ~3kPa-33 kPa. The surface was coated with extracellular matrix and MIN6 β-like cells were seeded. To independently examine a link between β-cell function and maturity, we examined islets from mice in which β-cells express CaMPARI, a photoconvertible fluorescent protein that in the presence of high Ca2+ activity changes from green to red. For all conditions in MIN6 cells and CAMPARI-expressing islets, we imaged Ca2+ dynamics via Fluo4 at low (2mM) and high glucose (11, 20mM). qPCR was conducted on MIN6 cells (6, 22 kPa) and flow-sorted CaMPARI-expressing β-cells (green, red) to examine expression of genes linked to β-cell function and maturity. With increased substrate stiffness, MIN6 cell Ca2+ oscillations were more robust, including a significant increase in duty cycle (p&amp;lt;0.01) and were more coordinated, consistent with increased excitability. With increasing stiffness, there was significantly decreased expression of Sur1, Kir6.2, Ins2, Pdx1, Fltp, and Ecad (p&amp;lt;0.01). Thus, the dynamics and coordination of Ca2+ activity is mechanoresponsive and may result from altered β-cell maturity. In CaMPARI islets, Ca2+ oscillations were more robust and coordinated for photo-converted red cells which marks cells with higher Ca2+, compared to green cells. In these red cells that show higher Ca2+, qPCR showed significantly increased expression of cFos, but decreased expression of Pdx1 and Ins2, again showing more excitable cells showed altered maturity. In conclusion, our results suggest there may be an inverse relationship between β-cell maturity and function. Further, that the maturity and function of β-cells is responsive to the mechanical environment. Disclosure K.Vazquez: None. R.K.Benninger: None. Funding National Institutes of Health (R01DK102950, R01DK106412)

The mitochondrial Ca2+ channel MCU is critical for tumor growth by supporting cell cycle progression and proliferation.

Introduction: The mitochondrial uniporter (MCU) Ca2+ ion channel represents the primary means for Ca2+ uptake by mitochondria. Mitochondrial matrix Ca2+ plays critical roles in mitochondrial bioenergetics by impinging upon respiration, energy production and flux of biochemical intermediates through the TCA cycle. Inhibition of MCU in oncogenic cell lines results in an energetic crisis and reduced cell proliferation unless media is supplemented with nucleosides, pyruvate or α-KG. Nevertheless, the roles of MCU-mediated Ca2+ influx in cancer cells remain unclear, in part because of a lack of genetic models. Methods: MCU was genetically deleted in transformed murine fibroblasts for study in vitro and in vivo. Tumor formation and growth were studied in murine xenograft models. Proliferation, cell invasion, spheroid formation and cell cycle progression were measured in vitro. The effects of MCU deletion on survival and cell-death were determined by probing for live/death markers. Mitochondrial bioenergetics were studied by measuring mitochondrial matrix Ca2+ concentration, membrane potential, global dehydrogenase activity, respiration, ROS production and inactivating-phosphorylation of pyruvate dehydrogenase. The effects of MCU rescue on metabolism were examined by tracing of glucose and glutamine utilization for fueling of mitochondrial respiration. Results: Transformation of primary fibroblasts in vitro was associated with increased MCU expression, enhanced MCU-mediated Ca2+ uptake, altered mitochondrial matrix Ca2+ concentration responses to agonist stimulation, suppression of inactivating-phosphorylation of pyruvate dehydrogenase and a modest increase of mitochondrial respiration. Genetic MCU deletion inhibited growth of HEK293T cells and transformed fibroblasts in mouse xenograft models, associated with reduced proliferation and delayed cell-cycle progression. MCU deletion inhibited cancer stem cell-like spheroid formation and cell invasion in vitro, both predictors of metastatic potential. Surprisingly, mitochondrial matrix [Ca2+], membrane potential, global dehydrogenase activity, respiration and ROS production were unaffected. In contrast, MCU deletion elevated glycolysis and glutaminolysis, strongly sensitized cell proliferation to glucose and glutamine limitation, and altered agonist-induced cytoplasmic Ca2+ signals. Conclusion: Our results reveal a dependence of tumorigenesis on MCU, mediated by a reliance on MCU for cell metabolism and Ca2+ dynamics necessary for cell-cycle progression and cell proliferation.

Cholinergic Stimulation Modulates the Functional Composition of CA3 Cell Types in the Hippocampus.

The functional heterogeneity of hippocampal CA3 pyramidal neurons has emerged as a key aspect of circuit function. Here, we explored the effects of long-term cholinergic activity on the functional heterogeneity of CA3 pyramidal neurons in organotypic slices obtained from male rat brains. Application of agonists to either AChRs generally, or mAChRs specifically, induced robust increases in network activity in the low-gamma range. Prolonged AChR stimulation for 48 h uncovered a population of hyperadapting CA3 pyramidal neurons that typically fired a single, early action potential in response to current injection. Although these neurons were present in control networks, their proportions were dramatically increased following long-term cholinergic activity. Characterized by the presence of a strong M-current, the hyperadaptation phenotype was abolished by acute application of either M-channel antagonists or the reapplication of AChR agonists. We conclude that long-term mAChR activation modulates the intrinsic excitability of a subset of CA3 pyramidal cells, uncovering a highly plastic cohort of neurons that are sensitive to chronic ACh modulation. Our findings provide evidence for the activity-dependent plasticity of functional heterogeneity in the hippocampus.SIGNIFICANCE STATEMENT The large heterogeneity of neuron types in the brain, each with its own specific functional properties, provides the rich cellular tapestry needed to account for the vast diversity of behaviors. By studying the functional properties of neurons in the hippocampus, a region of the brain involved in learning and memory, we find that exposure to the neuromodulator acetylcholine can alter the relative number of functionally defined neuron types. Our findings suggest that the heterogeneity of neurons in the brain is not a static feature but can be modified by the ongoing activity of the circuits to which they belong.