Cytoplasmic Ca2 Research Articles

Caution for Multidrug Therapy: Significant Baroreflex Afferent Neuroexcitation Coordinated by Multi-Channels/Pumps Under the Threshold Concentration of Yoda1 and Dobutamine Combination

Multi-drug therapies are common in cardiovascular disease intervention; however, io channel/pump coordination has not been tested electrophysiologically. Apparently, inward currents were not elicited by Yoda1/10 nM or Dobutamine/100 nM alone in Ah-type baroreceptor neurons, but were by their combination. To verify this, electroneurography and the whole-cell patch-clamp technique were performed. The results showed that Ah- and C-volley were dramatically increased by the combination at 0.5 V and 5 V, in contrast to A-volley, as consistent with repetitive discharge elicited by step and ramp with markedly reduced current injection/stimulus intensity. Notably, a frequency-dependent action potential (AP) duration was increased with Iberiotoxin-sensitive K+ component. Furthermore, an increased peak in AP measured in phase plots suggested enhanced Na+ influx, cytoplasmic Ca2+ accumulation through reverse mode of Na+/Ca2+ exchanger, and, consequently, functional KCa1.1 up-regulation. Strikingly, the Yoda1- or Dbtm-mediated small/transient Na+/K+-pump currents were robustly increased by their combination, implying a quick ion equilibration that may also be synchronized by hyperpolarization-induced voltage-sag, enabling faster repetitive firing. These novel findings demonstrate multi-channel/pump collaboration together to integrate neurotransmission at the cellular level for baroreflex, providing an afferent explanation in sexual dimorphic blood pressure regulation, and raising the caution regarding the individual drug concentration in multi-drug therapies to optimize efficacy and minimize toxicity.

Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE4/5/6/11 negatively regulate hydrotropism via phosphorylation of MIZU-KUSSEI1.

Hydrotropism facilitates the orientation of plant roots towards regions of elevated water potential, enabling them to absorb adequate water. Although calcium signaling plays a crucial role in plant response to water tracking, the exact regulatory mechanisms remain a mystery. Here, we employed the Arabidopsis (Arabidopsis thaliana) hydrotropism-specific protein MIZU-KUSSEI1 (MIZ1) as bait and found that calcium-dependent protein kinases4/5/6/11 (CPK4/5/6/11) interacted with MIZ1 in vitro and in vivo. The cpk4/5/6/11 mutant exhibited increased sensitivity to water potential and enhanced root tip curvature. Furthermore, CPK4/5/6/11 primarily phosphorylated MIZ1 at Ser14/36 residues. Additionally, CPK-mediated phosphorylation of MIZ1 relieved its inhibitory effect on the activity of the endoplasmic reticulum-localized Ca2+ pump ECA1, altering the balance between cytoplasmic Ca2+ inflow and outflow, thereby negatively regulating the hydrotropic growth of plants. Overall, our findings unveil the molecular mechanisms by which the CPK4/5/6/11-MIZ1 module functions in regulating plant hydrotropism responses and provide a theoretical foundation for enhancing plant water use efficiency and promoting sustainable agriculture.

Another-regulin regulates cardiomyocyte calcium handling via integration of neuroendocrine signaling with SERCA2a activity

Calcium (Ca2+) dysregulation is a hallmark feature of cardiovascular disease. Intracellular Ca2+ regulation is essential for proper heart function and is controlled by the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2a). Another-regulin (ALN) is a newly discovered cardiomyocyte-expressed SERCA2a inhibitor, suggesting cardiomyocyte Ca2+-handling is more complex than previously appreciated. To study the role of ALN in cardiomyocytes, we generated ALN null mice (knockout, KO) and found that cardiomyocytes from these animals displayed enhanced Ca2+ cycling and contractility compared to wildtype (WT) mice, indicating enhanced SERCA2a activity. In vitro and in vivo studies show that ALN is post-translationally modified via phosphorylation on Serine 19 (S19), suggesting this contributes to its ability to regulate SERCA2a. Immunoprecipitation and FRET analysis of ALN-WT, phospho-deficient ALN (S19A), or phosphomimetic ALN (S19D) revealed that S19 phosphorylation alters the SERCA2a-ALN interaction, leading to relief of its inhibitory effects. Adeno-associated virus mediated delivery of ALN-WT or phospho-mutant ALN-S19A/D in ALN KO mice showed that cardiomyocyte-specific expression of phospho-deficient ALN-S19A resulted in increased SERCA2a inhibition characterized by reduced rates of cytoplasmic Ca2+ clearance compared to ALN-WT and ALN-S19D expressing cells, further supporting a role for this phosphorylation event in controlling SERCA2a-regulation by ALN. Levels of ALN phosphorylation were markedly increased in cardiomyocytes in response to Gαq agonists (angiotensin II, endothelin-1, phenylephrine) and Gαq-mediated phosphorylation of ALN translated to increased Ca2+ cycling in cardiomyocytes from WT but not ALN KO mice. Collectively, these results indicate that ALN uniquely regulates Ca2+ handling in cardiomyocytes via integration of neuroendocrine signaling with SERCA2a activity.

RNA G-quadruplexes form scaffolds that promote neuropathological α-synuclein aggregation.

Synucleinopathies, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are triggered by α-synuclein aggregation, triggering progressive neurodegeneration. However, the intracellular α-synuclein aggregation mechanism remains unclear. Herein, we demonstrate that RNA G-quadruplex assembly forms scaffolds for α-synuclein aggregation, contributing to neurodegeneration. Purified α-synuclein binds RNA G-quadruplexes directly through the N terminus. RNA G-quadruplexes undergo Ca2+-induced phase separation and assembly, accelerating α-synuclein sol-gel phase transition. In α-synuclein preformed fibril-treated neurons, RNA G-quadruplex assembly comprising synaptic mRNAs co-aggregates with α-synuclein upon excess cytoplasmic Ca2+ influx, eliciting synaptic dysfunction. Forced RNA G-quadruplex assembly using an optogenetic approach evokes α-synuclein aggregation, causing neuronal dysfunction and neurodegeneration. The administration of 5-aminolevulinic acid, a protoporphyrin IX prodrug, prevents RNA G-quadruplex phase separation, thereby attenuating α-synuclein aggregation, neurodegeneration, and progressive motor deficits in α-synuclein preformed fibril-injected synucleinopathic mice. Therefore, Ca2+ influx-induced RNA G-quadruplex assembly accelerates α-synuclein phase transition and aggregation, potentially contributing to synucleinopathies.

African swine fever virus modulates the endoplasmic reticulum stress-ATF6-calcium axis to facilitate viral replication

ABSTRACT African swine fever (ASF), caused by African swine fever virus (ASFV), is a devastating infectious disease of domestic pigs and wild boar, which threatens the global pig industry. Endoplasmic reticulum (ER) is a multifunctional signaling organelle in eukaryotic cells that is involved in protein synthesis, processing, posttranslational modification and quality control. As intracellular parasitic organisms, viruses have evolved several strategies to modulate ER functions to favor their life cycles. We have previously demonstrated that the differentially expressed genes associated with unfolded protein response (UPR), which represents a response to ER stress, are significantly enriched upon ASFV infection. However, the correlation between the ER stress or UPR and ASFV replication has not been illuminated yet. Here, we demonstrated that ASFV infection induces ER stress both in target cells and in vivo, and subsequently activates the activating transcription factor 6 (ATF6) branch of the UPR to facilitate viral replication. Mechanistically, ASFV infection disrupts intracellular calcium (Ca2+) homeostasis, while the ATF6 pathway facilitates ASFV replication by increasing the cytoplasmic Ca2+ level. More specifically, we demonstrated that ASFV infection triggers ER-dependent Ca2+ release via the inositol triphosphate receptor (IP3R) channel. Notably, we showed that the ASFV B117L protein plays crucial roles in ER stress and the downstream activation of the ATF6 branch, as well as the disruption of Ca2+ homeostasis. Taken together, our findings reveal for the first time that ASFV modulates the ER stress-ATF6-Ca2+ axis to facilitate viral replication, which provides novel insights into the development of antiviral strategies for ASFV.

Role of oxytocin in bone.

Oxytocin (OT) is a posterior pituitary hormone that, in addition to its role in regulating childbirth and lactation, also exerts direct regulatory effects on the skeleton through peripheral OT and oxytocin receptor (OTR). Bone marrow mesenchymal stem cells (BMSCs), osteoblasts (OB), osteoclasts (OC), chondrocytes, and adipocytes all express OT and OTR. OT upregulates RUNX2, BMP2, ALP, and OCN, thereby enhancing the activity of BMSCs and promoting their differentiation towards OB rather than adipocytes. OT also directly regulates OPG/RANKL to inhibit adipocyte generation, increase the expression of SOX9 and COMP, and enhance chondrocyte differentiation. OB can secrete OT, exerting influence on the surrounding environment through autocrine and paracrine mechanisms. OT directly increases OC formation through the NκB/MAP kinase signaling pathway, inhibits osteoclast proliferation by triggering cytoplasmic Ca2+ release and nitric oxide synthesis, and has a dual regulatory effect on OCs. Under the stimulation of estrogen, OB synthesizes OT, amplifying the biological effects of estrogen and OT. Mediated by estrogen, the OT/OTR forms a feedforward loop with OB. Apart from estrogen, OT also interacts with arginine vasopressin (AVP), prostaglandins (PGE2), leptin, and adiponectin to regulate bone metabolism. This review summarizes recent research on the regulation of bone metabolism by OT and OTR, aiming to provide insights into their clinical applications and further research.

Collagen IV deficiency causes hypertrophic remodeling and endothelium-dependent hyperpolarization in small vessel disease with intracerebral hemorrhage

Genetic variants in COL4A1 and COL4A2 (encoding collagen IV alpha chain 1/2) occur in genetic and sporadic forms of cerebral small vessel disease (CSVD), a leading cause of stroke, dementia and intracerebral haemorrhage (ICH). However, the molecular mechanisms of CSVD with ICH and COL4A1/COL4A2 variants remain obscure. Vascular function and molecular investigations in mice with a Col4a1 missense mutation and heterozygous Col4a2 knock-out mice were combined with analysis of human brain endothelial cells harboring COL4A1/COL4A2 mutations, and brain tissue of patients with sporadic CSVD with ICH. Col4a1 missense mutations cause early-onset CSVD independent of hypertension, with enhanced vasodilation of small arteries due to endothelial dysfunction, vascular wall thickening and reduced stiffness. Mechanistically, the early-onset dysregulated endothelium-dependent hyperpolarization (EDH) is due to reduced collagen IV levels with elevated activity and levels of endothelial Ca2+-sensitive K+ channels. This results in vasodilation via the Na/K pump in vascular smooth muscle cells. Our data support this endothelial dysfunction preceding development of CSVD-associated ICH is due to increased cytoplasmic Ca2+ levels in endothelial cells. Moreover, cerebral blood vessels of patients with sporadic CSVD show genotype-dependent mechanisms with wall thickening and lower collagen IV levels in those harboring common non-coding COL4A1/COL4A2 risk alleles. COL4A1/COL4A2 variants act in genetic and sporadic CSVD with ICH via dysregulated EDH, and altered vascular wall thickness and biomechanics due to lower collagen IV levels and/or mutant collagen IV secretion. These data highlight EDH and collagen IV levels as potential treatment targets. MRC, Wellcome Trust, BHF.

L-Glutamate treatment alleviates chilling injury of prune (Prunus domestica L.) fruit by regulating ROS homeostasis, GABA shunt, and energy metabolism

The impacts of L-glutamate (L-Glu) treatment on chilling injury (CI), Ca2+ signaling, mitochondrial ultrastructure, and metabolisms of reactive oxygen species (ROS), γ-aminobutyric acid (GABA), energy of prune fruit under chilling stress were studied. The results found that the optimal concentration of L-Glu to suppress CI occurrence and maintain quality in prune fruit was 0.1 g L−1, which also enhanced the PdGLRs expression, cytoplasmic Ca2+ concentration, the contents of CaM, and CML under cold stress. Moreover, L-Glu treatment could reduce ROS accumulation and increase GABA content, and energy level, contributing to maintaining the integrity of the mitochondrial structure in cold-stored prune fruit. More importantly, PdGLRs expression and CaM/CML content positively correlated with antioxidant enzyme activities, GABA shunt, and energy status in prune fruit. These results indicated that the enhanced cold resistance of L-Glu-treated prunes might be attributed to the activated Ca2+ signaling, thus improving the antioxidant capacity, GABA, and energy levels.

Mitogen-Activated Protein Kinase Kinase OsMEK2 Positively Regulates Ca2+ Influx and Ferroptotic Cell Death during Rice Immune Responses.

Mitogen-activated protein (MAP) kinase (MAPK) signaling pathway is important in plant immune responses, involved in iron- and reactive oxygen species (ROS)-dependent ferroptotic cell death mediated by Ca2+. High Ca2+ influx triggered iron-dependent ROS accumulation, lipid peroxidation, and subsequent hypersensitive response (HR) cell death in rice (Oryza sativa). Apoplastic Ca2+ chelation by EGTA during avirulent Magnaporthe oryzae infection altered Ca2+, ROS, and Fe2+ accumulation, increasing rice susceptibility to infection. By contrast, acibenzolar-S-methyl (ASM), a plant defense activator, significantly enhanced Ca2+ influx, and H2O2 accumulation, triggering rice ferroptotic cell death during virulent Magnaporthe oryzae infection. Here, we report a novel role of the MAPK signaling pathway in regulating cytoplasmic Ca2+ increase during ferroptotic cell death in rice immunity, using the ΔOsmek2 knockout mutant rice. The knockout of rice OsMEK2 impaired the ROS accumulation, lipid peroxidation, and iron accumulation during avirulent M. oryzae infection. This study has shown that OsMEK2 could positively regulate iron- and ROS-dependent ferroptotic cell death in rice by modulating the expression of OsNADP-ME, OsRBOHB, OsPLC, and OsCNGC. This modulation indicates a possible mechanism for how OsMEK2 participates in Ca2+ regulation in rice ferroptotic cell death, suggesting its broader role in plant immune responses in response to M. oryzae infection.

ELD607 specifically traffics Orai1 to the lysosome leading to inhibition of store operated calcium entry

Orai1 is a plasma membrane Ca2+ channel involved in store operated calcium entry (SOCE). SOCE can regulate cell growth, exocytosis, gene expression and inflammation. We previously found that short palate lung and nasal epithelial clone 1′s (SPLUNC1) sixth α-helix (α6) bound Orai1 to inhibit SOCE. SPLUNC1 was not proteolytically stable, so we developed ELD607, an 11 amino acid peptide based on SPLUNC1’s α6 region which was more stable and more potent than SPLUNC1/α6. Here, we studied ELD607’s mechanism of action. We overexpressed either Orai1-HA or Orai1-YFP in HEK293T cells to probe ELD607-Orai1 interactions by confocal microscopy. We also measured changes in Fluo-4 fluorescence in a multiplate reader as a marker of cytoplasmic Ca2+ levels. ELD607 internalized Orai1 independently of STIM1. Both 15 min and 3 h exposure to ELD607 similarly depleted Orai1 in the plasma membrane. However, 3 h exposure to ELD607 yielded greater inhibition of SOCE. ELD607 continued to colocalize with Orai1 after internalization and this process was dependent on the presence of the ubiquitin ligase NEDD4.2. Similarly, ELD607 increased the colocalization between Orai1 and ubiquitin. ELD607 also increased the colocalization between Orai1 and Rab5 and 7, but not Rab11, suggesting that Orai1 trafficked through early and late but not recycling endosomes. Finally, ELD607 caused Orai1, but not Orai2, Orai3, or STIM1 to traffic to lysosomes. We conclude that ELD607 rapidly binds to Orai1 and works in an identical fashion as full length SPLUNC1 by internalizing Orai1 and sending it to lysosomes, leading to a decrease in SOCE.

NOX2-TRPM2 coupling promotes Zn2+ inhibition of complex III to exacerbate ROS production in a cellular model of Parkinson’s disease

Reactive oxygen species (ROS) serve vital physiological functions, but aberrant ROS production contributes to numerous diseases. Unfortunately, therapeutic progress targeting pathogenic ROS has been hindered by the limited understanding of whether the mechanisms driving pathogenic ROS differ from those governing physiological ROS generation. To address this knowledge gap, we utilised a cellular model of Parkinson’s disease (PD), as an exemplar of ROS-associated diseases. We exposed SH-SY5Y neuroblastoma cells to the PD-toxin, MPP+ (1-methyl-4-phenylpyridinium) and studied ROS upregulation leading to cell death, the primary cause of PD. We demonstrate: (1) MPP+ stimulates ROS production by raising cytoplasmic Ca2+ levels, rather than acting directly on mitochondria. (2) To raise the Ca2+, MPP+ co-stimulates NADPH oxidase-2 (NOX2) and the Transient Receptor Potential Melastatin2 (TRPM2) channel that form a positive feedback loop to support each other’s function. (3) Ca2+ exacerbates mitochondrial ROS (mtROS) production not directly, but via Zn2+. (4) Zn2+ promotes electron escape from respiratory complexes, predominantly from complex III, to generate mtROS. These conclusions are drawn from data, wherein inhibition of TRPM2 and NOX2, chelation of Ca2+ and Zn2+, and prevention of electron escape from complexes -all abolished the ability of MPP+ to induce mtROS production and the associated cell death. Furthermore, calcium ionophore mimicked the effects of MPP+, while Zn2+ ionophore replicated the effects of both MPP+ and Ca2+. Thus, we unveil a previously unrecognized signalling circuit involving NOX2, TRPM2, Ca2+, Zn2+, and complex III that drives cytotoxic ROS production. This circuit lies dormant in healthy cells but is triggered by pathogenic insults and could therefore represent a safe therapeutic target for PD and other ROS-linked diseases.

Aqueous extract of Descuraniae Semen attenuates lipopolysaccharide-induced inflammation by inhibiting ER stress and WNK4-SPAK-NKCC1 pathway.

Sepsis causes systemic inflammatory responses and acute lung injury (ALI). Despite modern treatments, sepsis-related ALI mortality remains high. Aqueous extract of Descuraniae Semen (AEDS) exerts anti-endoplasmic reticulum (ER) stress, antioxidant and anti-inflammatory effects. AEDS alleviates inflammation and oedema in ALI. Sodium-potassium-chloride co-transporter isoform 1 (NKCC1) is essential for regulating alveolar fluid and is important in ALI. The NKCC1 activity is regulated by upstream with-no-lysine kinase-4 (WNK4) and STE20/SPS1-related proline/alanine-rich kinase (SPAK). This study aimed to investigate the effects of AEDS on lipopolysaccharide (LPS)-induced ALI model in A549 cells, considering the regulation of ER stress, WNK4-SPAK-NKCC1 cascades, inflammation and apoptosis. Cell viability was investigated by the CCK-8 assay. The expressions of the proteins were assessed by immunoblotting analysis assays. The levels of pro-inflammatory cytokines were determined by ELISA. The expression of cytoplasmic Ca2+ in A549 cells was determined using Fluo-4 AM. AEDS attenuates LPS-induced inflammation, which is associated with increased pro-inflammatory cytokine expression and activation of the WNK4-SPAK-NKCC1 pathway. AEDS inhibits the WNK4-SPAK-NKCC1 pathway by regulating of Bcl-2, IP3R and intracellular Ca2+. WNK4 expression levels are significantly higher in the WNK4-overexpressed transfected A549 cells and significantly decrease after AEDS treatment. AEDS attenuates LPS-induced inflammation by inhibiting the WNK4-SPAK-NKCC1 cascade. Therefore, AEDS is regarded as a potential therapeutic agent for ALI.

Bimetallic Nanoplatforms for Prostate Cancer Treatment by Interfering Cellular Communication.

Cellular communication mediated by messenger molecules plays an important role in the progression of cancer. Herein, pH-sensitive zeolitic imidazolate framework-8 (ZIF-8) loaded with PtCl2(OH)2(NH3)2 [i.e., Pt(IV)] bimetallic nanoplatforms were developed for prostate cancer therapy by interfering inositol-1, 4, 5-trisphosphate (IP3)-mediated cellular communication. As an important messenger in cells, the function of IP3 was found to be efficiently interfered with by the Pt(IV)-binding inositol unit. This finding effect of Pt(IV) is totally different from its traditional function as a prodrug of cis-platinum for chemotherapy. The decreased IP3 signal further downregulated the cytoplasmic Ca2+ concentration and downstream signal transduction to inhibit proliferation and invasion of tumor cells. Meanwhile, Zn2+ released from ZIF-8 under an acidic tumor microenvironment decreased adenosine triphosphate biosynthesis, which could further limit the cellular communication. Such a proposed strategy of interfering cellular communication has demonstrated its feasibility in this study, which may provide new perspectives for cancer therapy.

CHCHD2 P14L, found in amyotrophic lateral sclerosis, exhibits cytoplasmic mislocalization and alters Ca2+ homeostasis.

CHCHD2 and CHCHD10, linked to Parkinson's disease and amyotrophic lateral sclerosis-frontotemporal dementia (ALS), respectively, are mitochondrial intermembrane proteins that form a heterodimer. This study aimed to investigate the impact of the CHCHD2 P14L variant, implicated in ALS, on mitochondrial function and its subsequent effects on cellular homeostasis. The missense variant of CHCHD2, P14L, found in a cohort of patients with ALS, mislocalized CHCHD2 to the cytoplasm, leaving CHCHD10 in the mitochondria. Drosophila lacking the CHCHD2 ortholog exhibited mitochondrial degeneration. In contrast, human CHCHD2 P14L, but not wild-type human CHCHD2, failed to suppress this degeneration, suggesting that P14L is a pathogenic variant. The mitochondrial Ca2+ buffering capacity was reduced in Drosophila neurons expressing human CHCHD2 P14L. The altered Ca2+-buffering phenotype was also observed in cultured human neuroblastoma SH-SY5Y cells expressing CHCHD2 P14L. In these cells, transient elevation of cytoplasmic Ca2+ facilitated the activation of calpain and caspase-3, accompanied by the processing and insolubilization of TDP-43. These observations suggest that CHCHD2 P14L causes abnormal Ca2+ dynamics and TDP-43 aggregation, reflecting the pathophysiology of ALS.

Baicalin induces cell death of non-small cell lung cancer cells via MCOLN3-mediated lysosomal dysfunction and autophagy blockage

BackgroundNon-small cell lung cancer (NSCLC) accounts for 85 % of lung cancer, becoming the most mortality of all cancers globally. Blockage of autophagy in NSCLC represents a promising therapeutic strategy that inhibits angiogenesis and overcomes drug resistance. Natural ingredients in anti-tumor adjuvants are increasingly reported to promote cell death with less side effects and the potential to increase chemotherapeutic drugs sensitivity. Baicalin, a Scutellaria baicalensis-extracted flavonoid glycoside, is reported to induce death of NSCLC cells, however, its effects on autophagy in NSCLC cells remain unclear. PurposeThis study aimed to investigate the effect of baicalin on autophagic flux in NSCLC cells, unraveling the underlying mechanism including potential target and its role in cell death of NSCLC cells. MethodsIn vitro anti-cancer effects of baicalin were verified by evaluating proliferation, clone formation, cell cycle, and cell migration in three NSCLC cell lines (A549, H1299, and PC-9). In vivo anti-tumor efficacies of baicalin were evaluated in subcutaneous xenograft tumor model in nude mice. Autophagy characterization in NSCLC cells included autophagic marker detection by western blot and immunofluorescence staining, subcellular structure observation by TEM, lysosomal function by RNA-seq and fluorescence staining (LysoTracker®, LysoSensor®, and acridine orange). Based on RNA-seq and molecular biological verification using apoptotic, autophagic, and lysosomal inhibitors, potential target molecule of baicalin was verified via Ca2+ flux assay, MCOLN3 knockdown by shRNA, and virtual molecular docking. ResultsBaicalin inhibited NSCLC cell proliferation and migration, and suppressed tumor growth in vivo. Baicalin blocked the autophagic flux via activating the membranal cation channel MCOLN3 of lysosome, which disrupted its Ca2+ balance and induced lysosome dysfunction, leading to failure of autolysosome degradation. The cytoplasmic Ca2+ imbalance further resulted in depolarization of mitochondrial membrane potentials and ROS accumulation in NSCLC cells, mediating autophagy-related apoptosis. ConclusionThis study demonstrated that baicalin inhibited autolysosome degradation by activating MCOLN3, leading to dysfunction in lysosomal pH elevation, thereby inhibiting autophagy in NSCLC, leading to apoptotic death of NSCLC cells. These findings enriched the existing theories of cancer therapy based on autophagy inhibition and underlying mechanisms of flavonoids as antitumor agents, paving the way for their clinical application in future.

Firing Patterns of Mitral Cells and Their Transformation in the Main Olfactory Bulb.

Mitral cells (MCs) in the main olfactory bulb relay odor information to higher-order olfactory centers by encoding the information in the form of action potentials. The firing patterns of these cells are influenced by both their intrinsic properties and their synaptic connections within the neural network. However, reports on MC firing patterns have been inconsistent, and the mechanisms underlying these patterns remain unclear. Using whole-cell patch-clamp recordings in mouse brain slices, we discovered that MCs exhibit two types of integrative behavior: regular/rhythmic firing and bursts of action potentials. These firing patterns could be transformed both spontaneously and chemically. MCs with regular firing maintained their pattern even in the presence of blockers of fast synaptic transmission, indicating this was an intrinsic property. However, regular firing could be transformed into bursting by applying GABAA receptor antagonists to block inhibitory synaptic transmission. Burst firing could be reverted to regular firing by blocking ionotropic glutamate receptors, rather than applying a GABAA receptor agonist, indicating that ionotropic glutamatergic transmission mediated this transformation. Further experiments on long-lasting currents (LLCs), which generated burst firing, also supported this mechanism. In addition, cytoplasmic Ca2+ in MCs was involved in the transformation of firing patterns mediated by glutamatergic transmission. Metabotropic glutamate receptors also played a role in LLCs in MCs. These pieces of evidence indicate that odor information can be encoded on a mitral cell (MC) platform, where it can be relayed to higher-order olfactory centers through intrinsic and dendrodendritic mechanisms in MCs.

Cavβ3 Contributes to the Maintenance of the Blood-Brain Barrier and Alleviates Symptoms of Experimental Autoimmune Encephalomyelitis.

Tight control of cytoplasmic Ca2+ concentration in endothelial cells is essential for the regulation of endothelial barrier function. Here, we investigated the role of Cavβ3, a subunit of voltage-gated Ca2+ (Cav) channels, in modulating Ca2+ signaling in brain microvascular endothelial cells (BMECs) and how this contributes to the integrity of the blood-brain barrier. We investigated the function of Cavβ3 in BMECs by Ca2+ imaging and Western blot, examined the endothelial barrier function in vitro and the integrity of the blood-brain barrier in vivo, and evaluated disease course after induction of experimental autoimmune encephalomyelitis in mice using Cavβ3-/- (Cavβ3-deficient) mice as controls. We identified Cavβ3 protein in BMECs, but electrophysiological recordings did not reveal significant Cav channel activity. In vivo, blood-brain barrier integrity was reduced in the absence of Cavβ3. After induction of experimental autoimmune encephalomyelitis, Cavβ3-/- mice showed earlier disease onset with exacerbated clinical disability and increased T-cell infiltration. In vitro, the transendothelial resistance of Cavβ3-/- BMEC monolayers was lower than that of wild-type BMEC monolayers, and the organization of the junctional protein ZO-1 (zona occludens-1) was impaired. Thrombin stimulates inositol 1,4,5-trisphosphate-dependent Ca2+ release, which facilitates cell contraction and enhances endothelial barrier permeability via Ca2+-dependent phosphorylation of MLC (myosin light chain). These effects were more pronounced in Cavβ3-/- than in wild-type BMECs, whereas the differences were abolished in the presence of the MLCK (MLC kinase) inhibitor ML-7. Expression of Cacnb3 cDNA in Cavβ3-/- BMECs restored the wild-type phenotype. Coimmunoprecipitation and mass spectrometry demonstrated the association of Cavβ3 with inositol 1,4,5-trisphosphate receptor proteins. Independent of its function as a subunit of Cav channels, Cavβ3 interacts with the inositol 1,4,5-trisphosphate receptor and is involved in the tight control of cytoplasmic Ca2+ concentration and Ca2+-dependent MLC phosphorylation in BMECs, and this role of Cavβ3 in BMECs contributes to blood-brain barrier integrity and attenuates the severity of experimental autoimmune encephalomyelitis disease.

Colonocyte keratins stabilize mitochondria and contribute to mitochondrial energy metabolism.

Keratin intermediate filaments form dynamic filamentous networks, which provide mechanical stability, scaffolding, and protection against stress to epithelial cells. Keratins and other intermediate filaments have been increasingly linked to the regulation of mitochondrial function and homeostasis in different tissues and cell types. While deletion of keratin 8 (K8-/-) in mouse colon elicits a colitis-like phenotype, epithelial hyperproliferation, and blunted mitochondrial ketogenesis, the role of K8 in colonocyte mitochondrial function and energy metabolism is unknown. We used two K8 knockout mouse models and CRISPR/Cas9 K8-/- colorectal adenocarcinoma Caco-2 cells to answer this question. The results show that K8-/- colonocyte mitochondria in vivo are smaller and rounder and that mitochondrial motility is increased in K8-/- Caco-2 cells. Furthermore, K8-/- Caco-2 cells displayed diminished mitochondrial respiration and decreased mitochondrial membrane potential compared with controls, whereas glycolysis was not affected. The levels of mitochondrial respiratory chain complex proteins and mitochondrial regulatory proteins mitofusin-2 and prohibitin were decreased both in vitro in K8-/- Caco-2 cells and in vivo in K8-/- mouse colonocytes, and reexpression of K8 into K8-/- Caco-2 cells normalizes the mitofusin-2 levels. Mitochondrial Ca2+ is an important regulator of mitochondrial energy metabolism and homeostasis, and Caco-2 cells lacking K8 displayed decreased levels and altered dynamics of mitochondrial matrix and cytoplasmic Ca2+. In summary, these novel findings attribute an important role for colonocyte K8 in stabilizing mitochondrial shape and movement and maintaining mitochondrial respiration and Ca2+ signaling. Further, how these metabolically compromised colonocytes are capable of hyperproliferating presents an intriguing question for future studies.NEW & NOTEWORTHY In this study, we show that colonocyte intermediate filament protein keratin 8 is important for stabilizing mitochondria and maintaining mitochondrial energy metabolism, as keratin 8-deficient colonocytes display smaller, rounder, and more motile mitochondria, diminished mitochondrial respiration, and altered Ca2+ dynamics. Changes in fusion-regulating proteins are rescued with reexpression of keratin 8. These alterations in colonocyte mitochondrial homeostasis contribute to keratin 8-associated colitis pathophysiology.

Ca2+ permeation through C-terminal cleaved, but not full-length human Pannexin1 hemichannels, mediates cell death

Pannexin1 hemichannels (Panx1 HCs) are found in the membrane of most mammalian cells and communicate the intracellular and extracellular spaces, enabling the passive transfer of ions and small molecules. They are involved in physiological and pathophysiological conditions. During apoptosis, the C-terminal tail of Panx1 is proteolytically cleaved, but the permeability features of hemichannels and their role in cell death remain elusive. To address these topics, HeLa cells transfected with full-length human Panx1 (fl-hPanx1) or C-terminal truncated hPanx1 (Δ371hPanx1) were exposed to alkaline extracellular saline solution, increasing the activity of Panx1 HCs. The Δ371hPanx1 HC was permeable to DAPI and Etd+, but not to propidium iodide, whereas fl-hPanx1 HC was only permeable to DAPI. Furthermore, the cytoplasmic Ca2+ signal increased only in Δ371hPanx1 cells, which was supported by bioinformatics approaches. The influx of Ca2+ through Δ371hPanx1 HCs was necessary to promote cell death up to about 95% of cells, whereas the exposure to alkaline saline solution without Ca2+ failed to induce cell death, and the Ca2+ ionophore A23187 promoted more than 80% cell death even in fl-hPanx1 transfectants. Moreover, cell death was prevented with carbenoxolone or 10Panx1 in Δ371hPanx1 cells, whereas it was undetectable in HeLa Panx1-/- cells. Pretreatment with Ferrostatin-1 and necrostatin-1 did not prevent cell death, suggesting that ferroptosis or necroptosis was not involved. In comparison, zVAD-FMK, a pancaspase inhibitor, reduced death by ~60%, suggesting the involvement of apoptosis. Therefore, alkaline pH increases the activity of Δ371hPanx1HCs, leading to a critical intracellular free-Ca2+ overload that promotes cell death.

PAR1-mediated Non-periodical Synchronized Calcium Oscillations in Human Mesangial Cells.

Mesangial cells offer structural support to the glomerular tuft and regulate glomerular capillary flow through their contractile capabilities. These cells undergo phenotypic changes, such as proliferation and mesangial expansion, resulting in abnormal glomerular tuft formation and reduced capillary loops. Such adaptation to the changing environment is commonly associated with various glomerular diseases, including diabetic nephropathy and glomerulonephritis. Thrombin-induced mesangial remodeling was found in diabetic patients, and expression of the corresponding protease-activated receptors (PARs) in the renal mesangium was reported. However, the functional PAR-mediated signaling in mesangial cells was not examined. This study investigated protease-activated mechanisms regulating mesangial cell calcium waves that may play an essential role in the mesangial proliferation or constriction of the arteriolar cells. Our results indicate that coagulation proteases such as thrombin induce synchronized oscillations in cytoplasmic Ca2+ concentration of mesangial cells. The oscillations required PAR1 G-protein coupled receptors-related activation, but not a PAR4, and were further mediated presumably through store-operated calcium entry and transient receptor potential canonical 3 (TRPC3) channel activity. Understanding thrombin signaling pathways and their relation to mesangial cells, contractile or synthetic (proliferative) phenotype may play a role in the development of chronic kidney disease and requires further investigation.