Regulation Of Calcium Influx Research Articles

Regulation of T Lymphocyte Functions through Calcium Signaling Modulation by Nootkatone.

Recent advancements in understanding the intricate molecular mechanisms underlying immunological responses have underscored the critical involvement of ion channels in regulating calcium influx, particularly in inflammation. Nootkatone, a natural sesquiterpenoid found in Alpinia oxyphylla and various citrus species, has gained attention for its diverse pharmacological properties, including anti-inflammatory effects. This study aimed to elucidate the potential of nootkatone in modulating ion channels associated with calcium signaling, particularly CRAC, KV1.3, and KCa3.1 channels, which play pivotal roles in immune cell activation and proliferation. Using electrophysiological techniques, we demonstrated the inhibitory effects of nootkatone on CRAC, KV1.3, and KCa3.1 channels in HEK293T cells overexpressing respective channel proteins. Nootkatone exhibited dose-dependent inhibition of channel currents, with IC50 values determined for each channel. Nootkatone treatment did not significantly affect cell viability, indicating its potential safety for therapeutic applications. Furthermore, we observed that nootkatone treatment attenuated calcium influx through activated CRAC channels and showed anti-proliferative effects, suggesting its role in regulating inflammatory T cell activation. These findings highlight the potential of nootkatone as a natural compound for modulating calcium signaling pathways by targeting related key ion channels and it holds promise as a novel therapeutic agent for inflammatory disorders.

Multifunctional graphene quantum dots: A therapeutic strategy for neurodegenerative diseases by regulating calcium influx, crossing the blood-brain barrier and inhibiting Aβ-protein aggregation

Multifunctional nanoparticles could be the hallmark for the treatment of neurodegenerative diseases. Dissociation of protein aggregates causing neuronal damage and transfer of specific drugs which can downregulate neuronal excitotoxicity by inhibiting glutamatergic N-Methyl-d-Aspartate-receptors (NMDA) and then reducing calcium influx are among the main factors to consider for proper therapy. Here, we present a multiplatform based on nitrogen-doped graphene quantum dots (NGQDs) with such functionalities. The NGQDs were functionalized with Memantine, the clinically used drug, via covalent and non-covalent coupling, and we confirmed that the pharmaceutical activity was not altered. Apart from that, using xCELLigence technology and flow cytometric analysis of ABC transporter function, we uncovered that the ABC transporters of the blood-brain barrier (BBB) do not affect the ability of NGQD to cross BBB. Surprisingly, this study found that NGQDs have an inhibitory effect on NMDA receptors, thus supporting the action of Memantine. Moreover, NGQDs and their derivatives demonstrated the potential to dissociate β-amyloid aggregates while possessing features suitable for bioimaging in various cell lines.

Bidirectional regulation mechanism of TRPM2 channel: role in oxidative stress, inflammation and ischemia-reperfusion injury.

Transient receptor potential melastatin 2 (TRPM2) is a non-selective cation channel that exhibits Ca2+ permeability. The TRPM2 channel is expressed in various tissues and cells and can be activated by multiple factors, including endogenous ligands, Ca2+, reactive oxygen species (ROS) and temperature. This article reviews the multiple roles of the TRPM2 channel in physiological and pathological processes, particularly on oxidative stress, inflammation and ischemia-reperfusion (I/R) injury. In oxidative stress, the excessive influx of Ca2+ caused by the activation of the TRPM2 channel may exacerbate cellular damage. However, under specific conditions, activating the TRPM2 channel can have a protective effect on cells. In inflammation, the activation of the TRPM2 channel may not only promote inflammatory response but also inhibit inflammation by regulating ROS production and bactericidal ability of macrophages and neutrophils. In I/R, the activation of the TRPM2 channel may worsen I/R injury to various organs, including the brain, heart, kidney and liver. However, activating the TRPM2 channel may protect the myocardium from I/R injury by regulating calcium influx and phosphorylating proline-rich tyrosine kinase 2 (Pyk2). A thorough investigation of the bidirectional role and regulatory mechanism of the TRPM2 channel in these physiological and pathological processes will aid in identifying new targets and strategies for treatment of related diseases.

Functional profiling of the G protein-coupled receptor C3aR1 reveals ligand-mediated biased agonism

G protein-coupled receptors (GPCRs) are leading druggable targets for several medicines, but many GPCRs are still untapped for their therapeutic potential due to poor understanding of specific signaling properties. The complement C3a receptor 1 (C3aR1) has been extensively studied for its physiological role in C3a-mediated anaphylaxis/inflammation, and in TLQP-21-mediated lipolysis, but direct evidence for the functional relevance of the C3a and TLQP-21 ligands and signal transduction mechanisms are still limited. In addition, C3aR1 G protein coupling specificity is still unclear, and whether endogenous ligands, or drug-like compounds, show ligand-mediated biased agonism is unknown. Here, we demonstrate that C3aR1 couples preferentially to Gi/o/z proteins and can recruit β-arrestins to cause internalization. Furthermore, we showed that in comparison to C3a63-77, TLQP-21 exhibits a preference toward Gi/o-mediated signaling compared to β-arrestin recruitment and internalization. We also show that the purported antagonist SB290157 is a very potent C3aR1 agonist, where antagonism of ligand-stimulated C3aR1 calcium flux is caused by potent β-arrestin-mediated internalization. Finally, ligand-mediated signaling bias impacted cell function as demonstrated by the regulation of calcium influx, lipolysis in adipocytes, phagocytosis in microglia, and degranulation in mast cells. Overall, we characterize C3aR1 as a Gi/o/z-coupled receptor, and demonstrate functional relevance of ligand-mediated signaling bias in key cellular models. Due to C3aR1 and its endogenous ligands being implicated in inflammatory and metabolic diseases, these results are of relevance toward future C3aR1 drug discovery.

SLC16A7 Facilitates aGVHD through Regulating Calcium Influx and Lipid Metabolism of CD4 + T Cells

T cell metabolic disorders in patients with acute graft-versus-host disease (aGVHD) have been studied, but the underlying mechanisms remain poorly understood. Solute carrier (SLC) transporters play a crucial role in cell metabolism, but their involvement in T cell-mediated aGVHD has not been investigated. In this study, we observed that SLC16A7 levels were down-regulated in CD4 + T cells of both aGVHD mice and patients, which in turn increased calcium (Ca 2+) influx in activated CD4 + T cells and promoted the differentiation of Th1 and Th17 cells, leading to the exacerbated aGVHD cascade. Moreover, deficiency of SLC16A7 promoted calcium overload in activated CD4 + T cells through regulating SERCA3 proteins and altered characteristics of lipid metabolism, decreasing levels of oleic acid, thereby aggravating aGVHD. Interestingly, supplementation of oleic acid rescued the survival of aGVHD mice. This study characterized a previously unknown role of SLC16A7 in T cell-mediated aGVHD and provided a promising therapeutic strategy to target T cell metabolism for the alleviation of aGVHD.

TRPC6 deficiency impairs middle cerebral artery myogenic response and brain spatial memory

Transient receptor potential channel 6 (TRPC6) is a nonselective cation channel belonging to the TRP ion channel family and expressed in vascular smooth muscle cells. TRPC6 regulates calcium influx in response to vascular wall mechanical stretch. A previous study reported that TRPC6 is critical for the myogenic response of the middle cerebral artery (MCA). Impaired MCA myogenic response is implicated in cerebral blood flow dysfunction and may promote cognitive deficits. In this study, we investigated whether TRPC6 contributes to the MCA myogenic response at pressure range (from 40 to 180 mmHg) in 12-week-old male wild-type (WT) and whole-body TRPC6 knock-out (TRPC6 KO) mice (n=5-8). Cognitive function (spatial learning and reference memory) was examined at 22 weeks of age using the Morris water maze test. Myogenic response in MCA was similar in WT and TRPC6 KO mice when perfusion pressure was increased from 40 to 80 mmHg. At higher pressures (>120 mmHg), however, TRPC6 KO mice displayed significantly reduced myogenic response compared to WT mice (3 % less of myogenic tone in TRPC6 KO compared to WT) which was further exacerbated when perfusion pressure was increased to 180 mmHg (9 % less of myogenic tone in TRPC6 KO compared to WT). There were no significant differences in wall thickness, wall-to-lumen ratio, vascular distensibility, incremental distensibility, or elastic modulus curves in MCA between WT and TRPC6 KO mice. Compared to WT mice, TRPC6 KO mice displayed similar spatial learning curves after 4 days of training but showed significantly impaired reference memory (14±1 % vs 29±4 % in the target quadrant) and reduced frequency (1.0±0.3 vs 2.1±0.4 in the target area crossing) compared to WT. These findings suggest that TRPC6 deficiency impairs myogenic response at higher perfusion pressures and spatial memory, even at a young age. (AG050049 (F.F), AG057842 (F.F), AG079336 (F.F), NIDDK R00DK113280, R01DK121411, NIGMS P20GM104357 and NIGMS U54GM115428) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Using CRISPR knock-in of fluorescent tags to examine isoform-specific expression of EGL-19 in C. elegans.

L-type voltage-gated calcium channels (VGCCs) regulate calcium influx and excitation-contraction coupling in many types of muscle cells. Thus, VGCC mutations can cause skeletal and cardiac muscle diseases in humans, such as Duchenne muscular dystrophy and Timothy syndrome. To better understand the genetics and native expression of VGCCs, we have chosen to use the microscopic roundworm, C. elegans . The egl-19 locus is the sole L-type VGCC gene and it encodes three distinct isoforms (a, b, and c). Isoform c is curious because the protein is truncated, lacking the transmembrane domains that form the physical calcium channel. In this study, we have characterized egl-19 expression using CRISPR/Cas9 genome editing to 'knock-in' fluorescent tags of differing colors, allowing us to distinguish the expression pattern of each isoform. Not surprisingly, we found that EGL-19 is expressed in all types of muscle. In addition, we provide evidence that the truncated c isoform is expressed. Finally, although we find evidence that specific isoforms can have unique subcellular distributions, we also observed some expression patterns that appear to be artifacts. Overall, our results show interesting patterns of egl-19 expression, but also highlight the need for caution when interpreting expression of reporter genes even when they represent endogenous tags.

Blockade of store-operated calcium entry sensitizes breast cancer cells to cisplatin therapy via modulating inflammatory response

Store-operated calcium entry (SOCE) is an important pathway for calcium signaling that regulates calcium influx across the plasma membrane upon the depletion of calcium stores in the endoplasmic reticulum. SOCE participates in regulating a number of physiological processes including cell proliferation and migration while SOCE dysregulation has been linked with pathophysiological conditions such as inflammation and cancer. The crosslink between cancer and inflammation has been well-established where abundant evidence demonstrate that inflammation plays a role in cancer pathophysiology and the response of cancer cells to chemotherapeutic agents including cisplatin. Indeed, the efficacy of cisplatin against cancer cells is reduced by inflammation. Interestingly, it was shown that SOCE enhances inflammatory signaling in immune cells. Therefore, the main objectives of this study are to examine the impact of SOCE inhibition on the cisplatin sensitivity of breast cancer cells and to explore its related mechanism in modulating the inflammatory response in breast cancer cells. Our findings showed that SOCE inhibitor (BTP2) enhanced cisplatin cytotoxicity against resistant breast cancer cells via inhibition of cell proliferation and migration as well as induction of apoptosis. We also found an upregulation in the gene expression of two major components of SOCE, STIM1 and ORAI1, in cisplatin-resistant breast cancer cells compared to cisplatin-sensitive breast cancer cells. In addition, cisplatin treatment increased the gene expression of STIM1 and ORAI1 in cisplatin-resistant breast cancer cells. Finally, this study also demonstrated that cisplatin therapy caused an increase in the gene expression of inflammatory mediators COX2, IL-8, and TNF-α as well as COX2 protein and upon SOCE inhibition using BTP2, the effect of cisplatin on the inflammatory mediators was reversed. Altogether, this study has proven the pivotal role of SOCE in cisplatin resistance of breast cancer cells and showed the importance of targeting this pathway in improving breast cancer therapy.

Change of intracellular calcium level causes acute neurotoxicity by antisense oligonucleotides via CSF route

Antisense oligonucleotides (ASOs) are promising therapeutics for intractable central nervous system (CNS) diseases. For this clinical application, neurotoxicity is one of the critical limitations. Therefore, an evaluation of this neurotoxicity from a behavioral perspective is important to reveal symptomatic dysfunction of the CNS and elucidate the underlying molecular mechanism. We here exploited a behavioral analysis method to categorize and quantify the acute neurotoxicity of mice administered with toxic ASOs via intracerebroventricular injection. The toxic ASOs were found to reduce consciousness and locomotor function in mice in a dose-dependent manner. Mechanistically, we analyzed the effects of modulators against receptors or channels, which regulate calcium influx of neurons, on the ASO neurotoxicity. Modulators promoting calcium influx mitigated, whereas those hindering calcium influx increased, invivo neurotoxicity of ASOs in mice. In an invitro assay to evaluate intracellular free calcium levels using rat primary cortical neurons, toxic ASOs reduced the calcium levels. The findings of this study demonstrated the behavioral characteristics of ASO-induced neurotoxicity and revealed that changes in intracellular free calcium levels are a part of the mechanism underlying the neurotoxic effects of ASO.

TNFα Enhances Calcium Influx by Interacting with AMPA Receptors in the Spinal Dorsal Horn Neurons.

Tumor necrosis factor alpha (TNFα) is a proinflammatory cytokine and has been implicated in pain regulation. Neuronal activity in the spinal dorsal horn contributes to nociceptive transmission. However, it is not fully understood how TNFα affects the activity of spinal dorsal horn neurons. In the present study, we used calcium imaging to characterize the mechanism by which TNFα regulates calcium influx in the cultured spinal dorsal horn neurons. We observed thatTNFα incubation caused an increase in fluorescent intensity of Fura-2, a specific intracellular calcium indicator, in the cultured spinal dorsal horn neurons, and such effect was significantly inhibited by co-incubation with R7050, a selective TNFα receptor antagonist, which suggests thatTNFα can enhance calcium influx and increase neuronal activity via activating TNFα receptors in the spinal dorsal horn. Using double immunofluorescence staining, we showed that TNFα receptors were co-expressed with a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor subunit GluA1 in the spinal dorsal horn neurons. We further observed that treatment with 1-naphthyl acetyl spermine (NASPM), a specific calcium-permeable AMPA receptor blocker, completely blocked the effect of TNFα incubation on calcium influx in the cultured neurons. Moreover, lipopolysaccharide (LPS)-induced calcium influx was inhibited by co-incubation with R7050. Together, our results suggest thatTNFα in the spinal dorsal horn can increase calcium-indicated neuronal activity through the interaction between its receptor and calcium-permeable AMPA receptors.

Bta-miR-6531 Regulates Calcium Influx in Bovine Leydig Cells and Is Associated with Sperm Motility.

MicroRNAs (miRNAs) play key roles in sperm as the regulatory factors involved in fertility and subsequent early embryonic development. Bta-miR-6531 is specifically a highly enriched miRNA in low-motility sperms in previous study. To investigate the mechanism of bta-miR-6531, 508 shared target genes of bta-miR-6531 were predicted using two miRNA target databases (TargetScan7 and miRWalk). According to the Kyoto Encyclopedia of Genes and Genomes (KEGG), the calcium and cAMP signaling pathways were the most enriched of the target genes. A dual-luciferase assay indicated that bta-miR-6531 targeted ATP2A2 mRNA by binding to the coding sequence region. In bovine Leydig cells, bta-miR-6531 overexpression affected the intracellular calcium concentration by restraining ATP2A2 expression. Moreover, we observed high calcium concentrations and high ATP2A2 protein levels in high-motility sperm compared with those in low-motility sperms. Furthermore, high-linkage single-nucleotide polymorphisms (SNPs) of the pre-bta-miR-6531 sequence were identified that related to sperm traits. Genotype TCTC of bta-miR-6531 showed high sperm motility and density and low deformity rate in Holstein bulls. However, the mutation in pre-miR-6531 did not significantly affect mature bta-miR-6531 expression in the sperm or cell models. Our results demonstrate that bta-miR-6531 might involve in sperm motility regulation by targeting ATP2A2 of the calcium signaling pathway in bovine spermatozoa.

I.10 Gene therapy and other novel treatment approaches for CMT

There is still no effective drug treatment available for Charcot-Marie-Tooth disease (CMT). Current management relies on rehabilitation therapy, surgery for skeletal deformities, and symptomatic treatment. The challenge is to find disease-modifying therapies. Several approaches, including gene silencing (by means of ASO, siRNA, CRISPR-Cas9 editing) to counteract the <i>PMP22</i> gene overexpression in the most frequent CMT1A type are under investigation. PXT3003 is the compound in the most advanced phase for CMT1A, as a second phase-III trial is ongoing. Gene therapy to substitute defective genes (particularly in recessive forms associated with loss-of-function mutations) or insert novel ones (e.g., <i>NT3</i> gene) are being developed and tested in animal models. Novel treatment approaches are also aimed at developing compounds acting on pathways important for different CMT types. Modulation of the Neuregulin pathway determining myelin thickness is promising for both hypo-demyelinating and hypermyelinating neuropathies; intervention on Unfolded Protein Response seems effective for rescuing misfolded myelin proteins such as P0 in CMT1B. HDAC6 inhibitors improved axonal transport and ameliorated phenotypes in different CMT models. Other potential therapeutic strategies include targeting macrophages, lipid metabolism, and Nav1.8 sodium channel in demyelinating CMT and the P2X7 receptor, which regulates calcium influx into Schwann cells, in CMT1A. Further approaches are aimed at correcting metabolic abnormalities, including the accumulation of sorbitol caused by biallelic mutations in the sorbitol dehydrogenase (SORD) gene and of neurotoxic glycosphingolipids in HSN1.

The integrin ligand SVEP1 regulates GPCR-mediated vasoconstriction via integrins α9β1 and α4β1.

Vascular tone is regulated by the relative contractile state of vascular smooth muscle cells (VSMCs). Several integrins directly modulate VSMC contraction by regulating calcium influx through L-type voltage-gated Ca2+ channels (VGCCs). Genetic variants in ITGA9, which encodes the α9 subunit of integrin α9β1, and SVEP1, a ligand for integrin α9β1, associate with elevated blood pressure; however, neither SVEP1 nor integrin α9β1 has reported roles in vasoregulation. We determined whether SVEP1 and integrin α9β1 can regulate VSMC contraction. SVEP1 and integrin binding were confirmed by immunoprecipitation and cell binding assays. Human induced pluripotent stem cell-derived VSMCs were used in in vitro [Ca2+ ]i studies, and aortas from a Svep1+/- knockout mouse model were used in wire myography to measure vessel contraction. We confirmed the ligation of SVEP1 to integrin α9β1 and additionally found SVEP1 to directly bind to integrin α4β1. Inhibition of SVEP1, integrin α4β1 or α9β1 significantly enhanced [Ca2+ ]i levels in isolated VSMCs to Gαq/11 -vasoconstrictors. This response was confirmed in whole vessels where a greater contraction to U46619 was seen in vessels from Svep1+/- mice compared to littermate controls or when integrin α4β1 or α9β1 was inhibited. Inhibition studies suggested that this effect was mediated via VGCCs, PKC and Rho A/Rho kinase dependent mechanisms. Our studies reveal a novel role for SVEP1 and the integrins α4β1 and α9β1 in reducing VSMC contractility. This could provide an explanation for the genetic associations with blood pressure risk at the SVEP1 and ITGA9 loci.

DOT1L Methyltransferase Regulates Calcium Influx in Erythroid Progenitor Cells in Response to Erythropoietin.

Erythropoietin (EPO) signaling plays a vital role in erythropoiesis by regulating proliferation and lineage-specific differentiation of murine hematopoietic progenitor cells (HPCs). An important downstream response of EPO signaling is calcium (Ca2+) influx, which is regulated by transient receptor potential channel (TRPC) proteins, particularly TRPC2 and TRPC6. While EPO induces Ca2+ influx through TRPC2, TRPC6 inhibits the function of TRPC2. Thus, interactions between TRPC2 and TRPC6 regulate the rate of Ca2+ influx in EPO-induced erythropoiesis. In this study, we observed that the expression of TRPC6 in KIT-positive erythroid progenitor cells was regulated by DOT1L. DOT1L is a methyltransferase that plays an important role in many biological processes during embryonic development including early erythropoiesis. We previously reported that Dot1l knockout (Dot1lKO) HPCs in the yolk sac failed to develop properly, which resulted in lethal anemia. In this study, we detected a marked downregulation of Trpc6 gene expression in Dot1lKO progenitor cells in the yolk sac compared to the wild type (WT). The promoter and the proximal regions of the Trpc6 gene locus exhibited an enrichment of H3K79 methylation, which is mediated solely by DOT1L. However, the expression of Trpc2, the positive regulator of Ca2+ influx, remained unchanged, resulting in an increased TRPC2/TRPC6 ratio. As the loss of DOT1L decreased TRPC6, which inhibited Ca2+ influx by TRPC2, Dot1lKO HPCs in the yolk sac exhibited accelerated and sustained elevated levels of Ca2+ influx. Such heightened Ca2+ levels might have detrimental effects on the growth and proliferation of HPCs in response to EPO.

FGL2-MCOLN3-Autophagy Axis-Triggered Neutrophil Extracellular Traps Exacerbate Liver Injury in Fulminant Viral Hepatitis.

Background & AimsFulminant viral hepatitis (FVH) is a life-threatening disease, but its pathogenesis is not fully understood. Neutrophil extracellular traps (NETs) were an unrecognized link between inflammation and coagulation, which are 2 main features of FVH. Here, we investigated the role and mechanism of NETs in the pathogenesis of FVH.MethodsA mouse model of FVH was established by murine hepatitis virus strain-3 infection. Liver leukocytes of infected or uninfected mice were used for single-cell RNA sequencing and whole-transcriptome sequencing. NETs depletion was achieved using DNase 1. Acetaminophen was used to establish a mouse model of non–virus-caused acute liver failure. Clinically, NETs-related markers in liver, plasma, and peripheral neutrophils were assessed in patients with hepatitis B virus (HBV)-related acute liver injury.ResultsIncreased hepatic NETs formation was observed in murine hepatitis virus strain-3–infected mice, but not in acetaminophen-treated mice. NETs depletion improved the liver damage and survival rate in FVH by inhibiting hepatic fibrin deposition and inflammation. An adoptive transfer experiment showed that neutrophil-specific fibrinogen-like protein 2 (FGL2) promoted NETs formation. FGL2 was found to directly interact with mucolipin 3, which regulated calcium influx and initiated autophagy, leading to NETs formation. Clinically, increased plasma NETs level was associated with coagulation dysfunction in patients with HBV acute liver injury. Colocalization of FGL2, NETs, and fibrin in liver was observed in these patients.ConclusionsNETs aggravated liver injury in FVH by promoting fibrin deposition and inflammation. NETs formation was regulated by the FGL2–mucolipin 3–autophagy axis. Targeting NETs may provide a new strategy for the treatment of FVH.

Luminometric Studies of Yeast Response to Complex Environmental Calcium Variations Demonstrate Sensing of External Calcium Ion Changes

The response of yeast to sharp environmental increases in calcium concentration has been extensively studied. However, systematic studies of the response under more general changes are still lacking. Only limited exploration of cellular responses has been conducted where calcium concentration is decreased. This article describes a set of luminometric experiments that monitor the cytosolic calcium concentration under changing external concentration conditions. As a decrease in external calcium concentrations requires the use of large sample volumes, the experiments require the use of equipment adapted for this purpose. We describe the modification of commercial luminometric equipment to make the exploration possible. We explore the yeast cellular behavior when an increase in external calcium concentration is followed by a decrease in external calcium concentration. We compare these results with those from the case of a double pulse of concentration increase. Results from the experiment show that the first, concentration increasing pulse produces the well-known sharp increase in cytosolic calcium followed by calcium sequestration to return to a cytosolic concentration near its initial condition. Surprisingly, the calcium decrease step shows similar results with a cytosolic increase followed by a return to lower levels. The results suggest the presence of a calcium sensing mechanism regulating calcium influx from external sources. This mechanism would produce channel opening as a response to any changes in external concentration, be it an enhancement or a depletion.

Challenges in Treating Charcot-Marie-Tooth Disease and Related Neuropathies: Current Management and Future Perspectives.

There is still no effective drug treatment available for Charcot-Marie-Tooth neuropathies (CMT). Current management relies on rehabilitation therapy, surgery for skeletal deformities, and symptomatic treatment of pain; fatigue and cramps are frequent complaints that are difficult to treat. The challenge is to find disease-modifying therapies. Several approaches, including gene silencing, to counteract the PMP22 gene overexpression in the most frequent CMT1A type are under investigation. PXT3003 is the compound in the most advanced phase for CMT1A, as a second-phase III trial is ongoing. Gene therapy to substitute defective genes or insert novel ones and compounds acting on pathways important for different CMT types are being developed and tested in animal models. Modulation of the Neuregulin pathway determining myelin thickness is promising for both hypo-demyelinating and hypermyelinating neuropathies; intervention on Unfolded Protein Response seems effective for rescuing misfolded myelin proteins such as P0 in CMT1B. HDAC6 inhibitors improved axonal transport and ameliorated phenotypes in different CMT models. Other potential therapeutic strategies include targeting macrophages, lipid metabolism, and Nav1.8 sodium channel in demyelinating CMT and the P2X7 receptor, which regulates calcium influx into Schwann cells, in CMT1A. Further approaches are aimed at correcting metabolic abnormalities, including the accumulation of sorbitol caused by biallelic mutations in the sorbitol dehydrogenase (SORD) gene and of neurotoxic glycosphingolipids in HSN1.

The Hidden Side of NCAM Family: NCAM2, a Key Cytoskeleton Organization Molecule Regulating Multiple Neural Functions.

Although it has been over 20 years since Neural Cell Adhesion Molecule 2 (NCAM2) was identified as the second member of the NCAM family with a high expression in the nervous system, the knowledge of NCAM2 is still eclipsed by NCAM1. The first studies with NCAM2 focused on the olfactory bulb, where this protein has a key role in axonal projection and axonal/dendritic compartmentalization. In contrast to NCAM1, NCAM2’s functions and partners in the brain during development and adulthood have remained largely unknown until not long ago. Recent studies have revealed the importance of NCAM2 in nervous system development. NCAM2 governs neuronal morphogenesis and axodendritic architecture, and controls important neuron-specific processes such as neuronal differentiation, synaptogenesis and memory formation. In the adult brain, NCAM2 is highly expressed in dendritic spines, and it regulates synaptic plasticity and learning processes. NCAM2’s functions are related to its ability to adapt to the external inputs of the cell and to modify the cytoskeleton accordingly. Different studies show that NCAM2 interacts with proteins involved in cytoskeleton stability and proteins that regulate calcium influx, which could also modify the cytoskeleton. In this review, we examine the evidence that points to NCAM2 as a crucial cytoskeleton regulation protein during brain development and adulthood. This key function of NCAM2 may offer promising new therapeutic approaches for the treatment of neurodevelopmental diseases and neurodegenerative disorders.

Abstract P498: Polycystin-1 Regulates Excitation-contraction Coupling In Atrial Cardiomyocytes

Patients with Autosomal Dominant Polycystic Kidney disease (ADPKD) have multiple cardiovascular manifestations, including increased susceptibility to arrhythmias. Mutations in polycystin-1 (PC1) encoding gene accounts for 85% cases of ADPKD, whereas mutations in polycystin-2 (PC2) only accounts for 15%. In kidney cells, PC1 interacts with PC2 to form a protein complex at the primary cilia to regulate calcium influx via PC2. However, cardiomyocytes are non-ciliated cells and the role of both PC1 and PC2 in atrial cardiomyocytes remains unknown. We have previously demonstrated that PC1 regulates action potentials and calcium handling to fine-tune ventricular cardiomyocyte contraction. Here, we hypothesize that PC1 regulates action potentials and calcium handling in atrial cardiomyocytes independent of PC2 actions. To test this hypothesis, we differentiated human induced pluripotent stem cells (iPSC) into atrial cardiomyocytes (iPSC-aCM) using previously published protocols. To determine the contribution of PC1/PC2 in atrial excitation-contraction coupling, protein expression was knocked down utilizing specific siRNA constructs, for each protein, or a universal control siRNA transfected using lipofectamine RNAiMAX. We measured action potentials using the potentiometric dye FluoVolt and intracellular calcium with Fura-2 AM or Fluo-4. Changes in fluorescence were monitored using a multiwavelength IonOptix system. iPSC-aCM were paced at 2 Hz to synchronize the beating pattern using field electrical stimulation. Our data shows that PC1 ablation significantly decreased action potential duration at 50% and 80% of repolarization, by 24% and 23%, respectively. Moreover, we observed that PC1 knockdown significantly reduced calcium transient amplitude elicited by field electrical stimulation without changes in calcium transient decay. Interestingly, PC2 knockdown did not modify calcium transients in atrial cardiomyocytes (iPSC-aCM). Our data suggest that PC1 regulates atrial excitation-contraction coupling independent of PC2 actions. This study warrants further investigation into atrial dysfunction in ADPKD patients with PC1 mutations.

MicroRNA-223 inhibits neutrophil extracellular traps formation through regulating calcium influx and small extracellular vesicles transmission

Modulation of miRNAs and neutrophil extracellular traps (NETs) formation are both implicated in inflammatory disorders. Adult-onset Still’s disease (AOSD) is a systemic autoinflammatory disease with neutrophilic leukocytosis and unknown etiology. Although the NETs formation is elevated in AOSD patients, the regulatory roles of miRNAs in NETs formation in AOSD remains unclear. We revealed that the circulating levels of IL-18, NETs, and miR-223 were significantly higher in active AOSD patients, compared with inactive AOSD patients or healthy controls (P < 0.005). Moreover, IL-18 increased calcium influx into neutrophils, which led to mitochondrial ROS (mROS) production and NETs formation. Elevated levels of NETs-DNA could induce miR-223 expression in neutrophils through activating Toll-like receptor 9. The upregulated miR-223 expression in neutrophils suppressed mROS production by blocking calcium influx, and subsequently inhibited IL-18-mediated NETs formation. Besides, the increased neutrophil-derived exosomal miR-223 levels were observed in active AOSD patients compared with healthy controls (P < 0.005). Our in vitro assays demonstrated that the neutrophil-derived small extracellular vesicles carried miR-223, which could repress IL-18 production in macrophages. Together, these results suggest a fine-tuned mechanism between inflammatory (IL-18 induced NETs) and anti-inflammatory (miR-223) factors in AOSD. MiR-223, mROS inhibitors, and calcium channel blockers are the potential therapeutics for autoinflammatory diseases such as AOSD.