Search results
Found 5065 matches for
Human myofibroblasts increase the arrhythmogenic potential of human induced pluripotent stem cell-derived cardiomyocytes.
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have the potential to remuscularize infarcted hearts but their arrhythmogenicity remains an obstacle to safe transplantation. Myofibroblasts are the predominant cell-type in the infarcted myocardium but their impact on transplanted hiPSC-CMs remains poorly defined. Here, we investigate the effect of myofibroblasts on hiPSC-CMs electrophysiology and Ca2+ handling using optical mapping of advanced human cell coculture systems mimicking cell-cell interaction modalities. Human myofibroblasts altered the electrophysiology and Ca2+ handling of hiPSC-CMs and downregulated mRNAs encoding voltage channels (KV4.3, KV11.1 and Kir6.2) and SERCA2a calcium pump. Interleukin-6 was elevated in the presence of myofibroblasts and direct stimulation of hiPSC-CMs with exogenous interleukin-6 recapitulated the paracrine effects of myofibroblasts. Blocking interleukin-6 reduced the effects of myofibroblasts only in the absence of physical contact between cell-types. Myofibroblast-specific connexin43 knockdown reduced functional changes in contact cocultures only when combined with interleukin-6 blockade. This provides the first in-depth investigation into how human myofibroblasts modulate hiPSC-CMs function, identifying interleukin-6 and connexin43 as paracrine- and contact-mediators respectively, and highlighting their potential as targets for reducing arrhythmic risk in cardiac cell therapy.
Mechanisms of SSRI Therapy and Discontinuation.
SSRIs are one of the most widely used drug therapies in primary care and psychiatry, and central to the management of the most common mental health problems in today's society. Despite this, SSRIs suffer from a slow onset of therapeutic effect and relatively poor efficacy as well as adverse effects, with recent concerns being focused on a disabling SSRI discontinuation syndrome. The mechanism underpinning their therapeutic effect has long shifted away from thinking that SSRIs act simply by increasing 5-HT in the synapse. Rather, a current popular view is that increased 5-HT is just the beginning of a series of complex downstream signalling events, which trigger changes in neural plasticity at the functional and structural level. These changes in plasticity are then thought to interact with neuropsychological processes to enhance re-learning of emotional experiences that ultimately brings about changes in mood. This compelling view of SSRI action is underpinning attempts to understand fast-acting antidepressants, such as ketamine and psychedelic drugs, and aid the development of future therapies. An important gap in the theory is evidence that changes in plasticity are causally linked to relevant behavioural effects. Also, predictions that the SSRI-induced neural plasticity might have applicability in other areas of medicine have not yet been borne out. In contrast to the sophisticated view of the antidepressant action of SSRIs, the mechanism underpinning SSRI discontinuation is little explored. Nevertheless, evidence of rebound increases in 5-HT neuron excitability immediately on cessation of SSRI treatment provide a starting point for future investigation. Indeed, this evidence allows formulation of a mechanistic explanation of SSRI discontinuation which draws on parallels with the withdrawal states of other psychotropic drugs.
Cholera intoxication of human enteroids reveals interplay between decoy and functional glycoconjugate ligands.
Prior research on cholera toxin (CT) binding and intoxication has relied on human colonic cancer derived epithelial cells. While these transformed cell lines have been beneficial, they neither derive from small intestine where intoxication occurs, nor represent the diversity of small intestinal epithelial cells (SI-ECs) and variation in glycoconjugate expression among individuals. Here, we used human enteroids, derived from jejunal biopsies of multipledonors to study CT binding and intoxication of human non-transformed SI-ECs. We modulated surface expression of glycosphingolipids, glycoproteins and specific glycans to distinguish the role of each glycan/glycoconjugate. Cholera-toxin-subunit-B (CTB) mutants were generated to decipher the preference of each glycoconjugate to different binding sites and the correlation between CT binding and intoxication. Human enteroids contain trace amounts of GM1, but other glycosphingolipids may be contributing to CT intoxication. We discovered that inhibition of either fucosylation or O-glycosylation sensitize enteroids to CT-intoxication. This can either be a consequence of the removal of fucosylated "decoy-like-ligands" binding to CTB's non-canonical site and/or increase in the availability of Gal/GalNAc-terminating glycoconjugates binding to the canonical site. Furthermore, simultaneous inhibition of fucosylation and O-glycosylation increased the availability of additional Gal/GalNAc-terminating glycoconjugates but counteracted the sensitization in CT intoxication caused by inhibiting O-glycosylation because of reduction in fucose. This implies a dual role of fucose as a functional glycan and a decoy, the interplay of which influences CT binding and intoxication. Finally, while the results were similar for enteroids from different donors, they were not identical, pointing to a role for human genetic variation in determining sensitivity to CT.
Continuous home cage monitoring of activity and sleep in mice during repeated paroxetine treatment and discontinuation.
RATIONALE: Non-invasive home cage monitoring is emerging as a valuable tool to assess the effects of experimental interventions on mouse behaviour. A field in which these techniques may prove useful is the study of repeated selective serotonin reuptake inhibitor (SSRI) treatment and discontinuation. SSRI discontinuation syndrome is an under-researched condition that includes the emergence of sleep disturbances following treatment cessation. OBJECTIVES: We used passive infrared (PIR) monitoring to investigate changes in activity, sleep, and circadian rhythms during repeated treatment with the SSRI paroxetine and its discontinuation in mice. METHODS: Male mice received paroxetine (10 mg/kg/day, s.c.) for 12 days, then were swapped to saline injections for a 13 day discontinuation period and compared to mice that received saline injections throughout. Mice were continuously tracked using the Continuous Open Mouse Phenotyping of Activity and Sleep Status (COMPASS) system. RESULTS: Repeated paroxetine treatment reduced activity and increased behaviourally-defined sleep in the dark phase. These effects recovered to saline-control levels within 24 h of paroxetine cessation, yet there was also evidence of a lengthening of sleep bouts in the dark phase for up to a week following discontinuation. CONCLUSIONS: This study provides the first example of how continuous non-invasive home cage monitoring can be used to detect objective behavioural changes in activity and sleep during and after drug treatment in mice. These data suggest that effects of paroxetine administration reversed soon after its discontinuation but identified an emergent change in sleep bout duration, which could be used as a biomarker in future preclinical studies to prevent or minimise SSRI discontinuation symptoms.
The TMEM16A anion channel as a versatile regulator of vascular tone.
The TMEM16A channel represents a key depolarizing mechanism in arterial smooth muscle and contractile pericytes, where it is activated by several endogenous contractile agonists. In this issue of Science Signaling, Mata-Daboin et al. demonstrate a previously unidentified role for TMEM16A in endothelial cells for acetylcholine-mediated vasorelaxation. Collectively, TMEM16A serves as a transducer of vasoactive stimuli to enable fine modulation of vessel tone.
Inhibition of striatal dopamine release by the L-type calcium channel inhibitor isradipine co-varies with risk factors for Parkinson's.
Ca2+ entry into nigrostriatal dopamine (DA) neurons and axons via L-type voltage-gated Ca2+ channels (LTCCs) contributes, respectively, to pacemaker activity and DA release and has long been thought to contribute to vulnerability to degeneration in Parkinson's disease. LTCC function is greater in DA axons and neurons from substantia nigra pars compacta than from ventral tegmental area, but this is not explained by channel expression level. We tested the hypothesis that LTCC control of DA release is governed rather by local mechanisms, focussing on candidate biological factors known to operate differently between types of DA neurons and/or be associated with their differing vulnerability to parkinsonism, including biological sex, α-synuclein, DA transporters (DATs) and calbindin-D28k (Calb1). We detected evoked DA release ex vivo in mouse striatal slices using fast-scan cyclic voltammetry and assessed LTCC support of DA release by detecting the inhibition of DA release by the LTCC inhibitors isradipine or CP8. Using genetic knockouts or pharmacological manipulations, we identified that striatal LTCC support of DA release depended on multiple intersecting factors, in a regionally and sexually divergent manner. LTCC function was promoted by factors associated with Parkinsonian risk, including male sex, α-synuclein, DAT and a dorsolateral co-ordinate, but limited by factors associated with protection, that is, female sex, glucocerebrosidase activity, Calb1 and ventromedial co-ordinate. Together, these data show that LTCC function in DA axons and isradipine effect are locally governed and suggest they vary in a manner that in turn might impact on, or reflect, the cellular stress that leads to parkinsonian degeneration.
Mutate and Conjugate: A Method to Enable Rapid In-Cell Target Validation.
Target validation remains a challenge in drug discovery, which leads to a high attrition rate in the drug discovery process, particularly in Phase II clinical trials. Consequently, new approaches to enhance target validation are valuable tools to improve the drug discovery process. Here, we report the combination of site-directed mutagenesis and electrophilic fragments to enable the rapid identification of small molecules that selectively inhibit the mutant protein. Using the bromodomain-containing protein BRD4 as an example, we employed a structure-based approach to identify the L94C mutation in the first bromodomain of BRD4 [BRD4(1)] as having a minimal effect on BRD4(1) function. We then screened a focused, KAc mimic-containing fragment set and a diverse fragment library against the mutant and wild-type proteins and identified a series of fragments that showed high selectivity for the mutant protein. These compounds were elaborated to include an alkyne click tag to enable the attachment of a fluorescent dye. These clickable compounds were then assessed in HEK293T cells, transiently expressing BRD4(1)WT or BRD4(1)L94C, to determine their selectivity for BRD4(1)L94C over other possible cellular targets. One compound was identified that shows very high selectivity for BRD4(1)L94C over all other proteins. This work provides a proof-of-concept that the combination of site-directed mutagenesis and electrophilic fragments, in a mutate and conjugate approach, can enable rapid identification of small molecule inhibitors for an appropriately mutated protein of interest. This technology can be used to assess the cellular phenotype of inhibiting the protein of interest, and the electrophilic ligand provides a starting point for noncovalent ligand development.
The expanding boundaries of sphingolipid lysosomal storage diseases; insights from Niemann-Pick disease type C.
Lysosomal storage diseases are inborn errors of metabolism that arise due to loss of function mutations in genes encoding lysosomal enzymes, protein co-factors or lysosomal membrane proteins. As a consequence of the genetic defect, lysosomal function is impaired and substrates build up in the lysosome leading to 'storage'. A sub group of these disorders are the sphingolipidoses in which sphingolipids accumulate in the lysosome. In this review, I will discuss how the study of these rare lysosomal disorders reveals unanticipated links to other rare and common human diseases using Niemann-Pick disease type C as an example.
The medial septum controls hippocampal supra-theta oscillations.
Hippocampal theta oscillations orchestrate faster beta-to-gamma oscillations facilitating the segmentation of neural representations during navigation and episodic memory. Supra-theta rhythms of hippocampal CA1 are coordinated by local interactions as well as inputs from the entorhinal cortex (EC) and CA3 inputs. However, theta-nested gamma-band activity in the medial septum (MS) suggests that the MS may control supra-theta CA1 oscillations. To address this, we performed multi-electrode recordings of MS and CA1 activity in rodents and found that MS neuron firing showed strong phase-coupling to theta-nested supra-theta episodes and predicted changes in CA1 beta-to-gamma oscillations on a cycle-by-cycle basis. Unique coupling patterns of anatomically defined MS cell types suggested that indirect MS-to-CA1 pathways via the EC and CA3 mediate distinct CA1 gamma-band oscillations. Optogenetic activation of MS parvalbumin-expressing neurons elicited theta-nested beta-to-gamma oscillations in CA1. Thus, the MS orchestrates hippocampal network activity at multiple temporal scales to mediate memory encoding and retrieval.
Integration of 3D-printed cerebral cortical tissue into an ex vivo lesioned brain slice.
Engineering human tissue with diverse cell types and architectures remains challenging. The cerebral cortex, which has a layered cellular architecture composed of layer-specific neurons organised into vertical columns, delivers higher cognition through intricately wired neural circuits. However, current tissue engineering approaches cannot produce such structures. Here, we use a droplet printing technique to fabricate tissues comprising simplified cerebral cortical columns. Human induced pluripotent stem cells are differentiated into upper- and deep-layer neural progenitors, which are then printed to form cerebral cortical tissues with a two-layer organization. The tissues show layer-specific biomarker expression and develop a structurally integrated network of processes. Implantation of the printed cortical tissues into ex vivo mouse brain explants results in substantial structural implant-host integration across the tissue boundaries as demonstrated by the projection of processes and the migration of neurons, and leads to the appearance of correlated Ca2+ oscillations across the interface. The presented approach might be used for the evaluation of drugs and nutrients that promote tissue integration. Importantly, our methodology offers a technical reservoir for future personalized implantation treatments that use 3D tissues derived from a patient's own induced pluripotent stem cells.
The biophysical properties of TRIC-A and TRIC-B and their interactions with RyR2.
Trimeric intracellular cation channels (TRIC-A and TRIC-B) are thought to provide counter-ion currents to enable charge equilibration across the sarco/endoplasmic reticulum (SR) and nuclear membranes. However, there is also evidence that TRIC-A may interact directly with ryanodine receptor type 1 (RyR1) and 2 (RyR2) to alter RyR channel gating. It is therefore possible that the reverse is also true, where the presence of RyR channels is necessary for fully functional TRIC channels. We therefore coexpressed mouse TRIC-A or TRIC-B with mouse RyR2 in HEK293 cells to examine if after incorporating membrane vesicles from these cells into bilayers, the presence of TRIC affects RyR2 function, and to characterize the permeability and gating properties of the TRIC channels. Importantly, we used no purification techniques or detergents to minimize damage to TRIC and RyR2 proteins. We found that both TRIC-A and TRIC-B altered the gating behavior of RyR2 and its response to cytosolic Ca2+ but that TRIC-A exhibited a greater ability to stimulate the opening of RyR2. Fusing membrane vesicles containing TRIC-A or TRIC-B into bilayers caused the appearance of rapidly gating current fluctuations of multiple amplitudes. The reversal potentials of bilayers fused with high numbers of vesicles containing TRIC-A or TRIC-B revealed both Cl- and K+ fluxes, suggesting that TRIC channels are relatively non-selective ion channels. Our results indicate that the physiological roles of TRIC-A and TRIC-B may include direct, complementary regulation of RyR2 gating in addition to the provision of counter-ion currents of both cations and anions.
Lipopolysaccharide triggers exacerbated microglial activation, excessive cytokine release and behavioural disturbances in mice with truncated Fused-in-Sarcoma Protein (FUS)
CNS inflammation, including microglial activation, in response to peripheral infections are known to contribute to the pathology of both familial and sporadic neurodegenerative disease. The relationship between Fused-in-Sarcoma Protein (FUS)-mediated disease in the transgenic FUS[1–359] animals and the systemic inflammatory response have not been explored. Here, we investigated microglial activation, inflammatory gene expression and the behavioural responses to lipopolysaccharide-induced (LPS; 0.1 mg/kg) systemic inflammation in the FUS[1–359] transgenic mice. The pathology of these mice recapitulates the key features of mutant FUS-associated familial frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Here, pre-symptomatic 8-week-old mutant or wild type controls were challenged with LPS or with saline and sucrose intake, novel cage exploration, marble burying and swimming behaviours were analyzed. The level of pro-inflammatory gene expression was also determined, and microglial activation was evaluated. In chronic experiments, to discover whether the LPS challenge would affect the onset of ALS-like paralysis, animals were evaluated for clinical signs from 5 to 7 weeks post-injection. Compared to controls, acutely challenged FUS[1–359]-tg mice exhibited decreased sucrose intake and increased floating behaviours. The FUS[1–359]-tg mice exhibited an increase in immunoreactivity for Iba1-positive cells in the prefrontal cortex and ventral horn of the spinal cord, which was accompanied by increased expression of interleukin-1β, tumour necrosis factor, cyclooxygenase-(COX)-1 and COX-2. However, the single LPS challenge did not alter the time to development of paralysis in the FUS[1–359]-tg mice. Thus, while the acute inflammatory response was enhanced in the FUS mutant animals, it did not have a lasting impact on disease progression.
Genes, inflammatory response, tolerance, and resistance to virus infections in migratory birds, bats, and rodents
Normally, the host immunological response to viral infection is coordinated to restore homeostasis and protect the individual from possible tissue damage. The two major approaches are adopted by the host to deal with the pathogen: resistance or tolerance. The nature of the responses often differs between species and between individuals of the same species. Resistance includes innate and adaptive immune responses to control virus replication. Disease tolerance relies on the immune response allowing the coexistence of infections in the host with minimal or no clinical signs, while maintaining sufficient viral replication for transmission. Here, we compared the virome of bats, rodents and migratory birds and the molecular mechanisms underlying symptomatic and asymptomatic disease progression. We also explore the influence of the host physiology and environmental influences on RNA virus expression and how it impacts on the whole brain transcriptome of seemingly healthy semipalmated sandpiper (Calidris pusilla) and spotted sandpiper (Actitis macularius). Three time points throughout the year were selected to understand the importance of longitudinal surveys in the characterization of the virome. We finally revisited evidence that upstream and downstream regulation of the inflammatory response is, respectively, associated with resistance and tolerance to viral infections.
Optical profiling of autonomous Ca2+ nanodomains generated by lysosomal TPC2 and TRPML1.
Multiple families of Ca2+-permeable channels co-exist on lysosomal Ca2+ stores but how each family couples to its own unique downstream physiology is unclear. We have therefore investigated the Ca2+-signalling architecture underpinning different channels on the same vesicle that drive separate pathways, using phagocytosis as a physiological stimulus. Lysosomal Ca2+-channels are a major Ca2+ source driving particle uptake in macrophages, but different channels drive different aspects of Fc-receptor-mediated phagocytosis: TPC2 couples to dynamin activation, whilst TRPML1 couples to lysosomal exocytosis. We hypothesised that they are driven by discrete local plumes of Ca2+ around open channels (Ca2+ nanodomains). To test this, we optimized Ca2+-nanodomain recordings by screening panels of genetically encoded Ca2+ indicators (GECIs) fused to TPC2 to monitor the [Ca2+] next to the channel. Signal calibration accounting for the distance of the GECI from the channel mouth reveals that, during phagocytosis, TPC2 generates local Ca2+ nanodomains around itself of up to 42 µM, nearly a hundred-fold greater than the global cytosolic [Ca2+] rise. We further show that TPC2 and TRPML1, though on the same lysosomes, generate autonomous Ca2+ nanodomains of high [Ca2+] that are largely insulated from one another, a platform allowing their discrete Ca2+-decoding to promote unique respective physiologies.
Depression is associated with reduced outcome sensitivity in a dual valence, magnitude learning task.
BACKGROUND: Learning from rewarded and punished choices is perturbed in depressed patients, suggesting that abnormal reinforcement learning may be a cognitive mechanism of the illness. However, previous studies have disagreed about whether this behavior is produced by alterations in the rate of learning or sensitivity to experienced outcomes. This previous work has generally assessed learning in response to binary outcomes of one valence, rather than to both rewarding and punishing continuous outcomes. METHODS: A novel drifting reward and punishment magnitude reinforcement-learning task was administered to patients with current (n = 40) and remitted depression (n = 39), and healthy volunteers (n = 40) to capture potential differences in learning behavior. Standard questionnaires were administered to measure self-reported depressive symptom severity, trait and state anxiety and level of anhedonic symptoms. RESULTS: Our findings demonstrate that patients with current depression adjust their learning behaviors to a lesser degree in response to trial-by-trial variations in reward and loss magnitudes than the other groups. Computational modeling revealed that this behavioral signature of current depressive state is better accounted for by reduced reward and punishment sensitivity (all p < 0.031), rather than a change in learning rate (p = 0.708). However, between-group differences were not related to self-reported symptom severity or comorbid anxiety disorders in the current depression group. CONCLUSION: These findings suggest that current depression is associated with reduced outcome sensitivity rather than altered learning rate. Previous findings reported in this domain mainly from binary learning tasks seem to generalize to learning from continuous outcomes.
Single-cell RNA sequencing of murine hearts for studying the development of the cardiac conduction system.
The development of the cardiac conduction system (CCS) is essential for correct heart function. However, critical details on the cell types populating the CCS in the mammalian heart during the development remain to be resolved. Using single-cell RNA sequencing, we generated a large dataset of transcriptomes of ~0.5 million individual cells isolated from murine hearts at six successive developmental corresponding to the early, middle and late stages of heart development. The dataset provides a powerful library for studying the development of the heart's CCS and other cardiac components. Our initial analysis identified distinct cell types between 20 to 26 cell types across different stages, of which ten are involved in forming the CCS. Our dataset allows researchers to reuse the datasets for data mining and a wide range of analyses. Collectively, our data add valuable transcriptomic resources for further study of cardiac development, such as gene expression, transcriptional regulation and functional gene activity in developing hearts, particularly the CCS.
Can in vitro studies aid in the development and use of antiseizure therapies? A report of the ILAE/AES Joint Translational Task Force
AbstractIn vitro preparations (defined here as cultured cells, brain slices, and isolated whole brains) offer a variety of approaches to modeling various aspects of seizures and epilepsy. Such models are particularly amenable to the application of anti‐seizure compounds, and consequently are a valuable tool to screen the mechanisms of epileptiform activity, mode of action of known anti‐seizure medications (ASMs), and the potential efficacy of putative new anti‐seizure compounds. Despite these applications, all disease models are a simplification of reality and are therefore subject to limitations. In this review, we summarize the main types of in vitro models that can be used in epilepsy research, describing key methodologies as well as notable advantages and disadvantages of each. We argue that a well‐designed battery of in vitro models can form an effective and potentially high‐throughput screening platform to predict the clinical usefulness of ASMs, and that in vitro models are particularly useful for interrogating mechanisms of ASMs. To conclude, we offer several key recommendations that maximize the potential value of in vitro models in ASM screening. This includes the use of multiple in vitro tests that can complement each other, carefully combined with in vivo studies, the use of tissues from chronically epileptic (rather than naïve wild‐type) animals, and the integration of human cell/tissue‐derived preparations.