Characterization of Negative Allosteric Modulators of the Calcium-Sensing Receptor for Repurposing as a Treatment of Asthma.

Asthma is still an incurable disease, and there is a recognized need for novel small-molecule therapies for people with asthma, especially those poorly controlled by current treatments. We previously demonstrated that calcium-sensing receptor (CaSR) negative allosteric modulators (NAMs), calcilytics, uniquely suppress both airway hyperresponsiveness (AHR) and inflammation in human cells and murine asthma surrogates. Here we assess the feasibility of repurposing four CaSR NAMs, which were originally developed for oral therapy for osteoporosis and previously tested in the clinic as a novel, single, and comprehensive topical antiasthma therapy. We address the hypotheses, using murine asthma surrogates, that topically delivered CaSR NAMs 1) abolish AHR; 2) are unlikely to cause unwanted systemic effects; 3) are suitable for topical application; and 4) inhibit airway inflammation to the same degree as the current standard of care, inhaled corticosteroids, and, furthermore, inhibit airway remodeling. All four CaSR NAMs inhibited poly-L-arginine-induced AHR in naïve mice and suppressed both AHR and airway inflammation in a murine surrogate of acute asthma, confirming class specificity. Repeated exposure to inhaled CaSR NAMs did not alter blood pressure, heart rate, or serum calcium concentrations. Optimal candidates for repurposing were identified based on anti-AHR/inflammatory activities, pharmacokinetics/pharmacodynamics, formulation, and micronization studies. Whereas both inhaled CaSR NAMs and inhaled corticosteroids reduced airways inflammation, only the former prevented goblet cell hyperplasia in a chronic asthma model. We conclude that inhaled CaSR NAMs are likely a single, safe, and effective topical therapy for human asthma, abolishing AHR, suppressing airways inflammation, and abrogating some features of airway remodeling. SIGNIFICANCE STATEMENT: Calcium-sensing receptor (CaSR) negative allosteric modulators (NAMs) reduce airway smooth muscle hyperresponsiveness, reverse airway inflammation as efficiently as topical corticosteroids, and suppress airway remodeling in asthma surrogates. CaSR NAMs, which were initially developed for oral therapy of osteoporosis proved inefficacious for this indication despite being safe and well tolerated. Here we show that structurally unrelated CaSR NAMs are suitable for inhaled delivery and represent a one-stop, steroid-free approach to asthma control and prophylaxis.

Induced pluripotent stem cells derived from the developing striatum as a potential donor source for cell replacement therapy for Huntington disease.

BACKGROUND: Cell replacement therapy (CRT) for Huntington disease (HD) requires a source of striatal (STR) progenitors capable of restoring the function lost due to STR degeneration. Authentic STR progenitors can be collected from the fetal putative striatum, or whole ganglionic eminence (WGE), but these tissues remain impractical for widespread clinical application, and alternative donor sources are required. Here we begin exploring the possibility that induced pluripotent stem cells (iPSC) derived from WGE may retain an epigenetic memory of their tissue of origin, which could enhance their ability to differentiate into STR cells. RESULTS: We generate four iPSC lines from human WGE (hWGE) and establish that they have a capacity similar to human embryonic stem cells with regard to their ability to differentiate toward an STR phenotype, as measured by expression and demethylation of key STR genes, while maintaining an overall different methylome. Finally, we demonstrate that these STR-differentiated hWGE iPSCs share characteristics with hWGE (i.e., authentic STR tissues) both in vitro and following transplantation into an HD model. Overall, iPSCs derived from human WGE show promise as a donor source for CRT for HD.

Kv7 channels are upregulated during striatal neuron development and promote maturation of human iPSC-derived neurons.

Kv7 channels determine the resting membrane potential of neurons and regulate their excitability. Even though dysfunction of Kv7 channels has been linked to several debilitating childhood neuronal disorders, the ontogeny of the constituent genes, which encode Kv7 channels (KNCQ), and expression of their subunits have been largely unexplored. Here, we show that developmentally regulated expression of specific KCNQ mRNA and Kv7 channel subunits in mouse and human striatum is crucial to the functional maturation of mouse striatal neurons and human-induced pluripotent stem cell-derived neurons. This demonstrates their pivotal role in normal development and maturation, the knowledge of which can now be harnessed to synchronise and accelerate neuronal differentiation of stem cell-derived neurons, enhancing their utility for disease modelling and drug discovery.

Forced cell cycle exit and modulation of GABAA, CREB, and GSK3ß signaling promote functional maturation of induced pluripotent stem cell-derived neurons.

Although numerous protocols have been developed for differentiation of neurons from a variety of pluripotent stem cells, most have concentrated on being able to specify effectively appropriate neuronal subtypes and few have been designed to enhance or accelerate functional maturity. Of those that have, most employ time courses of functional maturation that are rather protracted, and none have fully characterized all aspects of neuronal function, from spontaneous action potential generation through to postsynaptic receptor maturation. Here, we describe a simple protocol that employs the sequential addition of just two supplemented media that have been formulated to separate the two key phases of neural differentiation, the neurogenesis and synaptogenesis, each characterized by different signaling requirements. Employing these media, this new protocol synchronized neurogenesis and enhanced the rate of maturation of pluripotent stem cell-derived neural precursors. Neurons differentiated using this protocol exhibited large cell capacitance with relatively hyperpolarized resting membrane potentials; moreover, they exhibited augmented: 1) spontaneous electrical activity; 2) regenerative induced action potential train activity; 3) Na(+) current availability, and 4) synaptic currents. This was accomplished by rapid and uniform development of a mature, inhibitory GABAAreceptor phenotype that was demonstrated by Ca(2+) imaging and the ability of GABAAreceptor blockers to evoke seizurogenic network activity in multielectrode array recordings. Furthermore, since this protocol can exploit expanded and frozen prepatterned neural progenitors to deliver mature neurons within 21 days, it is both scalable and transferable to high-throughput platforms for the use in functional screens.

Improving and accelerating the differentiation and functional maturation of human stem cell-derived neurons: role of extracellular calcium and GABA.

Neurons differentiated from pluripotent stem cells using established neural culture conditions often exhibit functional deficits. Recently, we have developed enhanced media which both synchronize the neurogenesis of pluripotent stem cell-derived neural progenitors and accelerate their functional maturation; together these media are termed SynaptoJuice. This pair of media are pro-synaptogenic and generate authentic, mature synaptic networks of connected forebrain neurons from a variety of induced pluripotent and embryonic stem cell lines. Such enhanced rate and extent of synchronized maturation of pluripotent stem cell-derived neural progenitor cells generates neurons which are characterized by a relatively hyperpolarized resting membrane potential, higher spontaneous and induced action potential activity, enhanced synaptic activity, more complete development of a mature inhibitory GABAA receptor phenotype and faster production of electrical network activity when compared to standard differentiation media. This entire process - from pre-patterned neural progenitor to active neuron - takes 3 weeks or less, making it an ideal platform for drug discovery and disease modelling in the fields of human neurodegenerative and neuropsychiatric disorders, such as Huntington's disease, Parkinson's disease, Alzheimer's disease and Schizophrenia.

The Role of Kv1.2 Channel in Electrotaxis Cell Migration.

Voltage-gated potassium Kv1.2 channels play pivotal role in maintaining of resting membrane potential and, consequently, regulation of cellular excitability of neurons. Endogenously generated electric field (EF) have been proven as an important regulator for cell migration and tissue repair. The mechanisms of ion channel involvement in EF-induced cell responses are extensively studied but largely are poorly understood. In this study we generated three COS-7 clones with different expression levels of Kv1.2 channel, and confirmed their functional variations with patch clamp analysis. Time-lapse imaging analysis showed that EF-induced cell migration response was Kv1.2 channel expression level depended. Inhibition of Kv1.2 channels with charybdotoxin (ChTX) constrained the sensitivity of COS-7 cells to EF stimulation more than their motility. Immunocytochemistry and pull-down analyses demonstrated association of Kv1.2 channels with actin-binding protein cortactin and its re-localization to the cathode-facing membrane at EF stimulation, which confirms the mechanism of EF-induced directional migration. This study displays that Kv1.2 channels represent an important physiological link in EF-induced cell migration. The described mechanism suggests a potential application of EF which may improve therapeutic performance in curing injuries of neuronal and/or cardiac tissue repair, post operational therapy, and various degenerative syndromes.

The calcium-sensing receptor beyond extracellular calcium homeostasis: conception, development, adult physiology, and disease.

The extracellular calcium-sensing receptor (CaSR) is the first identified G protein-coupled receptor to be activated by an ion, extracellular calcium (Ca(2+)). Since the identification of the CaSR in 1993, genetic mutations in the CaSR gene, and murine models in which CaSR expression has been manipulated, have clearly demonstrated the importance of this receptor in the maintenance of stable, free, ionized Ca(2+) concentration in the extracellular fluids. These functions have been extensively reviewed elsewhere. However, the distribution pattern and expression of the CaSR in lower vertebrates strongly suggest that the CaSR must play a role that is independent of mineral cation metabolism. This review addresses the involvement of the CaSR in nutrient sensing; its putative and demonstrated functions during conception, embryonic development, and birth; and its contributions to adult physiology and disease, with reference to CaSR-based therapeutics. Recent ongoing developments concerning the role of the CaSR in stem cell differentiation are also reviewed.

Functional plasticity of the N-methyl-d-aspartate receptor in differentiating human erythroid precursor cells.

Calcium signaling is essential to support erythroid proliferation and differentiation. Precise control of the intracellular Ca(2+) levels in erythroid precursor cells (EPCs) is afforded by coordinated expression and function of several cation channels, including the recently identified N-methyl-d-aspartate receptor (NMDAR). Here, we characterized the changes in Ca(2+) uptake and electric currents mediated by the NMDARs occurring during EPC differentiation using flow cytometry and patch clamp. During erythropoietic maturation, subunit composition and properties of the receptor changed; in proerythroblasts and basophilic erythroblasts, fast deactivating currents with high amplitudes were mediated by the GluN2A subunit-dominated receptors, while at the polychromatic and orthochromatic erythroblast stages, the GluN2C subunit was getting more abundant, overriding the expression of GluN2A. At these stages, the currents mediated by the NMDARs carried the features characteristic of the GluN2C-containing receptors, such as prolonged decay time and lower conductance. Kinetics of this switch in NMDAR properties and abundance varied markedly from donor to donor. Despite this variability, NMDARs were essential for survival of EPCs in any subject tested. Our findings indicate that NMDARs have a dual role during erythropoiesis, supporting survival of polychromatic erythroblasts and contributing to the Ca(2+) homeostasis from the orthochromatic erythroblast stage to circulating red blood cells.

Functional expression of the multimodal extracellular calcium-sensing receptor in pulmonary neuroendocrine cells.

The Ca(2+)-sensing receptor (CaSR) is the master regulator of whole-body extracellular free ionized [Ca(2+)]o. In addition to sensing [Ca(2+)]o, CaSR integrates inputs from a variety of different physiological stimuli. The CaSR is also expressed in many regions outside the [Ca(2+)]o homeostatic system, including the fetal lung where it plays a crucial role in lung development. Here, we show that neuroepithelial bodies (NEBs) of the postnatal mouse lung express a functional CaSR. NEBs are densely innervated groups of neuroendocrine epithelial cells in the lung representing complex sensory receptors in the airways and exhibiting stem cell characteristics. qRT-PCR performed on laser microdissected samples from GAD67-GFP mouse lung cryosections revealed exclusive expression of the CaSR in the NEB microenvironment. CaSR immunoreactivity was present at NEB cells from postnatal day 14 onwards. Confocal imaging of lung slices revealed that NEB cells responded to an increase of [Ca(2+)]o with a rise in intracellular Ca(2+) ([Ca(2+)]i); an effect mimicked by several membrane-impermeant CaSR agonists (e.g. the calcimimetic R-568) and that was blocked by the calcilytic Calhex-231. Block of TRPC channels attenuated the CaSR-dependent increases in [Ca(2+)]i, suggesting that Ca(2+) influx through TRPC channels contributes to the total [Ca(2+)]i signal evoked by the CaSR in NEBs. CaSR also regulated baseline [Ca(2+)]i in NEBs and, through paracrine signaling from Clara-like cells, coordinated intercellular communication in the NEB microenvironment. These data suggest that the NEB CaSR integrates multiple signals converging on this complex chemosensory unit, and is a key regulator of this intrapulmonary airway stem cell niche.

Allele-specific RNA interference rescues the long-QT syndrome phenotype in human-induced pluripotency stem cell cardiomyocytes.

AIMS: Long-QT syndromes (LQTS) are mostly autosomal-dominant congenital disorders associated with a 1:1000 mutation frequency, cardiac arrest, and sudden death. We sought to use cardiomyocytes derived from human-induced pluripotency stem cells (hiPSCs) as an in vitro model to develop and evaluate gene-based therapeutics for the treatment of LQTS. METHODS AND RESULTS: We produced LQTS-type 2 (LQT2) hiPSC cardiomyocytes carrying a KCNH2 c.G1681A mutation in a IKr ion-channel pore, which caused impaired glycosylation and channel transport to cell surface. Allele-specific RNA interference (RNAi) directed towards the mutated KCNH2 mRNA caused knockdown, while leaving the wild-type mRNA unaffected. Electrophysiological analysis of patient-derived LQT2 hiPSC cardiomyocytes treated with mutation-specific siRNAs showed normalized action potential durations (APDs) and K(+) currents with the concurrent rescue of spontaneous and drug-induced arrhythmias (presented as early-afterdepolarizations). CONCLUSIONS: These findings provide in vitro evidence that allele-specific RNAi can rescue diseased phenotype in LQTS cardiomyocytes. This is a potentially novel route for the treatment of many autosomal-dominant-negative disorders, including those of the heart.

Carbon monoxide: an emerging regulator of ion channels.

Carbon monoxide is rapidly emerging as an important cellular messenger, regulating a wide range of physiological processes. Crucial to its role in both physiology and disease is its ability differentially to regulate several classes of ion channels, including examples from calcium-activated K(+) (BK(Ca)), voltage-activated K(+) (K(v)) and Ca(2+) channel (L-type) families, ligand-gated P2X receptors (P2X2 and P2X4), tandem P domain K(+) channels (TREK1) and the epithelial Na(+) channel (ENaC). The mechanisms by which CO regulates these ion channels are still unclear and remain somewhat controversial. However, available structure-function studies suggest that a limited range of amino acid residues confer CO sensitivity, either directly or indirectly, to particular ion channels and that cellular redox state appears to be important to the final integrated response. Whatever the molecular mechanism by which CO regulates ion channels, endogenous production of this gasotransmitter has physiologically important roles and is currently being explored as a potential therapeutic.

Cysteine residue 911 in C-terminal tail of human BK(Ca)α channel subunit is crucial for its activation by carbon monoxide.

The large conductance, voltage- and calcium-activated potassium channel, BK(Ca), is a known target for the gasotransmitter, carbon monoxide (CO). Activation of BK(Ca) by CO modulates cellular excitability and contributes to the physiology of a diverse array of processes, including vascular tone and oxygen-sensing. Currently, there is no consensus regarding the molecular mechanisms underpinning reception of CO by the BK(Ca). Here, employing voltage-clamped, inside-out patches from HEK293 cells expressing single, double and triple cysteine mutations in the BK(Ca) α-subunit, we test the hypothesis that CO regulation is conferred upon the channel by interactions with cysteine residues within the RCK2 domain. In physiological [Ca(2+)](i), all mutants carrying a cysteine substitution at position 911 (C911G) demonstrated significantly reduced CO sensitivity; the C911G mutant did not express altered Ca(2+)-sensitivity. In contrast, histidine residues in RCK1 domain, previously shown to ablate CO activation in low [Ca(2+)](i), actually increased CO sensitivity when [Ca(2+)](i) was in the physiological range. Importantly, cyanide, employed here as a substituent for CO at potential metal centres, occluded activation by CO; this effect was freely reversible. Taken together, these data suggest that a specific cysteine residue in the C-terminal domain, which is close to the Ca(2+) bowl but which is not involved in Ca(2+) activation, confers significant CO sensitivity to BK(Ca) channels. The rapid reversibility of CO and cyanide binding, coupled to information garnered from other CO-binding proteins, suggests that C911 may be involved in formation of a transition metal cluster which can bind and, thereafter, activate BK(Ca).

Alveolar epithelial CNGA1 channels mediate cGMP-stimulated, amiloride-insensitive, lung liquid absorption.

Impairment of lung liquid absorption can lead to severe respiratory symptoms, such as those observed in pulmonary oedema. In the adult lung, liquid absorption is driven by cation transport through two pathways: a well-established amiloride-sensitive Na(+) channel (ENaC) and, more controversially, an amiloride-insensitive channel that may belong to the cyclic nucleotide-gated (CNG) channel family. Here, we show robust CNGA1 (but not CNGA2 or CNGA3) channel expression principally in rat alveolar type I cells; CNGA3 was expressed in ciliated airway epithelial cells. Using a rat in situ lung liquid clearance assay, CNG channel activation with 1 mM 8Br-cGMP resulted in an approximate 1.8-fold stimulation of lung liquid absorption. There was no stimulation by 8Br-cGMP when applied in the presence of either 100 µM L: -cis-diltiazem or 100 nM pseudechetoxin (PsTx), a specific inhibitor of CNGA1 channels. Channel specificity of PsTx and amiloride was confirmed by patch clamp experiments showing that CNGA1 channels in HEK 293 cells were not inhibited by 100 µM amiloride and that recombinant αßγ-ENaC were not inhibited by 100 nM PsTx. Importantly, 8Br-cGMP stimulated lung liquid absorption in situ, even in the presence of 50 µM amiloride. Furthermore, neither L: -cis-diltiazem nor PsTx affected the ß(2)-adrenoceptor agonist-stimulated lung liquid absorption, but, as expected, amiloride completely ablated it. Thus, transport through alveolar CNGA1 channels, located in type I cells, underlies the amiloride-insensitive component of lung liquid reabsorption. Furthermore, our in situ data highlight the potential of CNGA1 as a novel therapeutic target for the treatment of diseases characterised by lung liquid overload.

An exon 5-less splice variant of the extracellular calcium-sensing receptor rescues absence of the full-length receptor in the developing mouse lung.

The authors have recently demonstrated that, in the developing mouse lung, fetal plasma Ca(2+) suppresses branching morphogenesis and cell proliferation while promoting fluid secretion via activation of the extracellular Ca(2+)-sensing receptor (CaSR). The aim of the current study was to further elucidate the role of Ca(2+) in lung development by studying the effects of extracellular Ca(2+) on fetal lung development in mice lacking the CaSR. These mice were produced by exon 5 deletion in the CaSR gene. Since such a maneuver has been known to induce the expression of an exon 5-less splice variant of the CaSR in some tissues, the molecular and functional expression of this splice variant in the developing mouse lung was also investigated. Whereas there was a mild in vivo phenotype observed in these mice, in vitro sensitivity of Casr(-/-) lung explants to specific activators of the CaSR was unaffected. These results imply that compensatory expression of an exon 5-less splice variant rescues CaSR function in this mouse model and therefore a lung-specific, complete CaSR knockout model must be developed to fully appreciate the role for this receptor in lung development and the contribution of its ablation to postnatal respiratory disease.

Purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by live cell imaging.

Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of complex sensory airway receptors involved in the regulation of breathing. Together with their surrounding Clara-like cells, they exhibit stem cell potential through their capacity to regenerate depopulated areas of the epithelium following lung injury. We have employed confocal live cell imaging microscopy and novel electrophysiological techniques in a new ex vivo lung slice model to unravel potential purinergic signaling pathways within the NEB microenvironment. Quinacrine histochemistry indicated high amounts of vesicular ATP in NEB cells. Using a "reporter-patching" method adapted to create a uniquely sensitive and selective biosensor for the direct detection of ATP release from NEBs ex vivo, we demonstrated quantal ATP release from NEBs following their depolarization. Enhancing enzymatic extracellular ATP hydrolysis or inhibiting P2 receptors confirmed the central role of ATP in paracrine interactions between NEB cells and Clara-like cells. Combined calcium imaging, pharmacology, and immunohistochemistry showed that ligand-binding to functional P2Y(2) receptors underpins the activation of Clara-like cells. Hence, NEB cells communicate with their cellular neighbors in the NEB microenvironment by releasing ATP, which rapidly evokes purinergic activation of surrounding Clara-like cells. Besides ATP acting on the P2X(3) receptor expressing vagal sensory nerve terminals between NEB cells, local paracrine purinergic signaling within this potential stem cell niche may be important to both normal airway function, airway epithelial regeneration after injury, and/or the pathogenesis of small cell lung carcinomas.

Drosophila taste neurons as an agonist-screening platform for P2X receptors.

The P2X receptor family of ATP-gated cation channels are attractive drug targets for pain and inflammatory disease, but no subtype-selective agonists, and few partially selective agonists have been described to date. As proof-of-concept for the discovery of novel P2X receptor agonists, here we demonstrate the use of Drosophila taste neurons heterologously expressing rat P2X2 receptors as a screening platform. We demonstrate that wild-type rat P2X2 expressed in Drosophila is fully functional (ATP EC50 8.7 µM), and that screening of small (2 µl) volumes of a library of 80 adenosine nucleotide analogues is rapid and straightforward. We have determined agonist potency and specificity profiles for rat P2X2 receptors; triphosphate-bearing analogues display broad activity, tolerating a number of substitutions, and diphosphate and monophosphate analogues display very little activity. While several ATP analogues gave responses of similar magnitude to ATP, including the previously identified agonists ATPγS and ATPαS, we were also able to identify a novel agonist, the synthetic analogue 2-fluoro-ATP, and to confirm its agonist activity on rat P2X2 receptors expressed in human cells. These data validate our Drosophila platform as a useful tool for the analysis of agonist structure-activity relationships, and for the screening and discovery of novel P2X receptor agonists.

Aberrant Development Corrected in Adult-Onset Huntington's Disease iPSC-Derived Neuronal Cultures via WNT Signaling Modulation.

Aberrant neuronal development and the persistence of mitotic cellular populations have been implicated in a multitude of neurological disorders, including Huntington's disease (HD). However, the mechanism underlying this potential pathology remains unclear. We used a modified protocol to differentiate induced pluripotent stem cells (iPSCs) from HD patients and unaffected controls into neuronal cultures enriched for medium spiny neurons, the cell type most affected in HD. We performed single-cell and bulk transcriptomic and epigenomic analyses and demonstrated that a persistent cyclin D1+ neural stem cell (NSC) population is observed selectively in adult-onset HD iPSCs during differentiation. Treatment with a WNT inhibitor abrogates this NSC population while preserving neurons. Taken together, our findings identify a mechanism that may promote aberrant neurodevelopment and adult neurogenesis in adult-onset HD striatal neurons with the potential for therapeutic compensation.

Novel regulatory aspects of the extracellular Ca2+-sensing receptor, CaR.

The capacity to sense and adapt to changes in environmental cues is of paramount importance for every living organism. From yeast to man, cells must be able to match cellular activities to growth environment and nutrient availability. Key to this process is the development of membrane-bound systems that can detect modifications in the extracellular environment and to translate these into biological responses. Evidence gathered over the last 15 years has demonstrated that many of these cell surface "sensors" belong to the G protein-coupled receptor superfamily. Crucial to our understanding of nutrient sensing in mammalian species has been the identification of the extracellular Ca(2+)/cation-sensing receptor, CaR. CaR was the first ion-sensing molecule identified in man and genetic studies in humans have revealed the importance of the CaR in mineral ion metabolism. Latter, it has become apparent that the CaR also plays an important role outside the Ca(2+) homeostatic system, as an integrator of multiple environmental signals for the regulation of many vital cellular processes, from cell-to-cell communication to secretion and cell survival/cell death. Recently, novel aspects of receptor function reveal an unexpected role for the CaR in the regulation of growth and development in utero.

Huntington's Disease Patient-Derived Astrocytes Display Electrophysiological Impairments and Reduced Neuronal Support.

In Huntington's disease (HD), while the ubiquitously expressed mutant Huntingtin (mtHTT) protein primarily compromises striatal and cortical neurons, glia also undergo disease-contributing alterations. Existing HD models using human induced pluripotent stem cells (iPSCs) have not extensively characterized the role of mtHTT in patient-derived astrocytes. Here physiologically mature astrocytes are generated from HD patient iPSCs. These human astrocytes exhibit hallmark HD phenotypes that occur in mouse models, including impaired inward rectifying K+ currents, lengthened spontaneous Ca2+ waves and reduced cell membrane capacitance. HD astrocytes in co-culture provided reduced support for the maturation of iPSC-derived neurons. In addition, neurons exposed to chronic glutamate stimulation are not protected by HD astrocytes. This iPSC-based HD model demonstrates the critical effects of mtHTT on human astrocytes, which not only broadens the understanding of disease susceptibility beyond cortical and striatal neurons but also increases potential drug targets.

Functional live cell imaging of the pulmonary neuroepithelial body microenvironment.

Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of neuroendocrine cells invariably accompanied by Clara-like cells. Together with NEBs, Clara-like cells form the so-called "NEB microenvironment," which recently has been assigned a potential pulmonary stem cell niche. Conclusive data on the nature of physiological stimuli for NEBs are lacking. This study aimed at developing an ex vivo mouse lung vibratome slice model for confocal live cell imaging of physiological reactions in identified NEBs and surrounding epithelial cells. Immunohistochemistry of fixed slices demonstrated that NEBs are almost completely shielded from the airway lumen by tight junction-linked Clara-like cells. Besides the unambiguous identification of NEBs, the fluorescent dye 4-Di-2-ASP allowed microscopic identification of ciliated cells, Clara cells, and Clara-like cells in live lung slices. Using the mitochondrial uncoupler FCCP and a mitochondrial membrane potential indicator, JC-1, increases in 4-Di-2-ASP fluorescence in NEB cells and ciliated cells were shown to represent alterations in mitochondrial membrane potential. Changes in the intracellular free calcium concentration ([Ca2+](i)) in NEBs and surrounding airway epithelial cells were simultaneously monitored using the calcium indicator Fluo-4. Application (5 s) of 50 mM extracellular potassium ([K+](o)) evoked a fast and reproducible [Ca2+](i) increase in NEB cells, while Clara-like cells displayed a delayed (+/- 4 s) [Ca2+](i) increase, suggestive of an indirect, NEB-mediated activation. The presented approach opens interesting new perspectives for unraveling the functional significance of pulmonary NEBs in control lungs and disease models, and for the first time allows direct visualization of local interactions within the NEB microenvironment.