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Cellular senescence is emerging as a driver of idiopathic pulmonary fibrosis (IPF), a progressive and fatal disease with limited effective therapies. The senescence-associated secretory phenotype (SASP), involving the release of inflammatory cytokines and profibrotic growth factors by senescent cells, is thought to be a product of multiple cell types in IPF, including lung fibroblasts. NF-κB is a master regulator of the SASP, and its activity depends on the phosphorylation of p65/RelA. The purpose of this study was to assess the role of Pim-1 kinase as a driver of NF-κB-induced production of inflammatory cytokines from low-passage IPF fibroblast cultures displaying markers of senescence. Our results demonstrate that Pim-1 kinase phosphorylates p65/RelA, activating NF-κB activity and enhancing IL-6 production, which in turn amplifies the expression of PIM1, generating a positive feedback loop. In addition, targeting Pim-1 kinase with a small molecule inhibitor dramatically inhibited the expression of a broad array of cytokines and chemokines in IPF-derived fibroblasts. Furthermore, we provide evidence that Pim-1 overexpression in low-passage human lung fibroblasts is sufficient to drive premature senescence, in vitro. These findings highlight the therapeutic potential of targeting Pim-1 kinase to reprogram the secretome of senescent fibroblasts and halt IPF progression.

Key points Rat somatosensory neurons express a junctional protein, junctophilin-4 (JPH4) JPH4 is necessary for the formation of store operated Ca2+ entry (SOCE) complex at the junctions between plasma membrane and endoplasmic reticulum in these neurons. Knockdown of JPH4 impairs endoplasmic reticulum Ca2+ store refill and junctional Ca2+ signalling in sensory neurons. In vivo knockdown of JPH4 in the dorsal root ganglion (DRG) sensory neurons significantly attenuated experimentally induced inflammatory pain in rats. Junctional nanodomain Ca2+ signalling maintained by JPH4 is an important contributor to the inflammatory pain mechanisms. Abstract Junctions of endoplasmic reticulum and plasma membrane (ER-PM junctions) form signalling nanodomains in eukaryotic cells. ER-PM junctions are present in peripheral sensory neurons and are important for the fidelity of G protein coupled receptor (GPCR) signalling. Yet little is known about the assembly, maintenance and physiological role of these junctions in somatosensory transduction. Using fluorescence imaging, proximity ligation, super-resolution microscopy, in vitro and in vivo gene knockdown we demonstrate that a member of the junctophilin protein family, junctophilin-4 (JPH4), is necessary for the formation of store operated Ca2+ entry (SOCE) complex at the ER-PM junctions in rat somatosensory neurons. Thus we show that JPH4 localises to the ER-PM junctional areas and co-clusters with SOCE proteins STIM1 and Orai1 upon ER Ca2+ store depletion. Knockdown of JPH4 impairs SOCE and ER Ca2+ store refill in sensory neurons. Furthermore, we demonstrate a key role of the JPH4 and junctional nanodomain Ca2+ signalling in the pain-like response induced by the inflammatory mediator bradykinin. Indeed, an in vivo knockdown of JPH4 in the dorsal root ganglion (DRG) sensory neurons significantly shortened the duration of nocifensive behaviour induced by hindpaw injection of bradykinin in rats. Since the ER supplies Ca2+ for the excitatory action of multiple inflammatory mediators, we suggest that junctional nanodomain Ca2+ signalling maintained by JPH4 is an important contributor to the inflammatory pain mechanisms.

The plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) regulates the activity of diverse ion channels to include the epithelial Na(+) channel ENaC. Whether PIP(2) regulation of ENaC is due to a direct phospholipid-protein interaction, remains obscure. To date, possible interaction of PIP(2) with ENaC primarily has been tested indirectly through assays of channel function. A fragment-based biochemical analysis approach is used here to directly quantify possible PIP(2)-ENaC interactions. We find using the CIBN-CRY2 optogenetic dimerization system that the phosphoryl group positioned at carbon 5 of PIP(2) is necessary for interaction with ENaC. Previous studies have implicated conserved basic residues in the cytosolic portions of β- and γ-ENaC subunits as being important for PIP(2)-ENaC interactions. To test this, we used synthetic peptides of these regions of β- and γ-ENaC. Steady-state intrinsic fluorescence spectroscopy demonstrated that phosphoinositides change the local conformation of the N terminus of β-ENaC, and two sites of γ-ENaC adjacent to the plasma membrane, suggesting direct interactions of PIP(2) with these three regions. Microscale thermophoresis elaborated PIP(2) interactions with the N termini of β- (K(d) ∼5.2 μm) and γ-ENaC (K(d) ∼13 μm). A weaker interaction site within the carboxyl terminus of γ-ENaC (K(d) ∼800 μm) was also observed. These results support that PIP(2) regulates ENaC activity by directly interacting with at least three distinct regions within the cytoplasmic domains of the channel that contain conserved basic residues. These interactions are probably electrostatic in nature, and are likely to bear a key structural role in support of channel activity.

M-type (KCNQ2/3) K(+) channels play dominant roles in regulation of active and passive neuronal discharge properties such as resting membrane potential, spike-frequency adaptation, and hyper-excitatory states. However, plasticity of M-channel expression and function in nongenetic forms of epileptogenesis are still not well understood. Using transgenic mice with an EGFP reporter to detect expression maps of KCNQ2 mRNA, we assayed hyperexcitability-induced alterations in KCNQ2 transcription across subregions of the hippocampus. Pilocarpine and pentylenetetrazol chemoconvulsant models of seizure induction were used, and brain tissue examined 48 hr later. We observed increases in KCNQ2 mRNA in CA1 and CA3 pyramidal neurons after chemoconvulsant-induced hyperexcitability at 48 hr, but no significant change was observed in dentate gyrus (DG) granule cells. Using chromogenic in situ hybridization assays, changes to KCNQ3 transcription were not detected after hyper-excitation challenge, but the results for KCNQ2 paralleled those using the KCNQ2-mRNA reporter mice. In mice 7 days after pilocarpine challenge, levels of KCNQ2 mRNA were similar in all regions to those from control mice. In brain-slice electrophysiology recordings, CA1 pyramidal neurons demonstrated increased M-current amplitudes 48 hr after hyperexcitability; however, there were no significant changes to DG granule cell M-current amplitude. Traumatic brain injury induced significantly greater KCNQ2 expression in the hippocampal hemisphere that was ipsilateral to the trauma. In vivo, after a secondary challenge with subconvulsant dose of pentylenetetrazole, control mice were susceptible to tonic-clonic seizures, whereas mice administered the M-channel opener retigabine were protected from such seizures. This study demonstrates that increased excitatory activity promotes KCNQ2 upregulation in the hippocampus in a cell-type specific manner. Such novel ion channel expressional plasticity may serve as a compensatory mechanism after a hyperexcitable event, at least in the short term. The upregulation described could be potentially leveraged in anticonvulsant enhancement of KCNQ2 channels as therapeutic target for preventing onset of epileptogenic seizures.

We investigated the contribution of excitatory transient receptor potential canonical (TRPC) cation channels to posttraumatic hyperexcitability in the brain 7 days following controlled cortical impact model of traumatic brain injury (TBI) to the parietal cortex in male adult mice. We investigated if TRPC1/TRPC4/TRPC5 channel expression is upregulated in excitatory neurons after TBI in contribution to epileptogenic hyperexcitability in key hippocampal and cortical circuits that have substantial cholinergic innervation. This was tested by measuring TRPC1/TRPC4/TRPC5 protein and messenger RNA (mRNA) expression, assays of cholinergic function, neuronal Ca2+ imaging in brain slices, and seizure susceptibility after TBI. We found region-specific increases in expression of TRPC1, TRPC4, and TRPC5 subunits in the hippocampus and cortex following TBI. The dentate gyrus, CA3 region, and cortex all exhibited robust upregulation of TRPC4 mRNA and protein. TBI increased cFos activity in dentate gyrus granule cells (DGGCs) and layer 5 pyramidal neurons both at the time of TBI and 7 days post-TBI. DGGCs displayed greater magnitude and duration of acetylcholine-induced rises in intracellular Ca2+ in brain slices from mice subjected to TBI. The TBI mice also exhibited greater seizure susceptibility in response to pentylenetetrazol-induced kindling. Blockade of TRPC4/TRPC5 channels with M084 reduced neuronal hyperexcitation and impeded epileptogenic progression of kindling. We observed that the time-dependent upregulation of TRPC4/TRPC5-containing channels alters cholinergic responses and activity of principal neurons acting to increase proexcitatory sensitivity. The underlying mechanism includes acutely decreased acetylcholinesterase function, resulting in greater Gq/11-coupled muscarinic receptor activation of TRPC channels. Overall, our evidence suggests that TBI-induced plasticity of TRPC channels strongly contributes to overt hyperexcitability and primes the hippocampus and cortex for seizures.

Cellular senescence is a plausible mediator of inflammation-related tissue dysfunction. In the aged brain, senescent cell identities and the mechanisms by which they exert adverse influence are unclear. Here we used high-dimensional molecular profiling, coupled with mechanistic experiments, to study the properties of senescent cells in the aged mouse brain. We show that senescence and inflammatory expression profiles increase with age and are brain region- and sex-specific. p16-positive myeloid cells exhibiting senescent and disease-associated activation signatures, including upregulation of chemoattractant factors, accumulate in the aged mouse brain. Senescent brain myeloid cells promote peripheral immune cell chemotaxis in vitro. Activated resident and infiltrating immune cells increase in the aged brain and are partially restored to youthful levels through p16-positive senescent cell clearance in female p16-InkAttac mice, which is associated with preservation of cognitive function. Our study reveals dynamic remodeling of the brain immune cell landscape in aging and suggests senescent cell targeting as a strategy to counter inflammatory changes and cognitive decline.

Nearly three million people in the USA suffer traumatic brain injury (TBI) yearly; however, there are no pre- or post-TBI treatment options available. KCNQ2-5 voltage-gated K+ channels underlie the neuronal “M current”, which plays a dominant role in the regulation of neuronal excitability. Our strategy towards prevention of TBI-induced brain damage is predicated on the suggested hyper-excitability of neurons induced by TBIs, and the decrease in neuronal excitation upon pharmacological augmentation of M/KCNQ K+ currents. Seizures are very common after a TBI, making further seizures and development of epilepsy disease more likely. Our hypothesis is that TBI-induced hyperexcitability and ischemia/hypoxia lead to metabolic stress, cell death and a maladaptive inflammatory response that causes further downstream morbidity. Using the mouse controlled closed-cortical impact blunt TBI model, we found that systemic administration of the prototype M-channel “opener”, retigabine (RTG), 30 min after TBI, reduces the post-TBI cascade of events, including spontaneous seizures, enhanced susceptibility to chemo-convulsants, metabolic stress, inflammatory responses, blood–brain barrier breakdown, and cell death. This work suggests that acutely reducing neuronal excitability and energy demand via M-current enhancement may be a novel model of therapeutic intervention against post-TBI brain damage and dysfunction.

PURPOSE: Triple-negative breast cancers (TNBCs) represent a heterogeneous group of tumors. The lack of targeted therapies combined with the inherently aggressive nature of TNBCs results in a higher relapse rate and poorer overall survival. We evaluated the heterogeneity of TNBC cell lines for TRPC channel expression and sensitivity to cation-disrupting drugs. METHODS: The TRPC1/4/5 agonist englerin A was used to identify a group of TNBC cell lines sensitive to TRPC1/4/5 activation and intracellular cation disruption. Quantitative RT-PCR, the sulforhodamine B assay, pharmacological inhibition, and siRNA-mediated knockdown approaches were employed. Epifluorescence imaging was performed to measure intracellular Ca(2+) and Na(+) levels. Mitochondrial membrane potential changes were monitored by confocal imaging. RESULTS: BT-549 and Hs578T cells express high levels of TRPC4 and TRPC1/4, respectively, and are exquisitely, 2000- and 430-fold, more sensitive to englerin A than other TNBC cell lines. While englerin A caused a slow Na(+) and nominal Ca(2+) accumulation in Hs578T cells, it elicited rapid increases in cytosolic Ca(2+) levels that triggered mitochondrial depolarization in BT-549 cells. Interestingly, BT-549 and Hs578T cells were also more sensitive to digoxin as compared to other TNBC cell lines. Collectively, these data reveal TRPC1/4 channels as potential biomarkers of TNBC cell lines with dysfunctional mechanisms of cation homeostasis and therefore sensitivity to cardiac glycosides. CONCLUSIONS: The sensitivity of BT-549 and Hs578T cells to englerin A and digoxin suggests a subset of TNBCs are highly susceptible to cation disruption and encourages investigation of TRPC1 and TRPC4 as potential new biomarkers of sensitivity to cardiac glycosides.

Triple-negative breast cancers (TNBC) are aggressive and heterogeneous cancers that lack targeted therapies. We implemented a screening program to identify new leads for subgroups of TNBC using diverse cell lines with different molecular drivers. Through this program, we identified an extract from Calotropis gigantea that caused selective cytotoxicity in BT-549 cells as compared to four other TNBC cell lines. Bioassay-guided fractionation of the BT-549 selective extract yielded nine cardenolides responsible for the selective activity. These included eight known cardenolides and a new cardenolide glycoside. Structure activity relationships among the cardenolides demonstrated a correlation between their relative potencies toward BT-549 cells and Na(+)/K(+) ATPase inhibition. Calotropin, the compound with the highest degree of selectivity for BT-549 cells, increased intracellular Ca(2+) in sensitive cells to a greater extent than in the resistant MDA-MB-231 cells. Further studies identified a second TNBC cell line, Hs578T, that is also highly sensitive to the cardenolides and mechanistic studies were conducted to identify commonalities among the sensitive cell lines. Experiments showed that both cardenolide-sensitive cell lines expressed higher mRNA levels of the Na(+)/Ca(2+) exchanger NCX1 than resistant TNBC cells. This suggests that NCX1 could be a biomarker to identify TNBC patients that might benefit from the clinical administration of a cardiac glycoside for anticancer indications.

K v 7 ("M-type," KCNQ) K+ currents, play dominant roles in controlling neuronal excitability. They act as a "brake" against hyperexcitable states in the central and peripheral nervous systems. Pharmacological augmentation of M current has been developed for controlling epileptic seizures, although current pharmacological tools are uneven in practical usefulness. Lately, however, M-current "opener" compounds have been suggested to be efficacious in preventing brain damage after multiple types of insults/diseases, such as stroke, traumatic brain injury, drug addiction and mood disorders. In this review, we will discuss what is known to date on these efforts and identify gaps in our knowledge regarding the link between M current and therapeutic potential for these disorders. We will outline the preclinical experiments that are yet to be performed to demonstrate the likelihood of success of this approach in human trials. Finally, we also address multiple pharmacological tools available to manipulate different K v 7 subunits and the relevant evidence for translational application in the clinical use for disorders of the central nervous system and multiple types of brain insults. We feel there to be great potential for manipulation of K v 7 channels as a novel therapeutic mode of intervention in the clinic, and that the paucity of existing therapies obligates us to perform further research, so that patients can soon benefit from such therapeutic approaches.

ANO1 (TMEM16A) is a Ca(2+)-activated Cl(−) channel (CaCC) expressed in peripheral somatosensory neurons that are activated by painful (noxious) stimuli. These neurons also express the Ca(2+)-permeable channel and noxious heat sensor TRPV1, which can activate ANO1. Here, we revealed an intricate mechanism of TRPV1-ANO1 channel coupling in rat dorsal root ganglion (DRG) neurons. Simultaneous optical monitoring of CaCC activity and Ca(2+) dynamics revealed that the TRPV1 ligand capsaicin activated CaCCs. However, depletion of endoplasmic reticulum (ER) Ca(2+) stores reduced capsaicin-induced Ca(2+) increases and CaCC activation, suggesting that ER Ca(2+) release contributed to TRPV1-induced CaCC activation. ER store depletion by plasma membrane-localized TRPV1 channels was demonstrated with an ER-localized Ca(2+) sensor in neurons exposed to a cell-impermeable TRPV1 ligand. Proximity ligation assays established that ANO1, TRPV1 and the IP(3) receptor IP(3)R1 were often found in close proximity to each other. Stochastic optical reconstruction microscopy (STORM) confirmed the close association between all three channels in DRG neurons. Together, our data reveal the existence of ANO1-containing multi-channel nanodomains in DRG neurons and suggest that coupling between TRPV1 and ANO1 requires ER Ca(2+) release, which may be necessary to enhance ANO1 activation.

Objective Subunit-specific positive allosteric modulators (PAMs) of gamma-aminobutyric acid-A (GABA-A) receptors are commonly used to uncover the role of GABA-A receptor isoforms in brain function. Recently, we have designed novel PAMs selective for β2/3-subunit containing GABA-A receptors (β2/3-selective PAMs) that are nonbenzodiazepine site-mediated and do not show an α-subunit isoform selectivity, yet exhibit anxiolytic efficacy with reduced potential for sedation, cognitive impairment, and tolerance. In this study, we used three novel β2/3-selective PAMs (2-261, 2-262, and 10029) with differential β2/3-subunit potency to identify the role of β2/3-selective receptor isoforms in limbic epileptogenesis. Methods Experimental epileptogenesis was induced in mice by daily hippocampus stimulations until each mouse showed generalized (stage 5) seizures. Patch-clamp electrophysiology was used to record GABA-gated currents. Brain levels of β2/3-selective PAMs were determined for mechanistic correlations. Results Treatment with the β2/3-selective PAMs 2-261 (30 mg/kg), 2-262 (10 mg/kg), and 10029 (30 mg/kg), 30 min prior to stimulations, significantly suppressed the rate of development of kindled seizure activity without affecting the afterdischarge (AD) signal, indicating their disease-modifying activity. The β2/3-selective agents suppressed chemical epileptogenesis in the pentylenetetrazol model. Test doses of these agents were devoid of acute antiseizure activity in the kindling model. Conclusion These findings demonstrate that β2/3-selective PAMs can moderately retard experimental epileptogenesis, indicating the protective role of β2/3-subunit GABA-A receptor isoforms in the development of epilepsy.

KCNQ (Kv7, “M-type”) K(+) channels and TRPC (transient receptor potential, “canonical”) cation channels are coupled to neuronal discharge properties and are regulated via G(q/11)-protein-mediated signals. Stimulation of G(q/11)-coupled receptors both consumes phosphatidylinositol 4,5-bisphosphate (PIP(2)) via phosphalipase Cβ hydrolysis and stimulates PIP(2) synthesis via rises in Ca(2+)(i) and other signals. Using brain-slice electrophysiology and Ca(2+) imaging from male and female mice, we characterized threshold K(+) currents in dentate gyrus granule cells (DGGCs) and CA1 pyramidal cells, the effects of G(q/11)-coupled muscarinic M(1) acetylcholine (M(1)R) stimulation on M current and on neuronal discharge properties, and elucidated the intracellular signaling mechanisms involved. We observed disparate signaling cascades between DGGCs and CA1 neurons. DGGCs displayed M(1)R enhancement of M-current, rather than suppression, due to stimulation of PIP(2) synthesis, which was paralleled by increased PIP(2)-gated G-protein coupled inwardly rectifying K(+) currents as well. Deficiency of KCNQ2-containing M-channels ablated the M(1)R-induced enhancement of M-current in DGGCs. Simultaneously, M(1)R stimulation in DGGCs induced robust increases in [Ca(2+)](i), mostly due to TRPC currents, consistent with, and contributing to, neuronal depolarization and hyperexcitability. CA1 neurons did not display such multimodal signaling, but rather M current was suppressed by M(1)R stimulation in these cells, similar to the previously described actions of M(1)R stimulation on M-current in peripheral ganglia that mostly involves PIP(2) depletion. Therefore, these results point to a pleiotropic network of cholinergic signals that direct cell-type-specific, precise control of hippocampal function with strong implications for hyperexcitability and epilepsy. SIGNIFICANCE STATEMENT At the neuronal membrane, protein signaling cascades consisting of ion channels and metabotropic receptors govern the electrical properties and neurotransmission of neuronal networks. Muscarinic acetylcholine receptors are G-protein-coupled metabotropic receptors that control the excitability of neurons through regulating ion channels, intracellular Ca(2+) signals, and other second-messenger cascades. We have illuminated previously unknown actions of muscarinic stimulation on the excitability of hippocampal principal neurons that include M channels, TRPC (transient receptor potential, “canonical”) cation channels, and powerful regulation of lipid metabolism. Our results show that these signaling pathways, and mechanisms of excitability, are starkly distinct between peripheral ganglia and brain, and even between different principal neurons in the hippocampus.

The fidelity of neuronal signaling requires organization of signaling molecules into macromolecular complexes, whose components are in intimate proximity. The intrinsic diffraction limit of light makes visualization of individual signaling complexes using visible light extremely difficult. However, using super-resolution stochastic optical reconstruction microscopy (STORM), we observed intimate association of individual molecules within signaling complexes containing ion channels (M-type K+, L-type Ca2+, or TRPV1 channels) and G protein-coupled receptors coupled by the scaffolding protein, A-kinase-anchoring protein (AKAP)79/150. Some channels assembled as multi-channel super-complexes. Surprisingly, we identified novel layers of interplay within macromolecular complexes containing diverse channel types at the single-complex level in sensory neurons, dependent on AKAP79/150. Electrophysiological studies revealed that such ion channels are functionally coupled as well. Our findings illustrate the novel role of AKAP79/150 as a molecular coupler of different channels which conveys cross-talk between channel activities within single microdomains in tuning the physiological response of neurons.

Synaptic GABAA receptors are primary mediators of rapid inhibition in the brain and play a key role in the pathophysiology of epilepsy and other neurologic disorders. The δ-subunit GABAA receptors are expressed extrasynaptically in the dentate gyrus and contribute to tonic inhibition, promoting network shunting as well as reducing seizure susceptibility. However, the neurosteroid structure-function relationship at δGABA(A) receptors within the native hippocampus neurons remains unclear. Here we report a structure-activity relationship for neurosteroid modulation of extrasynaptic GABAA receptor-mediated tonic inhibition in the murine dentate gyrus granule cells. We recorded neurosteroid allosteric potentiation of GABA as well as direct activation of tonic currents using a wide array of natural and synthetic neurosteroids. Our results shows that, for all neurosteroids, the C3α-OH group remains obligatory for extrasynaptic receptor functional activity, as C3β-OH epimers were inactive in activating tonic currents. Allopregnanolone and related pregnane analogs exhibited the highest potency and maximal efficacy in promoting tonic currents. Alterations at the C17 or C20 region of the neurosteroid molecule drastically altered the transduction kinetics of tonic current activation. The androstane analogs had the weakest modulatory response among the analogs tested. Neurosteroid potentiation of tonic currents was completely (approximately 95%) diminished in granule cells from δ-knockout mice, suggesting that δ-subunit receptors are essential for neurosteroid activity. The neurosteroid sensitivity of δGABA(A) receptors was confirmed at the systems level using a 6-Hz seizure test. A consensus neurosteroid pharmacophore model at extrasynaptic δGABA(A) receptors is proposed based on a structure-activity relationship for activation of tonic current and seizure protection.

Triple negative breast cancers (TNBCs) represent 15-20% of all breast cancers and are defined by lack of the estrogen and progesterone receptors and HER2 gene amplification. Therefore, TNBC patients do not benefit from available therapies targeting these receptors. The significant heterogeneity of TNBC precludes the expectation that a single molecular target will be identified for this disease. Our goal is to identify compounds with selective cytotoxic effects in subsets of TNBC cells to identify new therapeutic targets for TNBC subgroups. Our studies show that the TNBC cell lines BT-549 and Hs578T are distinct from other TNBC cells in that they are highly sensitive to 9 cardiac glycosides/cardenolides isolated from Calotropis gigantea as well as digoxin. BT-549 cells are 15-times more sensitive to the isolated cardiac glycoside calotropin and 9-times more sensitive to digoxin as compared to MDA-MB-231 TNBC cells. Cardiac glycosides bind to the Na+/K+ ATPase and inhibit its activity, leading to increased intracellular Na+, which results in higher intracellular Ca2+ through reversal of the Na+/Ca2+ exchanger (NCX). Notably, among the 10 cardiac glycosides/cardenolides, there was a significant correlation between potency for inhibition of purified Na+/K+ ATPase and cytotoxic potency and selectivity for BT-549 cells. These data suggest that inhibition of Na+/K+ ATPase is critical for the selective cytotoxic effects of these compounds. BT-549 and Hs578T cells express high levels of non-specific TRPC1/4 cation channels1 and low levels of SERCA2 Ca2+ pumps2 compared to other TNBC cells, suggesting that they have impaired ability to handle intracellular Ca2+. Consistent with this hypothesis, within 3 h, concentrations of calotropin and digoxin that are selectivity cytotoxic each initiated a significant increase in intracellular Ca2+ in BT-549 cells. To test whether sensitivity to cardiac glycosides was due to higher Na+/K+ ATPase expression, message and protein levels were evaluated in 10 TNBC cell lines. The results show that there is no correlation between levels of Na+/K+ ATPase and sensitivity to cardiac glycosides. In contrast, NCX-1 expression was found to be 120 and 60-fold higher in the sensitive BT-549 and Hs578T cells, respectively, as compared to the resistant TNBC cell lines. Ongoing studies are evaluating whether genetic manipulation of NCX-1 is sufficient to modulate sensitivity to cardiac glycosides. These findings demonstrate that NCX-1 expression is associated with the selective sensitivity of a subgroup of TNBC cell lines to the cytotoxic effects of cardiac glycosides. Our results suggest that NCX-1 could be a biomarker to identify TNBC patients that could ultimately benefit from use of a cardiac glycoside for anticancer indications. 1. Grant CV, et al. Breast Cancer Res Treat. 2019;177(2):345. 2. Varga K, et al. BMC Cancer. 2018;18(1):1029. Citation Format: Petra E. Pederson, Shengxin Cai, Chase M. Carver, Tanja Grkovic, Barry R. O'Keefe, April L. Risinger, Robert H. Cichewicz, Susan L. Mooberry. High expression of NCX-1 in triple negative breast cancer cell lines identifies a potential biomarker for sensitivity to cardiac glycosides [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1786.

OBJECTIVE Sex differences are evident in the antiseizure activity of neurosteroids; however, the potential mechanisms remain unclear. In this study, we sought to determine whether differences in target extrasynaptic δ-subunit γ-aminobutyric acid type A (GABA-A) receptor expression and function underlie the sex differences in seizure susceptibility and the antiseizure activity of neurosteroids. METHODS Sex differences in seizure susceptibility and protective activity of three distinct neurosteroids-allopregnanolone (AP), androstanediol (AD), and ganaxolone-were evaluated in the pilocarpine model of status epilepticus (SE) and kindling seizure test in mice. Immunocytochemistry was used for δGABA-A receptor expression analysis, and patch-clamp recordings in brain slices evaluated its functional currents. RESULTS Sex differences were apparent in kindling epileptogenic seizures, with males exhibiting a faster progression to a fully kindled state. Neurosteroids AP, AD, or ganaxolone produced dose-dependent protection against SE and acute partial seizures. However, female mice exhibited strikingly enhanced sensitivity to the antiseizure activity of neurosteroids compared to males. Sex differences in neurosteroid protection were unrelated to pharmacokinetic factors, as plasma levels of neurosteroids associated with seizure protection were similar between sexes. Mice lacking extrasynaptic δGABA-A receptors did not exhibit sex differences in neurosteroid protection. Consistent with a greater abundance of extrasynaptic δGABA-A receptors, AP produced a significantly greater potentiation of tonic currents in dentate gyrus granule cells in females than males; however, such enhanced AP sensitivity was diminished in δGABA-A receptor knockout female mice. SIGNIFICANCE Neurosteroids exhibit greater antiseizure potency in females than males, likely due to a greater abundance of extrasynaptic δGABA-A receptors that mediate neurosteroid-sensitive tonic currents and seizure protection. These findings indicate the potential to develop personalized gender-specific neurosteroid treatments for SE and epilepsy in men and women, including catamenial epilepsy.

A major challenge is to understand maladaptive changes in ion channels that sets neurons on a course towards epilepsy development. Voltage- and calcium-activated K+ (BK) channels contribute to early spike timing in neurons, and studies indicate that the BK channel plays a pathological role in increasing excitability early after a seizure. Here, we have investigated changes in BK channels and their accessory β4 subunit (KCNMB4) in dentate gyrus (DG) granule neurons of the hippocampus, key neurons that regulate excitability of the hippocampus circuit. Two days after pilocarpine-induced seizures, we found that the predominant effect is a downregulation of the β4 accessory subunit mRNA. Consistent with reduced expression, single channel recording and pharmacology indicate a switch in the subtype of channels expressed; from iberiotoxin-resistant, type II BK channels (BK α/β4) that have higher channel open probability and slow gating, to iberiotoxin-sensitive type I channels (BK α alone) with low open probability and faster gating. The switch to a majority of type I channel expression following seizure activity is correlated with a loss of BK channel function on spike threshold while maintaining the channel's contribution to increased early spike frequency. Using heterozygous β4 knockout mice, we find reduced expression is sufficient to increase seizure sensitivity. We conclude that seizure-induced downregulation of KCNMB4 is an activity dependent mechanism that increases the excitability of DG neurons. These novel findings indicate that BK channel subtypes are not only defined by cell-specific expression, but can also be plastic depending on the recent history of neuronal excitability.

Neurosteroids are endogenous regulators of neuronal excitability and seizure susceptibility. Neurosteroids, such as allopregnanolone (AP; 3α-hydroxy-5α-pregnan-20-one), exhibit enhanced anticonvulsant activity in perimenstrual catamenial epilepsy, a neuroendocrine condition in which seizures are clustered around the menstrual period associated with neurosteroid withdrawal (NSW). However, the molecular mechanisms underlying such enhanced neurosteroid sensitivity remain unclear. Neurosteroids are allosteric modulators of both synaptic (αβγ2-containing) and extrasynaptic (αβδ-containing) GABAAreceptors, but they display greater sensitivity toward δ-subunit receptors in dentate gyrus granule cells (DGGCs). Here we report a novel plasticity of extrasynaptic δ-containing GABAAreceptors in the dentate gyrus in a mouse perimenstrual-like model of NSW. In molecular and immunofluorescence studies, a significant increase occurred in δ subunits, but not α1, α2, β2, and γ2subunits, in the dentate gyrus of NSW mice. Electrophysiological studies confirmed enhanced sensitivity to AP potentiation of GABA-gated currents in DGGCs, but not in CA1 pyramidal cells, in NSW animals. AP produced a greater potentiation of tonic currents in DGGCs of NSW animals, and such enhanced AP sensitivity was not evident in δ-subunit knock-out mice subjected to a similar withdrawal paradigm. In behavioral studies, mice undergoing NSW exhibited enhanced seizure susceptibility to hippocampus kindling. AP has enhanced anticonvulsant effects in fully kindled wild-type mice, but not δ-subunit knock-out mice, undergoing NSW-induced seizures, confirming δ-linked neurosteroid sensitivity. These results indicate that perimenstrual NSW is associated with striking upregulation of extrasynaptic, δ-containing GABAAreceptors that mediate tonic inhibition and neurosteroid sensitivity in the dentate gyrus. These findings may represent a molecular rationale for neurosteroid therapy of catamenial epilepsy.

Identification of new molecular liabilities of a subset of triple-negative breast cancers through the investigation of englerin A There remains a need to identify targeted therapies for triple-negative breast cancers (TNBCs) but a major challenge has been the heterogeneity of these cancers. We initiated a screen to discover compounds that are selectively cytotoxic to cells representing distinct molecular subtypes of TNBC. The overall goal is to identify compounds with selective actions and new molecular liabilities for subtypes of TNBC. Englerin A was identified by cytotoxicity assay as a compound with a greater than 1,500-fold selectivity for BT-549 cells and 300-fold for Hs578T TNBC cells as compared to other TNBC cell lines. Interestingly, the treatment of Hs578T cells with englerin A produced a biphasic concentration response curve, which is unique among the TNBC cell lines but is seen in A-498 renal carcinoma cells where englerin A sensitivity was first noted.1 In renal cell carcinoma cell lines one strongly supported mechanism of action of englerin A is via activation of TRPC1/4/5 non-selective cation channels.2-4 In our assays, the TRPC1/4/5 antagonist Pico1455 decreases the potency of englerin A in BT-549 and Hs578T cells, but not more resistant TNBC cells, suggesting that the TRPC1/4/5 agonist activity of englerin A is likely responsible for the selective effects in the sensitive cell lines. BT-549 and Hs578T cells were found to have significantly higher expression of TRCP1 and TRPC4 subunit mRNA as compared to englerin A resistant cells. Furthermore, knockdown of TRPC4 expression in BT-549 cells decreased englerin A potency. Consistent with its function as an agonist of the TRPC1/4/5 cation channel, we found that englerin A caused a concentration-dependent increase in intracellular Ca2+ in BT-549 cells within 30 seconds of exposure, and this effect was inhibited by pre-treatment with Pico145. These studies led to the hypothesis that TNBC cells expressing high levels of TRPC4 might also be more sensitive to clinically approved classes of drugs that increase intracellular cation concentrations. Results show that BT-549 and Hs578T cells are more sensitive to digoxin than other TNBC cells. Overall, these studies suggest that a subgroup of TNBCs may be susceptible to treatments with selective targets of cation influx. Ongoing work is aimed at evaluating the sensitivity of these cell lines to other drugs that disrupt intracellular cation levels. 1. Ratnayake, R. et al. (2009). 2. Ludlow, M. J. et al. (2016). 3. Akbulut, Y. et al. (2015). 4. Carson, C. et al. (2015). 5. Rubaiy, H. N. et al. (2017). Citation Format: Corena V. Grant, Chase M. Carver, Shayne D. Hastings, April L. Risinger, John A. Beutler, Susan L. Mooberry. Identification of new molecular liabilities of a subset of triple-negative breast cancers through the investigation of englerin A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2143.

Zinc (Zn(2+)) is an essential cofactor in mammalian cells and neurons. Zn(2+) is released from synaptic vesicles of certain nerve terminals in the hippocampus during neuronal activity. Zn(2+) has been shown to inhibit synaptic GABAA receptors and alter the hippocampal network excitability. However, the ability of Zn(2+) to block extrasynaptic receptors remains unclear. Endogenous neurosteroids, such as allopregnanolone (AP), regulate neuronal excitability by allosteric activation of synaptic and extrasynaptic GABAA receptors. Neurosteroids activate extrasynaptic δGABAA receptor-mediated tonic inhibition in dentate gyrus granule cells (DGGCs), thereby contributing to the regulation of downstream circuit excitability. Here we report a novel inhibitory role of Zn(2+) at neurosteroid-sensitive, extrasynaptic δGABAA receptors by electrophysiological recordings in DGGCs from adult mice. Zn(2+) displayed a concentration-dependent, reversible noncompetitive blockade of AP-sensitive tonic current in DGGCs (IC50, 16 μm). Tonic current was fully blocked by Zn(2+), akin to the GABAA receptor antagonist gabazine. Zn(2+) inhibition of tonic current was lacking in DGGCs from δ-subunit knock-out mice. Moreover, AP-activated synaptic receptor-mediated phasic currents were not affected by Zn(2+) Finally, intrahippocampal infusion of Zn(2+) elicited rapid epileptiform activity and significantly blocked the antiseizure activity of AP in the kindling model of epilepsy. Thus, Zn(2+) inhibition of neurosteroid-sensitive, extrasynaptic GABAA receptors in the hippocampus has direct implications in many brain hyperexcitability conditions, such as seizures, epileptogenesis, and epilepsy. Zn(2+) interactions may aid to further understand the physiology of extrasynaptic GABAA receptors.Zn(2+) is most abundant in the synaptic vesicles of hippocampal mossy fibers. Zn(2+) release occurs with neuronal excitation, including seizure events, and exerts powerful excitability effects in the hippocampus circuits. Zn(2+) inhibits synaptic GABAA receptors, but its interaction is less well appreciated at the extrasynaptic receptors, which respond sensitively to endogenous neurosteroids. Here, we describe selective functional blockade by Zn(2+) of neurosteroid-sensitive, extrasynaptic GABAA receptors in the mouse hippocampus dentate gyrus, a key region associated with epilepsy and memory disorders. By demonstrating that extracellular Zn(2+) prevents neurosteroid augmentation of tonic current and protection against limbic seizures, our findings provide novel implications of this potential antagonistic interaction in a variety of neurological conditions.

TMEM16A (ANO1) is a Ca2+ activated Cl- channel (CaCC) expressed in peripheral somatosensory neurons responding to painful (noxious) stimuli; these neurons also express the noxious heat sensor TRPV1. Our previous findings indicate that in these neurons, TMEM16A is specifically coupled to Ca2+ release from intracellular stores due to close association of TMEM16A channels, G protein coupled receptors, and IP3 receptors in multiprotein complexes assembled at plasma membrane-endoplasmic reticulum (PM-ER) junctions. Like TRPV1, TMEM16A is activated by noxious heat and functional coupling between the two channels has also been shown. We used live confocal imaging of Cl- channel activity, proximity ligation and super-resolution microscopy to investigate physical and functional relationships between TMEM16A and TRPV1 channels, and IP3 receptors in rat dorsal root ganglion (DRG) neurons. (i) Simultaneous monitoring of Cl- channel activity and Ca2+ dynamics in DRG neurons transfected with a halide-sensitive EYFP mutant (H148Q/I152L) and loaded with fura-2 revealed that the TRPV1 ligand, capsaicin, can robustly activate CaCC. However, ER store depletion significantly reduced (but not abolished) the capsaicin-induced Ca2+ rises and CaCC activation, suggesting that Ca2+ release from the ER contributes significantly to TRPV1-mediated CaCC activation. (ii) Proximity ligation assays established that in DRG neurons, TMEM16A and TRPV1 is often in close (less than 40 nm) proximity; similarly, close proximity between TMEM16A and IP3R1 was also observed. (iii) Stochastic optical reconstruction (STORM) microscopy confirmed close association of TMEM16A and TRPV1 channels in DRG neurons, representing 21% of the total population of labeled channels. We also observed close proximity of TMEM16A and IP3R1, and between TRPV1 and IP3R1, within 100 nm, although these molecules could be somewhat further apart, compared to membrane-localized TMEM16A/TRPV1 complexes, consistent with the ER membrane localization of IP3R1. Taken together, our data reveal the composition of TMEM16A-containing signaling complexes in DRG neurons and suggest that coupling between TRPV1 and TMEM16A requires ER Ca2+ release, which may be necessary to boost activation of TMEM16A, as this channel has a relatively low Ca2+ affinity.

The ovarian cycle affects susceptibility to behavioral and neurologic conditions. The molecular mechanisms underlying these changes are poorly understood. Deficits in cyclical fluctuations in steroid hormones and receptor plasticity play a central role in physiologic and pathophysiologic menstrual conditions. It has been suggested that synaptic GABA(A) receptors mediate phasic inhibition in the hippocampus and extrasynaptic receptors mediate tonic inhibition in the dentate gyrus. Here we report a novel role of extrasynaptic δ-containing GABA(A) receptors as crucial mediators of the estrous cycle-related changes in neuronal excitability in mice, with hippocampus subfield specificity. In molecular and immunofluorescence studies, a significant increase occurred in δ-subunit, but not α4- and γ2-subunits, in the dentate gyrus during diestrus. However, δ-subunit upregulation was not evident in the CA1 region. The δ-subunit expression was undiminished by age and ovariectomy and in mice lacking progesterone receptors, but it was significantly reduced by finasteride, a neurosteroid synthesis inhibitor. Electrophysiologic studies confirmed greater potentiation of GABA currents by progesterone-derived neurosteroid allopregnanolone in dissociated dentate gyrus granule cells in diestrus than in CA1 pyramidal cells. The baseline conductance and allopregnanolone potentiation of tonic currents in dentate granule cells from hippocampal slices were higher than in CA1 pyramidal cells. In behavioral studies, susceptibility to hippocampus kindling epileptogenesis was lower in mice during diestrus. These results demonstrate the estrous cycle-related plasticity of neurosteroid-sensitive, δ-containing GABA(A) receptors that mediate tonic inhibition and seizure susceptibility. These findings may provide novel insight on molecular cascades of menstrual disorders like catamenial epilepsy, premenstrual syndrome, and migraine.

Neurosteroids are steroids synthesized within the brain with rapid effects on neuronal excitability. Allopregnanolone, allotetrahydrodeoxycorticosterone, and androstanediol are three widely explored prototype endogenous neurosteroids. They have very different targets and functions compared to conventional steroid hormones. Neuronal γ-aminobutyric acid (GABA) type A (GABA(A)) receptors are one of the prime molecular targets of neurosteroids.This review provides a critical appraisal of recent advances in the pharmacology of endogenous neurosteroids that interact with GABA(A) receptors in the brain. Neurosteroids possess distinct, characteristic effects on the membrane potential and current conductance of the neuron, mainly via potentiation of GABA(A) receptors at low concentrations and direct activation of receptor chloride channel at higher concentrations. The GABA(A) receptor mediates two types of inhibition, now characterized as synaptic (phasic) and extrasynaptic (tonic) inhibition. Synaptic release of GABA results in the activation of low-affinity γ2-containing synaptic receptors, while high-affinity δ-containing extrasynaptic receptors are persistently activated by the ambient GABA present in the extracellular fluid. Neurosteroids are potent positive allosteric modulators of synaptic and extrasynaptic GABA(A) receptors and therefore enhance both phasic and tonic inhibition. Tonic inhibition is specifically more sensitive to neurosteroids. The resulting tonic conductance generates a form of shunting inhibition that controls neuronal network excitability, seizure susceptibility, and behavior.The growing understanding of the mechanisms of neurosteroid regulation of the structure and function of the synaptic and extrasynaptic GABA(A) receptors provides many opportunities to create improved therapies for sleep, anxiety, stress, epilepsy, and other neuropsychiatric conditions.