Chemogenetic activation of mammalian brain neurons expressing insect Ionotropic Receptors by systemic ligand precursor administration.

Chemogenetic approaches employing ligand-gated ion channels are advantageous regarding manipulation of target neuronal population functions independently of endogenous second messenger pathways. Among them, Ionotropic Receptor (IR)-mediated neuronal activation (IRNA) allows stimulation of mammalian neurons that heterologously express members of the insect chemosensory IR repertoire in response to their cognate ligands. In the original protocol, phenylacetic acid, a ligand of the IR84a/IR8a complex, was locally injected into a brain region due to its low permeability of the blood-brain barrier. To circumvent this invasive injection, we sought to develop a strategy of peripheral administration with a precursor of phenylacetic acid, phenylacetic acid methyl ester, which is efficiently transferred into the brain and converted to the mature ligand by endogenous esterase activities. This strategy was validated by electrophysiological, biochemical, brain-imaging, and behavioral analyses, demonstrating high utility of systemic IRNA technology in the remote activation of target neurons in the brain.

The Neurotranscriptome of Monochamus alternatus.

The Japanese pine sawyer Monochamus alternatus serves as the primary vector for pine wilt disease, a devastating pine disease that poses a significant threat to the sustainable development of forestry in the Eurasian region. Currently, trap devices based on informational compounds have played a crucial role in monitoring and controlling the M. alternatus population. However, the specific proteins within M. alternatus involved in recognizing the aforementioned informational compounds remain largely unclear. To elucidate the spatiotemporal distribution of M. alternatus chemosensory-related genes, this study conducted neural transcriptome analyses to investigate gene expression patterns in different body parts during the feeding and mating stages of both male and female beetles. The results revealed that 15 genes in the gustatory receptor (GR) gene family exhibited high expression in the mouthparts, most genes in the odorant binding protein (OBP) gene family exhibited high expression across all body parts, 22 genes in the odorant receptor (OR) gene family exhibited high expression in the antennae, a significant number of genes in the chemosensory protein (CSP) and sensory neuron membrane protein (SNMP) gene families exhibited high expression in both the mouthparts and antennae, and 30 genes in the ionotropic receptors (IR) gene family were expressed in the antennae. Through co-expression analyses, it was observed that 34 genes in the IR gene family were co-expressed across the four developmental stages. The Antenna IR subfamily and IR8a/Ir25a subfamily exhibited relatively high expression levels in the antennae, while the Kainate subfamily, NMDA subfamily, and Divergent subfamily exhibited predominantly high expression in the facial region. MalIR33 is expressed only during the feeding stage of M. alternatus, the MalIR37 gene exhibits specific expression in male beetles, the MalIR34 gene exhibits specific expression during the feeding stage in male beetles, the MalIR8 and MalIR39 genes exhibit specific expression during the feeding stage in female beetles, and MalIR8 is expressed only during two developmental stages in male beetles and during the mating stage in female beetles. The IR gene family exhibits gene-specific expression in different spatiotemporal contexts, laying the foundation for the subsequent selection of functional genes and facilitating the full utilization of host plant volatiles and insect sex pheromones, thereby enabling the development of more efficient attractants.

Versatile Endogenous Editing of GluRIIA in Drosophila melanogaster.

Glutamate receptors at the postsynaptic side translate neurotransmitter release from presynapses into postsynaptic excitation. They play a role in many forms of synaptic plasticity, e.g., homeostatic scaling of the receptor field, activity-dependent synaptic plasticity and the induction of presynaptic homeostatic potentiation (PHP). The latter process has been extensively studied at Drosophila melanogaster neuromuscular junctions (NMJs). The genetic removal of the glutamate receptor subunit IIA (GluRIIA) leads to an induction of PHP at the synapse. So far, mostly imprecise knockouts of the GluRIIA gene have been utilized. Furthermore, mutated and tagged versions of GluRIIA have been examined in the past, but most of these constructs were not expressed under endogenous regulatory control or involved the mentioned imprecise GluRIIA knockouts. We performed CRISPR/Cas9-assisted gene editing at the endogenous locus of GluRIIA. This enabled the investigation of the endogenous expression pattern of GluRIIA using tagged constructs with an EGFP and an ALFA tag for super-resolution immunofluorescence imaging, including structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). All GluRIIA constructs exhibited full functionality and PHP could be induced by philanthotoxin at control levels. By applying hierarchical clustering algorithms to analyze the dSTORM data, we detected postsynaptic receptor cluster areas of ~0.15 µm2. Consequently, our constructs are suitable for ultrastructural analyses of GluRIIA.

The anterior retrosplenial cortex is required for short-term object in place recognition memory retrieval: Role of ionotropic glutamate receptors in male and female Long-Evans rats.

The anterior retrosplenial cortex (aRSC) integrates multimodal sensory information into cohesive associative recognition memories. Little is known about how information is integrated during different learning phases (i.e., encoding and retrieval). Additionally, sex differences are observed in performance of some visuospatial memory tasks; however, inconsistent findings warrant more research. We conducted three experiments using the 1-h delay object-in-place (1-h OiP) test to assess recognition memory retrieval in male and female Long-Evans rats. (i) We found both sexes performed equally in three repeated 1-h OiP test sessions. (ii) We showed infusions of a mixture of muscimol/baclofen (GABAA/B receptor agonists) into the aRSC ~15-min prior to the test phase disrupted 1-h OiP in both sexes. (iii) We assessed the role of aRSC ionotropic glutamate receptors in 1-h OiP retrieval using another squad of cannulated rats and confirmed that infusions of either the competitive AMPA/Kainate receptor antagonist CNQX (3 mM) or competitive NMDA receptor antagonist AP-5 (30 mM) (volumes = 0.50 uL/side) significantly impaired 1-h OiP retrieval in both sexes compared to controls. Taken together, findings challenge reported sex differences and clearly establish a role for aRSC ionotropic glutamate receptors in short-term visuospatial recognition memory retrieval. Thus, modulating neural activity in the aRSC may alleviate some memory processing impairments in related disorders.

Loss of activation by GABA in vertebrate delta ionotropic glutamate receptors.

Ionotropic glutamate receptors (iGluRs) mediate excitatory signals between cells by binding neurotransmitters and conducting cations across the cell membrane. In the mammalian brain, most of these signals are mediated by two types of iGluRs: AMPA and NMDA (i.e. iGluRs sensitive to 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid and N-methyl-D-aspartic acid, respectively). Delta-type iGluRs of mammals also form neurotransmitter-binding channels in the cell membrane, but in contrast, their channel is not activated by neurotransmitter binding, raising biophysical questions about iGluR activation and biological questions about the role of delta iGluRs. We therefore investigated the divergence of delta iGluRs from their iGluR cousins using molecular phylogenetics, electrophysiology, and site-directed mutagenesis. We find that delta iGluRs are found in numerous bilaterian animals (e.g., worms, starfish, and vertebrates) and are closely related to AMPA receptors, both genetically and functionally. Surprisingly, we observe that many iGluRs of the delta family are activated by the classical inhibitory neurotransmitter, γ-aminobutyric acid (GABA). Finally, we identify nine amino acid substitutions that likely gave rise to the inactivity of today's mammalian delta iGluRs, and these mutations abolish activity when engineered into active invertebrate delta iGluRs, partly by inducing receptor desensitization. These results offer biophysical insight into iGluR activity and point to a role for GABA in excitatory signaling in invertebrates.

[Effect of Suanzaoren Decoction on expression of ionotropic glutamate receptors and synaptic plasticity in hippocampus of anxiety rats].

This study investigated the effect of Suanzaoren Decoction on the expression of N-methyl-D-aspartate receptors(NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors(AMPAR) in the hippocampus and synaptic plasticity in rats with conditioned fear-induced anxiety. The effect of Suanzaoren Decoction on rat behaviors were evaluated through open field experiment, elevated plus maze experiment, and light/dark box experiment. Enzyme-linked immunosorbent assay(ELISA) was used to measure the levels of glutamate(Glu) and γ-aminobutyric acid(GABA) in the rat hippocampus. Real-time fluorescence quantitative PCR(qRT-PCR) and Western blot were employed to assess the gene and protein expression of ionotropic glutamate receptors in the hippocampal region. Transmission electron microscopy was utilized to observe the changes in the ultrastructure of synaptic neurons in the hippocampal region. Long-term potentiation(LTP) detection technique was employed to record the changes in population spike(PS) amplitude in the hippocampal region of mice in each group. The behavioral results showed that compared with the model group, the Suanzaoren Decoction group effectively increased the number of entries into open arms, time spent in open arms, percentage of time spent in open arms out of total movement time, number of entries into open arms out of total entries into both arms(P<0.01), and significantly increased the time spent in the light box and the number of shuttle crossings(P<0.01). There was an increasing trend in the number of grid crossings, entries into the center grid, and time spent in the center grid, indicating a significant anxiolytic effect. ELISA results showed that compared with the model group, the Suanzaoren Decoction group exhibited significantly reduced levels of Glu, Glu/GABA ratio(P<0.01), and significantly increased levels of GABA(P<0.01) in the rat hippocampus. Furthermore, Suanzaoren Decoction significantly decreased the gene and protein expression of NMDAR(GluN2B and GluN2A) and AMPAR(GluA1 and GluA2) compared with the model group. Transmission electron microscopy results demonstrated improvements in synapses, neuronal cells, and organelles in the hippocampal region of the Suanzaoren Decoction group compared with the model group. LTP detection results showed a significant increase in the PS amplitude changes in the hippocampal region of Suanzaoren Decoction group from 5 to 35 min compared with the model group(P<0.05, P<0.01). In conclusion, Suanzaoren Decoction exhibits significant anxiolytic effects, which may be attributed to the reduction in NMDAR and AMPAR expression levels and the improvement of synaptic plasticity.

Sulfate-modified nanosized polystyrene impairs memory by inhibiting ionotropic glutamate receptors and the cAMP-response element binding protein (CREB) pathway in Caenorhabditis elegans.

Nanoplastic contamination is an emerging environmental concern worldwide. In particular, sulfate anionic surfactants often appear along with nanosized plastic particles in personal care products, suggesting that sulfate-modified nanosized polystyrene (S-NP) may occur, remain, and spread into the environment. However, whether S-NP adversely affects learning and memory is unknown. In this study, we used a positive butanone training protocol to evaluate the effects of S-NP exposure on short-term associative memory (STAM) and long-term associative memory (LTAM) in Caenorhabditis elegans. We observed that long-term S-NP exposure impairs both STAM and LTAM in C. elegans. We also observed that mutations in the glr-1, nmr-1, acy-1, unc-43, and crh-1 genes eliminated the STAM and LTAM impairment induced by S-NP, and the mRNA levels of these genes were also decreased upon S-NP exposure. These genes encode ionotropic glutamate receptors (iGluRs), cyclic adenosine monophosphate (cAMP)/Ca2+ signaling proteins, and cAMP-response element binding protein (CREB)/CRH-1 signaling proteins. Moreover, S-NP exposure inhibited the expression of the CREB-dependent LTAM genes nid-1, ptr-15, and unc-86. Our findings provide new insights into long-term S-NP exposure and the impairment of STAM and LTAM, which involve the highly conserved iGluRs and CRH-1/CREB signaling pathways.

Ca2+ signaling in postsynaptic neurons: Neuroplastin-65 regulates the interplay between plasma membrane Ca2+ ATPases and ionotropic glutamate receptors.

Upon postsynaptic glutamate receptor activation, the cytosolic Ca2+ concentration rises and initiates signaling and plasticity in spines. The plasma membrane Ca2+ ATPase (PMCA) is a major player to limit the duration of cytosolic Ca2+ signals. It forms complexes with the glycoprotein neuroplastin (Np) isoforms Np55 and Np65 and functionally interplays with N-methyl-D-aspartate (NMDA)-type ionotropic glutamate receptors (iGluNRs). Moreover, binding of the Np65-specific extracellular domain to Ca2+-permeable GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type ionotropic glutamate receptors (iGluA1Rs) was found to be required for long-term potentiation (LTP). However, the link between PMCA and iGluRs function to regulate cytosolic Ca2+ signals remained unclear. Here, we report that Np65 coordinates PMCA and iGluRs' functions to modulate the duration and amplitude of cytosolic Ca2+ transients in dendrites and spines of hippocampal neurons. Using live-cell Ca2+ imaging, acute pharmacological treatments, and GCaMP5G-expressing hippocampal neurons, we discovered that endogenous or Np65-promoted PMCA activity contributes to the restoration of basal Ca2+ levels and that this effect is dependent on iGluR activation. Super-resolution STED and confocal microscopy revealed that electrical stimulation increases the abundance of synaptic neuroplastin-PMCA complexes depending on iGluR activation and that low-rate overexpression of Np65 doubled PMCA levels and decreased cell surface levels of GluN2A and GluA1 in dendrites and Shank2-positive glutamatergic synapses. In neuroplastin-deficient hippocampi, we observed reduced PMCA and unchanged GluN2B levels, while GluN2A and GluA1 levels were imbalanced. Our electrophysiological data from hippocampal slices argues for an essential interplay of PMCA with GluN2A- but not with GluN2B-containing receptors upon induction of synaptic plasticity. Accordingly, we conclude that Np65 may interconnect PMCA with core players of glutamatergic neurotransmission to fine-tune the Ca2+ signal regulation in basal synaptic function and plasticity.

Meeting a threat of the Anthropocene: Taste avoidance of metal ions by Drosophila.

The Anthropocene Epoch poses a critical challenge for organisms: they must cope with new threats at a rapid rate. These threats include toxic chemical compounds released into the environment by human activities. Here, we examine elevated concentrations of heavy metal ions as an example of anthropogenic stressors. We find that the fruit fly Drosophila avoids nine metal ions when present at elevated concentrations that the flies experienced rarely, if ever, until the Anthropocene. We characterize the avoidance of feeding and egg laying on metal ions, and we identify receptors, neurons, and taste organs that contribute to this avoidance. Different subsets of taste receptors, including members of both Ir (Ionotropic receptor) and Gr (Gustatory receptor) families contribute to the avoidance of different metal ions. We find that metal ions activate certain bitter-sensing neurons and inhibit sugar-sensing neurons. Some behavioral responses are mediated largely through neurons of the pharynx. Feeding avoidance remains stable over 10 generations of exposure to copper and zinc ions. Some responses to metal ions are conserved across diverse dipteran species, including the mosquito Aedes albopictus. Our results suggest mechanisms that may be essential to insects as they face challenges from environmental changes in the Anthropocene.

Opening of glutamate receptor channel to subconductance levels.

Ionotropic glutamate receptors (iGluRs) are tetrameric ligand-gated ion channels that open their pores in response to binding of the agonist glutamate1-3. An ionic current through a single iGluR channel shows up to four discrete conductance levels (O1-O4)4-6. Higher conductance levels have been associated with an increased number of agonist molecules bound to four individual ligand-binding domains (LBDs)6-10. Here we determine structures of a synaptic complex of AMPA-subtype iGluR and the auxiliary subunit γ2 in non-desensitizing conditions with various occupancy of the LBDs by glutamate. We show that glutamate binds to LBDs of subunits B and D only after it is already bound to at least the same number of LBDs that belong to subunits A and C. Our structures combined with single-channel recordings, molecular dynamics simulations and machine-learning analysis suggest that channel opening requires agonist binding to at least two LBDs. Conversely, agonist binding to all four LBDs does not guarantee maximal channel conductance and favours subconductance states O1 and O2, with O3 and O4 being rare and not captured structurally. The lack of subunit independence and low efficiency coupling of glutamate binding to channel opening underlie the gating of synaptic complexes to submaximal conductance levels, which provide a potential for upregulation of synaptic activity.

The subfornical organ drives hypertension in polycystic kidney disease via the hypothalamic paraventricular nucleus.

AIMS: Hypertension is a prevalent yet poorly understood feature of polycystic kidney disease. Previously, we demonstrated that increased glutamatergic neurotransmission within the hypothalamic paraventricular nucleus produces hypertension in the Lewis Polycystic Kidney (LPK) rat model of polycystic kidney disease. Here, we tested the hypothesis that augmented glutamatergic drive to the paraventricular nucleus in Lewis polycystic kidney rats originates from the forebrain lamina terminalis, a sensory structure that relays blood-borne information throughout the brain. METHODS AND RESULTS: Anatomical experiments revealed that 38% of paraventricular nucleus-projecting neurons in the subfornical organ of the lamina terminalis expressed Fos/Fra, an activation marker, in LPK rats while <1% of neurons were Fos/Fra+ in Lewis control rats (P = 0.01, n = 8). In anaesthetized rats, subfornical organ neuronal inhibition using isoguvacine produced a greater reduction in systolic blood pressure in LPK vs. Lewis rats (-21±4 vs. -7±2 mmHg, P < 0.01; n = 10), which could be prevented by prior blockade of paraventricular nucleus ionotropic glutamate receptors using kynurenic acid. Blockade of ionotropic glutamate receptors in the paraventricular nucleus produced an exaggerated depressor response in LPK relative to Lewis rats (-23±4 vs. -2±3 mmHg, P < 0.001; n = 13), which was corrected by prior inhibition of the subfornical organ with muscimol but unaffected by chronic systemic angiotensin II type I receptor antagonism or lowering of plasma hyperosmolality through high-water intake (P > 0.05); treatments that both nevertheless lowered blood pressure in LPK rats (P < 0.0001). CONCLUSION: Our data reveal multiple independent mechanisms contribute to hypertension in polycystic kidney disease, and identify high plasma osmolality, angiotensin II type I receptor activation and, importantly, a hyperactive subfornical organ to paraventricular nucleus glutamatergic pathway as potential therapeutic targets.

Expression of the ionotropic glutamate receptors on neuronostatin neurons in the periventricular nucleus of the hypothalamus.

BACKGROUND: Neuronostatin, a newly identified peptide, is accepted as an anorexigenic peptide since it suppresses food intake when given intracerebroventricularly. Although the effect mechanisms of neuronostatin have been shown in different studies, there are no reports in the literature describing the mechanisms controlling neuronostatin neurons. In this study, we aimed to determine the presence of the ionotropic glutamate receptor subunits (iGluRs) in neuronostatin neurons in the periventricular nucleus of the hypothalamus. MATERIALS AND METHODS: The presence of glutamate receptors in neuronostatin neurons was investigated by dual immunohistochemistry. Immunohistochemistry was performed on 40 µm thick coronal brain sections with antibodies against AMPA (GluA1-4), kainate (GluK1/2/3, and GluK5), and NMDA (GluN1 and GluN2A) receptor subunits. RESULTS: The results showed that the neuronostatin neurons expressed most of the NMDA and non-NMDA receptor subunits. The neuronostatin neurons in the anterior hypothalamic periventricular nucleus were particularly immunopositive for GluA1, GluA4, GluK1/2/3, GluK5 and GluN1 antibodies. No expression was observed for GluA2, GluA3 and GluN2A antibodies. CONCLUSIONS: For the first time in the literature, our study demonstrated that the neuronostatin neurons express glutamate receptor subunits which may form homomeric or heteromeric functional receptor complexes. Taken together, these results suggest that multiple subunits of iGluRs are responsible for glutamate transmission on neuronostatin neurons in the anterior hypothalamic periventricular nucleus.

2-Arachidonoylglycerol mobilization following brief synaptic stimulation in the dorsal lateral striatum requires glutamatergic and cholinergic neurotransmission.

Several forms of endocannabinoid (eCB) signaling have been described in the dorsal lateral striatum (DLS), however most experimental protocols used to generate eCBs do not recapitulate the firing patterns of striatal-projecting pyramidal neurons in the cortex or firing patterns of striatal medium spiny neurons. Therefore, it is unclear if current models of eCB signaling in the DLS provide a reliable description of mechanisms engaged under physiological conditions. To address this uncertainty, we investigated mechanisms of eCB mobilization following brief synaptic stimulation that mimics in vivo patterns of neural activity in the DLS. To monitor eCB mobilization, the novel genetically encoded fluorescent eCB biosensor, GRABeCB2.0, was expressed presynaptically in corticostriatal afferents of C57BL6J mice and evoked eCB transients were measured in the DLS using a brain slice photometry technique. We found that brief bouts of synaptic stimulation induce long lasting eCB transients that were generated predominantly by 2-arachidonoylglycerol (2-AG) mobilization. Efficient 2-AG mobilization required coactivation of AMPA and NMDA ionotropic glutamate receptors and muscarinic M1 receptors. Dopamine D2 receptors expressed on cholinergic interneurons inhibited 2-AG mobilization by inhibiting acetylcholine release. Collectively, these data uncover unrecognized mechanisms underlying 2-AG mobilization in the DLS.

Long-term effect of neonatal antagonism of ionotropic glutamate receptors on dendritic spines and cognitive function in rats.

Glutamate is the most abundant excitatory neurotransmitter in the hippocampus where mediates its actions by activating glutamate receptors. The activation of these receptors is essential for the maintenance and dynamics of dendritic spines and plasticity that correlate with learning and memory processes during neurodevelopment and adulthood. We studied in adults the effect of blocking ionotropic glutamate receptors (NMDAR, AMPAR, and KAR) functions at neonatal age (PD1-PD15) with their respective antagonists D-AP5, GYKI-53655 and UBP-302. We first evaluated memory using a new object recognition test in adults. Second, we evaluated the levels of glial fibrillary acidic protein, synaptophysin and actin with immunohistochemistry in the CA1, CA3, and dentate gyrus regions of the hippocampus and, finally, the number of dendritic spines and their dynamics using Golgi-Cox staining. We found that ionotropic glutamate receptor function blockade at neonatal age causes a reduction in short and long-term memory in adulthood and a reduction in the expression of synaptophysin and actin protein levels in the hippocampus regions studied. This blockade also reduced the number of dendritic spines and modified dendritic dynamics in the CA1 region. The antagonism of the three types of ionotropic glutamate receptors reduced the mushrooms and bifurcated types of spines and increased the thin spines. The number of stubby spines was reduced by D-AP5, increased by UPB-302, and not affected by GYKI-53655. Our results indicate that the blockade of neonatal ionotropic glutamate receptors produces alterations that persist until adulthood.

Ionotropic receptors mediate nitrogenous waste avoidance in Drosophila melanogaster.

Ammonia and its amine-containing derivatives are widely found in natural decomposition byproducts. Here, we conducted biased chemoreceptor screening to investigate the mechanisms by which different concentrations of ammonium salt, urea, and putrescine in rotten fruits affect feeding and oviposition behavior. We identified three ionotropic receptors, including the two broadly required IR25a and IR76b receptors, as well as the narrowly tuned IR51b receptor. These three IRs were fundamental in eliciting avoidance against nitrogenous waste products, which is mediated by bitter-sensing gustatory receptor neurons (GRNs). The aversion of nitrogenous wastes was evaluated by the cellular requirement by expressing Kir2.1 and behavioral recoveries of the mutants in bitter-sensing GRNs. Furthermore, by conducting electrophysiology assays, we confirmed that ammonia compounds are aversive in taste as they directly activated bitter-sensing GRNs. Therefore, our findings provide insights into the ecological roles of IRs as a means to detect and avoid toxic nitrogenous waste products in nature.

Glutamate receptor, ionotropic, N­methyl D­aspartate­associated protein 1 promotes colorectal cancer cell proliferation and metastasis, and is negatively regulated by miR­296­3p.

N­methyl D­aspartate receptors (NMDARs) are closely associated with the development, growth and metastasis of cancer. Glutamate receptor, ionotropic, N­methyl D­aspartate­associated protein 1 (GRINA) is a member of the of the NMDAR family, and its aberrant expression is associated with gastric cancer. However, the role of GRINA in colorectal cancer (CRC) is not completely understood. In the present study, expression profiles of GRINA in several CRC databases were obtained and further verified using clinical CRC samples. The effects of GRINA overexpression on CRC progression both in vivo and in vitro were assessed. Briefly, cell proliferation was detected using MTT assay, and cell migration and invasion ability were evaluated by wound healing and Transwell assay. In addition, the molecular mechanism underlying the upregulated expression of GRINA in CRC was investigated. The regulatory association between GRINA and miR­296­3p was detected by luciferase assay, reverse transcription­quantitative PCR and western blotting. The results demonstrated that GRINA expression levels were significantly increased in tumor samples compared with those in healthy samples, and upregulated expression of GRINA was associated with a less favorable prognostic outcome in patients with CRC. GRINA overexpression significantly increased CRC cell proliferation, invasion and migration. Additionally, it was determined that GRINA was post­transcriptionally regulated by microRNA (miR)­296­3p. Together, the results of the present study suggested the potential importance of the miR­296­3p/GRINA axis and highlighted potential novel targets for the management of CRC.

Computational Analysis of the Crystal and Cryo-EM Structures of P-Loop Channels with Drugs.

The superfamily of P-loop channels includes various potassium channels, voltage-gated sodium and calcium channels, transient receptor potential channels, and ionotropic glutamate receptors. Despite huge structural and functional diversity of the channels, their pore-forming domain has a conserved folding. In the past two decades, scores of atomic-scale structures of P-loop channels with medically important drugs in the inner pore have been published. High structural diversity of these complexes complicates the comparative analysis of these structures. Here we 3D-aligned structures of drug-bound P-loop channels, compared their geometric characteristics, and analyzed the energetics of ligand-channel interactions. In the superimposed structures drugs occupy most of the sterically available space in the inner pore and subunit/repeat interfaces. Cationic groups of some drugs occupy vacant binding sites of permeant ions in the inner pore and selectivity-filter region. Various electroneutral drugs, lipids, and detergent molecules are seen in the interfaces between subunits/repeats. In many structures the drugs strongly interact with lipid and detergent molecules, but physiological relevance of such interactions is unclear. Some eukaryotic sodium and calcium channels have state-dependent or drug-induced π-bulges in the inner helices, which would be difficult to predict. The drug-induced π-bulges may represent a novel mechanism of gating modulation.

Systemic Inflammation Induced Changes in Protein Expression of ABC Transporters and Ionotropic Glutamate Receptor Subunit 1 in the Cerebral Cortex of Familial Alzheimer`s Disease Mouse Model.

Alzheimer's disease (AD) is an incurable disease, with complex pathophysiology and a myriad of proteins involved in its development. In this study, we applied quantitative targeted absolute proteomic analysis for investigation of changes in potential AD drug targets, biomarkers, and transporters in cerebral cortices of lipopolysaccharide (LPS)-induced neuroinflammation mouse model, familial AD mice (APdE9) with and without LPS treatment as compared to age-matched wild type (WT) mice. The ABCB1, ABCG2 and GluN1 protein expression ratios between LPS treated APdE9 and WT control mice were 0.58 (95% CI 0.44-0.72), 0.65 (95% CI 0.53-0.77) and 0.61 (95% CI 0.52-0.69), respectively. The protein expression levels of other proteins such as MGLL, COX-2, CytC, ABCC1, ABCC4, SLC2A1 and SLC7A5 did not differ between the study groups. Overall, the study revealed that systemic inflammation can alter ABCB1 and ABCG2 protein expression in brain in AD, which can affect intra-brain drug distribution and play a role in AD development. Moreover, the inflammatory insult caused by peripheral infection in AD may be important factor triggering changes in GluN1 protein expression. However, more studies need to be performed in order to confirm these findings. The quantitative information about the expression of selected proteins provides important knowledge, which may help in the optimal use of the mouse models in AD drug development and better translation of preclinical data to humans.