Generation of a MOR‐CreER knock‐in mouse line to study cells and neural circuits involved in mu opioid receptor signaling

Mu opioid receptor (MOR) is involved in various brain functions, such as pain modulation, reward processing, and addictive behaviors, and mediates the main pharmacologic effects of morphine and other opioid compounds. To gain genetic access to MOR‐expressing cells, and to study physiological and pathological roles of MOR signaling, we generated a MOR‐CreER knock‐in mouse line, in which the stop codon of the Oprm1 gene was replaced by a DNA fragment encoding a T2A peptide and tamoxifen (Tm)‐inducible Cre recombinase. We show that the MOR‐CreER allele undergoes Tm‐dependent recombination in a discrete subtype of neurons that express MOR in the adult nervous system, including the olfactory bulb, cerebral cortex, striosome compartments in the striatum, hippocampus, amygdala, thalamus, hypothalamus, interpeduncular nucleus, superior and inferior colliculi, periaqueductal gray, parabrachial nuclei, cochlear nucleus, raphe nuclei, pontine and medullary reticular formation, ambiguus nucleus, solitary nucleus, spinal cord, and dorsal root ganglia. The MOR‐CreER mouse line combined with a Cre‐dependent adeno‐associated virus vector enables robust gene manipulation in the MOR‐enriched striosomes. Furthermore, Tm treatment during prenatal development effectively induces Cre‐mediated recombination. Thus, the MOR‐CreER mouse is a powerful tool to study MOR‐expressing cells with conditional gene manipulation in developing and mature neural tissues.     

Astrocytes mediate HIV‐1 Tat‐induced neuronal damage via ligand‐gated ion channel P2X7R

During human immunodeficiency virus (HIV)‐1 infection, perturbations in neuron–glia interactions may culminate in neuronal damage. Recently, purinergic receptors have been implicated in the promotion of virus‐induced neurotoxicity and supporting the viral life cycle at multiple stages. The astrocytes robustly express purinergic receptors. We therefore sought to examine if P2X7R, a P2X receptor subtype, can mediate HIV‐1 Tat‐induced neuronal apoptosis. Tat augmented the expression of P2X7R in astrocytes. Our data reveal the involvement of P2X7R in Tat‐mediated release of monocyte chemoattractant protein (MCP‐1) /chemokine (C‐C motif) ligand 2 (CCL2) from the astrocytes. P2X7R antagonists, such as the oxidized ATP, A438079, brilliant blue G, and broad spectrum P2 receptor antagonist suramin, attenuated Tat‐induced CCL2 release in a calcium‐ and extracellular signal‐regulated kinase (ERK)1/2‐dependent manner. Calcium chelators, (1,2‐bis(o‐aminophenoxy) ethane‐N,N,N',N'‐tetraacetic acid) acetoxymethyl ester and EGTA, and ERK1/2 inhibitor U0126 abolished chemokine (C‐C motif) ligand 2 release from astrocytes. Furthermore, in human neuronal cultures, we demonstrated P2X7R involvement in Tat‐mediated neuronal death. Importantly, in the TUNEL assay, the application of P2X7R‐specific antagonists or the knockdown of P2X7R in human astrocytes reduced HIV‐Tat‐induced neuronal death significantly, underlining the critical role of P2X7R in Tat‐mediated neurotoxicity. Our study provides novel insights into astrocyte‐mediated neuropathogenesis in HIV‐1 infection and a novel target for therapeutic management of neuroAIDS.

     

X1X1X2X2/X1X2Y sex chromosome systems in the Neotropical Gymnotiformes electric fish of the genus Brachyhypopomus

Several types of sex chromosome systems have been recorded among Gymnotiformes, including male and female heterogamety, simple and multiple sex chromosomes, and different mechanisms of origin and evolution. The X₁X₁X₂X₂/X₁X₂Y systems identified in three species of this order are considered homoplasic for the group. In the genus Brachyhypopomus, only B. gauderio presented this type of system. Herein we describe the karyotypes of Brachyhypopomus pinnicaudatus and B. n. sp. FLAV, which have an X₁X₁X₂X₂/X₁X₂Y sex chromosome system that evolved via fusion between an autosome and the Y chromosome. The morphology of the chromosomes and the meiotic pairing suggest that the sex chromosomes of B. gauderio and B. pinnicaudatus have a common origin, whereas in B . n. sp. FLAV the sex chromosome system evolved independently. However, we cannot discard the possibility of common origin followed by distinct processes of differentiation. The identification of two new karyotypes with an X₁X₁X₂X₂/X₁X₂Y sex chromosome system in Gymnotiformes makes it the most common among the karyotyped species of the group. Comparisons of these karyotypes and the evolutionary history of the taxa indicate independent origins for their sex chromosomes systems. The recurrent emergence of the X₁X₁X₂X₂/X₁X₂Y system may represent sex chromosomes turnover events in Gymnotiformes.     

Myosin‐X facilitates Shigella‐induced membrane protrusions and cell‐to‐cell spread

The intracellular pathogen Shigella flexneri forms membrane protrusions to spread from cell to cell. As protrusions form, myosin‐X (Myo10) localizes to Shigella. Electron micrographs of immunogold‐labelled Shigella‐infected HeLa cells reveal that Myo10 concentrates at the bases and along the sides of bacteria within membrane protrusions. Time‐lapse video microscopy shows that a full‐length Myo10 GFP‐construct cycles along the sides of Shigella within the membrane protrusions as these structures progressively lengthen. RNAi knock‐down of Myo10 is associated with shorter protrusions with thicker stalks, and causes a >80% decrease in confluent cell plaque formation. Myo10 also concentrates in membrane protrusions formed by another intracellular bacteria, Listeria, and knock‐down of Myo10 also impairs Listeria plaque formation. In Cos7 cells (contain low concentrations of Myo10), the expression of full‐length Myo10 nearly doubles Shigella‐induced protrusion length, and lengthening requires the head domain, as well as the tail‐PH domain, but not the FERM domain. The GFP‐Myo10‐HMM domain localizes to the sides of Shigella within membrane protrusions and the GFP‐Myo10‐PH domain localizes to host cell membranes. We conclude thatMyo10 generates the force to enhance bacterial‐induced protrusions by binding its head region to actin filaments and its PH tail domain to the peripheral membrane.     

Blocking elevated VEGF‐A attenuates non‐vasculature Fragile X syndrome abnormalities

Fragile X syndrome (FXS) is the most common form of inherited mental retardation. In exploring abnormalities associated with the syndrome, we have recently demonstrated abnormal vascular density in a FXS mouse model (Galvan and Galvez, 2012 ). One of the most prominent regulators of vascular growth is VEGF‐A (Vascular Endothelial Growth Factor A), suggesting that FXS is associated with abnormal VEGF‐A expression. In addition to its role in vascular regulation, VEGF‐A also induces cellular changes such as increasing cell proliferation, and axonal and neurite outgrowth independent of its effects on vasculature. These VEGF‐A induced cellular changes are consistent with FXS abnormalities such as increased axonal material, dendritic spine density, and cell proliferation. In support of these findings, the following study demonstrated that FXS mice exhibit increased expression of VEGF‐A in brain. These studies suggest that increased VEGF‐A expression in FXS is contributing to non‐vascular FXS abnormalities. To explore the role of VEGF‐A in mediating non‐vascular FXS abnormalities, the monoclonal antibody Bevacizumab was used to block free VEGF‐A. Bevacizumab treatment was found to decrease FXS Synapsin‐1 expression, a presynaptic marker for synapse density, and reduce FXS testicle weight to control levels. Blocking VEGF‐A also alleviated FXS abnormalities on novel object recognition, a test of cognitive performance. These findings demonstrate that VEGF‐A is elevated in FXS brain and suggest that its expression promotes non‐vascular FXS abnormalities. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 14–25, 2017     

SREBP‐1c,Pdx‐1, and GLP‐1R involved in palmitate–EPA regulated glucose‐stimulated insulin secretion in INS‐1 cells

Impairment of glucose‐stimulated insulin secretion (GSIS) caused by glucolipotoxicity is an essential feature in type 2 diabetes mellitus (T2DM). Palmitate and eicosapentaenoate (EPA), because of their lipotoxicity and protection effect, were found to impair or restore the GSIS in beta cells. Furthermore, palmitate was found to up‐regulate the expression level of sterol regulatory element‐binding protein (SREBP)‐1c and down‐regulate the levels of pancreatic and duodenal homeobox (Pdx)‐1 and glucagon‐like peptide (GLP)‐1 receptor (GLP‐1R) in INS‐1 cells. To investigate the underlying mechanism, the lentiviral system was used to knock‐down or over‐express SREBP‐1c and Pdx‐1, respectively. It was found that palmitate failed to suppress the expression of Pdx‐1 and GLP‐1R in SREBP‐1c‐deficient INS‐1 cells. Moreover, down‐regulation of Pdx‐1 could cause the low expression of GLP‐1R with/without palmitate treatment. Additionally, either SREBP‐1c down‐regulation or Pdx‐1 over‐expression could partially alleviate palmitate‐induced GSIS impairment. These results suggested that sequent SREBP‐1c‐Pdx‐1‐GLP‐1R signal pathway was involved in the palmitate‐caused GSIS impairment in beta cells. J. Cell. Biochem. 111: 634–642, 2010. © 2010 Wiley‐Liss, Inc.     

The activation of liver X receptors inhibits toll‐like receptor‐9‐induced foam cell formation

Toll‐like receptors (TLRs) are related to foam cell formation (FCF), key event in the establishment/progression of atherosclerosis. The activation of TLR2 and TLR4 can increase FCF. The aim of this study was to evaluate the role of TLR9 in FCF. Murine macrophages were treated with CpG‐ODN, TLR9 agonist, and oxidized particles of LDL (Paz‐PC) and FCF was analyzed by means of Oil Red O staining. The administration of CpG‐ODN plus Paz‐PC onto macrophages increased the amount of lipid droplets, correlated to increased levels of tumor necrosis factor (TNF)‐α, IFNβ, and IP‐10. The underlying mechanism by which TLR9 ligation influenced Paz‐PC in the FCF was NF‐κB‐ and IRF7‐dependent, as observed by higher levels of phosphorylated IκBα, increased nuclear translocation of the p65 subunit, lower levels of the total IKKα protein and higher release of interferon‐dependent cytokines, such as IP‐10. Liver X receptors (LXRs) regulate lipid cellular transport and negatively modulate TLR‐dependent signaling pathways. Indeed, the addition of GW3965, synthetic LXRs agonist, significantly reduced FCF after CpG‐ODN plus Paz‐PC stimulation. In this condition, we observed decreased levels of the nuclear translocation of the p65 subunit, related to the higher presence of LXRα into the nucleus. TNF‐α, IP‐10, and IFNβ levels were reduced by the administration of GW3965 following CpG‐ODN and Paz‐PC treatment. In conclusion, the activation of TLR9 facilitates the formation of foam cells in an NF‐κB‐ and IRF7‐dependent manner, countered by the activation of LXRs. This study further support LXRs as potential anti‐atherosclerotic target. J. Cell. Physiol. 223: 158–167, 2010. © 2009 Wiley‐Liss, Inc.     

Anticonvulsant Activity of Enantiomeric N‐trans‐Cinnamoyl Derivatives of 2‐Aminopropan‐1‐ols and 2‐Aminobutan‐1‐ols

Epilepsy, one of the most frequent neurological disorders, is still insufficiently treated in about 30% of patients. As a consequence, identification of novel anticonvulsant agents is an important issue in medicinal chemistry. In the present article we report synthesis, physicochemical, and pharmacological evaluation of N‐trans‐cinnamoyl derivatives of R and S‐2‐aminopropan‐1‐ol, as well as R and S‐2‐aminobutan‐1‐ol. The structures were confirmed by spectroscopy and for derivatives of 2‐aminopropan‐1‐ols the configuration was evaluated by means of crystallography. The investigated compounds were tested in rodent models of seizures: maximal electroshock (MES) and subcutaneous pentetrazol test (scPTZ), and also in a rodent model of epileptogenesis: pilocarpine‐induced status prevention. Additionally, derivatives of 2‐aminopropan‐1‐ols were tested in benzodiazepine‐resistant electrographic status epilepticus rat model as well as in vitro for inhibition of isoenzymes of cytochrome P450. All of the tested compounds showed promising anticonvulsant activity in MES. For R(–)‐(2E)‐N‐(1‐hydroxypropan‐2‐yl)‐3‐phenylprop‐2‐enamide pharmacological parameters were found as follows: ED₅₀ = 76.7 (68.2–81.3) mg/kg (MES, mice i.p., time = 0.5 h), ED₅₀ = 127.2 (102.1–157.9) mg/kg (scPTZ, mice i.p., time = 0.25 h), TD₅₀ = 208.3 (151.4–230.6) mg/kg (rotarod, mice i.p., time = 0.25 h). Evaluation in pilocarpine status prevention proved that all of the reported compounds reduced spontaneous seizure activity and act as antiepileptogenic agents. Both enantiomers of 2‐aminopropan‐1‐ols did not influence cytochrome P450 isoenzymes activity in vitro and are likely not to interact with CYP substrates in vivo. Chirality 28:482–488, 2016. © 2016 Wiley Periodicals, Inc.     

Re‐examining how Munc13‐1 facilitates opening of syntaxin‐1

Munc13‐1 is crucial for neurotransmitter release and, together with Munc18‐1, orchestrates assembly of the neuronal SNARE complex formed by syntaxin‐1, SNAP‐25, and synaptobrevin. Assembly starts with syntaxin‐1 folded into a self‐inhibited closed conformation that binds to Munc18‐1. Munc13‐1 is believed to catalyze the opening of syntaxin‐1 to facilitate SNARE complex formation. However, different types of Munc13‐1‐syntaxin‐1 interactions have been reported to underlie this activity, and the critical nature of Munc13‐1 for release may arise because of its key role in bridging the vesicle and plasma membranes. To shed light into the mechanism of action of Munc13‐1, we have used NMR spectroscopy, SNARE complex assembly experiments, and liposome fusion assays. We show that point mutations in a linker region of syntaxin‐1 that forms intrinsic part of the closed conformation strongly impair stimulation of SNARE complex assembly and liposome fusion mediated by Munc13‐1 fragments, even though binding of this linker region to Munc13‐1 is barely detectable. Conversely, the syntaxin‐1 SNARE motif clearly binds to Munc13‐1, but a mutation that disrupts this interaction does not affect SNARE complex assembly or liposome fusion. We also show that Munc13‐1 cannot be replaced by an artificial tethering factor to mediate liposome fusion. Overall, these results emphasize how very weak interactions can play fundamental roles in promoting conformational transitions and strongly support a model whereby the critical nature of Munc13‐1 for neurotransmitter release arises not only from its ability to bridge two membranes but also from an active role in opening syntaxin‐1 via interactions with the linker.     

Porphyromonas gingivalis‐nucleoside‐diphosphate‐kinase inhibits ATP‐induced reactive‐oxygen‐species via P2X7 receptor/NADPH‐oxidase signalling and contributes to persistence

Ligation of P2X₇ receptors with a ‘danger signal’, extracellular ATP (eATP), has recently been shown to result in production of intracellular reactive‐oxygen‐species (ROS) in macrophages. We show that primary gingival epithelial cells (GECs) produce sustained, robust cellular ROS upon stimulation by eATP. The induction of ROS was mediated by P2X₇ receptor signalling coupled with NADPH‐oxidase activation, as determined by pharmacological inhibition and RNA interference. Furthermore, Porphyromonas gingivalis, an oral opportunistic pathogen, upregulated the antioxidant glutathione response, modulated eATP‐induced cytosolic and mitochondrial ROS generated through P2X₇/NADPH‐oxidase interactome, and subsequently blocked oxidative stress in GECs via temporal secretion of a P. gingivalis effector, nucleoside‐diphosphate‐kinase (Ndk). An ndk‐deficient P. gingivalis mutant lacked the ability to inhibit ROS production and persist intracellularly following eATP stimulation. Treatment with recombinant Ndk significantly diminished eATP‐evoked ROS production. P. gingivalis infection elicited a strong, time‐dependent increase in anti‐oxidativemitochondrial UCP₂ levels, whereas ndk‐deficient mutant did not cause any change. The results reveal a novel signalling cascade that is tightly coupled with eATP signalling and ROS regulation. Ndk by P. gingivalis counteracts these antimicrobial signalling activities by secreting Ndk, thus contributing to successful persistence of the pathogen.     

Novel N‐Acyl‐1H‐imidazole‐1‐carbothioamides: Design,Synthesis, Biological and Computational Studies

The present study reports the convenient synthesis, spectroscopic characterization, bio‐assays and computational evaluation of a novel series of N‐acyl‐1H‐imidazole‐1‐carbothioamides. The screened derivatives displayed excellent antioxidant activity, moderate antibacterial and antifungal potential. The screened derivatives were found to be highly biocompatible against hRBCs. Molecular docking ascertained the mechanism and mode of action towards the molecular target delineating that ligands and complexes were stabilized at the active site by electrostatic and hydrophobic forces in accordance to the corresponding experimental results. Docking simulation provided additional information about the possibilities of inhibitory potential of the compounds against RNA. Computational evaluation predicted that N‐acyl‐1H‐imidazole‐1‐carbothioamides 5c and 5g can serve as potential surrogates for hit to lead generation and design of novel antioxidant and antibacterial agents.