Aktivierung und Desensibilsierung des olfaktorischen cAMP-gesteuerten-Kanal : Identifizerung von funktionellen Modulen

Der olfaktorische zyklisch-Nukleotid-gesteuerte Kanal (CNG-Kanal) wandelt in den Riechneuronen das chemische Duftstoffsignal in ein elektrisches Signal um und spielt somit eine wichtige Rolle bei der Generierung, aber auch bei der Terminierung des Rezeptorstroms. Der CNG-Kanal ist ein heterotetrameres Protein und setzt sich aus den drei Untereinheiten CNGA2, CNGA4 und CNGB1b wie folgt zusammen: A2-A2-A4-B1b. Der CNG-Kanal ist ein unspezifischer Kationenkanl, der hauptsächlich Kalzium in die Riechneuronen leitet. cAMP öffnet den CNG-Kanal kooperativ, und die Sensivität des CNG-Kanals für cAMP wird durch Calmodulin Kalzium-abhängig moduliert. In dieser Arbeit wurde die Beteiligung der einzelnen Untereinheiten bei der Aktivierung und der Desensibilisierung des CNG Kanals in einem heterologen Expressionssystem (HEK 293-Zellen) untersucht. Bei den patch-clamp Untersuchungen wurde festgestellt, dass HEK 293 Zellen, die mit den drei Untereinheiten transfiziert wurden, nicht eine einheitliche Kanalpopulation exprimierten, sondern Mischungen von allen möglichen Kanalzusammensetztungen (A24, A23-A41, A23-B1b1, und A22-A41-B1b1). Allerdings wurden zwei Ausschlusskriterien zur Identifizierung von inside-out Patches, die fast ausschließlich A2-A2-A4-B1b Kanäle hatten, bestimmt. Erstens können die verschiedenen CNG Kanäle anhand ihrer Sensivität für cAMP unterschieden werden. Die Spannungsabhängigkeit der Aktivierungskonstante (K½) für cAMP ist das zweite Kriterium. Für den CNG-Kanal wurde vor kurzem das erste adäquate kinetische Modell (C4L-Modell) für die Aktivierung beschrieben. Das C4L-Modell beschreibt eine sequentielle Bindung von cAMP an den homomeren CNGA2-Kanal. In der vorliegenden Arbeit konnte das C4L-Modell für den heteromeren A2-A2-A4-B1b-Kanal erweitert werden. Mit Mutagense wurden die Bindestellen für cAMP auf den einzelnen Untereinheiten funktionell ausgeschaltet. Somit wurden A2-A2-A4-B1b Kanäle mit zwei bis vier intakten Bindestellen erzeugt. Die Analyse der Dosis-Wirkungs-Beziehungen für die Kanalaktivierung ergab die Reihenfolge, in der die vier Kanaluntereinheiten cAMP binden. Die beiden CNGA2-Untereinheiten vermitteln die ersten beiden cAMP-Bindungen und öffnen den Kanal. Die Bindung des dritten und vierten cAMPs kann von CNGA4 oder CNGB1b gleich gut vermittelt werden, und stabilisieren den Kanal im offenen Zustand. Für die schnelle Desensibilisierung des A2-A2-A4-B1b Kanals wurde überprüft, ob eine der beiden LQ-Typ Calmodulin-Bindestellen von CNGA4 und CNGB1b ausreicht, um den Kanal zu desensibilisieren. Dafür wurden die beiden LQ-Typ Calmodulin-Bindestellen durch Mutagense funktionell ausgeschaltet. Der Effekt der iii Mutationen auf den Kanal wurde mit der patch-clamp Technik untersucht. Dabei wurden Dosis-Wirkungs-Beziehungen für cAMP unter desensibilisierenden Bedingungen, dass heißt in der Gegenwart von Kalzium, erstellt. Die Analyse der Daten ergab, dass CNGB1b notwenig und ausreichend ist, den A2-A2-A4-B1b Kanal zu desensibilisieren

Activation and desensitization of the olfactory cAMP-gated transduction channel: identification of functional modules

Olfactory receptor neurons respond to odor stimulation with a receptor potential that results from the successive activation of cyclic AMP (cAMP)-gated, Ca2+-permeable channels and Ca2+-activated chloride channels. The cAMP-gated channels open at micromolar concentrations of their ligand and are subject to a Ca2+-dependent feedback inhibition by calmodulin. Attempts to understand the operation of these channels have been hampered by the fact that the channel protein is composed of three different subunits, CNGA2, CNGA4, and CNGB1b. Here, we explore the individual role that each subunit plays in the gating process. Using site-directed mutagenesis and patch clamp analysis, we identify three functional modules that govern channel operation: a module that opens the channel, a module that stabilizes the open state at low cAMP concentrations, and a module that mediates rapid Ca2+-dependent feedback inhibition. Each subunit could be assigned to one of these functions that, together, define the gating logic of the olfactory transduction channel

Neuronal Expression of Glucosylceramide Synthase in Central Nervous System Regulates Body Weight and Energy Homeostasis

Abstract Hypothalamic neurons are main regulators of energy homeostasis. Neuronal function essentially depends on plasma membrane-located gangliosides. The present work demonstrates that hypothalamic integration of metabolic signals requires neuronal expression of glucosylceramide synthase (GCS; UDP-glucose:ceramide glucosyltransferase). As a major mechanism of central nervous system (CNS) metabolic control, we demonstrate that GCS-derived gangliosides interacting with leptin receptors (ObR) in the neuronal membrane modulate leptin-stimulated formation of signaling metabolites in hypothalamic neurons. Furthermore, ganglioside-depleted hypothalamic neurons fail to adapt their activity (c-Fos) in response to alterations in peripheral energy signals. Consequently, mice with inducible forebrain neuron-specific deletion of the UDP-glucose:ceramide glucosyltransferase gene (Ugcg) display obesity, hypothermia, and lower sympathetic activity. Recombinant adeno-associated virus (rAAV)-mediated Ugcg delivery to the arcuate nucleus (Arc) significantly ameliorated obesity, specifying gangliosides as seminal components for hypothalamic regulation of body energy homeostasis

Neuronal expression of glucosylceramide synthase in central nervous system regulates body weight and energy homeostasis.

Hypothalamic neurons are main regulators of energy homeostasis. Neuronal function essentially depends on plasma membrane-located gangliosides. The present work demonstrates that hypothalamic integration of metabolic signals requires neuronal expression of glucosylceramide synthase (GCS; UDP-glucose:ceramide glucosyltransferase). As a major mechanism of central nervous system (CNS) metabolic control, we demonstrate that GCS-derived gangliosides interacting with leptin receptors (ObR) in the neuronal membrane modulate leptin-stimulated formation of signaling metabolites in hypothalamic neurons. Furthermore, ganglioside-depleted hypothalamic neurons fail to adapt their activity (c-Fos) in response to alterations in peripheral energy signals. Consequently, mice with inducible forebrain neuron-specific deletion of the UDP-glucose:ceramide glucosyltransferase gene (Ugcg) display obesity, hypothermia, and lower sympathetic activity. Recombinant adeno-associated virus (rAAV)-mediated Ugcg delivery to the arcuate nucleus (Arc) significantly ameliorated obesity, specifying gangliosides as seminal components for hypothalamic regulation of body energy homeostasis

<i>Ugcg</i>^{f/f//CamKCreERT2} mice develop progressive obesity.

<p>Both female (A) and male (B) <i>Ugcg</i>^{f/f//CamKCreERT2} mice showed a progressive increase in body weight after tamoxifen induction (<i>n</i> = 6–9). (C) <i>Ugcg</i>^{f/f//CamKCreERT2} mice were larger than <i>Ugcg</i>^f/f littermates (16 wk p.i.), and body fat mass was prominently elevated. (D) Enlarged adipocytes in <i>Ugcg</i>^{f/f//CamKCreERT2} mice 9 wk p.i. (E) Increased weight of epigonadal WAT 9 wk p.i. in <i>Ugcg</i>^{f/f//CamKCreERT2} mice (<i>n</i> = 4–5). (F) NMR analysis revealed significant and progressive accumulation of body fat mass in <i>Ugcg</i>^{f/f//CamKCreERT2} mice (<i>n</i> = 9–10). *<i>p</i>≤0.05; **<i>p</i>≤0.01;***<i>p</i>≤0.001. Means ± SEM.</p

Hypothalamic neurons of <i>Ugcg</i>^{f/f//CamKCreERT2} mice are less responsive to peripheral leptin.

<p>(A–C) Brains of leptin-stimulated mice were analyzed for neuronal activity indicated by c-Fos immunofluorescence. Detailed pictures in the upper lane indicate regions of the Arc that are outlined in overview pictures (frames). Arrowheads mark c-Fos-positive neurons located in the VMH. Axis indicators were included indicating the medial (m) and ventral (v) axes. (A) <i>Ugcg</i>^{f/f//CamKCreERT2} mice showed leptin-induced neuronal activation comparable to <i>Ugcg</i>^f/f mice in the Arc 1–2 wk p.i. (B) Leptin response in the Arc was decreased in nonobese <i>Ugcg</i>^{f/f//CamKCreERT2} mice weight-matched to controls 3–4 weeks p.i. (C) Decreased c-Fos staining in the Arc was also observed in obese leptin-induced <i>Ugcg</i>^{f/f//CamKCreERT2} mice 6 wk p.i. The percentage of c-Fos-positive neurons per Arc section was depicted as values normalized to saline-injected <i>Ugcg</i>^f/f mice (<i>n</i> = 14–22 sections). Depicted sections are located between bregma levels −1.5 to −1.8. Quantification contains data from bregma levels −1.4 to −2.3. (D–F) <i>Ugcg</i>^{f/f//CamKCreERT2} mice retained leptin responsiveness in the VMH, as elevated c-Fos after leptin stimulation indicated (<i>n</i> = 8–20 sections). Quantification contains data from bregma levels −1.4 to −2.0. Datasets for each time point were acquired individually. Two (1–2 and 3–4 wk) or three (6 wk) independent animal groups were analyzed. Immunofluorescence and image acquisition for each dataset (treated and untreated controls and knockouts) were performed simultaneously. Scale bar: 75 µm; 3^rdv, 3^rd ventricle; *<i>p</i>≤0.05; **<i>p</i>≤0.01; ***<i>p</i>≤0.001. Means ± SEM.</p

GCS in hypothalamic neurons regulates neuronal leptin signaling at the plasma membrane.

<p>(A) Stat3 phosphorylation was markedly decreased in Arc sections of <i>Ugcg</i>^{f/f//CamKCreERT2} mice in response to peripheral leptin (5 mg/kg; 45 min) 6 wk p.i. (<i>n</i> = 16–33). Three independent animal groups were analyzed. (B) Serum leptin levels were unchanged 3 wk p.i. and increased prominently 7 wk p.i. in <i>Ugcg</i>^{f/f//CamKCreERT2} mice, reflecting increased body fat mass (<i>n</i> = 12–14). (C) mRNA expression analysis for suppressor of cytokine signaling 3 (SOCS-3) expression in Arc-enriched hypothalamic tissue was carried out 2, 6, and 9 wk p.i. <i>Socs-3</i> expression normalized to the housekeeping gene tubulin was unaltered (<i>n</i> = 3–5). (D) mRNA expression analysis for the long form of the leptin receptor, <i>Leprb</i>, in mediobasal hypothalamus was carried out 6 wk p.i. <i>Leprb</i> expression normalized to the housekeeping gene tubulin was unaltered at that time point (<i>n</i> = 4–5). (E) Immortalized mouse hypothalamic cells (N-41 cells) were analyzed for cell surface expression of ObR. Non-detergent-treated cells were fixed and simultaneously stained with two ObR antibodies. A proximity ligation assay (PLA) indicated quantifiable and unchanged ObR expression on the surface of controls and cells treated with the specific GCS inhibitor NB-DNJ (<i>n</i> = 41–47 cells). PLA principle is depicted on the right side. (F–G) N-41 cells were incubated with either saline or 100 ng/ml leptin (10 min). Close interactions between GCS-derived neuronal gangliosides GD1a/ObR (F) and GM1/ObR (G) were detected by PLA. Leptin treatment dynamically increased the GD1a/ObR and GM1/ObR PLA spots per cell (<i>n</i> = 48–67 cells). (H) Extracts from saline- and leptin-treated N-41 cells were immunoprecipitated with an ObR antibody, lipids were extracted, and GD1a and GM1 were visualized by immune overlay TLC. GD1a and GM1 co-immunoprecipitated (Co-IP) with ObR, which tended to be stronger in leptin-treated cells. Addition of a blocking peptide almost totally abolished ganglioside signals. Gangliosides GD1b and GT1b, expressed in mouse brain tissue, were not co-precipitated with ObR from hypothalamic tissue of <i>Ugcg</i>^f/f mice (5 mg/kg leptin, 45 min). (I) Co-IP showed significantly decreased leptin-induced complex formation between ObR and Jak in NB-DNJ-treated N-41 cells (<i>n</i> = 4). (J) Sustainable Jak phosphorylation could be induced in N-41 cells after 15 min of leptin treatment (0.5 µg/ml). NB-DNJ-treated cells showed a markedly delayed response to leptin. (K) Thirty minutes after leptin treatment, Jak phosphorylation was decreased in NB-DNJ-treated cells (<i>n</i> = 4). *<i>p</i>≤0.05; ***<i>p</i>≤0.001. Means ± SEM.</p

rAAV-mediated <i>Ugcg</i> gene delivery to the hypothalamic Arc ameliorates obesity and hyperleptinemia in <i>Ugcg</i>^{f/f//CamKCreERT2} mice.

<p>(A) Double immunofluorescence showed that Cre activity, indicated by beta galactosidase staining (b-gal), was targeted to Arc neurons expressing the long form of the ObR, as indicated by PStat3 staining in leptin-injected <i>R26R/Ugcg</i>^{f/+//CamKCreERT2} mice (5 mg/kg leptin, 120 min). (B) Stereotactic delivery of rAA viruses encoding <i>Ugcg</i> and <i>lacZ</i> to the Arc of <i>Ugcg</i>^{f/f//CamKCreERT2} mice resulted in a significant amelioration in body weight increase compared to rAAV-Empty/lacZ-injected <i>Ugcg</i>^{f/f//CamKCreERT2} mice (<i>n</i> = 6–8). (C) Serum leptin tended to be lower in rAAV-Ugcg/lacZ-injected <i>Ugcg</i>^{f/f//CamKCreERT2} mice 8 wk p.i. (<i>n</i> = 6–8). (D–F) Targeting of rAAV Ugcg/lacZ- and rAAV Empty/lacZ-injected animals that were included in the analyses. At the end of the experiments, brains were removed and stained for X-Gal to indicate vector delivery. Red marks depict exemplarily areas of strong X-Gal staining in animals considered as Arc targeted. Depicted are areas between bregma −1.9 (D), bregma −2.1 (E), and bregma −2.3 (F). (G) Restored ganglioside biosynthesis in the Arc of rAAV-Ugcg-injected <i>Ugcg</i>^{f/f//CamKCreERT2} mice, as shown by GD1a immunofluorescence 8 wk p.i. Scale bar: 18 µm. *<i>p</i>≤0.05. Means ± SEM.</p

POMC and NPY neurons of <i>Ugcg</i>^{f/f//CamKCreERT2} mice are less responsive to leptin.

<p>(A) Leptin engages POMC neurons in the Arc of control (<i>Ugcg</i>^f/f) mice and <i>Ugcg</i>^{f/f//CamKCreERT2} mice 1–2 wk p.i., as indicated by elevated c-Fos. This response was decreased in Ugcg^{f/f//CamKCreERT2} mice 6 wk p.i. (B) Elevated leptin-induced PStat3 levels in POMC neurons of <i>Ugcg</i>^f/f mice and <i>Ugcg</i>^{f/f///CamKCreERT2} mice 1–2 wk p.i. This response was blunted in Ugcg^{f/f//CamKCreERT2} mice 6 wk p.i. (C) Leptin slightly decreased the activity of NPY neurons in <i>Ugcg</i>^f/f mice and <i>Ugcg</i>^{f/f///CamKCreERT2} mice 1–2 wk p.i. This was not detected in <i>Ugcg</i>^{f/f///CamKCreERT2} mice 6 wk p.i. (D) Unlike 1–2 wk p.i., leptin did not elevate PStat3 in NPY neurons of <i>Ugcg</i>^{f/f///CamKCreERT2} mice 6 wk p.i. Datasets for each time point were acquired individually, and quantification contains normalized data from two (1–2 wk p.i.; <i>n</i> = 4–11) or three (6 wk p.i.; <i>n</i> = 18–27) independent animal groups. Immunofluorescence and image acquisition for each dataset (treated and untreated controls and knockouts) were performed simultaneously. Scale bar: 20 µm; *<i>p</i>≤0.05; **<i>p</i>≤0.01; ***<i>p</i>≤0.001. Means ± SEM.</p