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

Untersuchungen zu neuroanatomischen Veränderungen beim Gesangslernen des Zebrafinken Taeniopygia guttata

Der Gesangserwerb bei Singvögeln ist ein etabliertes Modell zur Erforschung von Lern- und Gedächtnisprozessen. Für die Kontrolle des Gesangsverhaltens von Singvögeln ist ein neuronales Netzwerk verantwortlich, das als Gesangssystem bezeichnet wird. Innerhalb dieses Gesangssystems unterscheidet man zwei Hauptstränge: eine prämotorische Bahn, die für die Steuerung des Gesangs verantwortlich ist, und eine anteriore Vorderhirnschleife („anterior forebrain pathway"; AFP), die mit der Niederlegung eines Gesangsmusters im Gedächtnis und mit dem Abgleich des gehörten Gesangs an dieses Gesangsmuster in Zusammenhang gebracht wird. Bei Zebrafinken (Taeniopygia guttata) lernen nur die Männchen singen, während Weibchen lediglich angeborene Kontaktrufe äußern. Gemeinsam ist beiden Geschlechtern, dass sie arteigenen Gesang, in Form eines Gesangsmusters im Gedächtnis speichern. In der vorliegenden Arbeit wurde gezeigt, dass die Konnektivitäten innerhalb der AFP in beiden Geschlechtern ähnlich verlaufen. Auf diesen Ergebnissen aufbauend wurden die Gesangsareale der AFP adulter Zebrafinken vergleichend zwischen den Geschlechtern bezüglich ihrer Volumenausbildung untersucht und die Anzahl, Dichte und Größe der Neurone und deren Zellkerne innerhalb dieser Gesangsareale bestimmt. Des Weiteren wurden adulte Zebrafinken auf Folgen gesangsdeprivierter Aufzucht untersucht, in der Annahme, dass morphologische Unterschiede zwischen sozial und gesangsdepriviert aufgezogenen Tieren Hinweise auf Ort und Art der Speicherung des erlernten Gesangsmusters geben könnten. Solche Veränderungen wurden bezüglich des Volumens des Nucleus dorsolateralis medialis des anterioren Thalamus (DLM) und der Neuronendichte im Nucleus robustus arcopallii (RA) gesangsdeprivierter Männchen nachgewiesen. Die bei Weibchen ermittelten morphologischen Unterschiede durch gesangsdeprivierte Aufzucht konzentrierten sich auf den RA, in dem sowohl das Arealvolumen als auch die Größe der Neurone und der Nuklei signifikant kleiner waren. Da Zebrafinkenweibchen lediglich angeborene Kontaktrufe äußern, an deren Produktion der RA nicht beteiligt ist, könnten diese deprivationsbedingten Veränderungen in Zusammenhang mit der Speicherung des arteigenen Gesangsmusters stehen.Song learning has emerged as one of the leading model systems for studying learning in vertebrates. In the avian brain there is a specific neuronal network, the so called song system, that controls song behaviour. This song system consists of two major pathways: the motor pathway which is responsible for song production, and the anterior forebrain pathway (AFP) which is important for song acquisition and adjustment of their vocalization to the learned song pattern. In zebra finches (Taeniopygia guttata) males learn to sing, whereas females only produce congenital contact calls. However, both sexes store a species specific song template in their memory. In this study it has been shown that there is high similarity between the connectivities of the AFP in males and females. Based on this, the song nuclei of the AFP of adult male and female zebra finches have been analyzed, concerning their volume, their neuronal number, density and size and whether differences between both sexes do exist. Furthermore, the impact of song deprivation in adult zebra finches has been examined. This study was based on the assumption that morphological differences between social and song deprived reared birds could give evidence for the place and mode of storing the learned song pattern. Differences between social and song deprived reared males were found in both the volume of the dorsolateral nucleus of the anterior thalamus (DLM) and the neuronal density in the robust nucleus of the arcopallium (RA). In females, morphological changes due to song deprivation were mainly found in RA. In this brain area the volume as well as the size of neurons and their nuclei were singnificantly reduced. Since females only produce congenital contact calls and since the production of these calls does not require the RA, the changes induced by song deprivation in females might be related to the storage of the conspecific song pattern

Deficiency in FTSJ1 Affects Neuronal Plasticity in the Hippocampal Formation of Mice

Simple Summary Neuronal plasticity refers to the brain’s ability to adapt in response to activity-dependent changes. This process, among others, allows the brain to acquire memory or to compensate for a neurocognitive deficit. We analyzed adult FTSJ1-deficient mice in order to gain insight into the role of FTSJ1 in neuronal plasticity. These mice displayed alterations in the hippocampus (a brain structure that is involved in memory and learning, among other functions) e.g., in the form of changes in dendritic spines. Changes in dendritic spines are considered to represent a morphological hallmark of altered neuronal plasticity, and thus FTSJ1 deficiency might have a direct effect upon the capacity of the brain to adapt to plastic changes. Long-term potentiation (LTP) is an electrophysiological correlate of neuronal plasticity, and is related to learning and to processes attributed to memory. Here we show that LTP in FTSJ1-deficient mice is reduced, hinting at disturbed neuronal plasticity. These findings suggest that FTSJ1 deficiency has an impact on neuronal plasticity not only morphologically but also on the physiological level. Abstract The role of the tRNA methyltransferase FTSJ1 in the brain is largely unknown. We analyzed whether FTSJ1-deficient mice (KO) displayed altered neuronal plasticity. We explored open field behavior (10 KO mice (aged 22–25 weeks)) and 11 age-matched control littermates (WT) and examined mean layer thickness (7 KO; 6 WT) and dendritic spines (5 KO; 5 WT) in the hippocampal area CA1 and the dentate gyrus. Furthermore, long-term potentiation (LTP) within area CA1 was investigated (5 KO; 5 WT), and mass spectrometry (MS) using CA1 tissue (2 each) was performed. Compared to controls, KO mice showed a significant reduction in the mean thickness of apical CA1 layers. Dendritic spine densities were also altered in KO mice. Stable LTP could be induced in the CA1 area of KO mice and remained stable at for at least 1 h, although at a lower level as compared to WTs, while MS data indicated differential abundance of several proteins, which play a role in neuronal plasticity. FTSJ1 has an impact on neuronal plasticity in the murine hippocampal area CA1 at the morphological and physiological levels, which, in conjunction with comparable changes in other cortical areas, might accumulate in disturbed learning and memory functions

Global Protein Profiling in Processed Immunohistochemistry Tissue Sections

Tissue sections, which are widely used in research and diagnostic laboratories and have already been examined by immunohistochemistry (IHC), may subsequently provide a resource for proteomic studies, even though only small amount of protein is available. Therefore, we established a workflow for tandem mass spectrometry-based protein profiling of IHC specimens and characterized defined brain area sections. We investigated the CA1 region of the hippocampus dissected from brain slices of adult C57BL/6J mice. The workflow contains detailed information on sample preparation from brain slices, including removal of antibodies and cover matrices, dissection of region(s) of interest, protein extraction and digestion, mass spectrometry measurement, and data analysis. The Gene Ontology (GO) knowledge base was used for further annotation. Literature searches and Gene Ontology annotation of the detected proteins verify the applicability of this method for global protein profiling using formalin-fixed and embedded material and previously used IHC slides

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

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

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