Genetic deletion of hepatic NCOR1 protects from atherosclerosis by promoting alternative bile acid-metabolism and sterol excretion

BACKGROUND The nuclear receptor corepressor 1 (NCOR1) plays an important role in the regulation of gene expression in immunometabolic conditions by connecting chromatin-modifying enzymes, coregulators and transcription factors. NCOR1 has been shown to be involved in cardiometabolic diseases. Recently, we demonstrated that the deletion of macrophage NCOR1 aggravates atherosclerosis by promoting CD36-triggered foam cell formation via PPARG derepression. PURPOSE Since NCOR1 modulates the function of several key regulators involved in hepatic lipid and bile acid metabolism, we hypothesized that its deletion in hepatocytes alters lipid metabolism and atherogenesis. METHODS To test this hypothesis, we generated hepatocyte-specific Ncor1 knockout mice on a Ldlr-/- background. Besides assessing the progression of the disease in thoracoabdominal aortae en face, we analyzed hepatic cholesterol and bile acid metabolism at expression and functional levels. RESULTS Our data demonstrate that liver-specific Ncor1 knockout mice on an atherosclerosis-prone background develop less atherosclerotic lesions than controls. Interestingly, under chow diet, plasma cholesterol levels of liver-specific Ncor1 knockout mice were slightly higher compared to control, but strongly reduced compared to control mice after feeding them an atherogenic diet for 12 weeks. Moreover, the hepatic cholesterol content was decreased in liver-specific Ncor1 knockout compared to control mice. Our mechanistic data revealed that NCOR1 reprograms the synthesis of bile acids towards the alternative pathway, which in turn reduce bile hydrophobicity and enhances fecal cholesterol excretion. CONCLUSIONS Our data suggest that hepatic Ncor1 deletion in mice decreases atherosclerosis development by reprograming bile acid metabolism and enhancing fecal cholesterol excretion

A Cepheid Distance to NGC 4603 in Centaurus

In an attempt to use Cepheid variables to determine the distance to the Centaurus cluster, we have obtained images of NGC 4603 with the Hubble Space Telescope on 9 epochs using WFPC2 and the F555W and F814W filters. This galaxy has been suggested to lie within the ``Cen30'' portion of the cluster and is the most distant object for which this method has been attempted. Previous distance estimates for Cen30 have varied significantly and some have presented disagreements with the peculiar velocity predicted from redshift surveys, motivating this investigation. Using our observations, we have found 61 candidate Cepheid variable stars; however, a significant fraction of these candidates are likely to be nonvariable stars whose magnitude measurement errors happen to fit a Cepheid light curve of significant amplitude for some choice of period and phase. Through a maximum likelihood technique, we determine that we have observed 43 +/- 7 real Cepheids and that NGC 4603 has a distance modulus of 32.61 +0.11/-0.10 (random, 1 sigma) +0.24/-0.25 (systematic, adding in quadrature), corresponding to a distance of 33.3 Mpc. This is consistent with a number of recent estimates of the distance to NGC 4603 or Cen30 and implies a small peculiar velocity consistent with predictions from the IRAS 1.2 Jy redshift survey if the galaxy lies in the foreground of the cluster.Comment: Accepted for publication in the Astrophysical Journal. 17 pages with 17 embedded figures and 3 tables using emulateapj.sty. Additional figures and images may be obtained from http://astro.berkeley.edu/~marc/n4603

Rapid Redistribution of Synaptic PSD-95 in the Neocortex In Vivo

Most excitatory synapses terminate on dendritic spines. Spines vary in size, and their volumes are proportional to the area of the postsynaptic density (PSD) and synaptic strength. PSD-95 is an abundant multi-domain postsynaptic scaffolding protein that clusters glutamate receptors and organizes the associated signaling complexes. PSD-95 is thought to determine the size and strength of synapses. Although spines and their synapses can persist for months in vivo, PSD-95 and other PSD proteins have shorter half-lives in vitro, on the order of hours. To probe the mechanisms underlying synapse stability, we measured the dynamics of synaptic PSD-95 clusters in vivo. Using two-photon microscopy, we imaged PSD-95 tagged with GFP in layer 2/3 dendrites in the developing (postnatal day 10–21) barrel cortex. A subset of PSD-95 clusters was stable for days. Using two-photon photoactivation of PSD-95 tagged with photoactivatable GFP (paGFP), we measured the time over which PSD-95 molecules were retained in individual spines. Synaptic PSD-95 turned over rapidly (median retention times τ (r) ~ 22–63 min from P10–P21) and exchanged with PSD-95 in neighboring spines by diffusion. PSDs therefore share a dynamic pool of PSD-95. Large PSDs in large spines captured more diffusing PSD-95 and also retained PSD-95 longer than small PSDs. Changes in the sizes of individual PSDs over days were associated with concomitant changes in PSD-95 retention times. Furthermore, retention times increased with developmental age (τ (r) ~ 100 min at postnatal day 70) and decreased dramatically following sensory deprivation. Our data suggest that individual PSDs compete for PSD-95 and that the kinetic interactions between PSD molecules and PSDs are tuned to regulate PSD size

GABAA receptor subtype involvement in addictive behaviour

GABAA receptors form the major class of inhibitory neurotransmitter receptors in the mammalian brain. This review sets out to summarise the evidence that variations in genes encoding GABAA receptor isoforms are associated with aspects of addictive behaviour in humans, while animal models of addictive behaviour also implicate certain subtypes of GABAA receptor. In addition to outlining the evidence for the involvement of specific subtypes in addiction, we summarise the particular contributions of these isoforms in control over the functioning of brain circuits, especially the mesolimbic system, and make a first attempt to bring together evidence from several fields to understanding potential involvement of GABAA Receptor Subtypes in addictive behaviour. While the weight of the published literature is on alcohol dependency, the underlying principles outlined are relevant across a number of different aspects of addictive behaviour

Modification of LIN-12 NOTCH signalling during vulval development in C. elegans

Zellkommunikation ist eine der wichtigsten Eigenschaften, die im Laufe der Evolution entwickelt worden ist, um vom Einzellstadium zu Organismen, die aus vielen verschiedenen Zellarten bestehen, zu wechseln. Deshalb wurden bereits sehr früh in der Geschichte der Vielzeller Signale erfunden, die von einer Zelle an die andere weitergeleitet werden, und deshalb sind diese Signalwege in Vielzellern stark konserviert. Das NOTCH Signal wird zum Beispiel von Zellen verwendet, die ihre benachbarten Zellen davon abbringen müssen, dasselbe Standard-Zellschicksal wie sie selbst anzunehmen. Beispiele für diese NOTCH-vermittelte laterale Hemmung findet man in der Entwicklung des Eiablage- und Kopulationsorgans von Fadenwürmern und während der Bildung von Nervenzellen in Drosophila. Das NOTCH Signal ist auch bei der Neubildung von Blutgefässen, der Entwicklung von Nervenzellen und der Spezifizierung von T-Zellen im Menschen wichtig. Der NOTCH-Signaltransduktionsweg kann als eine Art Schalter betrachtet werden, um schnell bestimmte Zellschicksale an- oder auszuschalten. Die spezifische Antwort der Zelle, die ein NOTCH Signal zugesendet bekommt, hängt von ihrer molekularen Zusammensetzung und den vorhandenen Zielfaktoren ab. Obwohl die Kernelemente des NOTCH Signals weitgehend bekannt sind, bleiben viele Zielfaktoren und Modifikatoren des NOTCH-Signaltransduktionsweges ungeklärt. Im ersten Teil dieser Doktorarbeit beschreibe ich einen neuen Zusammenhang zwischen dem Voranschreiten des Zellzykluses und dem NOTCH Signal in Caenorhabditis elegans: Ein bestimmter CDK/Cyclin Komplex (CDK-1/CYB-3), der während der G2 Phase des Zellzykluses aktiv ist, vermittelt nicht nur den Eintritt in die Mitose, sondern ist auch während des dritten Larvenstadiums für die korrekte Herabregulation des NOTCH Signals in der primären Vulvazelle wichtig. Zellzykluskomponenten, die während der G1 Phase aktiv sind, wie zum Beispiel die Cycline CYD-1 und CYE-1, haben dagegen einen positiven Einfluss auf das NOTCH Signal. Auf diese Weise ist die Spezifizierung von Zellschicksalen mittels des NOTCH- Signaltransduktionsweges eng verknüpft mit dem Voranschreiten des Zellzykluses. Ich schlage deshalb vor, dass das Durchlaufen des Zellzykluses zeitlich gesehen verschiedene intrazelluläre Zusammensetzungen schafft und somit einen Zeitgeber für Signaltransduktionsabläufe während bestimmter Entwicklungsphasen darstellt. Im zweiten Teil dieser Doktorarbeit zeige ich, dass das Gen ttr-11 ein Zielgen des NOTCH-Signaltransduktionsweges während der Entwicklung der Vulva von Caenorhabditis elegans ist. Das Gen ttr-11 wurde in einem Screen nach Kandidaten für NOTCH-Zielgene identifiziert und gehört zu einer großen Fadenwurm-spezifischen Genfamilie. Erst vor Kurzem haben Forscher begonnen die Funktion von manchen ttr Genen aufzudecken. Da Einzelmutationen in angehörigen Genen dieser Genfamilie nur geringe oder keine phänotypischen Effekte zeigen, und da das Genexpressionslevel von einzelnen ttr Genen oft niedrig ist, ist es schwierig die Aufgaben von ttr Genen in Fadenwürmern aufzuklären. Verschiedene Allele von ttr-11 haben milde aber gegenläufige Auswirkungen in verschiedenen mutanten Hintergründen zur Folge: Das NOTCH Signal von einer NOTCH Gain-of- Function-Mutante wird von wildtypischem ttr-11 gehemmt, wohingegen ttr- 11 in einem mutanten Hintergrund mit erhöhter EGFR/RAS/MAPK- Signaltransduktion einen positiven Einfluss auf das laterale Signal zeigt. Die milden Auswirkungen durch mutiertes ttr-11 könnten durch teilweise redundante Funktionen anderer Mitglieder der ttr Familie erklärt werden. Zum Beispiel zeigt ttr-57 hohe Sequenzähnlichkeit zu ttr-11. Jedoch halfen weder ttr-11 ttr-57 Doppelmutanten noch Überexpression von ttr-11 weiter die Aufgabe von ttr-11 während der Entwicklung der Vulva zu klären. Deshalb müssen weitere Experimente, wie zum Beispiel die Untersuchung der Funktionen von anderen nah verwandten ttr Genen in ttr-11 Mutanten, durchgeführt werden, um Licht ins Dunkel um die Funktion von ttr-11 zu bringen. SUMMARY Communication between cells is one of the most important features established during evolution to switch from a single cell organism to an organism made out of many different cell types. Therefore, signals that are sent from one cell to another have been invented quite early during the history of multicellular organisms and the signalling pathways transducing the signals are highly conserved in metazoans. For example, the NOTCH signal is used by cells to inhibit neighbouring cells from adopting the same, default cell fate. Examples for NOTCH-mediated lateral inhibition are nematode vulval development and Drosophila neurogenesis. NOTCH signalling is also important during human angiogenesis, neuronal development and T cell specification. The NOTCH signalling pathway can be regarded as a switch used to rapidly turn a specific fate on or off. The specific response of the cell receiving a NOTCH signal depends on the molecular environment and the downstream targets present in the cell. Although the roles of the core components of NOTCH signalling are well known, many downstream targets and modifiers of the NOTCH pathway remain to be elucidated. In the first part of this thesis, I describe a new connection between cell cycle progression and NOTCH signalling in the nematode Caenorhabditis elegans: One particular CDK/Cyclin complex (CDK-1/CYB-3) that is active during the G2 phase of the cell cycle not only mediates entry into mitosis, but it is also important for proper downregulation of the NOTCH signal in the primary vulval cell of the L3 larva. In contrast, cell cycle components active during the G1 phase, such as the cyclins CYD-1 and CYE-1, positively regulate NOTCH signalling in the vulval cells. Thus, cell fate specification via NOTCH signalling is tightly linked to cell cycle progression. I suggest that progression through cell cycle creates temporally different intracellular environments and therefore provides a timer for signalling events to occur during specific developmental phases. In a second part of this thesis, I show that the gene ttr-11 is a downstream target of NOTCH signalling during Caenorhabditis elegans vulval development. The gene ttr-11 was found in a screen for candidate NOTCH targets and belongs to a large nematode-specific gene family. Only recently, researchers have begun to uncover the function of some ttr genes. Since single mutants in members of this gene family show only a mild or no phenotype at all and because expression levels of individual ttr genes are often low, it is difficult to elucidate the role of ttr genes in nematodes. Different alleles of ttr-11 result in mild but contradictory effects in different mutant backgrounds: In a NOTCH gain-of-function background, wild-type ttr-11 inhibits the NOTCH signal, whereas ttr-11 shows a positive influence on lateral signalling in a background with elevated EGFR/RAS/MAPK signalling. The mild effects of mutated ttr-11 might be explained by partially redundant functions of other ttr-family members. For example, ttr-57 shows high sequence similarity to ttr-11. However, neither ttr-11 ttr-57 double mutants nor ttr-11 overexpression helped to clarify the role of ttr-11 during vulval development. Thus, further experiments, for example by examining the functions of other closely related ttr genes in ttr- 11 mutants, need to be performed to shed light on the function of ttr-11

Genetic deletion of hepatic NCOR1 protects from atherosclerosis by promoting alternative bile acid-metabolism and sterol excretion

Abstract Background The nuclear receptor corepressor 1 (NCOR1) plays an important role in the regulation of gene expression in immunometabolic conditions by connecting chromatin-modifying enzymes, coregulators and transcription factors. NCOR1 has been shown to be involved in cardiometabolic diseases. Recently, we demonstrated that the deletion of macrophage NCOR1 aggravates atherosclerosis by promoting CD36-triggered foam cell formation via PPARG derepression. Purpose Since NCOR1 modulates the function of several key regulators involved in hepatic lipid and bile acid metabolism, we hypothesized that its deletion in hepatocytes alters lipid metabolism and atherogenesis. Methods To test this hypothesis, we generated hepatocyte-specific Ncor1 knockout mice on a Ldlr-/- background. Besides assessing the progression of the disease in thoracoabdominal aortae en face, we analyzed hepatic cholesterol and bile acid metabolism at expression and functional levels. Results Our data demonstrate that liver-specific Ncor1 knockout mice on an atherosclerosis-prone background develop less atherosclerotic lesions than controls. Interestingly, under chow diet, plasma cholesterol levels of liver-specific Ncor1 knockout mice were slightly higher compared to control, but strongly reduced compared to control mice after feeding them an atherogenic diet for 12 weeks. Moreover, the hepatic cholesterol content was decreased in liver-specific Ncor1 knockout compared to control mice. Our mechanistic data revealed that NCOR1 reprograms the synthesis of bile acids towards the alternative pathway, which in turn reduce bile hydrophobicity and enhances fecal cholesterol excretion. Conclusions Our data suggest that hepatic Ncor1 deletion in mice decreases atherosclerosis development by reprograming bile acid metabolism and enhancing fecal cholesterol excretion

Deletion of fibroblast activation protein provides atheroprotection

AIMS Fibroblast activation protein (FAP) is upregulated at sites of tissue remodelling including chronic arthritis, solid tumours, and fibrotic hearts. It has also been associated with human coronary atherosclerotic plaques. Yet, the causal role of FAP in atherosclerosis remains unknown. To investigate the cause-effect relationship of endogenous FAP in atherogenesis, we assessed the effects of constitutive Fap deletion on plaque formation in atherosclerosis-prone apolipoprotein E (Apoe) or low-density lipoprotein receptor (Ldlr) knockout mice. METHODS AND RESULTS Using en face analyses of thoraco-abdominal aortae and aortic sinus cross-sections, we demonstrate that Fap deficiency decreased plaque formation in two atherosclerotic mouse models (-46% in Apoe and -34% in Ldlr knockout mice). As a surrogate of plaque vulnerability fibrous cap thickness was used; it was increased in Fap-deficient mice, whereas Sirius red staining demonstrated that total collagen content remained unchanged. Using polarized light, atherosclerotic lesions from Fap-deficient mice displayed increased FAP targets in terms of enhanced collagen birefringence in plaques and increased pre-COL3A1 expression in aortic lysates. Analyses of the Stockholm Atherosclerosis Gene Expression (STAGE) data revealed that FAP expression was increased in human atherosclerotic compared to non-atherosclerotic arteries. CONCLUSIONS Our data provide causal evidence that constitutive Fap deletion decreases progression of experimental atherosclerosis and increases features of plaque stability with decreased collagen breakdown. Thus, inhibition of FAP expression or activity may not only represent a promising therapeutic target in atherosclerosis but appears safe at the experimental level for FAP-targeted cancer therapies. TRANSLATIONAL PERSPECTIVE Fibroblast activation protein (FAP) is upregulated at sites of chronic tissue remodelling including rheumatoid arthritis and solid tumours. Indeed, depletion of FAP-positive cells inhibits tumour growth by increasing antitumour immunity. FAP has also been correlated with human coronary plaques, whereby its causal role remains unknown. Our data provide causal evidence that constitutive Fap deletion decreases progression of experimental atherosclerosis and increases features of plaque stability. Thus, inhibition of FAP expression or activity may not only represent a novel therapeutic target for atherosclerosis but appears safe - at the experimental level - for FAP-targeted cancer therapies

Macrophage NCOR1 protects from atherosclerosis by repressing a pro-atherogenic PPARγ signature

AIMS Nuclear receptors and their cofactors regulate key pathophysiological processes in atherosclerosis development. The transcriptional activity of these nuclear receptors is controlled by the nuclear receptor corepressors (NCOR), scaffolding proteins that form the basis of large corepressor complexes. Studies with primary macrophages demonstrated that the deletion of Ncor1 increases the expression of atherosclerotic molecules. However, the role of nuclear receptor corepressors in atherogenesis is unknown. METHODS AND RESULTS We generated myeloid cell-specific Ncor1 knockout mice and crossbred them with low-density lipoprotein receptor (Ldlr) knockouts to study the role of macrophage NCOR1 in atherosclerosis. We demonstrate that myeloid cell-specific deletion of nuclear receptor corepressor 1 (NCOR1) aggravates atherosclerosis development in mice. Macrophage Ncor1-deficiency leads to increased foam cell formation, enhanced expression of pro-inflammatory cytokines, and atherosclerotic lesions characterized by larger necrotic cores and thinner fibrous caps. The immunometabolic effects of NCOR1 are mediated via suppression of peroxisome proliferator-activated receptor gamma (PPARγ) target genes in mouse and human macrophages, which lead to an enhanced expression of the CD36 scavenger receptor and subsequent increase in oxidized low-density lipoprotein uptake in the absence of NCOR1. Interestingly, in human atherosclerotic plaques, the expression of NCOR1 is reduced whereas the PPARγ signature is increased, and this signature is more pronounced in ruptured compared with non-ruptured carotid plaques. CONCLUSIONS Our findings show that macrophage NCOR1 blocks the pro-atherogenic functions of PPARγ in atherosclerosis and suggest that stabilizing the NCOR1-PPARγ binding could be a promising strategy to block the pro-atherogenic functions of plaque macrophages and lesion progression in atherosclerotic patients