Scavenger receptor, class B, type I mediates the uptake and degradation of beta-VLDL particles in tissue culture

Das Ziel dieser Arbeit war, die Rolle von SR-BI im Stoffwechsel von ß-VLDL zu untersuchen. Da SR-BI kürzlich mit ß-VLDL Aufnahme in Verbindung gebracht wurde (Van Eck et al., 2007), war es von Interesse, die Wichtigkeit und Merkmale dieses Prozesses zu analysieren. Die Verwendung von zwei verschiedenen Zellsystemen, LDL-Rezeptor positive HepG2 and CHO Zellen und LDL-Rezeptor negative ldlA7 und ldlA7-SRBI Zellen, erlaubte eine Bewertung des Einflusses von SR-BI und des LDL-Rezeptors im Metabolismus von ß-VLDL. Die Experimente zeigten, dass die Mehrheit des assoziierten ß-VLDL von den Zellen aufgenommen wurde und dass die internalisierten Partikel nicht wieder von der Zelle abgegeben werden, was auf eine „Holopartikel“-Aufnahme hindeutet. Wir haben weiters herausgefunden, dass SR-BI Überexpression zu einer ~ 2-fach höheren ß-VLDL Zellassoziation führt. Die höhere ß-VLDL Assoziation in CHO Zellen verglichen mit ldlA7-SRBI Zellen untermauert die verhältnismäßige Wichtigkeit des LDL-Rezeptors. Diese wird bestätigt durch die Ergebnisse von SR-BI knockdown in HepG2 Zellen, welcher die ß-VLDL Assoziation nicht beeinträchtigt hat. Vorherige Studien entsprechen diesen Ergebnissen bezüglich der Wichtigkeit des LDL-Rezeptors im ß-VLDL Metabolismus. ß-VLDL wurde von den Zellen umgesetzt, was anhand des Anstiegs des zellulären Fettgehaltes (Cholesterin, Cholesterol Ester und Triglyceride) nach ß-VLDL Zufuhr gezeigt wurde. Die Abbaurate des Partikels war gering, aber messbar, was mit früheren Ergebnissen in Makrophagen übereinstimmt. Zusammenfassend kann gesagt werden, dass SR-BI die Bindung, Aufnahme und den Abbau von ß-VLDL in Zellkultursystemen vermittelt. Dennoch scheint SR-BI eine untergeordnete Funktion zu übernehmen, da der LDL-Rezeptor eine entscheidende Rolle im Metabolismus von ß-VLDL spielt, die aber an Wichtigkeit gewinnen könnte indem er beeinträchtigte pathways kompensiert.The present study was designed to elucidate the role of SR-BI in ß-VLDL metabolism. As SR-BI has recently been implicated in ß-VLDL uptake (Van Eck et al., 2007) it was of interest to further investigate the importance and characteristics of this process. The utilization of two different sets of cells, LDL-receptor positive HepG2 and CHO cells and LDL-receptor negative ldlA7 and ldlA7-SRBI cells, allowed an evaluation of the contribution of SR-BI and the LDL-receptor in ß-VLDL metabolism. Experiments illustrated that the majority of associated ß-VLDL is taken up by the cells and internalized particles can not be released back to the media, indicating an holoparticle uptake. We also found that SR-BI overexpression leads to a ~ 2-fold higher ß-VLDL cell association. The higher ß-VLDL association in CHO cells compared to ldlA7-SRBI cells provides evidence for the relative importance of the LDL-receptor. This is substantiated by the results from SR-BI knockdown in HepG2 cells, that did not impair ß-VLDL cell association. Former studies are in line with the present results concerning the importance of the LDL-receptor in ß-VLDL metabolism (Herijgers et al., 2000; Van Lenten et al., 1985; Perrey et al., 2001). ß-VLDL was utilized by the cells as indicated by an increase in the cellular lipid content (cholesterol, cholesteryl esters and triglycerides) after ß-VLDL feeding. The degradation of the particle was low but measurable, which is in accordance with former findings in macrophages (Tabas et al., 1990). In summary, SR-BI was shown to mediate the binding, uptake and degradation of ß- VLDL particles in tissue culture. However, SR-BI seems to play a subsidiary role, as the LDL-receptor is particularly prominent in ß-VLDL metabolism, that might gain importance in cases where it compensates for defective pathways

Differential basolateral–apical distribution of scavenger receptor, class B, type I in cultured cells and the liver

The high-density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediates selective cholesteryl ester uptake into the liver, which finally results in cholesterol secretion into the bile. Despite several reports, the distribution of hepatic SR-BI between the sinusoidal and canalicular membranes is still under debate. We present immunohistological data using specific markers showing that the bulk of SR-BI is present in sinusoidal membranes and, to a lesser extent, in canalicular membranes in murine and human liver sections. In addition, SR-BI was detected in preparations of rat liver canalicular membranes. We also compared the in vivo findings to HepG2 cells, a widely used in vitro hepatocyte model. Interestingly, SR-BI was enriched in bile canalicular-like (BC-like) structures in polarized HepG2 cells, which were cultivated either conventionally to form a monolayer or in Matrigel to form three-dimensional structures. Fluorescently labeled HDL was transported into close proximity of BC-like structures, whereas HDL labeled with the fluorescent cholesterol analog BODIPY-cholesterol was clearly detected within these structures. Importantly, similarly to human and mouse liver, SR-BI was localized in basolateral membranes in three-dimensional liver microtissues from primary human liver cells. Our results demonstrate that SR-BI is highly enriched in sinusoidal membranes and is also found in canalicular membranes. There was no significant basolateral–apical redistribution of hepatic SR-BI in fasting and refeeding experiments in mice. Furthermore, in vitro studies in polarized HepG2 cells showed explicit differences as SR-BI was highly enriched in BC-like structures. These structures are, however, functional and accumulated HDL-derived cholesterol. Thus, biological relevant model systems should be employed when investigating SR-BI distribution in vitro. Electronic supplementary material The online version of this article (doi:10.1007/s00418-014-1251-9) contains supplementary material, which is available to authorized users

APP‑BACE1 Interaction and Intracellular Localization Regulate Aβ Production in iPSC‑Derived Cortical Neurons

Alzheimer’s disease (AD) is characterized pathologically by amyloid β (Aβ)-containing plaques. Generation of Aβ from amyloid precursor protein (APP) by two enzymes, β- and γ-secretase, has therefore been in the AD research spotlight for decades. Despite this, how the physical interaction of APP with the secretases influences APP processing is not fully understood. Herein, we compared two genetically identical human iPSC-derived neuronal cell types: low Aβ-secreting neuroprogenitor cells (NPCs) and high Aβ-secreting mature neurons, as models of low versus high Aβ production. We investigated levels of substrate, enzymes and products of APP amyloidogenic processing and correlated them with the proximity of APP to β- and γ-secretase in endo-lysosomal organelles. In mature neurons, increased colocalization of full-length APP with the β-secretase BACE1 correlated with increased β-cleavage product sAPPβ. Increased flAPP/BACE1 colocalization was mainly found in early endosomes. In the same way, increased colocalization of APP-derived C-terminal fragment (CTF) with presenilin-1 (PSEN1), the catalytic subunit of γ-secretase, was seen in neurons as compared to NPCs. Furthermore, most of the interaction of APP with BACE1 in low Aβ-secreting NPCs seemed to derive from CTF, the remaining APP part after BACE1 cleavage, indicating a possible novel product-enzyme inhibition. In conclusion, our results suggest that interaction of APP and APP cleavage products with their secretases can regulate Aβ production both positively and negatively. β- and γ-Secretases are difficult targets for AD treatment due to their ubiquitous nature and wide range of substrates. Therefore, targeting APP-secretase interactions could be a novel treatment strategy for AD

TREM2 is down-regulated by HSV1 in microglia and involved in antiviral defense in the brain

Immunological control of viral infections in the brain exerts immediate protection and also long-term maintenance of brain integrity. Microglia are important for antiviral defense in the brain. Here, we report that herpes simplex virus type 1 (HSV1) infection of human induced pluripotent stem cell (hiPSC)-derived microglia down-regulates expression of genes in the TREM2 pathway. TREM2 was found to be important for virus-induced IFNB induction through the DNA-sensing cGAS-STING pathway in microglia and for phagocytosis of HSV1-infected neurons. Consequently, TREM2 depletion increased susceptibility to HSV1 infection in human microglia-neuron cocultures and in the mouse brain. TREM2 augmented STING signaling and activation of downstream targets TBK1 and IRF3. Thus, TREM2 is important for the antiviral immune response in microglia. Since TREM2 loss-of-function mutations and HSV1 serological status are both linked to Alzheimer's disease, this work poses the question whether genetic or virus-induced alterations of TREM2 activity predispose to post-infection neurological pathologies

ER stress induces caspase-2-tBID-GSDME-dependent cell death in neurons lytically infected with herpes simplex virus type 2

Neurotropic viruses, including herpes simplex virus (HSV) types 1 and 2, have the capacity to infect neurons and can cause severe diseases. This is associated with neuronal cell death, which may contribute to morbidity or even mortality if the infection is not controlled. However, the mechanistic details of HSV-induced neuronal cell death remain enigmatic. Here, we report that lytic HSV-2 infection of human neuron-like SH-SY5Y cells and primary human and murine brain cells leads to cell death mediated by gasdermin E (GSDME). HSV-2-induced GSDME-mediated cell death occurs downstream of replication-induced endoplasmic reticulum stress driven by inositol-requiring kinase 1α (IRE1α), leading to activation of caspase-2, cleavage of the pro-apoptotic protein BH3-interacting domain death agonist (BID), and mitochondria-dependent activation of caspase-3. Finally, necrotic neurons released alarmins, which activated inflammatory responses in human iPSC-derived microglia. In conclusion, lytic HSV infection in neurons activates an ER stress-driven pathway to execute GSDME-mediated cell death and promote inflammation.</p

Investigation of HDL endocytosis in endothelial cells and the influence of mTOR inhibition on the interplay of HDL and endothelial cells

High density lipoprotein (HDL) besitzt anti-atherogene Eigenschaften, die vermutlich für die umgekehrt proportionale Korrelation der HDL-Cholesterin Spiegel im Blut und dem Auftreten von kardiovaskulären Erkrankungen ausschlaggebend sind. Die wahrscheinlich am besten beschriebene dieser Eigenschaften ist die Beteiligung der HDL Partikel am Reversen Cholesterin Transport (RCT), bei welchem HDL überschüssiges Cholesterin von peripheren Zellen zur Leber transportiert, dem Ort an dem Cholesterin vom Körper ausgeschieden werden kann. Damit HDL Cholesterin, das sich in Schaumzellen in der Arterienwand angesammelt hat, aufnehmen und abtransportieren kann, müssen die HDL Partikel notwendigerweise das vaskuläre Endothel durchqueren. Die genauen Mechanismen dieser sogenannten HDL Transzytose sind nicht bekannt. In der vorliegenden Studie wollten wir zum einen den Aufnahmeprozess der HDL Partikel genau untersuchen und zum anderen den Einfluss des mammalian target of rapamycin (mTOR)-Inhibitors Rapamycin auf den HDL-Rezeptor scavenger receptor, class B, type I (SR-BI) und auf die Funktion der Endothelzellen charakterisieren. Wir haben die Aufnahme der HDL Partikel sowie den Transfer von Lipiden von HDL-Partikeln in primären Endothelzellen (human umbilical vein endothelial cells (HUVECs)) mittels Licht- und Elektronenmikoskopischer Techniken untersucht. Mit Hilfe neuer, fluoreszierender Cholesterin-Surrogate haben wir den Transfer von Cholesterin und Cholesterin Estern aus HDL Partikeln analysiert. Wir konnten in HUVECs zeigen, dass HDL verschiedene Stadien des Endozytose-Prozesses, nämlich clathrin-coated pits, tubuläre Endosomen und multivesikulären Endosomen, passiert. Zusätzlich haben wir die Aufnahme von HDL in vivo verfolgt, indem wir fluoreszenz-markiertes HDL in Mäuse injiziert haben. In der Leber war HDL vor allem in Endothelzellen lokalisiert, was die Bedeutung der HDL- Aufnahme im Endothel unterstreicht. Als Nächstes haben wir die Auswirkungen des mTOR-Inhibitors Rapamycin, der als immunsupprimierendes Medikament eingesetzt wird, auf Endothelzell-Migration, Stickstoffmonoxid (NO) Produktion sowie die Expression und Funktion des HDL-Rezeptors SR-BI untersucht. Die Kinase mTOR ist zentraler Bestandteil eines komplexen Netzwerkes, das den zellulären Stoffwechsel kontrolliert. mTOR-Inhibitoren haben viele positive Effekte aber auch unerwünschte systemische Nebenwirkungen, einschließlich Hyperlipidämie. Wir konnten zeigen, dass die SR-BI Expression in HUVECs durch Rapamycin herunterreguliert wird, was aber keinen Einfluss auf die HDL-Aufnahme hatte. Rapamycin hat weiters die durch HDL stimulierte Phosphorylierung von Akt und endothelial nitric oxide synthase (eNOS) beeinträchtigt und in der Folge die NO Produktion vermindert. Gleichermaßen war HDL stimulierte Endothelzell-Migration beeinträchtigt. Da SR-BI neben seiner Funktion bei der HDL-Aufnahme auch in die Aktivierung zellulärer Signaltransduktion durch HDL involviert ist, haben wir die Beteiligung von SR-BI an eben genannten Merkmalen endothelialer Dysfunktion, ausgelöst durch Rapamycin, analysiert. SR-BI Inaktivierung alleine hatte keinen Einfluss auf Zell-Migration oder eNOS Aktivierung, allerdings konnte SR-BI Überexprimierung der durch Rapamycin induzierten endothelialen Dysfunktion teilweise entgegenwirken. In dieser Arbeit haben wir die Aufnahme von HDL und den Lipidaustausch in humanen Endothelzellen in vitro und in vivo charakterisiert. Weiters haben wir die Auswirkungen des mTOR-Inhibitors Rapamycin auf SR-BI Expression und die durch HDL stimulierte NO Produktion und Zell-Migration in humanen Endothelzellen analysiert. Unsere Ergebnisse tragen zum Verständnis der HDL Endozytose in Endothelzellen bei und beschreiben die Effekte von Rapamycin auf endotheliale Dysfunktion, ein frühes Merkmal der Atherosklerose.High density lipoproteins (HDL) possess several anti-atherogenic properties which are likely responsible for the inverse correlation of HDL-cholesterol levels with the incidence of cardiovascular disease. The probably best described function of HDL particles is their contribution to reverse cholesterol transport (RCT) by facilitating the transport of excess cholesterol from peripheral tissues to the liver, the site of cholesterol excretion in the body. HDL transcytosis through the vascular endothelium has been proposed to be crucial for the removal of accumulating cholesterol from macrophage foam cells, residing in the subendothelial space of arteries, but the exact mechanisms are not fully understood. In this study we sought to identify on one hand the way stations of HDL uptake in endothelial cells and on the other hand the influence of the mammalian target of rapamycin (mTOR) inhibitor rapamycin on the regulation of the HDL receptor scavenger receptor, class B, type I (SR-BI) and on endothelial cell functions. We analyzed HDL particle uptake and HDL-derived lipid uptake in primary human umbilical vein endothelial cells (HUVECs) with light and electron microscopical methods. Using novel fluorescent cholesterol surrogates we followed the uptake of cholesterol and cholesterol esters from HDL particles. Moreover, we found HDL to be endocytosed via clathrin-coated pits, tubular endosomes and multivesicular bodies in HUVECs. In addition, we followed the fate of HDL in vivo, by injecting fluorescently labeled HDL into mice. In the liver, HDL was primarily found in endothelial cells, emphasizing the importance of the endothelium in HDL uptake. Next, we investigated the consequences of mTOR inhibition using rapamycin, which is an approved immunosuppressant drug, on endothelial cell migration, nitric oxide (NO) production and the expression and function of the HDL receptor SR-BI. Inhibition of the kinase mTOR, which is a pivotal regulator of cellular metabolism, has despite many beneficial outcomes severe systemic side effects as well, including hyperlipidemia. We found that rapamycin down-regulates SR-BI expression in HUVECs, but surprisingly did not impair HDL uptake. In addition, mTOR inhibition led to a decrease in HDL stimulated Akt and endothelial nitric oxide synthase (eNOS) phosphorylation, resulting in impaired nitric oxide (NO) production. Similarly, HDL induced endothelial cell migration was affected. Since SR-BI is not only responsible for HDL uptake but also HDL induced cellular signaling, the contribution of SR-BI to these features of endothelial dysfunction seen upon rapamycin treatment was assessed by SR-BI knockdown and overexpression. SR-BI knockdown alone could not mimic the rapamycin dependent effects on cell migration or eNOS activation, whereas SR-BI overexpression could partly counteract endothelial dysfunction. In summary, we have investigated HDL uptake and its lipid exchange in human endothelial cells in vitro and in murine liver in vivo. In addition, we have analyzed the effects of mTOR inhibition using rapamycin on SR-BI expression, HDL stimulated NO production, and cell migration in human endothelial cells. Our observations contribute to the understanding of HDL endocytosis in endothelial cells and the mechanisms by which rapamycin influences lipid metabolism and endothelial dysfunction, an early hallmark of atherosclerosis.submitted by Stefanie FruhwürthAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Med. Univ., Diss., 2014OeBB(VLID)171461

Bile acids reduce endocytosis of high-density lipoprotein (HDL) in HepG2 cells.

High-density lipoprotein (HDL) transports lipids to hepatic cells and the majority of HDL-associated cholesterol is destined for biliary excretion. Cholesterol is excreted into the bile directly or after conversion to bile acids, which are also present in the plasma as they are effectively reabsorbed through the enterohepatic cycle. Here, we provide evidence that bile acids affect HDL endocytosis. Using fluorescent and radiolabeled HDL, we show that HDL endocytosis was reduced in the presence of high concentrations of taurocholate, a natural non-cell-permeable bile acid, in human hepatic HepG2 and HuH7 cells. In contrast, selective cholesteryl-ester (CE) uptake was increased. Taurocholate exerted these effects extracellularly and independently of HDL modification, cell membrane perturbation or blocking of endocytic trafficking. Instead, this reduction of endocytosis and increase in selective uptake was dependent on SR-BI. In addition, cell-permeable bile acids reduced HDL endocytosis by farnesoid X receptor (FXR) activation: chenodeoxycholate and the non-steroidal FXR agonist GW4064 reduced HDL endocytosis, whereas selective CE uptake was unaltered. Reduced HDL endocytosis by FXR activation was independent of SR-BI and was likely mediated by impaired expression of the scavenger receptor cluster of differentiation 36 (CD36). Taken together we have shown that bile acids reduce HDL endocytosis by transcriptional and non-transcriptional mechanisms. Further, we suggest that HDL endocytosis and selective lipid uptake are not necessarily tightly linked to each other

Differential basolateral–apical distribution of scavenger receptor, class B, type I in cultured cells and the liver

The high-density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediates selective cholesteryl ester uptake into the liver, which finally results in cholesterol secretion into the bile. Despite several reports, the distribution of hepatic SR-BI between the sinusoidal and canalicular membranes is still under debate. We present immunohistological data using specific markers showing that the bulk of SR-BI is present in sinusoidal membranes and, to a lesser extent, in canalicular membranes in murine and human liver sections. In addition, SR-BI was detected in preparations of rat liver canalicular membranes. We also compared the in vivo findings to HepG2 cells, a widely used in vitro hepatocyte model. Interestingly, SR-BI was enriched in bile canalicular-like (BC-like) structures in polarized HepG2 cells, which were cultivated either conventionally to form a monolayer or in Matrigel to form three-dimensional structures. Fluorescently labeled HDL was transported into close proximity of BC-like structures, whereas HDL labeled with the fluorescent cholesterol analog BODIPY-cholesterol was clearly detected within these structures. Importantly, similarly to human and mouse liver, SR-BI was localized in basolateral membranes in three-dimensional liver microtissues from primary human liver cells. Our results demonstrate that SR-BI is highly enriched in sinusoidal membranes and is also found in canalicular membranes. There was no significant basolateral–apical redistribution of hepatic SR-BI in fasting and refeeding experiments in mice. Furthermore, in vitro studies in polarized HepG2 cells showed explicit differences as SR-BI was highly enriched in BC-like structures. These structures are, however, functional and accumulated HDL-derived cholesterol. Thus, biological relevant model systems should be employed when investigating SR-BI distribution in vitro.ISSN:0948-6143ISSN:1432-119

Bile acids and a non-steroidal FXR agonist reduce HDL endocytosis.

<p>(a) HepG2 cells were treated with the indicated concentrations of GW4064 or chenodeoxycholate (CDCA) in media containing lipoprotein-deficient serum (lpds) for 24 hours. Gene expression was analyzed by qRT-PCR and expression levels were normalized to GAPDH expression (n = 2). The increase in SHP mRNA indicates FXR activation. (b) HepG2 cells were incubated with 10 µM GW4064 or 100 µM CDCA in media containing lpds for 24 hours. Cells were then incubated with 50 µg/ml HDL-Alexa⁴⁸⁸ for 1 hour. Cells were fixed, counterstained with DAPI and imaged. Green: HDL; blue: nucleus; bar = 10 µm. (c) Quantification of fluorescence intensities of (b). (d) HepG2 cells were incubated with 10 µM GW4064 or 100 µM CDCA in media containing lpds for 24 hours. Cells were then incubated with 20 µg/ml ¹²⁵I-HDL for 1 hour. Uptake was determined after displacing cell surface bound HDL by a 100-fold excess at 4°C for 1 hour (n = 3).</p