A fluorescent sphingolipid binding domain peptide probe interacts with sphingolipids and cholesterol-dependent raft domains

We have designed a tagged probe [sphingolipid binding domain (SBD)] to facilitate the tracking of intracellular movements of sphingolipids in living neuronal cells. SBD is a small peptide consisting of the SBD of the amyloid precursor protein. It can be conjugated to a fluorophore of choice and exogenously applied to cells, thus allowing for in vivo imaging. Here, we present evidence to describe the characteristics of the SBD association with the plasma membrane. Our experiments demonstrate that SBD binds to isolated raft fractions from human neuroblastomas and insect neuronal cells. In protein-lipid overlay experiments, SBD interacts with a subset of glycosphingolipids and sphingomyelin, consistent with its raft association in neurons. We also provide evidence that SBD is taken up by neuronal cells in a cholesterol- and sphingolipid-dependent manner via detergent-resistant microdomains. Furthermore, using fluorescence correlation spectroscopy to assay the mobility of SBD in live cells, we show that SBD's behavior at the plasma membrane is similar to that of the previously described raft marker cholera toxin B, displaying both a fast and a slow component. Our data suggest that fluorescently tagged SBD can be used to investigate the dynamic nature of glycosphingolipid-rich detergent-resistant microdomains that are cholesterol-dependent.     

Sphingolipids as New Biomarkers for Assessment of Delayed-Type Hypersensitivity and Response to Triptolide

Background

Hypersensitivity diseases are associated with many severe human illnesses, including leprosy and tuberculosis. Emerging evidence suggests that the pathogenesis and pathological mechanisms of treating these diseases may be attributable to sphingolipid metabolism.

Methods

High performance liquid chromatography-tandem mass spectrometry was employed to target and measure 43 core sphingolipids in the plasma, kidneys, livers and spleens of BALB/c mice from four experimental groups: control, delayed-type hypersensitivity (DTH) model, DTH+triptolide, and control+triptolide. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to identify potential biomarkers associated with variance between groups. Relationships between the identified biomarkers and disease markers were evaluated by Spearman correlation.

Results

As a treatment to hypersensitivity disease, triptolide significantly inhibit the ear swelling and recover the reduction of splenic index caused by DTH. The sphingolipidomic result revealed marked alterations in sphingolipid levels between groups that were associated with the effects of the disease and triptolide treatment. Based on this data, 23 potential biomarkers were identified by OPLS-DA, and seven of these biomarkers correlated markedly with the disease markers (p<0.05) by Spearman correlation.

Conclusions

These data indicate that differences in sphingolipid levels in plasma and tissues are related to DTH and treatment with triptolide. Restoration of proper sphingolipid levels may attribute to the therapeutic effect of triptolide treatment. Furthermore, these findings demonstrate that targeted sphingolipidomic analysis followed by multivariate analysis presents a novel strategy for the identification of biomarkers in biological samples.     

Curcumin Mitigates the Intracellular Lipid Deposit Induced by Antipsychotics In Vitro

Scope

First- and second-generation antipsychotics (FGAs and SGAs, respectively), both inhibit cholesterol biosynthesis and impair the intracellular cholesterol trafficking, leading to lipid accumulation in the late endosome/lysosome compartment. In this study we examined if curcumin, a plant polyphenol that stimulates exosome release, can alleviate antipsychotic-induced intracellular lipid accumulation.

Methods

HepG2 hepatocarcinoma cells were treated with antipsychotics or placebo and DiI-labelled LDL for 18 h and then exposed to curcumin for the last 2 h. Cells and media were collected separately and used for biochemical analyses, electron microscopy and immunocytochemistry. Exosomes were isolated from the incubation medium by ultracentrifugation.

Results

Curcumin treatment reduced the number of heterolysosomes and shifted their subcellular localization to the periphery, as revealed by electron microscopy, and stimulated the release of lysosomal β-hexosaminidase and exosome markers flotillin-2 and CD63 into the media. The presence of DiI in exosomes released by cells preloaded with DiI-LDL demonstrated the endolysosomal origin of the microvesicles. Furthermore, curcumin increased the secretion of cholesterol as well as LDL-derived DiI and [³H]-cholesterol, in association with a decrease of intracellular lipids. Thus, the disruption of lipid trafficking induced by FGAs or SGAs can be relieved by curcumin treatment. This polyphenol, however, did not mitigate the reduction of cholesterol esterification induced by antipsychotics.

Conclusion

Curcumin stimulates exosome release to remove cholesterol (and presumably other lipids) accumulated within the endolysosomal compartment, thereby normalizing intracellular lipid homeostasis. This action may help minimize the adverse metabolic effects of antipsychotic treatment, which should now be evaluated in clinical trials.     

Metabolomics in early Alzheimer's disease: identification of altered plasma sphingolipidome using shotgun lipidomics

Background

The development of plasma biomarkers could facilitate early detection, risk assessment and therapeutic monitoring in Alzheimer''s disease (AD). Alterations in ceramides and sphingomyelins have been postulated to play a role in amyloidogensis and inflammatory stress related neuronal apoptosis; however few studies have conducted a comprehensive analysis of the sphingolipidome in AD plasma using analytical platforms with accuracy, sensitivity and reproducibility.

Methods and Findings

We prospectively analyzed plasma from 26 AD patients (mean MMSE 21) and 26 cognitively normal controls in a non-targeted approach using multi-dimensional mass spectrometry-based shotgun lipidomics [1], [2] to determine the levels of over 800 molecular species of lipids. These data were then correlated with diagnosis, apolipoprotein E4 genotype and cognitive performance. Plasma levels of species of sphingolipids were significantly altered in AD. Of the 33 sphingomyelin species tested, 8 molecular species, particularly those containing long aliphatic chains such as 22 and 24 carbon atoms, were significantly lower (p<0.05) in AD compared to controls. Levels of 2 ceramide species (N16:0 and N21:0) were significantly higher in AD (p<0.05) with a similar, but weaker, trend for 5 other species. Ratios of ceramide to sphingomyelin species containing identical fatty acyl chains differed significantly between AD patients and controls. MMSE scores were correlated with altered mass levels of both N20:2 SM and OH-N25:0 ceramides (p<0.004) though lipid abnormalities were observed in mild and moderate AD. Within AD subjects, there were also genotype specific differences.

Conclusions

In this prospective study, we used a sensitive multimodality platform to identify and characterize an essentially uniform but opposite pattern of disruption in sphingomyelin and ceramide mass levels in AD plasma. Given the role of brain sphingolipids in neuronal function, our findings provide new insights into the AD sphingolipidome and the potential use of metabolomic signatures as peripheral biomarkers.     

Different sphingolipids show differential partitioning into sphingolipid/cholesterol-rich domains in lipid bilayers

Two fluorescence-based approaches have been applied to examine the differential partitioning of fluorescent phospho- and sphingolipid molecules into sphingolipid-enriched domains modeling membrane "lipid rafts." Fluorescence-quenching measurements reveal that N-(diphenylhexatrienyl)propionyl- (DPH3:0-)-labeled gluco- and galactocerebroside partition into sphingolipid-enriched domains in sphingolipid/phosphatidylcholine/cholesterol bilayers with substantially higher affinity than do analogous sphingomyelin, ceramide, or phosphatidylcholine molecules. By contrast, the affinity of sphingomyelin and ceramide for such domains is only marginally greater than that of a phosphatidylcholine with similar hydrocarbon chains. By using direct measurements of molecular partitioning between vesicles of different compositions, we show that the relative affinities of different C(6)-NBD- and C(5)-Bodipy-labeled sphingolipids for sphingolipid-enriched domains are quantitatively, and in most circumstances even qualitatively, quite different from those found for species whose N-acyl chains more closely resemble the long saturated chains of cellular sphingolipids. These findings lend support in principle to previous suggestions that differential partitioning of different sphingolipids into "raft" domains could contribute to the differential trafficking of these species in eukaryotic cells. However, our findings also indicate that short-chain sphingolipid probes previously used to examine this phenomenon are in general ill-suited for such applications.     

New Evidence for the Role of Ceramide in the Development of Hepatic Insulin Resistance

Aim

There are few and contradictory data on the role of excessive accumulation of intracellular sphingolipids, particularly ceramides, in the development of hepatic insulin resistance. In our study we assessed accumulated sphingolipid fractions and clarify the mechanisms of hepatic insulin resistance development as well as involvement of fatty acid and ceramide transporters in this process.

Methods

In culture of primary rat hepatocytes, exposed to high concentration of palmitic acid (0.75mM) during short and prolonged incubation, high performance liquid chromatography was used to assess intra- and extracellular sphingolipid fractions content. Degree of palmitate-induced insulin resistance was estimated by measuring changes in phosphorylation of insulin pathway proteins by western blotting as well as changes in expression of different type of transporters.

Results

In our study short and prolonged exposure of primary hepatocytes to palmitic acid resulted in increased intracellular accumulation of ceramide which inhibited insulin signaling pathway. We observed a significant increase in the expression of fatty-acid transport protein (FATP2) and ceramide transfer protein (CERT) what is consistent with enhanced intracellular ceramide content. The content of extracellular ceramide was increased nearly threefold after short and twofold after long incubation period. Expression of microsomal triglyceride transfer protein (MTP) and ATP-binding cassette transporter (ABCA1) was increased significantly mainly after short palmitate incubation.

Conclusion

Our data showed that increase in intarcellular ceramide content contributes to the development of hepatic insulin resistance. We suggest pivotal role of transporters in facilitating fatty acid influx (FATP2), accumulation of ceramides (CERT) and export to the media (MTP and ABCA1).     

The structure of the NPC1L1 N-terminal domain in a closed conformation

Background

NPC1L1 is the molecular target of the cholesterol lowering drug Ezetimibe and mediates the intestinal absorption of cholesterol. Inhibition or deletion of NPC1L1 reduces intestinal cholesterol absorption, resulting in reduction of plasma cholesterol levels.

Principal Findings

Here we present the 2.8 Å crystal structure of the N-terminal domain (NTD) of NPC1L1 in the absence of cholesterol. The structure, combined with biochemical data, reveals the mechanism of cholesterol selectivity of NPC1L1. Comparison to the cholesterol free and bound structures of NPC1(NTD) reveals that NPC1L1(NTD) is in a closed conformation and the sterol binding pocket is occluded from solvent.

Conclusion

The structure of NPC1L1(NTD) reveals a degree of flexibility surrounding the entrance to the sterol binding pocket, suggesting a gating mechanism that relies on multiple movements around the entrance to the sterol binding pocket.     

A-RAF Kinase Functions in ARF6 Regulated Endocytic Membrane Traffic

Background

RAF kinases direct ERK MAPK signaling to distinct subcellular compartments in response to growth factor stimulation.

Methodology/Principal Findings

Of the three mammalian isoforms A-RAF is special in that one of its two lipid binding domains mediates a unique pattern of membrane localization. Specific membrane binding is retained by an N-terminal fragment (AR149) that corresponds to a naturally occurring splice variant termed DA-RAF2. AR149 colocalizes with ARF6 on tubular endosomes and has a dominant negative effect on endocytic trafficking. Moreover actin polymerization of yeast and mammalian cells is abolished. AR149/DA-RAF2 does not affect the internalization step of endocytosis, but trafficking to the recycling compartment.

Conclusions/Significance

A-RAF induced ERK activation is required for this step by activating ARF6, as A-RAF depletion or inhibition of the A-RAF controlled MEK-ERK cascade blocks recycling. These data led to a new model for A-RAF function in endocytic trafficking.     

Caveolar Fatty Acids and Acylation of Caveolin-1

Purpose

Caveolae are cholesterol and sphingolipids rich subcellular domains on plasma membrane. Caveolae contain a variety of signaling proteins which provide platforms for signaling transduction. In addition to enriched with cholesterol and sphingolipids, caveolae also contain a variety of fatty acids. It has been well-established that acylation of protein plays a pivotal role in subcellular location including targeting to caveolae. However, the fatty acid compositions of caveolae and the type of acylation of caveolar proteins remain largely unknown. In this study, we investigated the fatty acids in caveolae and caveolin-1 bound fatty acids.

Methods

Caveolae were isolated from Chinese hamster ovary (CHO) cells. The caveolar fatty acids were extracted with Folch reagent, methyl esterificated with BF3, and analyzed by gas chromatograph-mass spectrometer (GC/MS). The caveolin-1bound fatty acids were immunoprecipitated by anti-caveolin-1 IgG and analyzed with GC/MS.

Results

In contrast to the whole CHO cell lysate which contained a variety of fatty acids, caveolae mainly contained three types of fatty acids, 0.48 µg palmitic acid, 0.61 µg stearic acid and 0.83 µg oleic acid/caveolae preparation/5×10⁷ cells. Unexpectedly, GC/MS analysis indicated that caveolin-1 was not acylated by myristic acid; instead, it was acylated by palmitic acid and stearic acid.

Conclusion

Caveolae contained a special set of fatty acids, highly enriched with saturated fatty acids, and caveolin-1 was acylated by palmitic acid and stearic acid. The unique fatty acid compositions of caveolae and acylation of caveolin-1 may be important for caveolae formation and for maintaining the function of caveolae.     

Characterization of novel StAR (steroidogenic acute regulatory protein) mutations causing non-classic lipoid adrenal hyperplasia

Context

Steroidogenic acute regulatory protein (StAR) is crucial for transport of cholesterol to mitochondria where biosynthesis of steroids is initiated. Loss of StAR function causes lipoid congenital adrenal hyperplasia (LCAH).

Objective

StAR gene mutations causing partial loss of function manifest atypical and may be mistaken as familial glucocorticoid deficiency. Only a few mutations have been reported.

Design

To report clinical, biochemical, genetic, protein structure and functional data on two novel StAR mutations, and to compare them with published literature.

Setting

Collaboration between the University Children''s Hospital Bern, Switzerland, and the CIBERER, Hospital Vall d''Hebron, Autonomous University, Barcelona, Spain.

Patients

Two subjects of a non-consanguineous Caucasian family were studied. The 46,XX phenotypic normal female was diagnosed with adrenal insufficiency at the age of 10 months, had normal pubertal development and still has no signs of hypergonodatropic hypogonadism at 32 years of age. Her 46,XY brother was born with normal male external genitalia and was diagnosed with adrenal insufficiency at 14 months. Puberty was normal and no signs of hypergonadotropic hypogonadism are present at 29 years of age.

Results

StAR gene analysis revealed two novel compound heterozygote mutations T44HfsX3 and G221S. T44HfsX3 is a loss-of-function StAR mutation. G221S retains partial activity (∼30%) and is therefore responsible for a milder, non-classic phenotype. G221S is located in the cholesterol binding pocket and seems to alter binding/release of cholesterol.

Conclusions

StAR mutations located in the cholesterol binding pocket (V187M, R188C, R192C, G221D/S) seem to cause non-classic lipoid CAH. Accuracy of genotype-phenotype prediction by in vitro testing may vary with the assays employed.     

Assessing protein immunogenicity with a dendritic cell line-derived endolysosomal degradome

Background

The growing number of novel candidate molecules for the treatment of allergic diseases imposed a dramatic increase in the demand for animal experiments to select immunogenic vaccines, a pre-requisite for efficacy. Because no in vitro methods to predict the immunogenicity of a protein are currently available, we developed an in vitro assay that exploits the link between a protein''s immunogenicity and its susceptibility to endolysosomal proteolysis.

Methodology

We compared protein composition and proteolytic activity of endolysosomal fractions isolated from murine bone marrow- and human blood- derived dendritic cells, and from the dendritic cell line JAWS II. Three groups of structurally related antigen variants differing in their ability to elicit immune responses in vivo (Bet v 1.0101 and Bet v 1.0401, RNases A and S, holo- and apo-HRP) were subjected to in vitro simulated endolysosomal degradation. Kinetics and patterns of generated proteolytic peptides were evaluated by gel electrophoresis and mass spectrometry.

Results

Antigens displaying weak capacity of T cell priming in vivo were highly susceptible to endolysosomal proteases in vitro. As proteolytic composition, activity, and specificity of endolysosomal fractions derived from human and murine dendritic cells were comparable, the JAWS II cell line could be used as a substitute for freshly isolated human or murine cells in in vitro degradation assays.

Conclusions

Endolysosomal fractions prepared from the JAWS II cell line provide a reliable tool for in vitro estimation of protein immunogenicity. The rapid and simple assay described here is very useful to study the immunogenic properties of a protein, and can help to replace, reduce, and refine animal experiments in allergy research and vaccine development in general.     

Increased plasma membrane cholesterol in cystic fibrosis cells correlates with CFTR genotype and depends on de novo cholesterol synthesis

Background

Previous observations demonstrate that Cftr-null cells and tissues exhibit alterations in cholesterol processing including perinuclear cholesterol accumulation, increased de novo synthesis, and an increase in plasma membrane cholesterol accessibility compared to wild type controls. The hypothesis of this study is that membrane cholesterol accessibility correlates with CFTR genotype and is in part influenced by de novo cholesterol synthesis.

Methods

Electrochemical detection of cholesterol at the plasma membrane is achieved with capillary microelectrodes with a modified platinum coil that accepts covalent attachment of cholesterol oxidase. Modified electrodes absent cholesterol oxidase serves as a baseline control. Cholesterol synthesis is determined by deuterium incorporation into lipids over time. Incorporation into cholesterol specifically is determined by mass spectrometry analysis. All mice used in the study are on a C57Bl/6 background and are between 6 and 8 weeks of age.

Results

Membrane cholesterol measurements are elevated in both R117H and ΔF508 mouse nasal epithelium compared to age-matched sibling wt controls demonstrating a genotype correlation to membrane cholesterol detection. Expression of wt CFTR in CF epithelial cells reverts membrane cholesterol to WT levels further demonstrating the impact of CFTR on these processes. In wt epithelial cell, the addition of the CFTR inhibitors, Gly H101 or CFTR_inh-172, for 24 h surprisingly results in an initial drop in membrane cholesterol measurement followed by a rebound at 72 h suggesting a feedback mechanism may be driving the increase in membrane cholesterol. De novo cholesterol synthesis contributes to membrane cholesterol accessibility.

Conclusions

The data in this study suggest that CFTR influences cholesterol trafficking to the plasma membrane, which when depleted, leads to an increase in de novo cholesterol synthesis to restore membrane content.     

Glycolipid Trafficking in Drosophila Undergoes Pathway Switching in Response to Aberrant Cholesterol Levels

In lipid storage diseases, the intracellular trafficking of sphingolipids is altered by conditions of aberrant cholesterol accumulation. Drosophila has been used recently to model lipid storage diseases, but the effects of sterol accumulation on sphingolipid trafficking are not known in the fly, and the trafficking of sphingolipids in general has not been studied in this model organism. Here, we examined the uptake and intracellular distribution of a fluorescent glycolipid analog, BODIPY-lactosyl-ceramide, in Drosophila neurons. The uptake mechanism and intracellular trafficking route of this simple glycolipid are largely conserved. Our principle finding is that cholesterol steers trafficking of the glycolipid between Golgi, lysosome, and recycling compartments. Our analyses support the idea that cholesterol storage in Drosophila triggers a switch in glycolipid trafficking from the biosynthetic to the degradative endolysosomal pathway, whereas cholesterol depletion eliminates recycling of the glycolipid. Unexpectedly, we observe a novel phenomenon we term “hijacking,” whereby lactosyl-ceramide diverts the trafficking pathway of an endocytic cargo, dextran, completely away from its lysosomal target. This work establishes that glycolipid trafficking in Drosophila undergoes changes similar to those seen in mammalian cells under conditions of cholesterol storage and therefore validates Drosophila as a suitable model organism in which to study lipid storage diseases.     

Histidine domain-protein tyrosine phosphatase interacts with Grb2 and GrpL

Background

Histidine domain-protein tyrosine phosphatase (HD-PTP) plays a key role in vesicle trafficking and biogenesis. Although it is a large protein with at least five distinct structural domains, only a few of its interactors are presently known, and the significance of these interactions is largely obscure.

Methodology and Results

In this study we performed a yeast two-hybrid screening using a human colon cDNA library and found that Grb2 and GrpL are binding partners of HD-PTP. Co-immunoprecipitation, pull-down and immunocytochemistry experiments confirmed the interactions. We also discovered that the central proline-rich and histidine-rich domain of HD-PTP is responsible for these interactions.

Significance

The interaction of HD-PTP with two adapters of the Grb2 family, essential for numerous signaling pathways, suggests that HD-PTP might be important for signaling through a plethora of receptors.     

Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1H46R-Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking

Background

ALS2/alsin is a guanine nucleotide exchange factor for the small GTPase Rab5 and involved in macropinocytosis-associated endosome fusion and trafficking, and neurite outgrowth. ALS2 deficiency accounts for a number of juvenile recessive motor neuron diseases (MNDs). Recently, it has been shown that ALS2 plays a role in neuroprotection against MND-associated pathological insults, such as toxicity induced by mutant Cu/Zn superoxide dismutase (SOD1). However, molecular mechanisms underlying the relationship between ALS2-associated cellular function and its neuroprotective role remain unclear.

Methodology/Principal Findings

To address this issue, we investigated the molecular and pathological basis for the phenotypic modification of mutant SOD1-expressing mice by ALS2 loss. Genetic ablation of Als2 in SOD1^H46R, but not SOD1^G93A, transgenic mice aggravated the mutant SOD1-associated disease symptoms such as body weight loss and motor dysfunction, leading to the earlier death. Light and electron microscopic examinations revealed the presence of degenerating and/or swollen spinal axons accumulating granular aggregates and autophagosome-like vesicles in early- and even pre-symptomatic SOD1^H46R mice. Further, enhanced accumulation of insoluble high molecular weight SOD1, poly-ubiquitinated proteins, and macroautophagy-associated proteins such as polyubiquitin-binding protein p62/SQSTM1 and a lipidated form of light chain 3 (LC3-II), emerged in ALS2-deficient SOD1^H46R mice. Intriguingly, ALS2 was colocalized with LC3 and p62, and partly with SOD1 on autophagosome/endosome hybrid compartments, and loss of ALS2 significantly lowered the lysosome-dependent clearance of LC3 and p62 in cultured cells.

Conclusions/Significance

Based on these observations, although molecular basis for the distinctive susceptibilities to ALS2 loss in different mutant SOD1-expressing ALS models is still elusive, disturbance of the endolysosomal system by ALS2 loss may exacerbate the SOD1^H46R-mediated neurotoxicity by accelerating the accumulation of immature vesicles and misfolded proteins in the spinal cord. We propose that ALS2 is implicated in endolysosomal trafficking through the fusion between endosomes and autophagosomes, thereby regulating endolysosomal protein degradation in vivo.     

De novo sphingolipid synthesis is essential for viability, but not for transport of glycosylphosphatidylinositol-anchored proteins, in African trypanosomes

De novo sphingolipid synthesis is required for the exit of glycosylphosphatidylinositol (GPI)-anchored membrane proteins from the endoplasmic reticulum in yeast. Using a pharmacological approach, we test the generality of this phenomenon by analyzing the transport of GPI-anchored cargo in widely divergent eukaryotic systems represented by African trypanosomes and HeLa cells. Myriocin, which blocks the first step of sphingolipid synthesis (serine + palmitate --> 3-ketodihydrosphingosine), inhibited the growth of cultured bloodstream parasites, and growth was rescued with exogenous 3-ketodihydrosphingosine. Myriocin also blocked metabolic incorporation of [3H]serine into base-resistant sphingolipids. Biochemical analyses indicate that the radiolabeled lipids are not sphingomyelin or inositol phosphorylceramide, suggesting that bloodstream trypanosomes synthesize novel sphingolipids. Inhibition of de novo sphingolipid synthesis with myriocin had no adverse effect on either general secretory trafficking or GPI-dependent trafficking in trypanosomes, and similar results were obtained with HeLa cells. A mild effect on endocytosis was seen for bloodstream trypanosomes after prolonged incubation with myriocin. These results indicate that de novo synthesis of sphingolipids is not a general requirement for secretory trafficking in eukaryotic cells. However, in contrast to the closely related kinetoplastid Leishmania major, de novo sphingolipid synthesis is essential for the viability of bloodstream-stage African trypanosomes.     

Fatty Acid binding protein 4 is associated with carotid atherosclerosis and outcome in patients with acute ischemic stroke

Background and Purpose

Fatty acid binding protein 4 (FABP4) has been shown to play an important role in macrophage cholesterol trafficking and associated inflammation. To further elucidate the role of FABP4 in atherogenesis in humans, we examined the regulation of FABP4 in carotid atherosclerosis and ischemic stroke.

Methods

We examined plasma FABP4 levels in asymptomatic (n = 28) and symptomatic (n = 31) patients with carotid atherosclerosis, as well as in 202 subjects with acute ischemic stroke. In a subgroup of patients we also analysed the expression of FABP4 within the atherosclerotic lesion. In addition, we investigated the ability of different stimuli with relevance to atherosclerosis to regulate FABP4 expression in monocytes/macrophages.

Results

FABP4 levels were higher in patients with carotid atherosclerosis, both systemically and within the atherosclerotic lesion, with particular high mRNA levels in carotid plaques from patients with the most recent symptoms. Immunostaining of carotid plaques localized FABP4 to macrophages, while activated platelets and oxidized LDL were potent stimuli for FABP4 expression in monocytes/macrophages in vitro. When measured at the time of acute ischemic stroke, high plasma levels of FABP4 were significantly associated with total and cardiovascular mortality during follow-up, although we did not find that addition of FABP4 to the fully adjusted multivariate model had an effect on the prognostic discrimination for all-cause mortality as assessed by c-statistics.

Conclusions

FABP4 is linked to atherogenesis, plaque instability and adverse outcome in patients with carotid atherosclerosis and acute ischemic stroke.     

Murine Toll-Like Receptor 2 Activation Induces Type I Interferon Responses from Endolysosomal Compartments

Background

Toll-like receptors (TLRs) are among the first-line sentinels for immune detection and responsiveness to pathogens. The TLR2 subfamily of TLRs (TLR1, TLR2, TLR6) form heterodimers with each other and are thus able to recognize a broad range of components from several microbes such as yeast, Gram-positive bacteria and protozoa. Until now, TLR2 activation by bacterial ligands has long been associated with pro-inflammatory cytokines but not type I interferon responses.

Methodology/Principal Findings

Using a variety of transgenic mice, here we provide in vivo and in vitro data showing that TLR2 activation does in fact induce interferon-beta and that this occurs via MyD88-IRF1 and -IRF7 pathways. Interestingly, by microscopy we demonstrate that although a cell surface receptor, TLR2 dependent induction of type I interferons occurs in endolysosomal compartments where it is translocated to upon ligand engagement. Furthermore, we could show that blocking receptor internalization or endolysosomal acidification inhibits the ability of TLR2 to trigger the induction type I interferon but not pro-inflammatory responses.

Conclusion/Significance

The results indicate that TLR2 activation induces pro-inflammatory and type I interferon responses from distinct subcellular sites: the plasma membrane and endolysosomal compartments respectively. Apart from identifying and characterizing a novel pathway for induction of type I interferons, the present study offers new insights into how TLR signaling discriminates and regulates the nature of responses to be elicited against extracellular and endocytosed microbes. These findings may also have clinical implication. Excessive production of pro-inflammatory cytokines and type I IFNs following activation of TLRs is a central pathologic event in several hyper-inflammatory conditions. The discovery that the induction of pro-inflammatory and type I IFN responses can be uncoupled through pharmacological manipulation of endolysosomal acidification suggests new avenues for potential therapeutic intervention against inflammations and sepsis.     

A Temporal Threshold for Formaldehyde Crosslinking and Fixation

Background

Formaldehyde crosslinking is in widespread use as a biological fixative for microscopy and molecular biology. An assumption behind its use is that most biologically meaningful interactions are preserved by crosslinking, but the minimum length of time required for an interaction to become fixed has not been determined.

Methodology

Using a unique series of mutations in the DNA binding protein MeCP2, we show that in vivo interactions lasting less than 5 seconds are invisible in the microscope after formaldehyde fixation, though they are obvious in live cells. The stark contrast between live cell and fixed cell images illustrates hitherto unsuspected limitations to the fixation process. We show that chromatin immunoprecipitation, a technique in widespread use that depends on formaldehyde crosslinking, also fails to capture these transient interactions.

Conclusions/Significance

Our findings for the first time establish a minimum temporal limitation to crosslink chemistry that has implications for many fields of research.