Differential intracellular trafficking of extracellular vesicles in microglia and astrocytes.

Extracellular vesicles (EVs) have emerged as key players in cell-to-cell communication in both physiological and pathological processes in the Central Nervous System. Thus far, the intracellular pathways involved in uptake and trafficking of EVs within different cell types of the brain are poorly understood. In our study, the endocytic processes and subcellular sorting of EVs were investigated in primary glial cells, particularly linked with the EV-associated α-synuclein (α-syn) transmission. Mouse microglia and astrocytic primary cultures were incubated with DiI-stained mouse brain-derived EVs. The internalization and trafficking pathways were analyzed in cells treated with pharmacological reagents that block the major endocytic pathways. Brain-derived EVs were internalized by both glial cell types; however, uptake was more efficient in microglia than in astrocytes. Colocalization of EVs with early and late endocytic markers (Rab5, Lamp1) indicated that EVs are sorted to endo-lysosomes for subsequent processing. Blocking actin-dependent phagocytosis and/or macropinocytosis with Cytochalasin D or EIPA inhibited EV entry into glial cells, whereas treatment with inhibitors that strip cholesterol off the plasma membrane, induced uptake, however differentially altered endosomal sorting. EV-associated fibrillar α-Syn was efficiently internalized and detected in Rab5- and Lamp1-positive compartments within microglia. Our study strongly suggests that EVs enter glial cells through phagocytosis and/or macropinocytosis and are sorted to endo-lysosomes for subsequent processing. Further, brain-derived EVs serve as scavengers and mediate cell-to-glia transfer of pathological α-Syn which is also targeted to the endolysosomal pathway, suggesting a beneficial role in microglia-mediated clearance of toxic protein aggregates, present in numerous neurodegenerative diseases.

Yin and Yang of Biofilm Formation and Cyclic di-GMP Signaling of the Gastrointestinal Pathogen Salmonella enterica Serovar Typhimurium.

Within the last 60 years, microbiological research has challenged many dogmas such as bacteria being unicellular microorganisms directed by nutrient sources; these investigations produced new dogmas such as cyclic diguanylate monophosphate (cyclic di-GMP) second messenger signaling as a ubiquitous regulator of the fundamental sessility/motility lifestyle switch on the single-cell level. Successive investigations have not yet challenged this view; however, the complexity of cyclic di-GMP as an intracellular bacterial signal, and, less explored, as an extracellular signaling molecule in combination with the conformational flexibility of the molecule, provides endless opportunities for cross-kingdom interactions. Cyclic di-GMP-directed microbial biofilms commonly stimulate the immune system on a lower level, whereas host-sensed cyclic di-GMP broadly stimulates the innate and adaptive immune responses. Furthermore, while the intracellular second messenger cyclic di-GMP signaling promotes bacterial biofilm formation and chronic infections, oppositely, Salmonella Typhimurium cellulose biofilm inside immune cells is not endorsed. These observations only touch on the complexity of the interaction of biofilm microbial cells with its host. In this review, we describe the Yin and Yang interactive concepts of biofilm formation and cyclic di-GMP signaling using S. Typhimurium as an example.

Investigating the presence of doubly phosphorylated α-synuclein at tyrosine 125 and serine 129 in idiopathic Lewy body diseases.

Aggregation of the protein α-synuclein (α-syn) into insoluble intracellular assemblies termed Lewy bodies (LBs) is thought to be a critical pathogenic event in LB diseases such as Parkinson's disease and dementia with LBs. In LB diseases, the majority of α-syn is phosphorylated at serine 129 (pS129), suggesting that this is an important disease-related post-translational modification (PTM). However, PTMs do not typically occur in isolation and phosphorylation at the proximal tyrosine 125 (pY125) residue has received considerable attention and has been inconsistently reported to be present in LBs. Furthermore, the proximity of Y125 to S129 means that some pS129 antibodies may have epitopes that include Y125, in which case phosphorylation of Y125 will impede recognition of α-syn. This would potentially lead to underestimating LB pathology burdens if pY125 occurs alongside pS129. To address the apparent controversy in the literature regarding the detection of pY125, we investigated its presence in the LB pathology. We generated pS129 antibodies whose epitope includes or does not include Y125 and compared the extent of α-syn pathology recognized in mouse models of α-synucleinopathies, human brain tissue lysates and fixed post-mortem brain tissues. Our study demonstrated no difference in α-syn pathology recognized between pS129 antibodies, irrespective of whether Y125 was part of the epitope or not. Furthermore, evaluation with pY125 antibodies whose epitope does not include S129 demonstrated no labeling of LB pathology. This study reconciles disparate results in the literature and demonstrates pY125 is not a key component of LB pathology in murine models or human tissues in idiopathic LB diseases.

Childhood obesity and leucocyte telomere length.

BACKGROUND: Obesity in adulthood is associated with decreased leucocyte telomere length (LTL), which is associated with cardiovascular disease and diabetes mellitus type 2. The aim of our study was to investigate whether increased body mass index (BMI) is associated with decreased LTL in children and adolescents, and to identify other risk factors of shorter LTL in this population. MATERIALS AND METHODS: A cross-sectional study was conducted among 919 Greek children aged 9-13 years (The Healthy Growth Study). Participants were classified as obese (n = 124), overweight (n = 276) or of normal BMI (n = 519). LTL was determined by monochrome multiplex quantitative real-time polymerase chain reaction. Univariate and multivariable linear regression analyses were applied to determine the predictive factors of LTL. RESULTS: Both overweight and obese children had significantly shorter LTL than their normal-BMI counterparts. Following adjustment for age, sex, total daily energy intake and average weekly physical activity (average total steps per day), increasing weight category was inversely associated with LTL in children and adolescents (ß: -0.110 ± 0.035; P = .002). CONCLUSION: Overweight and obesity in childhood and adolescence are associated with shorter LTL, even following adjustment for potential confounding effects. Therefore, the increased BMI in childhood and adolescence may be associated with accelerated biological ageing and may have an adverse impact on future health in adulthood.

Cellular models of alpha-synuclein toxicity and aggregation.

Misfolding and aggregation of alpha-synuclein (α-synuclein) with concomitant cytotoxicity is a hallmark of Lewy body related disorders such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Although it plays a pivotal role in pathogenesis and disease progression, the function of α-synuclein and the molecular mechanisms underlying α-synuclein-induced neurotoxicity in these diseases are still elusive. Many in vitro and in vivo experimental models mimicking α-synuclein pathology such as oligomerization, toxicity and more recently neuronal propagation have been generated over the years. In particular, cellular models have been crucial for our comprehension of the pathogenic process of the disease and are beneficial for screening of molecules capable of modulating α-synuclein toxicity. Here, we review α-synuclein based cell culture models that reproduce some features of the neuronal populations affected in patients, from basic unicellular organisms to mammalian cell lines and primary neurons, to the cutting edge models of patient-specific cell lines. These reprogrammed cells known as induced pluripotent stem cells (iPSCs) have garnered attention because they closely reproduce the characteristics of neurons found in patients and provide a valuable tool for mechanistic studies. We also discuss how different cell models may constitute powerful tools for high-throughput screening of molecules capable of modulating α-synuclein toxicity and prevention of its propagation. This article is part of the Special Issue "Synuclein".

Transcriptomics in tissue glucocorticoid sensitivity.

BACKGROUND: Synthetic glucocorticoids are widely used in the treatment of several inflammatory, autoimmune and lymphoproliferative disorders. However, considerable variation in response to therapeutic doses of glucocorticoids has been documented among individuals. The aim of our study was to identify novel glucocorticoid sensitivity-determining genes using genome-wide expression profiling in healthy subjects. METHODS: One hundred one healthy subjects [mean age ± standard error of the mean (SEM); 26.52 ± 0.50 years] were given 0.25 mg dexamethasone at midnight, and serum cortisol concentrations were determined at 08:00 hours the following morning. Subjects were stratified into the 10% most glucocorticoid-sensitive and 10% most glucocorticoid-resistant according to the serum cortisol concentrations. Genomic DNA, RNA and plasma samples were obtained in the 22 subjects one month later. RESULTS: Transcriptomic analysis showed variability between glucocorticoid-resistant and glucocorticoid-sensitive subjects. One hundred thirty-three genes were upregulated and 49 downregulated in the glucocorticoid-resistant compared to the glucocorticoid-sensitive group. Further analysis revealed differences between 3 glucocorticoid-resistant and 3 glucocorticoid-sensitive subjects. The majority of the 1058 upregulated genes and 1139 downregulated genes were found to participate in telomere maintenance, systemic lupus erythematosus and Alzheimer's disease. Interestingly, Synuclein A, a key molecule in Parkinson's disease, was upregulated in the subgroup of glucocorticoid-sensitive subjects. CONCLUSIONS: We have identified differences in tissue sensitivity to glucocorticoids among healthy subjects at the transcriptomic level. These differences are associated with differential expression of genes related to autoimmune and neurological disorders.

The effect of intrauterine growth on leukocyte telomere length at birth.

Objective: Telomeres are specialized nucleoprotein structures located at the ends of chromosomes, which play a crucial role in genomic stability. Telomere shortening has been proposed as a biomarker for the onset of age-related diseases. This study aimed to determine whether restricted or increased intrauterine growth affects leukocyte telomere length (LTL) at birth. Materials and methods: One hundred sixty-five (n = 165) full-term neonates participated in the study. Fetuses were classified as intrauterine growth restriction (IUGR, n = 21), large-for-gestational-age (LGA, n = 15), or appropriate-for-gestational-age (AGA, n = 129), based on customized birth-weight standards. Mixed arteriovenous cord blood samples were collected for isolation of leukocyte DNA. The LTL was measured using multiplex monochrome quantitative real-time PCR and telomeric restriction fragments through Southern blot analysis (terminal restriction fragment [TRF]). Results: Despite differences among groups in birth weight, length and head circumference, LTL did not differ among AGA (6.78 ± 0.58), IUGR (10.54 ± 1.80), and LGA (11.95 ± 2.42) neonates (p = .098). Cord blood IGF-1 and IGFBP-3 concentrations were higher in the LGA group. LTL positively correlated with birth length (r = 0.176, p = .032). Conclusions: Intrauterine growth does not seem to affect LTL at birth. Further studies, comprising a larger sample size of IUGR, LGA, and AGA neonates, are required to determine whether growth at birth influences LTL.

Recent advances in the molecular mechanisms causing primary generalized glucocorticoid resistance.

Primary Generalized Glucocorticoid Resistance is a rare condition characterized by generalized, partial, target tissue insensitivity to glucocorticoids owing to inactivating mutations, insertions or deletions in the human glucocorticoid receptor (hGR) gene (NR3C1). Recent advances in molecular and structural biology have enabled us to elucidate the molecular mechanisms of action of the mutant receptors and to understand how certain conformational alterations of the defective hGRs result in generalized glucocorticoid resistance. Furthermore, our ever-increasing understanding of the molecular mechanisms of glucocorticoid action indicates that the glucocorticoid signaling pathway is a stochastic system that plays a fundamental role in maintaining both basal and stress-related homeostasis. In this review, we summarize the clinical manifestations and molecular pathogenesis of Primary Generalized Glucocorticoid Resistance, we present our recent findings from the functional characterization of three novel heterozygous point mutations in the NR3C1 gene, and we discuss the diagnostic approach and therapeutic management of the condition. When the condition is suspected, we recommend sequencing analysis of the NR3C1 gene as well as of other genes encoding proteins involved in the glucocorticoid signal transduction. The tremendous progress of next-generation sequencing will undoubtedly uncover novel hGR partners or cofactors.

Transient generalized glucocorticoid hypersensitivity.

BACKGROUND: Transient generalized glucocorticoid hypersensitivity is a rare disorder characterized by increased tissue sensitivity to glucocorticoids and compensatory hypo-activation of the hypothalamic-pituitary-adrenal axis. The condition itself and the underlying molecular mechanisms have not been elucidated. OBJECTIVE: To present the clinical manifestations, endocrinologic evaluation and transcriptomic profile in a patient with transient generalized glucocorticoid hypersensitivity. DESIGN AND RESULTS: A 9-year-old girl presented with an 8-month history of clinical manifestations suggestive of Cushing syndrome. Endocrinologic evaluation revealed undetectable 08:00 h ACTH (<1 pg/mL) and cortisol (0·025 µg/dL) concentrations, which remained decreased throughout the 24-h period and did not respond to stimulation with ovine CRH. The disease gradually resolved spontaneously over the ensuing 3 months. Sequencing of the human glucocorticoid receptor gene revealed no mutations or polymorphisms. Western blot analysis in peripheral blood mononuclear cells revealed equal protein expression of hGRα of the patient in the disease and postresolution phases compared with a control subject. Transcriptomic analysis in peripheral blood mononuclear cells in the disease and postresolution phases identified 903 differentially expressed genes. Of these, 106 genes were up-regulated and 797 were down-regulated in the disease compared with the resolution phase. Bioinformatics analysis on the differentially expressed gene networks revealed Nuclear Factor-κB as the predominant transcription factor influencing the expression of the majority of differentially expressed genes. CONCLUSIONS: Our findings indicate that a transient postreceptor defect, or a virus- or bacterium-encoded molecule, may have enhanced glucocorticoid signal transduction, leading to transient generalized glucocorticoid hypersensitivity and hypo-activation of the HPA axis.

Stress, the stress system and the role of glucocorticoids.

All living organisms have developed a highly conserved and regulatory system, the stress system, to cope with a broad spectrum of stressful stimuli that threaten, or are perceived as threatening, their dynamic equilibrium or homeostasis. This neuroendocrine system consists of the hypothalamic-pituitary-adrenal (HPA) axis and the locus caeruleus/norepinephrine-autonomic nervous system. In parallel with the evolution of the homeostasis and stress concepts from ancient Greek to modern medicine, significant advances in the field of neuroendocrinology have identified the physiologic biochemical effector molecules of the stress response. Glucocorticoids, the end-products of the HPA axis, play a fundamental role in the maintenance of both resting and stress-related homeostasis and, undoubtedly, influence the physiologic adaptive reaction of the organism against stressors. If the stress response is dysregulated in terms of magnitude and/or duration, homeostasis is turned into cacostasis with adverse effects on many vital physiologic functions, such as growth, development, metabolism, circulation, reproduction, immune response, cognition and behavior. A strong and/or long-lasting stressor may precipitate and/or cause many acute and chronic diseases. Moreover, stressors during pre-natal, post-natal or pubertal life may have a critical impact on our expressed genome. This review describes the central and peripheral components of the stress system, provides a comprehensive overview of the stress response, and discusses the role of glucocorticoids in a broad spectrum of stress-related diseases. © 2014 S. Karger AG, Basel.

Complex c-di-GMP signaling networks mediate transition between virulence properties and biofilm formation in Salmonella enterica serovar Typhimurium.

Upon Salmonella enterica serovar Typhimurium infection of the gut, an early line of defense is the gastrointestinal epithelium which senses the pathogen and intrusion along the epithelial barrier is one of the first events towards disease. Recently, we showed that high intracellular amounts of the secondary messenger c-di-GMP in S. typhimurium inhibited invasion and abolished induction of a pro-inflammatory immune response in the colonic epithelial cell line HT-29 suggesting regulation of transition between biofilm formation and virulence by c-di-GMP in the intestine. Here we show that highly complex c-di-GMP signaling networks consisting of distinct groups of c-di-GMP synthesizing and degrading proteins modulate the virulence phenotypes invasion, IL-8 production and in vivo colonization in the streptomycin-treated mouse model implying a spatial and timely modulation of virulence properties in S. typhimurium by c-di-GMP signaling. Inhibition of the invasion and IL-8 induction phenotype by c-di-GMP (partially) requires the major biofilm activator CsgD and/or BcsA, the synthase for the extracellular matrix component cellulose. Inhibition of the invasion phenotype is associated with inhibition of secretion of the type three secretion system effector protein SipA, which requires c-di-GMP metabolizing proteins, but not their catalytic activity. Our findings show that c-di-GMP signaling is at least equally important in the regulation of Salmonella-host interaction as in the regulation of biofilm formation at ambient temperature.

Virulence characteristics of translocating Escherichia coli and the interleukin-8 response to infection.

Four efficiently translocating Escherichia coli (TEC) strains isolated from the blood of humans (HMLN-1), pigs (PC-1) and rats (KIC-1 and KIC-2) were tested for their ability to adhere and translocate across human gut epithelial Caco-2 and HT-29 cells, to elicit a proinflammatory response and for the presence of 47 pathogenic E. coli virulence genes. HMLN-1 and PC-1 were more efficient in adhesion and translocation than rat strains, had identical biochemical phenotype (BPT) and serotype (O77:H18) and phylogenetic group (D). KIC-2 adhered more than KIC-1, belonged to different BPT and serotype but the same phylogenetic group as KIC-1. TEC strains elicited significantly higher IL-8 response in both cell lines (P < 0.05) and monocytic THP-1 (P < 0.0001) cells than non-TEC strains. KIC-2 induced the highest IL-8 response which may be associated with its immunostimulatory flagellin. Apart from adhesin genes fimH and bmaE that were carried by all strains, HMLN-1 and PC-1 carried capsule synthesis gene kpsMT III and KIC-2 carried the EAST1 toxin gene. The lack of known virulence genes and the ability of TEC to efficiently adhere and translocate whilst causing proinflammatory response suggests that these strains may carry as yet unidentified genes that enable their translocating ability.

Cyclic di-GMP signalling controls virulence properties of Salmonella enterica serovar Typhimurium at the mucosal lining.

Cyclic di-GMP (c-di-GMP), a novel secondary signalling molecule present in most bacteria, controls transition between motility and sessility. In Salmonella enterica serovar Typhimurium (S. typhimurium) high c-di-GMP concentrations favour the expression of a biofilm state through expression of the master regulator CsgD. In this work, we investigate the effect of c-di-GMP signalling on virulence phenotypes of S. typhimurium. After saturation of the cell with c-di-GMP by overexpression of a di-guanylate cyclase, we studied invasion and induction of a pro-inflammatory cytokine in epithelial cells, basic phenotypes that are major determinants of S. typhimurium virulence. Elevated c-di-GMP had a profound effect on invasion into and IL-8 production by the gastrointestinal epithelial cell line HT-29. Invasion was mainly inhibited through CsgD and the extracellular matrix component cellulose, while inhibition of the pro-inflammatory response occurred through CsgD, which inhibited the secretion of monomeric flagellin. Our results suggest that transition between biofilm formation and virulence in S. typhimurium at the epithelial cell lining is mediated by c-di-GMP signalling through CsgD and cellulose expression.