Exosomal small RNA profiling in first-trimester maternal blood explores early molecular pathways of preterm preeclampsia.

Introduction: Preeclampsia (PE) is a severe obstetrical syndrome characterized by new-onset hypertension and proteinuria and it is often associated with fetal intrauterine growth restriction (IUGR). PE leads to long-term health complications, so early diagnosis would be crucial for timely prevention. There are multiple etiologies and subtypes of PE, and this heterogeneity has hindered accurate identification in the presymptomatic phase. Recent investigations have pointed to the potential role of small regulatory RNAs in PE, and these species, which travel in extracellular vesicles (EVs) in the circulation, have raised the possibility of non-invasive diagnostics. The aim of this study was to investigate the behavior of exosomal regulatory small RNAs in the most severe subtype of PE with IUGR. Methods: We isolated exosomal EVs from first-trimester peripheral blood plasma samples of women who later developed preterm PE with IUGR (n=6) and gestational age-matched healthy controls (n=14). The small RNA content of EVs and their differential expression were determined by next-generation sequencing and further validated by quantitative real-time PCR. We also applied the rigorous exceRpt bioinformatics pipeline for small RNA identification, followed by target verification and Gene Ontology analysis. Results: Overall, >2700 small RNAs were identified in all samples and, of interest, the majority belonged to the RNA interference (RNAi) pathways. Among the RNAi species, 16 differentially expressed microRNAs were up-regulated in PE, whereas up-regulated and down-regulated members were equally found among the six identified Piwi-associated RNAs. Gene ontology analysis of the predicted small RNA targets showed enrichment of genes in pathways related to immune processes involved in decidualization, placentation and embryonic development, indicating that dysregulation of the induced small RNAs is connected to the impairment of immune pathways in preeclampsia development. Finally, the subsequent validation experiments revealed that the hsa_piR_016658 piRNA is a promising biomarker candidate for preterm PE associated with IUGR. Discussion: Our rigorously designed study in a homogeneous group of patients unraveled small RNAs in circulating maternal exosomes that act on physiological pathways dysregulated in preterm PE with IUGR. Therefore, our small RNA hits are not only suitable biomarker candidates, but the revealed biological pathways may further inform us about the complex pathology of this severe PE subtype.

One locus, several functional RNAs-emerging roles of the mechanisms responsible for the sequence variability of microRNAs.

With the development of modern molecular genetics, the original "one gene-one enzyme" hypothesis has been outdated. For protein coding genes, the discovery of alternative splicing and RNA editing provided the biochemical background for the RNA repertoire of a single locus, which also serves as an important pillar for the enormous protein variability of the genomes. Non-protein coding RNA genes were also revealed to produce several RNA species with distinct functions. The loci of microRNAs (miRNAs), encoding for small endogenous regulatory RNAs, were also found to produce a population of small RNAs, rather than a single defined product. This review aims to present the mechanisms contributing to the astonishing variability of miRNAs revealed by the new sequencing technologies. One important source is the careful balance of arm selection, producing sequentially different 5p- or 3p-miRNAs from the same pre-miRNA, thereby broadening the number of regulated target RNAs and the phenotypic response. In addition, the formation of 5', 3' and polymorphic isomiRs, with variable end and internal sequences also leads to a higher number of targeted sequences, and increases the regulatory output. These miRNA maturation processes, together with other known mechanisms such as RNA editing, further increase the potential outcome of this small RNA pathway. By discussing the subtle mechanisms behind the sequence diversity of miRNAs, this review intends to reveal this engaging aspect of the inherited "RNA world", how it contributes to the almost infinite molecular variability among living organisms, and how this variability can be exploited to treat human diseases.

Nucleotide binding is the critical regulator of ABCG2 conformational transitions.

ABCG2 is an exporter-type ABC protein that can expel numerous chemically unrelated xeno- and endobiotics from cells. When expressed in tumor cells or tumor stem cells, ABCG2 confers multidrug resistance, contributing to the failure of chemotherapy. Molecular details orchestrating substrate translocation and ATP hydrolysis remain elusive. Here, we present methods to concomitantly investigate substrate and nucleotide binding by ABCG2 in cells. Using the conformation-sensitive antibody 5D3, we show that the switch from the inward-facing (IF) to the outward-facing (OF) conformation of ABCG2 is induced by nucleotide binding. IF-OF transition is facilitated by substrates, and hindered by the inhibitor Ko143. Direct measurements of 5D3 and substrate binding to ABCG2 indicate that the high-to-low affinity switch of the drug binding site coincides with the transition from the IF to the OF conformation. Low substrate binding persists in the post-hydrolysis state, supporting that dissociation of the ATP hydrolysis products is required to reset the high substrate affinity IF conformation of ABCG2.

Partial Disturbance of Microprocessor Function in Human Stem Cells Carrying a Heterozygous Mutation in the DGCR8 Gene.

Maturation of microRNAs (miRNAs) begins by the "Microprocessor" complex, containing the Drosha endonuclease and its partner protein, "DiGeorge Syndrome Critical Region 8" (DGCR8). Although the main function of the two proteins is to coordinate the first step of precursor miRNAs formation, several studies revealed their miRNA-independent functions in other RNA-related pathways (e.g., in snoRNA decay) or, for the DGCR8, the role in tissue development. To investigate the specific roles of DGCR8 in various cellular pathways, we previously established a human embryonic stem-cell (hESC) line carrying a monoallelic DGCR8 mutation by using the CRISPR-Cas9 system. In this study, we genetically characterized single-cell originated progenies of the cell line and showed that DGCR8 heterozygous mutation results in only a modest effect on the mRNA level but a significant decrease at the protein level. Self-renewal and trilineage differentiation capacity of these hESCs were not affected by the mutation. However, partial disturbance of the Microprocessor function could be revealed in pri-miRNA processing along the human chromosome 19 miRNA cluster in several clones. With all these studies, we can demonstrate that the mutant hESC line is a good model to study not only miRNA-related but also other "noncanonical" functions of the DGCR8 protein.

A Novel Gene Controls a New Structure: PiggyBac Transposable Element-Derived 1, Unique to Mammals, Controls Mammal-Specific Neuronal Paraspeckles.

Although new genes can arrive from modes other than duplication, few examples are well characterized. Given high expression in some human brain subregions and a putative link to psychological disorders [e.g., schizophrenia (SCZ)], suggestive of brain functionality, here we characterize piggyBac transposable element-derived 1 (PGBD1). PGBD1 is nonmonotreme mammal-specific and under purifying selection, consistent with functionality. The gene body of human PGBD1 retains much of the original DNA transposon but has additionally captured SCAN and KRAB domains. Despite gene body retention, PGBD1 has lost transposition abilities, thus transposase functionality is absent. PGBD1 no longer recognizes piggyBac transposon-like inverted repeats, nonetheless PGBD1 has DNA binding activity. Genome scale analysis identifies enrichment of binding sites in and around genes involved in neuronal development, with association with both histone activating and repressing marks. We focus on one of the repressed genes, the long noncoding RNA NEAT1, also dysregulated in SCZ, the core structural RNA of paraspeckles. DNA binding assays confirm specific binding of PGBD1 both in the NEAT1 promoter and in the gene body. Depletion of PGBD1 in neuronal progenitor cells (NPCs) results in increased NEAT1/paraspeckles and differentiation. We conclude that PGBD1 has evolved core regulatory functionality for the maintenance of NPCs. As paraspeckles are a mammal-specific structure, the results presented here show a rare example of the evolution of a novel gene coupled to the evolution of a contemporaneous new structure.

Functional Characterization of the N-Terminal Disordered Region of the piggyBac Transposase.

The piggyBac DNA transposon is an active element initially isolated from the cabbage looper moth, but members of this superfamily are also present in most eukaryotic evolutionary lineages. The functionally important regions of the transposase are well described. There is an RNase H-like fold containing the DDD motif responsible for the catalytic DNA cleavage and joining reactions and a C-terminal cysteine-rich domain important for interaction with the transposon DNA. However, the protein also contains a ~100 amino acid long N-terminal disordered region (NTDR) whose function is currently unknown. Here we show that deletion of the NTDR significantly impairs piggyBac transposition, although the extent of decrease is strongly cell-type specific. Moreover, replacing the NTDR with scrambled but similarly disordered sequences did not rescue transposase activity, indicating the importance of sequence conservation. Cell-based transposon excision and integration assays reveal that the excision step is more severely affected by NTDR deletion. Finally, bioinformatic analyses indicated that the NTDR is specific for the piggyBac superfamily and is also present in domesticated, transposase-derived proteins incapable of catalyzing transposition. Our results indicate an essential role of the NTDR in the "fine-tuning" of transposition and its significance in the functions of piggyBac-originated co-opted genes.

Functional indications for transposase domestications - Characterization of the human piggyBac transposase derived (PGBD) activities.

Transposable elements are widespread in all living organisms. In addition to self-reproduction, they are a major source of genetic variation that drives genome evolution but our knowledge of the functions of human genes derived from transposases is limited. There are examples of transposon-derived, domesticated human genes that lost (SETMAR) or retained (THAP9) their transposase activity, however, several remnants in the human genome have not been thoroughly investigated yet. These include the five human piggyBac-derived sequences (PGBD1-5) which share ancestry with the Trichoplusia ni originated piggyBac (PB) transposase. Since PB is widely used in gene delivery applications, the potential activities of endogenous PGBDs are important to address. However, previous data is controversial, especially with the claimed transposition activity of PGBD5, it awaits further investigations. Here, we aimed to systematically analyze all five human PGBD proteins from several aspects, including phylogenetic conservation, potential transposase activity, expression pattern and their regulation in different stress conditions. Among PGBDs, PGBD5 is under the highest purifying selection, and exhibits the most cell type specific expression pattern. In a two-component vector system, none of the human PGBDs could mobilize either the insect PB transposon or the endogenous human PB-like MER75 and MER85 elements with intact terminal sequences. When cells were exposed to various stress conditions, including hypoxia, oxidative or UV stress, the expression profiles of all PGBDs showed different, often cell type specific responses; however, the pattern of PGBD5 in most cases had the opposite tendency than that of the other piggyBac-derived elements. Taken together, our results indicate that human PGBD elements did not retain their mobilizing activity, but their cell type specific, and cellular stress related expression profiles point toward distinct domesticated functions that require further characterization.

Posttranscriptional Regulation of the Human ABCG2 Multidrug Transporter Protein by Artificial Mirtrons.

ABCG2 is a membrane transporter protein that has been associated with multidrug resistance phenotype and tumor development. Additionally, it is expressed in various stem cells, providing cellular protection against endobiotics and xenobiotics. In this study, we designed artificial mirtrons to regulate ABCG2 expression posttranscriptionally. Applying EGFP as a host gene, we could achieve efficient silencing not only in luciferase reporter systems but also at the ABCG2 protein level. Moreover, we observed important new sequential-functional features of the designed mirtrons. Mismatch at the first position of the mirtron-derived small RNA resulted in better silencing than full complementarity, while the investigated middle and 3' mismatches did not enhance silencing. These latter small RNAs operated most probably via non-seed specific translational inhibition in luciferase assays. Additionally, we found that a mismatch in the first position has not, but a second mismatch in the third position has abolished target mRNA decay. Besides, one nucleotide mismatch in the seed region did not impair efficient silencing at the protein level, providing the possibility to silence targets carrying single nucleotide polymorphisms or mutations. Taken together, we believe that apart from establishing an efficient ABCG2 silencing system, our designing pipeline and results on sequential-functional features are beneficial for developing artificial mirtrons for other targets.

Tissue-specific and transcription-dependent mechanisms regulate primary microRNA processing efficiency of the human chromosome 19 MicroRNA cluster.

One of the longest human microRNA (miRNA) clusters is located on chromosome 19 (C19MC), containing 46 miRNA genes, which were considered to be expressed simultaneously and at similar levels from a common long noncoding transcript. Investigating the two tissue types where C19MC is exclusively expressed, we could show that there is a tissue-specific and chromosomal position-dependent decrease in mature miRNA levels towards the 3' end of the cluster in embryonic stem cells but not in placenta. Although C19MC transcription level is significantly lower in stem cells, this gradual decrease is not present at the primary miRNA levels, indicating that a difference in posttranscriptional processing could explain this observation. By depleting Drosha, the nuclease component of the Microprocessor complex, we could further enhance the positional decrease in stem cells, demonstrating that a tissue-specific, local availability of the Microprocessor complex could lie behind the phenomenon. Moreover, we could describe a tissue-specific promoter being exclusively active in placenta, and the epigenetic mark analysis suggested the presence of several putative enhancer sequences in this region. Performing specific chromatin immunoprecipitation followed by quantitative real-time PCR experiments we could show a strong association of Drosha with selected enhancer regions in placenta, but not in embryonic stem cells. These enhancers could provide explanation for a more efficient co-transcriptional recruitment of the Microprocessor, and therefore a more efficient processing of pri-miRNAs throughout the cluster in placenta. Our results point towards a new model where tissue-specific, posttranscriptional 'fine-tuning' can differentiate among miRNAs that are expressed simultaneously from a common precursor.

Establishing a human embryonic stem cell clone with a heterozygous mutation in the DGCR8 gene.

DiGeorge Syndrome (DGS) Critical Region 8 (DGCR8) is a primary candidate gene in they DGS. The DGCR8 microprocessor complex subunit is an essential cofactor in the canonical miRNA biogenesis which is involved in diverse cellular functions such as cell fate decisions, apoptosis and different signaling pathways. However, the role of DGCR8 in these processes or development of DGS is not fully understood. Here we present a heterozygous DGCR8 mutant human embryonic stem cell line (HuES9DGCR8+/-) created by the CRISPR/Cas9 system. The generated HuES9DGCR8+/- cells maintain normal karyotype, morphology, pluripotency and differentiation capacity into all three germ layers.

Precision-engineered reporter cell lines reveal ABCG2 regulation in live lung cancer cells.

Expression of the ABCG2 multidrug transporter is a marker of cancer stem cells and a predictor of recurrent malignant disease. Understanding how human ABCG2 expression is modulated by pharmacotherapy is crucial in guiding therapeutic recommendations and may aid rational drug development. Genome edited reporter cells are useful in investigating gene regulation and visualizing protein activity in live cells but require precise targeting to preserve native regulatory regions. Here, we describe a fluorescent reporter assay that allows the noninvasive assessment of ABCG2 regulation in human lung adenocarcinoma cells. Using CRISPR-Cas9 gene editing coupled with homology-directed repair, we targeted an EGFP coding sequence to the translational start site of ABCG2, generating ABCG2 knock-out and in situ tagged ABCG2 reporter cells. Using the engineered cell lines, we show that ABCG2 is upregulated by a number of anti-cancer medications, HDAC inhibitors, hypoxia-mimicking agents and glucocorticoids, supporting a model in which ABCG2 is under the control of a general stress response. To our knowledge, this is the first description of a fluorescent reporter assay system designed to follow the endogenous regulation of a human ABC transporter in live cells. The information gained may guide therapy recommendations and aid rational drug design.

Cellular expression and function of naturally occurring variants of the human ABCG2 multidrug transporter.

The human ABCG2 multidrug transporter plays a crucial role in the absorption and excretion of xeno- and endobiotics; thus the relatively frequent polymorphic and mutant ABCG2 variants in the population may significantly alter disease conditions and pharmacological effects. Low-level or non-functional ABCG2 expression may increase individual drug toxicity, reduce cancer drug resistance, and result in hyperuricemia and gout. In the present work we have studied the cellular expression, trafficking, and function of nine naturally occurring polymorphic and mutant variants of ABCG2. A comprehensive analysis of the membrane localization, transport, and ATPase activity, as well as retention and degradation in intracellular compartments was performed. Among the examined variants, R147W and R383C showed expression and/or protein folding defects, indicating that they could indeed contribute to ABCG2 functional deficiency. These studies and the applied methods should significantly promote the exploration of the medical effects of these personal variants, promote potential therapies, and help to elucidate the specific role of the affected regions in the folding and function of the ABCG2 protein.

The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/Dynamin.

Phagocytosis of various targets, such as apoptotic cells or opsonized pathogens, by macrophages is coordinated by a complex signaling network initiated by distinct phagocytic receptors. Despite the different initial signaling pathways, each pathway ends up regulating the actin cytoskeletal network, phagosome formation and closure, and phagosome maturation leading to degradation of the engulfed particle. Herein, we describe a new phagocytic function for the nucleoside diphosphate kinase 1 (NDK-1), the nematode counterpart of the first identified metastasis inhibitor NM23-H1 (nonmetastatic clone number 23) nonmetastatic clone number 23 or nonmetastatic isoform 1 (NME1). We reveal by coimmunoprecipitation, Duolink proximity ligation assay, and mass spectrometry that NDK-1/NME1 works in a complex with DYN-1/Dynamin (Caenorhabditis elegans/human homolog proteins), which is essential for engulfment and phagosome maturation. Time-lapse microscopy shows that NDK-1 is expressed on phagosomal surfaces during cell corpse clearance in the same time window as DYN-1. Silencing of NM23-M1 in mouse bone marrow-derived macrophages resulted in decreased phagocytosis of apoptotic thymocytes. In human macrophages, NM23-H1 and Dynamin are corecruited at sites of phagosome formation in F-actin-rich cups. In addition, NM23-H1 was required for efficient phagocytosis. Together, our data demonstrate that NDK-1/NME1 is an evolutionarily conserved element of successful phagocytosis.-Farkas, Z., Petric, M., Liu, X., Herit, F., Rajnavölgyi, É., Szondy, Z., Budai, Z., Orbán, T. I., Sándor, S., Mehta, A., Bajtay, Z., Kovács, T., Jung, S. Y., Afaq Shakir, M., Qin, J., Zhou, Z., Niedergang, F., Boissan, M., Takács-Vellai, K. The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/dynamin.

Circulating exosomal and Argonaute-bound microRNAs in preeclampsia.

INTRODUCTION: microRNAs (miRNAs) play important role in the regulation of placental development, and abnormal miRNA expression is associated with preeclampsia (PE). miRNAs are released from trophoblast cells to maternal blood flow, where they are highly stable, being encapsulated inside extracellular vesicles, like exosomes or bound to Argonaute proteins. In PE, placental dysfunction leads to aberrant extracellular miRNA secretion. hsa-miR-210 is a hypoxia-sensitive miRNA found to be upregulated in PE; however, it is unknown whether it is the cause or the consequence of the disease. OBJECTIVE: Our aim was to analyze the expression of several miRNAs, including hsa-miR-210 in placenta, exosome and Ago-bound fractions comparing normal (N) and PE pregnancies. We performed in vitro analyses of extracellular hsa-miR-210 secretion of trophoblast cell cultures (of villous and extravillous origin) under hypoxic condition. METHODS: PE and N placenta samples were collected from C-sections, and blood samples were drawn from each pregnant woman in the third trimester. HTR-8 and JAR cell lines were cultured in exosome-free media and treated with hypoxia-mimetic agents. Exosome and Ago-bound fractions were isolated by membrane affinity spin column method from plasma and cell media. Short RNAs were extracted from exosomes and vesicle-free fractions, and total-RNA was isolated from the placenta samples. The RNA purity and concentration were measured by spectrophotometry. Expression analysis was carried out by qPCR with specific primers to target and reference miRNAs. RESULTS: The level of hsa-miR-210 was significantly higher in PE placentas, which could cause a minor increase of exosomal and a high elevation of Ago-bound miR-210 in circulation. Hypoxia lead to intracellular hsa-miR-210 upregulation in trophoblast cell lines. In extravillous cell (HTR-8) media, only the level of exosomal hsa-miR-210 was increased but no change in Ago-bound hsa-miR-210 level was observed. In contrast, in villous cell (JAR) media, the level of exosomal hsa-miR-210 was increased and enhanced release of Ago-bound hsa-miR-210 was also observed. CONCLUSION: Based on our data, we postulate that in PE, exosomal hsa-miR-210 is secreted actively from the trophoblast, and by intercellular communication, it may have a role in disease etiology. In addition, there is a passive release of Ago-bound hsa-miR-210 into the circulation, which may represent by-products of cell-death and is thereby a possible consequence of the disease.

Transcription activity of transposon sequence limits Sleeping Beauty transposition.

Sleeping Beauty (SB) transposon based technology has been extensively applied in basic research and biotechnology for routine cell culture gene delivery and vertebrate transgenesis, and it is also investigated in various gene therapy applications. Cell tolerance for the transgene is a key factor during transgenesis and is modulated not only through the type but by the dose of expression. Our experimental results exemplify that transgenes regulated with high activity promoters can reduce the overall success of gene delivery. Observations connected to transposon donors regulated by different promoters have also revealed inverse correlation between transcription activity and the hyperactive variant SB100X excision efficiency. This competition between transcription and transposition was independent of the transgene coding sequence and did not alter the transgenic efficiency in general. However, promoters applied in the transgene cassette can produce different average copy numbers depending on the transcriptional activity of the transposon. Unlike the piggyBac (PB) transposon system, this phenomenon allows a fine balance of expression using the high copy potential SB system that adjusts the copy number of lower activity promoter driven transgenes to a higher expression level. All this contributes to a well-tolerated and satisfactory transgenesis, and would be important to consider in gene therapy applications.

Generation of multidrug resistant human tissues by overexpression of the ABCG2 multidrug transporter in embryonic stem cells.

The ABCG2 multidrug transporter provides resistance against various endo- and xenobiotics, and protects the stem cells against toxins and stress conditions. We have shown earlier that a GFP-tagged version of ABCG2 is fully functional and may be used to follow the expression, localization and function of this transporter in living cells. In the present work we have overexpressed GFP-ABCG2, driven by a constitutive (CAG) promoter, in HUES9 human embryonic stem cells. Stem cell clones were generated to express the wild-type and a substrate-mutant (R482G) GFP-ABCG2 variant, by using the Sleeping Beauty transposon system. We found that the stable overexpression of these transgenes did not change the pluripotency and growth properties of the stem cells, nor their differentiation capacity to hepatocytes or cardiomyocytes. ABCG2 overexpression provided increased toxin resistance in the stem cells, and protected the derived cardiomyocytes against doxorubicin toxicity. These studies document the potential of a stable ABCG2 expression for engineering toxin-resistant human pluripotent stem cells and selected stem cell derived tissues.

The nonstop decay and the RNA silencing systems operate cooperatively in plants.

Translation-dependent mRNA quality control systems protect the protein homeostasis of eukaryotic cells by eliminating aberrant transcripts and stimulating the decay of their protein products. Although these systems are intensively studied in animals, little is known about the translation-dependent quality control systems in plants. Here, we characterize the mechanism of nonstop decay (NSD) system in Nicotiana benthamiana model plant. We show that plant NSD efficiently degrades nonstop mRNAs, which can be generated by premature polyadenylation, and stop codon-less transcripts, which are produced by endonucleolytic cleavage. We demonstrate that in plants, like in animals, Pelota, Hbs1 and SKI2 proteins are required for NSD, supporting that NSD is an ancient and conserved eukaryotic quality control system. Relevantly, we found that NSD and RNA silencing systems cooperate in plants. Plant silencing predominantly represses target mRNAs through endonucleolytic cleavage in the coding region. Here we show that NSD is required for the elimination of 5' cleavage product of mi- or siRNA-guided silencing complex when the cleavage occurs in the coding region. We also show that NSD and nonsense-mediated decay (NMD) quality control systems operate independently in plants.

Characterization of calcium signals in human induced pluripotent stem cell-derived dentate gyrus neuronal progenitors and mature neurons, stably expressing an advanced calcium indicator protein.

Pluripotent stem cell derived human neuronal progenitor cells (hPSC-NPCs) and their mature neuronal cell culture derivatives may efficiently be used for central nervous system (CNS) drug screening, including the investigation of ligand-induced calcium signalization. We have established hippocampal NPC cultures derived from human induced PSCs, which were previously generated by non-integrating Sendai virus reprogramming. Using established protocols these NPCs were differentiated into hippocampal dentate gyrus neurons. In order to study calcium signaling without the need of dye loading, we have stably expressed an advanced calcium indicator protein (GCaMP6fast) in the NPCs using the Sleeping Beauty transposon system. We observed no significant effects of the long-term GCaMP6 expression on NPC morphology, gene expression pattern or neural differentiation capacity. In order to compare the functional properties of GCaMP6-expressing neural cells and the corresponding parental cells loaded with calcium indicator dye Fluo-4, a detailed characterization of calcium signals was performed. We found that the calcium signals induced by ATP, glutamate, LPA, or proteases - were similar in these two systems. Moreover, the presence of the calcium indicator protein allowed for a sensitive, repeatable detection of changes in calcium signaling during the process of neurogenesis and neuronal maturation.

Extensive astrocyte synchronization advances neuronal coupling in slow wave activity in vivo.

Slow wave activity (SWA) is a characteristic brain oscillation in sleep and quiet wakefulness. Although the cell types contributing to SWA genesis are not yet identified, the principal role of neurons in the emergence of this essential cognitive mechanism has not been questioned. To address the possibility of astrocytic involvement in SWA, we used a transgenic rat line expressing a calcium sensitive fluorescent protein in both astrocytes and interneurons and simultaneously imaged astrocytic and neuronal activity in vivo. Here we demonstrate, for the first time, that the astrocyte network display synchronized recurrent activity in vivo coupled to UP states measured by field recording and neuronal calcium imaging. Furthermore, we present evidence that extensive synchronization of the astrocytic network precedes the spatial build-up of neuronal synchronization. The earlier extensive recruitment of astrocytes in the synchronized activity is reinforced by the observation that neurons surrounded by active astrocytes are more likely to join SWA, suggesting causality. Further supporting this notion, we demonstrate that blockade of astrocytic gap junctional communication or inhibition of astrocytic Ca2+ transients reduces the ratio of both astrocytes and neurons involved in SWA. These in vivo findings conclusively suggest a causal role of the astrocytic syncytium in SWA generation.

A transgenic rat hepatocyte - Kupffer cell co-culture model for evaluation of direct and macrophage-related effect of poly(amidoamine) dendrimers.

Increasing number of papers demonstrate that Kupffer cells (KCs) play a role in the development of drug induced liver injury (DILI). Furthermore, elevated intracellular Ca2+ level of hepatocytes is considered as a common marker of DILI. Here we applied an in vitro model based on hepatocyte mono- and hepatocyte/KC co-cultures (H/KC) isolated from transgenic rats stably expressing the GCaMP2 fluorescent Ca2+ sensor protein to investigate the effects of polycationic (G5), polyanionic (G4.5) and polyethylene-glycol coated neutral (G5 Peg) dendrimers known to accumulate in the liver, primarily in KCs. Following dendrimer exposure, hepatocyte homeostasis was measured by MTT cytotoxicity assay and by Ca2+ imaging, while hepatocyte functions were studied by CYP2B1/2 inducibility, and bilirubin and taurocholate transport. G5 was significantly more cytotoxic than G4.5 for hepatocytes and induced Ca2+ oscillation and sustained Ca2+ signals at 1µM and10 µM, respectively both in hepatocytes and KCs. Dendrimer-induced Ca2+ signals in hepatocytes were attenuated by macrophages. Activation of KCs by lipopolysaccharide and G5 decreased the inducibility of CYP2B1/2, which was restored by depleting the KCs with gadolinium-chloride and pentoxyphylline, suggesting a role of macrophages in the hindrance of CYP2B1/2 induction by G5 and lipopolysaccharide. In the H/KC, but not in the hepatocyte mono-culture, G5 reduced the canalicular efflux of bilirubin and stimulated the uptake and canalicular efflux of taurocholate. In conclusion, H/KC provides a good model for the prediction of hepatotoxic potential of drugs, especially of nanomaterials known to be trapped by macrophages, activation of which presumably contributes to DILI.