Cell Type-specific Differences Research Articles

Singing modulates parvalbumin interneurons throughout songbird forebrain vocal control circuitry.

Across species, the performance of vocal signals can be modulated by the social environment. Zebra finches, for example, adjust their song performance when singing to females (‘female-directed’ or FD song) compared to when singing in isolation (‘undirected’ or UD song). These changes are salient, as females prefer the FD song over the UD song. Despite the importance of these performance changes, the neural mechanisms underlying this social modulation remain poorly understood. Previous work in finches has established that expression of the immediate early gene EGR1 is increased during singing and modulated by social context within the vocal control circuitry. Here, we examined whether particular neural subpopulations within those vocal control regions exhibit similar modulations of EGR1 expression. We compared EGR1 expression in neurons expressing parvalbumin (PV), a calcium buffer that modulates network plasticity and homeostasis, among males that performed FD song, males that produced UD song, or males that did not sing. We found that, overall, singing but not social context significantly affected EGR1 expression in PV neurons throughout the vocal control nuclei. We observed differences in EGR1 expression between two classes of PV interneurons in the basal ganglia nucleus Area X. Additionally, we found that singing altered the amount of PV expression in neurons in HVC and Area X and that distinct PV interneuron types in Area X exhibited different patterns of modulation by singing. These data indicate that throughout the vocal control circuitry the singing-related regulation of EGR1 expression in PV neurons may be less influenced by social context than in other neuron types and raise the possibility of cell-type specific differences in plasticity and calcium buffering.

Accelerated retinal aging in PACAP knock-out mice

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurotrophic and neuroprotective peptide. PACAP and its receptors are widely distributed in the retina. A number of reports provided evidence that PACAP is neuroprotective in retinal degenerations. The current study compared retina cell type-specific differences in young (3–4months) and aged adults (14–16months), of wild-type (WT) mice and knock-out (KO) mice lacking endogenous PACAP production during the course of aging. Histological, immunocytochemical and Western blot examinations were performed. The staining for standard neurochemical markers (tyrosine hydroxylase for dopaminergic cells, calbindin 28 kDa for horizontal cells, protein kinase Cα for rod bipolar cells) of young adult PACAP KO retinas showed no substantial alterations compared to young adult WT retinas, except for the specific PACAP receptor (PAC1-R) staining. We could not detect PAC1-R immunoreactivity in bipolar and horizontal cells in young adult PACAP KO animals. Some other age-related changes were observed only in the PACAP KO mice only. These alterations included horizontal and rod bipolar cell dendritic sprouting into the photoreceptor layer and decreased ganglion cell number. Also, Müller glial cells showed elevated GFAP expression compared to the aging WT retinas. Furthermore, Western blot analyses revealed significant differences between the phosphorylation state of ERK1/2 and JNK in KO mice, indicating alterations in the MAPK signaling pathway. These results support the conclusion that endogenous PACAP contributes to protection against aging of the nervous system.

Transient voltage-activated K+ currents in central antennal lobe neurons: cell type-specific functional properties.

In this study we analyzed transient voltage-activated K+ currents (IA) of projection neurons and local interneurons in the antennal lobe of the cockroach Periplaneta americana The antennal lobe is the first synaptic processing station for olfactory information in insects. Local interneurons are crucial for computing olfactory information and form local synaptic connections exclusively in the antennal lobe, whereas a primary task of the projection neurons is the transfer of preprocessed olfactory information from the antennal lobe to higher order centers in the protocerebrum. The different physiological tasks of these neurons require specialized physiological and morphological neuronal phenotypes. We asked if and how the different physiological phenotypes are reflected in the functional properties of IA, which is crucial for shaping intrinsic electrophysiological properties of neurons. Whole cell patch-clamp recordings from adult male P. americana showed that all their central antennal lobe neurons can generate IA The current exhibited marked cell type-specific differences in voltage dependence of steady-state activation and inactivation, and differences in inactivation kinetics during sustained depolarization. Pharmacological experiments revealed that IA in all neuron types was partially blocked by α-dendrotoxin and phrixotoxin-2, which are considered blockers with specificity for Shaker- and Shal-type channels, respectively. These findings suggest that IA in each cell type is a mixed current generated by channels of both families. The functional role of IA was analyzed in experiments under current clamp, in which portions of IA were blocked by α-dendrotoxin or phrixotoxin-2. These experiments showed that IA contributes significantly to the intrinsic electrophysiological properties, such as the action potential waveform and membrane excitability.NEW & NOTEWORTHY In the insect olfactory system, projection neurons and local interneurons have task-specific electrophysiological and morphological phenotypes. Voltage-activated potassium channels play a crucial role in shaping functional properties of these neurons. This study revealed marked cell type-specific differences in the biophysical properties of transient voltage-activated potassium currents in central antennal lobe neurons.

Differential cytotoxic and inflammatory potency of amorphous silicon dioxide nanoparticles of similar size in multiple cell lines

The likelihood of environmental and health impacts of silicon dioxide nanoparticles (SiNPs) has risen, due to their increased use in products and applications. The biological potency of a set of similarly-sized amorphous SiNPs was investigated in a variety of cells to examine the influence of physico-chemical and biological factors on their toxicity. Cellular LDH and ATP, BrdU incorporation, resazurin reduction and cytokine release were measured in human epithelial A549, human THP-1 and mouse J774A.1 macrophage cells exposed for 24 h to suspensions of 5–15, 10–20 and 12 nm SiNPs and reference particles. The SiNPs were characterized in dry state and in suspension to determine their physico-chemical properties. The dose-response data were simplified into particle potency estimates to facilitate the comparison of multiple endpoints of biological effects in cells. Mouse macrophages were the most sensitive to SiNP exposures. Cytotoxicity of the individual cell lines was correlated while the cytokine responses differed, supported by cell type-specific differences in inflammation-associated pathways. SiNP (12 nm), the most cytotoxic and inflammogenic nanoparticle had the highest surface acidity, dry-state agglomerate size, the lowest trace metal and organics content, the smallest surface area and agglomerate size in suspension. Particle surface acidity appeared to be the most significant determinant of the overall biological activity of this set of nanoparticles. Combined with the nanoparticle characterization, integration of the biological potency estimates enabled a comprehensive determination of the cellular reactivity of the SiNPs. The approach shows promise as a useful tool for first-tier screening of SiNP toxicity.

High-throughput downstream process development for cell-based products using aqueous two-phase systems (ATPS) - A case study.

The availability of preparative-scale downstream processing strategies for cell-based products presents a critical juncture between fundamental research and clinical development. Aqueous two-phase systems (ATPS) present a gentle, scalable, label-free, and cost-effective method for cell purification, and are thus a promising tool for downstream processing of cell-based therapeutics. Here, the application of a previously developed robotic screening platform that enables high-throughput cell partitioning analysis in ATPS is reported. In the present case study a purification strategy for two model cell lines based on high-throughput screening (HTS)-data and countercurrent distribution (CCD)-modeling, and validated the CCD-model experimentally is designed. The obtained data are shown an excellent congruence between CCD-model and experimental data, indicating that CCD-models in combination with HTS-data are a powerful tool in downstream process development. Finally, the authors are shown that while cell cycle phase significantly influences cell partitioning, cell type specific differences in surface properties are the main driving force in charge-dependent separation of HL-60 and L929 cells. In order to design a highly robust purification process it is, however, advisable to maintain constant growth conditions.

Isogenic iPSC Models of SRSF2-Mutant Myelodysplastic Syndrome Capture Disease Phenotypes, Splicing Defects and Drug Responses

A major recent advance for the biology of Myelodysplastic Syndrome (MDS) was the discovery of recurrent mutations in genes encoding splicing factors (SFs). SRSF2 is a SF that binds to RNA sequences called exonic splicing enhancers (ESEs) to promote inclusion of exons containing these motifs. Mutations of SRSF2 are found in 20-30% of MDS patients, are associated with adverse prognosis and are almost always heterozygous missense mutations (P95 L/R/H). This strongly suggests a gain or alteration of function mechanism, corroborated by recent findings by us and others in a knockin mouse model and in K562 cells expressing mutant SRSF2, showing that the SRSF2 P95H mutation changes the normal RNA binding specificity of SRSF2 (Kim et al. Cancer Cell, 2015; Zhang et al. PNAS, 2015). While mis-splicing of hematopoietic regulators, such as EZH2, was proposed as a potential downstream mechanism of SF mutations, systematically identifying the key downstream mediators remains a challenge. No human hematopoietic cell lines harboring SF mutations in the context of a normal diploid genome exist, primary MDS cells afford limited experimental opportunities and mouse modeling is likely to miss disease relevant targets, as only a quarter of regulated alternative splicing events are conserved between human and mouse.Our lab has pioneered the modeling of MDS with human induced pluripotent stem cells (iPSCs). We previously derived iPSCs with the SRSF2 P95L mutation and a chr7q deletion (del7q), as well as normal iPSCs from residual normal hematopoietic cells from the same MDS patient (Kotini et al. Nat. Biotech, 2015). To develop a model of mutant SRSF2, we used CRISPR/Cas9 technology to generate a panel of isogenic iPSCs with or without the SRSF2 P95 mutation, isolated or with the cooperating del(7q), all in the same genetic background, by both introducing the SRSF2 P95L mutation in normal iPSCs and correcting it in MDS-iPSCs from the same patient. Both patient-derived and genetically engineered SRSF2 P95L-iPSCs showed decreased growth and increased cell death of hematopoietic progenitor cells (HPCs), decreased clonogenic capacity and features of morphologic dysplasia. This phenotype is consistent with the SRSF2 mutation being an early, potentially initiating, event, supported by its frequent presence in the dominant clone and in individuals with clonal hematopoiesis of indeterminate potential (CHIP) without overt MDS. Using a competitive cell growth assay, we found that the splicing inhibitor E7107, as well as small molecule inhibitors of kinases modulating splicing, preferentially inhibit the growth of SRSF2 mutant, but not of isogenic normal, iPSC-derived HPCs.To investigate the effects of mutant SRSF2 in mRNA splicing, we performed RNA sequencing of purified CD34+, CD34+/CD45+ HPCs and undifferentiated iPSCs from mutant and isogenic wild-type (WT) iPSCs. SRSF2 mutant cells exhibited genome-wide alterations in ESE preferences, recapitulating the altered RNA binding found in patient cells, with mutant SRSF2 preferentially recognizing a CCNG motif versus a GGNG motif, while WT SRSF2 binds to both with similar affinity. Genes found mis-spliced in the SRSF2 mutant cells included previously reported genes of potential disease relevance, like EZH2 and FYN. While the majority of differentially spliced genes overlapped among the 3 cell states, cell type-specific differences were also noted, highlighting the importance of performing these analyses in the appropriate cell type. Importantly, iPSC-derived HPCs recapitulated a higher percentage of the mis-spliced events observed in patient cells than either the knockin mouse or the K562 models, thus capturing disease-relevant splicing alterations more faithfully than other models. To identify critical direct targets of mutant SRSF2, we used a second round of CRISPR/Cas9 gene editing to introduce a 3xFLAG epitope tag at the carboxyl terminus of the endogenous SRSF2 locus in isogenic SRSF2mutant and WT iPSCs. CLIP-seq experiments in undifferentiated cells and HPCs derived from them are underway to identify genes differentially bound by the mutant vs the WT SRSF2.The model we describe here will be valuable for dissecting the pathogenesis of MDS with SF mutations, testing drugs and identifying new therapeutic targets for drug development. DisclosuresNo relevant conflicts of interest to declare.

RNA Sequencing Reveals a Role of TonEBP Transcription Factor in Regulation of Pro-inflammatory Genes in Response to Hyperosmolarity in Healthy Nucleus Pulposus Cells: A HOMEOSTATIC RESPONSE?

Transcription factor tonicity-responsive enhancer-binding protein (TonEBP/NFAT5) is critical for osmo-adaptation and extracellular matrix homeostasis of nucleus pulposus (NP) cells in their hypertonic tissue niche. Recent studies implicate TonEBP signaling in inflammatory disease and rheumatoid arthritis pathogenesis. However, broader functions of TonEBP in the disc remain unknown. RNA sequencing was performed on NP cells with TonEBP knockdown under hypertonic conditions. 1140 TonEBP-dependent genes were identified and categorized using Ingenuity Pathway Analysis. Bioinformatic analysis showed enrichment of matrix homeostasis and cytokine/chemokine signaling pathways. C-C motif chemokine ligand 2 (CCL2), interleukin 6 (IL6), tumor necrosis factor (TNF), and nitric oxide synthase 2 (NOS2) were studied further. Knockdown experiments showed that TonEBP was necessary to maintain expression levels of these genes. Gain- and loss-of-function experiments and site-directed mutagenesis demonstrated that TonEBP binding to a specific site in the CCL2 promoter is required for hypertonic inducibility. Despite inhibition by dominant-negative TonEBP, IL6 and NOS2 promoters were not hypertonicity-inducible. Whole-disc response to hypertonicity was studied in an ex vivo organ culture model, using wild-type and haploinsufficient TonEBP mice. Pro-inflammatory targets were induced by hypertonicity in discs from wild-type but not TonEBP-haploinsufficient mice. Mechanistically, NF-κB activity increased with hypertonicity and was necessary for hypertonic induction of target genes IL6, TNF, and NOS2 but not CCL2 Although TonEBP maintains transcription of genes traditionally considered pro-inflammatory, it is important to note that some of these genes also serve anabolic and pro-survival roles. Therefore, in NP cells, this phenomenon may reflect a physiological adaptation to diurnal osmotic loading of the intervertebral disc.

In Vitro Toxicity Evaluation of Lignin-(Un)coated Cellulose Based Nanomaterials on Human A549 and THP-1 Cells.

A significant amount of research toward commercial development of cellulose based nanomaterials (CNM) is now in progress with some potential applications. Using human A549 and THP-1 cells, we evaluated the biological responses of various CNMs, made out of similar material but with functional and morphological variations. While A549 cells displayed minimal or no cytotoxic responses following exposure to CNMs, THP-1 cells were more susceptible to cytotoxicity, cellular damage and inflammatory responses. Further analysis of these biological responses evaluated using hierarchical clustering approaches was effective in discriminating (dis)-similarities of various CNMs studied and identified potential inflammatory factors contributing to cytotoxicity. No correlation between cytotoxicity and surface properties of CNMs was found. This study clearly highlights that, in addition to the source and characteristics of CNMs, cell type-specific differences in the recognition/uptake of CNMs along with their inherent capability to respond to external stimuli are crucial for assessing the toxicity of CNMs.

Identification of Cell Type-Specific Differences in Erythropoietin Receptor Signaling in Primary Erythroid and Lung Cancer Cells.

Lung cancer, with its most prevalent form non-small-cell lung carcinoma (NSCLC), is one of the leading causes of cancer-related deaths worldwide, and is commonly treated with chemotherapeutic drugs such as cisplatin. Lung cancer patients frequently suffer from chemotherapy-induced anemia, which can be treated with erythropoietin (EPO). However, studies have indicated that EPO not only promotes erythropoiesis in hematopoietic cells, but may also enhance survival of NSCLC cells. Here, we verified that the NSCLC cell line H838 expresses functional erythropoietin receptors (EPOR) and that treatment with EPO reduces cisplatin-induced apoptosis. To pinpoint differences in EPO-induced survival signaling in erythroid progenitor cells (CFU-E, colony forming unit-erythroid) and H838 cells, we combined mathematical modeling with a method for feature selection, the L1 regularization. Utilizing an example model and simulated data, we demonstrated that this approach enables the accurate identification and quantification of cell type-specific parameters. We applied our strategy to quantitative time-resolved data of EPO-induced JAK/STAT signaling generated by quantitative immunoblotting, mass spectrometry and quantitative real-time PCR (qRT-PCR) in CFU-E and H838 cells as well as H838 cells overexpressing human EPOR (H838-HA-hEPOR). The established parsimonious mathematical model was able to simultaneously describe the data sets of CFU-E, H838 and H838-HA-hEPOR cells. Seven cell type-specific parameters were identified that included for example parameters for nuclear translocation of STAT5 and target gene induction. Cell type-specific differences in target gene induction were experimentally validated by qRT-PCR experiments. The systematic identification of pathway differences and sensitivities of EPOR signaling in CFU-E and H838 cells revealed potential targets for intervention to selectively inhibit EPO-induced signaling in the tumor cells but leave the responses in erythroid progenitor cells unaffected. Thus, the proposed modeling strategy can be employed as a general procedure to identify cell type-specific parameters and to recommend treatment strategies for the selective targeting of specific cell types.

Cell Type-Specific Differences in Spike Timing and Spike Shape in the Rat Parasubiculum and Superficial Medial Entorhinal Cortex

SummaryThe medial entorhinal cortex (MEC) and the adjacent parasubiculum are known for their elaborate spatial discharges (grid cells, border cells, etc.) and the precessing of spikes relative to the local field potential. We know little, however, about how spatio-temporal firing patterns map onto cell types. We find that cell type is a major determinant of spatio-temporal discharge properties. Parasubicular neurons and MEC layer 2 (L2) pyramids have shorter spikes, discharge spikes in bursts, and are theta-modulated (rhythmic, locking, skipping), but spikes phase-precess only weakly. MEC L2 stellates and layer 3 (L3) neurons have longer spikes, do not discharge in bursts, and are weakly theta-modulated (non-rhythmic, weakly locking, rarely skipping), but spikes steeply phase-precess. The similarities between MEC L3 neurons and MEC L2 stellates on one hand and parasubicular neurons and MEC L2 pyramids on the other hand suggest two distinct streams of temporal coding in the parahippocampal cortex.

TDP-43 regulates endogenous retrovirus-K viral protein accumulation

The concomitant expression of neuronal TAR DNA binding protein 43 (TDP-43) and human endogenous retrovirus-K (ERVK) is a hallmark of ALS. Since the involvement of TDP-43 in retrovirus replication remains controversial, we sought to evaluate whether TDP-43 exerts an effect on ERVK expression. In this study, TDP-43 bound the ERVK promoter in the context of inflammation or proteasome inhibition, with no effect on ERVK transcription. However, over-expression of ALS-associated aggregating forms of TDP-43, but not wild-type TDP-43, significantly enhanced ERVK viral protein accumulation. Human astrocytes and neurons further demonstrated cell-type specific differences in their ability to express and clear ERVK proteins during inflammation and proteasome inhibition. Astrocytes, but not neurons, were able to clear excess ERVK proteins through stress granule formation and autophagy. In vitro findings were validated in autopsy motor cortex tissue from patients with ALS and neuro-normal controls. We further confirmed marked enhancement of ERVK in cortical neurons of patients with ALS. Despite evidence of enhanced stress granule and autophagic response in ALS cortical neurons, these cells failed to clear excess ERVK protein accumulation. This highlights how multiple cellular pathways, in conjunction with disease-associated mutations, can converge to modulate the expression and clearance of viral gene products from genomic elements such as ERVK. In ALS, ERVK protein aggregation is a novel aspect of TDP-43 misregulation contributing towards the pathology of this neurodegenerative disease.

Immuno and Affinity Cytochemical Analysis of Cell Wall Composition in the Moss Physcomitrella patens.

In contrast to homeohydric vascular plants, mosses employ a poikilohydric strategy for surviving in the dry aerial environment. A detailed understanding of the structure, composition, and development of moss cell walls can contribute to our understanding of not only the evolution of overall cell wall complexity, but also the differences that have evolved in response to selection for different survival strategies. The model moss species Physcomitrella patens has a predominantly haploid lifecycle consisting of protonemal filaments that regenerate from protoplasts and enlarge by tip growth, and leafy gametophores composed of cells that enlarge by diffuse growth and differentiate into several different types. Advantages for genetic studies include methods for efficient targeted gene modification and extensive genomic resources. Immuno and affinity cytochemical labeling were used to examine the distribution of polysaccharides and proteins in regenerated protoplasts, protonemal filaments, rhizoids, and sectioned gametophores of P. patens. The cell wall composition of regenerated protoplasts was also characterized by flow cytometry. Crystalline cellulose was abundant in the cell walls of regenerating protoplasts and protonemal cells that developed on media of high osmolarity, whereas homogalactuonan was detected in the walls of protonemal cells that developed on low osmolarity media and not in regenerating protoplasts. Mannan was the major hemicellulose detected in all tissues tested. Arabinogalactan proteins were detected in different cell types by different probes, consistent with structural heterogneity. The results reveal developmental and cell type specific differences in cell wall composition and provide a basis for analyzing cell wall phenotypes in knockout mutants.

High content screening application for cell-type specific behaviour in heterogeneous primary breast epithelial subpopulations.

BackgroundThe complex interaction between multiple cell types and the microenvironment underlies the diverse pathways to carcinogenesis and necessitates sophisticated approaches to in vitro hypotheses testing. The combination of mixed culture format with high content immunofluorescence screening technology provides a powerful platform for observation of cell type specific behavior.MethodsWe have developed a versatile, high-throughput method for assessing cell-type specific responses. In addition to the specificity and sensitivity offered traditionally by immunofluorescent detection in flow cytometry, the ‘in-cell’ analysis method we describe provides the added benefits of higher throughput and the ability to analyse protein subcellular localisation in situ. Furthermore, elimination of the cell dissociation step allows for more immediate analysis of responses to specific extrinsic stimuli. We applied this method to investigate ionising radiation treatment response in normal breast epithelial cells, measuring growth rate, cell cycle response and double-strand DNA breaks.ResultsThe ‘in-cell’ analysis approach elucidated several interesting donor and cell-type specific differences. Notably, in response to ionizing radiation we observed differential expression in luminal and basal-like cells of a member of the APOBEC enzyme family, recently identified as a critical driver of an oncogenic signature. Our findings suggest that this enzyme is active in the normal breast epithelium during DNA damage response.ConclusionsWe demonstrate the practical application of a new method for assessing cell-type specific change in mixed cultures, especially the analysis of normal primary cultures, overcoming a major technical issue of dissecting the response of multiple cell types in a heterogeneous population.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0681-9) contains supplementary material, which is available to authorized users.

Reciprocal changes in DNA methylation and hydroxymethylation and a broad repressive epigenetic switch characterize FMR1 transcriptional silencing in fragile X syndrome.

BackgroundFragile X syndrome (FXS) is the most common form of inherited intellectual disability, resulting from the loss of function of the fragile X mental retardation 1 (FMR1) gene. The molecular pathways associated with FMR1 epigenetic silencing are still elusive, and their characterization may enhance the discovery of novel therapeutic targets as well as the development of novel clinical biomarkers for disease status.ResultsWe have deployed customized epigenomic profiling assays to comprehensively map the FMR1 locus chromatin landscape in peripheral mononuclear blood cells (PBMCs) from eight FXS patients and in fibroblast cell lines derived from three FXS patient. Deoxyribonucleic acid (DNA) methylation (5-methylcytosine (5mC)) and hydroxymethylation (5-hydroxymethylcytosine (5hmC)) profiling using methylated DNA immunoprecipitation (MeDIP) combined with a custom FMR1 microarray identifies novel regions of DNA (hydroxy)methylation changes within the FMR1 gene body as well as in proximal flanking regions. At the region surrounding the FMR1 transcriptional start sites, increased levels of 5mC were associated to reciprocal changes in 5hmC, representing a novel molecular feature of FXS disease. Locus-specific validation of FMR1 5mC and 5hmC changes highlighted inter-individual differences that may account for the expected DNA methylation mosaicism observed at the FMR1 locus in FXS patients. Chromatin immunoprecipitation (ChIP) profiling of FMR1 histone modifications, together with 5mC/5hmC and gene expression analyses, support a functional relationship between 5hmC levels and FMR1 transcriptional activation and reveal cell-type specific differences in FMR1 epigenetic regulation. Furthermore, whilst 5mC FMR1 levels positively correlated with FXS disease severity (clinical scores of aberrant behavior), our data reveal for the first time an inverse correlation between 5hmC FMR1 levels and FXS disease severity.ConclusionsWe identify novel, cell-type specific, regions of FMR1 epigenetic changes in FXS patient cells, providing new insights into the molecular mechanisms of FXS. We propose that the combined measurement of 5mC and 5hmC at selected regions of the FMR1 locus may significantly enhance FXS clinical diagnostics and patient stratification.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0181-x) contains supplementary material, which is available to authorized users.

Differential DNA methylation patterns of homeobox genes in proximal and distal colon epithelial cells.

Region and cell-type specific differences in the molecular make up of colon epithelial cells have been reported. Those differences may underlie the region-specific characteristics of common colon epithelial diseases such as colorectal cancer and inflammatory bowel disease. DNA methylation is a cell-type specific epigenetic mark, essential for transcriptional regulation, silencing of repetitive DNA and genomic imprinting. Little is known about any region-specific variations in methylation patterns in human colon epithelial cells. Using purified epithelial cells and whole biopsies (n= 19) from human subjects, we generated epigenome-wide DNA methylation data (using the HELP-tagging assay), comparing the methylation signatures of the proximal and distal colon. We identified a total of 125 differentially methylated sites (DMS) mapping to transcription start sites of protein-coding genes, most notably several members of the homeobox (HOX) family of genes. Patterns of differential methylation were validated with MassArray EpiTYPER. We also examined DNA methylation in whole biopsies, applying a computational technique to deconvolve variation in methylation within cell types and variation in cell-type composition across biopsies. Including inferred epithelial proportions as a covariate in differential methylation analysis applied to the whole biopsies resulted in greater overlap with the results obtained from purified epithelial cells compared with when the covariate was not included. Results obtained from both approaches highlight region-specific methylation patterns of HOX genes in colonic epithelium. Regional variation in methylation patterns has implications for the study of diseases that exhibit regional expression patterns in the human colon, such as inflammatory bowel disease and colorectal cancer.

NKCC1-Mediated GABAergic Signaling Promotes Postnatal Cell Death in Neocortical Cajal-Retzius Cells.

During early development, a substantial proportion of central neurons undergoes programmed cell death. This activity-dependent process is essential for the proper structural and functional development of the brain. To uncover cell type-specific differences in the regulation of neuronal survival versus apoptosis, we studied activity-regulated cell death in Cajal-Retzius neurons (CRNs) and the overall neuronal population in the developing mouse cerebral cortex. CRNs in the upper neocortical layer represent an early-born neuronal population, which is important for cortical development and largely disappears by apoptosis during neonatal stages. In contrast to the overall neuronal population, activity blockade with tetrodotoxin improved survival of CRNs in culture. Activation of GABAA receptors also blocked spontaneous activity and caused overall cell death including apoptosis of CRNs. Blockade of the Na-K-Cl transporter NKCC1 in vitro or its genetic deletion in vivo rescued CRNs from apoptosis. This effect was mediated by blockade of the p75NTR receptor signaling pathway. In summary, we discovered a novel developmental death pathway mediated by NKCC1, via GABAA receptor-mediated membrane depolarization and p75NTR signaling in CRNs. This pathway controls apoptosis of CRNs and may be critically involved in neurodevelopmental disorders such as autism and schizophrenia.

IFNλ Stimulates MxA Production in Human Dermal Fibroblasts via a MAPK-Dependent STAT1-Independent Mechanism

IFNλ is important for epidermal defense against viruses. It is produced by, and acts on, keratinocytes, whereas fibroblasts were previously considered to be unresponsive to this type III IFN. Herein we report findings revealing cell type-specific differences in IFNλ signaling and function in skin resident cells. In dermal fibroblasts, IFNλ induced the expression of myxovirus protein A (MxA), a potent antiviral factor, but not other IFN signature genes as it does in primary keratinocytes. In contrast to its effect on keratinocytes, IFNλ did not phosphorylate signal transducer and activator of transcription 1 in fibroblasts, but instead activated mitogen activated protein kinases (MAPK). Accordingly, inhibition of MAPK activation (p38 and p42/44) blocked the expression of MxA protein in fibroblasts but not in keratinocytes. Functionally, IFNλ inhibited proliferation in keratinocytes but not in fibroblasts. Moreover, IFNλ upregulated the expression of Tumor growth factor beta 1 (TGFβ1)-induced collagens in fibroblasts. Taken together, our findings identify primary human dermal fibroblasts as responder cells to IFNλ. Our study shows cutaneous cell type-specific IFN signaling and suggests that IFNλ, although important for epidermal antiviral competence, may also have a regulatory role in the dermal compartment balancing type I IFN-induced inhibition of tissue repair processes.

CpG methylation differences between neurons and glia are highly conserved from mouse to human.

Understanding epigenetic differences that distinguish neurons and glia is of fundamental importance to the nascent field of neuroepigenetics. A recent study used genome-wide bisulfite sequencing to survey differences in DNA methylation between these two cell types, in both humans and mice. That study minimized the importance of cell type-specific differences in CpG methylation, claiming these are restricted to localized genomic regions, and instead emphasized that widespread and highly conserved differences in non-CpG methylation distinguish neurons and glia. We reanalyzed the data from that study and came to markedly different conclusions. In particular, we found widespread cell type-specific differences in CpG methylation, with a genome-wide tendency for neuronal CpG-hypermethylation punctuated by regions of glia-specific hypermethylation. Alarmingly, our analysis indicated that the majority of genes identified by the primary study as exhibiting cell type-specific CpG methylation differences were misclassified. To verify the accuracy of our analysis, we isolated neuronal and glial DNA from mouse cortex and performed quantitative bisulfite pyrosequencing at nine loci. The pyrosequencing results corroborated our analysis, without exception. Most interestingly, we found that gene-associated neuron vs. glia CpG methylation differences are highly conserved across human and mouse, and are very likely to be functional. In addition to underscoring the importance of independent verification to confirm the conclusions of genome-wide epigenetic analyses, our data indicate that CpG methylation plays a major role in neuroepigenetics, and that the mouse is likely an excellent model in which to study the role of DNA methylation in human neurodevelopment and disease.

Oxidative stress and its impact on neurons and glia in multiple sclerosis lesions

Oxidative injury plays a major role in brain damage in many age-related human brain diseases and is particularly pronounced in the progressive stage of multiple sclerosis. In the latter it is related to the chronic inflammatory process and is amplified by brain changes due to aging and accumulation of disease burden. It induces demyelination and neurodegeneration by direct oxidation of lipids, proteins and DNA as well as by the induction of mitochondrial injury, which results in energy deficiency and further amplification of oxygen radical production. It affects neurons and all types of glia cells, but neurons and oligodendrocytes are most vulnerable. Difference in the susceptibility for oxidative injury between different cellular components of the central nervous system appears to be due to cell type specific differences in anti-oxidant defense mechanisms, iron loading, cellular susceptibility to apoptosis induction and energy demand. This article is part of a Special Issue entitled: Neuro Inflammation edited by Helga E. de Vries and Markus Schwaninger.

ID: 194: Role of microRNAs in signal transduction pathways of the inflammatory cytokine Interleukin-6: Relevance for liver diseases

IL-6 plays important roles in regulation of liver functions and promotes hepatocarcinogenesis. As the contribution of IL-6-induced miRNAs to these effects is largely unknown, we investigate the effects of IL-6 on the miRNome of hepatoma cells and non-transformed hepatocytes. Moreover, little is known about miRNAs regulating key players of IL-6 signal transduction. Therefore, our aims were to identify such miRNAs in a miRNA mimics screen and to elucidate their effects on this pathway. Integration of the results of both approaches is expected to lead to the identification of regulatory circuits. While IL-6 (and hyperIL-6) induce the differential regulation of thousands of mRNAs in HepG2 and HuH-7 hepatoma cell lines, only selected miRNAs change their expression level significantly, including miR-21, miR-100, miR-145, miR-146b-5p. In contrast, we have observed previously that IFN-g has a profound effect on the miRNome of melanoma cells (Schmitt, Cell Comm. Sign. 2012). Therefore we investigate whether IL-6-type cytokines may have generally weaker effects on the miRNome compared to interferons and/or whether our observation is due to cell type-specific differences. To extend our in vitro findings, we analyse and correlate the expression levels of miRNAs and inflammatory cytokines in patients with HCC and advanced nonalcoholic steatohepatitis (NASH), the latter being at high risk to develop HCC. Bio-Plex cytokine immunoassays revealed that the serum level of IL-6 and HGF is higher in HCC patients than in healthy controls and NASH patients. Our work may contribute to the identification of novel biomarkers for the progression of chronic liver diseases.