Karin Weigelt

CRX ChIP-seq reveals the cis-regulatory architecture of mouse photoreceptors

Approximately 98% of mammalian DNA is noncoding, yet we understand relatively little about the function of this enigmatic portion of the genome. The cis-regulatory elements that control gene expression reside in noncoding regions and can be identified by mapping the binding sites of tissue-specific transcription factors. Cone-rod homeobox (CRX) is a key transcription factor in photoreceptor differentiation and survival, but its in vivo targets are largely unknown. Here, we used chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq) on CRX to identify thousands of cis-regulatory regions around photoreceptor genes in adult mouse retina. CRX directly regulates downstream photoreceptor transcription factors and their target genes via a network of spatially distributed regulatory elements around each locus. CRX-bound regions act in a synergistic fashion to activate transcription and contain multiple CRX binding sites which interact in a spacing- and orientation-dependent manner to fine-tune transcript levels. CRX ChIP-seq was also performed on Nrl(-/-) retinas, which represent an enriched source of cone photoreceptors. Comparison with the wild-type ChIP-seq data set identified numerous rod- and cone-specific CRX-bound regions as well as many shared elements. Thus, CRX combinatorially orchestrates the transcriptional networks of both rods and cones by coordinating the expression of photoreceptor genes including most retinal disease genes. In addition, this study pinpoints thousands of noncoding regions of relevance to both Mendelian and complex retinal disease.

Docosahexaenoic acid attenuates microglial activation and delays early retinal degeneration

Microgliosis is a common phenomenon in neurodegenerative disorders including retinal dystrophies. We performed a detailed characterization of activated microglia in the retinoschisin (Rs1h)‐deficient (Rs1h−/Y) mouse model of inherited retinal degeneration. To visualize and isolate microglia, we crossed Rs1h−/Y animals with transgenic MacGreen mice, which express green fluorescent protein under the control of the macrophage‐specific csf1r promoter. Activated microglia were detected in retinal sections and whole‐mounts of early postnatal MacGreen/Rs1h−/Y mice before the onset of overt neuronal cell death. These activated microglia contained prominent lipid droplets and analysis of the retinal lipid composition showed decreased docosahexaenoic acid (DHA) levels in Rs1h−/Y retinas. To establish a link between microglia activation, reduced DHA levels, and neurodegeneration, a dietary intervention study was performed. Female Rs1h−/− mice and their Rs1h−/Y litter were either subjected to a diet enriched with DHA, or a control chow lacking DHA. Supplementation with DHA enhanced photoreceptor survival and converted activated microglia to a quiescent phenotype. Furthermore, DHA, but not docosapentaenoic acid or adrenic acid reduced pro‐inflammatory gene expression, migration, and lipid accumulation of cultured BV‐2 microglia. We conclude that retinal DHA levels control the activity of microglia and thereby may affect the progression and extent of retinal degeneration.

Immune system dysregulation in first-onset postpartum psychosis

BACKGROUND Accumulating evidence suggests that dysregulation of the immune system represents an important vulnerability factor for mood disorders. Postpartum psychosis (PP) is a severe mood disorder occurring within 4 weeks after delivery, a period of heightened immune responsiveness and an altered endocrine set point. Therefore, the aim of this study was to examine immune activation in patients with first-onset PP at the level of monocytes, T cells, and serum cytokines/chemokines. METHODS We included 63 women admitted with first-onset PP. Control groups included healthy postpartum (n = 56) and nonpostpartum (n = 136) women. A quantitative-polymerase chain reaction monocyte gene expression analysis was performed with 43 genes previously identified as abnormally regulated in nonpostpartum mood disorder patients including the isoforms of the glucocorticoid receptor. Peripheral blood mononuclear cells percentages were measured by fluorescence-activated cell sorter analysis, whereas serum cytokines/chemokines were determined with a cytometric bead array. RESULTS In healthy women, postpartum T cell levels were significantly elevated compared with nonpostpartum. Patients with PP failed to show the normal postpartum T cell elevation. In contrast, these patients showed a significant elevation of monocyte levels and a significant upregulation of several immune-related monocyte genes compared with control subjects postpartum and nonpostpartum. Furthermore, the glucocorticoid receptor α/β gene expression ratio was decreased in monocytes of PP patients, strongly correlating with their immune activation. CONCLUSIONS This study demonstrates a robust dysregulation of the immuno-neuro-endocrine set point in PP, with a notable over-activation of the monocyte/macrophage arm of the immune system.

Immune and Neuroimmune Alterations in Mood Disorders and Schizophrenia

A large number of publications over the past 20 years have indicated that immune system function is altered in schizophrenia and mood disorder patients. This chapter reviews the evidence, which suggests that a proinflammatory state of the cytokine network induces psychopathologic symptoms and may be involved in the pathogenesis and pathophysiology of these major mental illnesses. The authors also present recent data, which relates immune activation to present theories on the influence of activated immune cells in altering brain function. They also focus on the role of the environment in immune activation and on the role of the microbiome and gut flora. Increased understanding of such factors could help in the development of novel treatment strategies and improved clinical management of mental disorders.

The Novel Activated Microglia/Macrophage WAP Domain Protein, AMWAP, Acts as a Counter-Regulator of Proinflammatory Response

Microgliosis is a common phenomenon in neurodegenerative disorders, including retinal dystrophies. To identify candidate genes involved in microglial activation, we used DNA-microarray analysis of retinal microglia from wild-type and retinoschisin-deficient (Rs1h−/Y) mice, a prototypic model for inherited retinal degeneration. Thereby, we cloned a novel 76 aa protein encoding a microglia/macrophage-restricted whey acidic protein (WAP) termed activated microglia/macrophage WAP domain protein (AMWAP). The gene consists of three exons and is located on mouse chromosome 11 in proximity to a chemokine gene cluster. mRNA expression of AMWAP was detected in microglia from Rs1h−/Y retinas, brain microglia, and other tissue macrophages. AMWAP transcription was rapidly induced in BV-2 microglia upon stimulation with multiple TLR ligands and IFN-γ. The TLR-dependent expression of AMWAP was dependent on NF-κB, whereas its microglia/macrophage-specific transcription was regulated by PU.1. Functional characterization showed that AMWAP overexpression reduced the proinflammatory cytokines IL-6 and IL-1β and concomitantly increased expression of the alternative activation markers arginase 1 and Cd206. Conversely, small interfering RNA knockdown of AMWAP lead to higher IL-6, IL-1β, and Ccl2 transcript levels, whereas diminishing arginase 1 and Cd206 expression. Moreover, AMWAP expressing cells had less migratory capacity and showed increased adhesion in a trypsin-protection assay indicating antiserine protease activity. In agreement with findings from other WAP proteins, micromolar concentrations of recombinant AMWAP exhibited significant growth inhibitory activity against Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis. Taken together, we propose that AMWAP is a counter-regulator of proinflammatory microglia/macrophage activation and a potential modulator of innate immunity in neurodegeneration.

BIOMARKERS OF NEUROLOGICAL AND PSYCHIATRIC DISEASE

A large number of publications over the past 20 years have indicated that immune system function is altered in schizophrenia and mood disorder patients. This chapter reviews the evidence, which suggests that a proinflammatory state of the cytokine network induces psychopathologic symptoms and may be involved in the pathogenesis and pathophysiology of these major mental illnesses. The authors also present recent data, which relates immune activation to present theories on the influence of activated immune cells in altering brain function. They also focus on the role of the environment in immune activation and on the role of the microbiome and gut flora. Increased understanding of such factors could help in the development of novel treatment strategies and improved clinical management of mental disorders.

Decreased serum level of miR-146a as sign of chronic inflammation in type 2 diabetic patients

Background There is increasing evidence that chronic inflammation is an important determinant in insulin resistance and in the pathogenesis of type 2 diabetes (T2D). MicroRNAs constitute a newly discovered system of cell regulation and in particular two microRNAs (miR-146a and miR-155) have been described as regulators and biomarkers of inflammation. Aim To determine a putative association between the levels of miR-146a and miR-155 in serum of T2D patients, clinical parameters and serological indicators of inflammation. Methods We performed quantitative Real Time PCR (qPCR) of microRNAs from serum (56 Ecuadorian T2D ambulatory patients and 40 non-diabetic controls). In addition, we evaluated T2D-related serum cytokines.chemokines and growth factors using a commercially available multi-analyte cytometric bead array system. We correlated outcomes to clinical parameters, including BMI, HbA1c and lipid state. Results The Ecuadorian non-diabetic controls appeared as overweight (BMI>25: patients 85%, controls 82.5%) and as dyslipidemic (hypercholesterolemia: patients 60.7%, controls 67.5%) as the patients. The serum levels of miR-146a were significantly reduced in T2D patients as compared to these non-diabetic, but obese/dyslipidemic control group (mean patients 0.61, mean controls set at 1; p = 0.042), those of miR-155 were normal. The serum levels of both microRNAs correlated to each other (r = 0.478; p<0.001) and to leptin levels. The microRNAs did not correlate to BMI, glycemia and dyslipidemia. From the tested cytokines, chemokines and growth factors, we found IL-8 and HGF significantly raised in T2D patients versus non-diabetic controls (p = 0.011 and 0.023 respectively). Conclusions This study shows decreased serum anti-inflammatory miR-146a, increased pro-inflammatory IL-8 and increased HGF (a vascular/insular repair factor) as discriminating markers of failure of glucose control occurring on the background of obesity and dyslipidemia.

Transcriptomic profiling identifies a PU.1 regulatory network in macrophages

PU.1 is a key transcription factor for hematopoiesis and macrophage differentiation. Using chromatin immunoprecipitation we have previously identified several PU.1 target genes in macrophages and microglia. With the aim to complement these studies, we performed a transcriptomic analysis of PU.1(-/-) progenitors after restoration of PU.1 activity. PUER cells committed to macrophage differentiation were analyzed with novel Affymetrix exon 1.0 ST arrays and Affymetrix 430 2.0 genome arrays for crosswise validation. We combined these genome-wide expression data with a publicly-available microarray dataset of PU.1-knockdown hematopoietic stem cells for an integrated analysis. Bibliographic gene connections, binding site prediction and ChIP-Chip data were used to define a multi-level PU.1 regulatory network in macrophages. Moreover, an alternative transcript of the novel PU.1 target gene Ptpro was identified by exon arrays and PU.1 binding to an intronic promoter was demonstrated. In conclusion, we present a PU.1 transcriptional network with novel validated PU.1 target genes.

Down-regulation of inflammation-protective microRNAs 146a and 212 in monocytes of patients with postpartum psychosis

BACKGROUND Postpartum psychosis (PP) is thought to belong to the bipolar spectrum. Recently we described an immune activation signature in monocytes of patients with PP using gene expression profiling. Immune activation genes are regulated by microRNAs (miRNAs). We therefore profiled miRNA expression in monocytes of PP patients to identify differentially expressed miRNAs between PP and the healthy state. METHODS In a profiling study we carried out miRNA profiling using TaqMan array human microRNA A cards v2.0 and monocytes of 8 PP patients. Data were analyzed against monocytes of healthy postpartum women (CP). Nine miRNAs were selected and tested using individual Q-PCR in a larger validation study on monocytes of 20 PP patients, 20 CP and 20 healthy non-postpartum women (HC). RESULTS In the validation study miR-146a expression was significantly down-regulated in the monocytes of first onset PP patients as compared to CP and HC; miR-212 expression was significantly down-regulated in PP patients with prior bipolar disorder. In silico miR-146a targeted 4 genes of the previously described monocyte activation signature in bipolar disorder; miR-212 targeted 2 of such genes. In a correlation study decreased expression of miR-146a in monocytes was related to decreased natural T regulator cells in PP patients; decreased miR-212 was correlated to increased Adrenomedulin and decreased IL-6 expression in monocytes and to higher Th2 cell levels. CONCLUSIONS This study identified changes in miR-146a and -212 expression in PP. Since these miRNAs are linked to inflammation, the study strengthens the view that PP is an inflammation-like condition.

Dap12 expression in activated microglia from retinoschisin- deficient retina and its PU.1-dependent promoter regulation

Several alterations in the expression of immune‐related transcripts were identified recently in the degenerating retina of the retinoschisin knockout (Rs1h−/Y) mouse, including the strong expression of the adaptor protein Dap12. As Dap12 is found in leukocytes, we hypothesized that its disease‐related expression may be confined to activated retinal microglia cells. To test this hypothesis, we established a procedure for isolation and culture of retinal microglia cells and performed genome‐wide expression profiling from Rs1h−/Y and control microglia. While retaining their activated state in culture, ex vivo microglia expressed high levels of Dap12 and the transcription factor PU.1. The activation‐dependent induction of Dap12 was also confirmed in the microglia cell line BV‐2 following in vitro stimulation. To examine the transcriptional regulation of Dap12 further, macrophage cell lines were transfected with several Dap12 reporter constructs. Promoter deletion assays and site‐directed mutagenesis experiments demonstrated an essential role of evolutionarily conserved PU.1 consensus sites in the proximal −104/+118 Dap12 promoter. In vitro and in vivo binding of PU.1 to this promoter region was demonstrated using EMSA and chromatin immunoprecipitation. Knockdown of PU.1 by RNA interference caused a significant reduction of endogenous Dap12 expression and re‐expression, and activation of PU.1 in PU.1−/− progenitor cells induced Dap12 transcription. Taken together, our results indicate that activated microglia from degenerating retinae express high levels of Dap12 and PU.1, and PU.1 controls the myeloid‐specific regulation of Dap12 directly and may also play a general role in microglia gene expression during retinal degeneration.