Endothelial Driven Inflammation in Metabolic Disease: A Dissertation

Obesity has been on the rise over the last 30 years, reaching worldwide epidemic proportions. Obesity has been linked to multiple metabolic disorders and co-morbidities such as Type 2 Diabetes Mellitus (T2DM), cardiovascular disease, non-alcoholic steatohepatitis and various cancers. Furthermore, obesity is associated with a chronic state of low-grade inflammation in adipose tissue (AT), and it is thought that insulin resistance (IR) and T2DM is associated with the inflammatory state of AT. Endothelial cells (ECs) mediate the migration of immune cells into underlying tissues during times of inflammation, including obesity- and cardiovascular disease-associated inflammation. Cytokines and chemoattractants released from inflamed tissues promote EC activation. Upon activation, ECs increase the expression of leukocyte adhesion molecules (LCAMs) including intercellular adhesion molecule 1 (ICAM-1), vascular adhesion molecule 1 (VCAM-1), E-selectin (E-sel) and P-selectin (P-sel). Increased expression of these LCAMs and increased infiltration of inflammatory cells such as macrophages, have been linked to IR, diabetes and atherosclerosis in obese individuals. Preliminary data from our lab suggests that lipolysis induced by the β-adrenergic receptor agonist CL 316,243 causes an increase in endothelial LCAM gene expression. In addition, histological analyses show increased content of immune cells within AT after the ECs become activated. Here, we demonstrate that CL 316,243-induced lipolysis causes infiltration of neutrophils in wild type (WT) but not E-sel knockout (KO) mice. Following EC activation, there was also a marked increase in cytokine gene expression including IL-1β, MCP-1, and TNF-α in an E-sel-dependent manner. In contrast, fasting-induced lipolysis was associated with increased macrophage infiltration into AT in the absence of EC activation in an E-sel-independent manner. We also examined the role of mitogen activated protein kinase kinase kinase kinase 4 (MAP4K4) as a potential contributor to endothelial activation and atherosclerosis. Here we demonstrate that deletion of MAP4K4 in ECs in vitro diminishes TNF-α-induced EC activation. Additionally, MAP4K4 depletion in primary ECs derived from lungs of mice expressing MAP4K4 shRNA decreases EC activation. Finally, endothelial specific depletion or loss of MAP4K4 reduced atherosclerotic plaque formation in vivo. Taken together, these results highlight the importance of the endothelium in modulating obesity-associated comorbidities. Furthermore, these data implicate endothelial MAP4K4 as a novel regulator of EC activation and consequently AT inflammation and atherosclerosis

Isolation of neuronal chromatin from brain tissue

<p>Abstract</p> <p>Background</p> <p>DNA-protein interactions in mature brain are increasingly recognized as key regulators for behavioral plasticity and neuronal dysfunction in chronic neuropsychiatric disease. However, chromatin assays typically lack single cell resolution, and therefore little is known about chromatin regulation of differentiated neuronal nuclei that reside in brain parenchyma intermingled with various types of non-neuronal cells.</p> <p>Results</p> <p>Here, we describe a protocol to selectively tag neuronal nuclei from adult brain – either by (anti-NeuN) immunolabeling or transgene-derived histone H2B-GFP fusion protein – for subsequent fluorescence-activated sorting and chromatin immunoprecipitation (ChIP). To illustrate an example, we compared histone H3 lysine 4 and 9 methylation marks at select gene promoters in neuronal, non-neuronal and unsorted chromatin from mouse forebrain and human cerebral cortex, and provide evidence for neuron-specific histone methylation signatures.</p> <p>Conclusion</p> <p>With the modifications detailed in this protocol, the method can be used to collect nuclei from specific subtypes of neurons from any brain region for subsequent ChIP with native/un-fixed or crosslinked chromatin preparations. Starting with the harvest of brain tissue, ChIP-ready neuronal nuclei can be obtained within one day.</p

Prefrontal dysfunction in schizophrenia involves mixed-lineage leukemia 1-regulated histone methylation at GABAergic gene promoters

Alterations in GABAergic mRNA expression play a key role for prefrontal dysfunction in schizophrenia and other neurodevelopmental disease. Here, we show that histone H3-lysine 4 methylation, a chromatin mark associated with the transcriptional process, progressively increased at GAD1 and other GABAergic gene promoters (GAD2, NPY, SST) in human prefrontal cortex (PFC) from prenatal to peripubertal ages and throughout adulthood. Alterations in schizophrenia included decreased GAD1 expression and H3K4-trimethylation, predominantly in females and in conjunction with a risk haplotype at the 5\u27 end of GAD1. Heterozygosity for a truncated, lacZ knock-in allele of mixed-lineage leukemia 1 (Mll1), a histone methyltransferase expressed in GABAergic and other cortical neurons, resulted in decreased H3K4 methylation at GABAergic gene promoters. In contrast, Gad1 H3K4 (tri)methylation and Mll1 occupancy was increased in cerebral cortex of mice after treatment with the atypical antipsychotic, clozapine. These effects were not mimicked by haloperidol or genetic ablation of dopamine D2 and D3 receptors, suggesting that blockade of D2-like signaling is not sufficient for clozapine-induced histone methylation. Therefore, chromatin remodeling mechanisms at GABAergic gene promoters, including MLL1-mediated histone methylation, operate throughout an extended period of normal human PFC development and play a role in the neurobiology of schizophrenia

Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis

Signalling pathways that control endothelial cell (EC) permeability, leukocyte adhesion and inflammation are pivotal for atherosclerosis initiation and progression. Here we demonstrate that the Sterile-20-like mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), which has been implicated in inflammation, is abundantly expressed in ECs and in atherosclerotic plaques from mice and humans. On the basis of endothelial-specific MAP4K4 gene silencing and gene ablation experiments in Apoe(-/-) mice, we show that MAP4K4 in ECs markedly promotes Western diet-induced aortic macrophage accumulation and atherosclerotic plaque development. Treatment of Apoe(-/-) and Ldlr(-/-) mice with a selective small-molecule MAP4K4 inhibitor also markedly reduces atherosclerotic lesion area. MAP4K4 silencing in cultured ECs attenuates cell surface adhesion molecule expression while reducing nuclear localization and activity of NFkappaB, which is critical for promoting EC activation and atherosclerosis. Taken together, these results reveal that MAP4K4 is a key signalling node that promotes immune cell recruitment in atherosclerosis

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research such as variabilities in autolysis time and tissue integrities are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan. Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research such as variabilities in autolysis time and tissue integrities are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain

Setdb1-mediated histone H3K9 hypermethylation in neurons worsens the neurological phenotype of Mecp2-deficient mice

Rett syndrome (RTT, OMIM # 312750), a neurodevelopmental disorder of early childhood, is primarily caused by mutations in the gene encoding methyl-CpG-binding protein 2 (MECP2). Various molecular functions have been ascribed to MECP2, including the regulation of histone modifications associated with repressive chromatin remodeling, but the role of these mechanisms for the pathophysiology of RTT remains unclear. Here, we explore whether or not neuronal expression of the histone H3-lysine 9 specific methyl-transferase, Setdb1 (Set domain, bifurcated 1)/Eset/Kmt1e, which is normally present only at low levels in differentiated neurons, rescues the RTT-like phenotype of Mecp2-deficient mice. A myc-tagged Setdb1 cDNA was expressed through the tau locus for ubiquitous expression in CNS neurons, or under control of the calcium/calmodulin-dependent protein kinase II (CK) promoter to selectively target postmitotic neurons in forebrain. However, the CK-Setdb1 transgene lead to an enhanced neurological deficit, and the tauSetdb1 allele further shortened life span of mice with a brain-wide deletion of Mecp2 during prenatal development. In contrast, no neurological deficits or premature death was observed in CK-Setdb1 and tauSetdb1 mice expressing wildtype Mecp2. However, levels of trimethylated H3K9 at pericentromeric repeats were fully maintained in differentiated neurons from symptomatic Mecp2 null mutant mice. Based on these results, we draw two conclusions: First, neuronal chromatin in RTT brain is not affected by a generalized deficit in H3K9 trimethylation. Second, artificial up-regulation of this repressive chromatin mark via Setdb1 gene delivery specifically to neurons is harmful for the Mecp2-deficient brain. This article is part of a Special Issue entitled \u27Trends in Neuropharmacology: In Memory of Erminio Costa\u27

Chromatin immunoprecipitation in postmortem brain

Methylation and other covalent modifications of nucleosome core histones are key regulators of chromatin structure and function, including epigenetic control of gene expression. For the human brain, however, very little is known about the regulation of histone modifications at specific genomic loci. Furthermore, chromatin immunoprecipitation protocols applicable to postmortem tissue are lacking, and the impact of potential confounds such as autolysis time or tissue pH is unknown. We treated cerebral cortex from human postmortem brain and mice by micrococcal nuclease digestion or, alternatively, by formaldehyde-crosslinking and sonication. We show that the bulk of nucleosomal DNA remains attached to histones during the first 30 h after death. Immunoprecipitation with antibodies against methylated histones was at least 10-fold more effective in unfixed, micrococcal nuclease-digested samples, in comparison to extracts prepared by fixation and sonication. Histone methylation differences across various genomic sites were maintained within a wide range of autolysis times and tissue pH. Therefore, immunoprecipitation of micrococcal nuclease-digested tissue extracts is a feasible approach to profile histone methylation at defined genomic loci in postmortem brain