Gene Expression and Functional Annotation of the Human Ciliary Body Epithelia

Purpose: The ciliary body (CB) of the human eye consists of the non-pigmented (NPE) and pigmented (PE) neuro-epithelia. We investigated the gene expression of NPE and PE, to shed light on the molecular mechanisms underlying the most important functions of the CB. We also developed molecular signatures for the NPE and PE and studied possible new clues for glaucoma. Methods: We isolated NPE and PE cells from seven healthy human donor eyes using laser dissection microscopy. Next, we performed RNA isolation, amplification, labeling and hybridization against 44×k Agilent microarrays. For microarray conformations, we used a literature study, RT-PCRs, and immunohistochemical stainings. We analyzed the gene expression data with R and with the knowledge database Ingenuity. Results: The gene expression profiles and functional annotations of the NPE and PE were highly similar. We found that the most important functionalities of the NPE and PE were related to developmental processes, neural nature of the tissue, endocrine and metabolic signaling, and immunological functions. In total 1576 genes differed statistically significantly between NPE and PE. From these genes, at least 3 were cell-specific for the NPE and 143 for the PE. Finally, we observed high expression in the (N)PE of 35 genes previously implicated in molecular mechanisms related to glaucoma. Conclusion: Our gene expression analysis suggested that the NPE and PE of the CB were quite similar. Nonetheless, cell-type specific differences were found. The molecular machineries of the human NPE and PE are involved in a range of neuro-endocrinological, developmental and immunological functions, and perhaps glaucoma

A New Strategy to Identify and Annotate Human RPE-Specific Gene Expression

Background: To identify and functionally annotate cell type-specific gene expression in the human retinal pigment epithelium (RPE), a key tissue involved in age-related macular degeneration and retinitis pigmentosa. Methodology: RPE, photoreceptor and choroidal cells were isolated from selected freshly frozen healthy human donor eyes using laser microdissection. RNA isolation, amplification and hybridization to 44 k microarrays was carried out according to Agilent specifications. Bioinformatics was carried out using Rosetta Resolver, David and Ingenuity software. Principal Findings: Our previous 22 k analysis of the RPE transcriptome showed that the RPE has high levels of protein synthesis, strong energy demands, is exposed to high levels of oxidative stress and a variable degree of inflammation. We currently use a complementary new strategy aimed at the identification and functional annotation of RPE-specific expressed transcripts. This strategy takes advantage of the multilayered cellular structure of the retina and overcomes a number of limitations of previous studies. In triplicate, we compared the transcriptomes of RPE, photoreceptor and choroidal cells and we deduced RPE specific expression. We identified at least 114 entries with RPE-specific gene expression. Thirty-nine of these 114 genes also show high expression in the RPE, comparison with the literature showed that 85% of these 39 were previously identified to be expressed in the RPE. In the group of 114 RPE specific genes there was an overrepresentation of genes involved in (membrane) transport, vision and ophthalmic disease. More fundamentally, we found RPE-specific involvement in the RAR-activation, retinol metabolism and GABA receptor signaling pathways. Conclusions: In this study we provide a further specification and understanding of the RPE transcriptome by identifying and analyzing genes that are specifically expressed in the RPE

Concerted changes in transcripts in the prefrontal cortex precede neuropathology in Alzheimer's disease

Using the Braak staging for neurofibrillary changes as an objective indicator of the progression of Alzheimer's disease, we have performed a systematic search for global gene expression changes in the prefrontal cortex during the course of Alzheimer's disease. In the prefrontal cortex, senile plaques and neurofibrillary changes start to appear around Braak stage III, allowing for the detection of changes in gene expression before, during and after the onset of Alzheimer's disease neuropathology. Two distinct patterns of tightly co-regulated groups of genes were observed: (i) an increase in expression in early Braak stages, followed by a decline in expression in later stages (the UPDOWN clusters; containing 865 genes) and (ii) a decrease in expression in early Braak stages, followed by an increase in expression in later stages (the DOWNUP clusters; containing 983 genes). The most profound changes in gene expression were detected between Braak stages II and III, just before or at the onset of plaque pathology and neurofibrillary changes in the prefrontal cortex. We also observed an increase in intracellular beta amyloid staining from Braak stages I to III and a clear decrease in Braak stages IV to VI. These data suggest a link between specific gene expression clusters and Alzheimer's disease-associated neuropathology in the prefrontal cortex. Gene ontology over-representation and functional gene network analyses indicate an increase in synaptic activity and changes in plasticity during the very early pre-symptomatic stage of the disease. In later Braak stages, the decreased expression of these genes suggests a reduction in synaptic activity that coincides with the appearance of plaque pathology and neurofibrillary changes and the clinical diagnosis of mild cognitive impairment. The interaction of the ApoE genotype with the expression levels of the genes in the UPDOWN and DOWNUP clusters demonstrates that the accelerating role of ApoE-ε4 in the progression of Alzheimer's disease is reflected in the temporal changes in gene expression presented here. Since the UPDOWN cluster contains several genes involved in amyloid precursor protein processing and beta amyloid clearance that increase in expression in parallel with increased intracellular beta amyloid load, just before the onset of plaque pathology in the prefrontal cortex, we hypothesize that the temporally orchestrated increase in genes involved in synaptic activity represents a coping mechanism against increased soluble beta amyloid levels. As these gene expression changes occur before the appearance of Alzheimer's disease-associated neuropathology, they provide an excellent starting point for the identification of new targets for the development of therapeutic strategies aimed at the prevention of Alzheimer's diseas

Comparison of human retinal pigment epithelium gene expression in macula and periphery highlights potential topographic differences in Bruch's membrane

Purpose: To describe gene expression differences between healthy, young human retinal pigment epithelium (RPE) cells from the macular area and RPE cells from two locations in the retinal periphery. Methods: RPE cells from six human donor eyes, ages 17-36, without histopathological abnormalities, were dissected by laser and isolated from cryosections. Total RNA was isolated, amplified, and hybridized to a custom made oligonucleotide array containing 22,000 genes. Bioinformatic analysis was carried out using the computer programs Rosetta Resolver and the webtools EASE/David and GoStat. Confirmatory real time PCR (RT-PCR) and immunohistochemistry were performed according to standard protocols. Results: Microarray and statistical analysis yielded 438 genes that were differentially expressed between macular RPE, and at least one out of two peripheral RPE locations. Out of these genes, 33 that showed fold changes of four, or higher, were selected for RT-PCR confirmation. For 17 genes (51%), a significant differential expression was found, while 11 additional genes (33%) showed a similar trend. Immuno-staining of one target (WFDC1) confirmed its differential expression on the protein level. Functional annotation and overrepresentation analysis independently defined extracellular matrix (ECM) genes as a statistically overrepresented class of genes in our RPE dataset. In total, 33 ECM genes were differentially expressed between macular and peripheral RPE regions. A subset of proteins corresponding to these genes is known to be present in Bruch's membrane. Conclusions: Our data showed that consistent topographical gene expression differences in the human RPE constitute around 1-5% of the RPE transcriptome. These changes may underlie topographical differences in RPE physiology, and pathology and may reflect local differences in the molecular composition and turnover of Bruch's membran

Decitabine mildly attenuates MLL‐rearranged acute lymphoblastic leukemia in vivo, and represents a poor chemo‐sensitizer

Abstract MLL‐rearranged acute lymphoblastic leukemia (ALL) represents a highly aggressive ALL subtype, characterized by aberrant DNA methylation patterns. DNA methyltransferase inhibitors, such as decitabine have previously been demonstrated to be effective in eradicating MLL‐rearranged ALL cells in vitro. Here, we assessed the in vivo anti‐leukemic potential of low‐dose DNA methyltransferase inhibitor decitabine using a xenograft mouse model of human MLL‐rearranged ALL. Furthermore, we explored whether prolonged exposure to low‐dose decitabine could chemo‐sensitize MLL‐rearranged ALL cells toward conventional chemotherapy as well as other known epigenetic‐based and anti‐neoplastic compounds. Our data reveal that decitabine prolonged survival in xenograft mice of MLL‐rearranged ALL by 8.5 days (P = .0181), but eventually was insufficient to prevent leukemia out‐growth, based on the examination of the MLLAF4 cell line SEM. Furthermore, we observe that prolonged pretreatment of low‐dose decitabine mildly sensitized toward the conventional drugs prednisolone, vincristine, daunorubicin, asparaginase, and cytarabine in a panel of MLL‐rearranged cell lines. Additionally, we assessed synergistic effects of decitabine with other epigenetic‐based or anticancer drugs using high‐throughput drug library screens. Validation of the top hits, including histone deacetylase inhibitor panobinostat, BCL2 inhibitor Venetoclax, MEK inhibitor pimasertib, and receptor tyrosine kinase foretinib, revealed additive and moderate synergistic effects for the combination of each drug together with decitabine in a cell line‐dependent manner

Loss of CRB2 results in gliosis and microglia activation.

<p>Immunohistochemistry pictures from P10 mouse retinae. Sections were stained with antibodies against: SOX9 and Glutamine synthetase (GS) (<b>A</b>, <b>B</b>), GFAP (<b>C</b>, <b>D</b>), CD45 (<b>E</b>, <b>F</b>), CD11b (<b>G</b>, <b>H</b>). The location of nuclei of Müller glia cells, stained with SOX9 was not altered in the mutant retinas (<b>B</b>), however disruption at the apical end feet of the Müller glia cells were observed at sites with photoreceptor protrusions (<b>B</b>). The mutant retinas showed activated Müller glia cells, detected by a moderate increase in the GFAP staining in the outer nuclear layer (arrowhead) (<b>D</b>). An increase in activated microglia cells in the outer nuclear layer, stained with anti-CD45 and anti-CD11b, was detected (<b>F</b> and <b>H</b>). No morphological changes were observed in the control retinae. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; OLM, outer limiting membrane; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bars: 20 µm.</p

Loss of CRB2 results in retinal disorganization in mice on C57BL/6J genetic background.

<p>Toluidine stained light microscopic pictures, of retina sections, from the control (<b>A</b>, <b>C</b> and <b>E</b>) and from the <i>Crb2</i>Chx10 cKO on C57BL/6J genetic background (<b>C</b>, <b>D</b> and <b>F</b>), at different ages, P10 - (<b>A</b>, <b>B</b>), 1M - (C, D), 3M - (<b>E</b>, <b>F</b>). At P10 (<b>B</b>), several photoreceptor nuclei were localized ectopically in the subretinal space. At 1M (<b>D</b>) protrusion of photoreceptor cell nuclei in the subretinal space and gaps in the outer limiting membrane were observed. At 3M (<b>F</b>) we observed thinner outer nuclear layers, with rows of photoreceptors cells protruding into the subretinal space through the outer limiting membrane and protrusions of inner nuclear layer cells into the outer nuclear layer. No abnormalities were observed in the control. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar: 50 µm.</p

Localization of the <i>loxP</i> sites and Crb2 probe in the Crb2 targeting construct.

<p>In the Crb2 targeting construct the <i>loxP</i> recombination sites are located in intron 9 and within exon 13 in the 3′ non-coding region. Upon Cre-mediated recombination the exons 10, 11, 12 and part of exon 13 are removed. The 60 bp probe for the Crb2 gene is located downstream of the 3’ <i>loxP</i>, before the polyadenylation signal. Two sets of primers were used to detect changes in Crb2 expression, they were located in exon 7 (primer 534/535) and in exon 11/12 (primer 536/537). In blue are represented the coding exons of the gene, in black the 3’ untranslated region. </p

Loss of CRB2 affects lamination of photoreceptor cells.

<p>Immunohistochemistry pictures from 10 days old mouse retinas. Sections were stained with antibodies against: recoverin (<b>A</b>, <b>B</b>), rhodopsin and cone arrestin (<b>C</b>, <b>D</b>), M-opsin (<b>E</b>, <b>F</b>), peanut agglutinin (PNA) (<b>G</b>, <b>H</b>). In the mutant retinas several photoreceptor nuclei localized in the subretinal space were positive for recoverin (<b>B</b>) and rhodopsin (<b>D</b>). Some cone arrestin (<b>D</b>), M-opsin (<b>F</b>) positive nuclei were also misplaced in the subretinal place, showing that also cone photoreceptors lamination was affected. Outer segment from the cone photoreceptor cells, stained with PNA, were present in both retinas (<b>G</b>, <b>H</b>). However, in the mutant retinas these segments were located between photoreceptor nuclei and not in contact with the retinal pigmented epithelium (<b>H</b>). GCL, ganglion cell layer; INL, inner nuclear layer; OLM, outer limiting membrane; ONL, outer nuclear layer. Scale bars: 25 µm.</p