The pivotal role of the complement system in aging and age-related macular degeneration: hypothesis re-visited.

During the past ten years, dramatic advances have been made in unraveling the biological bases of age-related macular degeneration (AMD), the most common cause of irreversible blindness in western populations. In that timeframe, two distinct lines of evidence emerged which implicated chronic local inflammation and activation of the complement cascade in AMD pathogenesis. First, a number of complement system proteins, complement activators, and complement regulatory proteins were identified as molecular constituents of drusen, the hallmark extracellular deposits associated with early AMD. Subsequently, genetic studies revealed highly significant statistical associations between AMD and variants of several complement pathway-associated genes including: Complement factor H (CFH), complement factor H-related 1 and 3 (CFHR1 and CFHR3), complement factor B (CFB), complement component 2 (C2), and complement component 3 (C3). In this article, we revisit our original hypothesis that chronic local inflammatory and immune-mediated events at the level of Bruch's membrane play critical roles in drusen biogenesis and, by extension, in the pathobiology of AMD. Secondly, we report the results of a new screening for additional AMD-associated polymorphisms in a battery of 63 complement-related genes. Third, we identify and characterize the local complement system in the RPE-choroid complex - thus adding a new dimension of biological complexity to the role of the complement system in ocular aging and AMD. Finally, we evaluate the most salient, recent evidence that bears directly on the role of complement in AMD pathogenesis and progression. Collectively, these recent findings strongly re-affirm the importance of the complement system in AMD. They lay the groundwork for further studies that may lead to the identification of a transcriptional disease signature of AMD, and hasten the development of new therapeutic approaches that will restore the complement-modulating activity that appears to be compromised in genetically susceptible individuals.

Technical and biological issues relevant to cell typing with aptamers.

A number of aptamers have been selected against cell surface biomarkers or against eukaryotic tissue culture cells themselves. To determine the general utility of aptamers for assessing the cell surface proteome, we developed a standardized flow cytometry assay and carried out a comprehensive study with 7 different aptamers and 14 different cell lines. By examining how aptamers performed with a variety of cell lines, we identified difficulties in using aptamers for cell typing. While there are some aptamers that show excellent correlation between cell surface binding and the expression of a biomarker on the cell surface, other aptamers showed nonspecific binding by flow cytometry. For example, it has recently been claimed that an anti-PTK7 (protein tyrosine kinase 7) aptamer identified a new biomarker for leukemia cells, but data with the additional cell lines shows that it is possible that the aptamer instead identifies a propensity for adherence. Better understanding and controlling for the role of background and nonspecific binding to cells should open the way to using arrays of aptamers for describing and quantifying the cell surface proteome.

NEIBank: genomics and bioinformatics resources for vision research.

NEIBank is an integrated resource for genomics and bioinformatics in vision research. It includes expressed sequence tag (EST) data and sequence-verified cDNA clones for multiple eye tissues of several species, web-based access to human eye-specific SAGE data through EyeSAGE, and comprehensive, annotated databases of known human eye disease genes and candidate disease gene loci. All expression- and disease-related data are integrated in EyeBrowse, an eye-centric genome browser. NEIBank provides a comprehensive overview of current knowledge of the transcriptional repertoires of eye tissues and their relation to pathology.

Oxidative stress-induced expression and modulation of Phosphatase of Regenerating Liver-1 (PRL-1) in mammalian retina.

The phosphatase of regenerating liver-1, PRL-1, gene was detected in a screen for foveal cone photoreceptor-associated genes. It encodes a small protein tyrosine phosphatase that was previously immunolocalized to the photoreceptors in primate retina. Here we report that in cones and cone-derived cultured cells both PRL-1 activity and PRL-1 gene expression are modulated under oxidative stress. Oxidation reversibly inhibited the phosphatase activity of PRL-1 due to the formation of an intramolecular disulfide bridge between Cys104 within the active site and another conserved Cys, Cys49. This modulation was observed in vitro, in cell culture and in isolated retinas exposed to hydrogen peroxide. The same treatment caused a rapid increase in PRL-1 expression levels in cultured cells which could be blocked by the protein translation inhibitor, cycloheximide. Increased PRL-1 expression was also observed in living rats subjected to constant light exposure inducing photooxidative stress. We further demonstrated that both oxidation and overexpression of PRL-1 upon oxidative stress are greatly enhanced by inhibition of the glutathione system responsible for cellular redox regulation. These findings suggest that PRL-1 is a molecular component of the photoreceptor's response to oxidative stress acting upstream of the glutathione system.

Lymphoid reservoirs of antigen-specific memory T helper cells.

How vaccines control the development of antigen-specific effector and memory T helper cells is central to protective immunity but remains poorly understood. Here we found that protein vaccination selected high-affinity, CXCR5+ICOS(hi) follicular B-helper T cells (T(FH) cells) that developed in draining lymphoid tissue to regulate B cell responses. In the memory phase, reservoirs of antigen-specific CXCR5+ICOS(lo) T(FH) cells persisted with less effector activity but accelerated antigen-recall ability. This new compartment of memory T(FH) cells was retained in draining lymphoid sites with antigen-specific memory B cells, persistent complexes of peptide and major histocompatibility complex class II and continued expression of CD69. Thus, protein vaccination promotes B cell immunity by selecting high-affinity effector T(FH) cells and creating lymphoid reservoirs of antigen-specific memory T(FH) cells in vivo.

Defining the human macula transcriptome and candidate retinal disease genes using EyeSAGE.

PURPOSE: To develop large-scale, high-throughput annotation of the human macula transcriptome and to identify and prioritize candidate genes for inherited retinal dystrophies, based on ocular-expression profiles using serial analysis of gene expression (SAGE). METHODS: Two human retina and two retinal pigment epithelium (RPE)/choroid SAGE libraries made from matched macula or midperipheral retina and adjacent RPE/choroid of morphologically normal 28- to 66-year-old donors and a human central retina longSAGE library made from 41- to 66-year-old donors were generated. Their transcription profiles were entered into a relational database, EyeSAGE, including microarray expression profiles of retina and publicly available normal human tissue SAGE libraries. EyeSAGE was used to identify retina- and RPE-specific and -associated genes, and candidate genes for retina and RPE disease loci. Differential and/or cell-type specific expression was validated by quantitative and single-cell RT-PCR. RESULTS: Cone photoreceptor-associated gene expression was elevated in the macula transcription profiles. Analysis of the longSAGE retina tags enhanced tag-to-gene mapping and revealed alternatively spliced genes. Analysis of candidate gene expression tables for the identified Bardet-Biedl syndrome disease gene (BBS5) in the BBS5 disease region table yielded BBS5 as the top candidate. Compelling candidates for inherited retina diseases were identified. CONCLUSIONS: The EyeSAGE database, combining three different gene-profiling platforms including the authors' multidonor-derived retina/RPE SAGE libraries and existing single-donor retina/RPE libraries, is a powerful resource for definition of the retina and RPE transcriptomes. It can be used to identify retina-specific genes, including alternatively spliced transcripts and to prioritize candidate genes within mapped retinal disease regions.

Human RPE expression of cell survival factors.

PURPOSE: To determine basal and tumor necrosis factor (TNF)-alpha-regulated expression of retinal pigment epithelial (RPE) cell survival factors and whether regulation is dependent on nuclear transcription factor (NF)-kappaB. METHODS: Cultured human RPE cells were infected with adenovirus encoding either mutant inhibitory (I)-kappaB or beta-galactosidase and treated with TNF-alpha for various times. Freshly prepared RPE/choroid and RPE samples were isolated from human donor eyes. Real-time reverse transcription-polymerase chain reaction, Western blot, and immunocytochemistry were used to determine survival factor gene expression, cellular protein levels, and localization, respectively. RESULTS: Multiple survival factor genes, including cellular inhibitor of apoptosis protein (c-IAP1), c-IAP2, TNF receptor-associated factor-1 (TRAF-1), TRAF-2, B-cell leukemia/lymphoma-2 (Bcl-2), Bcl-x, A1, and cellular Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme-like inhibitory protein (c-FLIP), were expressed in basal conditions in both cultured RPE cells and RPE cells in situ, whereas survivin was expressed only by cultured cells. TNF-alpha upregulated expression of TRAF-1, TRAF-2, c-IAP1, c-IAP2, c-FLIP, and A1. TRAF-1, c-FLIP, and to a lesser extent c-IAP2 protein levels were increased by TNF-alpha in a time-dependent manner, whereas c-IAP1, survivin, Bcl-x(L), and TRAF-2 protein levels were not influenced by TNF-alpha treatment at any time point tested. In contrast, Bcl-2 and A1 proteins were not detected under basal conditions or after TNF-alpha treatment. Overexpression of mutant IkappaB blocked TNF-alpha-induced TRAF-1, TRAF-2, c-IAP1, c-IAP2, c-FLIP, and A1 gene expression and downregulated TRAF-1 protein levels. TRAF-1 and Bcl-x(L) proteins were localized diffusely in RPE cytoplasm. CONCLUSIONS: Multiple RPE cell survival factors are expressed by human RPE cells. TNF-alpha regulates expression of some of these factors in an NF-kappaB-dependent manner, whereas others are not influenced by NF-kappaB. RPE cell survival factors may protect RPE cells from apoptosis normally and in diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR).

Expression of the blue-light receptor cryptochrome in the human retina.

PURPOSE: To analyze the patterns of expression of the cryptochromes, CRY1 and CRY2, in the human retina and to correlate expression of these putative blue-light receptors with nonvisual photoreceptor localization. METHODS: CRY1 and CRY2 mRNA expression was analyzed in 4-mm diameter punches of macula and midperipheral human retina by quantitative RT-PCR. CRY2 protein expression was examined by immunohistochemistry in cross sections of human retina, and its subcellular localization was determined by immunoblot analysis of fractionated human retinal extracts. RESULTS: CRY2 mRNA was 11 times more abundant than CRY1 throughout adult human retina. CRY2 immunoreactivity was detected in most cells in the ganglion cell layer (GCL) and in a subset of cells in the inner nuclear layer (INL) in both the macula and periphery. Immunoperoxidase staining further revealed that CRY2 was localized throughout the cytoplasm of cells in the GCL as well as within nuclei. This intracellular localization of CRY2 was confirmed by immunoblot analysis of fractionated human retinal extracts. CONCLUSIONS: Photopigments governing circadian photoreception have been localized to the inner retina. The relative abundance of CRY2 transcripts, coupled with CRY2 localization to the inner retina, supports a photoreceptive role for CRY2 in human retina. Furthermore, the discovery that CRY2 is also localized within the cytoplasm of some cells in the GCL, suggests it may perform a function separate from its known nuclear role in the transcriptional feedback loop underlying the molecular circadian clock.