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Strong evidence suggests that dysregulated lipid metabolism involving dysfunction of the retinal pigmented epithelium (RPE) underlies the pathogenesis of age-related macular degeneration (AMD), the leading cause of irreversible blindness in the elderly. A hallmark of AMD is the overproduction of lipid- and protein-rich extracellular deposits that accumulate in the extracellular matrix (Bruch's membrane (BrM)) adjacent to the RPE. We analyzed apolipoprotein A-1 (ApoA-1)-containing lipoproteins isolated from BrM of elderly human donor eyes and found a unique proteome, distinct from high-density lipoprotein (HDL) isolated from donor plasma of the same individuals. The most striking difference is higher concentrations of ApoB and ApoE, which bind to glycosaminoglycans. We hypothesize that this interaction promotes lipoprotein deposition onto BrM glycosaminoglycans, initiating downstream effects that contribute to RPE dysfunction/death. We tested this hypothesis using two potential therapeutic strategies to alter the lipoprotein/protein profile of these extracellular deposits. First, we used short heparan sulfate oligosaccharides to remove lipoproteins already deposited in both the extracellular matrix of RPE cells and aged donor BrM tissue. Second, an ApoA-1 mimetic, 5A peptide, was demonstrated to modulate the composition and concentration of apolipoproteins secreted from primary porcine RPE cells. Significantly, in a mouse model of AMD, this 5A peptide altered the proteomic profile of circulating HDL and ameliorated some of the potentially harmful changes to the protein composition resulting from the high-fat, high-cholesterol diet in this model. Together, these results suggest that targeting HDL interactions with BrM represents a new strategy to slow AMD progression in humans.

The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in the eye and its polarity is responsible for directional secretion and uptake of proteins, lipoprotein particles and extracellular vesicles (EVs). Such a secretional division dictates directed interactions between the systemic circulation (basolateral) and the retina (apical). Our goal is to define the polarized proteomes and physical characteristics of EVs released from the RPE. Primary cultures of porcine RPE cells were differentiated into polarized RPE monolayers on permeable supports. EVs were isolated from media bathing either apical or basolateral RPE surfaces, and two subpopulations of small EVs including exosomes, and dense EVs, were purified and processed for proteomic profiling. In parallel, EV size distribution and concentration were determined. Using protein correlation profiling mass spectrometry, a total of 631 proteins were identified in exosome preparations, 299 of which were uniquely released apically, and 94 uniquely released basolaterally. Selected proteins were validated by Western blot. The proteomes of these exosome and dense EVs preparations suggest that epithelial polarity impacts directional release. These data serve as a foundation for comparative studies aimed at elucidating the role of exosomes in the molecular pathophysiology of retinal diseases and help identify potential therapeutic targets and biomarkers.

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An imbalance between activation and inhibition of the complement system has been implicated in the etiologies of numerous common diseases. Allotypic variants of a key complement fluid-phase regulatory protein, complement factor H (CFH), are strongly associated with age-related macular degeneration (AMD), a leading cause of worldwide visual dysfunction, although its specific role in AMD pathogenesis is still not clear. CFH was isolated from individuals carrying combinations of two of the nonsynonymous coding variants most strongly associated with AMD risk, V62/H402 (risk haplotype variants), I62/Y402 (nonrisk haplotype variants), and V62/Y402. These proteins were used in two functional assays (cell surface- and fluid-phase–based) measuring cofactor activity of CFH in the factor I-mediated cleavage of C3b. Although no variant-specific differences in the cofactor activity were detected, when heparan sulfate (HS) was added to these assays, it accelerated the rate of C3b cleavage, and this effect could be modulated by degree of HS sulfation. Bruch’s membrane/choroid, a site of tissue damage in AMD, contains high concentrations of glycosaminoglycans, including HS. Addition of human Bruch’s membrane/choroid to the fluid-phase assay accelerated the C3b cleavage, and this effect was lost posttreatment of the tissue with heparinase III. Binding of CFH variants to Bruch’s membrane/choroid isolated from elderly, non-AMD donor eyes, was similar, as was the functional activity of bound CFH. These findings refine our understanding of interactions of HS and complement and support the hypothesis that these interactions play a role in the transition between normal aging and AMD in Bruch’s membrane/choroid.

Variations in the complement factor H (CFH) gene are tightly associated with age-related macular degeneration (AMD) across diverse populations. Of the many nonsynonymous coding variants in CFH, two are most strongly associated with increased risk of AMD: isoleucine 62 to valine (I62V) and tyrosine 402 to histidine (Y402H). Detection of these variations in a patient's blood is important for a risk assessment of AMD and disease prognosis. However, traditional methods of genetic analysis cannot be used for measuring CFH allotypes in some sources of human plasma and other biological fluids not containing DNA. The purpose was to develop a protein-based method of detecting CFH allotypes.A combination of a single-step affinity enrichment of CFH, gel separation, and mass spectrometry identification of the CFH peptides spanning amino acids at positions 62 and 402 was used to identify individual CFH allotypes.The CFH isoforms V62, I62, H402, and Y402 were reliably detected based on identification of tryptic peptides with masses of 1148.59 Da, 1162.60 Da, 2031.88 Da, and 2057.88 Da, respectively, using MALDI-TOF-TOF. The presence or absence pattern of these peptides in mass spectra of different CFH samples robustly correlated with all nine genotypes of CFH, as a result of variations at positions 62 and 402.A rapid and sensitive method has been developed for detection of V62, I62, H402, and Y402 variants of CFH in human plasma samples using mass spectrometry. This method can be used in clinical laboratories equipped with a basic inexpensive mass spectrometer capable of performing peptide fingerprinting.

Age-related macular degeneration (AMD) is a leading cause of visual dysfunction worldwide. Amyloid β (Aβ) peptides, Aβ1–40 (Aβ40) and Aβ1–42 (Aβ42), have been implicated previously in the AMD disease process. Consistent with a pathogenic role for Aβ, we show here that a mouse model of AMD that invokes multiple factors that are known to modify AMD risk (aged human apolipoprotein E 4 targeted replacement mice on a high-fat, cholesterol-enriched diet) presents with Aβ-containing deposits basal to the retinal pigmented epithelium (RPE), histopathologic changes in the RPE, and a deficit in scotopic electroretinographic response, which is reflective of impaired visual function. Strikingly, these electroretinographic deficits are abrogated in a dose-dependent manner by systemic administration of an antibody targeting the C termini of Aβ40 and Aβ42. Concomitant reduction in the levels of Aβ and activated complement components in sub-RPE deposits and structural preservation of the RPE are associated with anti-Aβ40/42 antibody immunotherapy and visual protection. These observations are consistent with the reduction in amyloid plaques and improvement of cognitive function in mouse models of Alzheimer's disease treated with anti-Aβ antibodies. They also implicate Aβ in the pathogenesis of AMD and identify Aβ as a viable therapeutic target for its treatment.

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.

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.