Spectral analysis by XANES reveals that GPNMB influences the chemical composition of intact melanosomes

GPNMB is a unique melanosomal protein. Unlike many melanosomal proteins, GPNMB has not been associated with any forms of albinism, and it is unclear whether GPNMB has any direct influence on melanosomes. Here, melanosomes from congenic strains of C57BL/6J mice mutant for Gpnmb are compared to strain-matched controls using standard transmission electron microscopy and synchrotron-based X-ray absorption near-edge structure analysis (XANES). Whereas electron microscopy did not detect any ultrastructural changes in melanosomes lacking functional GPNMB, XANES uncovered multiple spectral phenotypes. These results directly demonstrate that GPNMB influences the chemical composition of melanosomes and more broadly illustrate the potential for using genetic approaches in combination with nano-imaging technologies to study organelle biology.     

Intracellular sorting and targeting of melanosomal membrane proteins: identification of signals for sorting of the human brown locus protein, gp75

The structural and functional integrity of cytoplasmic organelles is maintained by intracellular mechanisms that sort and target newly synthesized proteins to their appropriate cellular locations. In melanocytic cells, melanin pigment is synthesized in specialized organelles, melanosomes. A family of melanocyte-specific proteins, known as tyrosinase-related proteins that regulate melanin pigment synthesis, is localized to the melanosomal membrane. The human brown locus protein, tyrosinase-related protein-1 or gp75, is the most abundant glycoprotein in melanocytic cells, and is a prototype for melanosomal membrane proteins. To investigate the signals that allow intracellular retention and sorting of glycoprotein (gp)75, we constructed protein chimeras containing the amino-terminal extracellular domain of the T lymphocyte surface protein CD8, and transmembrane and cytoplasmic domains of gp75. In fibroblast transfectants, chimeric CD8 molecules containing the 36-amino acid cytoplasmic domain of gp75 were retained in cytoplasmic organelles. Signals in the gp75 cytoplasmic tail alone, were sufficient for intracellular retention and targeting of the chimeric proteins to the endosomal/lysosomal compartment. Analysis of subcellular localization of carboxy-terminal deletion mutants of gp75 and the CD8/gp75 chimeras showed that deletion of up amino acids from the gp75 carboxyl terminus did not affect intracellular retention and sorting, whereas both gp75 and CD8/gp75 mutants lacking the carboxyl-terminal 27 amino acids were transported to the cell surface. This region contains the amino acid sequence, asn-gln-pro-leu-leu-thr, and this hexapeptide is conserved among other melanosomal proteins. Further evidence showed that this hexapeptide sequence is necessary for intracellular sorting of gp75 in melanocytic cells, and suggested that a signal for sorting melanosomal proteins along the endosomal/lysosomal pathway lies within this sequence. These data provide evidence for common signals for intracellular sorting of melanosomal and lysosomal proteins, and support the notion that lysosomes and melanosomes share a common endosomal pathway of biogenesis.     

Retinal pigment epithelial cell proliferation

The human retinal pigment epithelium forms early in development and subsequently remains dormant, undergoing minimal proliferation throughout normal life. Retinal pigment epithelium proliferation, however, can be activated in disease states or by removing retinal pigment epithelial cells into culture. We review the conditions that control retinal pigment epithelial proliferation in culture, in animal models and in human disease and interpret retinal pigment epithelium proliferation in context of the recently discovered retinal pigment epithelium stem cell that is responsible for most in vitro retinal pigment epithelial proliferation. Retinal pigment epithelial proliferation-mediated wound repair that occurs in selected macular diseases is contrasted with retinal pigment epithelial proliferation-mediated fibroblastic scar formation that underlies proliferative vitreoretinopathy. We discuss the role of retinal pigment epithelial proliferation in age-related macular degeneration which is reparative in some cases and destructive in others. Macular retinal pigment epithelium wound repair and regression of choroidal neovascularization are more pronounced in younger than older patients. We discuss the possibility that the limited retinal pigment epithelial proliferation and latent wound repair in older age-related macular degeneration patients can be stimulated to promote disease regression in age-related macular degeneration.     

Retinal dehydrogenase 12 (RDH12) mutations in leber congenital amaurosis

Leber congenital amaurosis (LCA), the most early-onset and severe form of all inherited retinal dystrophies, is responsible for congenital blindness. Ten LCA genes have been mapped, and seven of these have been identified. Because some of these genes are involved in the visual cycle, we regarded the retinal pigment epithelium and photoreceptor-specific retinal dehydrogenase (RDH) genes as candidate genes in LCA. Studying a series of 110 unrelated patients with LCA, we found mutations in the photoreceptor-specific RDH12 gene in a significant subset of patients (4.1%). Interestingly, all patients harboring RDH12 mutations had a severe yet progressive rod-cone dystrophy with severe macular atrophy but no or mild hyperopia.     

The dark side of lysosome-related organelles: specialization of the endocytic pathway for melanosome biogenesis

Melanosomes are lysosome-related organelles within which melanin pigments are synthesized and stored in melanocytes and retinal pigment epithelial cells. Early ultrastructural studies of pigment cells revealed that melanosomes consist of a complex series of organelles; more recently, these structures have been correlated with cargo constituents. By studying the fate of melanosomal and endosomal cargo in melanocytic cells, the effects of disease-related mutations on melanosomal morphology, and the genes affected by these mutations, we are beginning to gain novel insights into the biogenesis of these complex organelles and their relationship to the endocytic pathway. These insights demonstrate how specialized cells integrate unique and ubiquitous molecular mechanisms in subverting the endosomal system to generate cell-type specific structures and their associated functions. Further dissection of the melanosomal system will likely shed light not only on the biogenesis of lysosome-related organelles but also on general aspects of vesicular transport in the endosomal system.     

The differing embryonic origins of retinal and uveal (iris/ciliary body and choroid) melanosomes are mirrored by their phospholipid composition

The phospholipids present in uveal (iris/ciliary body and choroid) and retinal bovine ocular melanosomes were identified using mass spectrometry. Similar phospholipid content is found for the two types of uveal melanosome, with sphingomyelin being the major species. Significant differences are found between the uveal and retinal melanosome. Glycerophosphoethanolamine (GPEtn) is the major species in the retinal pigment epithelium (RPE); 93% of the GPEtn contain polyunsaturated fatty acids, notably docosahexanoic acid and arachidonic acid, in the sn-2 position. RPE melanosomes also contain detectable quantities of glycerophosphoserine and glycerophosphate; these species were not detected in the uveal samples. While the structural and functional roles of melanosomal lipids largely remain to be determined, these different lipid compositions reported herein offer new insights into the roles of melanosomes in the different ocular tissues.     

Biogenesis of melanosomes - the chessboard of pigmentation

Melanosomes are lysosome-related organelles in retinal pigment epithelial cells and epidermal melanocytes in which melanin pigments are synthesized and stored. Melanosomes are generated by multistep processes in which an immature unpigmented organelle forms and then subsequently matures. Such maturation requires inter-organellar transport of protein cargos required for pigment synthesis but also recruitment of effector proteins necessary for the correct transport of melanosomes to the cell periphery. Several studies have started to unravel the main pathways and mechanisms exploited by melanosomal proteins involved in melanosome structure and melanin synthesis. A major unexpected finding seen early in melanosome biogenesis showed the similarities between the fibrillar sheets of premelanosomes and amyloid fibrils. Late steps of melanosome formation are dependent on pathways regulated by proteins encoded by genes mutated in genetic diseases such as the Hermansky-Pudlak Syndrom (HPS) and different types of albinism. Altogether the findings from the past recent years have started to unravel how specialized cells integrate unique and ubiquitous molecular mechanisms in subverting the endosomal system to generate cell-type specific structures and their associated functions. Further dissection of the melanosomal system will likely shed light not only on the biogenesis of lysosome-related organelles but also on general aspects of vesicular transport in the endosomal system.     

Fas ligand (CD95 ligand) controls angiogenesis beneath the retina

A principal cause of blindness is subretinal neovascularization associated with age-related macular degeneration. Excised neovascular membranes from patients with age-related macular degeneration demonstrated a pattern of Fas+ new vessels in the center of the vascular complex, surrounded by FasL+ retinal pigment epithelial cells. In a murine model, Fas (CD95)-deficient (Ipr) and FasL-defective (gld) mice had a significantly increased incidence of neovascularization compared with normal mice. Furthermore, in gld mice there is massive subretinal neovascularization with uncontrolled growth of vessels. We found that cultured choroidal endothelial cells were induced to undergo apoptosis by retinal pigment epithelial cells through a Fas-FasL interaction. In addition, antibody against Fas prevented vascular tube formation of choroidal endothelial cells derived from the eye in a three-dimensional in vitro assay. Thus, FasL expressed on retinal pigment epithelial cells may control the growth and development of new subretinal vessels that can damage vision.     

Interaction of Complement Factor H and Fibulin3 in Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a major cause of vision loss. It is associated with development of characteristic plaque-like deposits (soft drusen) in Bruch’s membrane basal to the retinal pigment epithelium (RPE). A sequence variant (Y402H) in short consensus repeat domain 7 (SCR7) of complement factor H (CFH) is associated with risk for “dry” AMD. We asked whether the eye-targeting of this disease might be related to specific interactions of CFH SCR7 with proteins expressed in the aging human RPE/choroid that could contribute to protein deposition in drusen. Yeast 2-hybrid (Y2H) screens of a retinal pigment epithelium/choroid library derived from aged donors using CFH SCR7 baits detected an interaction with EFEMP1/Fibulin 3 (Fib3), which is the locus for an inherited macular degeneration and also accumulates basal to macular RPE in AMD. The CFH/Fib3 interaction was validated by co-immunoprecipitation of native proteins. Quantitative Y2H and ELISA assays with different recombinant protein constructs both demonstrated higher affinity for Fib3 for the disease-related CFH 402H variant. Immuno-labeling revealed colocalization of CFH and Fib3 in globular deposits within cholesterol-rich domains in soft drusen in two AMD donors homozygous for CFH 402H (H/H). This pattern of labeling was quite distinct from those seen in examples of eyes with Y/Y and H/Y genotypes. The CFH 402H/Fib3 interaction could contribute to the development of pathological aggregates in soft drusen in some patients and as such might provide a target for therapeutic intervention in some forms of AMD.     

Similar molecules spatially correlate with lipofuscin and N-retinylidene-N-retinylethanolamine in the mouse but not in the human retinal pigment epithelium

The accumulation of lipofuscin in the retinal pigment epithelium (RPE) has been implicated in the development of age-related macular degeneration (AMD) in humans. The exact composition of lipofuscin is not known but its best characterized component is N-retinylidene-N-retinylethanolamine (A2E), a byproduct of the retinoid visual cycle. Utilizing our recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI–IMS)-based technique to determine the spatial distribution of A2E, this study compares the relationships of lipofuscin fluorescence and A2E in the murine and human RPE on representative normal tissue. To identify molecules with similar spatial patterns, the images of A2E and lipofuscin were correlated with all the individual images in the MALDI–IMS dataset. In the murine RPE, there was a remarkable correlation between A2E and lipofuscin. In the human RPE, however, minimal correlation was detected. These results were reflected in the marked distinctions between the molecules that spatially correlated with the images of lipofuscin and A2E in the human RPE. While the distribution of murine lipofuscin showed highest similarities with some of the known A2E-adducts, the composition of human lipofuscin was significantly different. These results indicate that A2E metabolism may be altered in the human compared to the murine RPE.     

The Polarity of the Retinal Pigment Epithelium

The diversity of epithelia in the body permits a multitude of organ-specific functions. One of the foremost examples of this is the retinal pigment epithelium. Located between the photoreceptors of the retina and their principal blood supply, the choriocapillaris, the retinal pigment epithelium is critical for the survival and function of retinal photoreceptors. To serve this purpose, the retinal pigment epithelium cell has adapted the classic Golgi-to-cell-surface targeting pathways first described in such prototypic epithelial cell models as the Madin-Darby canine kidney cell, to arrive at a unique distribution of membrane and secreted proteins. More recent data suggest that the retinal pigment epithelium also takes advantage of its inherent asymmetry to augment the classical pathways of Golgi-to-cell-surface traffic. As retinal pigment epithelium transplants and gene therapy represent potential cures for retinal degenerative diseases, understanding the basis of the unique polarity properties of retinal pigment epithelium cells will be a critical issue for the development of future therapies.     

Autofluorescence method to measure macular pigment optical densities fluorometry and autofluorescence imaging

Non-invasive measurement of the optical density of the human macular pigment by the autofluorescence method takes advantage of the fluorescence of lipofuscin in the human retinal pigment epithelium. Measuring the intensity of fluorescence above 550 nm, where macular pigment has essentially zero absorption, and stimulating the fluorescence with two wavelengths, one well absorbed by macular pigment and the other minimally absorbed by macular pigment, provides a single-pass measurement of the macular pigment optical density. The method is implemented either by fluorometry of lipofuscin to yield the optical density of the macular pigment in a 2 degrees diameter central area, or by autofluorescence imaging to yield a high-resolution map of the macular pigment distribution.     

Progressive cone–rod dystrophy

Inherited macular dystrophies present with varying clinical signs. They possess significant overlap with other inherited retinal dystrophies and with several acquired retinal degenerations. A classification system for diagnostic purposes is crucial. This paper includes a case report of an extremely unusual presentation of a cone–rod dystrophy. Its challenge lies in the difficulty to differentiate it from not only inherited dystrophies of the photoreceptors and retinal pigment epithelium, but also from acquired degenerations of the macula and retina. This paper reviews the differential diagnoses and management considerations of cone–rod dystrophy.     

Mosaicism of the retinal pigment epithelium: seeing the small picture

The retinal pigment epithelium (RPE) is a monolayer of cells that appear phenotypically regular, but which exhibit striking cell-cell variability in content of melanin and lipofuscin granules, and in expression of many proteins. This naturally occurring cell heterogeneity likely arises by normal mechanisms regulating gene expression during development and postnatal aging. The consequence is a tissue in which individual cells may differ in their ability to support adjacent photoreceptors, and which may respond differentially to oxidative stress and other environmental influences that contribute to cell dysfunction during aging. The inherent variability of RPE cells is probably one factor contributing to the characteristically patchy pattern of retinal diseases like age-related macular degeneration.     

Vitronectin is a constituent of ocular drusen and the vitronectin gene is expressed in human retinal pigmented epithelial cells.

Age-related macular degeneration (AMD) leads to dysfunction and degeneration of retinal photoreceptor cells. This disease is characterized, in part, by the development of extracellular deposits called drusen. The presence of drusen is correlated with the development of AMD, although little is known about drusen composition or biogenesis. Drusen form within Bruch's membrane, a stratified extracellular matrix situated between the retinal pigmented epithelium and choriocapillaris. Because of this association, we sought to determine whether drusen contain known extracellular matrix constituents. Antibodies directed against a battery of extracellular matrix molecules were screened on drusen-containing sections from human donor eyes, including donors with clinically documented AMD. Antibodies directed against vitronectin, a plasma protein and extracellular matrix component, exhibit intense and consistent reactivity with drusen; antibodies to the conformationally distinct, heparin binding form of human vitronectin are similarly immunoreactive. No differences in vitronectin immunoreactivity between hard and soft drusen, or between macular and extramacular regions, have been observed. RT-PCR analyses revealed that vitronectin mRNA is expressed in the retinal pigmented epithelium (RPE)-choroidal complex and cultured RPE cells. These data document that vitronectin is a major constituent of human ocular drusen and that vitronectin mRNA is synthesized locally. Based on these data, we propose that vitronectin may participate in the pathogenesis of AMD.