Alan D. Marmorstein

Aberrant accumulation of EFEMP1 underlies drusen formation in Malattia Leventinese and age-related macular degeneration

Malattia Leventinese (ML), an inherited macular degenerative disease, is closely reminiscent of age-related macular degeneration (AMD), the most common cause of incurable blindness. Both ML and AMD are characterized by extracellular deposits known as drusen between the retinal pigment epithelium (RPE) and Bruchs membrane. The mechanism underlying drusen formation is unknown. An Arg to Trp mutation in a gene of unknown function, EFEMP1, is responsible for ML, indicating EFEMP1 may be important in drusen formation. Here, we show that wild-type EFEMP1 is a secreted protein whereas mutant EFEMP1 is misfolded, secreted inefficiently, and retained within cells. In normal eyes, EFEMP1 is not present at the site of drusen formation. However, in ML eyes, EFEMP1 accumulates within the RPE cells and between the RPE and drusen, but does not appear to be a major component of drusen. Furthermore, in AMD eyes, EFEMP1 is found to accumulate beneath the RPE immediately overlaying drusen, but not in the region where there is no apparent retinal pathology observed. These data present evidence that misfolding and aberrant accumulation of EFEMP1 may cause drusen formation and cellular degeneration and play an important role in the etiology of both ML and AMD.

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.

Protein Database, Human Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a single cell layer adjacent to the rod and cone photoreceptors that plays key roles in retinal physiology and the biochemistry of vision. RPE cells were isolated from normal adult human donor eyes, subcellular fractions were prepared, and proteins were fractionated by electrophoresis. Following in-gel proteolysis, proteins were identified by peptide sequencing using liquid chromatography tandem electrospray mass spectrometry and/or by peptide mass mapping using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Preliminary analyses have identified 278 proteins and provide a starting point for building a database of the human RPE proteome.

Saturation of, and competition for entry into, the apical secretory pathway

To investigate mechanisms of apical sorting in the secretory pathway of epithelial cells, we expressed varying amounts of the 165 amino acid isoform of vascular endothelial growth factor (VEGF(165)) and transforming growth factor beta1 (TGF-beta1) via replication defective adenoviruses. Apical sorting of both proteins was efficient at low expression levels but saturated or was reversed at high expression levels. High expression levels of TGF-beta1 were effective at competing VEGF(165) out of the apical pathway; however, VEGF(165) did not compete out TGF-beta1. Tunicamycin inhibition experiments showed that the apical polarity of VEGF(165) was independent of N-glycosylation. We conclude that the apical sorting of these two molecules is a saturable, signal-mediated process, involving competition for apical sorting receptors. The sorting of the two proteins does not appear to involve N-glycans as sorting signals, or lectin sorters. The observations are particularly relevant to gene therapy because they demonstrate that overexpression of a transgene can result in undesirable missorting of the encoded protein.

Retinal pigment epithelial cells exhibit unique expression and localization of plasma membrane syntaxins which may contribute to their trafficking phenotype

The SNARE membrane fusion machinery controls the fusion of transport vesicles with the apical and basolateral plasma-membrane domains of epithelial cells and is implicated in the specificity of polarized trafficking. To test the hypothesis that differential expression and localization of SNAREs may be a mechanism that contributes to cell-type-specific polarity of different proteins, we studied the expression and distribution of plasma-membrane SNAREs in the retinal pigment epithelium (RPE), an epithelium in which the targeting and steady-state polarity of several plasma membrane proteins differs from most other epithelia. We show here that retinal pigment epithelial cells both in vitro and in vivo differ significantly from MDCK cells and other epithelial cells in their complement of expressed t-SNAREs that are known — or suggested — to be involved in plasma membrane trafficking. Retinal pigment epithelial cells lack expression of the normally apical-specific syntaxin 3. Instead, they express syntaxins 1A and 1B, which are normally restricted to neurons and neuroendocrine cells, on their apical plasma membrane. The polarity of syntaxin 2 is reversed in retinal pigment epithelial cells, and it localizes to a narrow band on the lateral plasma membrane adjacent to the tight junctions. In addition, syntaxin 4 and the v-SNARE endobrevin/VAMP-8 localize to this sub-tight junctional domain, which suggests that this is a region of preferred vesicle exocytosis. Altogether, these data suggest that the unique polarity of many retinal pigment epithelial proteins results from differential expression and distribution of SNAREs at the plasma membrane. We propose that regulation of the expression and subcellular localization of plasma membrane SNAREs may be a general mechanism that contributes to the establishment of distinct sorting phenotypes among epithelial cell types.

Autosomal recessive bestrophinopathy is not associated with the loss of bestrophin-1 anion channel function in a patient with a novel BEST1 mutation

PURPOSE Mutations in BEST1, encoding bestrophin-1 (Best1), cause autosomal recessive bestrophinopathy (ARB). Encoding bestrophin-1 is a pentameric anion channel localized to the basolateral plasma membrane of the RPE. Here, we characterize the effects of the mutations R141H (CGC > CAC) and I366fsX18 (c.1098_1100+7del), identified in a patient in our practice, on Best1 trafficking, oligomerization, and channel activity. METHODS Currents of Cl- were assessed in transfected HEK293 cells using whole-cell patch clamp. Best1 localization was assessed by confocal microscopy in differentiated, human-induced pluripotent stem cell-derived RPE (iPSC-RPE) cells following expression of mutants via adenovirus-mediated gene transfer. Oligomerization was evaluated by coimmunoprecipitation in iPSC-RPE and MDCK cells. RESULTS Compared to Best1, Best1 I366fsX18 currents were increased while Best1 R141H Cl- currents were diminished. Coexpression of Best1 R141H with Best1 or Best1 I366fsX18 resulted in rescued channel activity. Overexpressed Best1, Best1 R141H, and Best1 I366fsX18 were all properly localized in iPSC-RPE cells; Best1 R141H and Best1 I366fsX18 coimmunoprecipitated with endogenous Best1 in iPSC-RPE cells and with each other in MDCK cells. CONCLUSIONS The first 366 amino acids of Best1 are sufficient to mediate channel activity and homo-oligomerization. The combination of Best1 and Best1 R141H does not cause disease, while Best1 R141H together with Best1 I366fsX18 causes ARB. Since both combinations generate comparable Cl- currents, this indicates that ARB in this patient is not caused by a loss of channel activity. Moreover, Best1 I366fsX18 differs from Best1 in that it lacks most of the cytosolic C-terminal domain, suggesting that the loss of this region contributes significantly to the pathogenesis of ARB in this patient.

Disease-causing mutations associated with four bestrophinopathies exhibit disparate effects on the localization, but not the oligomerization, of Bestrophin-1.

BEST1 encodes Bestrophin-1 (Best1), a homo-oligomeric, integral membrane protein localized to the basolateral plasma membrane of the retinal pigment epithelium. Mutations in BEST1 cause five distinct retinal degenerative diseases, including adult vitelliform macular dystrophy (AVMD), autosomal recessive bestrophinopathy (ARB), autosomal dominant vitreoretinochoroidopathy (ADVIRC), and retinitis pigmentosa (RP). The mechanisms underlying these diseases and why mutations cause one disease over another are, for the most part, unknown. To gain insights into these four diseases, we expressed 28 Best1 mutants fused to YFP in polarized MDCK monolayers and, via confocal microscopy and immunofluorescence, live-cell FRET, and reciprocal co-immunoprecipitation experiments, screened these mutants for defects in localization and oligomerization. All 28 mutants exhibited comparable FRET efficiencies to and co-immunoprecipitated with WT Best1, indicating unimpaired oligomerization. RP- and ADVIRC-associated mutants were properly localized to the basolateral plasma membrane of cells, while two AVMD and most ARB mutants were mislocalized. When co-expressed, all mislocalized mutants caused mislocalization of WT Best1 to intracellular compartments. Our current and past results indicate that mislocalization of Best1 is not an absolute feature of any individual bestrophinopathy, occurring in AVMD, BVMD, and ARB. Furthermore, some ARB mutants that do not also cause dominant disease cause mislocalization of Best1, indicating that mislocalization is not a cause of disease, and that absence of Best1 activity from the plasma membrane is tolerated. Lastly, we find that the ARB truncation mutants L174Qfs*57 and R200X can form oligomers with WT Best1, indicating that the first ∼174 amino acids of Best1 are sufficient for oligomerization to occur.

Noninvasive recording and response characteristics of the rat dc-electroretinogram

In response to light, the retinal pigment epithelium (RPE) generates a series of potentials that can be recorded using the dc-electroretinogram (dc-ERG). As these potentials can be related to specific cellular events, they provide information about RPE function and how that may be altered by disease or experimental manipulation. The purposes of the present study were to define a noninvasive means for recording the rat dc-ERG, to use this to define the stimulus-response properties of the major components, and to relate these results to measures of the rat electrooculogram (EOG). Parallel studies were conducted in two strains of rats (Long-Evans, LE; Sprague-Dawley, SD) that are commonly used in vision research. Rats were sedated with ketamine/xylazine and placed on a heating pad. Ag/AgCl wire electrodes were bridged with capillary tubes filled with Hanks balanced salt solution. The active electrode was placed in contact with the corneal surface and referenced to a second electrode placed within the orbit. The dc-ERG signal was amplified (dc-100 Hz), digitized, and stored offline. The duration of full-field flash stimuli was controlled using a mechanical shutter and flash luminance was controlled with neutral density filters. EOGs were recorded using subdermal platinum needle electrodes placed near the eye. In response to a 5-min light exposure, the dc-ERG of LE and SD rats included a distinct b-wave, after potential, c-wave, fast oscillation, and a slow potential of positive polarity the characteristics of which are consistent with a light peak. In LE rats, the final plateau of this slow positive potential was often lower than the prestimulus baseline; in SD rats, this potential achieved a level above the baseline. Analysis of EOGs recorded from these two strains yielded results consistent with the amplitude of the slow potential relative to the prestimulus baseline. Specifically, the amplitude of the EOG of SD rats increased when the eye was exposed to light. In LE rats, this increase did not occur, and in some cases light reduced the amplitude of the EOG. The two strains also differed with respect to c-wave implicit time, which was faster in SD rats. These results indicate that many of the major components of the dc-ERG are readily measured in the rat. Therefore, we believe that the rat may provide a useful animal model in which to conduct pharmacological analysis of nonneuronal responses and to develop animal models of human retinal disorders involving the RPE, such as Best Vitelliform Macular Dystrophy.

In vivo gene transfer as a means to study the physiology and morphogenesis of the retinal pigment epithelium in the rat.

Our understanding of the morphogenesis of epithelial phenotypes has been greatly advanced by the use of in vitro cell culture systems. However, cell cultures often do not faithfully reconstitute many of the differentiated properties of the cell from which they are derived and cannot be used to examine complex physiologic interactions between adjacent tissues. This is particularly true of the retinal pigment epithelium (RPE). Many plasma membrane proteins, in vivo, exhibit a reversed polarity with respect to other epithelia, and RPE-derived cell lines seldom exhibit these same polarity properties. Furthermore, the interaction between the RPE cell and the neuorsensory retina, or the underlying blood supply, the choroid, is absent in cell culture. Most epithelia are difficult to isolate and study in vivo. The RPE is an exception to this. We have explored several aspects of RPE protein transport properties, vision-related physiology, and disease-related pathophysiology in the eye using in vivo gene transfer and electrophysiologic techniques. By injecting replication-defective adenoviruses into the subretinal space of rat eyes, we have been able to easily direct the expression of a test protein and follow its sorting and physiologic effects on RPE cells and adjacent tissues. Due to binding and internalization of adenoviral vectors to integrins found on the RPE apical plasma membrane, expression in a healthy eye is essentially confined to the RPE cell, even under control of a cytomegalovirus promotor. The use of varying amounts of adenoviral vector allows for determination of dose-responsive effects and the comparison of multiple mutants of a protein. In addition, there are substantial savings with respect to time and money in comparison to standard transgenic approaches.

Control of Maintenance and Regeneration of Planarian Eyes by ovo.

PURPOSE Following decapitation, the planarian Schmidtea mediterranea regenerates its head and eyes. The gene ovo is required for eye maintenance and regeneration in response to wounding. In this study, we investigated whether eye regeneration in S. mediterranea could occur absent a wound healing response. METHODS One hundred twenty S. mediterranea were treated with ovo RNA interference (RNAi) or control (unc-22) RNAi by feeding double-stranded RNA (dsRNA). Following eye loss, ovo RNAi treatment was halted and replaced with control RNAi treatment. Quantitative real-time PCR (qPCR) was used to monitor ovo expression. Eye functionality was monitored via a phototaxis assay. Photoreceptor neurons were visualized via immunofluorescence staining of arrestin. RESULTS Treatment with ovo RNAi caused eyes to gradually shrink until they were completely absent. One hundred percent of ovo RNAi-treated planarians lost both eyes within 137 days of treatment onset. ovo RNAi-treated planarians were unable to regenerate eyes in response to decapitation. Upon removal of ovo RNAi, eyes became visible as small pigmented spots in the head within 28 days. The eyes slowly developed, appearing to gain pigmented cells first and then nonpigmented photoreceptors. Phototaxis assays demonstrated functional eye loss and eye restoration. ovo mRNA was significantly decreased following treatment with ovo RNAi and significantly increased following removal of ovo RNAi. Arrestin staining was present in the eyes, optic nerves, and optic chiasm of worms with regenerated eyes but not in eyeless worms. CONCLUSIONS S. mediterranea have the ability to generate functional eyes in the absence of a wound healing response. This ability requires the expression of ovo.