Gail M. Seigel

Evidence of apoptotic cell death in keratoconus

Purpose. To determine the potential role of apoptosis in the noninflammatory degeneration characteristic of keratoconus. Methods. Four normal corneas and 16 keratoconus corneas were obtained as archival specimens. Tissues were examined histopathologically for TUNEL immunoreactivity to detect the presence of DNA fragmentation. Tissues were also subjected to single-stranded DNA (ssDNA) analysis, a more apoptosis-specific stain. Results. Normal corneas exhibited fewer than five TUNEL-positive epithelial cells per section, these being very lightly stained. All 16 keratoconus corneas demonstrated extensive, intense TUNEL staining in at least one layer. Fifteen of 16 exhibited staining in the epithelial layer, 11 of 16 in the stromal layer, and 13 of 16 in the endothelial layer, whereas 10 of 16 keratoconus cases demonstrated TUNEL immunoreactivity in all three layers. The ssDNA stain was also positive and evident in all three layers of the cornea, although to a lesser degree than the TUNEL assay. Conclusions. The noninflammatory nature of keratoconus, coupled with the TUNEL in situ results, suggests apoptosis as a mode of cell death in this degenerative disease.

Multiple RIBEYE–RIBEYE Interactions Create a Dynamic Scaffold for the Formation of Synaptic Ribbons

Synaptic ribbons are large, dynamic structures in the active zone complex of ribbon synapses and important for the physiological properties of these tonically active synapses. RIBEYE is a unique and major protein component of synaptic ribbons. The aim of the present study was to understand how the synaptic ribbon is built and how the construction of the ribbon could contribute to its ultrastructural plasticity. In the present study, we demonstrate that RIBEYE self-associates using different independent approaches (yeast two-hybrid analyses, protein pull downs, synaptic ribbon–RIBEYE interaction assays, coaggregation experiments, transmission electron microscopy and immunogold electron microscopy). The A-domain [RIBEYE(A)] and B-domain [RIBEYE(B)] of RIBEYE contain five distinct sites for RIBEYE–RIBEYE interactions. Three interaction sites are present in the A-domain of RIBEYE and mediate RIBEYE(A)–RIBEYE(A) homodimerization and heterodimerization with the B-domain. The docking site for RIBEYE(A) on RIBEYE(B) is topographically and functionally different from the RIBEYE(B) homodimerization interface and is negatively regulated by nicotinamide adenine dinucleotide. The identified multiple RIBEYE–RIBEYE interactions have the potential to build the synaptic ribbon: heterologously expressed RIBEYE forms large electron-dense aggregates that are in part physically associated with surrounding vesicles and membrane compartments. These structures resemble spherical synaptic ribbons. These ribbon-like structures coassemble with the active zone protein bassoon, an interaction partner of RIBEYE at the active zone of ribbon synapses, emphasizing the physiological relevance of these RIBEYE-containing aggregates. Based on the identified multiple RIBEYE–RIBEYE interactions, we provide a molecular mechanism for the dynamic assembly of synaptic ribbons from individual RIBEYE subunits.

Intraretinal xenografts of differentiated human retinoblastoma cells integrate with the host retina

We report on the successful use of chemically modified Y79 human retinoblastoma cells for intraretinal xenografting into damaged adult mammalian eyes. Y79 cells were exposed in vitro to retinoic acid/butyrate to induce differentiation. Using a multisite transplantation method, the suspension was injected into the subretinal space of Fischer 344 rats. The survival, integration, and differentiation potential of these cells was studied, following their return to the intraocular milieu from which the progenitor cells originated. The grafted cells survived and differentiated into immature photoreceptor elements in the subretinal and intraretinal locations, as multiple clusters of rosette-forming cells intimately attached to the host neuroretina. The differentiation process included development of synaptic connectivity of the ribbon type with the surrounding neuropil. No signs of renewed cell division were found within grafts performed on 42 rat eyes, and there was no indication of cell-mediated host reaction against the transplants. This study indicates that tumorigenicity can be suppressed in mitotically arrested Y79 cells, and that these cells are capable of undergoing differentiation in vivo. This provides evidence of the remarkable differentiation properties of human retinoblastomas while indicating that Y79 cells may ultimately be able to substitute for fetal cells in experimental retinal transplantation.

Establishment and characterization of adenoviral E1A immortalized cell lines derived from the rat suprachiasmatic nucleus.

Primary cultured cells from the presumptive anlage of the rat suprachiasmatic nucleus (SCN) were immortalized by infection with a retroviral vector encoding the adenovirus 12S E1A gene. After drug selection, the resulting neural cell lines (SCN1.4 and SCN2.2) displayed (a) extended growth potential without evidence of transformed or tumorigenic properties, (b) expression of E1A protein within all cell nuclei, and (c) heterogeneous cell types in various stages of differentiation. A large proportion of the SCN1.4 and SCN2.2 cells were characterized by gliallike morphologies, but showed limited expression of corresponding cell type-specific antigens. In addition, both lines exhibited a stable population of cells with neuronlike characteristics. When treated so as to enhance differentiation, these cells were often distinguished by fine, long processes and immunocytochemical expression of neuronal markers and peptides found within SCN neurons in situ. Observations on SCN neuropeptide immunostaining, content, release, and mRNA expression followed a concordant pattern in which somatostatin and vasopressin cells were the most and least common peptidergic phenotypes in both lines, respectively. Since these results indicate that constituents of E1A-immortalized lines derived from the primordial SCN can differentiate into cells with phenotypes resembling parental peptidergic neurons, it will be critical to explore next whether these lines also retain the distinctive function of the SCN to generate circadian rhythms. Cloning of immortalized cell types could subsequently yield useful tools for studying the development of SCN glial and peptidergic cell types and delineating their distinct roles in mammalian circadian time-keeping.

Neuronal gene expression and function in the growth-stimulated R28 retinal precursor cell line

Purpose. Proliferative retinal progenitor cells that express neuronal characteristics are potentially useful for developmental studies and as experimental graft material. The continuously-growing R28 retinal cell line has been distributed to over 60 laboratories for a variety of studies, yet has not been fully characterized. In this study, we tested the hypothesis that the proliferative R28 retinal cell line contains subpopulations of cells that express neuronal mRNAs and proteins characteristic of CNS neurons. Methods. To this end, we sought to determine the potential retinal, neuronal, and growth-related characteristics of this retinal cell line through gene expression profiling, coupled with confirmatory immunocytochemistry and electrophysiology. Results. Despite expression of growth-stimulatory oncogenes and growth-promoting factors, subpopulations of R28 cells express abundant retinal and neuronal markers, as well as the functional capacity to respond to specific neurotransmitters such as dopamine, acetylcholine, serotonin, and glycine. Conclusion. Proliferative R28 cells retain functional neuronal properties that may prove useful in future studies of neuronal differentiation and development.

Differentiation of Y79 retinoblastoma cells with pigment epithelial-derived factor and interphotoreceptor matrix wash: effects on tumorigenicity.

We investigated the in vivo differentiation potential of Y79 human retinoblastoma cells following pre-treatment with two novel neurotrophic agents: PEDF (human recombinant pigmented-epithelial derived factor) or IPM (interphotoreceptor matrix) wash. These agents were able to induce a significant degree of morphological differentiation in vitro. However, 48 days after subretinal transplantation of pre-treated cells, massive tumor formation was apparent. In contrast, Y79 cells pre-treated with retinoic acid/sodium butyrate, which attain a lesser degree of morphological differentiation, did not produce tumors over a 30 to 60 day-survival time (del Cerro et al., Brain Research, 12-22, 1992). We conclude that for PEDF and IPM, the degree of in vitro differentiation and the degree of mitotic arrest are independent features.

RIBEYE Recruits Munc119, a Mammalian Ortholog of the Caenorhabditis elegans Protein unc119, to Synaptic Ribbons of Photoreceptor Synapses

Munc119 (also denoted as RG4) is a mammalian ortholog of the Caenorhabditis elegans protein unc119 and is essential for vision and synaptic transmission at photoreceptor ribbon synapses by unknown molecular mechanisms. Munc119/RG4 is related to the prenyl-binding protein PrBP/δ and expressed at high levels in photoreceptor ribbon synapses. Synaptic ribbons are presynaptic specializations in the active zone of these tonically active synapses and contain RIBEYE as a unique and major component. In the present study, we identified Munc119 as a RIBEYE-interacting protein at photoreceptor ribbon synapses using five independent approaches. The PrBP/δ homology domain of Munc119 is essential for the interaction with the NADH binding region of RIBEYE(B) domain. But RIBEYE-Munc119 interaction does not depend on NADH binding. A RIBEYE point mutant (RE(B)E844Q) that no longer interacted with Munc119 still bound NADH, arguing that binding of Munc119 and NADH to RIBEYE are independent from each other. Our data indicate that Munc119 is a synaptic ribbon-associated component. We show that Munc119 can be recruited to synaptic ribbons via its interaction with RIBEYE. Our data suggest that the RIBEYE-Munc119 interaction is essential for synaptic transmission at the photoreceptor ribbon synapse.

Oxysterol-induced toxicity in R28 and ARPE-19 cells.

Studies have shown an intimate relationship between cholesterol and retinal diseases; we examined the effects of cholesterol oxides on cultured cells. Using the rat retinal precursor cell line R28 and the human RPE cell line ARPE-19, we investigated the potential cytotoxicity of cholesterol oxides. Cultured R28 and ARPE-19 cells were treated with either 25-hydroxycholesterol and 7-ketocholesterol (0–50 µg/ml). Cell viability was determined by the WST-1 colorimetric assay. Production of reactive oxygen intermediate (ROI) was assessed by a fluorescent probe–based assay (2′,7′-dichlorodihydrofluorescein diacetate [H2DCFDA]). To detect the presence of apoptosis, DNA fragmentation gel analysis and Hoescht nuclear staining were performed. Both cholesterol oxides tested were toxic in a time- and dose-dependent fashion to the two cell lines used in this study. Treatment of R28 cells with either 25-hydroxycholesterol or 7-ketocholesterol at a concentration of 25 µg/ml resulted in greater than 50% loss of cell viability after 24 h. ARPE-19 cells were slightly less affected, with a loss of cell viability of approximately 20% and 40% after 24 h-exposure of 25-hydroxycholesterol and 7-ketocholesterol, respectively. DNA fragmentation and chromatin condensation demonstrated apoptotic events occurring in 7-ketocholesterol–treated cells. The fluorescent assay for ROI production showed that after an hour of exposure to 7-ketocholesterol, R28 cells responded with increased levels of ROIs, whereas no immediate production of ROIs were detected with treated ARPE-19 cells. These in vitro findings provide evidence that cholesterol oxides can directly damage cultured retinal and RPE cells. The oxysterol-induced oxidative stress in these cells may be a factor in the pathology of retinal degenerative diseases.

l-Deprenyl, blocking apoptosis and regulating gene expression in cultured retinal neurons

Apoptosis is the final pathway of many forms of retinal degeneration. E1A-NR3 is an immortalized retinal cell line that manifests specific phenotypes of retinal neurons. The present study induced apoptosis in these cells by two ischemic assaults, serum deprivation and hypoxia. The results demonstrated that both the assaults decreased viable cell numbers significantly by inducing apoptosis, as revealed by viable cell count, DNA fragmentation analysis, and in situ labeling of apoptotic cells by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method. l-Deprenyl is known to be a monoamine oxidase inhibitor, and it was found recently to have neurotrophic activities. We set out to determine the protective effect of l-deprenyl on retinal cells and delineate its mechanism independent of monoamine oxidase inhibition. At concentrations as low as 0.0001 and 0.001 microM, l-deprenyl significantly increased the numbers of surviving cells under serum-free and hypoxic conditions, respectively. This effect appeared to be dependent upon the l-deprenyl concentration within the range of 0.001 to 10 microM. The neurotrophic activity was via blocking apoptosis, as l-deprenyl decreased the fragmented DNA and the numbers of positively stained apoptotic cells under serum-free or hypoxic conditions. Using mRNA differential display, nine mRNAs were identified and confirmed by northern blot analysis to have altered expression levels at 8 hr of exposure to hypoxia. Five of them do not match any existing sequences in GenBank, whereas the other four represent known genes including c-jun, heat-shock protein hsp70, phosphoglycerate kinase (PGK), and calpactin I heavy chain. All of the four mRNAs were increased significantly by hypoxia. The c-jun, PGK, and calpactin mRNAs, but not hsp70, also were increased by serum withdrawal. l-Deprenyl partially reversed the increase in c-jun and hsp70 mRNA levels, but not in PGK and calpactin. These results suggest that l-deprenyl blocks apoptosis induced by hypoxia as well as by growth factor withdrawal and regulates the expression of apoptosis-related genes.

Toxicity of Indocyanine Green (ICG) in Combination with Light on Retinal Pigment Epithelial Cells and Neurosensory Retinal Cells

Purpose: To evaluate the toxicity of indocyanine green (ICG) in combination with light. Methods: Human retinal pigment epithelial cells (ARPE-19) and rat neurosensory retinal cells (R28) were treated with four different concentrations of ICG in combination with light exposure. Cell viability, mitochondrial function, and DNA synthesis were measured. Results: All concentrations of ICG with 10 min of light exposure caused a significant decrease in mitochondrial dehydrogenase activity in R28 and ARPE-19 cells. ICG without light exposure did not decrease mitochondrial dehydrogenase activity. In both cell lines, [3H]thymidine incorporation was increased when treated with ICG with or without light. R28 cells did not show any significant decrease in cell viability. Conclusions: The duration of light was a significant factor in ICG toxicity. ICG needs to be used with caution as it decreases the mitochondrial dehydrogenase activity and increases the DNA synthesis in retinal cells, markers for cell toxicity and dysfunction.