Reliability Assessment of a Rod Photoreceptor Outer Segment Grading System

Jablonski, Monica M.; Graney, Marshall J.; Kritchevsky, Stephen B.; Iannaccone, Alessandro

doi:10.1006/exer.2001.0987

Cited by 5 publications

(3 citation statements)

References 22 publications

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“…Details on the methods and technique used to grade the WB gels are provided in S1 Supporting Information . The method was developed and tested for reproducibility, based on a conceptually similar scale previously developed to assess other images [ 65 ] (see S1 Table and S2 Fig ). IR values were recorded in a database and analyzed as described in S1 Supporting Information .…”

Section: Methodsmentioning

confidence: 99%

Circulating Autoantibodies in Age-Related Macular Degeneration Recognize Human Macular Tissue Antigens Implicated in Autophagy, Immunomodulation, and Protection from Oxidative Stress and Apoptosis

et al. 2015

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BackgroundWe investigated sera from elderly subjects with and without age-related macular degeneration (AMD) for presence of autoantibodies (AAbs) against human macular antigens and characterized their identity.MethodsSera were collected from participants in the Age-Related Maculopathy Ancillary (ARMA) Study, a cross-sectional investigation ancillary to the Health ABC Study, enriched with participants from the general population. The resulting sample (mean age: 79.2±3.9 years old) included subjects with early to advanced AMD (n = 131) and controls (n = 231). Sera were tested by Western blots for immunoreactive bands against human donor macular tissue homogenates. Immunoreactive bands were identified and graded, and odds ratios (OR) calculated. Based on these findings, sera were immunoprecipitated, and subjected to 2D gel electrophoresis (GE). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify the targets recognized by circulating AAbs seen on 2D-GE, followed by ELISAs with recombinant proteins to confirm LC-MS/MS results, and quantify autoreactivities.ResultsIn AMD, 11 immunoreactive bands were significantly more frequent and 13 were significantly stronger than in controls. Nine of the more frequent bands also showed stronger reactivity. OR estimates ranged between 4.06 and 1.93, and all clearly excluded the null value. Following immunoprecipitation, 2D-GE and LC-MS/MS, five of the possible autoreactivity targets were conclusively identified: two members of the heat shock protein 70 (HSP70) family, HSPA8 and HSPA9; another member of the HSP family, HSPB4, also known as alpha-crystallin A chain (CRYAA); Annexin A5 (ANXA5); and Protein S100-A9, also known as calgranulin B that, when complexed with S100A8, forms calprotectin. ELISA testing with recombinant proteins confirmed, on average, significantly higher reactivities against all targets in AMD samples compared to controls.ConclusionsConsistent with other evidence supporting the role of inflammation and the immune system in AMD pathogenesis, AAbs were identified in AMD sera, including early-stage disease. Identified targets may be mechanistically linked to AMD pathogenesis because the identified proteins are implicated in autophagy, immunomodulation, and protection from oxidative stress and apoptosis. In particular, a role in autophagy activation is shared by all five autoantigens, raising the possibility that the detected AAbs may play a role in AMD via autophagy compromise and downstream activation of the inflammasome. Thus, we propose that the detected AAbs provide further insight into AMD pathogenesis and have the potential to contribute to disease biogenesis and progression.

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Section: Methodsmentioning

confidence: 99%

Circulating Autoantibodies in Age-Related Macular Degeneration Recognize Human Macular Tissue Antigens Implicated in Autophagy, Immunomodulation, and Protection from Oxidative Stress and Apoptosis

et al. 2015

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“…Because Xenopus retinas mature most rapidly in the posterior pole, assessment of outer segment structure was performed on sections taken exclusively from this area (Stiemke et al, 1994). The grading of nascent photoreceptor outer segments was performed in a masked fashion as described previously (Jablonski et al, 2001). Using this system, a grade of 4 represents proper folding of photoreceptor outer segment membranes, while a grade of 0 represents complete disorganization of membranes.…”

Section: Methodsmentioning

confidence: 99%

Cellular retinol binding protein 1 modulates photoreceptor outer segment folding in the isolated eye

Wang

Tong

Giorgianni

et al. 2010

Developmental Neurobiology

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In a previous study, we used differential proteomics to identify retinal proteins whose steady-state levels were altered in an experimental system in which photoreceptor outer segments were improperly folded. We determined that the steady-state level of cellular retinol binding protein 1 (CRBP1) was downregulated in eyes lacking organized outer segments. The purpose of this study was to determine if CRBP1 is a plausible candidate for regulating outer segment assembly. We used Morpholinos to directly test the hypothesis that a decreased level of CRBP1 protein was associated with the misfolding of outer segments. Results from these studies indicate that downregulation of CRBP1 protein resulted in aberrant assembly of outer segments. Because CRBP1 plays a dual role in the retina – retinal recycling and generation of retinoic acid – we evaluated both possibilities. Our data demonstrate that outer segment folding was not modified by 11-cis retinal supplementation, suggesting that CRBP1 influences outer segment assembly through a mechanism unrelated to rhodopsin regeneration. In contrast, retinoic acid is required for the proper organization of nascent outer segment membranes. The localization of CRBP1 within Muller cells and the RPE and its demonstrated role in modulating the proper folding of nascent outer segment membranes through retinoic acid further elucidates the role of these cells in directly influencing photoreceptor physiology.

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“…The assembly of the nascent photoreceptor outer segments under these four experimental conditions was then graded in a masked fashion using our previously described grading algorithm and six-step scale (Jablonski et al, 2001a). The key criterion of this grading scale is the amount of stacked and organized photoreceptor outer segment membrane that was associated with each evaluated photoreceptor cell.…”

Section: Methodsmentioning

confidence: 99%

Contribution of Müller cells toward the regulation of photoreceptor outer segment assembly

2004

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The assembly of photoreceptor outer segments into stacked discs is a complicated process, the precise regulation of which remains a mystery. It is known that the integrity of the outer segment is heavily dependent upon surrounding cell types including the retinal pigment epithelium and Müller cells; however the role played by Müller cells within this photoreceptor-specific process has not been fully explored. Using an RPE-deprived but otherwise intact Xenopus laevis eye rudiment preparation, we reveal that Müller cell involvement in outer segment assembly is dependent upon the stimulus provided to the retina. Pigment epithelium-derived factor is able to support proper membrane folding after inhibition of Müller cell metabolism by alpha-aminoadipic acid, while isopropyl beta-Dthiogalactoside, a permissive glycan, requires intact Müller cell function. These results demonstrate that both intrinsic and extrinsic redundant mechanisms exist to support the ability of photoreceptors to properly assemble their outer segments. Our study further suggests that the receptor for pigment epithelium-derived factor resides in photoreceptors themselves while that for permissive glycans is likely localized to Müller cells, which in turn communicate with photoreceptors to promote proper membrane assembly.

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Reliability Assessment of a Rod Photoreceptor Outer Segment Grading System

Cited by 5 publications

References 22 publications

Circulating Autoantibodies in Age-Related Macular Degeneration Recognize Human Macular Tissue Antigens Implicated in Autophagy, Immunomodulation, and Protection from Oxidative Stress and Apoptosis

Circulating Autoantibodies in Age-Related Macular Degeneration Recognize Human Macular Tissue Antigens Implicated in Autophagy, Immunomodulation, and Protection from Oxidative Stress and Apoptosis

Cellular retinol binding protein 1 modulates photoreceptor outer segment folding in the isolated eye

Contribution of Müller cells toward the regulation of photoreceptor outer segment assembly

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