Proteomic profiling of the retinal dysplasia and degeneration chick retina

Abstract

Purpose: In our previous paper we undertook proteomic analysis of the normal developing chick retina to identify proteins that were differentially expressed during retinal development. In the present paper we use the same proteomic approach to analyze the development and onset of degeneration in the retinal dysplasia and degeneration (rdd) chick. The pathology displayed by the rdd chick resembles that observed in some of the more severe forms of human retinitis pigmentosa.

Methods: Two-dimensional gel electrophoresis (pH 4-7), gel image analysis, and mass spectrometry were used to profile the developing and degenerating retina of the rdd and wild-type (wt) chick retina.

Results: Several proteins were identified by mass spectrometry that displayed differential expression between normal and rdd retina between embryonic day 12 (E12) and post-hatch day 1 (P1). Secernin 1 displayed the most significant variation in expression between rdd and wt retina; this may be due to differential phosphorylation in the rdd retina. Secernin 1 has dipeptidase activity and has been demonstrated to play a role in exocytosis; it has been shown to be overexpressed in certain types of cancer and has also been suggested as a potential neurotoxicologically relevant target. Its role in the retina and in particular its differential expression in the degenerate rdd retina remains unknown and will require further investigation. Other proteins that were differentially expressed in the rdd retina included valosin-containing protein, beta-synuclein, stathmin 1, nucleoside diphosphate kinase, histidine triad nucleotide-binding protein, and 40S ribosomal protein S12. These proteins are reported to be involved in several cellular processes, including the ubiquitin proteasome pathway, neuroprotection, metastatic suppression, transcriptional and translational regulation, and regulation of microtubule dynamics.

Conclusions: This proteomic study is the first such investigation of the rdd retina and represents a unique data set that has revealed several proteins that are differentially expressed during retinal degeneration in the rdd chick. Secernin 1 showed the most significant differences in expression during this degeneration period. Further investigation of the proteins identified may provide insight into the complex events underlying retinal degeneration in this animal model.

Figure 1

Retinal dysplasia and degeneration (rdd) retinal histology. Microtome sections of retina stained with hematoxylin and eosin. Normal retinal morphology is evident in the developing wt chick at embryonic day (E)13 (A), E18 (C), and post-hatch day (P)1 (E). At E13 the gross retinal morphology of the rdd retina (B) is similar to that of the wt retina; however, the degenerative changes are obvious in the rdd retina at E18 (D) and P1 (F). Magnified images of the outer plexiform layer (OPL) and outer nuclear layer (ONL) show the progressive disorganization of the outer retinal layers of the rdd chick from E13 (I), E18 (J), and P1 (K).

Figure 2

Protein profiles from wild type (wt) and retina dysplasia and degeneration (rdd) retina. A-D: Representative 2D protein profiles from embryonic day (E)12, E13, E17, E19, and post hatch day (P)1 rdd and wt chick retina displaying positions of proteins that were identified by MS (Table 1). The positions of two housekeeping proteins (β-tubulin and actin) are also shown. Proteins were extracted using 40 mM ammonium bicarbonate; 1 mg of retinal protein was separated in the first dimension on an 18-cm pH 4–7 IPG strip and in the second dimension on a 12% polyacrylamide gel. The protein spots were visualized with Coomassie brilliant blue G-colloidal. B: Levels of actin and tubulin in the rdd and wt retina. γ-Actin and β-tubulin were identified by MS and used as loading controls for the gels. There was no significant difference in the levels of actin or tubulin in the wt and rdd gels from E12 to P1 (B). Bar charts show age plotted against arbitrary units, with SEM.

Figure 3

Differential expression of secernin 1 in the retinal dysplasia and degeneration (rdd) retina. A: Representative 2D montage images generated using Progenesis 2D image analysis software, revealing the modulated expression of secernin 1 in the wt and rdd chick. Two isoforms of secernin 1 (isoforms “a” and “b”) were identified by MS (arrows). The expression of isoform “a” is significantly increased from E13 onwards in the rdd retina, while it is only present at very low levels in the wt retina. B: 3D images of the 2D gels generated using Progenesis of the two isoforms of secernin 1 identified by MS. The increased expression of isoform “a” is evident from E13 onwards in the rdd retina. C: Graphical representation of expression of secernin 1 in the wt and rdd chick displaying the expression of isoform “a” (right panel), isoform “b” (middle panel), and total expression of secernin 1 (left panel). Age is plotted against average normalized volume (n=3±SEM).