Year-end Sale % OFF 
Abstract
Smith-Lemli-Opitz syndrome (SLOS) is one of the most common recessive human disorders and is characterized by multiple congenital malformations as well as neurosensory and cognitive abnormalities. A rat model of SLOS has been developed that exhibits progressive retinal degeneration and visual dysfunction; however, the molecular events underlying the degeneration and dysfunction remain poorly understood. Here, we employed a well-controlled, ion-current-based approach to compare retinas from the SLOS rat model to retinas from age- and sex-matched control rats (n = 5/group). Retinas were subjected to detergent extraction and subsequent precipitation and on-pellet-digestion procedures and then were analyzed on a long, heated column (75 cm, with small particles) with a 7-h gradient. The high analytical reproducibility of the overall proteomics procedure enabled reliable expression profiling. In total, 1,259 unique protein groups, ~40% of which were membrane proteins, were quantified under highly stringent criteria, including a peptide false discovery rate of 0.4%, with high quality ion-current data (e.g. signal-to-noise ratio ≥ 10) obtained independently from at least two unique peptides for each protein. The ion-current-based strategy showed greater quantitative accuracy and reproducibility over a parallel spectral counting analysis. Statistically significant alterations of 101 proteins were observed; these proteins are implicated in a variety of biological processes, including lipid metabolism, oxidative stress, cell death, proteolysis, visual transduction, and vesicular/membrane transport, consistent with the features of the associated retinal degeneration in the SLOS model. Selected targets were further validated by Western blot analysis and correlative immunohistochemistry. Importantly, although photoreceptor cell death was validated by TUNEL analysis, Western blot and immunohistochemical analyses suggested a caspase-3-independent pathway. In total, these results provide compelling new evidence implicating molecular changes beyond the initial defect in cholesterol biosynthesis in this retinal degeneration model, and they might have broader implications with respect to the pathobiological mechanism underlying SLOS.
Highlights
From the ‡Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, New York 14260; §New York State Center of Excellence in Bioinformatics and Life Sciences, 701 Ellicott Street, Buffalo, New York 14203; ¶Research Service, Veterans Administration Western New York Healthcare System, Buffalo, New York 14215; ʈDepartments of Ophthalmology and Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York 14260; **SUNY Eye Institute, Buffalo, New York 14215
We previously described progressive retinal degeneration in this rat model of Smith-Lemli-Opitz syndrome (SLOS), which is characterized by the shortening of retinal rod outer segments, pyknosis and thinning of the outer nuclear layer (ONL) of the retina, and accumulation of membranous/lipid inclusions in the retinal pigment epithelium (RPE) [12, 13]
Development and Evaluation of the Ion-current-based Procedure for Profiling of Retinal Proteome—In order to achieve a reliable discovery of altered proteins in the AY9944-induced retinal degeneration in the SLOS rat model, we developed a reproducible, extensive, and well-controlled proteomic strategy for profiling of the retinal proteome with 10 animals
Summary
ApoA, apolipoprotein A; ApoE, apolipoprotein E; AUC, area under the curve; CATD, cathepsin D; cSREBP, cleaved sterol regulatory element-binding protein; DHC, dehydrocholesterol; GFAP, glial fibrillary acidic protein; ONL, outer nuclear layer; RPE, retinal pigment epithelium; SLOS, Smith-LemliOpitz syndrome; SREBP, sterol regulatory element-binding protein; Stat, signal transducer and activator of transcription 3; TBS, Trisbuffered saline; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. The molecular mechanisms that underlie the observed electrophysiological defects in the retina, the accumulation of membranous/lipid inclusions in the RPE, the shortening of retinal rod outer segments, and the initiation of ONL pyknosis in the SLOS rat model remain poorly understood. We performed a reproducible, wellcontrolled, ion-current-based comparative proteomic analysis of the retinas from AY9944-treated versus age/sex-matched control rats (n ϭ 5 animals per group). A high-concentration detergent mixture was used for the efficient extraction of proteins from retinas, and samples underwent a reproducible precipitation/on-pellet-digestion procedure and longcolumn, 7-h nano-LC-MS analysis. These approaches ensured extensive comparative analysis of retina samples with. The altered proteins were subjected to functional annotation, and selected groups of proteins of interest were further validated by means of Western blot and correlative immunohistochemical analysis
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
