Differential Retinal Protein Expression in Primary and Secondary Retinal Ganglion Cell Degeneration Identified by Integrated SWATH and Target-Based Proteomics

To investigate the retinal proteins associated with primary and secondary retinal ganglion cell (RGC) degeneration and explore their molecular pathways, SWATH label-free and target-based mass spectrometry was employed to identify the proteomes in various retinal locations in response to localized optic nerve injury. Unilateral partial optic nerve transection (pONT) was performed on adult Wistar rats and their retinas were harvested 2 weeks later. To confirm the separation of primary and secondary RGC degeneration, immunohistochemistry of RNA binding protein with multiple splicing (RBPMS) and glial fibrillary acidic protein (GFAP) was performed on retinal whole-mounts. Retinal proteomes in the temporal and nasal quadrants were evaluated with high resolution hybrid quadrupole time-of-flight mass spectrometry (QTOF-MS), and SWATH-based acquisition, and their expression was compared to the corresponding retinal quadrant in contralateral control eyes and further validated by multiple reaction monitoring mass spectrometry (MRM-MS). A total of 3641 proteins (FDR < 1%) were identified using QTOF-MS. The raw data are available via ProteomeXchange with the identifier PXD026783. Bioinformatics data analysis showed that there were 37 upregulated and 25 downregulated proteins in the temporal quadrant, whereas 20 and five proteins were upregulated and downregulated, respectively, in the nasal quadrant, respectively (n = 4, p < 0.05; fold change ≥ 1.4-fold or ≤0.7). Six proteins were regulated in both the temporal and the nasal quadrants, including CLU, GFAP, GNG5, IRF2BPL, L1CAM, and CPLX1. Linear regression analysis indicated a strong association between the data obtained by means of SWATH-MS and MRM-MS (temporal, R2 = 0.97; nasal, R2 = 0.96). Gene ontology analysis revealed statistically significant changes in the biological processes and cellular components of primary RGC degeneration. The majority of the significant changes in structural, signaling, and cell death proteins were associated with the loss of RGCs in the area of primary RGC degeneration. The combined use of SWATH-MS and MRM-MS methods detects and quantifies regional changes of retinal protein expressions after localized injury. Future investigation with this integrated approach will significantly increase the understanding of diverse processes of progressive RGC degeneration from a proteomic prospective.

SWATH Based Quantitative Proteomics Reveals Significant Lipid Metabolism in Early Myopic Guinea Pig Retina

Most of the previous myopic animal studies employed a single-candidate approach and lower resolution proteomics approaches that were difficult to detect minor changes, and generated limited systems-wide biological information. Hence, a complete picture of molecular events in the retina involving myopic development is lacking. Here, to investigate comprehensive retinal protein alternations and underlying molecular events in the early myopic stage, we performed a data-independent Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) based proteomic analysis coupled with different bioinformatics tools in pigmented guinea pigs after 4-day lens-induced myopia (LIM). Myopic eyes compared to untreated contralateral control eyes caused significant changes in refractive error and choroid thickness (p < 0.05, n = 5). Relative elongation of axial length and the vitreous chamber depth were also observed. Using pooled samples from all individuals (n = 10) to build a species-specific retinal ion library for SWATH analysis, 3202 non-redundant proteins (with 24,616 peptides) were identified at 1% global FDR. For quantitative analysis, the 10 individual retinal samples (5 pairs) were analyzed using a high resolution Triple-TOF 6600 mass spectrometry (MS) with technical replicates. In total, 37 up-regulated and 21 down-regulated proteins were found significantly changed after LIM treatment (log2 ratio (T/C) > 0.26 or < −0.26; p ≤ 0.05). Data are accepted via ProteomeXchange with identifier PXD025003. Through Ingenuity Pathways Analysis (IPA), “lipid metabolism” was found as the top function associated with the differentially expressed proteins. Based on the protein abundance and peptide sequences, expression patterns of two regulated proteins (SLC6A6 and PTGES2) identified in this pathway were further successfully validated with high confidence (p < 0.05) using a novel Multiple Reaction Monitoring (MRM) assay on a QTRAP 6500+ MS. In summary, through an integrated discovery and targeted proteomic approach, this study serves as the first report to detect and confirm novel retinal protein changes and significant biological functions in the early LIM mammalian guinea pigs. The study provides new workflow and insights for further research to myopia control.

Circadian Rhythm Disruption Results in Visual Dysfunction

Circadian rhythm disruption (CRD) contributes to the development of multiple metabolic and neurodegenerative diseases. However, its effect on vision is not understood. We evaluated the impact of CRD on retinal morphology, physiology, and vision after housing mice in a disruption inducing shorter light/dark cycle (L10:D10). Interestingly, the mice under L10:D10 exhibited three different entrainment behaviors; 'entrained', 'freerunning', and 'zigzagging.' These behavior groups under CRD exhibited reduced visual acuity, retinal thinning, and a decrease in the number of rod photoreceptors. Intriguingly, the electroretinogram response was decreased only in the mice exhibiting 'entrained' behavior. The retinal proteome showed distinct changes with each entrainment behavior. These results demonstrate that CRD leads to photoreceptor degeneration and visual dysfunction. We uniquely show the effect of entrainment behavior on retinal protein composition and physiology. Our data has broader implications in understanding and mitigating the effect of CRD on vision health.

Cryo-EM of retinoschisin branched networks suggests an intercellular adhesive scaffold in the retina

Mutations in the retinal protein retinoschisin (RS1) cause progressive loss of vision in young males, a form of macular degeneration called X-linked retinoschisis (XLRS). We previously solved the structure of RS1, a 16-mer composed of paired back-to-back octameric rings. Here, we show by cryo–electron microscopy that RS1 16-mers can assemble into extensive branched networks. We classified the different configurations, finding four types of interaction between the RS1 molecules. The predominant configuration is a linear strand with a wavy appearance. Three less frequent types constitute the branch points of the network. In all cases, the “spikes” around the periphery of the double rings are involved in these interactions. In the linear strand, a loop (usually referred to as spike 1) occurs on both sides of the interface between neighboring molecules. Mutations in this loop suppress secretion, indicating the possibility of intracellular higher-order assembly. These observations suggest that branched networks of RS1 may play a stabilizing role in maintaining the integrity of the retina.

2064 Regulation of retinal protein O-GlcNAcylation by angiotensin-(1-7) and cAMP

OBJECTIVES/SPECIFIC AIMS: Increased retinal protein O-GlcNAcylation occurs in response to hyperglycemia and contributes to diabetic retinopathy. Renin-angiotensin system (RAS) blockers reduce the incidence of diabetic retinopathy. Beneficial effects of RAS blockers are often attributed to production of angiotensin-(1-7) (Ang1-7). The objective here is to determine the impact of Ang1-7 on retinal protein O-GlcNAcylation. METHODS/STUDY POPULATION: C57/BL6 mice were fed a high-fat diet for 8 weeks and then treated for 3 weeks with either a vehicle control, the RAS blocker captopril, or captopril and the Ang1-7 receptor antagonist A779. R28 cells were used to assess levels of O-GlcNAcylated proteins in response to Ang1-7, and the role of cAMP was investigated with addition of forskolin, 6-Bnz-cAMP-AM, and 8-pCPT-2-O-Me-cAMP-AM to cell culture medium. RESULTS/ANTICIPATED RESULTS: Captopril attenuated retinal protein O-GlcNAcylation in mice fed a high-fat diet. This effect was reversed by A779. Ang1-7 attenuated protein O-GlcNAcylation and increased cAMP levels. Forskolin and the EPAC selective cAMP analog 8-pCPT-2-O-Me-cAMP-AM, but not the PKA selective cAMP analog 6-Bnz-cAMP-AM, attenuated O-GlcNAcylation. Inhibiting EPAC blocked the effect of forskolin, whereas inhibiting PKA did not. DISCUSSION/SIGNIFICANCE OF IMPACT: This study demonstrates a novel role for Ang1-7 in the retina and identifies a potential EPAC-dependent mechanism that regulates protein O-GlcNAcylation. Thus, future therapeutics targeted at an Ang1-7/EPAC axis in retina may be used to address DR.

Effect of point mutations on the ultrafast photo-isomerization of Anabaena sensory rhodopsin

Tuning excited state potentials and dynamics of a retinal protein through point mutations.