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.

Glutamate-induced retinal lipid and protein damage: the protective effects of catechin.

Glutamate toxicity has been implicated in various retinal diseases. Green tea leaf extract catechin has protective effects against cellular toxicity. This study investigated the effects of catechin on the glutamate-treated retina. Porcine retinal homogenates were incubated with glutamate (20 nmol) at 37 degrees C for 60 min. Catechin was co-incubated with the glutamate-treated retina in the same condition. The malondialdehyde (MDA) levels were determined as an index of lipid peroxidation (LPO). Differential protein expressions were derived from two-dimensional gel electrophoresis. Mass spectrometry was conducted to identify the proteins. Glutamate increased the retinal MDA (p<0.0001) and catechin reversed the effect (p<0.0001). There were significant changes in seven proteins after the glutamate treatment (p<0.05), namely, heterogeneous ribonucleoprotein, thioredoxin peroxidase, 5-hydroxytryptamine receptor, pyruvate dehydrogenase, ARHA protein, peroxiredoxin 6 and proteasome. Catechin significantly reversed the changes in thioredoxin peroxidase, 5-hydroxytryptamine receptor, peroxiredoxin 6 and pyruvate dehydrogenase (p<0.05). Our study shows that (a) retinal glutamate toxicity is mediated by LPO and protein modification, and (b) catechin ameliorates the toxicity.

Total retinal nitric oxide production is increased in intraocular pressure-elevated rats.

Nitric oxide (NO) is a well-known vaso-dilator but its regulation in the retina is unclear. This study was conducted to quantify total NO production and retinal ganglion cell (RGC) loss in an experimental glaucoma model. Three quarters of the peri-limbal/episcleral drainage vessels and anterior angle (right eyes) of Sprague-Dawley rats were thermally blocked using laser irradiation, while the left eyes served as controls. We measured the intraocular pressure (IOP) of both eyes using a digital tonometer (Tonopen) 21, 28 and 35 days after the laser treatment. After 35 days, we determined the total NO level in retinas and remaining ocular tissues for the laser-treated and control eyes using a spectro-photometric assay. The viable RGC numbers were also determined by counting the cell bodies stained retrogradely by fluoro-gold. The laser treatment significantly increased the IOP 2.0-2.6x throughout the whole periodof measurements (P<0.0001). The mean total RGC number decreased significantly from 98725+/-5383 (+/-S.E. (M.)) to 69276+/-5592, or 29.8% reduction, in the laser-treated eyes after 35 days (P=0.008). The mean total NO level in the laser-treated retina was significantly increased by 2.4x compared with controls (P=0.016), but no significant difference was found in the eyecups (P>0.05). Laser treatment resulted in significant IOP elevation and RGC loss, suggesting that thermal coagulation of the perilimbal region may provide an alternate protocol for glaucoma study. NO level was increased by two-fold in the retina but not in other ocular tissues. Since NO is capable of producing powerful peroxynitrite anions and hydroxyl radicals, elevated level of NO has a potential role in glaucoma.