Quantitative proteomic analysis reveals temporal regulation of aldehyde dehydrogenase 1A1 in the rat retina after partial optic nerve transection

Abstract

Purpose : To explore the retinal proteome profile at times after localized optic nerve injury and compare the protein expressions at the regions characterized by primary and secondary retinal ganglion cell (RGC) degeneration Methods : Unilateral partial optic nerve transection (pONT) was performed on the temporal side of the optic nerve in adult Wistar rats. The RGC density from 1 to 8 weeks after pONT was topographically quantified with Rbpms antibody. Temporal and nasal retinal samples were collected separately from the eyes after pONT and soluble proteins (n=4; 3 technical replicates) were subjected to profiling with a high resolution hybrid quadrupole time-of-flight MS running on label-free SWATHTM acquisition (SCIEX). An information dependent acquisition ion library was generated from all individual biological replicates for SWATHTM peptides quantification. MS spectra were searched for protein identification with ProteinPilot 5.0 (SCIEX) using the Paragon algorithm. Cellular localization of significantly regulated proteins (P<0.05; FC >1.4 or <0.7) using immunohistochemistry was performed. Results : There was 78.9±5.8% and 27.7±6.0% of RGC survival in the temporal quadrant at 1 and 8 weeks respectively indicating primary RGC degeneration. No change in RGC density was observed in the nasal quadrant at 1 week after pONT (n=8) but the percentage loss increased to 43.6±7.7% at 8 weeks (n=15; P=0.0001) demonstrating secondary RGC degeneration. A total of 3641 proteins (>25,000 peptides) with FDR<1% were identified in the rat retinas as an ion library. Compared to nasal retina, 6, 12 and 65 differentially expressed proteins were detected in the temporal retina at 1, 4, and 8 weeks respectively. Approximately 6, 4 and 3 folds upregulation of aldehyde dehydrogenase 1A1 (ALDH1A1) was noted in the temporal retina at 1, 4 and 8 weeks respectively. Immunohistochemistry showed that increased immunoreactivity of ALDH1A1 was predominately localized to Muller cells at the temporal retina at 1 week but not nasal retina, and the upregulation was diminished at 4 and 8 weeks. Conclusions : The finding demonstrated differential protein expression between primary and secondary RGC degeneration. The temporal change of ALDH1A1 suggests that Muller cells respond to localized injury and may play a role in detoxification during progressive RGC degeneration.