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Quantitative profiling of regional protein expression in rat retina after partial optic nerve transection using fluorescence difference two?dimensional gel electrophoresis.

ABSTRACT: To examine the difference between primary and secondary retinal ganglion cell (RGC) degeneration, the protein expression at four regions of retina including superior, temporal, inferior and nasal quadrant in a rat model of partial optic nerve transection (pONT) using 2?D Fluorescence Difference Gel Electrophoresis (DIGE) were investigated. Unilateral pONT was performed on the temporal side of optic nerves of adult Wistar rats to separate primary and secondary RGC loss. Topographical quantification of RGCs labeled by Rbpms antibody and analysis of axonal injury by grading of optic nerve damage at 1 week (n=8) and 8 weeks (n=15) after pONT demonstrated early RGC loss at temporal region, which is considered as primary RGC degeneration and progressing RGC loss at nasal region, which is considered as secondary RGC degeneration. Early protein expression in each retinal quadrant (n=4) at 2 weeks after pONT was compared with the corresponding quadrant in the contralateral control eye by DIGE. For all comparisons, 24 differentially expressed proteins (>1.2?fold; P<0.05; ?3 non?duplicated peptide matches) were identified by mass spectrometry (MS). Interestingly, in the nasal retina, serum albumin and members of crystallin family, including ?A, ?B, ?A2, ?A3, ?B2 and gamma S indicating stress response were upregulated. By contrast, only ?B and ?A2 crystallin proteins were altered in temporal quadrant. In the superior and inferior quadrants, ?B2 crystallin, keratin type I, S?arrestin and lamin?B1 were upregulated, while heat shock cognate 71 kDa protein and heterogeneous nuclear ribonucleoproteins A2/B1 were downregulated. In summary, the use of DIGE followed by MS is useful to detect early regional protein regulation in the retina after localized optic nerve injury.

SUBMITTER: Lam C

PROVIDER: S-EPMC6691257 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Quantitative profiling of regional protein expression in rat retina after partial optic nerve transection using fluorescence difference two‑dimensional gel electrophoresis.

Lam Chuen C Li King Kit KK Do Chi Wai CW Chan Henry H To Chi Ho CH Kwong Jacky Man Kwong JMK

Molecular medicine reports 20190725 3

To examine the difference between primary and secondary retinal ganglion cell (RGC) degeneration, the protein expression at four regions of retina including superior, temporal, inferior and nasal quadrant in a rat model of partial optic nerve transection (pONT) using 2‑D Fluorescence Difference Gel Electrophoresis (DIGE) were investigated. Unilateral pONT was performed on the temporal side of optic nerves of adult Wistar rats to separate primary and secondary RGC loss. Topographical quantificati ...[more]

PMID: 31524249

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Project description:PurposeA time-course analysis of gene regulation in the adult rat retina after intraorbital nerve crush (IONC) and intraorbital nerve transection (IONT).MethodsRNA was extracted from adult rat retinas undergoing either IONT or IONC at increasing times post-lesion. Affymetrix RAE230.2 arrays were hybridized and analyzed. Statistically regulated genes were annotated and functionally clustered. Arrays were validated by means of quantative reverse transcription polymerase chain reaction (qRT-PCR) on ten regulated genes at two times post-lesion. Western blotting and immunohistofluorescence for four pro-apoptotic proteins were performed on naïve and injured retinas. Finally, custom signaling maps for IONT- and IONC-induced death response were generated (MetaCore, Genego Inc.).ResultsHere we show that over time, 3,219 sequences were regulated after IONT and 1,996 after IONC. Out of the total of regulated sequences, 1,078 were commonly regulated by both injuries. Interestingly, while IONT mainly triggers a gene upregulation-sustained over time, IONC causes a transitory downregulation. Functional clustering identified the regulation of high interest biologic processes, most importantly cell death wherein apoptosis was the most significant cluster. Ten death-related genes upregulated by both injuries were used for array validation by means of qRT-PCR. In addition, western blotting and immunohistofluorescence of total and active Caspase 3 (Casp3), tumor necrosis factor receptor type 1 associated death domain (TRADD), tumor necrosis factor receptor superfamily member 1a (TNFR1a), and c-fos were performed to confirm their protein regulation and expression pattern in naïve and injured retinas. These analyses demonstrated that for these genes, protein regulation followed transcriptional regulation and that these pro-apoptotic proteins were expressed by retinal ganglion cells (RGCs). MetaCore-based death-signaling maps show that several apoptotic cascades were regulated in the retina following optic nerve injury and highlight the similarities and differences between IONT and IONC in cell death profiling.ConclusionsThis comprehensive time course retinal transcriptome study comparing IONT and IONC lesions provides a unique valuable tool to understand the molecular mechanisms underlying optic nerve injury and to design neuroprotective protocols.

Dataset Information

Quantitative profiling of regional protein expression in rat retina after partial optic nerve transection using fluorescence difference two?dimensional gel electrophoresis.

Publications

Quantitative profiling of regional protein expression in rat retina after partial optic nerve transection using fluorescence difference two‑dimensional gel electrophoresis.

Similar Datasets

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