Abstract
Purpose: :
To investigate the proteomic differences in retinal tissue after gene therapy in retinal degeneration 12 (rd12) mice.
Methods: :
1 μl scAAV5-smCBA-hRPE65 was injected subretinally in right eyes of rd12 mice at P14; left eyes remained uninjected as a control. Age-matched C57BL/6J was set up as a comparison group. Electroretinograms (ERG) in dark- and light-adapted conditions were recorded at P21 and P42. Total retinal protein content of each group was extracted at P14, P21 and P42. Protein expression was conducted by 2-DE. Proteins were identified by MALDI-TOF. Immunohistochemistry (IHC) was performed to locate the identified proteins, which were further verified by western blot and RT-PCR. The retinal reactive oxygen species (ROS) were detected by IHC, while TUNEL was used to detect apoptosis. Müller cells culture (MIO-M1) was used to understand the role of PRDX6, a protein of interest.
Results: :
Four weeks after injection (P42), the treated rd12 eyes demonstrated significant amplitudes increase in dark-adapted and light-adapted ERGs, while no rod ERG response was detected in untreated contralateral eyes. Proteomic profiling found differential proteins betweenuntreated -rd12 and treated -rd12 or wild-type eyes. Overall, 39 differential proteins were identified, 3 of which were selected for further tests. Alpha A crystallin (CRYAA), heat shock protein 70 (HSP70) and peroxiredoxin 6 (PRDX6) were up-regulated in untreated-rd12 eyes and not in treated-rd12 eyes or wild-type eyes. IHC confirmed the existence of these 3 proteins in retina, as well as the up-regulation in untreated-rd12 eyes, while Western blot confirmed the 2-DE results. RT-PCR results indicated that the untreated -rd12 eyes had increased HSP70 and PRDX6 mRNA levels, while CRYAA mRNA levels were decreased. ROS increased only in the untreated group, mainly in the outer nuclear layer where TUNEL indicated signs of apoptosis. The treated rd12 eyes did not show ROS increase or presence of apoptosis. MIO-M1 cells overexpressing PRDX6 displayed higher survival rate.
Conclusions: :
Proteomic analysis showed significant differences among groups at P42 with many differential proteins identified. Gene therapy could restore retinal function and protein expression in rd12 eyes. The up-regulation of CRYAA, HSP70 and PRDX6 in treated rd12 eyes could play a role in preventing cell death. PRDX6 could be an important antioxidant protector in the mouse retina.
Keywords: gene transfer/gene therapy • proteomics • retinal degenerations: hereditary