Leber congenital amaurosis (LCA) is one of the most severe forms of inherited retinal degeneration and can be caused by mutations in at least 15 different genes. To clarify the proteomic differences in LCA eyes, a cohort of retinal degeneration 12 (rd12) mice, an LCA2 model caused by a mutation in the RPE65 gene, were injected subretinally with an AAV vector (scAAV5-smCBA-hRPE65) in one eye, while the contralateral eye served as a control. Proteomics were compared between untreated rd12 and normal control retinas on P14 and P21, and among treated and untreated rd12 retinas and control retinas on P42. Gene therapy in rd12 mice restored retinal function in treated eyes, which was demonstrated by electroretinography (ERG). Proteomic analysis successfully identified 39 proteins expressed differently among the 3 groups. The expression of 3 proteins involved in regulation of apoptosis and neuroptotection (alpha A crystallin, heat shock protein 70 and peroxiredoxin 6) were investigated further. Immunofluorescence, Western blot and real-time PCR confirmed the quantitative changes in their expression. Furthermore, cell culture studies suggested that peroxiredoxin 6 could act in an antioxidant role in rd12 mice. Our findings support the feasibility of gene therapy in LCA2 patients and support a role for alpha A crystallin, heat shock protein 70 and peroxiredoxin 6 in the pathogenetic mechanisms involved in LCA2 disease process.
Tzekov RT, Liu Y, Sohocki MM, Zack DJ, Daiger SP, et al. (2001) Autosomal dominant retinal degeneration and bone loss in patients with a 12-bp deletion in the CRX gene. Invest Ophthalmol Vis Sci 42: 1319–1327.
Pelletier V, Jambou M, Delphin N, Zinovieva E, Stum M, et al. (2007) Comprehensive survey of mutations in RP2 and RPGR in patients affected with distinct retinal dystrophies: genotype-phenotype correlations and impact on genetic counseling. Hum Mutat 28: 81–91.
Morimura H, Fishman GA, Grover SA, Fulton AB, Berson EL, et al. (1998) Mutations in the RPE65 gene in patients with autosomal recessive retinitis pigmentosa or leber congenital amaurosis. Proc Natl Acad Sci U S A 95: 3088–3093.
Hamel CP, Tsilou E, Pfeffer BA, Hooks JJ, Detrick B, et al. (1993) Molecular cloning and expression of RPE65, a novel retinal pigment epithelium-specific microsomal protein that is post-transcriptionally regulated in vitro. J Biol Chem 268: 15751–15757.
Znoiko SL, Rohrer B, Lu K, Lohr HR, Crouch RK, et al. (2005) Downregulation of cone-specific gene expression and degeneration of cone photoreceptors in the Rpe65？/？ mouse at early ages. Invest Ophthalmol Vis Sci 46: 1473–1479.
Pang JJ, Chang B, Kumar A, Nusinowitz S, Noorwez SM, et al. (2006) Gene therapy restores vision-dependent behavior as well as retinal structure and function in a mouse model of RPE65 Leber congenital amaurosis. Mol Ther 13: 565–572.
Narfstrom K, Bragadottir R, Redmond TM, Rakoczy PE, van Veen T, et al. (2003) Functional and structural evaluation after AAV. RPE65 gene transfer in the canine model of Leber's congenital amaurosis. Adv Exp Med Biol 533: 423–430.
Le Meur G, Stieger K, Smith AJ, Weber M, Deschamps JY, et al. (2007) Restoration of vision in RPE65-deficient Briard dogs using an AAV serotype 4 vector that specifically targets the retinal pigmented epithelium. Gene Ther 14: 292–303.
Acland GM, Aguirre GD, Bennett J, Aleman TS, Cideciyan AV, et al. (2005) Long-term restoration of rod and cone vision by single dose rAAV-mediated gene transfer to the retina in a canine model of childhood blindness. Mol Ther 12: 1072–1082.
Hauswirth WW, Aleman TS, Kaushal S, Cideciyan AV, Schwartz SB, et al. (2008) Treatment of leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: short-term results of a phase I trial. Hum Gene Ther 19: 979–990.
Simonelli F, Ziviello C, Testa F, Rossi S, Fazzi E, et al. (2007) Clinical and molecular genetics of Leber's congenital amaurosis: a multicenter study of Italian patients. Invest Ophthalmol Vis Sci 48: 4284–4290.
Jacobson SG, Cideciyan AV, Ratnakaram R, Heon E, Schwartz SB, et al. (2012) Gene therapy for leber congenital amaurosis caused by RPE65 mutations: safety and efficacy in 15 children and adults followed up to 3 years. Arch Ophthalmol 130: 9–24.
Lai CM, Yu MJ, Brankov M, Barnett NL, Zhou X, et al. (2004) Recombinant adeno-associated virus type 2-mediated gene delivery into the Rpe65？/？ knockout mouse eye results in limited rescue. Genet Vaccines Ther 2: 3.
Pawlyk BS, Smith AJ, Buch PK, Adamian M, Hong DH, et al. (2005) Gene replacement therapy rescues photoreceptor degeneration in a murine model of Leber congenital amaurosis lacking RPGRIP. Invest Ophthalmol Vis Sci 46: 3039–3045.
Li W, Kong F, Li X, Dai X, Liu X, et al. (2009) Gene therapy following subretinal AAV5 vector delivery is not affected by a previous intravitreal AAV5 vector administration in the partner eye. Mol Vis 15: 267–275.
Li X, Li W, Dai X, Kong F, Zheng Q, et al. (2011) Gene therapy rescues cone structure and function in the 3-month-old rd12 mouse: a model for midcourse RPE65 leber congenital amaurosis. Invest Ophthalmol Vis Sci 52: 7–15.
Pang J, Boye SE, Lei B, Boye SL, Everhart D, et al. (2010) Self-complementary AAV-mediated gene therapy restores cone function and prevents cone degeneration in two models of Rpe65 deficiency. Gene Ther 17: 815–826.
Cavusoglu N, Thierse D, Mohand-Said S, Chalmel F, Poch O, et al. (2003) Differential proteomic analysis of the mouse retina: the induction of crystallin proteins by retinal degeneration in the rd1 mouse. Mol Cell Proteomics 2: 494–505.
Hidalgo-de-Quintana J, Evans RJ, Cheetham ME, van der Spuy J (2008) The Leber congenital amaurosis protein AIPL1 functions as part of a chaperone heterocomplex. Invest Ophthalmol Vis Sci 49: 2878–2887.
Fatma N, Kubo E, Sen M, Agarwal N, Thoreson WB, et al. (2008) Peroxiredoxin 6 delivery attenuates TNF-alpha-and glutamate-induced retinal ganglion cell death by limiting ROS levels and maintaining Ca2+ homeostasis. Brain Res 1233: 63–78.
Deretic D, Aebersold RH, Morrison HD, Papermaster DS (1994) Alpha A- and alpha B-crystallin in the retina. Association with the post-Golgi compartment of frog retinal photoreceptors. J Biol Chem 269: 16853–16861.
Whitlock NA, Lindsey K, Agarwal N, Crosson CE, Ma JX (2005) Heat shock protein 27 delays Ca2+-induced cell death in a caspase-dependent and -independent manner in rat retinal ganglion cells. Invest Ophthalmol Vis Sci 46: 1085–1091.