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S. Cottet, S. Métrailler, D. F. Schorderet; Triggering Bcl-2-Related Apoptotic Pathway in Rpe65-/- Mouse Model of Leber's Congenital Amaurosis. Invest. Ophthalmol. Vis. Sci. 2007;48(13):557.
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© ARVO (1962-2015); The Authors (2016-present)
Although apoptosis is a common pathway for photoreceptor degeneration in animal models of RP, the cellular and molecular events leading to cell death in the Rpe65-/- mouse model of LCA remain largely unknown. The Bcl-2 proteins can either promote cell survival or cell death, depending on the relative amount between pro- and anti-apoptotic members. Following cytotoxic signals the major Bax protein, Bax α isoform containing C-terminal transmembrane domain, translocates to mitochondria which triggers irreversible commitment to apoptosis. Altered regulation of Bcl-2-related proteins occurs in several forms of retinal injury, although the reported results remain controversial. It is yet not known whether these apoptotic regulators may impact on the death of photoreceptors in LCA pathology.
The amount of Bcl-2 proteins was assessed in wt and Rpe65-/- retinas from 2, 4, 6 and 12 month-old animals. Translocation of Bax to mitochondria was assessed by Western blot following subcellular fractionation of retinal cell extracts. Bax isoforms were identified by RT-PCR.
Expression of anti-apoptotic Bcl-2 is altered, as reflected by the decrease of protein level in 2 to 12 month-old mutant retina. Increased expression of both α and ß isoforms of pro-apoptotic Bax, as identified by RT-PCR, is also observed. Impaired equilibrium between anti- and pro-apoptotic proteins is illustrated by decreased ratio of Bcl-2 to Bax during progression of the disease. Bax α is increased in both cytosolic and mitochondria-enriched fractions, but the ratio of mitochondrial to cytoplasmic protein does not change between wt and ko retina. Surprisingly, simultaneous decrease of cytosolic and increase of mitochondrial Bax ß isoform, lacking C-terminus membrane anchor, is observed. Translocation of Bax ß to mitochondria increases as the disease progresses, as reflected by increased ratio of mitochondrial to cytoplasmic protein in retina from 2 to 6 month-old Rpe65-/- mice.
These results demonstrate that RPE65 defect leads to altered equilibrium between anti- and pro-apoptotic Bcl-2 members, thus favoring the balance towards apoptosis. Decreased expression of Bcl-2, paralleled with mitochondrial translocation of Bax suggest that Bcl-2-related intracellular pathway triggers apoptosis in Rpe65-deficient retina. Furthermore, specific translocation of Bax ß isoform to mitochondria might represent a new regulatory mechanism of Bax-dependent apoptosis in retinal degeneration.
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