Abstract
Pathogenesis in the Rpe65−/− mouse model of Leber's congenital amaurosis (LCA) is characterized by a slow and progressive degeneration of the rod photoreceptors. On the opposite, cones degenerate rapidly at early ages. Retinal degeneration in Rpe65 −/− mice, showing a null mutation in the gene encoding the retinal pigment epithelium 65-kDa protein (Rpe65), was previously reported to depend on continuous activation of a residual transduction cascade by unliganded opsin. However, the mechanisms of apoptotic signals triggered by abnormal phototransduction remain elusive. We previously reported that activation of a Bcl-2-dependent pathway was associated with apoptosis of rod photoreceptors in Rpe65−/− mice during the course of the disease. In this study we first assessed whether activation of Bcl-2-mediated apoptotic pathway was dependent on constitutive activation of the visual cascade through opsin apoprotein. We then challenged the direct role of pro-apoptotic Bax protein in triggering apoptosis of rod and cone photoreceptors.Quantitative PCR analysis showed that increased expression of pro-apoptotic Bax and decreased level of anti-apoptotic Bcl-2 were restored in Rpe65−/−/Gnat1−/− mice lacking the Gnat1 gene encoding rod transducin. Moreover, photoreceptor apoptosis was prevented as assessed by TUNEL assay. These data indicate that abnormal activity of opsin apoprotein induces retinal cell apoptosis through the Bcl-2-mediated pathway. Following immunohistological and real-time PCR analyses, we further observed that decreased expression of rod genes in Rpe65-deficient mice was rescued in Rpe65−/−/Bax−/− mice. Histological and TUNEL studies confirmed that rod cell demise and apoptosis in diseased Rpe65−/− mice were dependent on Bax-induced pathway. Surprisingly, early loss of cones was not prevented in Rpe65−/−/Bax−/− mice, indicating that pro-apoptotic Bax was not involved in the pathogenesis of cone cell death in Rpe65-deficient mice.This is the first report, to our knowledge, that a single genetic mutation can trigger two independent apoptotic pathways in rod and cone photoreceptors in Rpe65-dependent LCA disease. These results highlight the necessity to investigate and understand the specific death signaling pathways committed in rods and cones to develop effective therapeutic approaches to treat RP diseases.
Highlights
Mutations in the gene encoding the protein RPE65 are associated with several forms of inherited retinal dystrophies, such as autosomal recessive retinitis pigmentosa (RP) [1] and autosomal recessive childhood-onset severe retinal dystrophy [2] which are characterized by profound visual deficiency, night blindness and reduced or non detectable electroretinogram (ERG)
Related signaling pathway was associated with retinal degeneration in retinal pigment epithelium 65-kDa protein (Rpe65)-deficient mice, led us to investigate whether this apoptotic pathway was triggered by light-independent, constitutive activity of the phototransduction cascade
As depicted in figure 1A, whereas Bcl2-associated X protein (Bax) expression was increased by 1.5-fold in 6 and 12 month-old Rpe65deficient mice, transcriptional upregulation of the pro-apoptotic gene was abolished in Rpe652/2/Gnat12/2 mice in which phototransduction signaling was blocked in the absence of functional rod transducin
Summary
Mutations in the gene encoding the protein RPE65 are associated with several forms of inherited retinal dystrophies, such as autosomal recessive retinitis pigmentosa (RP) [1] and autosomal recessive childhood-onset severe retinal dystrophy [2] which are characterized by profound visual deficiency, night blindness and reduced or non detectable electroretinogram (ERG). LCA consists in a severe form of early-onset autosomal recessive RP and is due in 6 to 15% of the cases to mutations in the Rpe gene [3]. In the absence of the RPE65 protein, rod opsin apoprotein triggers light-independent, constitutive activation of the visual cascade resulting in photoreceptor cell loss [10]. Human RPE65-LCA is characterized as a rod-cone dystrophy with severe retinal defect in the first decades of life. The clinical outcome of this disease has mainly been attributed to rod and cone loss, the molecular pathways leading to retinal cell death still remain to be identified [11,12]
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